Nanoarchaeum genome, Nanoarchaeum polypeptides and nucleic acids encoding them and methods for making and using them

REFERENCE TO SEQUENCE LISTING SUBMITTED ON A COMPACT DISC

This application includes a compact disc (submitted in duplicate) containing a sequence listing. The entire content of the sequence listing is herein incorporated by reference. The sequence listing is identified on the compact disc as follows.

File Name
Date of Creation
Size (bytes)

Sequence Listing.txt
May 1, 2003
2,592,438

FIELD OF THE INVENTION

This invention relates generally to microbiology. In alternative aspects, the invention provides a genome of the hyperthermophile Nanoarchaeum equitans, polypeptides, including enzymes, structural proteins and binding proteins derived from this genome, polynucleotides encoding these polypeptides, methods of making and using the genome and these polynucleotides and polypeptides. The invention also provides isolated hyperthermophile Nanoarchaeum equitans.

BACKGROUND

Several theoretical and experimental studies have endeavored to derive the minimal set of genes that are necessary and sufficient to sustain a functioning cell under ideal conditions, that is, in the presence of unlimited amounts of all essential nutrients and in the absence of any adverse factors, including competition. A comparison of the first two completed bacterial genomes, those of the parasites Haemophilus influenzae and Mycoplasma genitalium, produced a version of the minimal gene set consisting of approximately 250 genes. Very similar estimates were obtained by analyzing viable gene knockouts in Bacillus subtilis, M. genitalium, and Mycoplasma pneumoniae. With the accumulation and comparison of multiple complete genome sequences, it became clear that only about 80 genes of the 250 in the original minimal gene set are represented by orthologs in all life forms. For approximately 15% of the genes from the minimal gene set, viable knockouts were obtained in M. genitalium. Unexpectedly, these included even some of the universal genes. Thus, some of the genes that were included in the first version of the minimal gene set, based on a limited genome comparison, could be, in fact, dispensable. The majority of these genes, however, are likely to encode essential functions but, in the course of evolution, are subject to non-orthologous gene displacement, that is, recruitment of unrelated or distantly related proteins for the same function. Further theoretical and experimental studies within the framework of the minimal-gene-set concept and the ultimate construction of a minimal genome are expected to advance our understanding of the basic principles of cell functioning by systematically detecting non-orthologous gene displacement and deciphering the roles of essential but functionally uncharacterized genes. Global knockout mutagenesis of the mycoplasmal genes, aimed at delineating a minimal gene set, has resulted in estimates that are very similar to those produced by original comparative genomic analysis but has also shown that even some of the universal or highly conserved genes can be dispensable. These results could indicate that even absolute evolutionary conservation does not automatically entail indispensability of a gene under any conditions, but their definitive interpretation requires further experiments.

SUMMARY OF THE INVENTION

The invention provides an isolated and/or a recombinant genome of a hyperthermophile Nanoarchaeum equitans (SEQ ID NO:1). The invention also provides an isolated hyperthermophile Nanoarchaeum equitans deposited as ATCC accession no. ______.

The invention provides isolated or recombinant nucleic acids comprising a nucleic acid sequence having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, and all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073, over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400, 2450, 2500, or more residues, encodes at least one polypeptide having an enzyme, structural or binding activity, and the sequence identities are determined by analysis with a sequence comparison algorithm or by a visual inspection.

In alternative aspects, the isolated or recombinant nucleic acid encodes a polypeptide comprising a sequence as set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, and all polypeptides disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:3 through SEQ ID NO:1073. In one aspect these polypeptides have an enzyme, structural or binding activity.

In one aspect, the sequence comparison algorithm is a BLAST algorithm, such as a BLAST version 2.2.2 algorithm. In one aspect, the filtering setting is set to blastall -p blastp -d “nr pataa”-F F and all other options are set to default.

In alternative aspects, the enzyme, structural or binding activity comprises a recombinase activity, a helicase activity, a DNA replication activity, a DNA recombination activity, an isomerase, a transisomerase activity or topoisomerase activity, a methyl transferase activity, an aminotransferase activity, a uracil-5-methyl transferase activity, a cysteinyl tRNA synthetase activity, a hydrolase, an esterase activity, a phosphoesterase activity, an acetylmuramyl pentapeptide phosphotransferase activity, a glycosyltransferase activity, an acetyltransferase activity, an acetylglucosamine phosphate transferase activity, a centromere binding activity, a telomerase activity or a transcriptional regulatory activity, a heat shock protein activity, a protease activity, a proteinase activity, a peptidase activity, a carboxypeptidase activity, an endonuclease activity, an exonuclease activity, a RecB family exonuclease activity, a polymerase activity, a carbamoyl phosphate synthetase activity, a methyl-thioadenine synthetase activity, an oxidoreductase activity, an Fe—S oxidoreductase activity, a flavodoxin reductase activity, a permease activity, a thymidylate activity, a dehydrogenase activity, a pyrophosphorylase activity, a coenzyme metabolism activity, a dinucleotide-utilizing enzyme activity, a molybdopterin or thiamine biosynthesis activity, a beta-lactamase activity, a ligand binding activity, an ion transport activity, an ion metabolism activity, a tellurite resistance protein activity, an inorganic ion transport activity, a nucleotide transport activity, a nucleotide metabolism activity, an actin or myosin activity, a lipase activity or a lipid acyl hydrolase (LAH) activity, a cell envelop biogenesis activity, an outer membrane synthesis activity, a ribosomal structure synthesis activity, a translational processing activity, a transcriptional initiation activity, a TATA-binding activity, a signal transduction activity, an energy metabolism activity, an ATPase activity, an information storage and/or processing activity, and/or any of the polypeptides activities as set forth in Table 3, below.

In one aspect, the isolated or recombinant nucleic acid encodes a polypeptide having an enzyme, structural or binding activity which is thermostable. The polypeptide can retain an enzyme, structural or binding activity under conditions comprising a temperature range of between about 37° C. to about 95° C.; between about 55° C. to about 85° C., between about 70° C. to about 95° C., or, between about 90° C. to about 95° C., about 96° C., about 97° C., about 98° C., or more. In another aspect, the isolated or recombinant nucleic acid encodes a polypeptide having an enzyme, structural or binding activity which is thermotolerant. The polypeptide can retain an enzyme, structural or binding activity after exposure to a temperature in the range from greater than 37° C. to about 95° C., about 96° C., about 97° C., about 98° C., or more or anywhere in the range from greater than 55° C. to about 85° C. In one aspect, the polypeptide retains an enzyme, structural or binding activity after exposure to a temperature in the range from greater than 90° C. to about 95° C., about 96° C., about 97° C., about 98° C., or more, at about pH 7, pH 6.5, pH 6.0, pH 5.5, pH 5, or pH 4.5.

The polypeptide can retain an enzyme, structural or binding activity under conditions comprising about pH 7, pH 6.5, pH 6.0, pH 5.5, pH 5, or pH 4.5. The polypeptide can retain an enzyme, structural or binding activity under these conditions comprising a temperature range of between about 40° C. to about 70° C., or, between about 90° C. to about 95° C., or more.

In one aspect, the isolated or recombinant nucleic acid comprises a sequence that hybridizes under stringent conditions to a sequence of the invention, wherein the nucleic acid encodes a polypeptide having an enzyme, structural or binding activity. The nucleic acid can at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or residues in length or the full length of the gene or transcript, with or without a signal sequence, or a propro sequence (e.g., as with a protease), as described herein. The stringent conditions can be highly stringent, moderately stringent or of low stringency, as described herein. The stringent conditions can include a wash step, e.g., a wash step comprising a wash in 0.2×SSC at a temperature of about 65° C. for about 15 minutes.

The invention provides a nucleic acid probe for identifying a nucleic acid encoding a polypeptide, e.g., with an enzyme, structural or binding activity, wherein the probe comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or more, consecutive bases of a sequence of the invention and the probe identifies the nucleic acid by binding or hybridization. The probe can comprise an oligonucleotide comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutive bases of a sequence of the invention.

The invention provides a nucleic acid probe for identifying a nucleic acid encoding a polypeptide with an enzyme, structural or binding activity, wherein the probe comprises a nucleic acid of the invention, e.g., a nucleic acid having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, and all additional nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073, or a subsequence thereof, over a region of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more consecutive residues, wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection.

The invention provides an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide having, e.g., an enzyme, structural or binding activity, wherein the primer pair is capable of amplifying a nucleic acid comprising a sequence of the invention, or fragments or subsequences thereof. One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.

The invention provides polypeptides generated by amplification, e.g., polymerase chain reaction (PCR), using an amplification primer pair of the invention. The invention provides methods of making a polypeptide by amplification, e.g., polymerase chain reaction (PCR), using an amplification primer pair of the invention. In one aspect, the amplification primer pair amplifies a nucleic acid from a library, e.g., a gene library, such as an environmental library.

The invention provides methods of amplifying a nucleic acid encoding a polypeptide comprising amplification of a template nucleic acid with an amplification primer sequence pair capable of amplifying a nucleic acid sequence of the invention, or fragments or subsequences thereof. The amplification primer pair can be an amplification primer pair of the invention.

The invention provides expression cassettes comprising a nucleic acid of the invention or a subsequence thereof. In one aspect, the expression cassette can comprise the nucleic acid that is operably linked to a promoter. The promoter can be a viral, bacterial, mammalian or plant promoter. In one aspect, the plant promoter can be a potato, rice, corn, wheat, tobacco or barley promoter. The promoter can be a constitutive promoter. The constitutive promoter can comprise CaMV35S. In another aspect, the promoter can be an inducible promoter. In one aspect, the promoter can be a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter. Thus, the promoter can be, e.g., a seed-specific, a leaf-specific, a root-specific, a stem-specific or an abscission-induced promoter. In one aspect, the expression cassette can further comprise a plant or plant virus expression vector.

The invention provides cloning vehicles comprising an expression cassette (e.g., a vector) of the invention or a nucleic acid of the invention. The cloning vehicle can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial chromosome. The viral vector can comprise an adenovirus vector, a retroviral vector or an adeno-associated viral vector. The cloning vehicle can comprise a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cell comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention, or a cloning vehicle of the invention. In one aspect, the transformed cell can be a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell. In one aspect, the plant cell can be a potato, wheat, rice, corn, tobacco or barley cell.

The invention provides transgenic non-human animals comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention. In one aspect, the animal is a mouse.

The invention provides transgenic plants comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention. The transgenic plant can be a corn plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed plant, a soybean plant, a rice plant, a barley plant or a tobacco plant. The invention provides transgenic seeds comprising a nucleic acid of the invention or an expression cassette (e.g., a vector) of the invention. The transgenic seed can be a corn seed, a wheat kernel, an oilseed, a rapeseed (a canola plant), a soybean seed, a palm kernel, a sunflower seed, a sesame seed, a peanut or a tobacco plant seed.

The invention provides an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid of the invention. The invention provides methods of inhibiting the translation of a polypeptide message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid of the invention.

The invention provides methods of inhibiting the translation of message in a cell comprising administering to the cell or expressing in the cell an antisense oligonucleotide comprising a nucleic acid sequence complementary to or capable of hybridizing under stringent conditions to a nucleic acid of the invention. The invention provides double-stranded inhibitory RNA (RNAi) molecules comprising a subsequence of a sequence of the invention. In one aspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. The invention provides methods of inhibiting the expression of a message in a cell comprising administering to the cell or expressing in the cell a double-stranded inhibitory RNA (iRNA), wherein the RNA comprises a subsequence of a sequence of the invention.

The invention provides isolated or recombinant polypeptides encoded by a nucleic acid of the invention. In alternative aspects, the polypeptide can have a sequence as set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, and all polypeptides disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:3 through SEQ ID NO:1073. The polypeptide can have an enzyme, structural or binding activity.

The invention provides isolated or recombinant polypeptides comprising a polypeptide of the invention lacking a signal sequence and/or a prepro sequence.

Another aspect of the invention provides an isolated or recombinant polypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutive bases of a polypeptide or peptide sequence of the invention, sequences substantially identical thereto, and the sequences complementary thereto. The peptide can be, e.g., an immunogenic fragment, a motif (e.g., a binding site) or an active site.

In one aspect, the isolated or recombinant polypeptide of the invention (with or without a signal sequence or a prepro sequence) has an enzyme, structural or binding activity.

In one aspect, the enzyme, structural or binding activity is thermostable. The polypeptide can retain an enzyme, structural or binding activity under conditions comprising a temperature range of between about 37° C. to about 95° C., between about 55° C. to about 85° C., between about 70° C. to about 95° C., or between about 90° C. to about 95° C. In another aspect, the enzyme, structural or binding activity can be thermotolerant. The polypeptide can retain an enzyme, structural or binding activity after exposure to a temperature in the range from greater than 37° C. to about 95° C., or in the range from greater than 55° C. to about 85° C. In one aspect, the polypeptide can retain an enzyme, structural or binding activity after exposure to a temperature in the range from greater than 90° C. to about 95° C. at pH 4.5.

In one aspect, the polypeptide can retain an enzyme, structural or binding activity under conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another aspect, the polypeptide can retain an enzyme, structural or binding activity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11.

In one aspect, the isolated or recombinant polypeptide can comprise the polypeptide of the invention that lacks a signal sequence and/or a prepro domain. In one aspect, the isolated or recombinant polypeptide can comprise the polypeptide of the invention comprising a heterologous signal sequence or a heterologous preprosequence, such as a heterologous enzyme or non-enzyme signal sequence.

The invention provides isolated or recombinant peptides comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or more sequence identity to a signal sequence and/or a prepro sequence of the invention, wherein the sequence identities are determined by analysis with a sequence comparison algorithm or by visual inspection. In one aspect, the peptides act as signal sequences on its endogenous polypeptide, on another enzyme, or a heterologous protein. In one aspect, the invention provides chimeric proteins comprising a first domain comprising a signal sequence of the invention and at least a second domain. The protein can be a fusion protein. The second domain can comprise an enzyme.

The invention provides chimeric polypeptides comprising at least a first domain comprising signal peptide (SP) and/or a prepro sequence of the invention or a catalytic domain (CD), or active site, of an enzyme of the invention and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP), prepro sequence or catalytic domain (CD). In one aspect, the heterologous polypeptide or peptide is not an enzyme. The heterologous polypeptide or peptide can be amino terminal to, carboxy terminal to or on both ends of the signal peptide (SP), prepro sequence or catalytic domain.

The invention provides isolated or recombinant nucleic acids encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises at least a first domain comprising signal peptide (SP), prepro sequence or a catalytic domain (CD), or active site, of a polypeptide of the invention, and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP), prepro sequence or catalytic domain (CD).

In one aspect, the enzyme, structural or binding activity comprises a specific activity at about 37° C. in the range from about 1 to about 1000 units, or about 10 to 100 units per milligram of protein. In another aspect, the enzyme, structural or binding activity comprises a specific activity from about 500 to about 750 units per milligram of protein. Alternatively, the enzyme, structural or binding activity comprises a specific activity at 37° C. in the range from about 1 to 1000, or about 500 to about 1200 units per milligram of protein. In one aspect, the enzyme, structural or binding activity comprises a specific activity at 37° C. in the range from about 750 to about 1000 units per milligram of protein. In another aspect, the thermotolerance comprises retention of at least half of the specific activity of the enzyme, structural or binding at 37° C. after being heated to the elevated temperature. Alternatively, the thermotolerance can comprise retention of specific activity at 37° C. in the range from about 500 to about 1200 units per milligram of protein after being heated to the elevated temperature.

The invention provides the isolated or recombinant polypeptide of the invention, wherein the polypeptide comprises at least one glycosylation site. In one aspect, glycosylation can be an N-linked glycosylation. In one aspect, the polypeptide can be glycosylated after being expressed in a P. pastoris or a S. pombe.

The invention provides protein preparations comprising a polypeptide of the invention, wherein the protein preparation comprises a liquid, a solid or a gel.

The invention provides heterodimers comprising a polypeptide of the invention and a second protein or domain. The second member of the heterodimer can be a different enzyme, a different enzyme or another protein. In one aspect, the second domain can be a polypeptide and the heterodimer can be a fusion protein. In one aspect, the second domain can be an epitope or a tag. In one aspect, the invention provides homodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having an enzyme, structural or binding activity, wherein the polypeptide comprises a polypeptide of the invention, a polypeptide encoded by a nucleic acid of the invention, or a polypeptide comprising a polypeptide of the invention and a second domain. In one aspect, the polypeptide can be immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube.

The invention provides arrays comprising an immobilized polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention. The invention provides arrays comprising an immobilized nucleic acid of the invention. The invention provides an array comprising an immobilized antibody of the invention.

The invention provides isolated or recombinant antibodies that specifically bind to a polypeptide of the invention or to a polypeptide encoded by a nucleic acid of the invention. The antibody can be a monoclonal or a polyclonal antibody. The invention provides hybridomas comprising an antibody of the invention.

The invention provides methods of isolating or identifying a polypeptide with an enzyme, structural or binding activity comprising the steps of: (a) providing an antibody of the invention; (b) providing a sample comprising polypeptides; and, (c) contacting the sample of step (b) with the antibody of step (a) under conditions wherein the antibody can specifically bind to the polypeptide, thereby isolating or identifying polypeptide. The invention provides methods of making an antibody comprising administering to a non-human animal a nucleic acid of the invention, or a polypeptide of the invention, in an amount sufficient to generate a humoral immune response, thereby making an antibody.

The invention provides methods of producing a recombinant polypeptide comprising the steps of: (a) providing a nucleic acid of the invention operably linked to a promoter; and, (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing a recombinant polypeptide. The method can further comprise transforming a host cell with the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in a transformed cell. The method can further comprise inserting into a host non-human animal the nucleic acid of step (a) followed by expressing the nucleic acid of step (a), thereby producing a recombinant polypeptide in the host non-human animal.

The invention provides methods for identifying a polypeptide having an enzyme activity comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention, or a fragment or variant thereof, (b) providing an enzyme substrate; and, (c) contacting the polypeptide or a fragment or variant thereof of step (a) with the substrate of step (b) and detecting an increase in the amount of substrate or a decrease in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product detects a polypeptide having an enzyme activity.

The invention provides methods for identifying an enzyme substrate comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test substrate; and, (c) contacting the polypeptide of step (a) with the test substrate of step (b) and detecting an increase in the amount of substrate or a decrease in the amount of reaction product, wherein a decrease in the amount of the substrate or an increase in the amount of the reaction product identifies the test substrate as an enzyme substrate.

The invention provides methods of determining whether a compound specifically binds to a polypeptide comprising the following steps: (a) expressing a nucleic acid or a vector comprising the nucleic acid under conditions permissive for translation of the nucleic acid to a polypeptide, wherein the nucleic acid and vector comprise a nucleic acid or vector of the invention; or, providing a polypeptide of the invention (b) contacting the polypeptide with the test compound; and, (c) determining whether the test compound specifically binds to the polypeptide, thereby determining that the compound specifically binds to the polypeptide.

The invention provides methods for identifying a modulator of a polypeptide activity comprising the following steps: (a) providing a polypeptide of the invention or a polypeptide encoded by a nucleic acid of the invention; (b) providing a test compound; (c) contacting the polypeptide of step (a) with the test compound of step (b); and, measuring an activity of the polypeptide, wherein a change in the polypeptide activity measured in the presence of the test compound compared to the activity in the absence of the test compound provides a determination that the test compound modulates the polypeptide activity.

In one aspect, the enzyme activity is measured by providing an enzyme substrate and detecting an increase in the amount of the substrate or a decrease in the amount of a reaction product. The decrease in the amount of the substrate or the increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of enzyme activity. The increase in the amount of the substrate or the decrease in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an inhibitor of enzyme activity.

The invention provides computer systems comprising a processor and a data storage device wherein said data storage device has stored thereon a polypeptide sequence of the invention or a nucleic acid sequence of the invention.

In one aspect, the computer system can further comprise a sequence comparison algorithm and a data storage device having at least one reference sequence stored thereon. The sequence comparison algorithm can comprise a computer program that indicates polymorphisms. The computer system can further comprising an identifier that identifies one or more features in said sequence.

The invention provides computer readable mediums having stored thereon a sequence comprising a polypeptide sequence of the invention or a nucleic acid sequence of the invention.

The invention provides methods for identifying a feature in a sequence comprising the steps of: (a) reading the sequence using a computer program which identifies one or more features in a sequence, wherein the sequence comprises a polypeptide sequence of the invention or a nucleic acid sequence of the invention; and, (b) identifying one or more features in the sequence with the computer program.

The invention provides methods for comparing a first sequence to a second sequence comprising the steps of: (a) reading the first sequence and the second sequence through use of a computer program which compares sequences, wherein the first sequence comprises a polypeptide sequence of the invention or a nucleic acid sequence of the invention; and, (b) determining differences between the first sequence and the second sequence with the computer program. In one aspect, the step of determining differences between the first sequence and the second sequence further comprises the step of identifying polymorphisms. In one aspect, the method further comprises an identifier (and use of the identifier) that identifies one or more features in a sequence. In one aspect, the method comprises reading the first sequence using a computer program and identifying one or more features in the sequence.

The invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide with an activity, e.g., an enzyme, structural or transcriptional control activity, from an environmental sample comprising the steps of: (a) providing an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide with an activity, wherein the primer pair is capable of amplifying a nucleic acid of the invention; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to the amplification primer pair; and, (c) combining the nucleic acid of step (b) with the amplification primer pair of step (a) and amplifying nucleic acid from the environmental sample, thereby isolating or recovering a nucleic acid encoding a polypeptide with an activity from an environmental sample. In one aspect, each member of the amplification primer sequence pair comprises an oligonucleotide comprising at least about 10 to 50 consecutive bases of a nucleic acid sequence of the invention. In one aspect, the amplification primer sequence pair is an amplification pair of the invention.

The invention provides methods for isolating or recovering a nucleic acid encoding a polypeptide with an activity, e.g., an enzyme, structural or transcriptional control activity, from an environmental sample comprising the steps of: (a) providing a polynucleotide probe comprising a nucleic acid sequence of the invention, or a subsequence thereof; (b) isolating a nucleic acid from the environmental sample or treating the environmental sample such that nucleic acid in the sample is accessible for hybridization to a polynucleotide probe of step (a); (c) combining the isolated nucleic acid or the treated environmental sample of step (b) with the polynucleotide probe of step (a); and, (d) isolating a nucleic acid that specifically hybridizes with the polynucleotide probe of step (a), thereby isolating or recovering a nucleic acid encoding a polypeptide with an activity from the environmental sample. In alternative aspects, the environmental sample comprises a water sample, a liquid sample, a soil sample, an air sample or a biological sample. In alternative aspects, the biological sample is derived from a bacterial cell, a protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acid encoding a polypeptide comprising the steps of: (a) providing a template nucleic acid comprising a nucleic acid of the invention; (b) modifying, deleting or adding one or more nucleotides in the template sequence, or a combination thereof, to generate a variant of the template nucleic acid. In one aspect, the method further comprises expressing the variant nucleic acid to generate a variant polypeptide. In alternative aspects, the modifications, additions or deletions are introduced by error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and/or a combination thereof. In alternative aspects, the modifications, additions or deletions are introduced by a method selected from the group consisting of recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and/or a combination thereof.

In one aspect, the method is iteratively repeated until a polypeptide (e.g., an enzyme) having an altered or different activity or an altered or different stability from that of a polypeptide encoded by the template nucleic acid is produced. In one aspect, the altered or different activity is a polypeptide (e.g., an enzyme) activity under an acidic condition, wherein the polypeptide encoded by the template nucleic acid is not active under the acidic condition. In one aspect, the altered or different activity is a polypeptide (e.g., an enzyme) activity under a high temperature, wherein the polypeptide encoded by the template nucleic acid is not active under the high temperature. In one aspect, the method is iteratively repeated until a polypeptide coding sequence having an altered codon usage from that of the template nucleic acid is produced. The method can be iteratively repeated until a gene having higher or lower level of message expression or stability from that of the template nucleic acid is produced.

The invention provides methods for modifying codons in a nucleic acid encoding an enzyme to increase its expression in a host cell, the method comprising (a) providing a nucleic acid of the invention encoding an enzyme; and, (b) identifying a non-preferred or a less preferred codon in the nucleic acid of step (a) and replacing it with a preferred or neutrally used codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acid encoding an enzyme, the method comprising (a) providing a nucleic acid of the invention encoding an enzyme; and, (b) identifying a codon in the nucleic acid of step (a) and replacing it with a different codon encoding the same amino acid as the replaced codon, thereby modifying codons in a nucleic acid encoding an enzyme.

The invention provides methods for modifying a codon in a nucleic acid encoding an enzyme to decrease its expression in a host cell, the method comprising (a) providing a nucleic acid of the invention encoding an enzyme; and, (b) identifying at least one preferred codon in the nucleic acid of step (a) and replacing it with a non-preferred or less preferred codon encoding the same amino acid as the replaced codon, wherein a preferred codon is a codon over-represented in coding sequences in genes in a host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell, thereby modifying the nucleic acid to decrease its expression in a host cell. In alternative aspects, the host cell is a bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acids encoding a plurality of modified enzyme active sites or substrate binding sites, wherein the modified active sites or substrate binding sites are derived from a first nucleic acid comprising a sequence encoding a first active site or a first substrate binding site the method comprising: (a) providing a first nucleic acid encoding a first active site or first substrate binding site, wherein the first nucleic acid sequence comprises a nucleic acid of the invention; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and, (c) using the set of mutagenic oligonucleotides to generate a set of active site-encoding or substrate binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified enzyme active sites or substrate binding sites. In alternative aspects, the method comprises mutagenizing the first nucleic acid of step (a) by a method comprising an optimized directed evolution system, gene site-saturation mutagenesis (GSSM), and synthetic ligation reassembly (SLR). The method can further comprise mutagenizing the first nucleic acid of step (a) or variants by a method comprising error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a combination thereof. The method can further comprise mutagenizing the first nucleic acid of step (a) or variants by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation and a combination thereof.

The invention provides methods for making a small molecule comprising the steps of: (a) providing a plurality of biosynthetic enzymes capable of synthesizing or modifying a small molecule, wherein one of the enzymes comprises an enzyme encoded by a nucleic acid of the invention; (b) providing a substrate for at least one of the enzymes of step (a); and, (c) reacting the substrate of step (b) with the enzymes under conditions that facilitate a plurality of biocatalytic reactions to generate a small molecule by a series of biocatalytic reactions.

The invention provides methods for modifying a small molecule comprising the steps: (a) providing a enzyme encoded by a nucleic acid of the invention; (b) providing a small molecule; and, (c) reacting the enzyme of step (a) with the small molecule of step (b) under conditions that facilitate an enzymatic reaction catalyzed by the enzyme, thereby modifying a small molecule by an enzymatic reaction. In one aspect, the method comprises providing a plurality of small molecule substrates for the enzyme of step (a), thereby generating a library of modified small molecules produced by at least one enzymatic reaction catalyzed by the enzyme. In one aspect, the method further comprises a plurality of additional enzymes under conditions that facilitate a plurality of biocatalytic reactions by the enzymes to form a library of modified small molecules produced by the plurality of enzymatic reactions. In one aspect, the method further comprises the step of testing the library to determine if a particular modified small molecule that exhibits a desired activity is present within the library. The step of testing the library can further comprises the steps of systematically eliminating all but one of the biocatalytic reactions used to produce a portion of the plurality of the modified small molecules within the library by testing the portion of the modified small molecule for the presence or absence of the particular modified small molecule with a desired activity, and identifying at least one specific biocatalytic reaction that produces the particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment of an enzyme comprising the steps of: (a) providing an enzyme comprising an amino acid sequence of the invention; and, (b) deleting a plurality of amino acid residues from the sequence of step (a) and testing the remaining subsequence for an enzyme activity, thereby determining a functional fragment of an enzyme. In one aspect, the enzyme activity is measured by providing an enzyme substrate and detecting an increase in the amount of the substrate or a decrease in the amount of a reaction product. In one aspect, a decrease in the amount of an enzyme substrate or an increase in the amount of the reaction product with the test compound as compared to the amount of substrate or reaction product without the test compound identifies the test compound as an activator of enzyme activity.

The invention provides methods for whole cell engineering of new or modified phenotypes by using real-time metabolic flux analysis, the method comprising the following steps: (a) making a modified cell by modifying the genetic composition of a cell, wherein the genetic composition is modified by addition to the cell of a nucleic acid comprising a sequence of the invention; (b) culturing the modified cell to generate a plurality of modified cells; (c) measuring at least one metabolic parameter of the cell by monitoring the cell culture of step (b) in real time; and, (d) analyzing the data of step (c) to determine if the measured parameter differs from a comparable measurement in an unmodified cell under similar conditions, thereby identifying an engineered phenotype in the cell using real-time metabolic flux analysis. In one aspect, the genetic composition of the cell is modified by a method comprising deletion of a sequence or modification of a sequence in the cell, or, knocking out the expression of a gene. The method can further comprise selecting a cell comprising a newly engineered phenotype. The method can further comprise culturing the selected cell, thereby generating a new cell strain comprising a newly engineered phenotype.

The invention provides isolated or recombinant signal sequences consisting of a sequence as set forth in residues 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30 or 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to 48, 1 to 49, 1 to 50, 1 to 51, 1 to 52, 1 to 53, 1 to 54, 1 to 55, of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, and all polypeptides disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:3 through SEQ ID NO:1073. The invention provides chimeric polypeptides comprising at least a first domain comprising signal peptide (SP) of the invention, and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP). In one aspect, the heterologous polypeptide or peptide is not an enzyme. In one aspect, the heterologous polypeptide or peptide is amino terminal to, carboxy terminal to or on both ends of the signal peptide (SP) or a catalytic domain (CD). The invention provides isolated or recombinant nucleic acids encoding a chimeric polypeptide, wherein the chimeric polypeptide comprises at least a first domain comprising signal peptide of the invention and at least a second domain comprising a heterologous polypeptide or peptide, wherein the heterologous polypeptide or peptide is not naturally associated with the signal peptide (SP).

The invention provides methods of increasing thermotolerance or thermostability of an enzyme polypeptide, the method comprising glycosylating an enzyme, wherein the polypeptide comprises at least thirty contiguous amino acids of a polypeptide of the invention, or a polypeptide encoded by a nucleic acid of the invention, thereby increasing the thermotolerance or thermostability of the enzyme. The invention provides methods of overexpressing a recombinant enzyme in a cell comprising expressing a vector comprising a nucleic acid sequence of the invention, wherein overexpression is effected by use of a high activity promoter, a dicistronic vector or by gene amplification of the vector.

The invention provides methods of making a transgenic plant comprising the following steps: (a) introducing a heterologous nucleic acid sequence into the cell, wherein the heterologous nucleic sequence comprises a sequence of the invention, thereby producing a transformed plant cell; (b) producing a transgenic plant from the transformed cell. In one aspect, step (a) further comprises introducing the heterologous nucleic acid sequence by electroporation or microinjection of plant cell protoplasts. In one aspect, step (a) step (a) comprises introducing the heterologous nucleic acid sequence directly to plant tissue by DNA particle bombardment or by using an Agrobacterium tumefaciens host.

The invention provides methods of expressing a heterologous nucleic acid sequence in a plant cell comprising the following steps: (a) transforming the plant cell with a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic sequence comprises a sequence of the invention; (b) growing the plant under conditions wherein the heterologous nucleic acids sequence is expressed in the plant cell.

The invention provides methods for a building a genome having a desired biological requirement, a desired biological property or a desired metabolic pathway comprising the following steps: (a) providing a minimal autonomous genome, wherein the genome comprises a sequence as set forth in SEQ ID NO:1; and (b) adding back to the minimal autonomous genome of step (a) one or more desired genes, thereby building a genome having a desired biological requirement, a desired biological property or a desired metabolic pathway. In one aspect the desired biological property comprises performing a metabolic function, synthesizing a structure or composition or regulating a cell cycle.

The invention provides methods for a building a minimal autonomous genome comprising the following steps: (a) providing a genome comprising a sequence as set forth in SEQ ID NO:1; and (b) performing global knockout mutagenesis on the genome, or adding genes to the genome, and determining whether a cell comprising the genome can survive or replicate autonomously, thereby building an autonomous minimal genome.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences and ATCC deposits, cited herein are hereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 is a block diagram of a computer system, as described in detail, below.

FIG. 3 is a flow diagram illustrating one embodiment of a process in a computer for determining whether two sequences are homologous, as described in detail, below.

FIG. 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence.

FIG. 5, summarizes data from the alanylation of unfractionated M. jannaschii tRNA by alanyl-tRNA synthetase, as described in detail, below.

FIG. 6 schematically illustrates the phylogenetic position of Nanoarchaeum equitans within the Archaea, as described in detail, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated and a recombinant genome of a hyperthermophile Nanoarchaeum equitans (SEQ ID NO:1). The invention also provides an isolated hyperthermophile Nanoarchaeum equitans deposited as ATCC accession no. ______. N. equitans is an obligate parasite growing on the crenarchaeon Ignicoccus. A ribosomal protein based phylogenetic analysis places its branching point closest to the root of the archaeal tree.

The invention also provides isolated and recombinant nucleic acids encoding polypeptides, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, etc., and all additional nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073. The invention also provides isolated and recombinant polypeptides, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, etc., and all polypeptides disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:3 through SEQ ID NO:1073.

The N. equitans genome (SEQ ID NO:1) is 490,885 base pairs and encodes the machinery for information processing and repair, but lacks that for lipid, cofactor, amino acid or nucleotide biosynthesis. Unusual features include a quite a few split genes, little evidence of lateral gene transfer, and few operons. The small genome size may be a consequence of genome reduction, and a lack of pseudogenes suggests an early adaptation to the parasitic lifestyle.

To shed light on the phylogenetic relationship among the Archaea, we concatenated and aligned the amino acid sequences of 35 ribosomal proteins. N. equitans was placed with high support at the most basal position within the Archaea in the maximum likelihood, maximum parsimony, and Bayseian trees suggesting that the Nanoarchaeota are the basal kingdom within the Archaea. In contrast to the bacterial small genome parasites, no free-living close relative to N. equitans could be detected so far. Therefore one cannot deduce the genome size of its possible precursor. The assumption of N. equitans being a non-reduced primitive genome would provide exciting possibilities for research about ancient features of parasitism, early organismal development, and also a starting point for the generation of a minimal autonomous genome.

The assumption of the genome of the invention (SEQ ID NO:1), an N. equitans genome, being a non-reduced primitive genome would provide exciting possibilities for research about ancient features of parasitism, early organismal development, and also a starting point for the generation of a minimal autonomous genome. Based on the fact that N. equitans possesses all the machinery for information processing and repair, it would be a perfect model to rebuild a universal genome based on different biological requirements. In contrast to minimize genomes, such as E. coli or Mycoplasma genitalium, in one aspect the invention provides methods for adding back to the genome of the invention (SEQ ID NO:1) all genes necessary to perform one or more desired metabolisms, synthesis of structures, performing cell cycles and other cellular and metabolic functions and the like.

The invention also provides methods using the genome of the invention (SEQ ID NO:1) for experimental studies within the framework of the minimal-gene-set concept and the ultimate construction of a minimal genome, as described, e.g., by Eugene V. Koonin, Annu. Rev. Genomics Hum. Genet. (2000) 01:99-116. These studies will advance our understanding of the basic principles of cell functioning by systematically detecting non-orthologous gene displacement and deciphering the roles of essential but functionally uncharacterized genes. The invention also provides methods using the genome of the invention (SEQ ID NO:1) in global knockout mutagenesis to analyze essential genes.

The invention's discovery of the archaeal kingdom Nanoarchaeota, and its sole species, Nanoarchaeum equitans, raised new questions about the evolution of the Archaea. Although ribosomal RNA sequences placed this species in a basal branch of the Archaea, analyses did not allow reliable placement of the organism's phylogenetic position in the 16S rRNA based tree of life. In addition, it was unclear whether N. equitans is a primitive or a derived member of the Archaea. SEQ ID NO:1, the genome of N. equitans, was deposited as GenBank accession no. ______. In one aspect, the genome of the invention has a single, circular chromosome of 490,885 basepairs (bp) and has an average G+C content of 31.6% (4). All 61 sense codons are used, but in line with the low G+C content the third codon position is mainly A or T. 539 open reading frames (ORFs) were identified that cover 92% of the genome; average ORF length is 835 bp. Functional roles were assigned to 66.8% of the annotated genes. Of the ORFs of unknown function only 18.3% have homologs in other organisms, while the remaining ones are unique to N. equitans (see Table 3, below).

Genes encoding single copies of 5S, 16S and 23S rRNA, and 35 tRNAs were identified. These stable RNAs exhibit much higher G+C content (65-80%) than the rest of the genome. This is likely due to the need to form stable secondary structures at the high growth temperature of N. equitans. The rRNA genes were not found in an operon. In fact, a low occurrence of operons is a notable feature of the N. equitans genome (SEQ ID NO:1). There is little evidence of significant lateral gene transfer in the genome; less than 5% of the N. equitans ORFs have close bacterial homologs and only about 14% have close crenarchaeal homologs. Finally, as no extrachromosomal elements could be detected (either by biochemical methods or during sequencing), the N. equitans genome of the invention (SEQ ID NO:1) represents the smallest sequenced genome of a cellular organism.

Given its small genome size it is not surprising that N. equitans lacks the metabolic capacity to synthesize many cell components, see Table 1.

TABLE 1

Comparison of N. equitans and other organisms with small genomes.

Nanoarchaeum

equitans

M. genitalium
^a

Buchnera sp. APS⁷

R. prowazokii
⁴

T. paliidum
⁹

Genome size
490,885
580,070
540,681
1,111,523
1,138,008

(base pairs)

G-C content
31.6%
31.6%
26.3%
29.1%
52.8%

Glycolysis
−
+
+
−
+

Pentose Phoshate
−
Limited
+
−
+

Pathway

TCA cycle
−
−
−
+
−

ATP production by
Limited
−
+
+
−

respiration

Amino acid synthesis
−
−
+
−
−

Nucleotide synthesis
−
−
+
−
−

Lipid synthesis
−
Limited
−
+
+

DNA recombination/
+
Limited
Limited
Limited
Limited

repair

(+) indicates all components of the pathway are present

(−) indicates that none of the components are present

^aLimited indicates that some but not all of the components are present.

Based on our current knowledge of metabolism, no ORFs were found that represent enzymes for the de novo synthesis of amino acids, nucleotides, cofactors or lipids, and known genes are absent for glycolysis, the pentose phosphate pathway and the citric acid cycle. This absence of metabolic capacity must necessitate the transport of many cellular components and metabolites from the host Ignicoccus, but it is currently unclear how this is accomplished. It remains enigmatic how N. equitans gains energy. At least a basic A1A0-type ATPase consisting of only 5 subunits as well as some components of an electron transport chain could be identified. Only five transport proteins have been identified in this genome: a Na+/Ca2+ exchange protein, a Mg2+- and Co2+-transporter, a C-4 dicarboxylate transporter, one ABC type transporter system involved in lipoprotein release, and an uncharacterized iron-regulated ABC transporter. N. equitans may acquire its lipids directly from its host Ignicoccus; a striking feature of this organism is the vast formation of vesicles at its cytoplasmic membrane, which may be part of a supply mechanism of cell components to N. equitans. (see, e.g., Huber (2000) Int. J. Syst. Evol. Microbiol. 50:2093). As similar features are found in parasites, the genome of N. equitans (SEQ ID NO:1) points to a parasitic life style. As a matter of fact, the presence of several N. equitans cells prevented multiplication of the Ignicoccus host.

A common feature of bacterial obligate parasites is their small genome size (see, e.g., Fraser (1995) Science 270:397; Shigenobu (2000) Nature 407:81; Andersson (1998) Nature 396:133; Fraser (1998) Science 281:375) as a consequence of genome reduction during adaptation to parasitic life style. This may also be true for N. equitans. The absence of many generally conserved operons (e.g., rRNAs, ATPase and RNA polymerase subunits) as well as the lack of duplicated genes is evidence for multiple genome rearrangements which usually accompany the reduction process (see, e.g., Andersson (1998) Trends Microbiol. 6:263).

In contrast to many obligate parasites, N. equitans has a repertoire of repair enzymes for base and nucleotide excision including endonucleases III, IV, and V (NEQ126a, NEQ398, NEQ077a, NEQ368, NEQ346a), rad2 (NEQ088) and rad25 (NEQ369). In addition, the presence of homologs of radA (NEQ426), Rad50 (NEQ256) and a Holliday-junction resolvase (NEQ424) may indicate that N. equitans can undergo homologous recombination. The presence of these enzymes sets N. equitans apart from other organisms with small genomes in significant ways. First, N. equitans may be able to repair damage to DNA that is likely to occur in its high temperature habitat. In addition, it appears to have the potential to acquire new genes via lateral gene transfer. In addition, despite its small genome N. equitans might have the capacity to adapt to changing environments. It is noteworthy that many organisms with small genomes have lost recombination/repair enzymes even though these losses incur significant negative effects. Organisms that lack these enzymes are at best evolutionarily stable (see, e.g., Tamas (2002) Science 296:2376) and at worst subject to the negative effects of increased occurrence and fixation of deleterious mutations (see, e.g., Moran (1996) Proc. Natl. Acad. Sci U.S.A. 93:2873).

N. equitans possesses a large and reasonably complete set of components for information processing (replication, transcription and translation) and completion of the cell cycle. For transcription, a4DNA-dependent RNA polymerase consisting of 14 subunits, and the archaeal genre proteins involved in transcription initiation, elongation and termination could be identified. The gene sets for DNA replication and cell cycle are similar to those found in Euryarchaeota and contain several components usually absent from Crenarchaeota (e.g., DNA polymerase II, two copies of ftsZ, and histones). The translational machinery of N. equitans is similar to other Archaea. However, three tRNA genes (for glutamate, histidine and tryptophan) were not found in N. equitans. These tRNA species may have an unusual sequence/structure (resembling e.g., mitochondrial tRNAs or M kandleri TRNA Glu) causing them to be missed by tRNA scan-SE (see, e.g., Lowe (1997) Nucleic Acids Res. 25:955). tRNA import from the host, the possibility of a tRNA species with dual function (based on different nucleotide modification) or of an anticodon change by RNA editing may also be plausible. Four tRNA species (for serine, tyrosine, isoleucine, and methionine) contain single introns in the expected position. Also present are two homologs of EndA (NEQ261, NEQ205), an intron excision enzyme that is found in all other Archaea. In some Euryarchaeota an in-frame gene duplication of EndA has occurred followed by a specialization of each domain (see, e.g., Lykke-Andersen (1997) EMBO J. 16:6290). While NEQ205 is most similar to Methanococcus jannaschii EndA (which does not have the gene duplication), NEQ261 shows more similarity to the N-terminal domain of the duplicated endA of Methanosarcina acetivorans. Also identified were some genes linked to snoRNA-dependent RNA modifications, including pseudouridine synthase (truB family, CBF5; NEQ454) and fibrillarin (NEQ125). This is in agreement with the recent reports that some archaea, possess snoRNAs (Omer (2000) Science 288:517). Thus, snoRNA metabolism may be an ancient characteristic of archaea that was present in a predecessor of all known archaeal phyla.

An unusual characteristic of the N. equitans genome (SEQ ID NO:1) is the high number of split genes, whose gene product is encoded by two physically separated ORFs, see Table 2.

TABLE 2

Split genes in N. equitans.

ORF encoding
ORF encoding
Distance between

Gene
N-terminal part
C-terminal part
ORFs

Reverse gyrase^a
NEQ434
NEQ318
95,625 bp

Topoisomerase I
NEQ045
NEQ324
241,346 bp

Glu-tRNA^Glaamidotransferase (gatE)
NEQ245
NEQ396
126,409 bp

Alanyl-tRNA synthetase
NEQ547
NEQ211
185,194 bp

DNA polymerase B^b
NEQ068
NEQ528
83,301 bp

RNA polymerase subunit B^c
NEQ173
NEQ156
13,388 bp

Large helicase related protein
NEQ003
NEQ409
134,395 bp

archaeosine tRNA-guanine transglycosylase
NEQ124
NEQ305
164,284 bp

Conserved hypothetical protein (RNA-binding
NEQ438
NEQ506
62,414 bp

protein homologous to eukaryotic snRNP)

^aalso split in Methanopyrus kandleri (different site)

^balso split in Methanothermobacter thermoautotrophicus

^calso split in methanogens, Archaeoglobales, and extreme halophiles

In most cases the site of the split lies between functional domains of the encoded proteins; thus it seems likely that the two separated ORFs encode parts of the corresponding enzyme, which assemble to a functional protein after independent expression. The split gene separated by the largest segment of DNA (see Table 2) encodes alanyl-tRNA synthetase. This gene provided the opportunity to test the idea that the individual protein parts are catalytically inactive, but that they reconstitute activity when combined (see, e.g., Burbaum (1991) Biochemistry 30:319). Only a combination of both parts of the split protein yielded a fully active enzyme as checked by the standard aminoacylation assay (FIG. 5, see also, discussion, below). Thus, in this case trans-splicing is not a prerequisite for enzyme activity (see, e.g., Ahel (2002) J. Biol. Chem. 277:34743).

The N. equitans reverse gyrase is split into two distinct genes encoding a helicase (NEQ434) and a topoisomerase (NEQ318) domain. Reverse gyrase appears to be the fusion product of a helicase and a5topoisomerase domain, and catalyzes positive supercoil formation in DNA (see, e.g., Krah (1996) Proc. Natl. Acad. Sci. U.S.A. 93:106; Forterre (2002) Trends Genet. 18:236). Since this enzyme is only present in hyperthermophiles, it was concluded that hyperthermophily appeared secondarily in the evolution of life (see, e.g., Forterre (2000) Trends Genet. 16:152). In light of the presence of independent helicase and topoisomerase domains in the deep rooted N. equitans, the evolution of hyperthermophily may have been a very early event in agreement with the view of a hot primeval earth (see, e.g., Stetter, in Commentarii Pontifica Academica Scientiarium, Vatican City (1997) vol. IV).

Another split gene encodes DNA-directed polymerase I. The N-terminal (NEQ068) and C-terminal (NEQ528) ORFs are separated by 83,301 bp and are located on opposite DNA strands. The C-terminal part (294 bp) of NEQ068 together with the N-terminal region (93 bp) of NEQ528 encode a mini-intein (129 aa). It is predicted that the two parts of the DNA polymerase are expressed separately and then covalently linked after the reassembled intein has been excised by a trans-splicing mechanism (see, e.g., Wu (1998) Proc. Natl. Acad. Sci. U.S.A. 95:9226).

It is unlikely that the split genes are solely the by-product of a small or reduced genome as the other known small genomes do not contain the number and type of split genes found in N. equitans. These split domain proteins may be early remnants of protein evolution that depended on domain fusion for the generation of modern/complex proteins (see, e.g., Doolittle (1995) Annu. Rev. Biochem. 64:287; Doolittle (1978) Nature 272:581; Gilbert (1997) Proc. Natl. Acad. Sci. U.S.A. 94:7698).

To shed light on the phylogenetic relationship among the Archaea the amino acid sequences of 35 ribosomal proteins were concatenated and aligned. A concatenated alignment of 35 ribosomal proteins was analyzed by maximum likelihood, Bayseian and parsimony analysis. N. equitans was placed with high support at the most basal position within the Archaea in the maximum likelihood, maximum parsimony, and Bayseian trees (see FIG. 6) suggesting that the Nanoarchaeota are the basal kingdom within the Archaea. FIG. 6 schematically illustrates the phylogenetic position of Nanoarchaeum equitans within the Archaea. The tree was determinedly the maximum likelihood method, based on 35 concatenated ribosomal protein sequences. Numbers indicate percentage of bootstrap resamplings. Scale bar corresponds to estimated substitutions per 100 positions.

Recent evidence shows that N. equitans is not the only extant member of the Nanoarchaeota. Two sequences from Caldera Uzon (Kamchatka) and Yellowstone National Park (USA) exhibited 83% sequence similarity to N. equitans and therefore represent a distinct group within the Nanoarchaeota (Hohn (2002) Syst. Appl. Micro. 25:551). Light microscopy and fluorescence in situ hybridization reveal that these novel Nanoarchaeota are tiny cocci like N. equitans attached to other archaeal species. Similar to Ignicoccus, these hosts may gain energy by S/H-autotrophy, a metabolism considered to be primitive (Fischer (1983) Nature 301:511). In contrast to the bacterial small genome parasites, no free-living close relative to N. equitans could be detected so far. Therefore one cannot deduce the genome size of its possible precursor. The assumption of N. equitans being a non-reduced primitive genome would provide exciting possibilities for research about ancient features of parasitism, early organismal development, and also a starting point for the generation of a minimal autonomous genome.

TABLE 3

SEQ

ID

Blast Hit/Manual
Genbank Definition
Blastp
Pfam & TIGR
EC

NO:
Name
Annotation
of top Blastp hit
Evalue
Domains
Number
COG Hit

1
N/A
N/A - SEQ ID NO: 1
N/A - SEQ ID NO: 1 is the DNA

N/A - SEQ ID NO: 1 is the DNA

is the DNA sequence
sequence of the whole genome

sequence of the whole genome

of the whole genome

2, 3
NEQ001
uncharacterized
conserved
5.00E−27
DUF57 Protein of unknown

Poorly

conserved protein
hypothetical

function DUF57 2.00e−06

characterized;

[Methanococcus

Function

jannaschii].

unknown; Uncharacterized

ACR 3e−28

4, 5
NEQ003
large helicase-
large helicase-related
0
DEAD DEAD/DEAH
3.6.1.—
Poorly

related protein
protein [Pyrococcus

box helicase 4.80e−47

characterized;

[Pyrococcus abyssi].

abyssi].

:helicase_-C

General

SPLIT See SEQ ID

Helicase conserved

function

NOS: 798, 799

C-terminal domain

prediction

1.70e−18 :recq recq

only; Lhr-like

ATP-dependent

helicases 0

DNA helicase RecQ

4.50e−06

6, 7
NEQ002
fkbp-type peptidyl-
fkbp-type peptidyl-
5.00E−13
0
5.2.1.8
Cellular

prolyl cis-
prolyl cis-transisomerase

processes; Posttranslational

transisomerase
[Methanothermobacter

modification,

thermautotrophicus].

protein

turnover,

chaperones; FKBP-

type

peptidyl-prolyl

cis-transisomerases 2

4e−14

8, 9
NEQ004
Uncharacterized
conserved
3.00E−20
DUF101 Protein of unknown

Poorly

conserved
hypothetical protein

function DUF101 2.90e−27

characterized;

protein[Methanopyrus
[Methanococcus

Function

kandleri AV19]

jannaschii].

unknown; Uncharacterized

ACR 3e−21

10,
NEQ005
hypothetical
288aa long
6.00E−29
DUF425 Protein of unknown

Poorly

11

carbamoylphosphate
hypothetical protein

function (DUF425) 1.60e−13

characterized;

synthetase
[Sulfolobus tokodaii].

Function

[Aeropyrum pernix].

unknown; Uncharacterized

ACR 2e−25

12,
NEQ007
methylated-DNA-
methylated-DNA-
3.00E−12
Methyltransf_-1 6-O-
2.1.1.
Information

13

[protein]-cysteineS-
[protein]-cysteine S-

methylguanine DNA
63
storage and

methyltransferase
methyltransferase

methyltransferase,

processing; DNA

(ogt)
(ogt) [Pyrobaculum

DNA binding domain

replication,

aerophilum].

1.60e−07 :ogt ogt

recombination

methylated-DNA-

and

protein-cysteine

repair; Methylated

methyltransferase

DNA-

9.60e−10

protein

cysteine

methyltransferase

8e−13

14,
NEQ006
hypothetical protein
hypothetical protein
3.00E−04
0

15

[Clostridium

perfringens].

16,
NEQ008
hypothetical 2-
418aa long conserved
2.00E−82
UPF0004
1.8.—.—
Information

17

methylthioadeninesynthase
hypothetical protein

Uncharacterized

storage and

[Sulfolobus tokodaii].

protein family

processing; Translation,

UPF0004 1.60e−22

ribosomal

:TIGR00089

structure and

TIGR00089

biogenesis; 2-

conserved

methylthioadenine

hypothetical protein

synthetase

3.30e−123

6e−80

:TIGR01125

TIGR01125

conserved

hypothetical protein

TIGR01125 6.70e−19

:TIGR01212

TIGR01212

conserved

hypothetical protein

TIGR01212 3.60e−3

:Radical_-SAM

Radical SAM

superfamily 5.10e−27

18,
NEQ009
hypothetical protein
conserved
8.00E−13
0

Poorly

19

hypothetical protein

characterized;

[Pyrobaculum

General

aerophilum].

function

prediction

only; Predicted

acetyltransferase

1e−07

20,
NEQ010
hypothetical protein
hypothetical protein
0.01
0

21

predicted by

GeneMark [Bacillus

anthracis A2012].

22,
NEQ011
desulfoferrodoxin
SUPEROXIDE
7.00E−27
Desulfoferrodox
1.15.—.—
Metabolism; Energy

23

[Pyrococcus abyssi]
REDUCTASE (SOR).

Desulfoferrodoxin

production

6.20e−24

and

:neela_-ferrous

conversion; Desulfoferrodoxin

neela_-ferrous

4e−28

desulfoferrodoxin

ferrous iron-binding

domain 2.00e−18

24,
NEQ012
glycosyltransferase
glycosyltransferase
2.00E−19
Glycos_-transf_-1 Glycosyl

Cellular

25

[Pyrococcusfuriosus
[Methanosarcina

transferases group 1 2.60e−06

processes; Cell

DSM 3638]

acetivorans str. C2A].

envelope

biogenesis,

outer

membrane; Predicted

glycosyltransferases

1e−13

26,
NEQ013
hypothetical
377aa long
6.00E−17
Radical_-SAM Radical SAM

Poorly

27

coenzyme PQQ
hypothetical

superfamily 3.20e−21

characterized;

synthesisprotein
coenzyme PQQ

General

[Sulfolobus tokodaii]
synthesis protein

function

[Sulfolobus tokodaii].

prediction

only; Predicted

Fe—S

oxidoreductases

8e−17

28,
NEQ014
C4-dicarboxylate
hypothetical protein
3.00E−60
tdt tdt C4-dicarboxylate

Cellular

29

transporter,
[Magnetococcus sp.

transporter/malic acid

processes; Inorganic

putative[Pyrococcus
MC-1].

transport protein 7.80e−33

ion

abyssi]

:C4dic_-mal_-tran C4-

transport and

dicarboxylate

metabolism; Tellurite

transporter/malic acid

resistance

transport protein 6.60e−32

protein and

related

permeases

3e−29

30,
NEQ015
hypothetical
257aa long conserved
2.00E−22
Thy1 Thymidylate synthase

Metabolism; Nucleotide

31

thymidylate
hypothetical protein

complementing protein

transport and

synthasecomplementing
[Sulfolobus tokodaii].

1.20e−09

metabolism; Predicted

protein

alternative

thymidylate

synthase 1e−16

32,
NEQ016
hypothetical protein
hypothetical protein
1.00E−12
DUF196 Uncharacterized

Poorly

33

[Methanosarcina

ACR, COG1343 7.60e−17

characterized;

barkeri].

Function

unknown; Uncharacterized

ACR 4e−13

34,
NEQ017
conserved
hypothetical protein
3.00E−60
DUF48 Protein of unknown

Poorly

35

hypothetical
[Pyrococcus

function DUF48 8.20e−96

characterized;

protein[Pyrococcus

horikoshii].

:TIGR00287 TIGR00287

Function

horikoshii]

conserved hypothetical

unknown; Uncharacterized

protein TIGR00287 3.80e−79

ACR 2e−61

36,
NEQ018
hypothetical protein
maturase [Euglena
9.00E−05
0

37

gracilis].

38,
NEQ019
conserved
hypothetical protein
1.00E−15

Poorly

39

hypothetical
[Pyrococcus

characterized;

protein[Pyrococcus

horikoshii].

Function

horikoshii]

unknown; Uncharacterized

ACR 7e−17

40,
NEQ020
hypothetical protein
hypothetical protein
6.00E−03
0

41

[Methanosarcina

mazei Goe1].

42,
NEQ022
ATP-dependent RNA
hypothetical protein
2.00E−53
DEAD DEAD/DEAH
2.7.7.—
Poorly

43

helicase[Fusobacterium
[Pyrococcus

box helicase 9.50e−06

characterized;

nucleatum]

horikoshii].

:helicase_-C

General

Helicase conserved

function

C-terminal domain

prediction

5.80e−4

only; Predicted

helicases

1e−54

44,
NEQ021
RecB family
hypothetical protein
1.00E−30
DUF83 Domain of unknown

Information

45

exonuclease
[Pyrococcus

function DUF83 2.50e−25

storage and

horikoshii].

:TIGR00372 TIGR00372

processing; DNA

conserved hypothetical

replication,

protein TIGR00372 3.80e−40

recombination

and

repair; RecB

family

exonuclease

1e−31

46,
NEQ023
protease
protease
5.00E−17
9.70E−16
3.4.—.—
Poorly

47

[Pyrobaculum
[Pyrobaculum

characterized;

aerophilum]

aerophilum].

General

function

prediction

only; Predicted

Zn-

dependent

peptidases

2e−13

48,
NEQ024
hypothetical sulfide
flavoprotein
2.00E−88
pyr_-redox Pyridine
1.6.99.3
Poorly

49

dehydrogenase[flavo
reductase, conjectural

nucleotide-

characterized;

cytochrome c]
[Pyrobaculum

disulphide

General

flavoprotein

aerophilum].

oxidoreductase

function

2.20e−05

prediction

only; Uncharacterized

NAD(FAD)-

dependent

dehydrogenases

3e−25

50,
NEQ025
glucose-1-
glucose-1-phosphate
4.00E−45
NTP_-transferase
2.7.7.
Cellular

51

phosphatethymidylyltransferase
thymidylyltransferase

Nucleotidyl
24
processes; Cell

(graD-1)
(graD-1)

transferase 1.10e−23

envelope

[A.
[Archaeoglobus

:galU galU UTP-

biogenesis,

fulgidus].

glucose-1-

outer

phosphate

membrane; Nucleoside-

uridylyltransferase

diphosphate-

1.90e−07 :rmlA rmlA

sugar

glucose-1-

pyrophosphorylases

phosphate

involved in

thymidylyltransferase

lipopolysaccharide

2.60e−3

biosynthesis/translation

:rmlA_-long

initiation

rmlA_-long glucose-

factor eIF2B

1-phosphate

subunits 3e−46

thymidyltransferase

4.10e−3

52,
NEQ026
hypothetical protein
hypothetical protein
0.31
0

53

[Clostridium

thermocellum ATCC

27405].

54,
NEQ028
hypothetical protein
hypothetical protein
2.00E−03
0

55

[Plasmodium yoelii

yoelii].

56,
NEQ027
hypothetical protein
putative bir1 protein
0.5
0

57

[Plasmodium yoelii

yoelii].

58,
NEQ029
hypothetical protein
protein disulfide
0.03
0

59

isomerase 4 [Giardia

intestinalis].

60,
NEQ030
holocytochrome-c
holocytochrome-c
3.00E−13
DsbD Cytochrome C

Cellular

61

synthase[Methanosarcina
synthase

biogenesis protein

processes; Posttranslational

acetivorans str.
[Methanosarcina

transmembrane region

modification,

C2A]

acetivorans str. C2A].

4.10e−4

protein

turnover,

chaperones; Cytochrome c

biogenesis

protein 2e−13

62,
NEQ031
Predicted RNA-
181aa long conserved
3.00E−18

Poorly

63

binding
hypothetical protein

characterized;

proteincontaining KH
[Sulfolobus tokodaii].

General

domain) [M. kandleri]

function

prediction

only; Predicted

RNA-

binding

protein

(contains KH

domains) 3e−16

64,
NEQ032
hypothetical protein
repeat organellar
4.00E−03
0

65

protein-related

[Plasmodium yoelii

yoelii].

66,
NEQ033
hypothetical protein
hypothetical protein
9.00E−07
0

67

[Cytophaga

hutchinsonii].

68,
NEQ034
hypothetical protein
similar to Plasmodium
1.00E−05
0

69

falciparum (isolate

3D7). Hypothetical

protein [Dictyostelium

discoideum].

70,
NEQ035
hypothetical protein
hypothetical protein
3.00E−03
0

71

[Lactobacillus

gasseri].

72,
NEQ036
hypothetical protein
Hypothetical protein
0.02
0

73

[Clostridium

acetobutylicum].

74,
NEQ038
transcription initiation
Ribosomal protein
5.00E−20
Ribosomal_-L37ae

Information

75

factor IID, TATA-box
L37AE/L43A

Ribosomal L37ae protein

storage and

binding protein (TBP)
[Methanopyrus

family 2.20e−26 :L37a L37a

processing; Translation,

kandleri AV19].

ribosomal protein L37a

ribosomal

9.10e−27

structure and

biogenesis; Ribosomal

protein

L37AE/L43A

7e−20

76,
NEQ037
Small nuclear
putative U6 snRNA-
9.00E−05
Sm Sm protein 1.30e−11

77

ribonucleoprotein(sn
ASSOCIATED SM-

RNP) homolog
LIKE PROTEIN

[Encephalitozoon

cuniculi].

78,
NEQ039
hypothetical protein
transcription initiation
1.00E−24
TBP Transcription factor

Information

79

factor IID, TATA-box

TFIID (or TATA-binding

storage and

binding protein

protein, TBP) 4.60e−21 :TBP

processing; Transcription;

[Methanococcus

Transcription factor TFIID (or

Transcription

jannaschii].

TATA-binding protein, TBP)

initiation

1.10e−06

factor TFIID

(TATA-

binding

protein) 1e−25

80,
NEQ041
hypothetical protein
predicted protein
0.36
0

81

[Methanosarcina

acetivorans str. C2A].

82,
NEQ040
hypothetical protein
hypothetical protein
6.00E−05
PAP2 PAP2 superfamily 6.00e−05

83

T13C5.6-

Caenorhabditis

elegans.

84,
NEQ042
thermostable
thermostable
1.00E−135
Peptidase_-M32
3.4.17.
Metabolism; Amino

85

carboxypeptidase
carboxypeptidase

Carboxypeptidase
19
acid

[P. aerophilum]
[Pyrobaculum

Taq (M32)

transport and

aerophilum].

metallopeptidase

metabolism; Zn-

2.80e−174

dependent

carboxypeptidases

1e−129

86,
NEQ043
hypothetical protein
ribosomal protein S26
0.09
0

87

[Spodoptera

frugiperda].

88,
NEQ044
O-sialoglycoprotein
O-sialoglycoprotein
4.00E−23
RIO1
3.4.24.
Cellular

89

endopeptidase[Methanosarcina
endopeptidase

RIO1/ZK632.3/MJ04
57
processes; Signal

acetivorans str. C2A]
[Methanosarcina

44 family 1.60e−4

transduction

acetivorans str. C2A].

mechanisms;

Mn2+-

dependent

serine/threonine

protein

kinase 1e−22

90,
NEQ045
DNA topoisomerase I
DNA topoisomerase I
6.00E−75
Topoisom_-bac DNA
5.99.
Information

91

[Pyrococcushorikoshii]
[Pyrococcus

topoisomerase
1.2
storage and

SPLIT see SEQ ID

horikoshii].

2.20e−23 :Toprim

processing; DNA

NOS: 632, 633

Toprim domain

replication,

4.00e−11 :topA_-bact

recombination

topA_-bact DNA

and

topoisomerase I

repair; Topoisomerase

2.90e−09 :topB topB

IA

DNA topoisomerase

4e−76

III 6.00e−05

:topA_-arch

topA_-arch DNA

topoisomerase I

1.10e−31

92,
NEQ047
hypothetical HESA
HESA protein
2.00E−10
ThiF ThiF family 3.00e−4

Metabolism; Coenzyme

93

protein[Aeropyrum
[Aeropyrum pernix].

metabolism; Dinucleotide-

pernix]

utilizing

enzymes

involved in

molybdopterin

and thiamine

biosynthesis

family 2 1e−11

94,
NEQ046
hypothetical protein
Unknown
0.03
0

95

[Streptococcus

agalactiae NEM316]

96,
NEQ048
hypothetical protein
hypothetical protein
5.00E−04
0

97

[Plasmodium

falciparum 3D7].

98,
NEQ050
hypothetical protein
hypothetical protein
4.00E−06
NTP_-transf_-2 Nucleotidyltransferase domain

99

[Plasmodium

falciparum 3D7].

100,
NEQ049
Predicted Fe—S
conserved
3.00E−14
TIGR01212 TIGR01212

Poorly

101

oxidoreductase[Thermoplasma
hypothetical protein

conserved hypothetical

characterized;

volcanium]
[Archaeoglobus

3.20e−4 protein TIGR01212 6.00e−3

General

fulgidus].

:Radical_-SAM Radical SAM

function

superfamily 4.10e−16

prediction

only; Predicted

Fe—S

oxidoreductases

2e−15

102,
NEQ051
ferredoxin-NADP
hypothetical protein
3.00E−17
FAD_-binding_-6
1.18.
Metabolism; Energy

103

reductase[Neisseria
[Cytophaga

Oxidoreductase
1.2
production

meningitidis MC58]

hutchinsonii].

FAD-binding domain

and

1.20e−13

conversion; Flavodoxin

:NAD_-binding_-1

reductases

Oxidoreductase

(ferredoxin-

NAD-binding

NADPH

domain 1.50e−06

reductases)

family 1 5e−14

104,
NEQ052
peptide chain
peptide chain release
1.00E−77
eRF1_-1 eRF1 domain 1

Information

105

release factor
factor aRF, subunit 1

2.40e−31 :eRF1_-2 eRF1

storage and

aRF, subunit 1
[Pyrococcus abyssi].

domain 2 1.40e−45 :eRF1_-3

processing; Translation,

[Pyrococcus abyssi]

eRF1 domain 3 9.70e−08

ribosomal

:eRF eRF peptide chain

structure and

release factor eRF/aRF,

biogenesis; Peptide

subunit 1 2.10e−67 :pelA

chain

pelA cell division protein

release factor

pelota 3.00e−3

eRF1 7e−79

106,
NEQ053
RNA
RNA
2.00E−86
ygcA ygcA RNA
2.1.1.—
Information

107

methyltransferase
methyltransferase

methyltransferase,

storage and

[Pyrococcushorikoshii]
[Pyrococcus

TrmA family 4.30e−55

processing; Translation,

horikoshii].

ribosomal

structure and

biogenesis; SAM-

dependent

methyltransferases

related

to tRNA

(uracil-5-)-

methyltransferase

1e−87

108,
NEQ054
Conserved
hypothetical protein
9.00E−07

Poorly

109

hypothetical
[Synechocystis sp.

characterized;

protein[Pyrococcus
PCC 6803].

Function

abyssi]

unknown; Predicted

membrane

protein 1e−07

110,
NEQ055
cysteinyl-tRNA
cysteinyl-tRNA
1.00E−117
tRNA-synt_-1e tRNA
6.1.1.
Information

111

synthetase[Pyrococcus
synthetase

synthetases class I
16
storage and

furiosus DSM
[Pyrococcus furiosus

(C) 3.70e−139 :metG

processing; Translation,

3638]
DSM 3638].

metG methionyl-

ribosomal

tRNA synthetase

structure and

2.90e−07 :cysS cysS

biogenesis; Cysteinyl-

cysteinyl-tRNA

tRNA

synthetase 1.70e−140

synthetase

1e−115

112,
NEQ057
Cell division control
398aa long
4.00E−61
AAA ATPase family

Information

113

6/orc1
hypothetical cell

associated with various

storage and

proteinhomolog
division control protein

cellular activities (AAA)

processing; DNA

(cdc6-1) [S solfataricus]
6 [Sulfolobus

1.10e−4

replication,

tokodaii].

recombination

and

repair; Cdc6-

related

protein, AAA

superfamily

ATPase 2e−61

114,
NEQ056
hypothetical protein
hypothetical protein
4.00E−03
0

115

[Plasmodium

falciparum 3D7].

116,
NEQ058
SSU ribosomal
SSU ribosomal
1.00E−49
Ribosomal_-S12 Ribosomal

Information

117

protein
protein S12P

protein S12 2.40e−57

storage and

S12P[Pyrococcus
[Pyrococcus abyssi].

:rpsL_-bact rpsL_-bact

processing; Translation,

abyssi]

ribosomal protein S12 1.30e−4

ribosomal

:S23_-S12_-E_-A

structure and

S23_-S12_-E_-A ribosomal

biogenesis; Ribosomal

protein S23 (S12) 3.60e−75

protein S12

1e−50

118,
NEQ059
Predicted ATPase
TWITCHING
1.00E−163
KH KH domain 7.40e−05

Poorly

119

(PilT family)
MOBILITY (PILT)

:PIN PIN domain 5.60e−08

characterized;

RELATED PROTEIN

General

(PILT) [Pyrococcus

function

abyssi].

prediction

only; ATPases

of the PilT

family 1e−164

120,
NEQ060
hypothetical protein
RNA helicase
0.11
0

121

[Plasmodium yoelii

yoelii].

122,
NEQ061
UDP-N-
UDP-N-
4.00E−21
Glycos_-transf_-4
2.7.8.
Cellular

123

acetylglucosamine-
acetylglucosamine-

Glycosyl transferase
15
processes; Cell

dolichyl-phosphateN-
dolichyl-phosphate N-

3.90e−10

envelope

acetylglucosaminephosphotransferase
acetylglucosaminephosphotransferase

biogenesis,

(gnptA) [Sulfolobus

outer

solfataricus].

membrane; UDP-

N-

acetylmuramyl

pentapeptide

phosphotransferase/

UDP-

N-

acetylglucosamine-

1-

phosphate

transferase

9e−21

124,
NEQ062
Hypothetical protein;
virulence factor
7.00E−05
0

125

permease?
homolog MviB

[Aquifex aeolicus].

126,
NEQ063
ribonuclease HII
ribonuclease HII
8.00E−34
RNase_-HII
3.1.26.4
Information

127

(rnhB)[Pyrobaculum
(rnhB) [Pyrobaculum

Ribonuclease HII

storage and

aerophilum]

aerophilum].

2.40e−34 :rnhC rnhC

processing; DNA

ribonuclease HIII

replication,

3.40e−3 :TIGR00729

recombination

TIGR00729

and

ribonuclease HII

repair; Ribonuclease

3.90e−45

HII 5e−34

128,
NEQ064
arylsulfatase,
conserved
1.00E−45
lactamase_-B Metallo-beta-

Poorly

129

putative
hypothetical protein

lactamase superfamily

characterized;

[Pyrococcusfuriosus
[Methanococcus

2.20e−06

General

DSM 3638]

jannaschii].

function

prediction

only; Metal-

dependent

hydrolases of

the beta-

lactamase

superfamily III

8e−47

130,
NEQ065
LSU ribosomal
82aa long
4.00E−12
Ribosomal_-L23 Ribosomal

Information

131

protein L23
hypothetical 50S

protein L23 1.40e−4

storage and

ribosomal protein L23

processing; Translation,

[Sulfolobus tokodaii].

ribosomal

structure and

biogenesis; Ribosomal

protein L23

8e−12

132,
NEQ066
hypothetical protein
hypothetical protein
3.00E−03
0

133

[Plasmodium

falciparum 3D7].

134,
NEQ067
Predicted hydrolase
conserved protein
7.00E−31

Poorly

135

of the metallo-beta-
[Methanosarcina

characterized;

lactamase

mazei Goe1].

General

superfamily [M.

function

prediction

only; Predicted

hydrolase of

the metallo-

beta-

lactamase

superfamily

3e−31

136,
NEQ068
DNA-directed DNA
DNA-directed DNA
6.00E−85
DNA_-pol_-B DNA
2.7.7.7
Information

137

polymerase I(family
polymerase

polymerase family B

storage and

B) [P furiosus] SPLIT
[Pyrococcus furiosus

3.70e−07

processing; DNA

see SEQ ID
DSM 3638].

:DNA_-pol_-B_-exo

replication,

NOS: 1028, 1029

DNA polymerase

recombination

family B,

and

exonuclease domain

repair; DNA

4.70e−40

polymerase

elongation

subunit

(family B) 5e−85

138,
NEQ069
SSU Ribosomal
Ribosomal protein
1.00E−46
Ribosomal_-S11 Ribosomal

Information

139

protein
S11 [Methanopyrus

protein S11 8.90e−50

storage and

S11[Methanopyrus

kandleri AV19].

processing; Translation,

kandleri AV19]

ribosomal

structure and

biogenesis; Ribosomal

protein S11

2e−46

140,
NEQ070
hypothetical protein
expressed protein
0.06
0

141

[Arabidopsis thaliana].

142,
NEQ072
hypothetical protein
P-type ATPase,
0.39
0

143

putative [Plasmodium

falciparum 3D7].

144,
NEQ071
conserved
conserved
1.00E−11
0

Cellular

145

hypothetical
hypothetical protein

processes; Signal

protein[Archaeoglobus
[Archaeoglobus

transduction

fulgidus]

fulgidus].

mechanisms;

Predicted

Ser/Thr

protein kinase

1e−12

146,
NEQ073
hypothetical protein
putative tyrosine-
0.46
0

147

protein kinase

[Salmonella enterica

subsp. enterica

serovar Typhi].

148,
NEQ074
ATPase subunit of
probable ATP-
2.00E−17
ABC_-tran ABC transporter

Poorly

149

an iron-
dependent transport

5.10e−09 :3a0501s02

characterized;

regulatedABC-type
protein [imported]-

3a0501s02 Type II

General

transporter [M. kandleri]

Pyrococcus furiosus.

(General) Secretory

function

Pathway (IISP) Family

prediction

protein 7.80e−3 :3a0106s01

only; Iron-

3a0106s01 sulfate transport

regulated

system permease protein

ABC

8.60e−4 :cbiO cbiO cobalt

transporter

transport protein ATP-

ATPase

binding subunit 1.30e−4

subunit SufC

:ccmA ccmA heme exporter

8e−18

protein CcmA 4.90e−05

150,
NEQ076
Predicted metal-
cleavage and
1.00E−144
lactamase_-B Metallo-beta-

Poorly

151

dependent
polyadenylation

lactamase superfamily

characterized;

RNase, consists of a
specifity factor protein

2.40e−14

General

metallo-beta-
[Pyrococcus furiosus

function

lactamase
DSM 3638].

prediction

only; Predicted

metal-

dependent

RNase,

consists of a

metallo-beta-

lactamase

domain and

an RNA-

binding KH

domain 1e−144

152,
NEQ075
LSU ribosomal
LSU ribosomal protein
1.00E−68
Ribosomal_-L18p Ribosomal

Information

153

protein
L18P [Methanococcus

L18p/L5e family 1.80e−35

storage and

L18P[Methanococcus

jannaschii].

processing; Translation,

jannaschii]

ribosomal

structure and

biogenesis; Ribosomal

protein L18

9e−70

154,
NEQ077
glutamate
Glutamate
1.00E−116
GLFV_-dehydrog
1.4.1.3
Metabolism; Amino

155

dehydrogenase[Thermoplasma
dehydrogenase

Glutamate/Leucine/

acid

volcanium]
[Thermoplasma

Phenylalanine/Valine

transport and

volcanium].

dehydrogenase

metabolism; Glutamate

1.40e−74

dehydrogenase/

:GLFV_-dehydrog_-N

leucine

Glu/Leu/Phe/Val

dehydrogenase

dehydrogenase,

1e−117

dimerisation domain

2.90e−61

156,
NEQ077a
Endonuclease IV
putative
2.00E−32
AP_-endonuc_-2 AP
3.1.21.2
Information

157

related protein
endonuclease IV

endonuclease family

storage and

[Pyrococcus abyssi]
[Pyrococcus furiosus

2 1.10e−4

processing; DNA

DSM 3638].

replication,

recombination

and

repair; Endonuclease

IV 2e−33

158,
NEQ078
conserved
hypothetical protein
1.00E−161
UPF0027 Uncharacterized

Poorly

159

hypothetical
[Aeropyrum pernix].

protein family UPF0027

characterized;

protein[Aeropyrum

9.40e−267

Function

pernix]

unknown; Uncharacterized

ACR 1e−162

160,
NEQ079
hypothetical protein
conserved
0.14
0

161

hypothetical protein

[Clostridium

perfringens].

162,
NEQ080
hypothetical protein
hypothetical protein
0.15
0

163

[Rhodopseudomonas

palustris].

164,
NEQ081
hypothetical protein
hypothetical protein
1.2
0

165

[Plasmodium yoelii

yoelii].

166,
NEQ082
translation
translation elongation
1.00E−161
GTP_EFTU
3.6.1.
Information

167

elongation factor eF-
factor eF-1, subunit

Elongation factor Tu
48
storage and

1, subunit alpha (tuf)
alpha (tuf)

GTP binding domain

processing; Translation,

[P furiosus]
[Pyrococcus furiosus

2.70e−92

ribosomal

DSM 3638].

:GTP_EFTU_D2

structure and

Elongation factor Tu

biogenesis; GTPases-

domain 2 8.50e−27

translation

:GTP_EFTU_D3

elongation

Elongation factor Tu

factors 1e−162

C-terminal domain

9.90e−38

:small_GTP

small_GTP small

GTP-binding protein

domain 7.40e−07

:selB selB

selenocysteine-

specific translation

elongation factor

9.80e−07 :EF-

1_alpha EF-1_alpha

translation

elongation factor

EF-1, subunit alpha

8.50e−242 :EF-Tu

EF-Tu translation

elongation factor Tu

1.10e−61 :FOLD979

No CATH

Annotation 5.70e−08

168,
NEQ083
SSU ribosomal
SSU ribosomal
1.00E−21
Ribosomal_S10 Ribosomal

Information

169

protein
protein S10AB

protein S10p/S20e 5.40e−30

storage and

S10AB(rps10AB)
(rps10AB) [Sulfolobus

:S10_Arc_S20_Euk

processing; Translation,

[Sulfolobus

solfataricus].

S10_Arc_S20_Euk

ribosomal

solfataricus]

ribosomal protein S10 7.10e−29

structure and

:rpsJ_bact rpsJ_bact

biogenesis; Ribosomal

ribosomal protein S10 8.50e−09

protein S10

2e−21

170,
NEQ084
coenzyme F420 -
coenzyme F420--
4.00E−19
CBS CBS domain 3.60e−3

Poorly

171

quinoneoxidoreductase
quinone

:CBS CBS domain 2.20e−4

characterized;

(EC1.6.5.-—) 41K
oxidoreductase (EC

General

chain
1.6.5.—) 41K chain

function

[validated] -

prediction

Archaeoglobus

only; CBS

fulgidus.

domains 3e−20

172,
NEQ085
hypothetical protein
GTA = Global
0.38
0

173

Transactivator = AcMN

PV orf42 [Bombyxmori

nuclear

polyhedrosis virus].

174,
NEQ086
hypothetical protein
hypothetical protein
3.00E−11
0

175

[Plasmodium

falciparum 3D7].

176,
NEQ088
DNA repair protein
DNA repair protein
1.00E−102
XPG_I XPG I-region 5.80e−42

Information

177

RAD2[Pyrococcus
RAD2 [Pyrococcus

:XPG_N XPG N-terminal

storage and

abyssi]

abyssi].

domain 3.40e−27

processing; DNA

replication,

recombination

and repair; 5′-

3′

exonuclease

(including N-

terminal

domain of

Poll) 1e−103

178,
NEQ087
lysyl-tRNA
lysyl-tRNA
5.00E−68
tRNA-synt_1f tRNA
6.1.1.6
Information

179

synthetase; LysS
synthetase; LysS

synthetases class I

storage and

(class-
[Halobacterium sp.

(K) 3.60e−86

processing; Translation,

1)[Halobacterium sp.
NRC-1].

:lysS_arch

ribosomal

NRC-1]

lysS_arch lysyl-

structure and

tRNA synthetase

biogenesis; Lysyl-

1.50e−75

tRNA

synthetase

class I 3e−69

180,
NEQ089
hypothetical protein
similar to midasin, a
0.18
0

181

large protein with an

N-terminal domain, a

central AAA domain

(with similarity to

dynein) composed of

6 tandem AAA

protomers, and a C-

terminal M-domain

containing MIDAS

(Metal Ion Dependent

Adhesion Site)

sequence motifs;

Mdn1p [Sacc

182,
NEQ090
polysaccharide
polysaccharide
1.00E−15
Polysacc_synt

Poorly

183

biosynthesis
biosynthesis protein,

Polysaccharide biosynthesis

characterized;

protein, putative
putative

protein 4.30e−3

General

[Archaeoglobus
[Archaeoglobus

function

fulgidus]

fulgidus].

prediction

only; Membrane

protein

involved in

the export of

O-antigen

and teichoic

acid 9e−17

184,
NEQ091
LSU ribosomal
LSU ribosomal protein
3.00E−46
Ribosomal_L10
4.2.99.
Information

185

protein
L10E [Methanococcus

Ribosomal protein
18
storage and

L10E[Methanococcus

jannaschii].

L10 1.30e−19

processing; Translation,

jannaschii]

ribosomal

structure and

biogenesis; Ribosomal

protein L10

2e−47

186,
NEQ092
LSU ribosomal
LSU ribosomal protein
3.00E−35
Ribosomal_L14 Ribosomal

Information

187

protein L14P
L14P (rplN)

protein L14p/L23e 2.60e−49

storage and

(rplN)[Methanococcus
[Methanococcus

:rplN_bact rplN_bact

processing; Translation,

jannaschii]

jannaschii].

ribosomal protein L14 9.50e−26

ribosomal

structure and

biogenesis; Ribosomal

protein L14

3e−36

188,
NEQ094
hypothetical protein
conserved protein
0.01
0

189

[Methanosarcina

mazei Goe1].

190,
NEQ093
LSU ribosomal
LSU ribosomal protein
6.00E−49
Ribosomal_L5 Ribosomal

Information

191

protein
L5P [Pyrococcus

protein L5 9.20e−19

storage and

L5P[Pyrococcus

abyssi].

:Ribosomal_L5_C ribosomal

processing; Translation,

abyssi]

L5P family C-terminus

ribosomal

1.80e−39

structure and

biogenesis; Ribosomal

protein L5 5e−50

192,
NEQ095
hypothetical protein
Cucumber Basic
0.33
DUF124 Protein of unknown

function DUF124 3.70e−3

193

Protein, A Blue

Copper Protein.

194,
NEQ096
Predicted P-loop
Predicted P-loop
7.00E−60
Acetyltransf

Poorly

195

ATPase fused to
ATPase fused to an

Acetyltransferase (GNAT)

characterized;

anacetyltransferase
acetyltransferase

family 2.70e−3

General

[M. kandler] SPLIT
[Methanopyrus

function

see SEQ ID NO: 964,

kandleri AV19].

prediction

965

only; Predicted

P-loop

ATPase fused

to an

acetyltransferase

1e−53

196,
NEQ097
nuclease, putative
nuclease, putative
2.00E−07
SNase Staphylococcal

Information

197

[Haemophilusinfluenzae
[Haemophilus

nuclease homologue 3.70e−07

storage and

Rd]

influenzae Rd].

processing; DNA

replication,

recombination

and

repair; Micrococcal

nuclease

(thermonuclease)

homologs 1e−08

198,
NEQ098
Predicted
hypothetical protein
5.00E−18
DUF118 Helix-turn-helix

Information

199

transcription
[Pyrococcus furiosus

family DUF118 9.60e−16

storage and

regulator[Halobacterium
DSM 3638].

processing; Transcription;

sp. NRC-1]

Predicted

transcriptional

regulators 3e−18

200,
NEQ099
hypothetical protein
wsv079 [shrimp white
0.5
0

201

spot syndrome virus].

202,
NEQ100
hypothetical protein
hemolectin
0.55
0

203

[Drosophila

melanogaster].

204,
NEQ101
LSU Ribosomal
Ribosomal protein
1.00E−41
Ribosomal_L11 Ribosomal

Information

205

protein
L11 [Methanopyrus

protein L11, RNA binding

storage and

L11[Methanopyrus

kandleri AV19].

domain 2.10e−17

processing; Translation,

kandleri AV19]

:Ribosomal_L11_N

ribosomal

Ribosomal protein L11, N-

structure and

terminal domain 1.90e−17

biogenesis; Ribosomal

protein L11

3e−41

206,
NEQ103
Archaeal/vacuolar-
V-TYPE ATP
1.00E−180
ATP-synt_ab ATP
3.6.3.
Metabolism; Energy

207

type H+-
SYNTHASE ALPHA

synthase alpha/beta
14
production

ATPasesubunit A
CHAIN (V-TYPE

family, nucleotide-

and

[Methanopyrus
ATPASE SUBUNIT

binding domain

conversion; Archaeal/

kandleri]
A).

4.30e−84 :ATP-

vacuolar-

synt_ab_C ATP

type H+-

synthase alpha/beta

ATPase

chain, C terminal

subunit A 1e−176

domain 2.90e−18

:ATP-synt_ab_N

ATP synthase

alpha/beta family,

beta-barrel domain

6.00e−17 :rho rho

transcription

termination factor

Rho 5.50e−3

:fliI_yscN fliI_yscN

ATPase FliI/YscN

family 6.00e−09

:atpD atpD ATP

synthase F1, beta

subunit 2.00e−11 :V-

ATPase_V1_B V-

ATPase_V1_B V-

type ATPase,

subunit B 3.00e−09

:ATP_syn_B_arch

ATP_syn_B_arch

ATP synthase

archaeal, B subunit

5.50e−10 :V-

ATPase_V1_A V-

ATPase_V1_A V-

type ATPase,

subunit A 2.10e−222

:ATP_syn_A_arch

ATP_syn_A_arch

ATP synthase

archaeal, A subunit

0.0

208,
NEQ102
histidyl-tRNA
histidyl-tRNA
8.00E−88
HGTP_anticodon
6.1.1.
Information

209

synthetase (class
synthetase

Anticodon binding
21
storage and

2)[Aeropyrum pernix]
[Aeropyrum pernix].

domain 2.40e−10

processing; Translation,

:tRNA-synt_2b tRNA

ribosomal

synthetase class II

structure and

core domain (G, H,

biogenesis; Histidyl-

P, S and T) 5.80e−32

tRNA

:hisS hisS

synthetase

histidyl-tRNA

5e−89

synthetase 4.40e−77

:hisS_second

hisS_second

histidyl-tRNA

synthetase 2,

putative 4.10e−33

210,
NEQ104
hypothetical protein
conserved
4.00E−05
0

211

hypothetical protein

[Methanococcus

jannaschii].

212,
NEQ105
SSU Ribosomal
Ribosomal protein
6.00E−26
Ribosomal_S6e Ribosomal

Information

213

protein S6E
S6E (S10)

protein S6e 1.20e−23

storage and

(S10)[Methanopyrus
[Methanopyrus

processing; Translation,

kandleri AV19].

kandleri AV19].

ribosomal

structure and

biogenesis; Ribosomal

protein S6E

(S10) 9e−24

214,
NEQ107
N2,N2-
hypothetical protein
8.00E−04
0

215

dimethylguanosine
[Plasmodium

tRNAmethyltransferase

falciparum 3D7].

[Aquifex aeolicus]

216,
NEQ109
conserved
cell division protein
2.00E−10
eRF1_1 eRF1 domain 1

Poorly

217

hypothetical
pelota (pelA),

8.40e−4: pelA pelA cell

characterized;

protein [Archaeoglobus
conjectural

division protein pelota 6.80e−3

General

fulgidus]
[Pyrobaculum

function

aerophilum].

prediction

only; Predicted

RNA-

binding

proteins 2e−10

218,
NEQ108
cell division protein
N2,N2-
4.00E−56
TRM N2,N2-
2.1.1.
Information

219

pelota
dimethylguanosine

dimethylguanosine
32
storage and

(pelA), conjectural
tRNA

tRNA

processing; Translation,

[Pyrobaculum
methyltransferase

methyltransferase

ribosomal

aerophilum]
[Aquifex aeolicus].

1.10e−80 :TRM1

structure and

TRM1 N2,N2-

biogenesis; N2,

dimethylguanosine

N2-

tRNA

dimethylguanosine

methyltransferase

tRNA

1.30e−65

methyltransferase

3e−57

220,
NEQ110
Predicted exosome
conserved
1.00E−09

Poorly

221

subunit,
hypothetical protein

characterized;

predictedexoribonuclease
[Archaeoglobus

General

related to

fulgidus].

function

RNase PH

prediction

only; Archaeal

serine

proteases 7e−10

222,
NEQ111
GTP-binding protein,
conserved
2.00E−26
RNase_PH 3′
2.7.7.
Information

223

gtp1/obg
hypothetical protein

exoribonuclease
56
storage and

family[Pyrococcus
[Archaeoglobus

family, domain 1

processing; Translation,

furiosus DSM 3638]

fulgidus].

1.20e−12

ribosomal

:RNase_PH_C 3′

structure and

exoribonuclease

biogenesis; RNase

family, domain 2

PH-

6.30e−4

related

exoribonuclease

1e−27

224,
NEQ112
hypothetical protein
GTP-binding protein,
8.00E−50
GTP1_OBG GTP1/OBG

Poorly

225

[Pyrococcusfuriosus
gtp1/obg family

family 1.70e−3 :TGS TGS

characterized;

DSM 3638]
[Pyrococcus furiosus

domain 1.20e−22

General

DSM 3638].

function

prediction

only; Predicted

GTPase 2e−49

226,
NEQ113
hypothetical
hypothetical protein
4.00E−06
DUF54 Protein of unknown

function DUF54 1.80e−3

227

nucleotidyltransferase
[Pyrococcus furiosus

[Pyrococcus
DSM 3638].

furiosus DSM 3638]

228,
NEQ114
Tryptophanyl-tRNA
399aa long
2.00E−39
HD HD domain 3.00e−08

Poorly

229

synthetase
hypothetical

characterized;

(trpS) (class 1b)
interferon-gamma

General

[Sulfolobus
inducible protein

function

solfataricus]
[Sulfolobus tokodaii].

prediction

only; HD

superfamily

phosphohydrolases

1e−40

230,
NEQ115
hypothetical protein
Tryptophanyl-tRNA
1.00E−111
tRNA-synt_1b tRNA
6.1.1.2
Information

231

synthetase (trpS)

synthetases class I

storage and

[Sulfolobus

(W and Y) 7.10e−40

processing; Translation,

solfataricus].

:trpS trpS

ribosomal

tryptophanyl-tRNA

structure and

synthetase 5.40e−55

biogenesis; Tryptophanyl-

tRNA

synthetase

1e−102

232,
NEQ116
Predicted nucleotide
olfactory receptor 1-5
0.88
0

233

kinase related
[Takifugu rubripes].

toCMP and AMP

kinase

[Methanopyrus

234,
NEQ118
cell division inhibitor
phosphoesterase
3.00E−04
DHH DHH family 6.50e−07

235

minD
[Methanosarcina

homolog [Pyrococcus

acetivorans str. C2A].

furiosus DSM 3638]

236,
NEQ117
phosphoesterase
hypothetical protein
3.00E−25
SKI Shikimate kinase 2.70e−3

Metabolism; Nucleotide

237

[Methanosarcinaacetivorans
[Pyrococcus

transport and

str. C2A]

horikoshii].

metabolism; Predicted

nucleotide

kinase

(related to

CMP and

AMP kinases)

2e−26

238,
NEQ119
hypothetical protein
cell division inhibitor
6.00E−36
ArsA_ATPase Anion-

Cellular

239

minD homolog

transporting ATPase 4.50e−3

processes; Cell

[Pyrococcus furiosus

:fer4_NifH 4Fe-4S iron sulfur

division and

DSM 3638].

cluster binding proteins,

chromosome

NifH/frxC family 6.10e−3

partitioning; ATPases

:ParA ParA family ATPase

involved in

2.40e−17 :eps_fam eps_fam

chromosome

capsular exopolysaccharide

partitioning

family 5.90e−08

3e−34

240,
NEQ120
hypothetical protein
similar to Plasmodium
0.83
0

241

falciparum.

Asparagine-rich

antigen [Dictyostelium

discoideum].

242,
NEQ121
hypothetical protein
hypothetical protein
4.00E−06
0

243

[Plasmodium

falciparum 3D7].

244,
NEQ123
hypothetical protein
claustrin-chicken.
2.00E−09
0

Information

245

storage and

processing; Translation,

ribosomal

structure and

biogenesis; Translation

initiation

factor 2

(GTPase) 1e−07

246,
NEQ122
Metal-dependent
METAL DEPENDENT
1.00E−40
lactamase_B Metallo-beta-

Poorly

247

hydrolase of thebeta-
HYDROLASE

lactamase superfamily

characterized;

lactamase
[Pyrococcus abyssi].

3.30e−07

General

superfamily

function

prediction

only; Metal-

dependent

hydrolases of

the beta-

lactamase

superfamily I

1e−41

248,
NEQ124
Queuine/archaeosine
queuine trna-
4.00E−87
TGT Queuine tRNA-
2.4.2.
Information

249

tRNA-
ribosyltransferase

ribosyltransferase
29
storage and

ribosyltransferase [Methanopyrus
[Pyrococcus furiosus

1.20e−37

processing; Translation,

DSM 3638].

:Q_tRNA_tgt

ribosomal

Q_tRNA_tgt

structure and

queuine tRNA-

biogenesis; Queuine/

ribosyltransferase

archaeosine

1.80e−10

tRNA-

:arcsn_tRNA_tgt

ribosyltransferase

arcsn_tRNA_tgt

4e−86

archaeosine tRNA-

ribosyltransferase

2.00e−15

:tgt_general

tgt_general tRNA-

guanine

transglycosylases,

various specificities

2.00e−60

250,
NEQ125
fibrillarin-like pre-
fibrillarin-like pre-
1.00E−65
Fibrillarin Fibrillarin 9.60e−96

Information

251

rRNA
rRNA processing

storage and

processingprotein
protein [Pyrococcus

processing; Translation,

[Pyrococcus

furiosus DSM 3638].

ribosomal

furiosus]

structure and

biogenesis; Fibrillarin-

like

rRNA

methylase 8e−67

252,
NEQ126
L-asparaginase
L-asparaginase
2.00E−70
Asparaginase
3.5.1.1
Metabolism; Amino

253

[Pyrococcushorikoshii] = GatD
[Pyrococcus

Asparaginase

acid

horikoshii].

7.80e−14 :asnASE_I

transport and

asnASE_I L-

metabolism; L-

asparaginases, type

asparaginase/

I 1.40e−81

archaeal Glu-

:asnASE_II

tRNAGln

asnASE_II L-

amidotransferase

asparaginases, type

subunit D

II 2.20e−08

1e−71

254,
NEQ126a
PUTATIVE
PUTATIVE
5.00E−25
DUF123 Domain of
4.2.99.
Poorly

255

ENDONUCLEASE III
ENDONUCLEASE.

unknown function
18
characterized;

[Pyrococcus abyssi]
[Pyrococcus abyssi].

DUF123 3.70e−33

Function

SPLIT see SEQ ID

unknown; Uncharacterized

NOS: 778, 779

ACR 3e−26

256,
NEQ127
Predicted carbamoyl
nodulation protein
0
CmcH_NodU
2.—.—.—
Cellular

257

transferase, NodU
[Methanococcus

Carbamoyltransferase

processes; Posttranslational

family

jannaschii].

2.10e−147

modification,

[Methanopyrus

protein

turnover,

chaperones; Predicted

carbamoyl

transferase,

NodU family 0

258,
NEQ128
conserved
conserved
2.00E−13
0

Poorly

259

hypothetical
hypothetical protein

characterized;

protein[Thermotoga
[Thermotoga

General

maritima]

maritima].

function

prediction

only; Uncharacterized

proteins of

the AP

superfamily

2e−14

260,
NEQ130
translation initiation
translation initiation
1.00E−14
eIF-1a Eukaryotic initiation

Information

261

factor aIF-
factor aIF-1A

factor 1A 2.00e−14 :eIF-1A

storage and

1A [Methanococcus
[Methanococcus

eIF-1A translation initiation

processing; Translation,

jannaschii]

jannaschii].

factor eIF-1A 7.40e−18

ribosomal

structure and

biogenesis; Translation

initiation

factor IF-1 8e−16

262,
NEQ129
Predicted membrane
hypothetical protein
7.00E−18
UPF0051 Uncharacterized

Poorly

263

components ofan
[Desulfovibrio

protein family (UPF0051)

characterized;

uncharacterized iron-

desulfuricans G20].

1.60e−07

General

regulated

function

prediction

only; Predicted

membrane

components

of an

uncharacterized

iron-

regulated

ABC-type

transporter

SufB 9e−16

264,
NEQ131
Predicted RNA-
conserved
5.00E−43
Translin Translin family

Information

265

binding protein of
hypothetical protein

3.70e−09

storage and

thetranslin family [M kandleri]
[Pyrobaculum

processing; DNA

aerophilum].

replication,

recombination

and

repair; Translin

(RNA-

binding

protein,

recombination

hotspot

binding in

eukaryotes)

1e−16

266,
NEQ133
cell division protein
cell division protein
1.00E−102
tubulin Tubulin/FtsZ
3.4.24.—
Cellular

267

FtsZ
FtsZ [Pyrococcushorikoshii].

family, GTPase

processes; Cell

[Pyrococcushorikoshii]

domain 2.40e−77

division and

:ftsZ ftsZ cell

chromosome

division protein FtsZ

partitioning; Cell

1.20e−125

division

:tubulin_C

GTPase 1e−103

Tubulin/FtsZ family,

C-terminal domain

2.90e−16

268,
NEQ132
hypothetical protein
RNA polymerase
2.6
0

269

beta-prime subunit

[Candidatus

Carsonella ruddii].

270,
NEQ134
hypothetical protein
hypothetical protein
1
0

271

[Plasmodium yoelii

yoelii].

272,
NEQ135
hypothetical protein
unknown protein-
3.00E−03
0

273

related [Plasmodium

yoelii yoelii].

274,
NEQ136
hypothetical protein
putative retroelement
2.4
0

275

pol polyprotein

[Arabidopsis thaliana].

276,
NEQ139
hypothetical protein
hypothetical protein
0.01
0

277

[Plasmodium

falciparum 3D7].

278,
NEQ139
hypothetical protein
ymf77 [Tetrahymena
2.00E−06
0

279

thermophila].

280,
NEQ138
hypothetical protein
521aa long
0.05
0

281

hypothetical protein

[Sulfolobus tokodaii].

282,
NEQ140
hypothetical protein
ORF MSV156
2.00E−03
0

283

hypothetical protein

[Melanoplus

sanguinipes

entomopoxvirus].

284,
NEQ141
HSP60 family
HSP60 family
0
cpn60_TCP1 TCP-
2.7.1.
Cellular

285

chaperonin[Methanopyrus
chaperonin

1/cpn60 chaperonin
68
processes; Posttranslational

kandleri AV19]
[Methanopyrus

family 4.30e−219

modification,

kandleri AV19].

protein

turnover,

chaperones; Chaperonin

GroEL

(HSP60

family) 0

286,
NEQ144
DNA topoisomerase
DNA topoisomerase
1.00E−107
HATPase_c
5.99.
Information

287

VI, subunit B (top6B)
VI, subunit B (top6B)

Histidine kinase-,
1.3
storage and

[Archaeoglobus
[Archaeoglobus

DNA gyrase B-, and

processing; DNA

fulgidus]

fulgidus].

HSP90-like ATPase

replication,

3.70e−11 :top6b

recombination

top6b DNA

and

topoisomerase VI, B

repair; DNA

subunit 7.80e−160

topoisomerase

VI, subunit

B 1e−108

288,
NEQ143
Predicted
165aa long conserved
4.00E−18
HTH_3 Helix-turn-helix

Information

289

transcription
hypothetical protein

4.00e−16 :TIGR00270

storage and

factor, homolog of
[Sulfolobus tokodaii].

TIGR00270 conserved

processing; Transcription;

eukaryotic MBF1 [M.

hypothetical protein

Predicted

TIGR00270 1.10e−15

transcription

factor,

homolog of

eukaryotic

MBF1 5e−17

290,
NEQ146
LSU Ribosomal
Ribosomal protein L4
2.00E−55
Ribosomal_L4 Ribosomal

Information

291

protein
[Methanopyrus

protein L4/L1 family 4.60e−69

storage and

L4 [Methanopyrus

kandleri AV19].

processing; Translation,

kandleri AV19]

ribosomal

structure and

biogenesis; Ribosomal

protein L4 1e−52

292,
NEQ145
hypothetical protein
hypothetical protein
0.36
0

293

[Clostridium

thermocellum ATCC

27405].

294,
NEQ147
conserved
conserved
7.00E−35
UPF0047 Uncharacterised

Poorly

295

hypothetical
hypothetical protein

protein family UPF0047

characterized;

protein [Archaeoglobus
[Archaeoglobus

2.40e−48 :TIGR00149

Function

fulgidus]

fulgidus].

TIGR00149 conserved

unknown; Uncharacterized

hypothetical protein

ACR 4e−36

TIGR00149 3.30e−34

296,
NEQ148
Fe—S oxidoreductase
hypothetical protein
1.00E−163
B12-binding B12
1.97.
Metabolism; Energy

297

family
[Pyrococcus

binding domain
1.4
production

protein [Methanopyrus

horikoshii].

8.90e−19

and

kandleri AV19]

:TIGR00089

conversion; Fe—S

TIGR00089

oxidoreductases

conserved

family 2

hypothetical protein

1e−164

2.10e−06

:Radical_SAM

Radical SAM

superfamily 2.00e−26

298,
NEQ150
DNA-binding protein
hypothetical protein
2.00E−07
DUF122 Protein of unknown

Poorly

299

[Aeropyrumpernix]
[Pyrococcus

function DUF122 1.20e−05

characterized;

horikoshii].

General

function

prediction

only; DNA-

binding

protein 1e−08

300,
NEQ149
adenylate kinase
adenylate kinase
2.00E−12

2.7.4.3
Metabolism; Nucleotide

301

(ATP-
(ATP-AMP

transport and

AMPtransphosphorylase)
transphosphorylase)

metabolism; Archaeal

[Pyrococcus
[Pyrococcus furiosus

adenylate

DSM 3638].

kinase 2e−13

302,
NEQ151
hypothetical protein
AotM [Aeromonas
0.11
0

303

hydrophila].

304,
NEQ152
tRNA
413aa long
4.00E−32
NTP_transf_2
2.7.7.
Information

305

nucleotidyltransferase
hypothetical tRNA

Nucleotidyltransferase
25
storage and

(tRNAadenylyltransferase)
nucleotidyltransferase

domain 5.80e−06

processing; Translation,

(tRNA CCA-
[Sulfolobus tokodaii].

ribosomal

pyrophosphorylase)

structure and

biogenesis; tRNA

nucleotidyltransferase

(CCA-adding

enzyme) 4e−31

306,
NEQ153
hypothetical protein
hypothetical protein
0.06
0

307

[Mycoplasma

pneumoniae].

308,
NEQ154
hypothetical protein
RKST1.
0.07
0

309

310,
NEQ155
oligosaccharyl
hypothetical protein
6.00E−16
0

Poorly

311

transferase stt3
[Pyrococcus

characterized;

subunitrelated

horikoshii].

General

protein [P. furiosus]

function

prediction

only; Uncharacterized

membrane

protein,

required for

N-linked

glycosylation

4e−17

312,
NEQ156
DNA-directed RNA
DNA-directed RNA
0
RNA_pol_Rpb2_6
2.7.7.6
Information

313

polymerase, subunit
polymerase, subunit B

RNA polymerase

storage and

B′ (rpoB1) [M. jannaschii]
[Pyrococcus abyssi].

Rpb2, domain 6

processing; Transcription;

1.90e−158

DNA-

:RNA_pol_Rpb2_7

directed

RNA polymerase

RNA

Rpb2, domain 7

polymerase

3.50e−31

beta

:RNA_pol_Rpb2_4

subunit/140 kD

RNA polymerase

subunit

Rpb2, domain 4

(split gene in

1.40e−30

Mjan, Mthe,

:RNA_pol_Rpb2_5

Aful) 0

RNA polymerase

Rpb2, domain 5

7.10e−17

314,
NEQ157
GTP-binding protein,
hypothetical protein
4.00E−44
MMR_HSR1 GTPase of

Poorly

315

GTP1/OBG-
[Pyrococcus

unknown function 5.40e−05

characterized;

family [Archaeoglobus

horikoshii].

:small_GTP small_GTP

General

fulgidus]

small GTP-binding protein

function

domain 1.10e−08

prediction

only; Predicted

GTPase 3e−45

316,
NEQ158
conserved
hypothetical protein
2.00E−41
DUF437 Protein of unknown

Poorly

317

hypothetical
[Pyrococcus furiosus

function (DUF437) 4.90e−27

characterized;

protein [Pyrococcus
DSM 3638].

Function

horikoshii]

unknown; Uncharacterized

ACR 1e−41

318,
NEQ159
Predicted
149aa long conserved
1.00E−19
PBP

Poorly

319

phospholipid-
hypothetical protein

Phosphatidylethanolamine-

characterized;

bindingprotein
[Sulfolobus tokodaii].

binding protein 7.60e−07

General

[Thermoplasma

:TIGR00481 TIGR00481

function

volcanium]

conserved hypothetical

prediction

protein TIGR00481 8.10e−13

only; Phospholipid-

binding

protein 4e−18

320,
NEQ160
hypothetical protein

M. jannaschii

0.01
0

321

predicted coding

region MJ0793

[Methanococcus

jannaschii].

322,
NEQ162
conserved putative
hypothetical protein
6.00E−29
UPF0118 Domain of

Poorly

323

membrane
[Aquifex aeolicus].

unknown function DUF20

characterized;

protein, possibly a

4.90e−38

General

permease [P. abyssi]

function

prediction

only; Predicted

permease

4e−30

324,
NEQ161
hypothetical protein
hypothetical protein
2.00E−06
0

325

[Plasmodium yoelii

yoelii].

326,
NEQ163
hypothetical protein
EVM140 (Ectromelia
1.2
0

327

virus].

328,
NEQ164
hypothetical
asoB protein
8.00E−04
0

329

protein maybe
[Methanococcus

membran protein

jannaschii].

330,
NEQ165
Predicted RNA
hypothetical protein
8.00E−21
GidB Glucose
2.1.1.—
Information

331

methylase[Methanopyrus
[Pyrococcus furiosus

inhibited division

storage and

kandleri AV19]
DSM 3638].

protein 5.30e−3

processing; Translation,

:gidB gidB glucose-

ribosomal

inhibited division

structure and

protein B 5.50e−05

biogenesis; Predicted

RNA

methylase 6e−20

332,
NEQ166
H(+)-transporting
H(+)-transporting ATP
2.00E−15
ATP-synt_D ATP
3.6.3.
Metabolism; Energy

333

ATP synthasesubunit
synthase subunit D

synthase subunit D
14
production

D (V-type ATPase
[Pyrococcus

1.60e−09

and

subunit D)

horikoshii].

:V_ATPase_subD

conversion; Archaeal/

V_ATPase_subD

vacuolar-

V-type ATPase,

type H+-

subunit D 1.90e−18

ATPase

subunit D 2e−16

334,
NEQ168
Preprotein
Preprotein
9.00E−60
secY eubacterial secY

Cellular

335

translocase subunit
translocase subunit

protein 9.90e−09

processes; Cell

SecY[Methanopyrus
SecY [Methanopyrus

:3a0501s007 3a0501s007

motility and

kandleri AV19]

kandleri AV19].

preprotein translocase, SecY

secretion; Preprotein

subunit 1.20e−15

translocase

subunit SecY

8e−61

336,
NEQ170
activator 1,
activator 1, replication
1.00E−102
AAA ATPase family
2.7.7.7
Information

337

replication factor C,
factor C, 35 KD

associated with

storage and

35 KD subunit
subunit

various cellular

processing; DNA

[Archaeoglobus
[Archaeoglobus

activities (AAA)

replication,

fulgidus]

fulgidus].

1.30e−15 :ruvB ruvB

recombination

Holliday junction

and

DNA helicase RuvB

repair; ATPase

5.50e−3 :holB holB

involved in

DNA polymerase III,

DNA

delta prime subunit

replication 1e−103

1.00e−3

338,
NEQ169
type II secretion
type II secretion
1.00E−104
GSPII_E Type II/IV secretion

Cellular

339

system protein(gspE-
system protein (gspE-

system protein 7.10e−06

processes; Cell

1) [Archaeoglobus
1) [Archaeoglobus

motility and

fulgidus]

fulgidus].

secretion; Type

IV secretory

pathway,

VirB11

components,

and related

ATPases

involved in

archaeal

flagella

biosynthesis

1e−106

340,
NEQ171
hypothetical protein
similar to Plasmodium
3.00E−05
0

341

falciparum.

Hypothetical protein

[Dictyostelium

discoideum].

342,
NEQ173
DNA-directed RNA
DNA-DIRECTED RNA
8.00E−96
RNA_pol_Rpb2_2
2.7.7.6
Information

343

polymerase, subunit
POLYMERASE

RNA polymerase

storage and

B″ (rpoB2) [M. jannaschii]
SUBUNIT B.

Rpb2, domain 2

processing; Transcription;

3.50e−07

DNA-

:RNA_pol_Rpb2_1

directed

RNA polymerase

RNA

beta subunit 5.50e−33

polymerase

:RNA_pol_Rpb2_3

beta

RNA polymerase

subunit/140 kD

Rpb2, domain 3

subunit

4.60e−21

(split gene in

Mjan, Mthe,

Aful) 1e−94

344,
NEQ172
{putative
Eps7F [Streptococcus
2.00E−12
Glycos_transf_2 Glycosyl

Cellular

345

glycosyltransferase}

thermophilus].

transferase 2.50e−24

processes; Cell

envelope

biogenesis,

outer

membrane; Glycosyltransferases

involved

in cell wall

biogenesis

3e−12

346,
NEQ175
ABC-type transport
hypothetical protein
1.00E−60
DUF214 Predicted

Poorly

347

systems, involvedin
[Methanococcus

permease 1.30e−33

characterized;

lipoprotein release,

jannaschii].

General

permease

function

prediction

only; ABC-

type transport

systems,

involved in

lipoprotein

release,

permease

components

7e−62

348,
NEQ174
RecA-superfamily
hypothetical protein
1.00E−96

Cellular

349

ATPase implicatedin
[Pyrococcus

processes; Signal

signal transduction

horikoshii].

transduction

[Mp. kandleri]

mechanisms;

RecA-

superfamily

ATPases

implicated in

signal

transduction

1e−97

350,
NEQ176
SSU ribosomal
SSU ribosomal
9.00E−16
Ribosomal_S27 Ribosomal

Information

351

protein
protein S27AE;

protein S27a 4.10e−21

storage and

S27AE; (rps27AE)
(rps27AE)

processing; Translation,

[Pyrococcus
[Pyrococcus furiosus

ribosomal

furiosus]
DSM 3638].

structure and

biogenesis; Ribosomal

protein

S27AE 5e−16

352,
NEQ177
threonyl-tRNA
threonyl-tRNA
1.00E−151
HGTP_anticodon
6.1.1.3
Information

353

synthetase (class
synthetase

Anticodon binding

storage and

2)[Pyrobaculum
[Pyrobaculum

domain 1.20e−37

processing; Translation,

aerophilum]

aerophilum].

:tRNA-synt_2b tRNA

ribosomal

synthetase class II

structure and

core domain (G, H,

biogenesis; Threonyl-

P, S and T) 2.10e−06

tRNA

:glyS_dimeric

synthetase

glyS_dimeric glycyl-

1e−108

tRNA synthetase

1.30e−3 :proS_fam_l

proS_fam_l prolyl-

tRNA synthetase

3.00e−4 :thrS thrS

threonyl-tRNA

synthetase 5.10e−50

354,
NEQ178
LSU ribosomal
LSU ribosomal protein
6.00E−20
KOW KOW motif 2.80e−05

Information

355

protein
L14E [Methanococcus

storage and

L14E[Methanococcus

jannaschii].

processing; Translation,

jannaschii].

ribosomal

structure and

biogenesis; Ribosomal

protein L14E

3e−21

356,
NEQ179
LSU ribosomal
LSU ribosomal protein
2.00E−13
Ribosomal_L7Ae Ribosomal

Information

357

protein
L30E [Methanococcus

protein

storage and

L30E[Methanococcus

jannaschii].

L7Ae/L30e/S12e/Gadd45

processing; Translation,

jannaschii]

family 1.80e−10

ribosomal

structure and

biogenesis; Ribosomal

protein L30E

9e−15

358,
NEQ180
transcription
NUSA PROTEIN
6.00E−09
0

Information

359

termination factor
HOMOLOG.

storage and

nusA-

processing; Transcription;

Methanococcus

Transcription

vannielii

elongation

factor 2e−08

360,
NEQ182
DNA-directed RNA
DNA-DIRECTED RNA
1.00E−10
RNA_pool_L RNA
2.7.7.6
Information

361

polymerasesubunit L
POLYMERASE

polymerases L/13

storage and

[Sulfolobus
SUBUNIT L.

to 16 kDa subunit

processing; Transcription;

acidocaldarius]

2.90e−12

DNA-

directed

RNA

polymerase,

subunit L 2e−08

362,
NEQ181
LSU ribosomal
LSU ribosomal protein
2.00E−58
Ribosomal_L15e Ribosomal

Information

363

protein
L15E; (rpl15E)

L15 1.60e−78

storage and

L15E; (rpl15E)
[Pyrococcus furiosus

processing; Translation,

[Pyrococcus
DSM 3638].

ribosomal

furiosus]

structure and

biogenesis; Ribosomal

protein L15E

9e−59

364,
NEQ183
LSU ribosomal
50S ribosomal protein
4.00E−16
Ribosomal_L44 Ribosomal

Information

365

protein
L44 related protein

protein L44 2.00e−11

storage and

L44E; (rpl44E)
[Thermoplasma

processing; Translation,

[Pyrococcus

acidophilum].

ribosomal

furiosus]

structure and

biogenesis; Ribosomal

protein L44E

2e−17

366,
NEQ184
Predicted exosome
conserved
1.00E−23
KH KH domain 2.00e−06

Information

367

subunit, RNA-
hypothetical protein

storage and

bindingprotein Rrp4
[Archaeoglobus

processing; Translation,

(contain S1

fulgidus].

ribosomal

structure and

biogenesis; RNA-

binding

protein Rrp4

and related

proteins

(contain S1

domain and

KH domain)

7e−25

368,
NEQ185
Glu-tRNA
Aspartyl/glutamyl-
1.00E−77
DUF186 GatB/Yqey
6.3.5.—
Information

369

amidotransferase,
tRNA(Asn/Gln)

domain 4.40e−4

storage and

subunitB (gatB-2)
amidotransferase

:GatB_N PET112

processing; Translation,

[Archaeoglobus
subunit B (Asp/Glu-

family, N terminal

ribosomal

fulgidus]
ADT subunit B).

region 5.40e−106

structure and

:gatB gatB

biogenesis; Asp-

glutamyl-tRNA(Gln)

tRNAAsn/Glu-

amidotransferase, B

tRNAGln

subunit 4.40e−104

amidotransferase

:gatB_rel gatB_rel

B subunit

aspartyl-tRNA(Asn)

(PET112

amidotransferase, B

homolog) 7e−79

subunit, putative

1.10e−23 :gatB_rel

gatB_rel aspartyl-

tRNA(Asn)

amidotransferase, B

subunit, putative

5.00e−4 :gatB_rel

gatB_rel aspartyl-

tRNA(Asn)

amidotransferase, B

subunit, putative

3.80e−3

370,
NEQ186
ATP-dependent 26S
Proteasome-activating
1.00E−111
AAA ATPase family
3.6.1.3
Cellular

371

proteasomeregulatory
nucleotidase

associated with

processes; Posttranslational

subunit [M. kandleri
(Proteasome

various cellular

modification,

regulatory subunit).

activities (AAA)

protein

8.10e−89 :FtsH_fam

turnover,

FtsH_fam ATP-

chaperones; ATP-

dependent

dependent

metalloprotease

26S

FtsH 4.70e−21

proteasome

:26Sp45 26Sp45

regulatory

26S proteasome

subunit 1e−107

subunit P45 family

7.10e−182

372,
NEQ188
hypothetical protein
rubrerythrin (rr1)
0.04
0

373

[Archaeoglobus

fulgidus].

374,
NEQ187
SSU ribosomal
SSU ribosomal
3.00E−37
Ribosomal_S19e Ribosomal

Information

375

protein
protein S19E;

protein S19e 9.00e−45

storage and

S19E; (rps19E)
(rps19E) [Pyrococcus

processing; Translation,

[Pyrococcus

furiosus DSM 3638].

ribosomal

furiosus]

structure and

biogenesis; Ribosomal

protein S19E

(S16A) 5e−38

376,
NEQ190
branched-chain
branched-chain amino
3.00E−68
aminotran_4
2.6.1.
Metabolism; Amino

377

amino
acid aminotransferase

Aminotransferase
42
acid

acidaminotransferase
(ilvE) [Pyrobaculum

class IV 5.60e−72

transport and

(ilvE) [Pb

aerophilum].

:D_amino_aminoT

metabolism; Branched-

D_amino_aminoT

chain amino

D-amino acid

acid

aminotransferase

aminotransferase/

6.10e−11 :ilvE_I

4-amino-

ilvE_I branched-

4-

chain amino acid

deoxychorismate

aminotransferase

lyase 1e−62

3.20e−107 :ilvE_II

ilvE_II branched-

chain amino acid

aminotransferase

2.20e−21

378,
NEQ189
hemolysin-related
probable hemolysin-
8.00E−31
CBS CBS domain 1.00e−05

Cellular

379

protein[Thermotoga maritima]
related protein

:DUF21 Domain of unknown

processes; Cell

[Clostridium

function DUF21 3.90e−16

motility and

perfringens].

:CorC_HlyC Transporter

secretion; Hemolysins

associated domain 3.90e−14

and

related

proteins

containing

CBS domains

7e−31

380,
NEQ191
alkyl hydroperoxide
alkyl hydroperoxide
3.00E−90
AhpC-TSA
1.6.4.—
Cellular

381

reductase[Methanococcus
reductase

AhpC/TSA family

processes; Posttranslational

jannaschii]
[Methanococcus

2.40e−39

modification,

jannaschii].

protein

turnover,

chaperones; Peroxiredoxin

2e−91

382,
NEQ193
hypothetical protein
hypothetical protein
0.12
0

383

[Clostridium

thermocellum ATCC

27405].

384,
NEQ192
chorismate
chorismate
2.00E−87
ACT ACT domain
4.2.1.
Metabolism; Amino

385

mutase/prephenatedehydratase
mutase/prephenate

1.30e−10
51
acid

(pheA)
dehydratase (pheA)

:Chorismate_mut

transport and

[A. fulgidus]
[Archaeoglobus

Chorismate mutase

metabolism; Prephenate

fulgidus].

1.50e−06 :PDH

dehydratase

Prephenate

9e−54

dehydrogenase

3.10e−05 :PDT

Prephenate

dehydratase 2.50e−51

386,
NEQ195
putative
putative
2.00E−16
Glycos_transf_1
2.4.1.—
Cellular

387

glycosyltransferase
glycosyltransferase

Glycosyl

processes; Cell

[Actinobacillus
[Actinobacillus

transferases group 1

envelope

actinomycetemcomitans].

1.50e−21

biogenesis,

outer

membrane; Predicted

glycosyltransferases

1e−15

388,
NEQ196
hypothetical protein
Conserved
0.53
0

389

hypothetical protein

[Sulfolobus

solfataricus].

390,
NEQ194
hypothetical protein
NADH
0.29
0

391

dehydrogenase

subunit 5

[Caenorhabditis

elegans].

392,
NEQ197
hypothetical protein
SecY-independent
0.02
0

393

transporter protein

[Chondrus crispus].

394,
NEQ198
putative inner
conserved
1.00E−107
MS_channel

Cellular

395

membrane protein
hypothetical protein

Mechanosensitive ion

processes; Cell

[Methanococcus

channel 1.60e−86

envelope

jannaschii].

biogenesis,

outer

membrane; Small-

conductance

mechanosensitive

channel

1e−108

396,
NEQ199
replication factor A
replication factor A
1.00E−07
tRNA_anti OB-fold nucleic

Information

397

related
related protein

acid binding domain 7.40e−10

storage and

protein[Methanococcus
[Methanococcus

processing; DNA

jannaschii].

jannaschii].

replication,

recombination

and

repair; Replication

factor A

large subunit

and related

ssDNA-

binding

proteins 1e−08

398,
NEQ200
hypothetical protein
hypothetical protein
0.18
0

399

[Plasmodium

falciparum 3D7].

400,
NEQ201
LSU ribosomal
LSU ribosomal protein
1.00E−30
60s_ribosomal 60s Acidic

Information

401

protein L12A(rpl12A)
L12A [Pyrococcus

ribosomal protein 9.80e−19

storage and

[Pyrococcus abyssi]

abyssi].

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein

L12E/L44/L45/

RPP1/RPP2

6e−32

402,
NEQ204
LSU Ribosomal
Ribosomal protein
4.00E−22
Ribosomal_L22 Ribosomal

Information

403

protein
L22 [Methanopyrus

protein L22p/L17e 1.20e−09

storage and

L22[Methanopyrus

kandleri AV19].

:L22_arch L22_arch

processing; Translation,

kandleri AV19]

ribosomal protein L22 1.20e−16

ribosomal

structure and

biogenesis; Ribosomal

protein L22

7e−17

404,
NEQ203
proteasome, subunit
proteasome, subunit
1.00E−33
proteasome
3.4.25.1
Cellular

405

beta
beta (psmB)

Proteasome A-type

processes; Posttranslational

(psmB)(Multicatalytic
[Methanococcus

and B-type 6.80e−44

modification,

endopeptidase

jannaschii].

protein

turnover,

chaperones; Proteasome

protease

subunit 9e−35

406,
NEQ205
tRNA intron
Chain A, Crystal
4.00E−21
tRNA_int_endo
3.1.27.9
Information

407

endonuclease
Structure Of The Trna

tRNA intron

storage and

(endA) [M. jannaschii]
Splicing

endonuclease,

processing; Translation,

Endonuclease From

catalytic C-terminal

ribosomal

Methanococcus

domain 7.00e−19

structure and

Jannaschii.

:endA endA tRNA

biogenesis; tRNA

intron endonuclease

splicing

1.20e−10

endonuclease

3e−22

408,
NEQ206
conserved protein
conserved protein
1.00E−20
PUA PUA domain 2.60e−17

Information

409

with predicted
with predicted RNA

:unchar_dom_2

storage and

RNAbinding PUA
binding PUA domain

unchar_dom_2

processing; Translation,

domain [Pb
[Pyrobaculum

uncharacterized domain 2

ribosomal

aerophilum]

aerophilum].

9.40e−17

structure and

biogenesis; PUA

domain

(predicted

RNA-binding

domain) 1e−14

410,
NEQ208
arginyl-tRNA
hypothetical protein
2.00E−80
tRNA-synt_1d tRNA
6.1.1.
Information

411

synthetase (class
[Lactobacillus

synthetases class I
19
storage and

1)[Streptococcus

gasseri].

(R) 8.70e−60 :argS

processing; Translation,

pneumoniae TIGR4]

argS arginyl-tRNA

ribosomal

synthetase 4.70e−71

structure and

biogenesis; Arginyl-

tRNA

synthetase

9e−71

412,
NEQ207
LSU Ribosomal
Ribosomal protein
5.00E−33
Ribosomal_L13 Ribosomal

Information

413

protein
L13 [Methanopyrus

protein L13 5.60e−28

storage and

L13[Methanopyrus

kandleri AV19].

:rpIM_bact rpIM_bact

processing; Translation,

kandleri AV19]

ribosomal protein L13 3.60e−05

ribosomal

:L13_A_E L13_A_E

structure and

ribosomal protein L13 6.20e−47

biogenesis; Ribosomal

protein L13

7e−29

414,
NEQ209
hypothetical purine
virulent strain
4.00E−11
0

Information

415

NTPase [Phorikoshii]
associated lipoprotein

storage and

[Borrelia burgdorferi].

processing; DNA

replication,

recombination

and

repair; ATPase

involved in

DNA repair

4e−11

416,
NEQ210
Prolyl-tRNA
Prolyl-tRNA
6.00E−95
HGTP_anticodon
6.1.1.
Information

417

synthetase (class
synthetase

Anticodon binding
15
storage and

2)[Methanopyrus
[Methanopyrus

domain 7.40e−24

processing; Translation,

kandleri AV19]

kandleri AV19].

:tRNA-synt_2b tRNA

ribosomal

synthetase class II

structure and

core domain (G, H,

biogenesis; Prolyl-

P, S and T) 8.40e−33

tRNA

:proS_fam_I

synthetase

proS_fam_I prolyl-

6e−62

tRNA synthetase

3.00e−145

:proS_fam_II

proS_fam_II prolyl-

tRNA synthetase

3.00e−05

418,
NEQ212
Predicted nucleic
hypothetical protein
1.00E−11
PIN PIN domain 4.70e−3

Poorly

419

acid-binding
[Pyrococcus furiosus

characterized;

protein[Thermoplasma
DSM 3638].

General

volcanium]

function

prediction

only; Predicted

nucleic

acid-binding

protein,

consists of a

PIN domain

and a Zn-

ribbon

module 2e−12

420,
NEQ211
alanyl-tRNA
alanyl-tRNA
7.00E−76
tRNA-synt_2c tRNA
6.1.1.7
Information

421

synthetase
synthetase

synthetases class II

storage and

[Pyrococcus abyssi].
[Pyrococcus abyssi].

(A) 5.90e−11

processing; Translation,

ribosomal

structure and

biogenesis; Alanyl-

tRNA

synthetase

5e−77

422,
NEQ213
histidine triad protein
histidine triad protein
4.00E−18
HIT HIT family 1.00e−11

Metabolism; Nucleotide

423

(HIT familyprotein)
[Methanosarcina

transport and

[Ms acetivorans]

acetivorans str. C2A].

metabolism; Diadenosine

tetraphosphate

(Ap4A)

hydrolase and

other HIT

family

hydrolases

9e−16

424,
NEQ214
hypothetical
DNA GyrAse a-
2.00E−03
0

425

proteinS-Layer
subunit, putative

domain?
[Plasmodium

falciparum 3D7].

426,
NEQ215
hypothetical protein
hypothetical protein
0.08
0

427

[Staphylococcus

aureus subsp. aureus

Mu50].

428,
NEQ216
hypothetical protein
integral membrane
1.00E−03
0

429

protein [Plasmodium

falciparum 3D7].

430,
NEQ217
H+-transporting ATP
Archaeal/vacuolar-
5.00E−11
ATP-synt_C ATP
3.6.3.
Metabolism; Energy

431

synthase, subunit K
type H+-ATPase

synthase subunit C
14
production

(atpK-1/atpK-2)
subunit K

8.70e−20

and

homolog -
[Methanopyrus

:ATP_synt_c

conversion; F0

kandleri AV19].

ATP_synt_c ATP

F1-type ATP

synthase, F0

synthase c

subunit c 9.50e−3

subunit/Archaeal/

vacuolar-

type H+-

ATPase

subunit K 4e−12

432,
NEQ218
hypothetical protein
conserved
0.04

433

hypothetical protein

[Clostridium

perfringens].

434,
NEQ219
SSU ribosomal
Ribosomal protein
1.00E−14
Ribosomal_S27e Ribosomal

Information

435

protein S27E
S27E [Methanopyrus

protein S27 7.10e−23

storage and

kandleri AV19].

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein S27E

6e−12

436,
NEQ220
Translation
Translation elongation
1.00E−07
EF1BD EF-1 guanine

Information

437

elongation factor EF-
factor EF-1beta

nucleotide exchange domain

storage and

1beta[Methanopyrus
[Methanopyrus

1.50e−09 :aEF-1_beta aEF-

processing; Translation,

kandleri AV19]

kandleri AV19].

1_beta translation

ribosomal

elongation factor aEF-1 beta

structure and

6.10e−10

biogenesis; Translation

elongation

factor EF-

1beta 2e−08

438,
NEQ221
hypothetical protein
hypothetical protein
0.69
0

439

T26A5.5-

Caenorhabditis

elegans.

440,
NEQ222
hypothetical protein
132aa long
0.03
0

441

hypothetical protein

[Sulfolobus tokodaii].

442,
NEQ223
agmatinase (speB)
agmatinase (speB)
2.00E−16
arginase Arginase
3.5.3.
Metabolism; Amino

443

(agmatineureohydrolase)
[Archaeoglobus

family 1.70e−07
11
acid

[Archaeoglobus

fulgidus].

:hutG hutG

transport and

formiminoglutamase

metabolism; Arginase/

9.50e−05

agmatinase/

:agmatinase

formimionoglutamate

agmatinase

hydrolase,

agmatinase,

arginase

putative 4.90e−12

family 1e−17

0

444,
NEQ224
hypothetical protein
MYOSIN HEAVY
0.03
0

445

CHAIN

[Encephalitozoon

cuniculi].

446,
NEQ225
hypothetical protein
hypothetical protein
1.00E−03
0

447

[Plasmodium

falciparum 3D7].

448,
NEQ226
putative diphthamide
hypothetical protein
2.00E−31
Diphthamide_syn Putative

Information

449

synthesis
[Ferroplasma

diphthamide synthesis

storage and

protein[Methanosarcina

acidarmanus].

protein 9.50e−13 :diphth2_R

processing; Translation,

acetivorans]

diphth2_R diphthamide

ribosomal

biosynthesis protein 2-

structure and

related domain 9.80e−22

biogenesis; Diphthamide

synthase

subunit DPH2

2e−31

450,
NEQ227
SSU Ribosomal
SSU ribosomal
1.00E−13
Ribosomal_S14 Ribosomal

Information

451

protein S14
protein S14AB

protein S14p/S29e 4.30e−06

storage and

(rps14AB) [Sulfolobus

processing; Translation,

solfataricus].

ribosomal

structure and

biogenesis; Ribosomal

protein S14

6e−14

452,
NEQ229
transcriptional
Transcriptional
2.00E−25
ASNC_trans_reg AsnC

Information

453

regulatory
regulator Ptr2.

family 1.30e−21

storage and

protein, AsnC family

processing; Transcription;

[M. jannaschii]

Transcriptional

regulators 2e−26

454,
NEQ228
met-10+ protein
met-10+ protein
2.00E−32
Met_10 Met-10+
2.1.1.—
Poorly

455

[Pyrococcus
[Pyrococcus furiosus

like-protein 4.20e−26

characterized;

furiosus DSM 3638]
DSM 3638].

General

function

prediction

only; Predicted

methyltransferase

8e−28

456,
NEQ230
isoleucyl-tRNA
isoleucyl-tRNA
1.00E−175
tRNA-synt_1 tRNA
6.1.1.5
Information

457

synthetase (class
synthetase

synthetases class I

storage and

1a)[Pyrococcus
[Pyrococcus furiosus

(I, L, M and V)

processing; Translation,

furiosus DSM 3638]
DSM 3638].

4.80e−188 :ileS ileS

ribosomal

isoleucyl-tRNA

structure and

synthetase 3.80e−199

biogenesis; Isoleucyl-

:leuS_bact

tRNA

leuS_bact leucyl-

synthetase

tRNA synthetase

1e−175

2.20e−05 :metG

metG methionyl-

tRNA synthetase

7.30e−07 :valS valS

valyl-tRNA

synthetase 9.80e−25

458,
NEQ231
DNA-directed RNA
DNA-directed RNA
9.00E−10
RpoE2 Archaeal
2.7.7.6
Information

459

polymerase, subunit
polymerase, subunit E

DNA-directed RNA

storage and

E″ (rpoE2) [S
(rpoE2) [Sulfolobus

polymerase subunit

processing; Transcription;

solfataricus]

solfataricus].

E″ (RpoE″ or

DNA-

RpoE2) 1.90e−17

directed

RNA

polymerase

subunit E′ 2e−07

460,
NEQ233
hypothetical protein
hypothetical protein
0.22
0

461

[Plasmodium yoelii

yoelii].

462,
NEQ232
hypothetical protein
hypothetical protein
1.00E−04
0

463

[Pyrococcus

horikoshii].

464,
NEQ234
methanol
methanol
8.00E−73
AAA ATPase family

Poorly

465

dehydrogenase
dehydrogenase

associated with various

characterized;

regulator; (moxR)
regulator; (moxR)

cellular activities (AAA)

General

[Pyrococcus
[Pyrococcus furiosus

6.90e−4 :Mg_chelatase

function

furiosus]
DSM 3638].

Magnesium chelatase,

prediction

subunit ChII 4.20e−09

only; MoxR-

like ATPases

2e−73

466,
NEQ235
NMD protein
conserved protein
1.00E−17
1.30E−18

Information

467

affecting
[Methanothermobacter

storage and

ribosomestability and

thermautotrophicus].

processing; Translation,

mRNA decay

ribosomal

structure and

biogenesis; NMD

protein

affecting

ribosome

stability and

mRNA decay

1e−18

468,
NEQ236
hypothetical protein
ELM2 domain,
3.00E−09
0

469

putative [Plasmodium

yoelii yoelii].

470,
NEQ237
hypothetical
conserved
1.00E−04
0

471

proteinmaybe
hypothetical protein

membran protein
[Methanosarcina

acetivorans str. C2A].

472,
NEQ238
protoporphyrinogen
protoporphyrinogen
2.00E−26
hemK_rel_arch

Information

473

oxidase
oxidase (hemK)

hemK_rel_arch methylase,

storage and

(hemK)[Methanococcus
[Methanococcus

putative 5.80e−41

processing; Translation,

jannaschii]

jannaschii].

ribosomal

structure and

biogenesis; Predicted

rRNA

or tRNA

methylase 1e−27

474,
NEQ239
leucyl-tRNA
leucyl-tRNA
1.00E−168
tRNA-synt_1 tRNA
6.1.1.4
Information

475

synthetase (leuS)
synthetase (leuS)

synthetases class I

storage and

(class1a)
[Methanococcus

(I, L, M and V)

processing; Translation,

[Methanococcus

jannaschii].

1.60e−12 :ileS ileS

ribosomal

jannaschii]

isoleucyl-tRNA

structure and

synthetase 1.00e−06

biogenesis; Leucyl-

:leuS_arch

tRNA

leuS_arch leucyl-

synthetase

tRNA synthetase

1e−169

6.10e−170

:leuS_bact

leuS_bact leucyl-

tRNA synthetase

1.50e−3 :metG metG

methionyl-tRNA

synthetase 1.50e−06

:valS valS valyl-

tRNA synthetase

4.10e−07

476,
NEQ240
DNA polymerase II
DNA polymerase
3.00E−67
Metallophos
2.7.7.7
Information

477

small subunit (PolII)
delta small subunit

Calcineurin-like

storage and

[Mc. jannaschii]
[Methanococcus

phosphoesterase

processing; DNA

jannaschii].

4.80e−13 :tRNA_anti

replication,

OB-fold nucleic acid

recombination

binding domain

and

2.80e−10

repair; DNA

polymerase II

small subunit

(predicted

phosphatase)

2e−68

478,
NEQ241
LSU ribosomal
50S ribosomal protein
2.00E−38
Ribosomal_L6 Ribosomal

Information

479

protein
L6 [Pyrococcus

protein L6 2.60e−17

storage and

L6[Pyrococcus

horikoshii].

:Ribosomal_L6 Ribosomal

processing; Translation,

horikoshii]

protein L6 2.70e−06

ribosomal

structure and

biogenesis; Ribosomal

protein L6 1e−39

480,
NEQ242
SSU Ribosomal
Ribosomal protein S7
8.00E−52
Ribosomal_S7 Ribosomal

Information

481

protein
[Methanopyrus

protein S7p/S5e 9.00e−50

storage and

S7[Methanopyrus

kandleri AV19].

:S7_S5_E_A S7_S5_E_A

processing; Translation,

kandleri AV19]

ribosomal protein S7 4.20e−95

ribosomal

:rpsG_bact rpsG_bact

structure and

ribosomal protein S7 2.70e−09

biogenesis; Ribosomal

protein S7 8e−51

482,
NEQ243
hypothetical protein
hypothetical protein
4.00E−04
0

483

[Ferroplasma

acidarmanus].

484,
NEQ245
Glutamyl-tRNA-Gln
628aa long conserved
2.00E−80
GatB_N PET112
6.3.5.—
Information

485

amidotransferase
hypothetical protein

family, N terminal

storage and

(gatE) [Sulfolobus
[Sulfolobus tokodaii].

region 2.30e−90

processing; Translation,

solfataricus] SPLIT

:gatB gatB

ribosomal

See SEQ ID

glutamyl-tRNA(Gln)

structure and

NOS: 772, 773

amidotransferase, B

biogenesis; Archaeal

subunit 1.90e−27

Glu-

:gatB_rel gatB_rel

tRNAGln

aspartyl-tRNA(Asn)

amidotransferase

amidotransferase, B

subunit E

subunit, putative

(contains

5.10e−109

GAD domain)

1e−79

486,
NEQ244
conserved
conserved
1.00E−29
UPF0099 Domain of

Poorly

487

hypothetical
hypothetical protein

unknown function UPF0099

characterized;

protein[Thermoplasma
[Thermoplasma

1.50e−37 :TIGR00283

Function

acidophilum]

acidophilum].

TIGR00283 conserved

unknown; Uncharacterized

hypothetical protein

ACR 8e−31

TIGR00283 6.30e−41

488,
NEQ246
hypothetical protein
hypothetical protein
0.09
0

489

[Plasmodium

falciparum 3D7].

490,
NEQ247
SSU ribosomal
30S ribosomal protein
4.00E−49
S4 S4 domain 1.60e−18

Information

491

protein
S4 [Pyrococcus

:rpsD_bact rpsD_bact

storage and

S4P[Pyrococcus

horikoshii].

ribosomal protein S4 7.20e−4

processing; Translation,

horikoshii]

:rpsD_arch rpsD_arch

ribosomal

ribosomal protein S4 3.00e−79

structure and

biogenesis; Ribosomal

protein S4

and related

proteins 3e−50

492,
NEQ248
Predicted exosome
ribonuclease ph (rph)
1.00E−42
RNase_PH 3′
2.7.7.
Information

493

subunit, RNase
[Pyrococcus furiosus

exoribonuclease
56
storage and

PH[Methanopyrus
DSM 3638].

family, domain 1

processing; Translation,

kandleri AV19]

1.10e−27

ribosomal

:RNase_PH_C 3′

structure and

exoribonuclease

biogenesis; RNase

family, domain 2

PH 9e−44

1.30e−3

494,
NEQ249
hypothetical protein

495

496,
NEQ250
hypothetical protein
putative hemolysin III
0.04

497

[Streptococcus

mutans UA159].

498,
NEQ252
valyl-tRNA
valyl-tRNA synthetase
1.00E−166
tRNA-synt_1 tRNA
6.1.1.9
Information

499

synthetase (class
[Aeropyrum pernix].

synthetases class I

storage and

1a)[Aeropyrum

(I, L, M and V)

processing; Translation,

pernix]

3.20e−73 :ileS ileS

ribosomal

isoleucyl-tRNA

structure and

synthetase 5.00e−21

biogenesis; Valyl-

:leuS_bact

tRNA

leuS_bact leucyl-

synthetase

tRNA synthetase

1e−167

1.70e−07 :metG

metG methionyl-

tRNA synthetase

1.20e−09 :valS valS

valyl-tRNA

synthetase 1.70e−109

500,
NEQ254
hypothetical protein
K10D2.3.p
0.25
0

501

[Caenorhabditis

elegans].

502,
NEQ253
hypothetical protein
thyrotropin-releasing
0.02
0

503

hormone receptor

[Homo sapiens].

504,
NEQ256
DNA double-strand
DNA double-strand
6.00E−68
SMC_N
3.1.11.—
Information

505

break repair
break repair rad50

RecF/RecN/SMC N

storage and

rad50ATPase
ATPase.

terminal domain

processing; DNA

1.90e−07

replication,

recombination

and

repair; ATPase

involved in

DNA repair

3e−66

506,
NEQ255
hypothetical protein
unknown
0.32
0

507

[Fusobacterium

nucleatum subsp.

nucleatum ATCC

25586].

508,
NEQ257
LSU ribosomal
LSU ribosomal protein
1.00E−29
KOW KOW motif 8.90e−08

Information

509

protein
L24P [Pyrococcus

:rplX_A_E rplX_A_E

storage and

L24P[Pyrococcus

abyssi]

ribosomal protein L24 2.00e−40

processing; Translation,

abyssi]

ribosomal

structure and

biogenesis; Ribosomal

protein L24

1e−30

510,
NEQ258
hypothetical protein
conserved
3.00E−05
8.50E−04

511

hypothetical protein

[Archaeoglobus

fulgidus].

512,
NEQ259
hypothetical protein
putative peptidoglycan
2.00E−04
0

513

bound protein

(LPXTG motif)

[Listeria innocua].

514,
NEQ261
tRNA intron
hypothetical protein
1.00E−05
0

515

endonuclease
[Methanosarcina

(endA)

barkeri].

[M. acetovorans]

516,
NEQ260
hypothetical protein
hypothetical protein 4-
0.04
0

517

Trypanosoma brucei

mitochondrion.

518,
NEQ262
LSU Ribosomal
hypothetical protein
8.00E−06
Ribosomal_L29 Ribosomal

519

protein L29
[Clostridium

L29 protein 1.80e−06 :L29

thermocellum ATCC

L29 ribosomal protein L29 4.40e−15

27405].

520,
NEQ264
Translation initiation
Translation initiation
4.00E−12
SUI1 Translation initiation

Information

521

factor
factor [Pyrococcus

factor SUI1 9.70e−10

storage and

(SUI1)[Pyrococcus

horikoshii].

:SUI1_rel SUI1_rel

processing; Translation,

horikoshii]

translation initation factor

ribosomal

SUI1, putative 1.10e−10

structure and

biogenesis; Translation

initiation

factor (SUI1)

2e−13

522,
NEQ263
V-type H+-
H+-transporting ATP
1.00E−100
ATP-synt_ab ATP
3.6.3.
Metabolism; Energy

523

transporting ATP
synthase, subunit B

synthase alpha/beta
14
production

synthasebeta chain
(atpB)

family, nucleotide-

and

(V-type ATPase
[Methanococcus

binding domain

conversion; Archaeal/

subunit B)

jannaschii].

2.10e−68 :ATP-

vacuolar-

synt_ab_C ATP

type H+-

synthase alpha/beta

ATPase

chain, C terminal

subunit B 1e−101

domain 3.40e−08

:atpA atpA ATP

synthase F1, alpha

subunit 1.20e−3

:flil_yscN flil_yscN

ATPase Flil/YscN

family 2.00e−11

:atpD atpD ATP

synthase F1, beta

subunit 2.70e−11 :V-

ATPase_V1_B V-

ATPase_V1_B V-

type ATPase,

subunit B 2.70e−111

:ATP_syn_B_arch

ATP_syn_B_arch

ATP synthase

archaeal, B subunit

4.70e−132

:ATP_syn_A_arch

ATP_syn_A_arch

ATP synthase

archaeal, A subunit

6.30e−3

524,
NEQ265
Hypothetical protein
predicted cytoskeletal
1.00E−06
0

525

protein [Mycoplasma

penetrans]

526,
NEQ266
hypothetical protein
hypothetical protein
0.01
0

527

[Plasmodium

falciparum 3D7].

528,
NEQ267
SPLIT see SEQ ID
conserved
8.00E−20

Poorly

529

NOS: 530, 531;
hypothetical protein

characterized;

flagella
[Methanococcus

Function

accessoryprotein

jannaschii].

unknown; Predicted

[Methanococcus

membrane

voltae]

protein 5e−21

530,
NEQ268
SPLIT see SEQ ID
conserved
2.00E−26
DUF110 Integral membrane

Poorly

531

NOS: 528, 529;
hypothetical protein

protein DUF110 8.30e−3

characterized;

FLAGELLAACCESSORY
[Archaeoglobus

Function

PROTEIN J

fulgidus].

unknown; Predicted

membrane

protein 1e−27

532,
NEQ270
translation initiation
Chain A, Structure Of
1.00E−104
GTP_EFTU
3.6.1.
Information

533

factor aIF-2, subunit
The Wild-Type Large

Elongation factor Tu
48
storage and

gamma [Pyrococcus
Gamma Subunit Of

GTP binding domain

processing; Translation,

abyssi]
Initiation Factor Eif2

1.60e−44

ribosomal

From Pyrococcus

:GTP_EFTU_D2

structure and

Abyssi Complexed

Elongation factor Tu

biogenesis; GTPases-

With Gdp-Mg2+.

domain 2 2.00e−06

translation

:small_GTP

elongation

small_GTP small

factors 1e−106

GTP-binding protein

domain 4.00e−06

:selB selB

selenocysteine-

specific translation

elongation factor

8.20e−07 :EF-

1_alpha EF-1_alpha

translation

elongation factor

EF-1, subunit alpha

3.70e−06 :EF-Tu EF-

Tu translation

elongation factor Tu

3.80e−09 :FOLD979

No CATH

Annotation 1.60e−127

534,
NEQ271
hypothetical protein
purine NTPase
5.00E−06
0

Information

535

[Methanococcus

storage and

jannaschii].

processing; DNA

replication,

recombination

and

repair; ATPase

involved in

DNA repair

3e−07

536,
NEQ273
hypothetical protein
NADH
0.04
0

537

dehydrogenase I, J

subunit [Brucella suis

1330].

538,
NEQ275
hypothetical protein
hypothetical protein
3.00E−06
0

539

[Plasmodium yoelii

yoelii].

540,
NEQ274
SSU ribosomal
SSU ribosomal
2.00E−27
Ribosomal_S8 Ribosomal

Information

541

protein S8P;
protein S8P; (rps8E)

protein S8 7.00e−32

storage and

(rps8E)[Pyrococcus
[Pyrococcus furiosus

processing; Translation,

furiosus DSM 3638]
DSM 3638].

ribosomal

structure and

biogenesis; Ribosomal

protein S8 5e−28

542,
NEQ276
transcription initiation
transcription initiation
2.00E−69
transcript_fac2 Transcription

Information

543

factor IIB(TFIIB)
factor IIB [Pyrococcus

factor TFIIB repeat 1.60e−17

storage and

(TFB)[Pyrococcus

abyssi]

:transcript_fac2

processing; Transcription;

abyssi]

Transcription factor TFIIB

Transcription

repeat 8.40e−14

initiation

factor IIB 1e−70

544,
NEQ277
hypothetical protein
hypothetical protein
1.00E−03
0

545

[Archaeoglobus fulgidus]
[Archaeoglobus

fulgidus].

546,
NEQ278
hypothetical protein
putative outer
0.35
0

547

membrane protein;

probably involved in

nutrient binding

[Bacteroides

thetaiotaomicron VPI-

5482]

548,
NEQ279
hypothetical protein
hypothetical protein
4.00E−04
0

549

[Plasmodium

falciparum 3D7].

550,
NEQ281
hypothetical protein
hypothetical protein
1.00E−10

Poorly

551

[Pyrococcus

characterized;

horikoshii].

General

function

prediction

only; Archaeal

serine

proteases 8e−12

552,
NEQ280
hypothetical protein
hypothetical protein
0.14
0

553

[Plasmodium

falciparum 3D7].

554,
NEQ282
minichromosome
DNA replication
1.00E−121
MCM MCM2/3/5 family

Information

555

maintenance(MCM)
initiator

1.50e−172 :TIGR00368

storage and

protein [Sulfolobus
(Cdc21/Cdc54)

TIGR00368 Mg chelatase-

processing; DNA

[Methanothermobacter

related protein 9.00e−3

replication,

thermautotrophicus].

recombination

and

repair; Predicted

ATPase

involved in

replication

control,

Cdc46/Mcm

family 1e−122

556,
NEQ283
Predicted ATPase of
conserved
6.00E−48
PAPS_reduct

Cellular

557

the PP-
hypothetical protein

Phosphoadenosine

processes; Cell

loopsuperfamily
[Archaeoglobus

phosphosulfate reductase

division and

implicated in cell

fulgidus].

family 8.60e−4 :UPF0021

chromosome

cycle

Uncharacterized protein

partitioning; Predicted

family UPF0021 4.50e−15

ATPase of

:TIGR00269 TIGR00269

the PP-loop

conserved hypothetical

superfamily

protein TIGR00269 3.70e−4

implicated in

cell cycle

control 4e−49

558,
NEQ284
hypothetical protein
methylase ermT
0.02
0

559

[Plasmid p121BS].

560,
NEQ285
conserved
hypothetical protein
1.00E−25
0

561

hypothetical
[Methanosarcina

protein[Sulfolobus

barkeri].

tokodaii]

562,
NEQ288
putative archeal
archaeal histone
1.00E−09
CBFD_NFYB_HMF Histone-

Information

563

histone
[Methanococcus

like transcription factor

storage and

jannaschii].

(CBF/NF-Y) and archaeal

processing; DNA

histone 1.40e−12

replication,

recombination

and

repair; Histones

H3 and H4

6e−11

564,
NEQ290
hypothetical protein
putative transport
0.11
0

565

protein [Buchnera

aphidicola (Baizongia

pistaciae)]

566,
NEQ289
hypothetical protein
agCP6807
0.06
0

567

[Anopheles gambiae

str. PEST].

568,
NEQ291
hypothetical protein
Uncharacterized
3.00E−03
0

569

protein conserved in

bacteria

[Wigglesworthia

brevipalpis].

570,
NEQ292
hypothetical protein
HEP27 PROTEIN
6.9
0

571

(PROTEIN D).

572,
NEQ293
conserved
hypothetical protein
2.00E−40
UPF0024 Uncharacterized

Poorly

573

hypothetical
[Aquifex aeolicus].

protein family UPF0024

characterized;

protein[Aquifex

2.20e−46 :TIGR00094

Function

aeolicus]

TIGR00094 conserved

unknown; Uncharacterized

Electrontransfer

hypothetical protein

ACR 1e−41

TIGR00094 3.30e−19

:TIGR00094 TIGR00094

conserved hypothetical

protein TIGR00094 5.40e−16

574,
NEQ294
n-type ATP
hypothetical protein
1.00E−35
DUF71 Domain of unknown

Poorly

575

pyrophosphatasesuperfamily
[Pyrococcus

function DUF71 5.10e−34

characterized;

[Pyrococcus

horikoshii].

:TIGR00289 TIGR00289

General

furiosus]

conserved hypothetical

function

protein TIGR00289 2.50e−21

prediction

:MJ0570_dom MJ0570_dom

only; Predicted

MJ0570-related

ATPases of

uncharacterized domain

PP-loop

3.80e−31

superfamily

1e−36

576,
NEQ295
putative nucleolar
NUCLEOLAR
2.00E−45
Nol1_Nop2_Sun
2.1.1.—
Information

577

protein III (nol1-
PROTEIN

NOL1/NOP2/sun

storage and

nop2-sun family)
[Pyrococcus abyssi].

family 1.90e−38

processing; Translation,

:nop2p nop2p

ribosomal

NOL1/NOP2/sun

structure and

family putative RNA

biogenesis; tRNA

methylase 5.90e−53

and rRNA

:rsmB rsmB Sun

cytosine-C5-

protein 5.50e−06

methylases

1e−46

578,
NEQ297
LSU Ribosomal
LSU ribosomal protein
6.00E−17
Ribosomal_L34e Ribosomal

Information

579

protein L34E
L34E [Methanococcus

protein L34e 8.70e−15

storage and

jannaschii].

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein L34E

3e−18

580,
NEQ296
hypothetical protein
MURF2 protein (AA 1-
2.00E−04
0

581

348) [Crithidia

fasciculata].

582,
NEQ298
Acetyltransferase
hypothetical protein
1.00E−10
0

583

[Fusobacteriumnucleatum
[Pyrococcus

subsp.

horikoshii].

nucleatum]

584,
NEQ299
Predicted ABC-class
transport protein
1.00E−155
ABC_tran ABC
1.8.—.—
Poorly

585

ATPase, RNaseL
[Pyrococcus

transporter 1.80e−23

characterized;

inhibitor homolog [M. kandleri]

horikoshii].

:ABC_tran ABC

General

transporter 2.90e−18

function

:fer4 4Fe-4S binding

prediction

domain 4.40e−09

only; RNase L

:3a0501s02

inhibitor

3a0501s02 Type II

homolog,

(General) Secretory

predicted

Pathway (IISP)

ATPase 1e−156

Family protein

5.30e−3 :3a0106s01

3a0106s01 sulfate

transport system

permease protein

4.90e−05 :cbiO cbiO

cobalt transport

protein ATP-binding

subunit 1.00e−05

:ntrCD ntrCD nitrate

transport ATP-

binding subunits C

and D 7.90e−4 :drrA

drrA daunorubicin

resistance ABC

transporter ATP-

binding subunit

5.00e−05 :thiQ thiQ

ABC transporter,

ATP-binding protein,

ThiQ subfamily

7.20e−3 :RLI

Possible metal-

binding domain in

RNase L inhibitor,

RLI 3.70e−14

586,
NEQ300
hypothetical surface
surface layer protein
3.00E−06
0

587

layer protein
[Methanothermococcus

[Mcthermolithotrophicus]

thermolithotrophicus].

588,
NEQ301
hypothetical protein
EsV-1-147
0.04
0

589

[Ectocarpus

siliculosus virus].

590,
NEQ302
glutamyl-tRNA
glutamyl-tRNA
1.00E−122
tRNA-synt_1c tRNA
6.1.1.
Information

591

synthetase (class
synthetase

synthetases class I
17
storage and

1)[Pyrococcus
[Pyrococcus abyssi].

(E and Q), catalytic

processing; Translation,

abyssi]

domain 2.50e−80

ribosomal

:glnS glnS

structure and

glutaminyl-tRNA

biogenesis; Glutamyl-

synthetase 3.00e−36

and

:gltX_arch gltX_arch

glutaminyl-

glutamyl-tRNA

tRNA

synthetase 5.20e−147

synthetases

:gltX_bact

1e−123

gltX_bact glutamyl-

tRNA synthetase

1.40e−13 :tRNA-

synt_1c_C tRNA

synthetases class I

(E and Q), anti-

codon binding

domain 2.00e−07

592,
NEQ303
LSU Ribosomal
LSU ribosomal protein
1.00E−19
Ribosomal_L35Ae

Information

593

protein L35AE
L35AE. [Pyrococcus

Ribosomal protein L35Ae

storage and

abyssi]

1.00e−20

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein

L35AE/L33A

7e−21

594,
NEQ304
hypothetical

M. jannaschii

0.01
0

595

predicted coding

region MJ0944

[Methanococcus

jannaschii].

596,
NEQ305
Archaeosine tRNA-
archaeosine tRNA-
2.00E−08
0

Cellular

597

ribosyltransferase[T
ribosyltransferase

processes; Posttranslational

volcanium] SPLIT
[Methanosarcina

modification,

See SEQ ID

mazei Goe1].

protein

NOS: 248, 249

turnover,

chaperones; Prefoldin,

molecular

chaperone

implicated in

de novo

protein

folding, alpha

subunit 7e−08

598,
NEQ306
3-oxoacyl-[acyl-
3-oxoacyl-[acyl-
1.00E−30
adh_short short
1.1.1.
Metabolism; Secondary

599

carrier-
carrier-protein]

chain
100
metabolites

protein]reductase
reductase [Aquifex

dehydrogenase

biosynthesis,

[Aquifex aeolicus]

aeolicus].

5.90e−45

transport and

catabolism; Dehydrogenases

with

different

specificities

(related to

short-chain

alcohol

dehydrogenases)

1e−31

600,
NEQ307
nucleoside
Nucleoside
8.00E−41
NDK Nucleoside
2.7.4.6
Metabolism; Nucleotide

601

diphosphate kinase
diphosphate kinase

diphosphate kinase

transport and

(ndk)[Pyrobaculum
(NDK) (NDP kinase)

1.10e−22

metabolism; Nucleoside

aerophilum]
(Nucleoside-2-P

diphosphate

kinase).

kinase 1e−26

602,
NEQ309
hypothetical protein
hypothetical protein
0.45
0

603

[Pyrococcus furiosus

DSM 3638].

604,
NEQ308
Seryl-tRNA
453aa long
1.00E−77
Seryl_tRNA_N
6.1.1.
Information

605

synthetase (class
hypothetical seryl-

Seryl-tRNA
11
storage and

2)[Sulfolobus
tRNA synthetase

synthetase N-

processing; Translation,

tokodaii]
[Sulfolobus tokodaii].

terminal domain

ribosomal

2.90e−10 :tRNA-

structure and

synt_2b tRNA

biogenesis; Seryl-

synthetase class II

tRNA

core domain (G, H,

synthetase

P, S and T) 5.60e−42

1e−76

:serS serS seryl-

tRNA synthetase

1.90e−81

606,
NEQ310
hypothetical protein
hypothetical protein
2.00E−14
0

Poorly

607

[Pyrococcusabyssi]
[Pyrococcus abyssi].

characterized;

Function

unknown; Uncharacterized

ACR 3e−15

608,
NEQ312
hypothetical protein
hypothetical protein
0.9
0

609

[Plasmodium

falciparum 3D7].

610,
NEQ311
LSU ribosomal
LSU ribosomal protein
1.00E−44
Ribosomal_L30 Ribosomal

Information

611

protein L30P
L30P (rpmD)

protein L30p/L7e 7.90e−09

storage and

(rpmD)[Methanococcus
[Methanococcus

:L30P_arch L30P_arch

processing; Translation,

jannaschii]

jannaschii].

ribosomal protein L30P

ribosomal

7.90e−81 :L7 L7 60S

structure and

ribosomal protein L7 3.00e−3

biogenesis; Ribosomal

protein

L30/L7E 1e−45

612,
NEQ314
hypothetical protein
conserved
2.00E−06
5.30E−04

Poorly

613

hypothetical protein

characterized;

[Archaeoglobus

Function

fulgidus].

unknown; Uncharacterized

ACR 1e−07

614,
NEQ313
conserved
conserved protein
6.00E−06
0

615

hypothetical
[Methanosarcina

protein[Methanosarcina

mazei Goe1].

acetivorans str.

C2A]

616,
NEQ316
putative dUTPase
putative
2.00E−38
dUTPase dUTPase
3.5.4.
Metabolism; Nucleotide

617

deoxynucleotide

1.40e−10 :dut dut
13
transport and

triphosphate

deoxyuridine 5′-

metabolism; Deoxycytidine

deaminase

triphosphate

deaminase

[Streptomyces

nucleotidohydrolase

8e−37

coelicolor A3(2)].

(dut) 1.30e−3

618,
NEQ315
proteinase IV -
hypothetical protein
3.00E−23
Peptidase_U7
3.4.21.—
Cellular

619

[Methanococcusjannaschii]
[Prochlorococcus

Peptidase family U7

processes; Cell

Periplasmic

marinus subsp.

7.80e−25

motility and

serine

pastoris str.

:SppA_dom

secretion; Periplasmic

CCMP1378].

SppA_dom signal

serine

peptide peptidase

proteases

SppA, 36K type

(ClpP class)

2.50e−27

3e−23

620,
NEQ317
LSU ribosomal
50S RIBOSOMAL
7.00E−15

Information

621

protein
PROTEIN L15P.

storage and

L15[Sulfolobus

processing; Translation,

acidocaldarius]

ribosomal

structure and

biogenesis; Ribosomal

protein L15

7e−15

622,
NEQ318
reverse gyrase
reverse gyrase
1.00E−175
Topoisom_bac DNA
5.99.
Information

623

[Pyrococcus abyssi]
[Pyrococcus abyssi].

topoisomerase
1.3
storage and

SPLIT See SEQ ID

5.60e−59 :Toprim

processing; DNA

NOS: 848, 849

Toprim domain

replication,

1.00e−46 :topA_bact

recombination

topA_bact DNA

and

topoisomerase I

repair; Reverse

4.30e−32 :rgy rgy

gyrase 1e−176

reverse gyrase

2.20e−36 :topB topB

DNA topoisomerase

III 2.50e−06

:topA_arch

topA_arch DNA

topoisomerase I

6.20e−10

624,
NEQ319
SSU hypothetical
126aa long
2.00E−46
Ribosomal_L7Ae Ribosomal

Information

625

30S ribosomalprotein
hypothetical 30S

protein

storage and

HS6 [Sulfolobus
ribosomal protein HS6

L7Ae/L30e/S12e/Gadd45

processing; Translation,

tokodaii]
[Sulfolobus tokodaii].

family 1.40e−45

ribosomal

structure and

biogenesis; Ribosomal

protein HS6-

type

(S12/L30/L7a)

4e−46

626,
NEQ321
C4-type Zn-finger-
hypothetical protein
1.00E−17
ZPR1 ZPR1 zinc-finger

Poorly

627

containing
[Pyrococcus furiosus

domain 1.30e−11 :ZPR1_znf

characterized;

protein[Methanopyrus
DSM 3638].

ZPR1_znf ZPR1 zinc finger

General

kandleri AV19]

domain 5.50e−06 :zpr1_rel

function

zpr1_rel ZPR1-related zinc

prediction

finger protein 2.00e−06

only; C4-type

Zn finger 3e−18

628,
NEQ320
SSU Ribosomla
S ribosomal protein
2.00E−13
0

Information

629

protein S17E
S17E [Pyrococcus

storage and

horikoshii].

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein S17E

1e−14

630,
NEQ322
hypothetical protein
Putative Nonclathrin
4.9
0

631

coat protein gamma-

like protein [Oryza

sativa (japonica

cultivar-group)].

632,
NEQ324
DNA topoisomerase I
DNA topoisomerase I
9.00E−34

5.99.
Information

633

[Pyrococcus horikoshii]
[Pyrococcus

1.2
storage and

SPLIT see SEQ ID

horikoshii].

processing; DNA

NOS: 90, 91

replication,

recombination

and

repair; Topoisomerase

IA

8e−35

634,
NEQ323
translation initiation
translation initiation
1.00E−25
eIF5_eIF2B Domain found in

Information

635

factor eIF-2
factor eIF-2 beta

IF2B/IF5 3.30e−27 :aIF-

storage and

beta[Pyrococcus
[Pyrococcus

2beta aIF-2beta translation

processing; Translation,

horikoshii]

horikoshii].

initiation factor aIF-2, beta

ribosomal

subunit, putative 7.90e−26

structure and

biogenesis; Translation

initiation

factor eIF-2,

beta

subunit/eIF-5

N-terminal

domain 1e−26

636,
NEQ325
conserved
hypothetical protein
2.00E−19
DUF207 Uncharacterized

Poorly

637

hypothetical
[Pyrococcus furiosus

ACR, COG1590 1.50e−27

characterized;

protein[Pyrococcus
DSM 3638].

Function

furiosus DSM 3638]

unknown; Uncharacterized

ACR 3e−20

638,
NEQ326
SSU ribosomal
30S ribosomal protein
8.00E−26
Ribosomal_S17 Ribosomal

Information

639

protein
S17 [Pyrococcus

protein S17 3.70e−23

storage and

S17[Pyrococcus

horikoshii].

processing; Translation,

horikoshii]

ribosomal

structure and

biogenesis; Ribosomal

protein S17

4e−27

640,
NEQ328
transcriptional
transcriptional
6.00E−12
ASNC_trans_reg AsnC

Information

641

regulatory
regulatory protein,

family 7.40e−3

storage and

protein, asnC family
asnC family

processing; Transcription;

[Pyrococcus
[Pyrococcus furiosus

Transcriptional

furiosus]
DSM 3638].

regulators 4e−13

642,
NEQ327
hypothetical protein
cell division protein
0.05
0

643

[Chaetosphaeridium

globosum].

644,
NEQ329
dCTP deaminase
Deoxycytidine
2.00E−10
0
3.5.4.
Metabolism; Nucleotide

645

[Methanosarcinaacetivorans
triphosphate

13
transport and

str. C2A]
deaminase

metabolism; Deoxycytidine

[Methanosarcinamazei

deaminase

Goe1].

2e−07

646,
NEQ330
hypothetical protein

0

647

648,
NEQ331
hypothetical protein
Similar to protein
1.00E−03
0

649

kinase C substrate

80K-H [Mus

musculus].

650,
NEQ332
hypothetical protein

0

651

652,
NEQ334
Predicted RNA-
DNA-binding protein
2.00E−42
1.00E−67

Information

653

binding
[Pyrococcus furiosus

storage and

protein[Methanopyrus
DSM 3638].

processing; Translation,

kandleri AV19]

ribosomal

structure and

biogenesis; Predicted

RNA-

binding

protein 8e−42

654,
NEQ333
pseudouridylate
tRNA pseudouridine
4.00E−10
PseudoU_synth_1
4.2.1.
Information

655

synthase I
synthase A [Bacillus

tRNA pseudouridine
70
storage and

(truA)[Methanococcus

cereus ATCC 14579]

synthase 7.20e−07

processing; Translation,

jannaschii]

:hisT_truA hisT_truA

ribosomal

tRNA pseudouridine

structure and

synthase A 7.60e−22

biogenesis; Pseudouridylate

synthase

(tRNA psi55)

6e−11

656,
NEQ335
hypothetical protein
hypothetical protein
0.01
0

657

[Arabidopsis thaliana].

658,
NEQ336
hypothetical protein
Superfamily II
0.02
0

659

DNA/RNA helicase,

SNF2 family

[Clostridium

acetobutylicum].

660,
NEQ338
DNA-directed RNA
DNA-directed RNA
6.00E−17
RNA_pol_N RNA
2.7.7.6
Information

661

polymerase, subunit
polymerase, subunit N

polymerases N/8 kDa

storage and

N [Aeropyrum pernix]
[Aeropyrum pernix].

subunit 3.60e−25

processing; Transcription;

DNA-

directed

RNA

polymerase,

subunit N

(RpoN/RPB10)

3e−18

662,
NEQ337
L-isoaspartyl protein
L-isoaspartyl protein
8.00E−12
0
2.1.1.
Information

663

carboxylmethyltransferase
carboxyl

.77
storage and

isolog (pimT)
methyltransferase

processing; Translation,

isolog (pimT)

ribosomal

[Methanococcus

structure and

jannaschii].

biogenesis; Predicted

SAM-

dependent

methyltransferase

involved

in tRNA-Met

maturation

6e−13

664,
NEQ340
Predicted hydrolase
phosphoglycolate
3.00E−03
Hydrolase haloacid dehalogenase-like

665

(HADsuperfamily)
phosphatase [Nostoc

hydrolase 1.10e−07

[Thermoplasma
sp. PCC 7120].

666,
NEQ339
conserved
conserved
1.00E−09
Peptidase_M50 Peptidase

Poorly

667

hypothetical
hypothetical integral

family M50 6.20e−05

characterized;

integralmembrane
membrane protein

General

protein [M jannaschii]
[Methanococcus

function

jannaschii].

prediction

only; Zn-

dependent

proteases 8e−11

668,
NEQ341
primase DnaG-like
hypothetical protein
3.00E−86
Toprim Toprim
2.7.7.—
Information

669

[Pyrococcusabyssi]
[Pyrococcus furiosus

domain 1.10e−06

storage and

DSM 3638].

processing; DNA

replication,

recombination

and

repair; DNA

primase

(bacterial

type) 1e−85

670,
NEQ342
hypothetical
409aa long
1.00E−63
Nop Putative snoRNA

Information

671

nucleolar protein
hypothetical nucleolar

binding domain 9.50e−72

storage and

NOP56[Sulfolobus
protein [Sulfolobus

processing; Translation,

tokodaii]; snoRNA

tokodaii].

ribosomal

structure and

biogenesis; Protein

implicated in

ribosomal

biogenesis,

Nop56p

homolog 2e−63

672,
NEQ344
small heat shock
small heat shock
4.00E−13
HSP20 Hsp20/alpha

Cellular

673

protein (class
protein (class I)

crystallin family 1.10e−11

processes; Posttranslational

I)[Aquifex aeolicus]
[Aquifex aeolicus].

modification,

and other

protein

turnover,

chaperones;

Molecular

chaperone

(small heat

shock protein)

4e−14

674,
NEQ343
intracellular protease
intracellular protease
3.00E−07

Poorly

675

(Protease
[Pyrococcus abyssi].

characterized;

I)[Pyrococcus abyssi]

General

function

prediction

only; Putative

intracellular

protease/amidase

2e−08

676,
NEQ345
anaerobic
hypothetical protein

0
1.17.
Metabolism; Nucleotide

677

ribonucleoside-
[Pyrococcus

4.2
transport and

triphosphatereductase

horikoshii].

metabolism; Oxygen-

[Pyrococcus

sensitive

abyssi]

ribonucleoside-

triphosphate

reductase 0

678, 679

putative
hypothetical protein
8.00E−28
Methyltransf_1 6-O-
3.1.—.—
Information

endonuclease V
[Ferroplasma

methylguanine DNA

storage and

acidarmanus].

methyltransferase,

processing; DNA

DNA binding domain

replication,

2.30e−3 :Endonuc_V

recombination

Endonuclease V

and

2.20e−29

repair; Deoxyinosine

3′endonuclease

(endonuclease

V) 3e−20

680,
NEQ346
LSU Ribosomal
50S ribosomal protein
7.00E−09
Ribosomal_LX Ribosomal

Information

681

protein L20A
LX [Pyrococcus

LX protein 3.80e−10

storage and

horikoshii].

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein L20A

(L18A) 4e−10

682,
NEQ347
Predicted
conserved
9.00E−44
UPF0103 Protein of

Poorly

683

dioxygenase
hypothetical protein

unknown function DUF52

characterized;

[Methanopyruskandleri
[Methanosarcina

4.10e−61

General

AV19]

acetivorans str. C2A].

function

prediction

only; Predicted

dioxygenase

4e−39

684,
NEQ348
putative archeal
archaeal histone A2
7.00E−12
CBFD_NFYB_HMF Histone-

Information

685

histone
[Methanococcus

like transcription factor

storage and

jannaschii].

(CBF/NF-Y) and archaeal

processing; DNA

histone 1.40e−21

replication,

recombination

and

repair; Histones

H3 and H4

4e−13

686,
NEQ349
ATP-dependent
ATP-dependent
1.00E−177
Sigma54_activat
3.4.21.
Cellular

687

protease La
protease Lon

Sigma-54 interaction
53
processes; Posttranslational

(lon)[Archaeoglobus
[Thermococcus

domain 5.40e−3

modification,

fulgidus]

kodakaraensis].

:lon_rel lon_rel

protein

ATP-dependent

turnover,

protease, putative

chaperones; Predicted

8.40e−219 : 8.60e−11

ATP-

dependent

protease 1e−173

688,
NEQ350
Predicted aromatic
Predicted aromatic
8.00E−15
DUF59 Domain of
2.3.1.
Poorly

689

ring
ring hydroxylating

unknown function
30
characterized;

hydroxylatingenzyme
enzyme

DUF59 2.50e−15

General

[Thermoplasma
[Thermoplasma

function

volcanium]

volcanium].

prediction

only; Predicted

metal-sulfur

cluster

biosynthetic

enzyme 5e−16

690,
NEQ351
Predicted
hypothetical protein
8.00E−03
0

691

transcriptional
[Pyrococcus furiosus

regulator
DSM 3638].

692,
NEQ352
putative ABC
hypothetical protein
1.00E−13
DUF63 Membrane protein of

Poorly

693

transporter, transmembrane
[Pyrococcus furiosus

unknown function DUF63

characterized;

component
DSM 3638].

3.40e−4

Function

[P.

unknown; Predicted

membrane

proteins 2e−12

694,
NEQ353
hypothetical protein
hypothetical protein
5.00E−27
0

Poorly

695

[Archaeoglobus

characterized;

fulgidus].

General

function

prediction

only; Predicted

nucleic acid

binding

protein

containing the

AN1-type Zn-

finger 3e−28

696,
NEQ354
hypothetical protein
hypothetical protein
1.00E−03
0

697

[Plasmodium

falciparum 3D7].

698,
NEQ356
hypothetical
hypothetical protein
0.01
0

699

proteinputative
[Plasmodium

vitamin B12

falciparum 3D7].

transporter?

700,
NEQ355
predicted
conserved
8.00E−11
0

Information

701

transcriptional
hypothetical protein

storage and

regulator
[Archaeoglobus

processing; Transcription;

fulgidus].

Predicted

transcriptional

regulator

containing an

HTH domain

fused to a Zn-

ribbon 4e−12

702,
NEQ358
Predicted calcineurin
hypothetical protein
6.00E−52
Metallophos Calcineurin-like

Poorly

703

superfamilyphosphoesterase
[Pyrococcus abyssi].

phosphoesterase 4.80e−07

characterized;

[M. kandleri]

:TIGR00024 TIGR00024

General

conserved hypothetical

function

protein TIGR00024 5.00e−38

prediction

only; Predicted

ICC-like

phosphoesterases

4e−53

704,
NEQ357
hypothetical protein

M. jannaschii

0.05
0

705

predicted coding

region MJECL28

[Methanococcus

jannaschii].

706,
NEQ359
SSU Ribosomnal
SSU ribosomal
1.00E−20
Ribosomal_S28e Ribosomal

Information

707

protein S28E
protein S28E

protein S28e 3.70e−32

storage and

[Pyrococcus abyssi].

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein

S28E/S33 8e−22

708,
NEQ361
LSU ribosomal
LSU ribosomal protein
4.00E−66
Ribosomal_L2 Ribosomal

Information

709

protein
L2P [Pyrococcus

Proteins L2, RNA binding

storage and

L2P[Pyrococcus

abyssi]

domain 1.70e−10 :rplB_bact

processing; Translation,

abyssi]

rplB_bact ribosomal protein

ribosomal

L2 3.00e−08 :FOLD1900 No

structure and

CATH Annotation 2.20e−12

biogenesis; Ribosomal

:Ribosomal_L2_C

protein L2 3e−67

Ribosomal Proteins L2, C-

terminal domain 1.10e−46

710,
NEQ360
Glu-tRNA
Glu-tRNA
3.00E−80
Amidase Amidase
6.3.5.—
Information

711

amidotransferase,
amidotransferase,

5.80e−87 :gatA gatA

storage and

subunitA (gatA-2)
subunit A (gatA-2)

glutamyl-tRNA(Gln)

processing; Translation,

[Archaeoglobus
[Archaeoglobus

amidotransferase, A

ribosomal

fulgidus]

fulgidus].

subunit 6.20e−105

structure and

biogenesis; Asp-

tRNAAsn/Glu-

tRNAGln

amidotransferase

A subunit

and related

amidases 2e−81

712,
NEQ362
hypothetical protein
hypothetical protein
4.00E−03
NTP_transf_2 Nucleotidyltransferase

713

[Pyrococcus furiosus

domain 2.60e−3

DSM 3638].

714,
NEQ363
Archaea-specific
hypothetical protein
5.00E−20
DUF78 Protein of unknown

Information

715

DNA-bindingprotein
[Pyrococcus

function DUF78 5.40e−34

storage and

[Methanopyrus

horikoshii].

:TIGR00285 TIGR00285

processing; Transcription;

kandleri]

conserved hypothetical

Archaeal

protein TIGR00285 3.90e−35

DNA-

binding

protein 3e−21

716,
NEQ364
LSU Ribosomal
LSU ribosomal protein
2.00E−12
Ribosomal_L31e Ribosomal

Information

717

proten L31E
L31E [Methanococcus

protein L31e 1.20e−11

storage and

jannaschii].

processing; Translation,

ribosomal

structure and

biogenesis; Ribosomal

protein L31E

1e−13

718,
NEQ365
hypothetical protein
hypothetical protein
9.00E−15

3.1.11.—
Information

719

[Plasmodium

storage and

falciparum 3D7].

processing; DNA

replication,

recombination

and

repair; ATPase

involved in

DNA repair

9e−14

720,
NEQ366
GTP-binding protein
GTP-binding protein
1.00E−39
MMR_HSR1 GTPase of

Poorly

721

[Pyrococcushorikoshii]
[Pyrococcus

unknown function 4.00e−25

characterized;

horikoshii].

:MG442 MG442 GTP-

General

binding conserved

function

hypothetical protein 1.50e−13

prediction

only; Predicted

GTPases

7e−41

722,
NEQ367
nucleotidyltransferase
176aa long conserved
9.00E−03
CTP_transf_2 Cytidylyltransferase 6.60e−06

723

[Pyrococcus furiosus
hypothetical protein

DSM 3638]
[Sulfolobus tokodaii].

724,
NEQ368
DNA-(apurinic or

M. jannaschii

4.00E−48
AP_endonuc_2 AP endonuclease

725

apyrimidinic
predicted coding

family 2 1.80e−3

site)lyase
region MJ1455

(endonuclease IV)
[Methanococcus

jannaschii].

726,
NEQ369
DNA repair protein
ATP-dependent
3.00E−33
DEAD DEAD/DEAH
2.7.7.—
Information

727

RAD25[Pyrococcus
helicase, putative

box helicase 1.60e−11

storage and

abyssi]
[Sulfolobus

:helicase_C

processing; DNA

solfataricus].

Helicase conserved

replication,

C-terminal domain

recombination

8.30e−10 :SNF2_N

and

SNF2 family N-

repair; DNA or

terminal domain

RNA

8.00e−3

helicases of

superfamily II

5e−25

728,
NEQ370
DNA-directed RNA
DNA-directed RNA
5.00E−39
S1 S1 RNA binding
2.7.7.6
Information

729

polymerase, subunit
polymerase, subunit E

domain 1.90e−10

storage and

E′ (rpoE1) [M. jannaschii]
(rpoE1)

:rpoE rpoE DNA-

processing; Transcription;

[Methanococcus

directed RNA

DNA-

jannaschii].

polymerase 2.80e−47

directed

:RNA_pol_Rpb7_N

RNA

RNA polymerase

polymerase

Rpb7, N-terminal

subunit E 4e−40

domain 4.60e−22

730,
NEQ372
hypothetical DNA-
220aa long
5.00E−57
UDG Uracil DNA
2.7.7.7
Information

731

directed
hypothetical DNA-

glycosylase

storage and

DNApolymerase,
directed DNA

superfamily 1.20e−37

processing; DNA

bacteriophage-type
polymerase,

:SPO1polNrel

replication,

bacteriophage-type

SPO1polNrel phage

recombination

[Sulfolobus tokodaii].

SPO1 DNA

and

polymerase-related

repair; Uracil-

protein 8.30e−94

DNA

glycosylase

6e−54

732,
NEQ371
conserved
Hypothetical protein
7.00E−25

3.1.3.3
Poorly

733

hypothetical
[Sulfolobus

characterized;

protein[Sulfolobus

solfataricus].

Function

solfataricus]

unknown; Uncharacterized

archaeal

coiled-coil

domain 4e−24

734,
NEQ373
ferredoxin1 [3Fe-4S] -
FERREDOXIN.
3.00E−07
fer4 4Fe-4S binding domain

Metabolism; Energy

735

Thermococcuslitoralis

3.00e−3

production

and

conversion; Ferredoxin

1 6e−07

736,
NEQ375
SSU Ribosomal
Ribosomal protein
2.00E−23
Ribosomal_S3Ae Ribosomal

Information

737

protein
S3AE [Methanopyrus

S3Ae family 5.80e−15

storage and

S3AE[Methanopyrus

kandleri AV19].

processing; Translation,

kandleri AV19]

ribosomal

structure and

biogenesis; Ribosomal

protein S3AE

3e−23

738,
NEQ376
hypothetical protein
Unknown (protein for
0.29
0

739

MGC: 27006) [Homo

sapiens].

740,
NEQ377
DNA-directed RNA
112aa long
8.00E−23
RNA_POL_M_15 KD
2.7.7.6
Information

741

polymerasesubunit
hypothetical DNA-

RNA polymerases

storage and

M [Sulfolobus
directed RNA

M/15 Kd subunit

processing; Transcription;

tokodaii]
polymerase subunit M

7.60e−08 :TFIIS

DNA-

[Sulfolobus tokodaii].

Transcription factor

directed

S-II (TFIIS) 2.50e−15

RNA

polymerase

subunit

M/Transcription

elongation

factor TFIIS

5e−21

742,
NEQ378
Predicted Zn-
hypothetical protein
1.00E−54
lactamase_B Metallo-beta-

Poorly

743

dependent hydrolase
[Pyrococcus abyssi].

lactamase superfamily

characterized;

ofthe beta-lactamase

9.80e−11

General

superfamily

function

prediction

only; Predicted

Zn-

dependent

hydrolases of

the beta-

lactamase

fold 7e−56

744,
NEQ379
LSU ribosomal
50S ribosomal protein
2.00E−42
Ribosomal_L19e Ribosomal

Information

745

protein
L19 [Pyrococcus

protein L19e 6.10e−67

storage and

L19E[Pyrococcus

horikoshii].

processing; Translation,

horikoshii]

ribosomal

structure and

biogenesis; Ribosomal

protein L19E

3e−43

746,
NEQ380
hypothetical protein
hypothetical protein
4.00E−03
0

747

[Plasmodium

falciparum 3D7].

748,
NEQ381
pyruvate fromate-
PYRUVATE
4.00E−71
Radical_SAM
1.97.
Cellular

749

lyase
FORMATE-LYASE

Radical SAM
1.4
processes; Posttranslational

activatingenzyme
ACTIVATING

superfamily 4.30e−15

modification,

related protein [P. abyssi]
ENZYME RELATED

protein

PROTEIN

turnover,

[Pyrococcus abyssi].

chaperones; Pyruvate-

formate

lyase-

activating

enzyme 3e−72

750,
NEQ383
DNA repair protein
hypothetical protein
1.00E−13
Metallophos Calcineurin-like

Information

751

RAD32[Pyrococcus
[Pyrococcus

phosphoesterase 7.10e−15

storage and

abyssi]

horikoshii].

:sbcd sbcd exonuclease

processing; DNA

SbcD 5.60e−05

replication,

recombination

and

repair; DNA

repair

exonuclease

8e−15

752,
NEQ382
Predicted Fe—S
hypothetical protein
1.00E−73
Radical_SAM Radical SAM

Metabolism; Energy

753

oxidoreductase[Thermoplasma
[Pyrococcus furiosus

superfamily 7.50e−15

production

volcanium]
DSM 3638].

and

conversion; Fe—S

oxidoreductases

2e−74

754,
NEQ384
SpoU rRNA
spoU protein homolog
1.00E−36
SpoU_methylase
2.1.1.—
Information

755

methylase[Methanos
[imported] -

SpoU rRNA

storage and

arcina acetivorans

Pyrococcus sp.

Methylase family

processing; Translation,

str. C2A]

1.60e−07

ribosomal

:rRNA_methyl_1

structure and

rRNA_methyl_1

biogenesis; rRNA

RNA

methylase

methyltransferase,

5e−32

TrmH family, group

1 5.10e−40

:rRNA_methyl_2

rRNA_methyl_2

RNA

methyltransferase,

TrmH family, group

2 2.30e−3

756,
NEQ386
hypothetical protein
conserved
7.00E−06
DUF153 MTH1175-like domain

757

[Pyrococcusabyssi]
hypothetical protein

(DUF153/COG1433) 2.70e−05

[Thermoanaerobacter

tengcongensis].

758,
NEQ385
conserved
hypothetical protein
1.00E−05
DUF232 Putative transcriptional

759

hypothetical
[Aeropyrum pernix].

regulator 4.40e−11

protein[Aeropyrum

pernix]

760,
NEQ387
ATP-dependent RNA
ATP-dependent RNA
1.00E−127
DEAD DEAD/DEAH
2.7.7.—
Information

761

helicase, EIF-
helicase, EIF-4A

box helicase 8.30e−08

storage and

4AFAMILY
FAMILY [Pyrococcus

:ERCC4 ERCC4

processing; DNA

[Pyrococcus abyssi]

abyssi].

domain 8.70e−23

replication,

:helicase_C

recombination

Helicase conserved

and

C-terminal domain

repair; ERCC4-

9.30e−25 :SNF2_N

like helicases

SNF2 family N-

3e−97

terminal domain

1.40e−3

762,
NEQ388
SSU ribosomal
ribosomal protein S5
2.00E−55
Ribosomal_S5 Ribosomal

Information

763

protein
[Pyrobaculum

protein S5, N-terminal

storage and

S5[Pyrobaculum

aerophilum].

domain 1.40e−10 :rpsE_arch

processing; Translation,

aerophilum]

rpsE_arch ribosomal protein

ribosomal

S5 4.10e−97 :rpsE_bact

structure and

rpsE_bact ribosomal protein

biogenesis; Ribosomal

S5 4.80e−12

protein S5 2e−56

:Ribosomal_S5_C

Ribosomal protein S5, C-

terminal domain 7.30e−21

764,
NEQ389
tyrosyl-tRNA
tyrosyl-tRNA
1.00E−100
tRNA-synt_1b tRNA
6.1.1.1
Information

765

synthetase (class
synthetase

synthetases class I

storage and

1b)[Pyrococcus
[Pyrococcus furiosus

(W and Y) 7.60e−54

processing; Translation,

furiosus]
DSM 3638].

:tyrS tyrS tyrosyl-

ribosomal

tRNA synthetase

structure and

6.40e−17

biogenesis; Tyrosyl-

tRNA

synthetase

1e−100

766,
NEQ392
hypothetical protein
potassium channel
0.14
0

767

subunit [Gallus

gallus].

768,
NEQ391
hypothetical protein
AMV156 [Amsacta
7.00E−10
0

769

moorei

entomopoxvirus].

770,
NEQ393
translation initiation
translation initiation
1.00E−19
eIF_5A eIF_5A translation

Information

771

factor eIF-5A(eif5A)
factor eIF-5A (eif5A)

initiation factor eIF-5A 8.80e−21

storage and

[Archaeoglobus
[Archaeoglobus

processing; Translation,

fulgidus]

fulgidus].

ribosomal

structure and

biogenesis; Translation

elongation

factor

P/translation

initiation

factor eIF-5A

2e−20

772,
NEQ396
Glu-tRNA
Glu-tRNA
3.00E−49
GAD GAD domain
6.3.5.—
Information

773

amidotransferase
amidotransferase

1.30e−4 :GatB

storage and

(gatE)
(gatB)

PET112 family, C

processing; Translation,

[Methanococcus

terminal region

ribosomal

jannaschii].

5.50e−06 :gatB_rel

structure and

gatB_rel aspartyl-

biogenesis; Archaeal

tRNA(Asn)

Glu-

amidotransferase, B

tRNAGln

subunit, putative

amidotransferase

1.70e−46

subunit E

(contains

GAD domain)

2e−50

774,
NEQ395
hypothetical protein
conserved
4.00E−05
primase_sml primase_sml DNA primase,

775

hypothetical protein

eukaryotic-type, small subunit,

[Thermoplasma

putative 4.30e−4

acidophilum].

776,
NEQ397
LSU ribosomal
LSU ribosomal protein
9.00E−25
Ribosomal_L21e Ribosomal

Information

777

protein
L21E; (rpl21E)

protein L21e 2.20e−25

storage and

L21E; (rpl21E)
[Pyrococcus furiosus

processing; Translation,

[Pyrococcus
DSM 3638].

ribosomal

furiosus]

structure and

biogenesis; Ribosomal

protein L21E

2e−25

778,
NEQ398
endonuclease III
endonuclease III
4.00E−40
HhH-GPD HhH-
4.2.99.
Information

779

[Aquifexaeolicus]Split;
[Aquifex aeolicus].

GPD superfamily
18
storage and

see SEQ ID

base excision DNA

processing; DNA

NOS: 254, 255

repair protein 2.00e−17

replication,

:nth nth

recombination

endonuclease III

and

2.80e−40

repair; Predicted

EndoIII-

related

endonuclease

3e−41

780,
NEQ399
methionine
methionine
2.00E−58
Peptidase_M24
3.4.11.
Information

781

aminopeptidase
aminopeptidase (map)

metallopeptidase
18
storage and

(map)(EC 3.4.11.18)
(EC 3.4.11.18)

family M24 6.00e−12

processing; Translation,

[Pyrococcus abyssi]
[Pyrococcus abyssi].

:crvDNA_42K

ribosomal

crvDNA_42K DNA-

structure and

binding protein, 42 kDa

biogenesis; Methionine

3.60e−4

aminopeptidase

:met_pdase_I

2e−59

met_pdase_I

methionine

aminopeptidase,

type I 1.80e−11

:met_pdase_II

met_pdase_II

methionine

aminopeptidase,

type II 8.20e−81

782,
NEQ401
Mg-chelatase
Mg-chelatase subunit
2.00E−77
AAA ATPase family

Metabolism; Coenzyme

783

subunit Chll and
Chll and Chld (MoxR-

associated with various

metabolism; Mg-

Chld(MoxR-like
like ATPase and vWF

cellular activities (AAA)

chelatase

ATPase and vWF
domain)

6.90e−06 :Mg_chelatase

subunit Chll

[Methanopyrus

Magnesium chelatase,

5e−36

kandleri AV19].

subunit Chll 3.60e−08

:Sigma54_activat Sigma-54

interaction domain 3.10e−3

784,
NEQ403
hypothetical protein
conserved
4.00E−05
vwa von Willebrand factor type

785

hypothetical protein

A domain 1.70e−3

[Chlorobium tepidum

TLS].

786,
NEQ402
deoxyhypusine
deoxyhypusine
4.00E−89
DS Deoxyhypusine
2.5.1.
Information

787

synthase
synthase related

synthase 5.90e−73
46
storage and

(catalyzingalso the
protein

:dhys dhys

processing; Translation,

synthesis of
[Thermoplasma

deoxyhypusine

ribosomal

homospermidine)

acidophilum].

synthase, putative

structure and

8.50e−67

biogenesis; Deoxyhypusine

synthase 3e−90

788,
NEQ404
hypothetical protein
Uncharacterized
0.01
0

789

protein conserved in

bacteria

[Wigglesworthia

brevipalpis].

790,
NEQ405
Translation initiation
Translation initiation
1.00E−30
S1 S1 RNA binding domain

Information

791

factor eIF2-
factor eIF2-alpha

7.60e−07

storage and

alpha[Methanopyrus
[Methanopyrus

processing; Translation,

kandleri AV19]

kandleri AV19].

ribosomal

structure and

biogenesis; Translation

initiation

factor

eIF2alpha 2e−29

792,
NEQ406
hypothetical
similar to
3.00E−06
0

793

proteinsimilar to
phosphoenolpyruvate

PEP-kinase
synthase [Nostoc sp.

PCC 7120].

794,
NEQ408
hypothetical protein
hypothetical protein
0.01
0

795

[Aeropyrum pernix].

796,
NEQ407
hypothetical protein
hypothetical protein
2.00E−04
0

797

[Streptococcus

mutans UA159].

798,
NEQ409
large helicase-
large helicase-related
8.00E−68
0
3.6.1.—
Poorly

799

related protein; (lhr-
protein; (lhr-2)

characterized;

2)
[Pyrococcus furiosus

General

DSM 3638].

function

prediction

only; Lhr-like

helicases 4e−68

800,
NEQ410
V-type ATP synthase
hypothetical protein
8.00E−34
V_ATPase_sub_a
3.6.3.
Metabolism; Energy

801

subunit I (V-
[Clostridium

V-type ATPase
14
production

typeATPase subunit

thermocellum ATCC

116 kDa subunit

and

I) [Pc. abyssi]
27405].

family 7.40e−11

conversion; Archaeal/

vacuolar-

type H+-

ATPase

subunit I 8e−33

802,
NEQ411
DNA-directed RNA
Chain F, Structure Of
4.00E−06
RNA_pol_Rpb4 RNA

Poorly

803

polymerase, subunit
An Archeal Homolog

polymerase Rpb4 1.60e−4

characterized;

F (rpoF) [S. solfataricus]
Of The Eukaryotic

Function

Rna Polymerase li

unknown; Uncharacterized

Rpb4RPB7

ArCR 2e−07

COMPLEX.

804,
NEQ412
leucine
leucine
1.00E−67
Peptidase_M17
3.4.11.1
Metabolism; Amino

805

aminopeptidase
aminopeptidase

Cytosol

acid

[Clostridiumperfringens]
[Clostridium

aminopeptidase

transport and

perfringens].

family, catalytic

metabolism; Leucyl

domain 5.10e−123

aminopeptidase

5e−64

806,
NEQ413
hypothetical protein
hypothetical protein
0.91
0

807

[Enterococcus

faecium].

808,
NEQ415
hypothetical protein
185aa long
2.00E−11
0

809

[Sulfolobus tokodaii]
hypothetical protein

[Sulfolobus tokodaii].

810,
NEQ414
Archaeal ATP
hypothetical protein
5.00E−20
0

Poorly

811

dependent
[Pyrococcus furiosus

characterized;

serineprotease
DSM 3638].

General

{secretory signal

function

sequence}

prediction

only; Archaeal

serine

proteases 5e−21

812,
NEQ416
hypothetical protein
Predicted ATPase
0.06
0

813

involved in biogenesis

of flagella

[Thermoplasma

volcanium].

814,
NEQ417
glycyl-tRNA
glycyl-tRNA
2.00E−98
HGTP_anticodon
6.1.1.
Information

815

synthetase (class
synthetase

Anticodon binding
14
storage and

2)[Pyrococcus
[Pyrococcus abyssi].

domain 4.00e−30

processing; Translation,

abyssi]

:tRNA-synt_2b tRNA

ribosomal

synthetase class II

structure and

core domain (G, H,

biogenesis; Glycyl-

P, S and T) 3.40e−41

tRNA

:glyS_dimeric

synthetase,

glyS_dimeric glycyl-

class II 1e−99

tRNA synthetase

2.00e−151

816,
NEQ418
hypothetical protein
hypothetical protein
0.02
0

817

[Plasmodium

falciparum 3D7].

818,
NEQ420
DNA polymerase II
DNA polymerase II
0
polC polC DNA
2.7.7.7
Information

819

subunit 2
subunit 2 [Pyrococcus

polymerase II, large

storage and

(largesubunit)

furiosus DSM 3638].

subunit DP2 0.0

processing; DNA

[Pyrococcus

:PolC_DP2 DNA

replication,

furiosus]

polymerase II large

recombination

subunit DP2 4.60e−283

and repair; NA

polymerase II

large subunit 0

820,
NEQ419
hypothetical protein
hypothetical protein
3.00E−07
0

Poorly

821

[Pyrococcus horikoshii]
[Pyrococcus

characterized;

horikoshii].

Function

unknown; Uncharacterized

ArCR 2e−08

822,
NEQ421
ATPase subunit of a
ABC transporter,
1.00E−56
ABC_tran ABC
1.8.—.—
Poorly

823

ABC-typetransport
ATP-binding protein

transporter 2.00e−58

characterized;

system involved in
[Methanosarcina

:3a0501s02

General

acetivorans str. C2A].

3a0501s02 Type II

function

(General) Secretory

prediction

Pathway (IISP)

only; ABC-

Family protein

type transport

1.80e−55

systems,

:3a0106s01

involved in

3a0106s01 sulfate

lipoprotein

transport system

release,

permease protein

ATPase

3.70e−46 :cbiO cbiO

components

cobalt transport

1e−55

protein ATP-binding

subunit 2.80e−16

:ntrCD ntrCD nitrate

transport ATP-

binding subunits C

and D 8.00e−20

:proV proV glycine

betaine/L-proline

transport ATP

binding subunit

4.10e−09 :potA potA

spermidine/putrescine

ABC transporter

ATP-binding subunit

1.10e−09 :drrA drrA

daunorubicin

resistance ABC

transporter ATP-

binding subunit

1.90e−10 :ccmA

ccmA heme

exporter protein

CcmA 3.80e−14

:thiQ thiQ ABC

transporter, ATP-

binding protein,

ThiQ subfamily

3.80e−16 :nodl nodl

nodulation ABC

transporter Nodl

2.70e−05

824,
NEQ422
DIPHTIN
DIPHTIN SYNTHASE
5.00E−22
TP_methylase
2.1.1.
Information

825

SYNTHASE[Encephalitozoon
[Encephalitozoon

Tetrapyrrole
98
storage and

cuniculi]

cuniculi].

(Corrin/Porphyrin)

processing; Translation,

Methylases 5.70e−4

ribosomal

:dph5 dph5

structure and

diphthine synthase

biogenesis; Diphthamide

3.40e−13

biosynthesis

methyltransferase

DPH5

8e−20

826,
NEQ424
Holliday-junction
conserved protein
1.00E−08
Hjc Archaeal holliday

Information

827

resolvase[Sulfolobus
[Methanothermobacter

junction resolvase (hjc)

storage and

solfataricus]

thermautotrophicus].

1.70e−18

processing; DNA

replication,

recombination

and

repair; Holliday

junction

resolvase -

archaeal type

6e−10

828,
NEQ423
Probable thiamine
Thiamine biosynthesis
2.00E−47
Thil Thiamine biosynthesis

Metabolism; Coenzyme

829

biosynthesisprotein
ATP pyrophosphatase

protein (Thil) 5.40e−19

metabolism; Thiamine

thil [Archaeoglobus
[Thermoanaerobacter

:THUMP THUMP domain

biosynthesis

fulgidus]

tengcongensis].

7.10e−18 :TIGR00342

ATP

TIGR00342 thiamine

pyrophosphatase

biosynthesis protein Thil

8e−43

2.20e−39

830,
NEQ425
type IV secretion
type IV secretion
7.00E−76
GSPII_E Type II/IV secretion

Cellular

831

system
system protein

system protein 2.40e−3

processes; Cell

protein[Methanosarcina
[Methanosarcina

motility and

acetivorans str.

acetivorans str. C2A].

secretion; Type

C2A]

IV secretory

pathway,

VirB11

components,

and related

ATPases

involved in

archaeal

flagella

biosynthesis

1e−34

832,
NEQ426
recombinase, radA
recombinase, radA
9.00E−97
HHH Helix-hairpin-
3.6.1.—
Information

833

[Pyrococcusfuriosus
[Pyrococcus furiosus

helix motif 7.40e−05

storage and

DSM 3638]
DSM 3638].

processing; DNA

replication,

recombination

and

repair; RecA/RadA

recombinase

5e−97

834,
NEQ427
DNA-directed RNA
DNA-directed RNA
9.00E−81
1.10E−23
2.7.7.6
Information

835

polymerase, subunit
polymerase, subunit A

storage and

A″ (rpoA2)
[Pyrococcus abyssi].

processing; Transcription;

[Pyrococcus

DNA-

directed

RNA

polymerase

beta'

subunit/160 kD

subunit

(split gene in

archaea and

Syn) 6e−82

836,
NEQ428
Predicted RNA-
conserved
8.00E−10
UPF0044 Uncharacterised

Information

837

binding
hypothetical protein

protein family UPF0044

storage and

proteincontaining KH
[Methanococcus

1.20e−13 :TIGR00253

processing; Translation,

domain, possibly

jannaschii].

TIGR00253 conserved

ribosomal

hypothetical protein

structure and

TIGR00253 4.20e−05

biogenesis; Predicted

RNA-

binding

protein

containing KH

domain,

possibly

ribosomal

protein 4e−11

838,
NEQ429
hypothetical protein
hypothetical protein
6.00E−03
0

839

[Plasmodium

falciparum 3D7].

840,
NEQ430
activator 1
activator 1 (replication
8.00E−73
AAA ATPase family
2.7.7.7
Information

841

(replication factor C),
factor C), 53 KD

associated with

storage and

53 KD subunit [Mc
subunit

various cellular

processing; DNA

jannaschii]
[Methanococcus

activities (AAA)

replication,

jannaschii].

2.60e−11 :Rad17

recombination

Rad17 cell cycle

and

checkpoint protein

repair; ATPase

3.50e−3

involved in

DNA

replication 5e−74

842,
NEQ431
conserved
hypothetical protein
5.00E−31
DUF99 Protein of unknown

Poorly

843

hypothetical
[Methanosarcina

function DUF99 1.80e−49

characterized;

protein[Pyrococcus

barkeri].

Function

furiosus DSM 3638]

unknown; Uncharacterized

ACR 3e−27

844,
NEQ433
LSU ribosomal
ribosomal protein L3
5.00E−71
Ribosomal_L3 Ribosomal

Information

845

protein L3 (E. coli
(E. coli L3)

protein L3 1.30e−63

storage and

L3)[Mtb
[Methanothermobacter

processing; Translation,

thermautotrophicus]

thermautotrophicus].

ribosomal

structure and

biogenesis; Ribosomal

protein L3 3e−72

846,
NEQ432
Type I signal
Type I signal
3.00E−08
0

Cellular

847

peptidase[Methanopyrus
peptidase

processes; Cell

kandleri AV19]
[Methanopyrus

motility and

kandleri AV19].

secretion; Signal

peptidase

I 6e−07

848,
NEQ434
reverse gyrase
reverse gyrase
1.00E−132
DEAD DEAD/DEAH
5.99.
Information

849

[Pyrococcus
[Pyrococcus

box helicase 2.60e−06
1.3
storage and

horikoshii].

:rgy rgy reverse

processing; DNA

gyrase 1.20e−08

replication,

recombination

and

repair; Reverse

gyrase 1e−134

850,
NEQ435
hypothetical protein
hypothetical protein
7.00E−25
DUF460 Protein of unknown

Poorly

851

[Archaeoglobusfulgidus]
[Archaeoglobusfulgidus].

function (DUF460) 6.10e−18

characterized;

Function

unknown; Uncharacterized

ACR 5e−26

852,
NEQ436
protein translocase,
protein translocase,
2.00E−46
SecD_SecF Protein export

Cellular

853

subunit SECD(secD)
subunit SECD (secD)

membrane protein 9.00e−4

processes; Cell

[Methanococcus
[Methanococcus

:2A0604s01 2A0604s01

motility and

jannaschii]

jannaschii].

protein-export membrane

secretion; Preprotein

protein (SecDF) Family

translocase

2.20e−08 :3a0501s07

subunit SecD

3a0501s07 protein-export

2e−47

membrane protein SecF

1.50e−05 :secD secD

protein-export membrane

protein SecD 2.60e−10

854,
NEQ437
protein translocase,
protein translocase,
4.00E−30
SecD_SecF Protein export

Cellular

855

subunit SECF(secF)
subunit SECF (secF)

membrane protein 1.10e−09

processes; Cell

[Methanococcus
[Methanococcus

:2A0604s01 2A0604s01

motility and

jannaschii]

jannaschii].

protein-export membrane

secretion; Preprotein

protein (SecDF) Family

translocase

2.00e−06 :3a0501s07

subunit SecF

3a0501s07 protein-export

3e−31

membrane protein SecF

2.20e−3

856,
NEQ438
predicted RNA-
hypothetical protein
3.00E−30
0

Information

857

binding protein
[Pyrococcus abyssi].

storage and

homologous to

processing; Transcription;

eukaryotic snRNP

Predicted

[Methanopyrus

RNA-

kandleri AV19]

binding

SPLIT see SEQ ID

protein

NOS: 982, 983

homologous

to eukaryotic

snRNP 2e−31

858,
NEQ440
Predicted DNA-
methyltransferase
4.00E−30
UPF0020 Putative
2.1.1.—
Information

859

modificationmethylase
related protein

RNA methylase

storage and

[Methanopyrus
[Methanothermobacter

family UPF0020

processing; DNA

kandleri]

thermautotrophicus].

2.00e−30

replication,

:TIGR01177

recombination

TIGR01177

and

conserved

repair; Predicted

hypothetical protein

DNA

TIGR01177 1.20e−30

modification

methylase 3e−31

860,
NEQ442
hypothetical protein
hypothetical protein
0.02
0

861

[Plasmodium

falciparum 3D7].

862,
NEQ441
Uncharacterized
hypothetical protein
8.00E−53
DUF51 Protein of unknown

Poorly

863

conserved
[Aquifex aeolicus].

function DUF51 4.60e−85

characterized;

protein[Methanopyrus

:TIGR00296 TIGR00296

Function

kandleri AV19]

conserved hypothetical

unknown; Uncharacterized

protein TIGR00296 3.50e−52

ACR 6e−54

864,
NEQ443
hypothetical protein
hypothetical protein
6.00E−03
0

865

[Plasmodium

falciparum 3D7].

866,
NEQ444
hypothetical protein
haemagglutinin
0.02
0

867

[Mycoplasma

gallisepticum].

868,
NEQ445
hypothetical protein
possible HNRNP
0.21
0

869

arginine n-

methyltransferase

[Plasmodium yoelii

yoelii].

870,
NEQ446
SSU Ribosomal
SSU ribosomal
5.00E−27
Ribosomal_S9 Ribosomal

Information

871

protein
protein S9P; (rps9P)

protein S9/S16 1.30e−29

storage and

S9[Methanopyrus
[Pyrococcus furiosus

processing; Translation,

kandleri AV19]
DSM 3638].

ribosomal

structure and

biogenesis; Ribosomal

protein S9 7e−28

872,
NEQ448
hypothetical protein
OUTER CAPSID
0.21
0

873

PROTEIN VP4

(HEMAGGLUTININ)

(OUTER LAYER

PROTEIN VP4)

[CONTAINS: OUTER

CAPSID PROTEINS

VP5 AND VP8].

874,
NEQ447
hypothetical protein

M. jannaschii

9.00E−03
0

875

predicted coding

region MJ0027

[Methanococcus

jannaschii].

876,
NEQ449
hypothetical protein
hypothetical protein
5.00E−08
0

Cellular

877

[Sulfolobustokodaii]
[Plasmodium

processes; Cell

falciparum 3D7].

division and

chromosome

partitioning; Chromosome

segregation

ATPases 2e−07

878,
NEQ450
ubiquinol-
ubiquinol-cytochrome
7.00E−25
Ribosomal_L10e Ribosomal

Information

879

cytochrome C
C reductase complex,

L10 7.20e−31 :L10e L10e

storage and

reductasecomplex,
subunit VI requiring

ribosomal protein L10.e

processing; Translation,

subunit VI requiring
protein

2.30e−32

ribosomal

protein
[Archaeoglobus

structure and

fulgidus].

biogenesis; Ribosomal

protein

L16/L10E 6e−26

880,
NEQ451
Predicted Zn-
hypothetical protein
1.00E−21

Poorly

881

dependent
[Aquifex aeolicus].

characterized;

protease[Methanopyrus

General

kandleri AV19]

function

prediction

only; Predicted

Zn-

dependent

proteases 1e−22

882,
NEQ452
DNA-directed RNA
DNA-directed RNA
3.00E−19
RNA_pol_A_bac
2.7.7.6
Information

883

polymerase, subunit
polymerase, subunit D

Bacterial RNA

storage and

D (rpoD)
[Pyrococcus abyssi].

polymerase, alpha

processing; Transcription;

[Pyrococcus abyssi]

chain, N terminal

DNA-

domain 1.50e−05

directed

RNA

polymerase

alpha

subunit/40 kD

subunit 2e−20

884,
NEQ453
Predicted
Predicted
7.00E−10
V4R V4R domain 1.50e−05

Poorly

885

transcriptional
transcriptional

characterized;

regulatorconsisting
regulator consisting of

General

of a V4R domain and a
a V4R domain and a

function

DNA-binding HTH

prediction

domain

only; Predicted

[Methanopyrus

hydrocarbon

kandleri AV19].

binding

protein

(contains V4R

domain) 3e−08

886,
NEQ454
centromere binding
centromere binding
1.00E−112
PUA PUA domain
4.2.1.70
Information

887

proteinhomolog/pseudouridine
protein

1.20e−25 :TruB_N

storage and

synthase
homolog/pseudouridine

TruB family

processing; Translation,

of
synthase

pseudouridylate

ribosomal

[Pyrococcus furiosus

synthase (N terminal

structure and

DSM 3638].

domain) 2.40e−57

biogenesis; Pseudouridine

:CBF5 CBF5 rRNA

synthase 1e−112

pseudouridine

synthase, putative

2.40e−170 :TruB

TruB tRNA

pseudouridine

synthase B 8.60e−26

:unchar_dom_2

unchar_dom_2

uncharacterized

domain 2 4.80e−4

888,
NEQ455
Predicted membrane
hypothetical protein
1.00E−18
DUF112 Integral membrane

Poorly

889

protein[Methanopyrus
[Pyrococcus abyssi].

protein DUF112 9.90e−4

characterized;

kandleri AV19]

Function

unknown; Predicted

membrane

protein 9e−20

890,
NEQ457
methionyl-tRNA
methionyl-tRNA
1.00E−155
tRNA-synt_1 tRNA
6.1.1.
Information

891

synthetase (class
synthetase

synthetases class I
10
storage and

1a)[Pyrococcus
[Pyrococcus

(I, L, M and V)

processing; Translation,

horikoshii]

horikoshii].

2.20e−09

ribosomal

:tRNA_bind Putative

structure and

tRNA binding

biogenesis; Methionyl-

domain 2.60e−40

tRNA

:metG metG

synthetase

methionyl-tRNA

1e−115

synthetase 2.00e−152

:metG_C_term

metG_C_term

methionyl-tRNA

synthetase C-

terminal region, beta

subunit 2.20e−28

:pheT_bact

pheT_bact

phenylalanyl-tRNA

synthetase, beta

subunit 2.30e−4

892,
NEQ456
Mg-dependent
hypothetical protein
6.00E−38
TatD_DNase TatD
3.1.21.—
Information

893

DNase
[Acidianus

related DNase

storage and

[Methanopyruskandleri

ambivalens].

2.80e−69

processing; DNA

AV19]

:TIGR00010

replication,

TIGR00010

recombination

deoxyribonuclease,

and

TatD family 6.60e−53

repair; Mg-

dependent

DNase 3e−28

894,
NEQ458
hypothetical protein
hypothetical protein
1.00E−03
0

895

[Plasmodium

falciparum 3D7].

896,
NEQ459
glutaredoxin-like
glutaredoxin-like
7.00E−23

1.6.4.5
Cellular

897

protein
protein [Pyrococcus

processes; Posttranslational

[Pyrococcusfuriosus

furiosus DSM 3638].

modification,

DSM 3638]

protein

turnover,

chaperones; Thiol-

disulfide

isomerase

and

thioredoxins

1e−23

898,
NEQ460
Predicted
Predicted
2.00E−39
RIO1 RIO1/ZK632.3/MJ0444

Cellular

899

serine/threonine
serine/threonine

family 2.10e−57

processes; Signal

proteinkinase
protein kinase

transduction

[Methanopyrus
[Methanopyrus

mechanisms;

kandleri AV19]

kandleri AV19].

Predicted

serine/threonine

protein

kinases 2e−38

900,
NEQ461
inorganic pyrophosphatase[Sulfolobus tokodaii]

Pyrophosphatase Inorganic
3.6.1.1
Metabolism; Energy

901

pyrophosphatase 2.20e−63

production

and

conversion; Inorganic

pyrophosphatase

7e−48

902,
NEQ462
hypothetical protein
hypothetical protein
6.00E−04
0

903

[Plasmodium

falciparum 3D7].

904,
NEQ464
RIO1-like
RIO1-like
3.00E−44
RIO1 RIO1/ZK632.3/MJ0444

Cellular

905

serine/threonine
serine/threonine

family 1.10e−22

processes; Signal

proteinkinase fused
protein kinase fused

transduction

to an N-terminal
to an N-terminal DNA-

mechanisms;

DNA-binding
binding HTH domain

RIO-like

[Methanopyrus

serine/threonine

kandleri AV19].

protein

kinase fused

to N-terminal

HTH domain

3e−27

906,
NEQ463
Predicted GTPase,
GTP binding protein
1.00E−104
TGS TGS domain 7.60e−11

Poorly

907

probabletranslation
[Sulfolobus

:TIGR00092 TIGR00092

characterized;

factor [Methanopyrus

solfataricus].

conserved hypothetical

General

protein TIGR00092 1.70e−10

function

prediction

only; Predicted

GTPase 4e−95

908,
NEQ466
Predicted hydrolase
conserved
3.00E−98
lactamase_B
3.—.—.—
Poorly

909

of the metallo-beta-
hypothetical protein

Metallo-beta-

characterized;

lactamase
[Methanococcus

lactamase

General

superfamily

jannaschii].

superfamily 1.10e−19

function

:MG423 MG423

prediction

conserved

only; Predicted

hypothetical protein

hydrolase of

2.10e−61

the metallo-

beta-

lactamase

superfamily

2e−99

910,
NEQ467
SSU Ribosomal
Ribosomal protein
7.00E−37
Ribosomal_S13 Ribosomal

Information

911

protein
S13 [Methanopyrus

protein S13/S18 4.30e−41

storage and

S13[Methanopyrus

kandleri AV19].

:FOLD1946 No CATH

processing; Translation,

kandleri AV19]

Annotation 1.00e−4

ribosomal

structure and

biogenesis; Ribosomal

protein S13

9e−34

912,
NEQ468
hypothetical protein
hypothetical protein
8.00E−05
0

913

[Plasmodium yoelii

yoelii].

914,
NEQ469
SSU ribosomal
SSU ribosomal
8.00E−35
Ribosomal_S8e Ribosomal

Information

915

protein S8E
protein S8E (rps8E)

protein S8e 1.70e−43 :S8e

storage and

(rps8E)[Archaeoglobus
[Archaeoglobus

S8e ribosomal protein S8.e

processing; Translation,

fulgidus]

fulgidus].

8.30e−39

ribosomal

structure and

biogenesis; Ribosomal

protein S8E

5e−36

916,
NEQ470
hypothetical protein
Uncharacterized
3.00E−06
0

Poorly

917

conserved protein

characterized;

[Thermoplasma

Function

volcanium].

unknown; Uncharacterized

ArCR 4e−07

918,
NEQ471
hypothetical protein
TraD-like protein
5.00E−03
0

919

[Haemophilus

influenzae biotype

aegyptius].

920,
NEQ472
Calcineurin
conserved protein
3.00E−12
Metallophos Calcineurin-like

Poorly

921

superfamilyphosphoesterase
[Methanothermobacter

phosphoesterase 6.30e−18

characterized;

[Methanopyrus

thermautotrophicus].

:TIGR00040 TIGR00040

General

conserved hypothetical

function

protein TIGR00040 6.60e−05

prediction

only; Predicted

phosphoesterases,

related

to the lcc

protein 2e−13

922,
NEQ473
cell division protein
cell division protein
9.00E−77
tubulin Tubulin/FtsZ
3.4.24.—
Cellular

923

(ftsZ-
(ftsZ-1)

family, GTPase

processes; Cell

1)[Archaeoglobus
[Archaeoglobus

domain 5.80e−57

division and

fulgidus]

fulgidus].

:ftsZ ftsZ cell

chromosome

division protein FtsZ

partitioning; Cell

1.70e−103

division

:tubulin_C

GTPase 6e−78

Tubulin/FtsZ family,

C-terminal domain

1.20e−15

924,
NEQ475
cell division control
cell division control
0
AAA ATPase family
2.7.1.—
Cellular

925

protein 48,
protein 48, aaa family;

associated with

processes; Posttranslational

aaafamily; (cdc48-2)
(cdc48-2) [Pyrococcus

various cellular

modification,

[P. furiosus]

furiosus DSM 3638].

activities (AAA)

protein

1.70e−94 :AAA

turnover,

ATPase family

chaperones; ATPases

associated with

of the

various cellular

AAA+ class 0

activities (AAA)

8.30e−92 :cdc48_2

Cell division protein

48 (CDC48), domain

2 2.40e−15

:cdc48_N Cell

division protein 48

(CDC48), N-terminal

domain 1.20e−32

:Sigma54_activat

Sigma-54 interaction

domain 3.20e−3

:FtsH_fam

FtsH_fam ATP-

dependent

metalloprotease

FtsH 1.70e−22

:26Sp45 26Sp45

26S proteasome

subunit P45 family

1.10e−79 :CDC48

CDC48 AAA family

ATPase, CDC48

subfamily 0.0

926,
NEQ474
hypothetical protein
hypothetical protein
0.21
0

927

[Clostridium

thermocellum ATCC

27405].

928,
NEQ476
hypothetical protein

M. jannaschii

0.04
0

929

predicted coding

region MJ1254

[Methanococcus

jannaschii].

930,
NEQ477
hypothetical protein
cytochrome c oxidase
0.49
0

931

subunit III (aa3 type)

[Oceanobacillus

iheyensis].

932,
NEQ478
SSU ribosomal
ribosomal protein S4
3.00E−47
KOW KOW motif 4.50e−06

Information

933

protein
[Methanothermobacter

:Ribosomal_S4e Ribosomal

storage and

S4E[Methanothermobacter

thermautotrophicus].

family S4e 2.70e−18 :S4 S4

processing; Translation,

domain 2.10e−07

ribosomal

structure and

biogenesis; Ribosomal

protein S4E

2e−48

934,
NEQ479
phenylalanyl-tRNA
phenylalanyl-tRNA
4.00E−78
pheT_arch
6.1.1.
Information

935

synthetase beta-
synthetase beta-chain

pheT_arch
20
storage and

chain[Pyrococcus
[Pyrococcus furiosus

phenylalanyl-tRNA

processing; Translation,

furiosus]
DSM 3638].

synthetase, beta

ribosomal

subunit 3.10e−89

structure and

:pheT_bact

biogenesis; Phenylalanyl-

pheT_bact

tRNA

phenylalanyl-tRNA

synthetase

synthetase, beta

beta subunit

subunit 2.20e−09

4e−78

:B3_4 B3/4 domain

8.10e−06 :B5 tRNA

synthetase B5

domain 3.90e−4

936,
NEQ480
SSU ribosomal
SSU ribosomal
2.00E−45
Ribosomal_S19 Ribosomal

Information

937

protein
protein S19P

protein S19 4.10e−30

storage and

S19P[Pyrococcus
[Pyrococcus abyssi].

:rpsS_arch rpsS_arch

processing; Translation,

abyssi]

ribosomal protein S19 1.70e−67

ribosomal

:rpsS_bact rpsS_bact

structure and

ribosomal protein S19 5.50e−19

biogenesis; Ribosomal

protein S19

3e−46

938,
NEQ481
SSU ribosomal
SSU ribosomal
3.00E−31
Ribosomal_S3_C Ribosomal

Information

939

protein S3P
protein S3P (rpsC)

protein S3, C-terminal

storage and

(rpsC)[Methanococcus
[Methanococcus

domain 3.70e−4 :rpsC_E_A

processing; Translation,

jannaschii]

jannaschii].

rpsC_E_A ribosomal protein

ribosomal

S3 1.20e−36 :rpsC_bact

structure and

rpsC_bact ribosomal protein

biogenesis; Ribosomal

S3 3.00e−05

protein S3 2e−32

940,
NEQ482
transcription initiation
conserved protein
2.00E−08
TFIIE_alpha TFIIE alpha

Information

941

factor IIE, subunit
[Methanothermobacter

subunit 4.40e−05

storage and

alpha (TFE) [Mc.

thermautotrophicus].

:TIGR00373 TIGR00373

processing; Transcription;

jannaschii]

conserved hypothetical

Transcription

protein TIGR00373 2.40e−05

initiation

factor IIE,

large subunit

2e−09

942,
NEQ483
hypothetical protein
hypothetical protein
0.01
0

943

[Cytophaga

hutchinsonii].

944,
NEQ485
hypothetical protein
hypothetical protein
3.00E−06
0

945

[Plasmodium

falciparum 3D7].

946,
NEQ484
hypothetical protein
hypothetical protein
0.01
0

947

[Cytophaga

hutchinsonii].

948,
NEQ486
Na+/Ca+ exchanging
hypothetical
2.00E−41
Na_Ca_Ex Sodium/calcium

Cellular

949

protein
conserved protein

exchanger protein 2.50e−28

processes; Inorganic

related[Pyrococcus
[Oceanobacillus

:Na_Ca_Ex Sodium/calcium

ion

abyssi]

iheyensis].

exchanger protein 4.50e−26

transport and

:TIGR00367 TIGR00367

metabolism; Ca2+/

K+-dependent Na+/Ca+

Na+

exchanger related-protein

antiporter 8e−42

2.10e−20

950,
NEQ487
SSU Ribosomal
Ribosomal protein
2.00E−43
Ribosomal_S15 Ribosomal

Information

951

protein
S15P/S13E

protein S15 4.00e−14

storage and

S15P/S13E[Methanopyrus
[Methanopyrus

processing; Translation,

kandleri AV19]

kandleri AV19].

ribosomal

structure and

biogenesis; Ribosomal

protein

S15P/S13E

3e−44

952,
NEQ489
LSU ribosomal
LSU ribosomal protein
2.00E−20
L15 Ribosomal protein L15

Information

953

protein L18E
L18E (rpl18E)

1.20e−06

storage and

(rpl18E)[Sulfolobus
[Sulfolobus

processing; Translation,

solfataricus]

solfataricus].

ribosomal

structure and

biogenesis; Ribosomal

protein L18E

1e−19

954,
NEQ490
hypothetical protein
Ubie_methyltran,
2.00E−07
0

955

ubiE/COQ5

methyltransferase

family [Bacillus

anthracis A2012].

956,
NEQ491
NADH
324aa long
2.00E−70
pyr_redox Pyridine
1.6.4.5
Cellular

957

oxidase/thioredoxin
hypothetical

nucleotide-

processes; Posttranslational

reductase[Sulfolobus
thioredoxin reductase

disulphide

modification,

solfataricus]

[Sulfolobus tokodaii].

oxidoreductase

protein

2.80e−72 :gidA gidA

turnover,

glucose-inhibited

chaperones; Thioredoxin

division protein A

reductase 3e−69

8.10e−07

:TRX_reduct

TRX_reduct

thioredoxin

reductase 9.00e−128

:gltA gltA glutamate

synthase (NADPH),

homotetrameric

1.10e−3

:GOGAT_sm_gam

GOGAT_sm_gam

glutamate

synthases,

NADH/NADPH,

small subunit 6.10e−3

958,
NEQ492
hypothetical protein
ADHESIN AIDA-I
2.00E−03
0

959

PRECURSOR.

960,
NEQ493
O-sialoglycoprotein
O-sialoglycoprotein
1.00E−75
Peptidase_M22
3.4.24.
Cellular

961

endopeptidase
endopeptidase

Glycoprotease
57
processes; Posttranslational

[Pyrococcus
[Pyrococcus

family 5.90e−61 :gcp

modification,

horikoshii].

horikoshii].

gcp

protein

metalloendopeptidase,

turnover,

putative,

chaperones;

glycoprotease family

Metal-

1.20e−81

dependent

proteases

with possible

chaperone

activity 1e−76

962,
NEQ494
pyruvate formate-
351aa long conserved
1.00E−104
Radical_SAM
1.97.
Cellular

963

lyase
hypothetical protein

Radical SAM
1.4
processes; Posttranslational

activatingenzyme
[Sulfolobus tokodaii].

superfamily 5.00e−25

modification,

homolog [Pb

protein

aerophilum]

turnover,

chaperones; Pyruvate-

formate

lyase-

activating

enzyme 8e−96

964,
NEQ495
Predicted P-loop
hypothetical protein
1.00E−92
1.10E−62

Poorly

965

ATPase fused to
[Pyrococcus

characterized;

anacetyltransferase

horikoshii].

General

[M. kandleri] SPLIT

function

see SEQ ID

prediction

NOS: 194, 195

only; Predicted

P-loop

ATPase fused

to an

acetyltransferase

9e−94

966,
NEQ498
Translation initiation
Translation initiation
1.00E−166
GTP_EFTU
3.6.1.
Information

967

factor 2,
factor 2, GTPase

Elongation factor Tu
48
storage and

GTPase[Methanopyrus
[Methanopyrus

GTP binding domain

processing; Translation,

kandleri AV19]

kandleri AV19].

1.70e−40 :mobB

ribosomal

mobB

structure and

molybdopterin-

biogenesis; Translation

guanine dinucleotide

initiation

biosynthesis protein

factor 2

MobB 7.50e−3

(GTPase) 1e−150

:small_GTP

small_GTP small

GTP-binding protein

domain 8.50e−15

:selB selB

selenocysteine-

specific translation

elongation factor

1.20e−4 :EF-Tu EF-

Tu translation

elongation factor Tu

6.90e−3 :IF-2 IF-2

translation initiation

factor IF-2 4.40e−08

:alF-2 alF-2

translation initiation

factor alF-2 5.50e−213

:FOLD979 No

CATH Annotation

2.50e−3

968,
NEQ497
hypothetical protein
unnamed protein
3.00E−04
0

969

product, putative

[Plasmodium yoelii

yoelii].

970,
NEQ499
hypothetical protein
hypothetical protein
4.00E−03
0

971

[Pyrococcusabyssi]
[Pyrococcus abyssi].

972,
NEQ500
hypothetical protein
predicted protein
1.00E−05
0

973

[Methanosarcina

acetivorans str. C2A].

974,
NEQ502
2′-5′ RNA ligase
conserved
1.00E−26
2_5_ligase 2′,5′
6.5.1.—
Information

975

[Methanopyruskandleri
hypothetical protein

RNA ligase family

storage and

AV19]
[Methanococcus

6.70e−20

processing; Translation,

jannaschii].

:2_5_ligase 2′,5′

ribosomal

RNA ligase family

structure and

3.40e−05

biogenesis; 2′-

5′ RNA ligase

1e−27

976,
NEQ501
magnesium and
magnesium and
1.00E−06
0

Cellular

977

cobalt
cobalt transport

processes; Inorganic

transportprotein
protein (corA)

ion

(corA) [Haemophilus
[Haemophilus

transport and

influenzae Rd].

metabolism; Mg2+

and

Co2+

transporters

9e−08

978,
NEQ504
hypothetical protein
Unknown (protein for
0.06
0

979

MGC: 55710) [Danio

rerio].

980,
NEQ503
DNA-directed RNA
DNA-directed RNA
0
RNA_pol_Rpb1_2
2.7.7.6
Information

981

polymerase, subunit
polymerase, subunit A

RNA polymerase

storage and

A′ (rpoA1) [A. fulgidus]
(rpoA1)

Rpb1, domain 2

processing; Transcription;

[Archaeoglobus

1.10e−93 : 2.00e−39

DNA-

fulgidus].

: 3.60e−91 : 8.40e−30

directed

RNA

polymerase

beta′

subunit/160 kD

subunit

(split gene in

archaea and

Syn) 0

982,
NEQ506
Predicted RNA-
hypothetical protein
1.00E−41
0

Information

983

binding
[Pyrococcus furiosus

storage and

proteinhomologous
DSM 3638].

processing; Transcription;

to eukaryotic snRNP

Predicted

RNA-

binding

protein

homologous

to eukaryotic

snRNP 8e−41

984,
NEQ505
Phenylalanyl-tRNA
Phenylalanyl-tRNA
1.00E−74
tRNA-synt_2d tRNA
6.1.1.
Information

985

synthetase
synthetase alpha

synthetases class II
20
storage and

alphasubunit (pheS)
subunit (pheS)

core domain (F)

processing; Translation,

[S. solfataricus]
[Sulfolobus

4.30e−61 :pheS

ribosomal

solfataricus].

pheS phenylalanyl-

structure and

tRNA synthetase,

biogenesis; Phenylalanyl-

alpha subunit 9.20e−66

tRNA

synthetase

alpha subunit

7e−64

986,
NEQ507
DNA-directed RNA
DNA-directed RNA
2.00E−16
RNA_pol_Rpb5_C
2.7.7.6
Information

987

polymerase, subunit
polymerase, subunit H

RNA polymerase

storage and

H (rpoH) [Mc
(rpoH)

Rpb5, C-terminal

processing; Transcription;

jannaschii]
[Methanococcus

domain 4.00e−26

DNA-

jannaschii].

directed

RNA

polymerase,

subunit H,

RpoH/RPB5

1e−17

988,
NEQ508
SSU ribosomal
30S ribosomal protein
1.00E−57
Ribosomal_S2 Ribosomal

Information

989

protein
S2 [Pyrococcus

protein S2 3.20e−30

storage and

S2[Pyrococcus

horikoshii].

:rpsB_bact rpsB_bact

processing; Translation,

horikoshii]

ribosomal protein S2 4.40e−06

ribosomal

:Sa_S2_E_A Sa_S2_E_A

structure and

ribosomal protein S2 3.70e−98

biogenesis; Ribosomal

protein S2 8e−59

990,
NEQ509
DNA ligase; ATP
DNA ligase
1.00E−139
DNA_ligase ATP
6.5.1.1
Information

991

dependent[Pyrococcus
[Pyrococcus abyssi].

dependent DNA

storage and

abyssi]

ligase domain

processing; DNA

7.30e−70 :dnl1 dnl1

replication,

DNA ligase I, ATP-

recombination

dependent (dnl1)

and

5.60e−163 : 4.10e−08

repair; ATP-

: 1.10e−67

dependent

DNA ligase

1e−140

992,
NEQ510
hypothetical protein
putative histidine
0.02
0

993

kinase, possibly

involved in

competence

[Streptococcus

pyogenes].

994,
NEQ511
desuccinylase
desuccinylase
8.00E−28
Peptidase_M20
3.5.1.
Metabolism; Amino

995

[Pyrococcus
[Pyrococcus

Peptidase family
18
acid

horikoshii]

horikoshii].

M20/M25/M40

transport and

1.10e−63

metabolism; Acetylornithine

:dapE_proteo

deacetylase/Succinyl-

dapE_proteo

diaminopimelate

succinyl-

desuccinylase

diaminopimelate

and related

desuccinylase

deacylases

1.30e−11

6e−29

996,
NEQ512
Predicted P-loop
conserved
3.00E−37
0

Poorly

997

ATPase[Methanopyrus
hypothetical protein

characterized;

kandleri AV19]
[Methanosarcina

General

acetivorans str. C2A].

function

prediction

only; Predicted

ATPase 3e−09

998,
NEQ513
hypothetical Glu-
Glu-tRNA
1.00E−05
gatC gatC glutamyl-

Information

999

tRNA
amidotransferase,

tRNA(Gln)

storage and

amidotransferase,
subunit C (gatC)

amidotransferase, C subunit

processing; Translation,

subunit C
[Archaeoglobus

2.20e−4

ribosomal

fulgidus].

structure and

biogenesis; Asp-

tRNAAsn/Glu-

tRNAGln

amidotransferase

C subunit

7e−07

1000,
NEQ514
hypothetical protein
exodeoxyribonuclease
9.00E−03
0

1001

V, beta chain (recB)

[Borrelia burgdorferi].

1002,
NEQ515
Uncharacterized
hypothetical protein
4.00E−38

1003

protein conserved
[Pyrococcus

inarchaea

horikoshii].

[Methanopyrus

kandleri]

1004,
NEQ516
hypothetical protein
hypothetical protein
1.00E−11
0

Cellular

1005

[Pyrococcus abyssi].

processes; Posttranslational

modification,

protein

turnover,

chaperones; Prefoldin,

chaperonin

cofactor 7e−13

1006,
NEQ518
hypothetical protein
U30 [Human
0.25
0

1007

herpesvirus 7].

1008,
NEQ517
translation initiation
223aa long
4.00E−16
eIF6 eIF-6 family 2.30e−09

Information

1009

factor 6[Sulfolobus
hypothetical

:eIF-6 eIF-6 translation

storage and

tokodaii]
translation initiation

initiation factor eIF-6,

processing; Translation,

factor 6 [Sulfolobus

putative 2.70e−12

ribosomal

tokodaii].

structure and

biogenesis; Eukaryotic

translation

initiation

factor 6

(EIF6) 4e−12

1010,
NEQ519
histidine triad (HIT)
HIT family protein (hit)
5.00E−29
HIT HIT family
3.6.1.
Metabolism; Nucleotide

1011

protein MJ0866(cell
[Methanococcus

2.10e−44
17
transport and

cycle regulation)-M. jannaschii

jannaschii].

metabolism; Diadenosine

tetraphosphate

(Ap4A)

hydrolase and

other HIT

family

hydrolases

5e−30

1012,
NEQ520
hypothetical protein
Sulfur transfer protein
1.4
0

1013

involved in thiamine

biosynthesis [Aquifex

aeolicus].

1014,
NEQ521
Proteasome alpha
Protease subunit of
8.00E−60
proteasome
3.4.25.1
Cellular

1015

subunit(Multicatalytic
the proteasome

Proteasome A-type

processes; Posttranslational

endopeptidase
[Methanopyrus

and B-type 9.90e−40

modification,

kandleri AV19].

protein

turnover,

chaperones; Proteasome

protease

subunit 6e−57

1016,
NEQ523
n-type ATP
hypothetical protein
7.00E−59
UPF0021 Uncharacterized

Cellular

1017

pyrophosphatasesupperfamily
[Pyrococcus abyssi].

protein family UPF0021

processes; Cell

[Pyrococcus

2.00e−17 :TIGR00269

division and

furiosus];

TIGR00269 conserved

chromosome

hypothetical protein

partitioning; Predicted

TIGR00269 4.90e−13

ATPase of

the PP-loop

superfamily

implicated in

cell cycle

control 5e−60

1018,
NEQ522
Predicted N6-
conserved
2.00E−37
THUMP THUMP
2.1.1.—
Information

1019

adenine-specific
hypothetical protein

domain 1.50e−11

storage and

RNAmethylase
[Methanococcus

:UPF00200 Putative

processing; DNA

containing THUMP

jannaschii].

RNA methylase

replication,

family UPF0020

recombination

1.00e−37

and

:TIGR01177

repair; Predicted

TIGR01177

N6-

conserved

adenine-

hypothetical protein

specific DNA

TIGR01177 4.30e−19

methylases

1e−38

1020,
NEQ525
3′-
n-type ATP
2.00E−33
PAPS_reduct
1.8.99.4
Metabolism; Amino

1021

phosphoadenosine
pyrophosphatase

Phosphoadenosine

acid

5′-
superfamily

phosphosulfate

transport and

phosphosulfatesulfotransferase
[Pyrococcus furiosus

reductase family

metabolism; 3′-

DSM 3638].

9.30e−18

phosphoadenosine

5′-

phosphosulfate

sulfotransferase

(PAPS

reductase)/FAD

synthetase

and related

enzymes 1e−30

1022,
NEQ524
Predicted exosome
Predicted exosome
6.00E−48
UPF0023 Uncharacterized

Information

1023

subunit[Methanopyrus
subunit

protein family UPF0023

storage and

kandleri AV19]
[Methanopyrus

8.90e−24 :TIGR00291

processing; Translation,

kandleri AV19].

TIGR00291 conserved

ribosomal

hypothetical protein

structure and

TIGR00291 2.20e−53

biogenesis; Predicted

exosome

subunit 2e−47

1024,
NEQ526
hypothetical
hypothetical protein
6.00E−12
0

Information

1025

protein(similar to
[Thermoplasma

storage and

Queuine tRNA-

volcanium].

processing; Translation,

ribosyltransferase)

ribosomal

structure and

biogenesis; Queuine

tRNA-

ribosyltransferases,

contain

PUA domain

4e−13

1026,
NEQ527
Predicted P-loop
495aa long conserved
1.00E−23
DUF87 Domain of unknown

Poorly

1027

ATPase[Methanopyrus
hypothetical protein

function DUF87 2.30e−07

characterized;

kandleri AV19]
[Sulfolobus tokodaii].

General

function

prediction

only; Predicted

ATPase 1e−17

1028,
NEQ528
DNA-directed DNA
DNA POLYMERASE.
4.00E−50
DNA_pol_B DNA
2.7.7.7
Information

1029

polymerase I(family

polymerase family B

storage and

B) SPLIT see SEQ

4.80e−4

processing; DNA

ID NO: 136, 137

replication,

recombination

and

repair; DNA

polymerase

elongation

subunit

(family B) 1e−46

1030,
NEQ529
hypothetical protein

DUF457 Predicted membrane-bound

metal-dependent

1031

hydrolase (DUF457) 6.00e−06

1032,
NEQ531
putative integral
hypothetical protein
2.00E−27
MS_channel

Cellular

1033

membrane
[Nostoc sp. PCC

Mechanosensitive ion

processes; Cell

protein. [Streptomyces
7120].

channel 1.40e−35

envelope

coelicolor A3(2)]

biogenesis,

outer

membrane; Small-

conductance

mechanosensitive

channel

1e−22

1034,
NEQ530
LSU ribosomal
LSU ribosomal protein
9.00E−32
Ribosomal_L32e Ribosomal

Information

1035

protein
L32E [Pyrococcus

protein L32 6.60e−40

storage and

L32E[Pyrococcus

abyssi].

processing; Translation,

abyssi]

ribosomal

structure and

biogenesis; Ribosomal

protein L32E

5e−33

1036,
NEQ532
hypothetical protein
similar to cell wall
4.00E−04
0

1037

binding proteins

[Listeria

monocytogenes EGD-

e].

1038,
NEQ533
hypothetical protein
similar to Plasmodium
1.4
0

1039

falciparum.

Hypothetical protein

[Dictyostelium

discoideum].

1040,
NEQ534
Predicted
hypothetical protein
6.00E−33
0

Information

1041

transcriptional
[Pyrococcus

storage and

regulator[Methanopyrus

horikoshii].

processing; Transcription;

kandleri AV19]

Predicted

transcriptional

regulators 8e−34

1042,
NEQ535
aspartyl-tRNA
aspartyl-tRNA
2.00E−92
tRNA-synt_2 tRNA
6.1.1.
Information

1043

synthetase (class
synthetase

synthetases class II
12
storage and

2)[Aeropyrum pernix]
[Aeropyrum pernix].

(D, K and N) 3.90e−69

processing; Translation,

:tRNA-synt_2d

ribosomal

tRNA synthetases

structure and

class II core domain

biogenesis; Aspartyl/

(F) 3.70e−3

asparaginyl-

:tRNA_anti OB-fold

tRNA

nucleic acid binding

synthetases

domain 6.00e−16

2e−93

:asnS asnS

asparaginyl-tRNA

synthetase 8.00e−39

:aspS_arch

aspS_arch aspartyl-

tRNA synthetase

3.50e−109

:aspS_bact

aspS_bact aspartyl-

tRNA synthetase

3.40e−11 :genX

genX lysyl-tRNA

synthetase-related

protein 1.60e−05

:lysS_bact

lysS_bact lysyl-

tRNA synthetase

2.00e−05

1044,
NEQ536
putative nucleolar
putative nucleolar
2.00E−78
Nol1_Nop2_Sun
2.1.1.—
Information

1045

protein I (nol1-nop2-
protein I (nol1-nop2-

NOL1/NOP2/sun

storage and

sun family)
sun family)

family 2.90e−49

processing; Translation,

[Pyrococcus
[Pyrococcus furiosus

:nop2p nop2p

ribosomal

DSM 3638].

NOL1/NOP2/sun

structure and

family putative RNA

biogenesis; tRNA

methylase 7.50e−101

and rRNA

:rsmB rsmB

cytosine-C5-

Sun protein 7.70e−09

methylases

3e−77

1046,
NEQ538
acetylpolyamine
acetylpolyamine
4.00E−40
Hist_deacetyl
3.5.1.
Cellular

1047

aminohydrolase, putative
aminohydrolase,

Histone deacetylase
48
processes; Signal

[Pyrobaculum
putative [Pyrobaculum

family 2.40e−21

transduction

aerophilum]

aerophilum].

mechanisms;

Deacetylases,

including

yeast histone

deacetylase

and acetoin

utilization

protein 2e−37

1048,
NEQ537
proliferating-cell
proliferating-cell
1.00E−37
PCNA Proliferating cell

Information

1049

nuclear
nuclear antigen

nuclear antigen, N-terminal

storage and

antigen[Pyrococcus
[Pyrococcus abyssi].

domain 5.00e−4 :PCNA_C

processing; DNA

abyssi]

Proliferating cell nuclear

replication,

antigen, C-terminal domain

recombination

2.00e−05 :pcna pcna

and

proliferating cell nuclear

repair; DNA

antigen (pcna) 3.60e−14

polymerase

sliding clamp

subunit

(PCNA

homolog) 8e−39

1050,
NEQ539
hypothetical protein
hypothetical protein
3.00E−08
0

Cellular

1051

[Pyrococcusfuriosus
[Pseudomonas

processes; Cell

DSM 3638]

syringae pv. syringae

envelope

B728a].

biogenesis,

outer

membrane; Small-

conductance

mechanosensitive

channel

3e−07

1052,
NEQ540
transcription
transcription
4.00E−27
KOW KOW motif 2.10e−07

Information

1053

antitermination
antitermination proteinnusG

:L26e_arch L26e_arch

storage and

proteinnusG
[Pyrococcus

ribosomal protein L24 5.70e−25

processing; Transcription;

[Pyrococcus

furiosus DSM 3638].

Transcription

furiosus]

antiterminator

2e−27

1054,
NEQ541
hypothetical
135aa long
800E−07
0

1055

transcriptional
hypothetical

regulator[Sulfolobus
transcriptional

tokodaii]
regulator [Sulfolobus

tokodaii].

1056,
NEQ543
elongation factor 2
elongation factor 2
0
EFG_C Elongation
3.6.1.
Information

1057

(EF-2); (EF-
(EF-2); (EF-2)

factor G C-terminus
48
storage and

2)[Pyrococcus
[Pyrococcus furiosus

1.00e−32

processing; Translation,

furiosus]
DSM 3638].

:GTP_EFTU

ribosomal

Elongation factor Tu

structure and

GTP binding domain

biogenesis; Translation

7.10e−76

elongation

:GTP_EFTU_D2

and release

Elongation factor Tu

factors

domain 2 6.40e−14

(GTPases) 0

:small_GTP

small_GTP small

GTP-binding protein

domain 5.20e−12

:selB selB

selenocysteine-

specific translation

elongation factor

6.70e−3 :EF-G EF-G

translation

elongation factor G

3.90e−52 :EF-Tu EF-

Tu translation

elongation factor Tu

2.30e−4 :aEF-2 aEF-

2 translation

elongation factor

aEF-2 0.0 :prfC prfC

peptide chain

release factor 3

4.30e−05 :EFG_IV

Elongation factor G,

domain IV 8.70e−24

1058,
NEQ542
DNA topoisomerase
type II DNA
1.00E−110
TP6A_N Type IIB
5.99.
Information

1059

VI, subunit A(top6A)
topoisomerase

DNA topoisomerase
1.3
storage and

[Pyrococcus abyssi]
subunit a [Pyrococcus

9.00e−30

processing; DNA

furiosus DSM 3638].

replication,

recombination

and

repair; DNA

topoisomerase

VI, subunit

A 1e−111

1060,
NEQ545
hypothetical protein

0

1061

1062,
NEQ544
Predicted
Predicted membrane-
8.00E−29
Peptidase_M50 Peptidase

Cellular

1063

membrane-
associated Zn-

family M50 9.90e−25

processes; Cell

associated Zn-
dependent protease

envelope

dependentprotease
[Methanopyrus

biogenesis,

[M. kandleri]

kandleri AV19].

outer

membrane; Predicted

membrane-

associated

Zn-dependent

proteases 1

3e−24

1064,
NEQ546
LSU ribosomal
LSU ribosomal protein
1.00E−35
Ribosomal_L1 Ribosomal

Information

1065

protein L1P
L1P (rpl1P)

protein L1p/L10e family

storage and

(rpl1P)[Pyrococcus
[Pyrococcus abyssi].

8.10e−39 :rplA_bact

processing; Translation,

abyssi]

rplA_bact ribosomal protein

ribosomal

L1 1.40e−07

structure and

biogenesis; Ribosomal

protein L1 7e−37

1066,
NEQ548
SSU ribosomal
SSU ribosomal
1.00E−10
Ribosomal_S24e Ribosomal

Information

1067

protein
protein S24E

protein S24e 3.60e−11

storage and

S24E[Methanococcus
[Methanococcus

processing; Translation,

jannaschii]

jannaschii].

ribosomal

structure and

biogenesis; Ribosomal

protein S24E

7e−12

1068,
NEQ547
alanyl-tRNA
alanyl-tRNA
3.00E−64
tRNA-synt_2c tRNA
6.1.1.7
Information

1069

synthetase [Giardia
synthetase [Giardia

synthetases class II

storage and

intestinalis].

(A) 6.50e−3

processing; Translation,

ribosomal

structure and

biogenesis; Alanyl-

tRNA

synthetase

6e−61

1070,
NEQ549
hypothetical protein
DNA helicase II
4.00E−09
0

Information

1071

[Fusobacterium

storage and

nucleatum subsp.

processing; DNA

nucleatum ATCC

replication,

25586].

recombination

and

repair; RecB

family

exonuclease

8e−07

1072,
NEQ550
hypothetical protein
hypothetical protein
0.01
0

1073

[Plasmodium

falciparum 3D7].

The invention for the first time has isolated and cultivated “Nanoarchaeum equitans,” a new nanosized hyperthermophilic archaeon derived from a submarine hot vent. This archaeon cannot be attached to any known Archaeal group, including the phyla Crenarchaeota, Korarchaeota or Euryarchaeota and therefore must represent an unknown phylum which we have named ‘Nanoarchaeota’ and species, which we name ‘Nanoarchaeum equitans’.

Cells of N. equitans are spherical, and only about 400 nm in diameter. They grow attached to the surface of a specific archaeal host, a new member of the genus Ignicoccus. The natural distribution of the ‘Nanoarchaeota’ is so far unknown. Owing to their unusual single-stranded (ss)rRNA sequence, members remained undetectable by commonly used ecological studies based on the polymerase chain reaction. ‘N. equitans’ harbors the smallest archaeal genome (SEQ ID NO:1); it is only 0.5 megabases in size. The organism of the invention will provide insight into the evolution of thermophily, of tiny genomes and of interspecies communication.

DEFINITIONS

The term “antibody” includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.”

The terms “array” or “microarray” or “biochip” or “chip” as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface, as discussed in further detail, below.

As used herein, the terms “computer,” “computer program” and “processor” are used in their broadest general contexts and incorporate all such devices, as described in detail, below.

A “coding sequence of” or a “sequence encodes” a particular polypeptide or protein, is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences.

The term “expression cassette” as used herein refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as an enzyme of the invention) in a host compatible with such sequences. Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers. “Operably linked” as used herein refers to linkage of a promoter upstream from a DNA sequence such that the promoter mediates transcription of the DNA sequence. Thus, expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. A “vector” comprises a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. The vector optionally comprises viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and includes both the expression and non-expression plasmids. Where a recombinant microorganism or cell culture is described as hosting an “expression vector” this includes both extra-chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.

“Plasmids” are designated by a lower case “p” preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.

The term “gene” means the segment of DNA involved in producing a polypeptide chain, including, inter alia, regions preceding and following the coding region, such as leader and trailer, promoters and enhancers, as well as, where applicable, intervening sequences (introns) between individual coding segments (exons).

The phrases “nucleic acid” or “nucleic acid sequence” as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., double stranded iRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156.

“Amino acid” or “amino acid sequence” as used herein refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.

The terms “polypeptide” and “protein” as used herein, refer to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain modified amino acids other than the 20 gene-encoded amino acids. The term “polypeptide” also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides. The peptides and polypeptides of the invention also include all “mimetic” and “peptidomimetic” forms, as described in further detail, below.

As used herein, the term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. As used herein, an isolated material or composition can also be a “purified” composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition. Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity. In alternative aspects, the invention provides nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid is adjacent to a “backbone” nucleic acid to which it is not adjacent in its natural environment. In one aspect, nucleic acids represent 5% or more of the number of nucleic acid inserts in a population of nucleic acid “backbone molecules.” “Backbone molecules” according to the invention include nucleic acids such as expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids, and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. In one aspect, the enriched nucleic acids represent 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the number of nucleic acid inserts in the population of recombinant backbone molecules. “Recombinant” polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein. “Synthetic” polypeptides or protein are those prepared by chemical synthesis, as described in further detail, below.

A promoter sequence is “operably linked to” a coding sequence when RNA polymerase which initiates transcription at the promoter will transcribe the coding sequence into mRNA, as discussed further, below.

“Oligonucleotide” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

The phrase “substantially identical” in the context of two nucleic acids or polypeptides, refers to two or more sequences that have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide or amino acid residue (sequence) identity, when compared and aligned for maximum correspondence, as measured using one any known sequence comparison algorithm, as discussed in detail below, or by visual inspection. In alternative aspects, the invention provides nucleic acid and polypeptide sequences having substantial identity to an exemplary sequence of the invention over a region of at least about 100 residues, 150 residues, 200 residues, 300 residues, 400 residues, or a region ranging from between about 50 residues to the full length of the nucleic acid or polypeptide. Nucleic acid sequences of the invention can be substantially identical over the entire length of a polypeptide coding region.

Additionally a “substantially identical” amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine). One or more amino acids can be deleted, for example, from a polypeptide of the invention, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for the polypeptide's biological activity can be removed. Modified polypeptide sequences of the invention can be assayed for a biological activity (e.g., enzymatic, binding, structural, and the like) by any number of methods, including contacting the modified polypeptide sequence with an enzyme substrate and determining whether the modified polypeptide decreases the amount of specific substrate in the assay or increases the bioproducts of the enzymatic reaction of a functional enzyme with the substrate, as discussed further, below.

“Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Suitably stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below. In alternative aspects, nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein.

The term “variant” refers to polynucleotides or polypeptides of the invention modified at one or more base pairs, codons, introns, exons, or amino acid residues (respectively) yet still retain the biological activity of a polypeptide (e.g., an enzyme) of the invention. Variants can be produced by any number of means included methods such as, for example, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSM and any combination thereof. Techniques for producing variant a polypeptide (e.g., an enzyme) having activity at a pH or temperature, for example, that is different from a wild-type a polypeptide (e.g., an enzyme), are included herein.

The term “saturation mutagenesis” or “GSSM” includes a method that uses degenerate oligonucleotide primers to introduce point mutations into a polynucleotide, as described in detail, below.

The term “optimized directed evolution system” or “optimized directed evolution” includes a method for reassembling fragments of related nucleic acid sequences, e.g., related genes, and explained in detail, below.

The term “synthetic ligation reassembly” or “SLR” includes a method of ligating oligonucleotide fragments in a non-stochastic fashion, and explained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids (e.g., the exemplary SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073), including expression cassettes such as expression vectors, encoding polypeptides (e.g., enzymes) of the invention. The invention also includes methods for discovering new polypeptide (e.g., enzyme) sequences using the nucleic acids of the invention. Also provided are methods for modifying the nucleic acids of the invention by, e.g., synthetic ligation reassembly, optimized directed evolution system and/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. In practicing the methods of the invention, homologous genes can be modified by manipulating a template nucleic acid, as described herein. The invention can be practiced in conjunction with any method or protocol or device known in the art, which are well described in the scientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used to practice the methods of the invention is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them. A polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification. Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of the invention operatively linked to expression (e.g., transcriptional or translational) control sequence(s), e.g., promoters or enhancers, to direct or modulate RNA synthesis/expression. The expression control sequence can be in an expression vector. Exemplary bacterial promoters include lac, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I.

Promoters suitable for expressing a polypeptide in bacteria include the E. coli lac or trp promoters, the lacI promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter, the lambda PL promoter, promoters from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter. Eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, heat shock promoters, the early and late SV40 promoter, LTRs from retroviruses, and the mouse metallothionein-I promoter. Other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses may also be used.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding polypeptides (e.g., enzymes) of the invention. Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast). Vectors of the invention can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Exemplary vectors are include: bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid or other vector may be used so long as they are replicable and viable in the host. Low copy number or high copy number vectors may be employed with the present invention.

The expression vector may comprise a promoter, a ribosome-binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression. Mammalian expression vectors can comprise an origin of replication, any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking non-transcribed sequences. In some aspects, DNA sequences derived from the SV40 splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectable marker genes to permit selection of host cells containing the vector. Such selectable markers include genes encoding dihydrofolate reductase or genes conferring neomycin resistance for eukaryotic cell culture, genes conferring tetracycline or ampicillin resistance in E. coli, and the S. cerevisiae TRP1 gene. Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryotic cells may also contain enhancers to increase expression levels. Enhancers are cis-acting elements of DNA, usually from about 10 to about 300 bp in length that act on a promoter to increase its transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancers.

A DNA sequence may be inserted into a vector by a variety of procedures. In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, blunt ends in both the insert and the vector may be ligated. A variety of cloning techniques are known in the art, e.g., as described in Ausubel and Sambrook. Such procedures and others are deemed to be within the scope of those skilled in the art.

The vector may be in the form of a plasmid, a viral particle, or a phage. Other vectors include chromosomal, non-chromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by, e.g., Sambrook.

Particular bacterial vectors which may be used include the commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any other vector may be used as long as it is replicable and viable in the host cell.

Host Cells and Transformed Cells

The invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding polypeptides (e.g., enzymes) of the invention, a vector of the invention. The host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells. Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus. Exemplary insect cells include Drosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art.

The vector may be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Depending upon the host employed in a recombinant production procedure, the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated. Polypeptides of the invention may or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce a polypeptide of the invention. Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof. In some aspects, the DNA construct may be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.

The expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids encoding the polypeptides of the invention, or modified nucleic acids, can be reproduced by, e.g., amplification. The invention provides amplification primer sequence pairs for amplifying nucleic acids encoding polypeptides (e.g., enzymes) of the invention. In one aspect, the primer pairs are capable of amplifying nucleic acid sequences of the invention, e.g., including the exemplary SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073, or a subsequence thereof, etc. One of skill in the art can design amplification primer sequence pairs for any part of or the full length of these sequences.

The invention provides an amplification primer sequence pair for amplifying a nucleic acid encoding a polypeptide of the invention, wherein the primer pair is capable of amplifying a nucleic acid comprising a sequence of the invention, or fragments or subsequences thereof. One or each member of the amplification primer sequence pair can comprise an oligonucleotide comprising at least about 10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.

The invention provides amplification primer pairs, wherein the primer pair comprises a first member having a sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of a nucleic acid of the invention, and a second member having a sequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of the complementary strand of the first member. The invention provides polypeptides (e.g., enzymes) generated by amplification, e.g., polymerase chain reaction (PCR), using an amplification primer pair of the invention. The invention provides methods of making polypeptides (e.g., enzymes) by amplification, e.g., polymerase chain reaction (PCR), using an amplification primer pair of the invention. In one aspect, the amplification primer pair amplifies a nucleic acid from a library, e.g., a gene library, such as an environmental library.

Amplification reactions can also be used to quantify the amount of nucleic acid in a sample (such as the amount of message in a cell sample), label the nucleic acid (e.g., to apply it to an array or a blot), detect the nucleic acid, or quantify the amount of a specific nucleic acid in a sample. In one aspect of the invention, message isolated from a cell or a cDNA library are amplified. The skilled artisan can select and design suitable oligonucleotide amplification primers. Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) J. Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplification assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary nucleic acid of the invention (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073, and nucleic acids encoding SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, etc., and all polypeptides disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:3 through SEQ ID NO:1073) over a region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues. The invention provides polypeptides comprising sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an exemplary polypeptide of the invention. The extent of sequence identity (homology) may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.

In alternative embodiments, the sequence identify can be over a region of at least about 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400 consecutive residues, or the full length of the nucleic acid or polypeptide. The extent of sequence identity (homology) may be determined using any computer program and associated parameters, including those described herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default parameters.

Homologous sequences also include RNA sequences in which uridines replace the thymines in the nucleic acid sequences. The homologous sequences may be obtained using any of the procedures described herein or may result from the correction of a sequencing error. It will be appreciated that the nucleic acid sequences as set forth herein can be represented in the traditional single character format (see, e.g., Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) or in any other format which records the identity of the nucleotides in a sequence.

Various sequence comparison programs identified herein are used in this aspect of the invention. Protein and/or nucleic acid sequence identities (homologies) may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

Homology or identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications. The terms “homology” and “identity” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection. For sequence comparison, one sequence can act as a reference sequence (e.g., an exemplary sequence SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073) to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous residues. For example, in alternative aspects of the invention, contiguous residues ranging anywhere from 20 to the full length of an exemplary sequence of the invention are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. If the reference sequence has the requisite sequence identity to an exemplary sequence of the invention, e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence of the invention, that sequence is within the scope of the invention. In alternative embodiments, subsequences ranging from about 20 to 600, about 50 to 200, and about 100 to 150 are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequence for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of person & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection. Other algorithms for determining homology or identity include, for example, in addition to a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local Content Program), MACAW (Multiple Alignment Construction & Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such alignment programs can also be used to screen genome databases to identify polynucleotide sequences having substantially identical sequences. A number of genome databases are available, for example, a substantial portion of the human genome is available as part of the Human Genome Sequencing Project (Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium (Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae (Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast (S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al., 2000). Significant progress has also been made in sequencing the genomes of model organism, such as mouse, C. elegans, and Arabadopsis sp. Databases containing genomic information annotated with some functional information are maintained by different organization, and are accessible via the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms are also used to practice the invention. They are described, e.g., in Altschul (1977) Nuc. Acids Res. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul (1990) supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure of similarity provided by BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a references sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. In one aspect, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”). For example, five specific BLAST programs can be used to perform the following task: (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database; (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and, (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).

In one aspect of the invention, to determine if a nucleic acid has the requisite sequence identity to be within the scope of the invention, the NCBI BLAST 2.2.2 programs is used. default options to blastp. There are about 38 setting options in the BLAST 2.2.2 program. In this exemplary aspect of the invention, all default values are used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a “-F F” setting is used, which disables filtering. Use of default filtering often results in Karlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the invention include:

- “Filter for low complexity: ON
- >Word Size: 3
- >Matrix: Blosum62
- >Gap Costs: Existence: 11
- >Extension: 1”

Other default settings are: filter for low complexity OFF, word size of 3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and a gap extension penalty of −1.

An exemplary NCBI BLAST 2.2.2 program setting is set forth in Example 1, below. Note that the “-W” option defaults to 0. This means that, if not set, the word size defaults to 3 for proteins and 11 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies, motifs and the like in silico the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention, e.g., an exemplary sequence of the invention. As used herein, the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.

Another aspect of the invention is a computer readable medium having recorded thereon at least one nucleic acid and/or polypeptide sequence of the invention. Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems), particularly computer systems, which store and manipulate the sequences and sequence information described herein. One example of a computer system 100 is illustrated in block diagram form in FIG. 1. As used herein, “a computer system” refers to the hardware components, software components, and data storage components used to analyze a nucleotide or polypeptide sequence of the invention. The computer system 100 can include a processor for processing, accessing and manipulating the sequence data. The processor 105 can be any well-known type of central processing unit, such as, for example, the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq, AMD or International Business Machines. The computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.

In one aspect, the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon. The computer system 100 can further include one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or a modem capable of connection to a remote data storage system (e.g., via the internet) etc. In some embodiments, the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.

The computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100. Software for accessing and processing the nucleotide or amino acid sequences of the invention can reside in main memory 115 during execution.

In some aspects, the computer system 100 may further comprise a sequence comparison algorithm for comparing a nucleic acid sequence of the invention. The algorithm and sequence(s) can be stored on a computer readable medium. A “sequence comparison algorithm” refers to one or more programs which are implemented (locally or remotely) on the computer system 100 to compare a nucleotide sequence with other nucleotide sequences and/or compounds stored within a data storage means. For example, the sequence comparison algorithm may compare the nucleotide sequences of an exemplary sequence, e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073, stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies or structural motifs.

The parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. In some aspects, the parameters may be the default parameters used by the algorithms in the absence of instructions from the user.

FIG. 2 is a flow diagram illustrating one aspect of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK that is available through the Internet. The process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100. As discussed above, the memory could be any type of memory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison. The process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database. Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system.

Once a comparison of the two sequences has been performed at the state 210, a determination is made at a decision state 210 whether the two sequences are the same. Of course, the term “same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as “same” in the process 200. If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name of the stored sequence is displayed to the user, the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.

It should be noted that if a determination had been made at the decision state 212 that the sequences were not homologous, then the process 200 would move immediately to the decision state 218 in order to determine if any other sequences were available in the database for comparison. Accordingly, one aspect of the invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid sequence of the invention and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify structural motifs, or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes.

FIG. 3 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous. The process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state 256. The process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it can be a single letter amino acid code so that the first and sequence sequences can be easily compared. A determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read. If there are not any more characters to read, then the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence to a sequence of the invention to determine whether the sequences differ at one or more positions. The program can record the length and identity of inserted, deleted or substituted nucleotides or amino acid residues with respect to the sequence of either the reference or the invention. The computer program may be a program which determines whether a reference sequence contains a single nucleotide polymorphism (SNP) with respect to a sequence of the invention, or, whether a sequence of the invention comprises a SNP of a known sequence. Thus, in some aspects, the computer program is a program which identifies SNPs. The method may be implemented by the computer systems described above and the method illustrated in FIG. 3. The method can be performed by reading a sequence of the invention and the reference sequences through the use of the computer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier for identifying features within a nucleic acid or polypeptide of the invention. An “identifier” refers to one or more programs which identifies certain features within a nucleic acid sequence. For example, an identifier may comprise a program which identifies an open reading frame (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagram illustrating one aspect of an identifier process 300 for detecting the presence of a feature in a sequence. The process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100. The process 300 then moves to a state 306 wherein a database of sequence features is opened. Such a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be “Initiation Codon” and the attribute would be “ATG”. Another example would be the feature name “TAATAA Box” and the feature attribute would be “TAATAA”. An example of such a database is produced by the University of Wisconsin Genetics Computer Group. Alternatively, the features may be structural polypeptide motifs such as alpha helices, beta sheets, or functional polypeptide motifs such as enzymatic active sites, helix-turn-helix motifs or other motifs known to those skilled in the art. Once the database of features is opened at the state 306, the process 300 moves to a state 308 wherein the first feature is read from the database. A comparison of the attribute of the first feature with the first sequence is then made at a state 310. A determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name of the found feature is displayed to the user. The process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence. If the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database. Thus, in one aspect, the invention provides a computer program that identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, a sequence can be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparison algorithms, identifiers, or sources of reference nucleotide sequences or polypeptide sequences to be compared to a nucleic acid sequence of the invention. The programs and databases used to practice the invention include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius2.DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwent's World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.

Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to a sequence of the invention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073, or a nucleic acid that encodes a polypeptide of the invention, e.g., SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, etc., and all polypeptides disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:3 through SEQ ID NO:1073. The stringent conditions can be highly stringent conditions, medium stringent conditions, low stringent conditions, including the high and reduced stringency conditions described herein. In alternative embodiments, nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of the molecule, e.g., an exemplary nucleic acid of the invention. For example, they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400 residues in length. Nucleic acids shorter than full length are also included. These nucleic acids are useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA (single or double stranded), antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like.

In one aspect, nucleic acids of the invention are defined by their ability to hybridize under high stringency comprises conditions of about 50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30° C. to 35° C. Alternatively, nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA). In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide. A specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of “low stringency” hybridization conditions is when the above hybridization is conducted at 10% formamide.

The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions are well known in the art. Hybridization conditions are discussed further, below.

Oligonucleotides Probes and Methods for Using Them

The invention also provides nucleic acid probes for identifying nucleic acids encoding polypeptides (e.g., enzymes) of the invention, and polypeptides having the same activity as a polypeptide of the invention. In one aspect, the probe comprises at least 10 consecutive bases of a sequence of the invention, e.g., the exemplary SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073. Alternatively, a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a sequence of the invention. The probes identify a nucleic acid by binding or hybridization. The probes can be used in arrays of the invention, see discussion below, including, e.g., capillary arrays. The probes of the invention can also be used to isolate other nucleic acids or polypeptides.

The probes of the invention can be used to determine whether a biological sample, such as a soil sample, contains an organism having a nucleic acid sequence of the invention or an organism from which the nucleic acid was obtained. In such procedures, a biological sample potentially harboring the organism from which the nucleic acid was isolated is obtained and nucleic acids are obtained from the sample. The nucleic acids are contacted with the probe under conditions which permit the probe to specifically hybridize to any complementary sequences present in the sample. Where necessary, conditions which permit the probe to specifically hybridize to complementary sequences may be determined by placing the probe in contact with complementary sequences from samples known to contain the complementary sequence, as well as control sequences which do not contain the complementary sequence. Hybridization conditions, such as the salt concentration of the hybridization buffer, the formamide concentration of the hybridization buffer, or the hybridization temperature, may be varied to identify conditions which allow the probe to hybridize specifically to complementary nucleic acids (see discussion on specific hybridization conditions).

If the sample contains the organism from which the nucleic acid was isolated, specific hybridization of the probe is then detected. Hybridization may be detected by labeling the probe with a detectable agent such as a radioactive isotope, a fluorescent dye or an enzyme capable of catalyzing the formation of a detectable product. Many methods for using the labeled probes to detect the presence of complementary nucleic acids in a sample are familiar to those skilled in the art. These include Southern Blots, Northern Blots, colony hybridization procedures, and dot blots. Protocols for each of these procedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable of specifically hybridizing to any complementary sequences which are present in the nucleic acid sample), may be used in an amplification reaction to determine whether the sample contains an organism containing a nucleic acid sequence of the invention (e.g., an organism from which the nucleic acid was isolated). In one aspect, the probes comprise oligonucleotides. In one aspect, the amplification reaction may comprise a PCR reaction. PCR protocols are described in Ausubel and Sambrook (see discussion on amplification reactions). In such procedures, the nucleic acids in the sample are contacted with the probes, the amplification reaction is performed, and any resulting amplification product is detected. The amplification product may be detected by performing gel electrophoresis on the reaction products and staining the gel with an intercalator such as ethidium bromide. Alternatively, one or more of the probes may be labeled with a radioactive isotope and the presence of a radioactive amplification product may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acid sequence of the invention can also be used in chromosome walking procedures to identify clones containing additional, e.g., genomic sequences. Such methods allow the isolation of genes which encode additional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used as probes to identify and isolate related nucleic acids. In some aspects, the so-identified related nucleic acids may be cDNAs or genomic DNAs from organisms other than the one from which the nucleic acid of the invention was first isolated. In such procedures, a nucleic acid sample is contacted with the probe under conditions which permit the probe to specifically hybridize to related sequences. Hybridization of the probe to nucleic acids from the related organism is then detected using any of the methods described above.

In nucleic acid hybridization reactions, the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter. Hybridization may be carried out under conditions of low stringency, moderate stringency or high stringency. As an example of nucleic acid hybridization, a polymer membrane containing immobilized denatured nucleic acids is first prehybridized for 30 minutes at 45° C. in a solution consisting of 0.9 M NaCl, 50 mM NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid. Approximately 2×107 cpm (specific activity 4−9×108 cpm/ug) of ³²P end-labeled oligonucleotide probe are then added to the solution. After 12-16 hours of incubation, the membrane is washed for 30 minutes at room temperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh 1×SET at Tm−10° C. for the oligonucleotide probe. The membrane is then exposed to auto-radiographic film for detection of hybridization signals.

By varying the stringency of the hybridization conditions used to identify nucleic acids, such as cDNAs or genomic DNAs, which hybridize to the detectable probe, nucleic acids having different levels of homology to the probe can be identified and isolated. Stringency may be varied by conducting the hybridization at varying temperatures below the melting temperatures of the probes. The melting temperature, Tm, is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly complementary probe. Very stringent conditions are selected to be equal to or about 5° C. lower than the Tm for a particular probe. The melting temperature of the probe may be calculated using the following exemplary formulas. For probes between 14 and 70 nucleotides in length the melting temperature (Tm) is calculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fraction G+C)−(600/N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature may be calculated using the equation: Tm=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)-(600/N) where N is the length of the probe. Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA or 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg denatured fragmented salmon sperm DNA, 50% formamide. Formulas for SSC and Denhardt's and other solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to the prehybridization solutions listed above. Where the probe comprises double stranded DNA, it is denatured before addition to the hybridization solution. The filter is contacted with the hybridization solution for a sufficient period of time to allow the probe to hybridize to cDNAs or genomic DNAs containing sequences complementary thereto or homologous thereto. For probes over 200 nucleotides in length, the hybridization may be carried out at 15-25° C. below the Tm. For shorter probes, such as oligonucleotide probes, the hybridization may be conducted at 5-10° C. below the Tm. In one aspect, hybridizations in 6×SSC are conducted at approximately 68° C. In one aspect, hybridizations in 50% formamide containing solutions are conducted at approximately 42° C. All of the foregoing hybridizations would be considered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove any non-specifically bound detectable probe. The stringency used to wash the filters can also be varied depending on the nature of the nucleic acids being hybridized, the length of the nucleic acids being hybridized, the degree of complementarity, the nucleotide sequence composition (e.g., GC v. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examples of progressively higher stringency condition washes are as follows: 2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency); 0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderate stringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between the hybridization temperature and 68° C. (high stringency); and 0.15M NaCl for 15 minutes at 72° C. (very high stringency). A final low stringency wash can be conducted in 0.1×SSC at room temperature. The examples above are merely illustrative of one set of conditions that can be used to wash filters. One of skill in the art would know that there are numerous recipes for different stringency washes.

Nucleic acids which have hybridized to the probe can be identified by autoradiography or other conventional techniques. The above procedure may be modified to identify nucleic acids having decreasing levels of homology to the probe sequence. For example, to obtain nucleic acids of decreasing homology to the detectable probe, less stringent conditions may be used. For example, the hybridization temperature may be decreased in increments of 5° C. from 68° C. to 42° C. in a hybridization buffer having a Na+ concentration of approximately 1M. Following hybridization, the filter may be washed with 2×SSC, 0.5% SDS at the temperature of hybridization. These conditions are considered to be “moderate” conditions above 50° C. and “low” conditions below 50° C. An example of “moderate” hybridization conditions is when the above hybridization is conducted at 55° C. An example of “low stringency” hybridization conditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as 6×SSC, containing formamide at a temperature of 42° C. In this case, the concentration of formamide in the hybridization buffer may be reduced in 5% increments from 50% to 0% to identify clones having decreasing levels of homology to the probe. Following hybridization, the filter may be washed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide. A specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide. A specific example of “low stringency” hybridization conditions is when the above hybridization is conducted at 10% formamide.

These probes and methods of the invention can be used to isolate nucleic acids having a sequence with at least about 99%, 98%, 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% homology to a nucleic acid sequence of the invention comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, or 500 consecutive bases thereof, and the sequences complementary thereto. Homology may be measured using an alignment algorithm, as discussed herein. For example, the homologous polynucleotides may have a coding sequence which is a naturally occurring allelic variant of one of the coding sequences described herein. Such allelic variants may have a substitution, deletion or addition of one or more nucleotides when compared to nucleic acids of the invention.

Additionally, the probes and methods of the invention may be used to isolate nucleic acids which encode polypeptides having at least about 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence identity (homology) to a polypeptide of the invention comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof as determined using a sequence alignment algorithm (e.g., such as the FASTA version 3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 program with exemplary settings as set forth herein).

Inhibiting Expression of Polypeptides

The invention further provides for nucleic acids complementary to (e.g., antisense sequences to) the nucleic acids of the invention, e.g., polypeptide-encoding nucleic acids. Antisense sequences are capable of inhibiting the transport, splicing or transcription of polypeptide-encoding genes. The inhibition can be effected through the targeting of genomic DNA or messenger RNA. The transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage. One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind polypeptide-coding gene or message, in either case preventing or inhibiting the production or function of the polypeptide. The association can be though sequence-specific hybridization. Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of polypeptide message. The oligonucleotide can have activity which causes such cleavage, such as ribozymes. The oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One may screen a pool of many different such oligonucleotides for those with the desired activity. Inhibition of polypeptide (e.g, enzyme) expression can have a variety of industrial applications. These compositions also can be expressed by the plant (e.g., a transgenic plant) or another organism (e.g., a bacterium or other microorganism transformed with an enzyme gene of the invention). The compositions of the invention for the inhibition of polypeptide (e.g., enzyme) expression (e.g., antisense, iRNA, ribozymes, antibodies) can be used as pharmaceutical compositions.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of binding message which can inhibit polypeptide activity by targeting mRNA. Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such oligonucleotides using the novel reagents of the invention. For example, gene walking/RNA mapping protocols to screen for effective antisense oligonucleotides are well known in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183, describing an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection. See also Smith (2000) Eur. J. Pharm. Sci. 11:191-198.

Naturally occurring nucleic acids are used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl)glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense enzyme sequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides for with ribozymes capable of binding polypeptide-coding message which can inhibit polypeptide activity by targeting mRNA. Strategies for designing ribozymes and selecting the polypeptide-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide. This potential advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.

The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNaseP-like RNA (in association with an RNA guide sequence). Examples of such hammerhead motifs are described by Rossi (1992) Aids Research and Human Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis delta virus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif by Guerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S. Pat. No. 4,987,071. The recitation of these specific motifs is not intended to be limiting; those skilled in the art will recognize that an enzymatic RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, a so-called “RNAi” molecule, comprising an polypeptide-encoding sequence of the invention. The RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAi can inhibit expression of polypeptide-coding gene, e.g., an enzyme-encoding gene. In one aspect, the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In one aspect, the RNAi's of the invention are used in gene-silencing therapeutics, see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one aspect, the invention provides methods to selectively degrade RNA using the RNAi's of the invention. The process may be practiced in vitro, ex vivo or in vivo. In one aspect, the RNAi molecules of the invention can be used to generate a loss-of-function mutation in a cell, an organ or an animal. Methods for making and using RNAi molecules for selectively degrade RNA are well known in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleic acids of the invention, e.g., those encoding a polypeptide. These methods can be repeated or used in various combinations to generate enzymes having an altered or different activity or an altered or different stability from that of an enzyme encoded by the template nucleic acid. These methods also can be repeated or used in various combinations, e.g., to generate variations in gene/message expression, message translation or message stability. In another aspect, the genetic composition of a cell is altered by, e.g., modification of a homologous gene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. For example, random or stochastic methods, or, non-stochastic, or “directed evolution,” methods.

Methods for random mutation of genes are well known in the art, see, e.g., U.S. Pat. No. 5,830,696. For example, mutagens can be used to randomly mutate a gene. Mutagens include, e.g., ultraviolet light or gamma irradiation, or a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated psoralens, alone or in combination, to induce DNA breaks amenable to repair by recombination. Other chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other mutagens are analogues of nucleotide precursors, e.g., nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. These agents can be added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA 89:5467-5471; or, combinatorial multiple cassette mutagenesis, see, e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleic acids, e.g., genes, can be reassembled after random, or “stochastic,” fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. In alternative aspects, modifications, additions or deletions are introduced by error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR), recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or a combination of these and other methods.

The following publications describe a variety of recursive recombination procedures and/or methods which can be incorporated into the methods of the invention: Stemmer (1999) “Molecular breeding of viruses for targeting and other clinical properties” Tumor Targeting 4:1-4; Ness (1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of a cytokine using DNA family shuffling” Nature Biotechnology 17:793-797; Minshull (1999) “Protein evolution by molecular breeding” Current Opinion in Chemical Biology 3:284-290; Christians (1999) “Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNA shuffling of a family of genes from diverse species accelerates directed evolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of an arsenate detoxification pathway by DNA shuffling,” Nature Biotechnology 15:436-438; Zhang (1997) “Directed evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening” Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNA Shuffling to Pharmaceuticals and Vaccines” Current Opinion in Biotechnology 8:724-733; Crameri et al. (1996) “Construction and evolution of antibody-phage libraries by DNA shuffling” Nature Medicine 2:100-103; Crameri et al. (1996) “Improved green fluorescent protein by molecular evolution using DNA shuffling” Nature Biotechnology 14:315-319; Gates et al. (1996) “Affinity selective isolation of ligands from peptide libraries through display on a lac repressor headpiece dimer” Journal of Molecular Biology 255:373-386; Stemmer (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCH Publishers, New York. pp. 447-457; Crameri and Stemmer (1995) “Combinatorial multiple cassette mutagenesis creates all the permutations of mutant and wildtype cassettes” BioTechniques 18:194-195; Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmid form large numbers of oligodeoxyribonucleotides” Gene, 164:49-53; Stemmer (1995) “The Evolution of Molecular Computation” Science 270: 1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology 13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNA shuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751. Mutational methods of generating diversity include, for example, site-directed mutagenesis (Ling et al. (1997) “Approaches to DNA mutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al. (1996) “Oligonucleotide-directed random mutagenesis using the phosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “In vitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985) “Strategies and applications of in vitro mutagenesis” Science 229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J. 237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directed mutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis using uracil containing templates (Kunkel (1985) “Rapid and efficient site-specific mutagenesis without phenotypic selection” Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficient site-specific mutagenesis without phenotypic selection” Methods in Enzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressors with new DNA-binding specificities” Science 242:240-245); oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982) “Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983) “Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987) “Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template” Methods in Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor et al. (1985) “The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor et al. (1985) “The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis” Nucl. Acids Res. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “The gapped duplex DNA approach to oligonucleotide-directed mutation construction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol. “Oligonucleotide-directed construction of mutations via gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations” Nucl. Acids Res. 16: 7207; and Fritz et al. (1988) “Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols used in the methods of the invention include point mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell 38:879-887), mutagenesis using repair-deficient host strains (Carter et al. (1985) “Improved oligonucleotide site-directed mutagenesis using M13 vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improved oligonucleotide-directed mutagenesis using M13 vectors” Methods in Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Use of oligonucleotides to generate large deletions” Nucl. Acids Res. 14: 5115), restriction-selection and restriction-selection and restriction-purification (Wells et al. (1986) “Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984) “Total synthesis and cloning of a gene coding for the ribonuclease S protein” Science 223: 1299-1301; Sakamar and Khorana (1988) “Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985) “Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites” Gene 34:315-323; and Grundstrom et al. (1985) “Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316), double-strand break repair (Mandecki (1986); Arnold (1993) “Protein engineering for unusual environments” Current Opinion in Biotechnology 4:450-455. “Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis” Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many of the above methods can be found in Methods in Enzymology Volume 154, which also describes useful controls for trouble-shooting problems with various mutagenesis methods.

See also U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methods for In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep. 22, 1998) “Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by Random Fragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods and Compositions for Cellular and Metabolic Engineering;” WO 95/22625, Stemmer and Crameri, “Mutagenesis by Random Fragmentation and Reassembly;” WO 96/33207 by Stemmer and Lipschutz “End Complementary Polymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methods for Generating Polynucleotides having Desired Characteristics by Iterative Selection and Recombination;” WO 97/35966 by Minshull and Stemmer, “Methods and Compositions for Cellular and Metabolic Engineering;” WO 99/41402 by Punnonen et al. “Targeting of Genetic Vaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen Library Immunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine Vector Engineering;” WO 99/41368 by Punnonen et al. “Optimization of Immunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmer and Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;” EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by Recursive Sequence Recombination;” WO 99/23107 by Stemmer et al., “Modification of Virus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 by Apt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayre et al. “Evolution of Whole Cells and Organisms by Recursive Sequence Recombination;” WO 98/27230 by Patten and Stemmer, “Methods and Compositions for Polypeptide Engineering;” WO 98/27230 by Stemmer et al., “Methods for Optimization of Gene Therapy by Recursive Sequence Shuffling and Selection,” WO 00/00632, “Methods for Generating Highly Diverse Libraries,” WO 00/09679, “Methods for Obtaining in Vitro Recombined Polynucleotide Sequence Banks and Resulting Sequences,” WO 98/42832 by Arnold et al., “Recombination of Polynucleotide Sequences Using Random or Defined Primers,” WO 99/29902 by Arnold et al., “Method for Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 by Vind, “An in Vitro Method for Construction of a DNA Library,” WO 98/41622 by Borchert et al., “Method for Constructing a Library Using DNA Shuffling,” and WO 98/42727 by Pati and Zarling, “Sequence Alterations using Homologous Recombination.”

Certain U.S. applications provide additional details regarding various diversity generating methods, including “SHUFFLING OF CODON ALTERED GENES” by Patten et al. filed Sep. 28, 1999, (U.S. Ser. No. 09/407,800); “EVOLUTION OF WHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre et al., filed Jul. 15, 1998 (U.S. Ser. No. 09/166,188), and Jul. 15, 1999 (U.S. Ser. No. 09/354,922); “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., filed Sep. 28, 1999 (U.S. Ser. No. 09/408,392), and “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID RECOMBINATION” by Crameri et al., filed Jan. 18, 2000 (PCT/US00/01203); “USE OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., filed Sep. 28, 1999 (U.S. Ser. No. 09/408,393); “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g. “METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000 (U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FOR USE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan. 18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACID TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” by Affholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549).

Non-stochastic, or “directed evolution,” methods include, e.g., saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a combination thereof are used to modify the nucleic acids of the invention to generate polypeptides with new or altered properties (e.g., activity under highly acidic or alkaline conditions, high temperatures, and the like). Polypeptides encoded by the modified nucleic acids can be screened for an activity before testing for an enzyme or other activity. Any testing modality or protocol can be used, e.g., using a capillary array platform. See, e.g., U.S. Pat. Nos. 6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

In one aspect of the invention, non-stochastic gene modification, a “directed evolution process,” is used to generate polypeptides with new or altered properties. Variations of this method have been termed “gene site-saturation mutagenesis,” “site-saturation mutagenesis,” “saturation mutagenesis” or simply “GSSM.” It can be used in combination with other mutagenization processes. See, e.g., U.S. Pat. Nos. 6,171,820; 6,238,884. In one aspect, GSSM comprises providing a template polynucleotide and a plurality of oligonucleotides, wherein each oligonucleotide comprises a sequence homologous to the template polynucleotide, thereby targeting a specific sequence of the template polynucleotide, and a sequence that is a variant of the homologous gene; generating progeny polynucleotides comprising non-stochastic sequence variations by replicating the template polynucleotide with the oligonucleotides, thereby generating polynucleotides comprising homologous gene sequence variations.

In one aspect, codon primers containing a degenerate N,N,G/T sequence are used to introduce point mutations into a polynucleotide, so as to generate a set of progeny polypeptides in which a full range of single amino acid substitutions is represented at each amino acid position, e.g., an amino acid residue in an enzyme active site or ligand binding site targeted to be modified. These oligonucleotides can comprise a contiguous first homologous sequence, a degenerate N,N,G/T sequence, and, optionally, a second homologous sequence. The downstream progeny translational products from the use of such oligonucleotides include all possible amino acid changes at each amino acid site along the polypeptide, because the degeneracy of the N,N,G/T sequence includes codons for all 20 amino acids. In one aspect, one such degenerate oligonucleotide (comprised of, e.g., one degenerate N,N,G/T cassette) is used for subjecting each original codon in a parental polynucleotide template to a full range of codon substitutions. In another aspect, at least two degenerate cassettes are used—either in the same oligonucleotide or not, for subjecting at least two original codons in a parental polynucleotide template to a full range of codon substitutions. For example, more than one N,N,G/T sequence can be contained in one oligonucleotide to introduce amino acid mutations at more than one site. This plurality of N,N,G/T sequences can be directly contiguous, or separated by one or more additional nucleotide sequence(s). In another aspect, oligonucleotides serviceable for introducing additions and deletions can be used either alone or in combination with the codons containing an N,N,G/T sequence, to introduce any combination or permutation of amino acid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In another aspect, degenerate cassettes having less degeneracy than the N,N,G/T sequence are used. For example, it may be desirable in some instances to use (e.g. in an oligonucleotide) a degenerate triplet sequence comprised of only one N, where said N can be in the first second or third position of the triplet. Any other bases including any combinations and permutations thereof can be used in the remaining two positions of the triplet. Alternatively, it may be desirable in some instances to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets) allows for systematic and easy generation of a full range of possible natural amino acids (for a total of 20 amino acids) into each and every amino acid position in a polypeptide (in alternative aspects, the methods also include generation of less than all possible substitutions per amino acid residue, or codon, position). For example, for a 100 amino acid polypeptide, 2000 distinct species (i.e. 20 possible amino acids per position X 100 amino acid positions) can be generated. Through the use of an oligonucleotide or set of oligonucleotides containing a degenerate N,N,G/T triplet, 32 individual sequences can code for all 20 possible natural amino acids. Thus, in a reaction vessel in which a parental polynucleotide sequence is subjected to saturation mutagenesis using at least one such oligonucleotide, there are generated 32 distinct progeny polynucleotides encoding 20 distinct polypeptides. In contrast, the use of a non-degenerate oligonucleotide in site-directed mutagenesis leads to only one progeny polypeptide product per reaction vessel. Nondegenerate oligonucleotides can optionally be used in combination with degenerate primers disclosed; for example, nondegenerate oligonucleotides can be used to generate specific point mutations in a working polynucleotide. This provides one means to generate specific silent point mutations, point mutations leading to corresponding amino acid changes, and point mutations that cause the generation of stop codons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel contains polynucleotides encoding at least 20 progeny polypeptide (e.g., enzyme) molecules such that all 20 natural amino acids are represented at the one specific amino acid position corresponding to the codon position mutagenized in the parental polynucleotide (other aspects use less than all 20 natural combinations). The 32-fold degenerate progeny polypeptides generated from each saturation mutagenesis reaction vessel can be subjected to clonal amplification (e.g. cloned into a suitable host, e.g., E. coli host, using, e.g., an expression vector) and subjected to expression screening. When an individual progeny polypeptide is identified by screening to display a favorable change in property (when compared to the parental polypeptide, such as increased enzyme activity under alkaline or acidic conditions), it can be sequenced to identify the correspondingly favorable amino acid substitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in a parental polypeptide using saturation mutagenesis as disclosed herein, favorable amino acid changes may be identified at more than one amino acid position. One or more new progeny molecules can be generated that contain a combination of all or part of these favorable amino acid substitutions. For example, if 2 specific favorable amino acid changes are identified in each of 3 amino acid positions in a polypeptide, the permutations include 3 possibilities at each position (no change from the original amino acid, and each of two favorable changes) and 3 positions. Thus, there are 3×3×3 or 27 total possibilities, including 7 that were previously examined—6 single point mutations (i.e. 2 at each of three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together with another stochastic or non-stochastic means to vary sequence, e.g., synthetic ligation reassembly (see below), shuffling, chimerization, recombination and other mutagenizing processes and mutagenizing agents. This invention provides for the use of any mutagenizing process(es), including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed “synthetic ligation reassembly,” or simply “SLR,” a “directed evolution process,” to generate polypeptides with new or altered properties. SLR is a method of ligating oligonucleotide fragments together non-stochastically. This method differs from stochastic oligonucleotide shuffling in that the nucleic acid building blocks are not shuffled, concatenated or chimerized randomly, but rather are assembled non-stochastically. See, e.g., U.S. patent application Ser. No. 09/332,835 entitled “Synthetic Ligation Reassembly in Directed Evolution” and filed on Jun. 14, 1999 (“U.S. Ser. No. 09/332,835”). In one aspect, SLR comprises the following steps: (a) providing a template polynucleotide, wherein the template polynucleotide comprises sequence encoding a homologous gene; (b) providing a plurality of building block polynucleotides, wherein the building block polynucleotides are designed to cross-over reassemble with the template polynucleotide at a predetermined sequence, and a building block polynucleotide comprises a sequence that is a variant of the homologous gene and a sequence homologous to the template polynucleotide flanking the variant sequence; (c) combining a building block polynucleotide with a template polynucleotide such that the building block polynucleotide cross-over reassembles with the template polynucleotide to generate polynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology between polynucleotides to be rearranged. Thus, this method can be used to non-stochastically generate libraries (or sets) of progeny molecules comprised of over 10¹⁰⁰different chimeras. SLR can be used to generate libraries comprised of over 10¹⁰⁰⁰different progeny chimeras. Thus, aspects of the present invention include non-stochastic methods of producing a set of finalized chimeric nucleic acid molecule shaving an overall assembly order that is chosen by design. This method includes the steps of generating by design a plurality of specific nucleic acid building blocks having serviceable mutually compatible ligatable ends, and assembling these nucleic acid building blocks, such that a designed overall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid building blocks to be assembled are considered to be “serviceable” for this type of ordered assembly if they enable the building blocks to be coupled in predetermined orders. Thus the overall assembly order in which the nucleic acid building blocks can be coupled is specified by the design of the ligatable ends. If more than one assembly step is to be used, then the overall assembly order in which the nucleic acid building blocks can be coupled is also specified by the sequential order of the assembly step(s). In one aspect, the annealed building pieces are treated with an enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks is obtained by analyzing a set of progenitor nucleic acid sequence templates that serve as a basis for producing a progeny set of finalized chimeric polynucleotides. These parental oligonucleotide templates thus serve as a source of sequence information that aids in the design of the nucleic acid building blocks that are to be mutagenized, e.g., chimerized or shuffled.

In one aspect of this method, the sequences of a plurality of parental nucleic acid templates are aligned in order to select one or more demarcation points. The demarcation points can be located at an area of homology, and are comprised of one or more nucleotides. These demarcation points are preferably shared by at least two of the progenitor templates. The demarcation points can thereby be used to delineate the boundaries of oligonucleotide building blocks to be generated in order to rearrange the parental polynucleotides. The demarcation points identified and selected in the progenitor molecules serve as potential chimerization points in the assembly of the final chimeric progeny molecules. A demarcation point can be an area of homology (comprised of at least one homologous nucleotide base) shared by at least two parental polynucleotide sequences. Alternatively, a demarcation point can be an area of homology that is shared by at least half of the parental polynucleotide sequences, or, it can be an area of homology that is shared by at least two thirds of the parental polynucleotide sequences. Even more preferably a serviceable demarcation points is an area of homology that is shared by at least three fourths of the parental polynucleotide sequences, or, it can be shared by at almost all of the parental polynucleotide sequences. In one aspect, a demarcation point is an area of homology that is shared by all of the parental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustively in order to generate an exhaustive library of progeny chimeric polynucleotides. In other words, all possible ordered combinations of the nucleic acid building blocks are represented in the set of finalized chimeric nucleic acid molecules. At the same time, in another embodiment, the assembly order (i.e. the order of assembly of each building block in the 5′ to 3 sequence of each finalized chimeric nucleic acid) in each combination is by design (or non-stochastic) as described above. Because of the non-stochastic nature of this invention, the possibility of unwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performed systematically. For example, the method is performed in order to generate a systematically compartmentalized library of progeny molecules, with compartments that can be screened systematically, e.g. one by one. In other words this invention provides that, through the selective and judicious use of specific nucleic acid building blocks, coupled with the selective and judicious use of sequentially stepped assembly reactions, a design can be achieved where specific sets of progeny products are made in each of several reaction vessels. This allows a systematic examination and screening procedure to be performed. Thus, these methods allow a potentially very large number of progeny molecules to be examined systematically in smaller groups. Because of its ability to perform chimerizations in a manner that is highly flexible yet exhaustive and systematic as well, particularly when there is a low level of homology among the progenitor molecules, these methods provide for the generation of a library (or set) comprised of a large number of progeny molecules. Because of the non-stochastic nature of the instant ligation reassembly invention, the progeny molecules generated preferably comprise a library of finalized chimeric nucleic acid molecules having an overall assembly order that is chosen by design. The saturation mutagenesis and optimized directed evolution methods also can be used to generate different progeny molecular species. It is appreciated that the invention provides freedom of choice and control regarding the selection of demarcation points, the size and number of the nucleic acid building blocks, and the size and design of the couplings. It is appreciated, furthermore, that the requirement for intermolecular homology is highly relaxed for the operability of this invention. In fact, demarcation points can even be chosen in areas of little or no intermolecular homology. For example, because of codon wobble, i.e. the degeneracy of codons, nucleotide substitutions can be introduced into nucleic acid building blocks without altering the amino acid originally encoded in the corresponding progenitor template. Alternatively, a codon can be altered such that the coding for an originally amino acid is altered. This invention provides that such substitutions can be introduced into the nucleic acid building block in order to increase the incidence of intermolecularly homologous demarcation points and thus to allow an increased number of couplings to be achieved among the building blocks, which in turn allows a greater number of progeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which the building blocks are generated allows the design and introduction of nucleotides (e.g., one or more nucleotides, which may be, for example, codons or introns or regulatory sequences) that can later be optionally removed in an in vitro process (e.g. by mutagenesis) or in an in vivo process (e.g. by utilizing the gene splicing ability of a host organism). It is appreciated that in many instances the introduction of these nucleotides may also be desirable for many other reasons in addition to the potential benefit of creating a serviceable demarcation point.

In one aspect, a nucleic acid building block is used to introduce an intron. Thus, functional introns are introduced into a man-made gene manufactured according to the methods described herein. The artificially introduced intron(s) can be functional in a host cells for gene splicing much in the way that naturally-occurring introns serve functionally in gene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed “optimized directed evolution system” to generate polypeptides with new or altered properties. Optimized directed evolution is directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of nucleic acids through recombination. Optimized directed evolution allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence. This method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.

In addition, this method provides a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems. Previously, if one generated, for example, 10¹³chimeric molecules during a reaction, it would be extremely difficult to test such a high number of chimeric variants for a particular activity. Moreover, a significant portion of the progeny population would have a very high number of crossover events which resulted in proteins that were less likely to have increased levels of a particular activity. By using these methods, the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events. Thus, although one can still generate 10¹³chimeric molecules during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events. Because the resulting progeny population can be skewed to have a predetermined number of crossover events, the boundaries on the functional variety between the chimeric molecules is reduced. This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is to create oligonucleotides corresponding to fragments or portions of each parental sequence. Each oligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. Additional information can also be found in U.S. Ser. No. 09/332,835. The number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created. For example, three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature. As one example, a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences. The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a ⅓ chance (assuming 3 parents) that an oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined to predict the population of crossover events that are likely to occur during each step in a ligation reaction given a set number of parental variants, a number of oligonucleotides corresponding to each variant, and the concentrations of each variant during each step in the ligation reaction. The statistics and mathematics behind determining the PDF is described below. By utilizing these methods, one can calculate such a probability density function, and thus enrich the chimeric progeny population for a predetermined number of crossover events resulting from a particular ligation reaction. Moreover, a target number of crossover events can be predetermined, and the system then programmed to calculate the starting quantities of each parental oligonucleotide during each step in the ligation reaction to result in a probability density function that centers on the predetermined number of crossover events. These methods are directed to the use of repeated cycles of reductive reassortment, recombination and selection that allow for the directed molecular evolution of a nucleic acid encoding an polypeptide through recombination. This system allows generation of a large population of evolved chimeric sequences, wherein the generated population is significantly enriched for sequences that have a predetermined number of crossover events. A crossover event is a point in a chimeric sequence where a shift in sequence occurs from one parental variant to another parental variant. Such a point is normally at the juncture of where oligonucleotides from two parents are ligated together to form a single sequence. The method allows calculation of the correct concentrations of oligonucleotide sequences so that the final chimeric population of sequences is enriched for the chosen number of crossover events. This provides more control over choosing chimeric variants having a predetermined number of crossover events.

In addition, these methods provide a convenient means for exploring a tremendous amount of the possible protein variant space in comparison to other systems. By using the methods described herein, the population of chimerics molecules can be enriched for those variants that have a particular number of crossover events. Thus, although one can still generate 10¹³chimeric molecules during a reaction, each of the molecules chosen for further analysis most likely has, for example, only three crossover events. Because the resulting progeny population can be skewed to have a predetermined number of crossover events, the boundaries on the functional variety between the chimeric molecules is reduced. This provides a more manageable number of variables when calculating which oligonucleotide from the original parental polynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotide sequence by creating oligonucleotides corresponding to fragments or portions of each parental sequence. Each oligonucleotide preferably includes a unique region of overlap so that mixing the oligonucleotides together results in a new variant that has each oligonucleotide fragment assembled in the correct order. See also U.S. Ser. No. 09/332,835.

The number of oligonucleotides generated for each parental variant bears a relationship to the total number of resulting crossovers in the chimeric molecule that is ultimately created. For example, three parental nucleotide sequence variants might be provided to undergo a ligation reaction in order to find a chimeric variant having, for example, greater activity at high temperature. As one example, a set of 50 oligonucleotide sequences can be generated corresponding to each portions of each parental variant. Accordingly, during the ligation reassembly process there could be up to 50 crossover events within each of the chimeric sequences. The probability that each of the generated chimeric polynucleotides will contain oligonucleotides from each parental variant in alternating order is very low. If each oligonucleotide fragment is present in the ligation reaction in the same molar quantity it is likely that in some positions oligonucleotides from the same parental polynucleotide will ligate next to one another and thus not result in a crossover event. If the concentration of each oligonucleotide from each parent is kept constant during any ligation step in this example, there is a ⅓ chance (assuming 3 parents) that a oligonucleotide from the same parental variant will ligate within the chimeric sequence and produce no crossover.

Determining Crossover Events

Embodiments of the invention include a system and software that receive a desired crossover probability density function (PDF), the number of parent genes to be reassembled, and the number of fragments in the reassembly as inputs. The output of this program is a “fragment PDF” that can be used to determine a recipe for producing reassembled genes, and the estimated crossover PDF of those genes. The processing described herein is preferably performed in MATLAB® (The Mathworks, Natick, Mass.) a programming language and development environment for technical computing.

Iterative Processes

In practicing the invention, these processes can be iteratively repeated. For example a nucleic acid (or, the nucleic acid) responsible for an altered polypeptide (e.g., enzyme) phenotype is identified, re-isolated, again modified, re-tested for activity. This process can be iteratively repeated until a desired phenotype is engineered. For example, an entire biochemical anabolic or catabolic pathway can be engineered into a cell, including enzyme activity.

Similarly, if it is determined that a particular oligonucleotide has no affect at all on the desired trait (e.g., a new enzyme phenotype), it can be removed as a variable by synthesizing larger parental oligonucleotides that include the sequence to be removed. Since incorporating the sequence within a larger sequence prevents any crossover events, there will no longer be any variation of this sequence in the progeny polynucleotides. This iterative practice of determining which oligonucleotides are most related to the desired trait, and which are unrelated, allows more efficient exploration all of the possible protein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention that provide variants of polypeptides of the invention, e.g., antibodies, enzymes, and the like. In vivo shuffling can be performed utilizing the natural property of cells to recombine multimers. While recombination in vivo has provided the major natural route to molecular diversity, genetic recombination remains a relatively complex process that involves 1) the recognition of homologies; 2) strand cleavage, strand invasion, and metabolic steps leading to the production of recombinant chiasma; and finally 3) the resolution of chiasma into discrete recombined molecules. The formation of the chiasma requires the recognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybrid polynucleotide from at least a first polynucleotide and a second polynucleotide. The invention can be used to produce a hybrid polynucleotide by introducing at least a first polynucleotide and a second polynucleotide which share at least one region of partial sequence homology into a suitable host cell. The regions of partial sequence homology promote processes which result in sequence reorganization producing a hybrid polynucleotide. The term “hybrid polynucleotide”, as used herein, is any nucleotide sequence which results from the method of the present invention and contains sequence from at least two original polynucleotide sequences. Such hybrid polynucleotides can result from intermolecular recombination events which promote sequence integration between DNA molecules. In addition, such hybrid polynucleotides can result from intramolecular reductive reassortment processes which utilize repeated sequences to alter a nucleotide sequence within a DNA molecule.

Producing Sequence Variants

The invention also provides methods of making sequence variants of the nucleic acid and polypeptide sequences of the invention or isolating polypeptides, e.g., enzymes, sequence variants using the nucleic acids and polypeptides of the invention. In one aspect, the invention provides for variants of an polypeptide-encoding gene of the invention, which can be altered by any means, including, e.g., random or stochastic methods, or, non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also be created in vitro. Variants may be created using genetic engineering techniques such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives may be created using chemical synthesis or modification procedures. Other methods of making variants are also familiar to those skilled in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids which encode polypeptides having characteristics which enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. These nucleotide differences can result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In error prone PCR, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. Error prone PCR is described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992. Briefly, in such procedures, nucleic acids to be mutagenized are mixed with PCR primers, reaction buffer, MgCl2, MnCl2, Taq polymerase and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction may be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 110 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciated that these parameters may be varied as appropriate. The mutagenized nucleic acids are cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids is evaluated.

Variants may also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized. Clones containing the mutagenized DNA are recovered and the activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, e.g., U.S. Pat. No. 5,965,408.

Still another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different but highly related DNA sequence in vitro, as a result of random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction. Sexual PCR mutagenesis is described, e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, in such procedures a plurality of nucleic acids to be recombined are digested with DNase to generate fragments having an average size of 50-200 nucleotides. Fragments of the desired average size are purified and resuspended in a PCR mixture. PCR is conducted under conditions which facilitate recombination between the nucleic acid fragments. For example, PCR may be performed by resuspending the purified fragments at a concentration of 10-30 ng/ul in a solution of 0.2 mM of each dNTP, 2.2 mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5 units of Taq polymerase per 100:1 of reaction mixture is added and PCR is performed using the following regime: 94° C. for 60 seconds, 94° C. for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45 times) and 72° C. for 5 minutes. However, it will be appreciated that these parameters may be varied as appropriate. In some aspects, oligonucleotides may be included in the PCR reactions. In other aspects, the Klenow fragment of DNA polymerase I may be used in a first set of PCR reactions and Taq polymerase may be used in a subsequent set of PCR reactions. Recombinant sequences are isolated and the activities of the polypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some embodiments, random mutations in a sequence of interest are generated by propagating the sequence of interest in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways. Such “mutator” strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for in vivo mutagenesis are described, e.g., in PCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassette mutagenesis a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide “cassette” that differs from the native sequence. The oligonucleotide often contains completely and/or partially randomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.

In some embodiments, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described, e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random and site-directed mutagenesis are described, e.g., in Arnold (1993) Current Opinion in Biotechnology 4:450-455.

In some embodiments, the variants are created using shuffling procedures wherein portions of a plurality of nucleic acids which encode distinct polypeptides are fused together to create chimeric nucleic acid sequences which encode chimeric polypeptides as described in, e.g., U.S. Pat. Nos. 5,965,408; 5,939,250.

The invention also provides variants of polypeptides of the invention comprising sequences in which one or more of the amino acid residues (e.g., of an exemplary polypeptide of the invention) are substituted with a conserved or non-conserved amino acid residue (e.g., a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Thus, polypeptides of the invention include those with conservative substitutions of sequences of the invention, including but not limited to the following replacements: replacements of an aliphatic amino acid such as Alanine, Valine, Leucine and Isoleucine with another aliphatic amino acid; replacement of a Serine with a Threonine or vice versa; replacement of an acidic residue such as Aspartic acid and Glutamic acid with another acidic residue; replacement of a residue bearing an amide group, such as Asparagine and Glutamine, with another residue bearing an amide group; exchange of a basic residue such as Lysine and Arginine with another basic residue; and replacement of an aromatic residue such as Phenylalanine, Tyrosine with another aromatic residue. Other variants are those in which one or more of the amino acid residues of the polypeptides of the invention includes a substituent group.

Other variants within the scope of the invention are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide, for example, polyethylene glycol.

Additional variants within the scope of the invention are those in which additional amino acids are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.

In some aspects, the variants, fragments, derivatives and analogs of the polypeptides of the invention retain the same biological function or activity as the exemplary polypeptides, e.g., an enzyme activity, as described herein. In other aspects, the variant, fragment, derivative, or analog includes a proprotein, such that the variant, fragment, derivative, or analog can be activated by cleavage of the proprotein portion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in Host Cells

The invention provides methods for modifying polypeptide-encoding nucleic acids to modify codon usage. In one aspect, the invention provides methods for modifying codons in a nucleic acid encoding a polypeptide to increase or decrease its expression in a host cell. The invention also provides nucleic acids encoding a polypeptide modified to increase its expression in a host cell, polypeptides so modified, and methods of making the modified polypeptides. The method comprises identifying a “non-preferred” or a “less preferred” codon in polypeptide-encoding nucleic acid and replacing one or more of these non-preferred or less preferred codons with a “preferred codon” encoding the same amino acid as the replaced codon and at least one non-preferred or less preferred codon in the nucleic acid has been replaced by a preferred codon encoding the same amino acid. A preferred codon is a codon over-represented in coding sequences in genes in the host cell and a non-preferred or less preferred codon is a codon under-represented in coding sequences in genes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes and vectors of the invention include bacteria, yeast, fungi, plant cells, insect cells and mammalian cells. Thus, the invention provides methods for optimizing codon usage in all of these cells, codon-altered nucleic acids and polypeptides made by the codon-altered nucleic acids. Exemplary host cells include gram negative bacteria, such as Escherichia coli and Pseudomonas fluorescens; gram positive bacteria, such as Streptomyces diversa, Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis. Exemplary host cells also include eukaryotic organisms, e.g., various yeast, such as Saccharomyces sp., including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and mammalian cells and cell lines and insect cells and cell lines. Thus, the invention also includes nucleic acids and polypeptides optimized for expression in these organisms and species.

For example, the codons of a nucleic acid encoding an polypeptide isolated from a bacterial cell are modified such that the nucleic acid is optimally expressed in a bacterial cell different from the bacteria from which the polypeptide was derived, a yeast, a fungi, a plant cell, an insect cell or a mammalian cell. Methods for optimizing codons are well known in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188; Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect. Immun. 69:7250-7253, describing optimizing codons in mouse systems; Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizing codons in yeast; Feng (2000) Biochemistry 39:15399-15409, describing optimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif. 20:252-264, describing optimizing codon usage that affects secretion in E. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleic acid, a polypeptide, an expression cassette or vector or a transfected or transformed cell of the invention. The transgenic non-human animals can be, e.g., goats, rabbits, sheep, pigs, cows, rats and mice, comprising the nucleic acids of the invention. These animals can be used, e.g., as in vivo models to study polypeptide activity, or, as models to screen for modulators of polypeptide activity in vivo. The coding sequences for the polypeptides to be expressed in the transgenic non-human animals can be designed to be constitutive, or, under the control of tissue-specific, developmental-specific or inducible transcriptional regulatory factors. Transgenic non-human animals can be designed and generated using any method known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and using transformed cells and eggs and transgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods 231:147-157, describing the production of recombinant proteins in the milk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating the production of transgenic goats. U.S. Pat. No. 6,211,428, describes making and using transgenic non-human mammals which express in their brains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No. 5,387,742, describes injecting cloned recombinant or synthetic DNA sequences into fertilized mouse eggs, implanting the injected eggs in pseudo-pregnant females, and growing to term transgenic mice whose cells express proteins related to the pathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describes making and using a transgenic mouse whose genome comprises a disruption of the gene encoding amyloid precursor protein (APP).

“Knockout animals” can also be used to practice the methods of the invention. For example, in one aspect, the transgenic or modified animals of the invention comprise a “knockout animal,” e.g., a “knockout mouse,” engineered not to express or to be unable to express a polypeptide.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleic acid, a polypeptide (e.g., an enzyme), an expression cassette or vector or a transfected or transformed cell of the invention. The invention also provides plant products, e.g., oils, seeds, leaves, extracts and the like, comprising a nucleic acid and/or a polypeptide (e.g., an enzyme) of the invention. The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). The invention also provides methods of making and using these transgenic plants and seeds. The transgenic plant or plant cell expressing a polypeptide of the invention may be constructed in accordance with any method known in the art. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can be introduced into a plant cell by any means. For example, nucleic acids or expression constructs can be introduced into the genome of a desired plant host, or, the nucleic acids or expression constructs can be episomes. Introduction into the genome of a desired plant can be such that the host's enzyme production is regulated by endogenous transcriptional or translational control elements. The invention also provides “knockout plants” where insertion of gene sequence by, e.g., homologous recombination, has disrupted the expression of the endogenous gene. Means to generate “knockout” plants are well-known in the art, see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao (1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traits on essentially any plant, e.g., on oil-seed containing plants, such as soybeans, rapeseed, sunflower seeds, sesame and peanuts. Nucleic acids of the invention can be used to manipulate metabolic pathways of a plant in order to optimize or alter host's expression of a polypeptide (e.g., an enzyme). This can change the polypeptide's activity (e.g., enzyme activity) in a plant. Alternatively, polypeptides of the invention can be used in production of a transgenic plant to produce a compound not naturally produced by that plant. This can lower production costs or create a novel product.

In one aspect, the first step in production of a transgenic plant involves making an expression construct for expression in a plant cell. These techniques are well known in the art. They can include selecting and cloning a promoter, a coding sequence for facilitating efficient binding of ribosomes to mRNA and selecting the appropriate gene terminator sequences. One exemplary constitutive promoter is CaMV35S, from the cauliflower mosaic virus, which generally results in a high degree of expression in plants. Other promoters are more specific and respond to cues in the plant's internal or external environment. An exemplary light-inducible promoter is the promoter from the cab gene, encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greater expression in a plant cell. For example, a sequence of the invention is likely to have a higher percentage of A-T nucleotide pairs compared to that seen in a plant, some of which prefer G-C nucleotide pairs. Therefore, A-T nucleotides in the coding sequence can be substituted with G-C nucleotides without significantly changing the amino acid sequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order to identify plant cells or tissues that have successfully integrated the transgene. This may be necessary because achieving incorporation and expression of genes in plant cells is a rare event, occurring in just a few percent of the targeted tissues or cells. Selectable marker genes encode proteins that provide resistance to agents that are normally toxic to plants, such as antibiotics or herbicides. Only plant cells that have integrated the selectable marker gene will survive when grown on a medium containing the appropriate antibiotic or herbicide. As for other inserted genes, marker genes also require promoter and termination sequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporating sequences of the invention and, optionally, marker genes into a target expression construct (e.g., a plasmid), along with positioning of the promoter and the terminator sequences. This can involve transferring the modified gene into the plant through a suitable method. For example, a construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the constructs can be introduced directly to plant tissue using ballistic methods, such as DNA particle bombardment. For example, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use of particle bombardment to introduce transgenes into wheat; and Adam (1997) supra, for use of particle bombardment to introduce YACs into plant cells. For example, Rinehart (1997) supra, used particle bombardment to generate transgenic cotton plants. Apparatus for accelerating particles is described U.S. Pat. No. 5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000 particle acceleration instrument; see also, John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describing particle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with nucleic acids, e.g., an expression construct. Although plant regeneration from protoplasts is not easy with cereals, plant regeneration is possible in legumes using somatic embryogenesis from protoplast derived callus. Organized tissues can be transformed with naked DNA using gene gun technique, where DNA is coated on tungsten microprojectiles, shot 1/100th the size of cells, which carry the DNA deep into cells and organelles. Transformed tissue is then induced to regenerate, usually by somatic embryogenesis. This technique has been successful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in to plant cells using recombinant viruses. Plant cells can be transformed using viral vectors, such as, e.g., tobacco mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use of viral replicons for the expression of genes in plants,” Mol. Biotechnol. 5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well described in the scientific literature. See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell is contained in the bacterial chromosome as well as in another structure known as a Ti (tumor-inducing) plasmid. The Ti plasmid contains a stretch of DNA termed T-DNA (˜20 kb long) that is transferred to the plant cell in the infection process and a series of vir (virulence) genes that direct the infection process. A. tumefaciens can only infect a plant through wounds: when a plant root or stem is wounded it gives off certain chemical signals, in response to which, the vir genes of A. tumefaciens become activated and direct a series of events necessary for the transfer of the T-DNA from the Ti plasmid to the plant's chromosome. The T-DNA then enters the plant cell through the wound. One speculation is that the T-DNA waits until the plant DNA is being replicated or transcribed, then inserts itself into the exposed plant DNA. In order to use A. tumefaciens as a transgene vector, the tumor-inducing section of T-DNA have to be removed, while retaining the T-DNA border regions and the vir genes. The transgene is then inserted between the T-DNA border regions, where it is transferred to the plant cell and becomes integrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plants using the nucleic acids of the invention, including important cereals, see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148, discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stable integration of a DNA comprising a gene that is functional in a cell of a cereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration of whole plants capable of transmitting the incorporated target gene to the next generation. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants from transgenic tissues such as immature embryos, they can be grown under controlled environmental conditions in a series of media containing nutrients and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenic plants, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. Since transgenic expression of the nucleic acids of the invention leads to phenotypic changes, plants comprising the recombinant nucleic acids of the invention can be sexually crossed with a second plant to obtain a final product. Thus, the seed of the invention can be derived from a cross between two transgenic plants of the invention, or a cross between a plant of the invention and another plant. The desired effects (e.g., expression of the polypeptides of the invention to produce a plant in which flowering behavior is altered) can be enhanced when both parental plants express the polypeptides (e.g., an enzyme) of the invention. The desired effects can be passed to future plant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in or inserted in any plant or seed. Transgenic plants of the invention can be dicotyledonous or monocotyledonous. Examples of monocot transgenic plants of the invention are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples of dicot transgenic plants of the invention are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana. Thus, the transgenic plants and seeds of the invention include a broad range of plants, including, but not limited to, species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

In alternative embodiments, the nucleic acids of the invention are expressed in plants (e.g., as transgenic plants), such as oil-seed containing plants, e.g., soybeans, rapeseed, sunflower seeds, sesame and peanuts. The nucleic acids of the invention can be expressed in plants which contain fiber cells, including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and flax. In alternative embodiments, the transgenic plants of the invention can be members of the genus Gossypium, including members of any Gossypium species, such as G. arboreum; G. herbaceum, G. barbadense, and G. hirsutum.

The invention also provides for transgenic plants to be used for producing large amounts of the polypeptides (e.g., an enzyme or antibody) of the invention. For example, see Palmgren (1997) Trends Genet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producing human milk protein beta-casein in transgenic potato plants using an auxin-inducible, bidirectional mannopine synthase (mas 1′,2′) promoter with Agrobacterium tumefaciens-mediated leaf disc transformation methods).

Using known procedures, one of skill can screen for plants of the invention by detecting the increase or decrease of transgene mRNA or protein in transgenic plants. Means for detecting and quantitation of mRNAs or proteins are well known in the art.

Polypeptides and Peptides

The invention provides isolated or recombinant polypeptides having a sequence identity (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity) to an exemplary sequence of the invention, e.g., SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, and all polypeptides disclosed in the SEQ ID listing, which include all odd numbered SEQ ID NO:s from SEQ ID NO:3 through SEQ ID NO:1073. As discussed above, the identity can be over the full length of the polypeptide, or, the identity can be over a subsequence thereof, e.g., a region of at least about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more residues. Polypeptides of the invention can also be shorter than the full length of exemplary polypeptides. In alternative embodiment, the invention provides polypeptides (peptides, fragments) ranging in size between about 5 and the full length of a polypeptide, e.g., an enzyme, of the invention; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400 or more residues, e.g., contiguous residues of the exemplary polypeptide of the invention. Peptides of the invention can be useful as, e.g., labeling probes, antigens, toleragens, motifs, enzyme active sites.

In one aspect, the polypeptides, e.g., enzymes, of the invention are active at a high and/or at a low temperature, or, over a wide range of temperature, e.g., they can be active in the temperatures ranging between 20° C. to 90° C., between 30° C. to 80° C., or between 40° C. to 70° C. The invention also provides polypeptides, e.g., enzymes, having activity at alkaline pHs or at acidic pHs, e.g., low water acidity. In alternative aspects, the polypeptides, e.g., enzymes, of the invention can have activity in acidic pHs as low as pH 6.5, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0 and pH 3.5. In alternative aspects, the polypeptides, e.g., enzymes, of the invention can have activity in alkaline pHs as high as pH 7.5, pH 8.0, pH 8.5, pH 9.0, and pH 9.5. In one aspect, the polypeptide, e.g., enzymes, of the invention are active in the temperature range of between about 40° C. to about 70° C. under conditions of low water activity (low water content).

The invention also provides methods for further modifying the exemplary polypeptides of the invention to generate polypeptide (e.g., enzymes) with desirable properties. For example, enzymes generated by the methods of the invention can have altered substrate specificities, substrate binding specificities, substrate cleavage patterns, thermal stability, pH/activity profile, pH/stability profile (such as increased stability at low, e.g. pH<6 or pH<5, or high, e.g. pH>9, pH values), stability towards oxidation, Ca²⁺ dependency, specific activity and the like. The invention provides for altering any property of interest. For instance, the alteration may result in a variant which, as compared to a parent enzyme, has altered pH and temperature activity profile.

Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa. For example, peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol. 289:3□13) and automated synthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

The peptides and polypeptides of the invention can also be glycosylated. The glycosylation can be added post-translationally either chemically or by cellular biosynthetic mechanisms, wherein the later incorporates the use of known glycosylation motifs, which can be native to the sequence or can be added as a peptide or added in the nucleic acid coding sequence. The glycosylation can be O-linked or N-linked.

The peptides and polypeptides of the invention, as defined above, include all “mimetic” and “peptidomimetic” forms. The terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. As with polypeptides of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, in one aspect, a mimetic composition is within the scope of the invention if it has the same activity as a polypeptide of the invention.

Polypeptide mimetic compositions of the invention can contain any combination of non-natural structural components. In alternative aspect, mimetic compositions of the invention include one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide of the invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH2-NH), ethylene, olefin (CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by, e.g., non-carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R′—N—C—N—R′) such as, e.g., 1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile derivative (e.g., containing the CN-moiety in place of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues. Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but also can be referred to as the R— or S— form.

The invention also provides methods for modifying the polypeptides of the invention by either natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc), or by chemical modification techniques, and the resulting modified polypeptides. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. See, e.g., Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used to synthesize the polypeptide or fragments of the invention. Such method have been known in the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recently been employed in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally utilized the teachings of H. M. Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing peptides upon the tips of a multitude of “rods” or “pins” all of which are connected to a single plate. When such a system is utilized, a plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the pin's or rod's tips. By repeating such a process step, i.e., inverting and inserting the rod's and pin's tips into appropriate solutions, amino acids are built into desired peptides. In addition, a number of available FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an Applied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques.

Enzymes

The invention provides novel enzymes, nucleic acids encoding them, antibodies that bind them, peptides representing the enzyme's antigenic sites (epitopes) and active sites, and methods for making and using them. In one aspect, polypeptides of the invention have an enzyme activity, as described herein, see, e.g., Table 3. In alternative aspects, the enzymes of the invention have activities that have been modified from those of the exemplary enzymes described herein. The invention includes enzymes with and without signal sequences and the signal sequences themselves. The invention includes immobilized enzymes, anti-enzyme antibodies and fragments thereof. The invention includes heterocomplexes, e.g., fusion proteins, heterodimers, etc., comprising the enzymes of the invention.

Any of the many enzyme activity assays known in the art can be used to determine if a polypeptide has an enzyme activity and is within the scope of the invention. Routine protocols for determining enzyme activities are well known in the art.

Determining peptides representing the enzyme's antigenic sites (epitopes), active sites, binding sites, signal sequences, and the like can be done by routine screening protocols.

The enzymes of the invention are highly selective catalysts. As with other enzymes, they catalyze reactions with exquisite stereo-, regio-, and chemo-selectivities that are unparalleled in conventional synthetic chemistry. Moreover, the enzymes of the invention are remarkably versatile. They can be tailored to function in organic solvents, operate at extreme pHs (for example, high pHs and low pHs) extreme temperatures (for example, high temperatures and low temperatures), extreme salinity levels (for example, high salinity and low salinity), and catalyze reactions with compounds that are structurally unrelated to their natural, physiological substrates. Enzymes of the invention can be designed to be reactive toward a wide range of natural and unnatural substrates, thus enabling the modification of virtually any organic lead compound. Enzymes of the invention can also be designed to be highly enantio- and regio-selective. The high degree of functional group specificity exhibited by these enzymes enables one to keep track of each reaction in a synthetic sequence leading to a new active compound. Enzymes of the invention can also be designed to catalyze many diverse reactions unrelated to their native physiological function in nature.

The present invention exploits the unique catalytic properties of enzymes. Whereas the use of biocatalysts (i.e., purified or crude enzymes, non-living or living cells) in chemical transformations normally requires the identification of a particular biocatalyst that reacts with a specific starting compound. The present invention uses selected biocatalysts, i.e., the enzymes of the invention, and reaction conditions that are specific for functional groups that are present in many starting compounds. Each biocatalyst is specific for one functional group, or several related functional groups, and can react with many starting compounds containing this functional group. The biocatalytic reactions produce a population of derivatives from a single starting compound. These derivatives can be subjected to another round of biocatalytic reactions to produce a second population of derivative compounds. Thousands of variations of the original compound can be produced with each iteration of biocatalytic derivatization.

Enzymes react at specific sites of a starting compound without affecting the rest of the molecule, a process that is very difficult to achieve using traditional chemical methods. This high degree of biocatalytic specificity provides the means to identify a single active enzyme within a library. The library is characterized by the series of biocatalytic reactions used to produce it, a so-called “biosynthetic history”. Screening the library for biological activities and tracing the biosynthetic history identifies the specific reaction sequence producing the active compound. The reaction sequence is repeated and the structure of the synthesized compound determined. This mode of identification, unlike other synthesis and screening approaches, does not require immobilization technologies, and compounds can be synthesized and tested free in solution using virtually any type of screening assay. It is important to note, that the high degree of specificity of enzyme reactions on functional groups allows for the “tracking” of specific enzymatic reactions that make up the biocatalytically produced library.

The invention also provides methods of discovering new enzymes using the nucleic acids, polypeptides and antibodies of the invention. In one aspect, lambda phage libraries are screened for expression-based discovery of enzymes. Use of lambda phage libraries in screening allows detection of toxic clones; improved access to substrate; reduced need for engineering a host, by-passing the potential for any bias resulting from mass excision of the library; and, faster growth at low clone densities. Screening of lambda phage libraries can be in liquid phase or in solid phase. Screening in liquid phase gives greater flexibility in assay conditions; additional substrate flexibility; higher sensitivity for weak clones; and ease of automation over solid phase screening.

Many of the procedural steps are performed using robotic automation enabling the execution of many thousands of biocatalytic reactions and screening assays per day as well as ensuring a high level of accuracy and reproducibility (see discussion of arrays, below). As a result, a library of derivative compounds can be produced in a matter of weeks. For further teachings on modification of molecules, including small molecules, see PCT/US94/09174.

Signal Sequences, Prepro Domains and Catalytic Domains

The invention provides signal sequences (e.g., signal peptides (SPs)), prepro domains, and catalytic domains (CDs). The invention provides nucleic acids encoding these catalytic domains (CDs) and signal sequences (SPs, e.g., a peptide having a sequence comprising/consisting of amino terminal residues of a polypeptide of the invention). In one aspect, the invention provides a signal sequence comprising a peptide comprising/consisting of a sequence as set forth in residues 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32 or 1 to 33 of a polypeptide of the invention, e.g., SEQ ID NO:3, SEQ NO:5, etc. as set forth in the SEQ ID listing.

Exemplary signal sequences are set forth as follows (e.g., SEQ ID NO:1038 encodes signal peptide SEQ ID NO:1039, etc.):

SEQ ID NO:
Signalp Prediction
Signal Sequence

1038, 1039
Cleavage site:
AA1 28 AA2 29
MIELLLYGLFYILGIFLISFFASHSYSA

Probability:
0.824

108, 109
Cleavage site:
AA1 36 AA2 37
MDILKYFKLLLAAILSILIILIIFKLLYYIAFLL

Probability:
0.674
YT

146, 147
Cleavage site:
AA1 34 AA2 35
MDRSIELSLHLIVYAILSLLVLSLVFMFFSKS

Probability:
0.876
QS

172, 173
Cleavage site:
AA1 24 AA2 25
MKRTIILLLSALALGVALSQIAFL

Probability:
1

196, 197
Cleavage site:
AA1 25 AA2 26
MQRIIPFLFLILVLVFLSLIPKSCK

Probability:
0.599

202, 203
Cleavage site:
AA1 67 AA2 68
MHYRRAMSITFKNSKHKRNMRRSIELPISVV

Probability:
1
LFAIIGLMVFSILLGFIPKVLGSLLGGFMSSVS

ATK

220, 221
Cleavage site:
AA1 27 AA2 28
MNSKDILIYASISSLLLMSFILFQTHR

Probability:
0.524

270, 271
Cleavage site:
AA1 30 AA2 31
MRGAISTIVYILIGLIGAAFSLILMNSVFE

Probability:
0.759

274, 275
Cleavage site:
AA1 29 AA2 30
MFRFSKMISWLGSLIILIMFLGVVSIVFN

Probability:
0.998

276, 277
Cleavage site:
AA1 39 AA2 40
MRALSNIIWLISAIVVALMSIAIISFSFFKTVN

Probability:
0.999
PLAALS

282, 283
Cleavage site:
AA1 21 AA2 22
MFFIFKIFRLLFALPLIMLLA

Probability:
0.889

308, 309
Cleavage site:
AA1 38 AA2 39
MIAINYMNLEMRDVVLGLVFVIAMAVAAVI

Probability:
0.973
GAPSLALA

310, 311
Cleavage site:
AA1 38 AA2 39
MAQTKTKTQIRLKMLEKIEKYKEPVFLILLF

Probability:
0.796
LSGFLFK

318, 319
Cleavage site:
AA1 28 AA2 29
MEKKVLIAIPLLLSVGFIFYYFSPPSNN

Probability:
0.962

322, 323
Cleavage site:
AA1 33 AA2 34
MKLPILILLAVVLIIVFFILLPFIPYIATGAVIA

Probability:
0.978

328, 329
Cleavage site:
AA1 25 AA2 26
MDYNIFINIVLSSFVVALASSLVTV

Probability:
0.782

378, 379
Cleavage site:
AA1 19 AA2 20
MLPLILILLSALFSSYETA

Probability:
0.953

390, 391
Cleavage site:
AA1 24 AA2 25
MFALIIQLSSYALAFILSPLFVLS

Probability:
0.876

394, 395
Cleavage site:
AA1 18 AA2 19
MKSLIAFIAFIITGFLAT

Probability:
0.567

430, 431
Cleavage site:
AA1 31 AA2 32
MDLALASALAIGLAAFGSAIAQGLAASAAA

Probability:
1
A

440, 441
Cleavage site:
AA1 20 AA2 21
MRKLLSLPLITFFVLGLSVG

Probability:
0.864

468, 469
Cleavage site:
AA1 20 AA2 21
MKKMFVPLMAAMPFLAIGLA

Probability:
0.997

488, 489
Cleavage site:
AA1 25 AA2 26
MRRALESVNYLILLALSLFIALFVA

Probability:
0.954

500, 501
Cleavage site:
AA1 19 AA2 20
MILLLLLPLALSAVVYTNT

Probability:
0.999

506, 507
Cleavage site:
AA1 28 AA2 29
MTLNMKNLRKKVWLFSLILLGIVLVFLS

Probability:
0.95

524, 525
Cleavage site:
AA1 22 AA2 23
MKTRFTLLLSLLLLNSIPIAIS

Probability:
0.997

534, 535
Cleavage site:
AA1 16 AA2 17
MKKVFLSLLLVSTAFA

Probability:
0.997

546, 547
Cleavage site:
AA1 34 AA2 35
MFGFYFKVYGALALLIAIALFAGYLFIDPNT

Probability:
0.795
REK

550, 551
Cleavage site:
AA1 30 AA2 31
MNKSDIVILAFFFVSLGLGILTLLPANTVK

Probability:
0.907

58, 59
Cleavage site:
AA1 22 AA2 23
MKAKKLLIFVPLLLLPLLTLLM

Probability:
0.608

586, 587
Cleavage site:
AA1 24 AA2 25
MKTLAKAQVVLASGMLLAGAVAQN

Probability:
0.998

608, 609
Cleavage site:
AA1 26 AA2 27
MRAFYSIALFSILTLISLLIAHVLIT

Probability:
0.972

64, 65
Cleavage site:
AA1 55 AA2 56
MMNVIDRIMRKIPLPKKFVYKIEEKAKNYIIK

Probability:
0.984
EGPKLAPKLASVLTILAGAGLAMA

646, 647
Cleavage site:
AA1 36 AA2 37
MSVKKWQEQLKNFLNNLDKHSILIGVAAGII

Probability:
0.974
LAVSA

66, 67
Cleavage site:
AA1 23 AA2 24
MLRLIILFLTIIGALAINVNVYS

Probability:
0.986

70, 71
Cleavage site:
AA1 16 AA2 17
MKRFLPAVVVLGLALA

Probability:
1

718, 719
Cleavage site:
AA1 20 AA2 21
MKKLFWVLAIPLALSAVQLK

Probability:
0.963

738, 739
Cleavage site:
AA1 25 AA2 26
MRAVSWVLGLVVAIVLSLISFFIVS

Probability:
0.922

746, 747
Cleavage site:
AA1 46 AA2 47
MNLILLAEILFSLTSIFFAALILKPFLVFNIFAT

Probability:
0.713
LFGKQFACKSFA

768, 769
Cleavage site:
AA1 18 AA2 19
MRHYFLASLLIFTPIAVS

Probability:
0.946

796, 797
Cleavage site:
AA1 18 AA2 19
MLKFIALLIVSYIMELLA

Probability:
0.538

80, 81
Cleavage site:
AA1 20 AA2 21
MHELVFALISILFLSLIAFK

Probability:
0.657

806, 807
Cleavage site:
AA1 42 AA2 43
MMKKKFEEALTKFEEFGLVTLALAFVFLVL

Probability:
0.83
VLYPLFMGVAFA

810, 811
Cleavage site:
AA1 22 AA2 23
MKLNTLKNLLPIIFFFLGYFFA

Probability:
0.859

854, 855
Cleavage site:
AA1 23 AA2 24
MKKKLIGLAFSLSILLFSIYVLV

Probability:
1

866, 867
Cleavage site:
AA1 22 AA2 23
MFRRSITPIIAVVMMLMMTVMA

Probability:
1

868, 869
Cleavage site:
AA1 24 AA2 25
MKSQSHLIEFVLVIGIALAGLSSA

Probability:
0.838

888, 889
Cleavage site:
AA1 27 AA2 28
MGILTKILIKHKLLPFILFIIMGYLFA

Probability:
0.979

894, 895
Cleavage site:
AA1 20 AA2 21
MRGINALIAIIVLVAIVGLA

Probability:
0.991

902, 903
Cleavage site:
AA1 29 AA2 30
MKYSKFLFTWLVLSSALSLVWPANFPLVS

Probability:
0.969

912, 913
Cleavage site:
AA1 26 AA2 27
MRKINMYYLLLLGILFMLSGCVNLSN

Probability:
0.998

930, 931
Cleavage site:
AA1 19 AA2 20
MRRSSIFTLVFYALVGATA

Probability:
0.814

948, 949
Cleavage site:
AA1 22 AA2 23
MLVSTLFNSFLILVSLFFLLFG

Probability:
0.86

958, 959
Cleavage site:
AA1 19 AA2 20
MRTKVGLILFALPIVPALA

Probability:
1

96, 97
Cleavage site:
AA1 29 AA2 30
MRSVSLTLNTIVMIALAISVFSILFIVLS

Probability:
0.97

972, 973
Cleavage site:
AA1 20 AA2 21
MRSITPIIAIVILLLVTISA

Probability:
0.941

The signal sequences of the invention can be isolated peptides, or, sequences joined to another polypeptide, e.g., as a fusion protein. In one aspect, the invention provides polypeptides comprising signal sequences of the invention. In one aspect, polypeptides comprising signal sequences of the invention comprise sequences heterologous to an enzyme of the invention (e.g., a fusion protein comprising a signal sequence of the invention and sequences from another polypeptide or a non-enzyme protein). In one aspect, the invention provides polypeptides of the invention with heterologous signal sequences, e.g., sequences with a yeast signal sequence. An enzyme of the invention can comprise a heterologous signal sequence, e.g., in a vector, e.g., a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In one aspect, the signal sequences of the invention are identified following identification of novel polypeptides, e.g., enzymes. The pathways by which proteins are sorted and transported to their proper cellular location are often referred to as protein targeting pathways. One of the most important elements in all of these targeting systems is a short amino acid sequence at the amino terminus of a newly synthesized polypeptide called the signal sequence. This signal sequence directs a protein to its appropriate location in the cell and is removed during transport or when the protein reaches its final destination. Most lysosomal, membrane, or secreted proteins have an amino-terminal signal sequence that marks them for translocation into the lumen of the endoplasmic reticulum. More than 100 signal sequences for proteins in this group have been determined. The signal sequences can vary in length from 13 to 36 amino acid residues. Various methods of recognition of signal sequences are known to those of skill in the art. For example, in one aspect, novel polypeptide signal peptides are identified by a method referred to as SignalP. SignalP uses a combined neural network which recognizes both signal peptides and their cleavage sites. (Nielsen, et al., “Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.” Protein Engineering, vol. 10, no. 1, p. 1-6 (1997).

It should be understood that in some aspects polypeptides of the invention may not have signal sequences. In one aspect, the invention provides the polypeptides of the invention lacking all or part of a signal sequence. In one aspect, the invention provides a nucleic acid sequence encoding a signal sequence from one polypeptide operably linked to a nucleic acid sequence of a different.

The invention also provides isolated or recombinant polypeptides comprising signal sequences (SPs) and catalytic domains (CDs) of the invention and heterologous sequences. The heterologous sequences are sequences not naturally associated (e.g., to an enzyme) with an SP and/or CD. The sequence to which the SP and/or CD are not naturally associated can be on the SP's, and/or CD's amino terminal end, carboxy terminal end, and/or on both ends of the SP and/or CD. In one aspect, the invention provides an isolated or recombinant polypeptide comprising (or consisting of) a polypeptide comprising a signal sequence (SP) and/or catalytic domain (CD) of the invention with the proviso that it is not associated with any sequence to which it is naturally associated (e.g., an enzyme sequence). Similarly in one aspect, the invention provides isolated or recombinant nucleic acids encoding these polypeptides. Thus, in one aspect, the isolated or recombinant nucleic acid of the invention comprises coding sequence for a signal sequence (SP) and/or catalytic domain (CD) of the invention and a heterologous sequence (i.e., a sequence not naturally associated with the a signal sequence (SP) and/or catalytic domain (CD) of the invention). The heterologous sequence can be on the 3′ terminal end, 5′ terminal end, and/or on both ends of the SP and/or CD coding sequence.

Hybrid (Chimeric) Polypeptides and Peptide Libraries

In one aspect, the invention provides hybrid polypeptides and fusion proteins, including peptide libraries, comprising sequences of the invention. The peptide libraries of the invention can be used to isolate peptide modulators (e.g., activators or inhibitors) of targets, such as enzyme substrates, receptors, enzymes. The peptide libraries of the invention can be used to identify formal binding partners of targets, such as ligands, e.g., cytokines, hormones and the like. In one aspect, the invention provides chimeric proteins comprising a signal sequence (SP) and/or catalytic domain (CD) of the invention and a heterologous sequence (see above).

The invention also provides methods for generating “improved” and hybrid polypeptides using the nucleic acids and polypeptides of the invention. The invention provides methods for generating hybrid polypeptides (e.g., hybrid enzymes).

In one aspect, the methods of the invention produce new hybrid polypeptides by utilizing cellular processes that integrate the sequence of a first polynucleotide such that resulting hybrid polynucleotides encode polypeptides demonstrating activities derived from the first biologically active polypeptides. For example, the first polynucleotides can be an exemplary nucleic acid sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, etc., including all nucleic acids disclosed in the SEQ ID listing, which include all even numbered SEQ ID NO:s from SEQ ID NO:2 through SEQ ID NO:1073) encoding an exemplary polypeptide of the invention. The first nucleic acid can encode a polypeptide from one organism that functions effectively under a particular environmental condition, e.g. high salinity. It can be “integrated” with an polypeptide encoded by a second polynucleotide from a different organism that functions effectively under a different environmental condition, such as extremely high temperatures. For example, when the two nucleic acids can produce a hybrid molecule by e.g., recombination and/or reductive reassortment. A hybrid polynucleotide containing sequences from the first and second original polynucleotides may encode an polypeptide that exhibits characteristics of both polypeptides encoded by the original polynucleotides. Thus, the polypeptide encoded by the hybrid polynucleotide may function effectively under environmental conditions shared by each of the polypeptides encoded by the first and second polynucleotides, e.g., high salinity and extreme temperatures.

Alternatively, a hybrid polypeptide resulting from this method of the invention may exhibit specialized polypeptide activity not displayed in the original polypeptides. For example, following recombination and/or reductive reassortment of polynucleotides encoding polypeptide activities, the resulting hybrid polypeptide encoded by a hybrid polynucleotide can be screened for specialized activities obtained from each of the original polypeptides, for example, the type of bond on which an enzyme acts and the temperature at which the enzyme functions. Thus, for example, an enzyme may be screened to ascertain those chemical functionalities which distinguish the hybrid enzyme from the original enzymes, such as: (a) amide (peptide bonds), i.e., enzymes; (b) ester bonds, i.e., amylases and lipases; (c) acetals, i.e., glycosidases and, for example, the temperature, pH or salt concentration at which the hybrid polypeptide functions.

Sources of the polynucleotides to be “integrated” with nucleic acids of the invention may be isolated from individual organisms (“isolates”), collections of organisms that have been grown in defined media (“enrichment cultures”), or, uncultivated organisms (“environmental samples”). The use of a culture-independent approach to derive polynucleotides encoding novel bioactivities from environmental samples is most preferable since it allows one to access untapped resources of biodiversity. “Environmental libraries” are generated from environmental samples and represent the collective genomes of naturally occurring organisms archived in cloning vectors that can be propagated in suitable prokaryotic hosts. Because the cloned DNA is initially extracted directly from environmental samples, the libraries are not limited to the small fraction of prokaryotes that can be grown in pure culture. Additionally, a normalization of the environmental DNA present in these samples could allow more equal representation of the DNA from all of the species present in the original sample. This can dramatically increase the efficiency of finding interesting genes from minor constituents of the sample that may be under-represented by several orders of magnitude compared to the dominant species.

For example, gene libraries generated from one or more uncultivated microorganisms are screened for an activity of interest. Potential pathways encoding bioactive molecules of interest are first captured in prokaryotic cells in the form of gene expression libraries. Polynucleotides encoding activities of interest are isolated from such libraries and introduced into a host cell. The host cell is grown under conditions that promote recombination and/or reductive reassortment creating potentially active biomolecules with novel or enhanced activities.

The microorganisms from which hybrid polynucleotides may be prepared include prokaryotic microorganisms, such as Eubacteria and Archaebacteria, and lower eukaryotic microorganisms such as fungi, some algae and protozoa. Polynucleotides may be isolated from environmental samples. Nucleic acid may be recovered without culturing of an organism or recovered from one or more cultured organisms. In one aspect, such microorganisms may be extremophiles, such as hyperthermophiles, psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles. In one aspect, polynucleotides encoding polypeptides isolated from extremophilic microorganisms are used to make hybrid polypeptides. Such polypeptides may function at temperatures above 100° C. in, e.g., terrestrial hot springs and deep sea thermal vents, at temperatures below 0° C. in, e.g., arctic waters, in the saturated salt environment of, e.g., the Dead Sea, at pH values around 0 in, e.g., coal deposits and geothermal sulfur-rich springs, or at pH values greater than 11 in, e.g., sewage sludge. For example, polypeptides cloned and expressed from extremophilic organisms can show high activity throughout a wide range of temperatures and pHs.

Polynucleotides selected and isolated as described herein, including at least one nucleic acid of the invention, are introduced into a suitable host cell. A suitable host cell is any cell that is capable of promoting recombination and/or reductive reassortment. The selected polynucleotides can be in a vector that includes appropriate control sequences. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or preferably, the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al., 1986).

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; and plant cells. The selection of an appropriate host for recombination and/or reductive reassortment or just for expression of recombinant protein is deemed to be within the scope of those skilled in the art from the teachings herein. Mammalian cell culture systems that can be employed for recombination and/or reductive reassortment or just for expression of recombinant protein include, e.g., the COS-7 lines of monkey kidney fibroblasts, described in “SV40-transformed simian cells support the replication of early SV40 mutants” (Gluzman, 1981), the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors can comprise an origin of replication, a suitable promoter and enhancer, and necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking non-transcribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

Host cells containing the polynucleotides of interest (for recombination and/or reductive reassortment or just for expression of recombinant protein) can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The clones which are identified as having the specified polypeptide activity may then be sequenced to identify the polynucleotide sequence encoding polypeptides having the enhanced activity.

In another aspect, the nucleic acids and methods of the present invention can be used to generate novel polynucleotides for biochemical pathways, e.g., pathways from one or more operons or gene clusters or portions thereof. For example, bacteria and many eukaryotes have a coordinated mechanism for regulating genes whose products are involved in related processes. The genes are clustered, in structures referred to as “gene clusters,” on a single chromosome and are transcribed together under the control of a single regulatory sequence, including a single promoter which initiates transcription of the entire cluster. Thus, a gene cluster is a group of adjacent genes that are either identical or related, usually as to their function.

Gene cluster DNA can be isolated from different organisms and ligated into vectors, particularly vectors containing expression regulatory sequences which can control and regulate the production of a detectable protein or protein-related array activity from the ligated gene clusters. Use of vectors which have an exceptionally large capacity for exogenous DNA introduction are particularly appropriate for use with such gene clusters and are described by way of example herein to include the f-factor (or fertility factor) of E. coli. This f-factor of E. coli is a plasmid which affects high-frequency transfer of itself during conjugation and is ideal to achieve and stably propagate large DNA fragments, such as gene clusters from mixed microbial samples. “Fosmids,” cosmids or bacterial artificial chromosome (BAC) vectors can be used as cloning vectors. These are derived from E. coli f-factor which is able to stably integrate large segments of genomic DNA. When integrated with DNA from a mixed uncultured environmental sample, this makes it possible to achieve large genomic fragments in the form of a stable “environmental DNA library.” Cosmid vectors were originally designed to clone and propagate large segments of genomic DNA. Cloning into cosmid vectors is described in detail in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989). Once ligated into an appropriate vector, two or more vectors containing different polyketide synthase gene clusters can be introduced into a suitable host cell. Regions of partial sequence homology shared by the gene clusters will promote processes which result in sequence reorganization resulting in a hybrid gene cluster. The novel hybrid gene cluster can then be screened for enhanced activities not found in the original gene clusters.

Thus, in one aspect, the invention relates to a method for producing a biologically active hybrid polypeptide using a nucleic acid of the invention and screening the polypeptide for an activity (e.g., enhanced activity) by:

(1) introducing at least a first polynucleotide (e.g., a nucleic acid of the invention) in operable linkage and a second polynucleotide in operable linkage, said at least first polynucleotide and second polynucleotide sharing at least one region of partial sequence homology, into a suitable host cell;

(2) growing the host cell under conditions which promote sequence reorganization resulting in a hybrid polynucleotide in operable linkage;

(3) expressing a hybrid polypeptide encoded by the hybrid polynucleotide;

(4) screening the hybrid polypeptide under conditions which promote identification of the desired biological activity (e.g., enhanced enzyme activity); and

(5) isolating the a polynucleotide encoding the hybrid polypeptide.

Methods for screening for various polypeptide (e.g., enzyme) activities are known to those of skill in the art and are discussed throughout the present specification. Such methods may be employed when isolating the polypeptides and polynucleotides of the invention.

In vivo reassortment can be focused on “inter-molecular” processes collectively referred to as “recombination.” In bacteria it is generally viewed as a “RecA-dependent” phenomenon. The invention can rely on recombination processes of a host cell to recombine and re-assort sequences, or the cells' ability to mediate reductive processes to decrease the complexity of quasi-repeated sequences in the cell by deletion. This process of “reductive reassortment” occurs by an “intra-molecular”, RecA-independent process. Thus, in one aspect of the invention, using the nucleic acids of the invention novel polynucleotides are generated by the process of reductive reassortment. The method involves the generation of constructs containing consecutive sequences (original encoding sequences), their insertion into an appropriate vector, and their subsequent introduction into an appropriate host cell. The reassortment of the individual molecular identities occurs by combinatorial processes between the consecutive sequences in the construct possessing regions of homology, or between quasi-repeated units. The reassortment process recombines and/or reduces the complexity and extent of the repeated sequences, and results in the production of novel molecular species.

Various treatments may be applied to enhance the rate of reassortment. These could include treatment with ultra-violet light, or DNA damaging chemicals, and/or the use of host cell lines displaying enhanced levels of “genetic instability”. Thus the reassortment process may involve homologous recombination or the natural property of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in genetic instability. “Quasi-repeats” are repeats that are not restricted to their original unit structure. Quasi-repeated units can be presented as an array of sequences in a construct; consecutive units of similar sequences. Once ligated, the junctions between the consecutive sequences become essentially invisible and the quasi-repetitive nature of the resulting construct is now continuous at the molecular level. The deletion process the cell performs to reduce the complexity of the resulting construct operates between the quasi-repeated sequences. The quasi-repeated units provide a practically limitless repertoire of templates upon which slippage events can occur. The constructs containing the quasi-repeats thus effectively provide sufficient molecular elasticity that deletion (and potentially insertion) events can occur virtually anywhere within the quasi-repetitive units. When the quasi-repeated sequences are all ligated in the same orientation, for instance head to tail or vice versa, the cell cannot distinguish individual units. Consequently, the reductive process can occur throughout the sequences. In contrast, when for example, the units are presented head to head, rather than head to tail, the inversion delineates the endpoints of the adjacent unit so that deletion formation will favor the loss of discrete units. Thus, in one aspect of the invention, the sequences to be reassorted are in the same orientation. Random orientation of quasi-repeated sequences will result in the loss of reassortment efficiency, while consistent orientation of the sequences will offer the highest efficiency. However, while having fewer of the contiguous sequences in the same orientation decreases the efficiency, it may still provide sufficient elasticity for the effective recovery of novel molecules. Constructs can be made with the quasi-repeated sequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of a variety of methods, including the following: a) Primers that include a poly-A head and poly-T tail which when made single-stranded would provide orientation can be utilized. This is accomplished by having the first few bases of the primers made from RNA and hence easily removed RNase H. b) Primers that include unique restriction cleavage sites can be utilized. Multiple sites, a battery of unique sequences, and repeated synthesis and ligation steps would be required. c) The inner few bases of the primer could be thiolated and an exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identification of cloning vectors with a reduced repetitive index (R1). The re-assorted encoding sequences can then be recovered by amplification. The products are re-cloned and expressed. The recovery of cloning vectors with reduced R1 can be affected by: 1) The use of vectors only stably maintained when the construct is reduced in complexity. 2) The physical recovery of shortened vectors by physical procedures. In this case, the cloning vector would be recovered using standard plasmid isolation procedures and size fractionated on either an agarose gel, or column with a low molecular weight cut off utilizing standard procedures. 3) The recovery of vectors containing interrupted genes which can be selected when insert size decreases. 4) The use of direct selection techniques with an expression vector and the appropriate selection.

Encoding sequences (for example, genes) from related organisms may demonstrate a high degree of homology and encode quite diverse protein products. These types of sequences are particularly useful in the present invention as quasi-repeats. However, this process is not limited to such nearly identical repeats.

The following is an exemplary method of the invention. Encoding nucleic acid sequences (quasi-repeats) are derived from three (3) species, including a nucleic acid of the invention. Each sequence encodes a protein with a distinct set of properties, including a polypeptide, e.g., an enzyme, of the invention. Each of the sequences differs by a single or a few base pairs at a unique position in the sequence. The quasi-repeated sequences are separately or collectively amplified and ligated into random assemblies such that all possible permutations and combinations are available in the population of ligated molecules. The number of quasi-repeat units can be controlled by the assembly conditions. The average number of quasi-repeated units in a construct is defined as the repetitive index (R1). Once formed, the constructs may, or may not be size fractionated on an agarose gel according to published protocols, inserted into a cloning vector, and transfected into an appropriate host cell. The cells are then propagated and “reductive reassortment” is effected. The rate of the reductive reassortment process may be stimulated by the introduction of DNA damage if desired. Whether the reduction in R1 is mediated by deletion formation between repeated sequences by an “intra-molecular” mechanism, or mediated by recombination-like events through “inter-molecular” mechanisms is immaterial. The end result is a reassortment of the molecules into all possible combinations. In one aspect, the method comprises the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind or otherwise interact, or catalyze a particular reaction (e.g., such as catalytic domain of an enzyme) with a predetermined macromolecule, such as for example a proteinaceous receptor, an oligosaccharide, virion, or other predetermined compound or structure. The polypeptides, e.g., enzymes, that are identified from such libraries can be used for various purposes, e.g., the industrial processes described herein and/or can be subjected to one or more additional cycles of shuffling and/or selection.

In another aspect, it is envisioned that prior to or during recombination or reassortment, polynucleotides generated by the method of the invention can be subjected to agents or processes which promote the introduction of mutations into the original polynucleotides. The introduction of such mutations would increase the diversity of resulting hybrid polynucleotides and polypeptides encoded therefrom. The agents or processes which promote mutagenesis can include, but are not limited to: (+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N3-Adenine (See Sun and Hurley, (1992); an N-acetylated or deacetylated 4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis (See, for example, van de Poll et al. (1992)); or a N-acetylated or deacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis (See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium, a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNA adduct capable of inhibiting DNA replication, such as 7-bromomethyl-benz[a]anthracene (“BMA”), tris(2,3-dibromopropyl)phosphate (“Tris-BP”), 1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA), benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II) halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline (“N-hydroxy-IQ”), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine (“N-hydroxy-PhIP”). Especially preferred means for slowing or halting PCR amplification consist of UV light (+)-CC-1065 and (+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA adducts or polynucleotides comprising the DNA adducts from the polynucleotides or polynucleotides pool, which can be released or removed by a process including heating the solution comprising the polynucleotides prior to further processing.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus and methodologies can be used to in conjunction with the polypeptides and nucleic acids of the invention, e.g., to screen polypeptides for enzyme activity, to screen compounds as potential modulators of activity (e.g., potentiation or inhibition of enzyme activity), for antibodies that bind to a polypeptide of the invention, for nucleic acids that hybridize to a nucleic acid of the invention, and the like.

Immobilized Polypeptide Solid Supports

The polypeptides, fragments thereof and nucleic acids that encode the enzymes and fragments can be affixed to a solid support. This is often economical and efficient in the use of polypeptides, e.g., antibodies, enzymes, ligands, in industrial processes. For example, a consortium or cocktail of polypeptides (e.g., enzymes or active fragments thereof), which are used in a specific chemical reaction, can be attached to a solid support and dunked into a process vat. The enzymatic reaction can occur. Then, the solid support can be taken out of the vat, along with the enzymes affixed thereto, for repeated use. In one embodiment of the invention, an isolated nucleic acid of the invention is affixed to a solid support. In another embodiment of the invention, the solid support is selected from the group of a gel, a resin, a polymer, a ceramic, a glass, a microelectrode and any combination thereof.

For example, solid supports useful in this invention include gels. Some examples of gels include Sepharose, gelatin, glutaraldehyde, chitosan-treated glutaraldehyde, albumin-glutaraldehyde, chitosan-Xanthan, toyopearl gel (polymer gel), alginate, alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magnetic agarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEG hydrogel, phosphorylated polyvinyl alcohol (PVA), monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.

Another solid support useful in the present invention are resins or polymers. Some examples of resins or polymers include cellulose, acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™ XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50, polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.

Another type of solid support useful in the present invention is ceramic. Some examples include non-porous ceramic, porous ceramic, SiO₂, Al₂O₃. Another type of solid support useful in the present invention is glass. Some examples include non-porous glass, porous glass, aminopropyl glass or any combination thereof. Another type of solid support that can be used is a microelectrode. An example is a polyethyleneimine-coated magnetite. Graphitic particles can be used as a solid support.

Another example of a solid support is a cell, such as a red blood cell.

Methods of Immobilization

There are many methods that would be known to one of skill in the art for immobilizing polypeptides (e.g., antibodies, enzymes, binding proteins, or fragments thereof), or nucleic acids, onto a solid support. Some examples of such methods include, e.g., electrostatic droplet generation, electrochemical means, via adsorption, via covalent binding, via cross-linking, via a chemical reaction or process, via encapsulation, via entrapment, via calcium alginate, or via poly(2-hydroxyethyl methacrylate). Like methods are described in Methods in Enzymology, Immobilized Enzymes and Cells, Part C. 1987. Academic Press. Edited by S. P. Colowick and N, O. Kaplan. Volume 136; and Immobilization of Enzymes and Cells. 1997. Humana Press. Edited by G. F. Bickerstaff. Series: Methods in Biotechnology, Edited by J. M. Walker.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, San Diego, Calif., can be used to in the methods of the invention. Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array, including capillary arrays. Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention. Capillary arrays provide another system for holding and screening samples. For example, a sample screening apparatus can include a plurality of capillaries formed into an array of adjacent capillaries, wherein each capillary comprises at least one wall defining a lumen for retaining a sample. The apparatus can further include interstitial material disposed between adjacent capillaries in the array, and one or more reference indicia formed within of the interstitial material. A capillary for screening a sample, wherein the capillary is adapted for being bound in an array of capillaries, can include a first wall defining a lumen for retaining the sample, and a second wall formed of a filtering material, for filtering excitation energy provided to the lumen to excite the sample.

A polypeptide or nucleic acid, e.g., a ligand, can be introduced into a first component into at least a portion of a capillary of a capillary array. Each capillary of the capillary array can comprise at least one wall defining a lumen for retaining the first component. An air bubble can be introduced into the capillary behind the first component. A second component can be introduced into the capillary, wherein the second component is separated from the first component by the air bubble. A sample of interest can be introduced as a first liquid labeled with a detectable particle into a capillary of a capillary array, wherein each capillary of the capillary array comprises at least one wall defining a lumen for retaining the first liquid and the detectable particle, and wherein the at least one wall is coated with a binding material for binding the detectable particle to the at least one wall. The method can further include removing the first liquid from the capillary tube, wherein the bound detectable particle is maintained within the capillary, and introducing a second liquid into the capillary tube.

The capillary array can include a plurality of individual capillaries comprising at least one outer wall defining a lumen. The outer wall of the capillary can be one or more walls fused together. Similarly, the wall can define a lumen that is cylindrical, square, hexagonal or any other geometric shape so long as the walls form a lumen for retention of a liquid or sample. The capillaries of the capillary array can be held together in close proximity to form a planar structure. The capillaries can be bound together, by being fused (e.g., where the capillaries are made of glass), glued, bonded, or clamped side-by-side. The capillary array can be formed of any number of individual capillaries, for example, a range from 100 to 4,000,000 capillaries. A capillary array can form a microtiter plate having about 100,000 or more individual capillaries bound together.

Arrays, or “BioChips”

Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array. Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention. For example, in one aspect of the invention, a monitored parameter is transcript expression of a polypeptide-encoding (e.g., an enzyme) gene. One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or “biochip.” By using an “array” of nucleic acids on a microchip, some or all of the transcripts of a cell can be simultaneously quantified. Alternatively, arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention. “Polypeptide arrays” can also be used to simultaneously quantify a plurality of proteins.

The present invention can be practiced with any known “array,” also referred to as a “microarray” or “nucleic acid array” or “polypeptide array” or “antibody array” or “biochip,” or variation thereof. Arrays are generically a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as described, for example, in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics Supp. 21:25-32. See also published U.S. patent applications Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated or recombinant antibodies that specifically bind to a polypeptide (e.g., an enzyme) of the invention. These antibodies can be used to isolate, identify or quantify the polypeptides of the invention or related polypeptides. These antibodies can be used to inhibit the activity of a polypeptide (e.g., an enzyme) of the invention. These antibodies can be used to isolated polypeptides related to those of the invention, e.g., related polypeptides.

The antibodies can be used in immunoprecipitation, staining (e.g., FACS), immunoaffinity columns, and the like. If desired, nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of the invention.

Alternatively, the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased. Furthermore, the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York. Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides can be used to generate antibodies which bind specifically to the polypeptides of the invention. The resulting antibodies may be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample. In such procedures, a protein preparation, such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the polypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solid support, such as a bead or other column matrix. The protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of the invention. After a wash to remove non-specifically bound proteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibody may be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding may be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the sample may be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of the invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, for example, a nonhuman. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies which may bind to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to the polypeptides of the invention. Alternatively, transgenic mice may be used to express humanized antibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may be used in screening for similar polypeptides from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody and those polypeptides which specifically bind the antibody are detected. Any of the procedures described above may be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, polypeptides (e.g., enzymes) and/or antibodies of the invention. The kits also can contain instructional material teaching the methodologies and industrial uses of the invention, as described herein.

The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES
Example 1
Blast Program Used for Sequence Identify Profiling

This example describes an exemplary sequence identity program to determine if a nucleic acid is within the scope of the invention. An NCBI BLAST 2.2.2 program is used, default options to blastp. All default values were used except for the default filtering setting (i.e., all parameters set to default except filtering which is set to OFF); in its place a “-F F” setting is used, which disables filtering. Use of default filtering often results in Karlin-Altschul violations due to short length of sequence. The default values used in this example:

- “Filter for low complexity: ON
- >Word Size: 3
- >Matrix: Blosum62
- >Gap Costs: Existence: 11
- >Extension: 1”

Other default settings were: filter for low complexity OFF, word size of 3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and a gap extension penalty of −1. The “-W” option was set to default to 0. This means that, if not set, the word size defaults to 3 for proteins and 11 for nucleotides. The settings read:

<<README.bls.txt>>

>
--------------------------------------------------------------------------

>
blastall arguments:

>

>
-p Program Name [String]

>
-d Database [String]

>
default = nr

>
-i Query File [File In]

>
default = stdin

>
-e Expectation value (E) [Real]

>
default = 10.0

>
-m alignment view options:

>
0 = pairwise,

>
1 = query-anchored showing identities,

>
2 = query-anchored no identities,

>
3 = flat query-anchored, show identities,

>
4 = flat query-anchored, no identities,

>
5 = query-anchored no identities and blunt ends,

>
6 = flat query-anchored, no identities and blunt ends,

>
7 = XML Blast output,

>
8 = tabular,

>
9 tabular with comment lines [Integer]

>
default = 0

>
-o BLAST report Output File [File Out] Optional

>
default = stdout

>
-F Filter query sequence (DUST with blastn, SEG with others)

[String]

>
default = T

>
-G Cost to open a gap (zero invokes default behavior) [Integer]

>
default = 0

>
-E Cost to extend a gap (zero invokes default behavior) [Integer]

>
default = 0

>
-X X dropoff value for gapped alignment (in bits) (zero invokes

>
default behavior) [Integer]

>
default = 0

>
-I Show GI's in deflines [T/F]

>
default = F

>
-q Penalty for a nucleotide mismatch (blastn only) [Integer]

>
default = −3

>
-r Reward for a nucleotide match (blastn only) [Integer]

>
default = 1

>
-v Number of database sequences to show one-line descriptions for

(V)

>
[Integer]

>
default = 500

>
-b Number of database sequence to show alignments for (B)

[Integer]

>
default = 250

>
-f Threshold for extending hits, default if zero [Integer]

>
default = 0

>
-g Perform gapped alignment (not available with tblastx) [T/F]

>
default = T

>
-Q Query Genetic code to use [Integer]

>
default = 1

>
-D DB Genetic code (for tblast[nx] only) [Integer]

>
default = 1

>
-a Number of processors to use [Integer]

>
default = 1

>
-O SeqAlign file [File Out] Optional

>
-J Believe the query defline [T/F]

>
default = F

>
-M Matrix [String]

>
default = BLOSUM62

>
-W Word size, default if zero [Integer]

>
default = 0

>
-z Effective length of the database (use zero for the real size)

>
[String]

>
default = 0

>
-K Number of best hits from a region to keep (off by default, if

>
used a value of 100 is recommended) [Integer]

>
default = 0

>
-P 0 for multiple hits 1-pass, 1 for single hit 1-pass, 2 for 2-pass

>
[Integer]

>
default = 0

>
-Y Effective length of the search space (use zero for the real size)

>
[Real]

>
default = 0

>
-S Query strands to search against database (for blast[nx], and

>
tblastx). 3 is both, 1 is top, 2 is bottom [Integer]

>
default = 3

>
-T Produce HTML output [T/F]

>
default = F

>
-l Restrict search of database to list of GI's [String] Optional

>
-U Use lower case filtering of FASTA sequence [T/F] Optional

>
default = F

>
-y Dropoff (X) for blast extensions in bits (0.0 invokes default

>
behavior) [Real]

>
default = 0.0

>
-Z X dropoff value for final gapped alignment (in bits) [Integer]

>
default = 0

>
-R PSI-TBLASTN checkpoint file [File In] Optional

>
-n MegaBlast search [T/F]

>
default = F

>
-L Location on query sequence [String] Optional

>
-A Multiple Hits window size (zero for single hit algorithm)

[Integer]

>
default = 40

Example 2
Isolation and Characterization of the Hyperthermophile Nanoarchaeum equitans and its Genome

This example describes the isolation and characterization of the hyperthermophile Nanoarchaeum equitans of the invention and its genome (SEQ ID NO:1).

To investigate hot submarine vent microbial communities, experiments were carried out to cultivate hyperthermophiles from samples of originally hot rocks and gravel taken at the Kolbeinsey ridge, north of Iceland, see, e.g., Fricke (1989) “Hydrothermal vent communities at the shallow subpolar mid-Atlantic ridge.” Mar. Biol. 102, 425-429. By anaerobic incubation at 90° C. in the presence of S, H2 and CO₂, a new autotrophic sulphur-reducing species of the archaeal genus Ignicoccus could be enriched. In contrast to known species, several large spherical Ignicoccus cells were covered by very tiny cocci which could be stained by DNA-specific fluorescence microscopy, using DAPI, see, e.g., Huber (1985) Syst. Appl. Microbiol. 6:105-106. Few such tiny cocci existed in the free state. These tiny cocci could be physically isolated either by using ‘optical tweezers’ (see, e.g., Ashkin (1987) Nature 330:769-771; Huber (1995) Nature 376:57-58; or, or by ultrafiltration, pore width, 0.45 mm. All attempts to grow these organisms in pure culture employing various inorganic and organic energy sources failed. However, isolation of a combination of a tiny coccus attached to an Ignicoccus sphere after incubation under enrichment conditions, resulted in a defined co-culture in which about half of the Ignicoccus cells appeared to be colonized by the tiny cocci. The final density of both the tiny cocci and the Ignicoccus cells was about 3×10⁷cells ml⁻¹resulting in about two tiny cocci per Ignicoccus cell on average. The purified co-culture was used in all further investigations.

Cloning of single Ignicoccus cells gave rise to cultures which never contained tiny cocci. By electron microscopy, a close attachment of the tiny cocci to the surface of the Ignicoccus cells became evident. The tiny cocci consistently exhibited a cell diameter of about 400 nm. In contrast to Ignicoccus, they were covered by a regular surface layer (S-layer) with six-fold symmetry and a lattice constant of 15 nm. Ultra-thin sections showed the presence of cytoplasmic membranes inside the tiny cocci and the Ignicoccus cells. No specific attachment structures could be detected at the sites of contact which suggested a loose connection of the tiny cocci with the Ignicoccus cells. In line with this assumption, the tiny cocci could be removed by mild sonification (30W).

The phylogenetic relationships of the tiny cocci were investigated by ss rRNA sequence comparisons (see, e.g., Woese (1987) Microbiol. Rev. 51:221-271). Total DNA of the co-culture was extracted and ss rRNA genes were PCR-amplified using primers addressed to highly conserved gene sections. Surprisingly, however, only the Ignicoccus ss rRNA gene sequence was amplified even when primers considered general for all Archaea as well as for all organisms (‘universal’) had been employed. More directly, the co-culture-derived DNA for ss rRNA genes was examined by Southern blot hybridization (see, e.g., Sambrook, J. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, New York, 1989), taking advantage of the generally high sequence homology of all ss rRNA genes. Two different hybridization signals became visible, indicating two non-identical ss rRNA genes. As a control, DNA isolated from the separate Ignicoccus sp. by the same procedure yielded only one hybridization signal which was identical with one from the mixed culture. Therefore, the tiny cocci must have given rise to the second hybridization signal, indicating that they hold a single-stranded (ss)rRNA gene different from Ignicoccus.

The corresponding DNA fragment was cloned and sequenced. Its sequence turned out to be unique, harboring many base exchanges even in the so-called ‘highly conserved regions’ that are usually employed as primer targets for ss rDNA PCR. This explains our initial failure to amplify this gene by PCR. Comparison of the new single-stranded (ss)rRNA sequence with those of other microorganisms established the tiny cocci as previously unknown members of the archaeal domain indicated by sequence identities of 0.69 to 0.81 with the Archaea in contrast to sequence identities of only 0.59 to 0.70 with bacterial species.

The secondary structure of the ss rRNA sequence conformed to the standard two-dimensional structural model (see, e.g., Woese (1987) supra). It exhibited features characteristic of Archaea, especially helices numbers 17, 18, 36 and 47. The sequence identities to the Crenarchaeota (0.73-0.81), the Euryarchaeota (0.69-0.81), and the ‘Korarchaeota’ (0.73-0.75) were in the same range as those between these three known phyla of Archaea (0.69-0.83). Therefore, the tiny cocci represent a new archaeal phylum. On the basis of its extremely small cell size, we named it ‘Nanoarchaeota’ (the dwarf archaea) and the corresponding species ‘Nanoarchaeum equitans’ (riding the fire sphere).

In ss rRNA phylogenetic trees that are founded on the maximum parsimony, distance matrix, and maximum likelihood methods (see, e.g., Ludwig (1998) supra), ‘N. equitans’ represents an isolated, very deeply branching lineage. However, because of its unique ss rRNA, a large variation of its branching point, challenged by insignificant bootstrap values below 50%, was observed. Therefore, the determination of the accurate branching position of the ‘Nanoarchaeota’ must await the discovery of further members of this group. Owing to their great divergences in ss rRNA, in contrast to the Ignicoccus host, cells of the ‘Nanoarchaeota’ did not stain by fluorescence in situ hybridization using ss rRNA-targeted oligonucleotide probes directed against Crenarchaeota and Euryarchaeota). However, after redesigning these oligonucleotide probes on the basis of the ‘Nanoarchaeota’ sequence (SEQ ID NO:1), the tiny cocci exhibited a bright fluorescence, indicating that they possessed ribosomal RNA harboring the target sequence.

Few physiological properties of the ‘Nanoarchaeum’ strain are known thus far. Isolated cells did not grow on cell homogenates of Ignicoccus sp., so they require an actively growing Ignicoccus culture. Within pure cultures of Ignicoccus, ‘Nanoarchaeum’ cells locally separated by dialysis bags were unable to propagate. Therefore, a direct cell-cell contact with the host appears to be a prerequisite for growth. No negative influence on Ignicoccus, such as slower growth, lower final cell density, or increased cell lysis, could be observed in the co-culture compared to the pure culture, rendering a parasitic mode of living of ‘Nanoarchaeum’ unlikely. Therefore, the organism is most probably symbiotic. So far, archaeal symbionts are only known at mesophilic temperatures in association with eukaryotes (see, e.g., Preston (1996) Proc. Natl. Acad. Sci. USA 93, 6241-6246) and bacteria (see, e.g., Boetius (2000) Nature 407:623-626). Co-cultures of ‘Nanoarchaeum’ grew within the same temperature range (70° C. to 98° C.) as its host. During mass-culturing in a 300-litre enamel-protected fermentor3, final cell concentrations of ‘Nanoarchaeum’ could be improved about ten-fold by an increased gassing rate (20 liter/min; H₂:CO₂=80:20), while that of Ignicoccus remained unchanged. This procedure improved hydrogen supply and efficiently removed H₂S, the main metabolic end product of Ignicoccus. During the late exponential growth phase, about 80% of the ‘Nanoarchaeum’ cells detached from their host cells and occurred freely in suspension. They could be collected (for example, 1.5 g wet weight) by high-speed centrifugation, after removal of the Ignicoccus cells at low speed. By this method, cell masses of ‘Nanoarchaeum’ became available for biochemical and molecular investigations.

A first analysis of its genome size by adding up the sizes of restriction fragments determined after pulse field gel electrophoresis resulted in only 500 kilobases (kb), one of the smallest genomes of all prokaryotes known so far. With its tiny cell and genome size, ‘Nanoarchaeum’ resembles an intermediate between the smallest living organisms like Mycoplasma genitalium (see, e.g., Fraser (1995) Science 270:397-403) and big viruses like the pox virus and Chlorella virus CVK2 (see, e.g., Rohozinski (1989) Virology 168:363-369; Murphy, F. A. et al. (eds) Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses, Springer, Vienna/New York, 1995. Nature Vol. 417. 2 MAY 2002), and is close to the theoretical minimum genome size calculated for a living being (see, e.g., Hutchinson (1999) Science 286:2165-2169).

Small cell size and reduced genomes are common features observed in many symbiotic or parasitic bacteria, but have so far not been reported for Archaea or hyperthermophiles. No final conclusions can be drawn about a primitive or derived state of evolution of ‘Nanoarchaeum’. However, its high growth temperature and anaerobic mode of life correlates with probable early environmental conditions which suggest that the ‘Nanoarchaeota’ are possibly still a primitive form of microbial life.

Cultivation conditions: Cultures were grown anaerobically in serum bottles as described by Huber (2000) Int. J. Syst. Evol. Microbiol. 50:2093-2100. Mass culturing was carried out in the same medium in a 300-litre enamel-protected fermentor (gassing rate routinely 2 liter/min H₂:CO₂=80:20). Cell mixtures were collected by centrifugation (9,000 r.p.m.; 30 min using rotor GS3 (Sorvall)) and resuspended in sulphur-free culture medium. The Ignicoccus cells were removed by centrifugation at 2,000 r.p.m. for 30 min using rotor SS34 (Sorvall). Then the ‘Nanoarchaeum’ cells were precipitated at 15,000 r.p.m. for 15 min using rotor SS34. Axenic cultivation of ‘Nanoarchaeum equitans’ was tested autotrophically in the presence of H₂:CO₂(80:20) and elemental sulphur, nitrate, nitrite, and sulphate (each 0.1% w/v) as possible electron acceptors, and heterotrophically on single and complex organic nutrients such as sugars, amino acids, yeast extract, peptone, bacterial and archaeal (for example, from Ignicoccus) cell extracts (each 0.1% w/v; gas phase: H₂:CO₂=80:20 and N₂:CO₂=80:20).

Light and electron microscopy: Phase-contrast and electron microscopy were carried out as described by Huber (2000) supra. Confocal image series were recorded on a LEICA TCS SP2 laser scanning microscope using the laser wavelengths 488 nm (argon) and 543 nm (HeNe) excitation. Fluorescence in situ hybridization (FISH) was performed as described15. CY3- and rhodamine-green-labeled oligonucleotides were obtained from Metabion (Germany).

Southern blot experiments: DNAs were digested with restriction endonucleases EcoRI and HindIII. The fragments were separated by electrophoresis (1% Seakem ME agarose) and transferred to a positively charged nylon membrane by Southern blot13. The membrane was hybridized with a digoxigenine-labelled single-stranded DNA fragment of an archaeal ss rRNA gene (Metallosphaera sedula; length 600 nucleotides, position 519 to 1119, Escherichia coli).

Estimation of the genome size: For pulse field gel electrophoresis ‘Nanoarchaeum’ cells were embedded in 100-ml agarose plugs (0.8% Incert agarose, FMC; final concentration: 3£108 cells per plug). After cell lysis according to lysis method ‘three’ (see, e.g., Baumann (1998) Extremophiles 2:101-108) genomic DNA was digested using the restriction enzymes AscI, BssHII, NotI, or SacII (each 100 units, 18 h). The restriction fragments were separated as described by Baumann (1998) supra.

Small subunit rDNA sequence analysis: The nearly complete ss rRNA gene of the new Ignicoccus species was PCR-amplified and sequenced as described by Huber (2000) supra. To obtain the ss rRNA gene of ‘N. equitans’, genomic DNA of the co-culture was digested with EcoRI and separated as described (see ‘Southern blot’). DNA fragments of the correct size, identified by Southern blot, were extracted, cloned into a plasmid (pBluescript SK(2)) and amplified in Escherichia coli (strain Dh5a). These inserts were sequenced by primer walking. The new sequences were aligned with approximately 10,000 homologous sequences available in public databases using the automatic alignment tools of the ARB package (Technical University of Munich, Del.). Distance matrix (Jukes-Cantor correction), maximum parsimony, and maximum likelihood (fastDNAml) methods were used for tree reconstruction as implemented in the ARB package. The secondary structure of the ss rRNA of ‘N. equitans’ was displayed using the program RnaViz28.

Library Construction and DNA Sequencing: N. equitans was grown in a 300 liter fermentor in a co-culture with Ignicoccus sp. and the N. equitans cells were purified from Ignicoccus as described above. The cell pellet was lysed by enzymatic and chemical digestion, followed by the isolation and purification of genomic DNA, as described by Zhou (1996) Appl. Environ. Microbiol. 62:316; Robertson (1996) Soc. Indust. Microbiol. News 46:3; Short (1997) Nat. Biotechnol. 15:1322. Genomic DNA was either digested with restriction enzymes or sheared to provide clonable fragments. Two plasmid libraries were made by subcloning randomly sheared fragments of this DNA into a high-copy number vector (˜2.8 kbp library) or low-copy number vector (˜6.3 kbp library). DNA sequence was obtained from both ends of the plasmid inserts to create ‘mate-pairs’—a pair of reads from a single clone that should be adjacent to one another in the genome. Library construction, DNA sequencing and assembly methods were essentially as described by Adams (2000) Science 287:2185; Myers (2000) Science 287:2196; Venter (2001) Science 291:1304. The assembly procedures resulted in a single scaffold of four contigs comprising 489,082 base pairs. The gaps between the four contigs were then sequenced with 10× coverage which resulted in a single circular sequence. A set of computational methods was applied to the N. equitans genome of the invention (SEQ ID NO:1). Two gene prediction programs, Glimmer (see, e.g., Delcher (1999) Nucleic Acids Res. 27:4636) and Critica (see, e.g., Badger (1999) Mol. Biol. Evol. 16:4512), were run on the assembled sequences. The results of the two programs were merged to generate a unique set of genes. When the two programs selected different start codons for genes with the same stop codon, the longer gene was included in the set for further analysis. This unique set of genes was then translated into amino acid sequences and subjected to BlastP searches (with an E-value cutoff of 1e-10) against the non-redundant amino acid (nraa) protein database, as described by the NCBI, NLM, NIH website. The predicted protein set was then searched against the InterPro database release 3.1 (see, e.g., Apweiler (2001) Nucleic Acids Res. 29, 37) using software modified from the original ipr scan programs provided by InterPro (The European Bioinformatics Institute (EBI), European Molecular Biology Laboratory (EMBL), Cambridge, UK). The predicted protein set was also searched against the NCBI Clusters of Othologous Groups (COGs) database mid-2001 update (see, e.g., Tatusov (2001) Nucleic Acids Res. 29:22). Lastly, gene family analysis was performed using the NCBI blast clust program. tRNA genes were identified by the tRNA scan-SE program (see, e.g., Lowe (1997) Nucleic Acids Res. 25:955) and rRNAs were identified by searching the genomic sequences against a set of known rRNAs with BlastN and verified by profile alignment to the multiple alignments from known rRNA sequences. Protein sets from the main scaffold and small scaffolds were compared to the protein sequences from all finished genomes deposited in the GenBank using the blast program.

Preparation of alanyl-tRNA synthetases and aminoacylation assay: The methods were adapted from Ahel et al., as described in Ahel (2002) J. Biol. Chem. 277:34743. N. equitans alaS1 (NEQ547), N. equitans alaS2 (NEQ211), and M. jannaschii alaS genes were PCR amplified from the respective genomic DNA and cloned into the pCR2.1 TOPO vector. Correct sequences were subsequently re-cloned into pET11a (Invitrogen) for expression of the proteins in the E. coli BL21-Codon Plus (DE3)-RIL strain. Cultures were grown at 37° C. in Luria-Bertani medium supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. Expression of the recombinant proteins was induced for 3 hours (h) at 30° C. by addition of 1 mM isopropyl-1-thio-D-galactopyranoside before cell harvesting. Cells were resuspended in buffer containing 50 mM Tris-HCl, pH 7.5, and 300 mM NaCl, and broken by sonication. S-100 fractions were extensively flocculated at 70° C. for 45 min, and then centrifuged for 30 min at 20,000×g. Supernatants were collected and stored at 4° C. before use in aminoacylation assays.

Aminoacylation was performed in a 0.1 ml reaction at 70° C. in 50 mM HEPES, pH 7.2, 50 mM KCl, 10 mM ATP, 50 μM [³H] alanine (52 Ci/ml), 15 mM MgCl₂and 5 mM 2-mercaptoethanol, unfractionated M. jannaschii tRNA (3 mg per ml of reaction) and the different alanyl-tRNA synthetases (100 nM). Aliquots of 20 μl were removed at the time intervals indicated in FIG. 5 and radioactivity measured as described. FIG. 5 summarizes data from the alanylation of unfractionated M. jannaschii tRNA by alanyl-tRNA synthetase. The purification and aminoacylation procedures were adapted from Ahel (2002) J. Biol. Chem. 277:34743. The enzymes used are: M. jannaschii AlaRS (filled squares), N. equitans AlaRS1—N-terminal part (open circles), N. equitans AlaRS2—C-terminal part (filled triangles), N. equitans AlaRS1+AlaRS2 (filled circles).

Phylogenetic Analysis: A concatenated alignment of 35 ribosomal proteins was obtained from Matte-Tailliez (see e.g., Matte-Tailliez (2002) Mol. Biol. Evol. 13:631). To this alignment we added the N. equitans, Methanopyrus kandleri and the eukaryotic outgroup sequences (Arabidopsis thaliana and Saccharomyces cerevisiae). The alignment was then recalculated with ClustalW (see, e.g., Thompson (1994) Nucleic Acids Res. 22:4673) and optimized by hand using BioEdit (Hall (1999) Nucleic Acids Symp. Ser. 41:95; North Carolina State University, Raleigh, N.C.). The program RASA (University of Massachusetts, Lowell, Mass.) was used to evaluate the alignment for the presence of long branches (see, e.g., Lyons-Weiler (1996) Mol. Biol. Evol. 13:749). Maximum likelihood analysis was performed with Protml from PHYLIP v. 3.6a2.3 (University of Washington, Seattle, Wash.). Parameters used were: the Jones-Taylor-Thornton model, rate variation among sites, constant rate of change, global rearrangements, randomized input order of sequences with three jumbles. One hundred bootstrap resamplings were performed to assess the support for individual branches. Bayseian analysis of the dataset was done with the MRBAYES software (University of California, San Diego, Calif.; Huelsenbeck (2001) Bioinformatics 17:754). Four simultaneous MCMC chains were run for two hundred thousand generations after the convergence of the likelihood values, using the default settings of the program. A 50% majority-rule consensus tree was generated based on the resulted 2000 trees and the bipartition values (percentage representation of a particular clade) were recorded at the nodes. The program PAUP 4.*™ (see, e.g., Swafford (1996) PAUP*. Sinauer Associates, Sunderland, Mass.) was used for the parsimony analysis. The alignment was sampled for 500 bootstrap replicates. Each bootstrap replicate was analyzed with ten random addition sequence replicates with TBR branch swapping and equal weighting for all sites.

Nanoarchaeum genome, Nanoarchaeum polypeptides and nucleic acids encoding them and methods for making and using them

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