Helicobacter aminoacyl-tRNA synthetase proteins, nucleic acids and strains comprising same

BACKGROUND OF THE INVENTION
H. pylori is a gram-negative, microaerophilic bacterium that infects the gastric mucosa of humans, and is one of the most common infections in humans worldwide. In industralized countries, about 50% of population is infected with H. pylori during their lifetime, and in nonindustrialized countries, this ratio is even higher (for review, see Goodwin, C. S. and B. W. Worsley (eds.), 1993, Helicobacter pylori: Biology and Clinical Practice, (CRC Press, Boca Raton, Fla.); Hunt, R. H. and G. N. J. Tytgat (eds.), 1994, Helicobacter pylori: Basic Mechanisms to Clinical Cure, (Kluwer Academic Publishers, Dordrecht, Netherlands).
H. pylori infection has been invariably found associated with chronic gastritis (Warren, J. D. and B. J. Marshall, Lancet i: 1273-1275 (1983); Marshall, B. J. and J. R. Warren, Lancet ii: 1311-1314 (1984)). Administration of Helicobacter species to humans and to animals leads to development of gastritis (Marshall, B. J. et al., Med. J. Aust. 142: 436-439 (1985); Morris, A. and G. Nicholson, Am. J. Gastroenterol. 82: 192-199 (1987); Krakowka, S. et al., Infect. Immun. 55: 2789 (1987)). H. pylori infection is also responsible for about 80% of gastric ulcers, and almost 100% of duodenal ulcers. Successful treatment of the infection leads to healing of the diseases (Coghlan, J. G. et al., Lancet ii: 1109-1111 (1987); Labenz, J. et al., Am. J. Gastroenterol., 88: 491-495 (1993). In addition, various studies found that H. pylori infection increases gastric cancer by 4 to 6-fold (Correa, P., Cancer Research 52: 6735-6740 (1992); Nomura, A. et al., N. Engl. J. Med., 325: 1132-1136 (1991); Parsonnet, J. et al., J. Natl. Cancer Inst., 83: 640-643 (1991)).
The current most commonly used therapies to eradicate H. pylori infection are so-called triple therapies, in which patients are administered two different antibiotics and an anti-acid secretion drug simultaneously (for review, see O'Morain, C. and H. Lamouliatte, 1994, "Eradication", In: The year in Helicobacter pylori, P. Malfertheiner et al., (Eds.), (Current Science Ltd., London, UK) pp. 46-52). The efficacy of the therapies varies from clinical center to clinical center. Resistance to current antibiotics, in particular to metronidazole and other commonly used antibiotics for eradication of H. pylori infection, is well documented (for review, see Goodwin, C.S., 1993, "The susceptibility of Helicobacter pylori to antibiotics", In: Helicobacter pylori: biology and clinical practice, Goodwin, C. S. and B. W. Worsley (Eds.), (CRC Press, Boca Raton, Fla.) pp. 343-349). In addition, side effects of current antibiotic therapies are quite common and sometimes cause termination of the therapies before complete healing of the infection. Because of the development of resistance to antibiotics and adverse side-effects of current therapies for helicobacter infection, there is a continuing need for new drug targets and new antibiotics.
SUMMARY OF THE INVENTION
The invention relates to isolated and/or recombinant nucleic acids which encode aminoacyl-tRNA synthetases of helicobacter origin. The invention also relates to recombinant DNA constructs and vectors containing DNA having a sequence which encodes an aminoacyl-tRNA synthetase of helicobacter origin, or portions of the enzyme. These nucleic acids and constructs can be used to produce recombinant aminoacyl-tRNA synthetases of helicobacter origin.
A further embodiment of the invention is antisense nucleic acid which can hybridize to the nucleic acid which encodes an aminoacyl-tRNA synthetase of helicobacter. In cells, antisense nucleic acid can inhibit the function of an RNA which encodes an aminoacyl-tRNA synthetase of helicobacter.
The invention also relates to proteins or polypeptides, referred to herein as isolated and/or recombinant helicobacter aminoacyl-tRNA synthetases. These proteins are useful in the identification of inhibitors of aminoacyl-tRNA synthetase function (including inhibitors having antimicrobial activity), in biochemical separations of the amino acid which they specifically recognize, and in quantitations of the amino acid and ATP. Antibodies which bind to these enzymes can be made and can be used in the purification and study of the enzyme.
The recombinant helicobacter aminoacyl-tRNA synthetases can be produced in host cells using cells and methods described herein. Tester strains, which are cells engineered to rely on the function of the tRNA synthetase encoded by an introduced cloned gene, are also an embodiment of the invention. Tester strains can be used to test the effectiveness of drug candidates in the inhibition of the essential tRNA synthetase enzyme encoded by the introduced cloned gene. In this way, potential inhibitors of the enzyme can be screened for antimicrobial or antibiotic effects, without requiring the culture of pathogenic strains of helicobacter, such as Helicobacter pylori.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph illustrating the aminoacylation activity (cpm, counts per minute of �.sup.3 H!isoleucyl-tRNA) over time (minutes) of the GST-H. pylori Ile tRNA synthetase as compared with that of purified E. coli Ile tRNA synthetase, using crude total E. coli tRNA as a substrate (Example 6). O, E. coli IleRS; .quadrature., GST-H. pylori Ile tRNA synthetase; .diamond., GST-H. pylori-Ile tRNA synthetase minus tRNA.
FIG. 2 is a graph illustrating the aminoacylation activity (cpm, counts per minute of �.sup.3 H!methionyl-tRNA) over time (minutes) of the GST-H. pylori Met tRNA synthetase as compared with that of purified E. coli Met tRNA synthetase, using E. coli tRNA.sup.fMet as a substrate (Example 6). .largecircle., E. coli MetRS; .quadrature., GST-H. pylori Met tRNA synthetase; .diamond., no enzyme; X, GST-H. pylori-Ile tRNA synthetase minus tRNA.
FIG. 3 is a graph illustrating the aminoacylation activity (cpm, counts per minute of �.sup.3 H!seryl-tRNA) over time of the GST-H. pylori Ser tRNA synthetase as compared with that of partially purified E. coli Ser tRNA synthetase, using 90 .mu.M E. coli total tRNA (Boehringer-Mannheim) as a substrate (Example 8). .largecircle., E. coli SerRS; .circle-solid., GST-H. pylori Ser tRNA synthetase; A, GST-H. pylori-Ser tRNA synthetase minus tRNA.
FIG. 4 is a graph illustrating the aminoacylation activity (cpm, counts per minute of �.sup.3 H!lysyl-tRNA) over time of the GST-H. pylori Lys tRNA synthetase as compared with that of partially purified E. coli Lys tRNA synthetase, using 90 .mu.M E. coli total tRNA (Boehringer-Mannheim) as a substrate (Example 8). .diamond-solid., E. coli LysRS; .circle-solid., GST-H. pylori Lys tRNA synthetase; A, GST-H. pylori-Lys tRNA synthetase minus tRNA.
FIG. 5 is a schematic diagram of the structure of vector pQB211, a construct which directs the expression of an H. pylori lysyl-tRNA synthetase in S. cerevisiae, and includes a presequence for targeting the protein encoded by the inserted gene to mitochondria. (The circular plasmid is presented in linear form; PADH, alcohol dehydrogenase (ADH1) gene promoter; CoxIV, sequence encoding the first 22 amino acids of the cytochrome oxidase IV presequence; Hp Lys, H. pylori lysyl-tRNA synthetase; 2.mu. Ori, 2 micron origin of replication; Leu2, LEU2 selectable marker; AP.sup.R, ampicillin resistance).

DETAILED DESCRIPTION OF THE INVENTION
The aminoacyl-tRNA synthetases are enzymes with the common general function of catalyzing the following reaction:
aaRS+aa+ATP.rarw..fwdarw.aaRS.aa-AMP+PP.sub.i aaRS.aa-AMP+tRNA.rarw..fwdarw.aa-tRNA+aaRS+AMP
(aaRS=aminoacyl-tRNA synthetase; aa=amino acid; ATP=adenosine 5'-triphospate; AMP=adenosine 5'-monophosphate; PP.sub.i =inorganic pyrophosphate) The second (aminoacylation) step is often referred to as "charging" the tRNA.
Generally, in each bacterial organism, there are 20 aminoacyl-tRNA synthetases, each specific for a different amino acid. Eucaryotic organisms also typically encode 20 cytoplasmic aaRSs, one specific for each amino acid. In addition, eucaryotic organisms generally encode a separate set of mitochondrial aaRSs. In the yeast Saccharomyces cerevisiae, the cytoplasmic and mitochondrial enzymes are encoded by separate nuclear genes, with the exception of histidyl- and valyl-tRNA synthetases (Natsoulis, G., et al. Cell 46:235-243 (1986); Chatton, B. et al., J. Biol. Chem. 263:52-57 (1988)). Each aminoacyl-tRNA synthetase enzyme recognizes and reacts with a specific amino acid and with one or more tRNAs that recognize the codons specific for that amino acid (cognate tRNAs). The specificity of the aaRS for the amino acid is determined by protein-amino acid interactions, and the specificity of the aaRS for the tRNA is determined by protein-RNA interactions, using different sites on the aaRS.
The tRNA synthetases can be subdivided into two groups of enzymes, class I and class II, based on short regions of sequence homology as well as distinct active site core tertiary structures (Eriani, G., et al., Nature 347:203-206 (1990); Moras, D., Trends Biochem. Sci. 17:159-164 (1992)). The twenty-one aminoacyl-tRNA synthetases from E. coli have been divided into two classes (see, e.g., Burbaum, J. J. and P. Schimmel, J. Biol Chem. 266(26) :16965-16968 (1991)).
Nucleic Acids, Constructs and Vectors
The present invention relates to isolated and/or recombinant (including, e.g., essentially pure) nucleic acids having sequences which encode a helicobacter aminoacyl-tRNA synthetase, or a portion of a helicobacter aminoacyl-tRNA synthetase. In one embodiment, the nucleic acid or portion thereof encodes a protein or polypeptide having at least one function characteristic of a helicobacter aminoacyl-tRNA synthetase specific for a selected amino acid, such as a catalytic activity (e.g., catalysis of aminoacyl-adenylate formation, catalysis of aminoacylation of a tRNA with the amino acid), and/or binding function (e.g., tRNA-, amino acid- or ATP-binding), and/or antigenic function (e.g., binding of antibodies that also bind to non-recombinant helicobacter aaRS), and/or oligomerization function. Oligomerization activity is the ability of a protein subunit or protein fragment to bind together with one or more other protein subunits or protein fragments, thus altering the quaternary structure of the resulting complex. For example, "adhesive" fragments with oligomerization activity can bind to another fragment with no catalytic activity of its own to restore or partially restore enzymatic activity (Jasin, M., et al., U.S. Pat. No. 4,952,501). The present invention also relates more specifically to isolated and/or recombinant nucleic acids or a portion thereof having sequences which encode an aminoacyl-tRNA synthetase of Helicobacter pylori origin, or a portion thereof.
The invention further relates to isolated and/or recombinant nucleic acids that are characterized by (1) their ability to hybridize to (a) a nucleic acid encoding a helicobacter aminoacyl-tRNA synthetase specific for a selected amino acid, such as a nucleic acid having the sequence of SEQ ID NO:1 (having a GTG initiation codon as shown, or an ATG initiation codon as appropriate), SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11, (b) the complement of any one of (a), or (c) portions of either of the foregoing (e.g., a portion comprising the open reading frame); or (2) by their ability to encode a polypeptide having the amino acid sequence of a helicobacter aminoacyl-tRNA synthetase (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12), or functional equivalents thereof (e.g., a polypeptide which aminoacylates the isoaccepting cognate aminoacyl-tRNAs (such as tRNA.sup.Ile, tRNA.sup.Met, tRNA.sup.Leu, tRNA.sup.Val, tRNA.sup.Lys or tRNA.sup.Ser of H. pylori) with a selected amino acid); or (3) by both characteristics. In one embodiment, the percent amino acid sequence similarity between the polypeptides encoded by SEQ ID NO:2, 4, 6, 8, 10, and 12, and the respective functional equivalents of these polypeptides is at least about 80% (.gtoreq.80%). In a preferred embodiment, the percent amino acid sequence similarity between the polypeptides encoded by SEQ ID NO:2, 4, 6, 8, 10, and 12, and their respective functional equivalents is at least about 85% (.gtoreq.85%). More preferably, the percent amino acid sequence similarity between the polypeptides encoded by SEQ ID NO:2, 4, 6, 8, 10, and 12, and their respective functional equivalents is at least about 90%, and still more preferably, at least about 95%.
Isolated and/or recombinant nucleic acids meeting these criteria comprise nucleic acids having sequences identical to sequences of naturally occurring helicobacter aaRS genes and portions thereof, or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues are modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.
Such nucleic acids can be detected and isolated under high stringency conditions or moderate stringency conditions, for example. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 (see particularly 2.10.8-11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 1, Suppl. 26, 1991), the teachings of which are hereby incorporated by reference. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Thus, high or moderate stringency conditions can be determined empirically, depending in part upon the characteristics of the known DNA to which other unknown nucleic acids are being compared for sequence similarity.
Isolated and/or recombinant nucleic acids that are characterized by their ability to hybridize to (a) a nucleic acid encoding a helicobacter aminoacyl-tRNA synthetase (for example, those nucleic acids depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11, (b) the complement of such nucleic acids, (c) or a portion thereof (e.g. under high or moderate stringency conditions), may further encode a protein or polypeptide having at least one function characteristic of a helicobacter aminoacyl-tRNA synthetase specific for a selected amino acid, such as a catalytic activity (e.g., aminoacyl-adenylate formation, aminoacylation of a tRNA with amino acid), binding function (e.g., tRNA-, amino acid-, or ATP-binding), antigenic function (e.g., binding of antibodies that also bind to non-recombinant helicobacter aaRS), and/or oligomerization function. The catalytic or binding function of a protein or polypeptide encoded by hybridizing nucleic acid may be detected by standard enzymatic assays for activity or binding (e.g., assays which monitor aminoacyl-adenylate formation, aminoacylation of tRNA). Functions characteristic of the aminoacyl-tRNA synthetase may also be assessed by in vivo complementation activity or other suitable methods. Enzymatic assays, complementation tests, or other suitable methods can also be used in procedures for the identification and/or isolation of nucleic acids which encode a polypeptide such as a polypeptide of the amino acid sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, or functional equivalents of these polypeptides. The antigenic properties of proteins or polypeptides encoded by hybridizing nucleic acids can be determined by immunological methods employing antibodies that bind to a helicobacter aminoacyl-tRNA synthetase, such as immunoblot, immunoprecipitation and radioimmunoassay.
Nucleic acids of the present invention can be used in the production of proteins or polypeptides. For example, DNA containing all or part of the coding sequence for a helicobacter aminoacyl-tRNA synthetase such as lysyl-tRNA synthetase, or DNA which hybridizes to DNA having the sequence SEQ ID NO:9, can be incorporated into various constructs and vectors created for further manipulation of sequences or for production of the encoded polypeptide in suitable host cells. For expression in E. coli and other organisms, a GTG initiation codon can be altered to ATG as appropriate.
Nucleic acids referred to herein as "isolated" are nucleic acids separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. "Isolated" nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids which are isolated. Nucleic acids referred to herein as "recombinant" are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial recombination, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. "Recombinant" nucleic acids are also those that result from recombination events that occur through the natural mechanisms of cells, but are selected for after the introduction to the cells of nucleic acids designed to allow and make probable a desired recombination event.
Portions of the isolated nucleic acids which code for polypeptides having a certain function can be identified and isolated by, for example, the method of Jasin, M., et al., U.S. Pat. No. 4,952,501. The aminoacyl-tRNA synthetases are known to have different quaternary structures, including both monomeric and multimeric structures (e.g., homodimers, tetramers and heteromultimeric .alpha..sub.2 .beta..sub.2 forms) . Thus, as used herein, a nucleic acid which encodes a portion of a helicobacter aminoacyl-tRNA synthetase can also refer to one of two or more distinct subunits of said tRNA synthetase.
A further embodiment of the invention is antisense nucleic acid, which is complementary, in whole or in part, to a target molecule comprising a sense strand, and can hybridize with the target molecule. The target can be DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart). When introduced into a cell, antisense nucleic acid can inhibit the expression of the gene encoded by the sense strand. Antisense nucleic acids can be produced by standard techniques.
In a particular embodiment, the antisense nucleic acid is wholly or partially complementary to and can hybridize with a target nucleic acid, wherein the target nucleic acid can hybridize to a nucleic acid having the sequence of the complement of the top strand shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11). For example, antisense nucleic acid can be complementary to a target nucleic acid having the sequence shown as the top strand of the open reading frame in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, or to a portion thereof sufficient to allow hybridization. In another embodiment, the antisense nucleic acid is wholly or partially complementary to and can hybridize with a target nucleic acid which encodes a helicobacter aminoacyl-tRNA synthetase.
H. pylori is the most important human pathogen among helicobacter species. Because advances in the understanding and treatment of H. pylori infection would be of tremendous benefit, it was the species selected for most of the experimental work described herein. As described in the Exemplification, PCR fragments of H. pylori aaRS genes were isolated, cloned and used as probes to screen an ordered cosmid genomic library of H. pylori (Bukanov, N. O. and D. E. Berg, Mol. Microbiol. 11(3): 509-523 (1994)). Nucleic acids encoding complete aminoacyl-tRNA synthetases from H. pylori were isolated and characterized. These isolated genes, encoding isoleucyl-, methionyl-, leucyl-, valyl-, lysyl- and seryl-tRNA synthetases, are representatives of a broader class of helicobacter aminoacyl-tRNA synthetase genes, including synthetase genes encoding enzymes specific for each amino acid and derived from various species of helicobacter. These additional genes can also be used to express helicobacter aminoacyl-tRNA synthetases, with utilities corresponding to those described herein, and can be used in the production of host cells and tester strains comprising recombinant helicobacter aminoacyl-tRNA synthetase genes using methods described herein. The approaches described herein, including, but not limited to, the approaches to isolate and manipulate the isoleucyl-, methionyl-, leucyl-, valyl-, lysyl- and seryl-tRNA synthetase genes of H. pylori, to construct vectors and host strains, and to produce and use the proteins, to produce antibodies, etc., can be applied to other members of the genus Helicobacter, including, but not limited to, pathogenic species such as H. cinaedi and H. fennelliae, intestinal organisms which cause diarrheal illnesses (particularly in HIV-infected individuals), H. mustelae (in ferrets), H. felis (in dogs and cats), H. muridarum (in mice), H. nemestrinae (in nonhuman primates), and H. acinonyx (in cheetahs). For example, the H. pylori aminoacyl-tRNA synthetase genes described here, or sufficient portions thereof, whether isolated and/or recombinant or synthetic, including fragments produced by PCR, can be used as probes or primers to detect and/or recover homologous genes of the other helicobacter species (e.g., by hybridization, PCR or other suitable techniques). Similarly, genes encoding H. pylori aminoacyl-tRNA synthetases specific for other amino acids can be isolated from an ordered cosmid library, other genomic libraries (e.g., libraries constructed in .lambda.gt11 and pbluescript SK as described by Covaccio et al., Proc. Natl. Acad. Sci. USA, 90: 5791-5795 (1993), or lambda ZAPII as described by Evans et al., J. Bacteriol., 175: 674-683 (1993) and Tummuru et al., Infection and Inmunity, 61: 1799-1809 (1993) ) or other Helicobacter libraries, according to methods described herein or other suitable methods.
Proteins
The invention also relates to proteins or polypeptides encoded by nucleic acids of the present invention. The proteins and polypeptides of the present invention can be isolated and/or recombinant. Proteins or polypeptides referred to herein as "isolated" are proteins or polypeptides purified to a state beyond that in which they exist in cells. "Isolated" proteins or polypeptides include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, including essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis, or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. Proteins or polypeptides referred to herein as "recombinant" are proteins or polypeptides produced by the expression of recombinant nucleic acids.
In a preferred embodiment, the protein or portion thereof has at least one function characteristic of a helicobacter aminoacyl-tRNA synthetase specific for a selected amino acid, for example, catalytic activity (e.g., catalysis of aminoacyl-adenylate formation, catalysis of aminoacylation of a tRNA with amino acid), binding function (e.g., tRNA-, amino acid-, or ATP-binding), antigenic function (e.g., binding of antibodies that also bind to non-recombinant helicobacter aminoacyl-tRNA synthetase), and/or oligomerization activity. As such, these proteins are referred to as aminoacyl-tRNA synthetases of helicobacter origin or helicobacter aminoacyl-tRNA synthetases, and include, for example, naturally occurring helicobacter aminoacyl-tRNA synthetases, variants (e.g. mutants) of those proteins and/or portions thereof. Such variants include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides in which one or more residues are modified, and mutants comprising one or more modified residues.
In a particularly preferred embodiment, like naturally occurring helicobacter aminoacyl-tRNA synthetases, isolated and/or recombinant helicobacter aminoacyl-tRNA synthetases of the present invention aminoacylate the isoaccepting cognate tRNAs of the helicobacter organism with a selected amino acid in a two-step reaction. For example, in the case of H. pylori, an isolated, recombinant lysyl-tRNA synthetase is able to aminoacylate each of the isoaccepting species of cognate tRNA.sup.Lys of H. pylori with lysine. In the first step, the lysyl-tRNA synthetase catalyzes the covalent linkage of lysine to ATP to form an adenylate complex (lysyl-adenylate) with the release of pyrophosphate, and, in a second step, catalyzes the covalent linkage of lysine to a specific tRNA recognized by the enzyme, releasing AMP.
The invention further relates to fusion proteins, comprising a helicobacter aminoacyl-tRNA synthetase (as described above) as a first moiety, linked to second moiety not occurring in the helicobacter aaRS as found in nature. Thus, the second moiety can be an amino acid or polypeptide. The first moiety can be in an N-terminal location, C-terminal location or internal to the fusion protein. In one embodiment, the fusion protein comprises a H. pylori aminoacyl-tRNA synthetase as the first moiety, and a second moiety comprising a linker sequence and affinity ligand.
Fusion proteins can be produced by a variety of methods. For example, a fusion protein can be produced by the insertion of an aaRS gene or portion thereof into a suitable expression vector, such as Bluescript SK +/- (Stratagene), pGEX-4T-2 (Pharmacia) and pET-15b (Novagen). The resulting construct is then introduced into a suitable host cell for expression. Upon expression, fusion protein can be purified from a cell lysate by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)).
The invention also relates to isolated and/or recombinant portions of an aminoacyl-tRNA synthetase of helicobacter origin. For example, a portion of an aminoacyl-tRNA synthetase can also refer to one of two or more distinct subunits of said tRNA synthetase. Portions of the enzyme can be made which have full or partial function on their own, or which when mixed together (though fully, partially, or nonfunctional alone), spontaneously assemble with one or more other polypeptides to reconstitute a functional protein having at least one function characteristic of an aminoacyl-tRNA synthetase. (See, e.g., Shiba, K. and Schimmel, P., J. Biol. Chem. 267:22703-22706 (1992) for an example of three inactive peptides from E. coli IleRS spontaneously assembling in vivo to reconstitute active enzyme; see also, Burbaum, J. and Schimmel, P., Biochemistry 30(2): 319-324 (1991), describing non-overlapping segments of E. coli MetRS that can fold together to reconstitute an active enzyme capable of recognizing and charging tRNA in vitro and in vivo; see also Jasin, M. et al. (U.S. Pat. No. 4,952,501) describing deletion studies of E. coli alanyl-tRNA synthetase which showed that large portions of the protein were unnecessary for specific aminoacylation activity.) Based on this type of analysis, portions of a helicobacter aaRS can be made which have at least one function characteristic of a helicobacter aminoacyl-tRNA synthetase, such as a catalytic function, binding function, antigenic function and/or oligomerization function. Studies on the structure and function of the aaRSs provide the basis for being able to divide the helicobacter aaRS enzymes into functional domains (Schimmel, P., Current Biology 1:811-816 (1991)).
The sequences and structures of the catalytic domains of several tRNA synthetases which have been purified and studied led to the identification of two distinct classes designated class I and class II (Schimmel, P., Ann. Rev. Biochem. 56:125-158 (1987); Webster, T. A., et al., Science 226:1315-1317 (1984); Eriani, G. et al , Nature 347:203-206 (1990) and Cusack, S., et al., Nature 347:249-255 (1990)). Class I enzymes have a well-conserved N-terminal nucleotide binding fold responsible for amino acid binding, aminoacyl-adenylate formation, and tRNA acceptor helix docking. The N-terminal nucleotide binding fold is comprised of alternating .beta.-strands and .alpha.-helices. The C-terminal domain is rich in .alpha.-helices and contains residues needed for interactions with the parts of the tRNA distal to the amino acid attachment site (Shepard, A., et al., Proc. Natl. Acad. Sci. U.S.A. 89:9964-9968 (1992); Hou, Y.-M.,et al., Proc. Natl. Acad. Sci. U.S.A. 88:976-980 (1991)). Five enzymes--cysteinyl-, isoleucyl-, leucyl-, methionyl-, and valyl-tRNA synthetases--have been grouped together because they are more closely related in sequence and arrangement of their domains to each other than to the other five members of class I (Hou, Y.-M., et al., Proc. Natl. Acad. Sci. U.S.A. 88:976-980 (1991); Eriani, G., et al., Nucleic Acids Res. 19:265-269 (1991)). Furthermore, the C-terminal domains of isoleucyl-, leucyl-, methionyl-, cysteinyl- and valyl-tRNA synthetases appear to have a common origin, which is distinct from the C-terminal domain found in other class I enzymes (Shiba, K., et al., Proc. Natl. Acad. Sci. USA 89:1880-1884 (1992); Shepard, A., et al., Proc. Natl. Acad. Sci. U.S.A. 89:9964-9968 (1992)). In E. coli, these five enzymes of class I vary in size from 461 to 951 amino acids and are active as monomers. The size variation is in large part explained by the variability in the lengths of the two insertions designated connective polypeptide 1 (CP1), which is inserted between the second .alpha.-helix and third .beta.-strand of the nucleotide binding fold, and CP2, which is placed between the third a-helix and fourth .beta.-strand (Starzyk, R. M., et al., Science 237:1614-1618 (1987)). In all of these enzymes, CP1 is the larger of the two insertions and varies in E. coli from 61 in cysteinyl-tRNA synthetase to 300 amino acids in isoleucyl-tRNA synthetase (Hou, Y.-M., et al., Proc. Natl. Acad. Sci. USA 88:976-980 (1991)). While a portion of CP1 may be deleted from isoleucyl-tRNA synthetase without loss of function (Starzyk, R. M., et al., Science 237:1614-1618 (1987)), this insertion is known to facilitate acceptor helix interactions in the related glutaminyl-tRNA synthetase whose three dimensional structure in complex with tRNA.sup.Gln has been determined by X-ray crystallography (Rould, M. A et al., Science 246:1135-1142 (1989)). In some tRNA synthetases, this second domain interacts directly with the anticodon (Rould, M. A. et al., Science 246:1135-1142 (1989) and Cavarelli, J., et al., Nature 362:181-184 (1993)), while in other enzymes there is no contact made between the second domain and the anticodon (Biou, V.,et al., Science 263:1404-1410 (1994)). To a first approximation, the two domains in class I tRNA synthetases interact with the two distinct domains of the L-shaped tRNA structure. Thus, the recognition elements of the tRNA synthetase and of the tRNA which are needed for the operational RNA code are segregated into discrete protein and RNA domains.
The primary sequence of the class II enzymes is generally characterized by three conserved motifs. These motifs are designated in the order they occur in the sequence as motif 1, motif 2, and motif 3. Although the motifs have a conserved core, they vary in length and are marked by as little as a single invariant amino acid residue. The motif sequences are defined as follows:
Motif 1: g.PHI.xx.PHI.xxP.PHI..PHI.
Motif 2: (F/Y/H)Rx(E/D)(4-12x)(R/H)xxxFxxx(D/E)
Motif 3: .lambda.x.PHI.g.PHI.g.PHI.eR.PHI..PHI..PHI..PHI..PHI.
The abbreviations are: x, variant; .PHI., hydrophobic; and .lambda., small amino acids. Lower case letters indicate that the amino acid is partially conserved. None of these motifs are found in the class I family. With the exception of E. coli Gly- and Phe-tRNA synthetases which only contain a discernible motif 3, class II enzymes characterized to date incorporate all three motifs (Ribas de Pouplana, L. et al., Protein Science 2:2259-2262 (1993)).
The second class of tRNA synthetases was firmly defined when the crystal structure of the E. coli Ser-tRNA synthetase active site was shown to have no relationship to the Rossmann fold of class I enzymes (Cusack, S. C., et al., Nature 347:249-255 (1990)). X-ray diffraction investigations with an ATP-bound Ser-tRNA synthetase co-crystal from T. thermophilus revealed the details of a novel ATP binding site (Cusack, S., et al., In The Translational Apparatus, K. H. Nierhaus et al., eds., Plenum Press, New York, pp. 1-9, 1993; Belrhali, H., et al., Science 263:1432-1436 (1994); Biou, V., et al., Science 263:1404-1410 (1994)).
Motif 3 is comprised of a .beta.-strand followed by an .alpha. helix and is characterized by a GLER sequence. This motif is the only one that has been universally detected in all of the class II enzymes studied. The crystal structures of yeast Ser- and Asp- (Ruff, M. S. et al., Science 252:1682-1689 (1991)) tRNA synthetases suggest a role for motif 3 in amino acid and ATP binding. Mutations in this region have resulted in a reduction in binding and/or a high K.sub.m for amino acid or ATP binding (Eriani, G., et al., Nature 347:203-206 (1993); Anselme, J. and Hartlein, M., FEBS Lett. 280:163-166 (1991); Kast, P. and Hennecke, H., J. Mol. Biol., 222:99-124 (1991); Kast, P. et al., FEBS Lett. 293:160-163 (1991); Lanker, S., et al., Cell 70:647-657 (1992)).
Yeast Asp-tRNA synthetase was the first class II enzyme to be co-crystallized with its cognate tRNA (Ruff, M., et al., Science 252:1682-1689 (1991)). The yeast Asp-tRNA synthetase contains a nucleotide binding structure similar to that found in Ser-tRNA synthetase. The combination of these two class II crystal structures provides a model for the active sites of all of the class II tRNA synthetases.
Because motif 1 is at the dimer interface in the crystal structures of yeast Asp-tRNA synthetase (Ruff, M. S., et al., Science 252:1682-1689 (1991)), E. coli Ser-tRNA synthetase (Cusack, S., et al., Nature 347:249-255 (1990); Cusack, S., et al., In The Translational Apparatus, K. H. Nierhaus et al., eds., Plenum Press, New York, pp. 1-9, 1993; Price, S., et al., FEBS Lett. 324:167-170 (1993)) and T. thermophilus Ser-tRNA synthetase (Cusack, S., et al., In The Translational Apparatus, K. H. Nierhaus et al., eds., Plenum Press, New York, pp. 1-9, 1993; Belrhali, H., et al., Science 263:1432-1436 (1994); Biou, V., et al., Science 263:1404-1410 (1994)), motif 1 was thought to be important for dimerization. This motif was identified in the N-terminal region of E. coli Ala-tRNA synthetase (Ribas de Pouplana, et al., Protein Science 2:2259-2262 (1993)), but a series of deletion mutations had also previously demonstrated that a region at the C-terminus of the protein is needed for oligomerization (Jasin, M., et al., Nature 306:441-447 (1983); Jasin, et al., Cell 36:1089-1095 (1984)). Thus, motif 1 is not sufficient for oligomerization of this enzyme.
An idiographic representation of the predicted eight-stranded .beta.-structure with three a-helices of the E. coli Ala-tRNA synthetase has been constructed (Ribas de Pouplana, L., et al., Protein Science 2:2259-2262 (1993)); Shi, J.-P., et al., Biochemistry 33:5312-5318 (1994)).
Collectively, over 40 mutations in motif 2 and the region between motif 2 and 3 were individually constructed and tested (Davis, M. W., et al., Biochemistry 33:9904-9911 (1994); Shi, J.-P., et al., Biochemistry 33:5312-5318 (1994)). These mutations were mostly at conserved residues with chemical functional groups. Although motif 2 is of a different size and has only two identical amino acid residues with its counterpart in yeast Asp- and T. thermophilus Ser-tRNA synthetases, the mutational analysis of this motif can be explained in terms of those structures, and shows the importance of predicted motif 2 for adenylate synthesis (Ribas de Pouplana, L., et al., Protein Science 2:2259-2262 (1993)). A study of the products of random mutagenesis of this region also demonstrated the importance of motif 2 for adenylate transfer (Lu, Y. and Hill, K. A. W., J. Biol. Chem. 269:12137-12141 (1994)). Mutagenesis of specific residues in motif 2 of E. coli Ala-tRNA synthetase and mutagenesis of their predicted counterparts in motif 2 of yeast Asp-tRNA synthetase yielded similar results with regard to loss of function (Cavarelli, J., et al., EMBO J. 13:327-337 (1994); Davis, M. W., et al., Biochemistry 33:9904-9911 (1994)). Evidence was obtained for sequence context determining how the energy of adenylate binding is partitioned between ground and transition states in the two enzymes. In addition, a conserved aspartate residue among Ala-tRNA synthetases at the beginning of motif 3 was shown to be important for the adenylate synthesis and particularly for the adenylate transfer reaction (Davis, M. W., et al., Biochemistry 33:9904-9911 (1994)). The functional significance of motif 3 for adenylate synthesis has been demonstrated by mutagenesis in the yeast Asp-tRNA synthetase system (Cavarelli, J., et al., EMBO J. 13:327-337 (1994)).
Upon consideration of this information, with the remaining teachings of the specification, H. pylori tRNA synthetase derivatives can be constructed which possess at least one function characteristic of a helicobacter aminoacyl-tRNA synthetase.
Method of Producing Recombinant aaRSs
Another aspect of the invention relates to a method of producing a helicobacter aminoacyl-tRNA synthetase or a portion thereof, and to expression systems and host cells containing a vector appropriate for expression of a helicobacter aminoacyl-tRNA synthetase.
Cells that express a recombinant aminoacyl-tRNA synthetase or a portion thereof can be made and maintained in culture to produce protein for isolation and purification. These cells can be procaryotic or eucaryotic. Examples of procaryotic cells that can be used to express helicobacter aminoacyl-tRNA synthetases include Escherichia coli, Bacillus subtilis and other bacteria. Examples of eucaryotic cells that can be used to express the aminoacyl-tRNA synthetases include yeasts such as Saccharomyces cerevisiae, S. pombe, Pichia pastoris, and other lower eucaryotic cells, as well as cells of higher eucaryotes, such as those from insects and mammals. (See, e.g., Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., (1993)).
To make host cells that produce a recombinant aaRS protein or portion thereof for isolation and purification, as a first step the gene encoding the enzyme can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon, which can be present in a single copy or multiple copies, or the gene can be integrated in a host cell chromosome. Such a suitable replicon contains all or part of the coding sequence for aminoacyl-tRNA synthetase operably linked to one or more expression control sequences whereby the coding sequence is under the control of transcription signals and linked to appropriate translation signals to permit translation of the aaRS, portion thereof, or of a fusion protein comprising an aaRS or portion thereof. As a second step, the vector can be introduced into cells by a method appropriate to the type of host cells (e.g., transformation, electroporation, infection). In a third step, for expression from the aaRS gene, the host cells can be maintained under appropriate conditions, e.g., in the presence of inducer, normal growth conditions, etc.).
As a particular example of the above approach to producing active helicobacter aminoacyl-tRNA synthetase, a gene encoding the H. pylori aaRS can be integrated into the genome of a virus that enters the host cells. By infection of the host cells, the components of a system which permits the transcription and translation of the helicobacter aaRS gene are present in the host cells. Alternatively, an RNA polymerase gene, inducer, or other component required to complete such a gene expression system may be introduced into the host cells already containing the helicobacter aaRS gene, for example, by means of a virus that enters the host cells and contains the required component. The aaRS gene can be under the control of an inducible or constitutive promoter. The promoter can be one that is recognized by the host cell RNA polymerase. The promoter can, alternatively, be one that is recognized by a viral RNA polymerase and is transcribed following infection of the host cells with a virus.
Antibodies
The invention further relates to antibodies that bind to an isolated and/or recombinant helicobacter aminoacyl-tRNA synthetase, including portions of antibodies (e.g., a peptide), which can specifically recognize and bind to the tRNA synthetase. These antibodies can be used in methods to purify the enzyme or portion thereof by various methods of immunoaffinity chromatography, or to selectively inactivate one of the enzyme's active sites, or to study other aspects of enzyme structure, for example.
The antibodies of the present invention can be polyclonal or monoclonal, and the term antibody is intended to encompass both polyclonal and monoclonal antibodies. Antibodies of the present invention can be raised against an appropriate immunogen, including proteins or polypeptides of the present invention, such as an isolated and/or recombinant helicobacter aminoacyl-tRNA synthetase or portion thereof, or synthetic molecules, such as synthetic peptides. The immunogen, for example, can be a protein having at least one function of a helicobacter aminoacyl-tRNA synthetase, as described herein.
The term antibody is also intended to encompass single chain antibodies, chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single chain antibodies, comprising portions from more than one species. For example, the chimeric antibodies can comprise portions of proteins derived from two different species, joined together chemically by conventional techniques or prepared as a contiguous protein using genetic engineering techniques (e.g., DNA encoding the protein portions of the chimeric antibody can be expressed to produce a contiguous protein chain). See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; and Winter, European Patent No. 0,239,400 B1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988)) regarding single chain antibodies.
Whole antibodies and biologically functional fragments thereof are also encompassed by the term antibody. Biologically functional antibody fragments which can be used include those fragments sufficient for binding of the antibody fragment to a helicobacter aaRS to occur, such as Fv, Fab, Fab' and F(ab').sub.2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For instance, papain or pepsin cleavage can generate Fab or F(ab').sub.2 fragments, respectively. Alternatively, antibodies can be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab').sub.2 heavy chain portion can be designed to include DNA sequences encoding the CH.sub.1 domain and hinge region of the heavy chain.
Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)). Generally, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as SP2/0) with antibody producing cells. The antibody producing cell, preferably those obtained from the spleen or lymph nodes, are obtained from animals immunized with the antigen of interest. The fused cells (hybridomas) are isolated using selective culture conditions, and cloned by limiting dilution. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Assays for Inhibitors and Tester Strains
The enzymatic assays, binding assays, and construction of tester strains described below, which rely upon the nucleic acids and proteins of the present invention, can be used, alone or in combination with each other or other suitable methods, to identify inhibitors of one or more helicoabacter aminoacyl-tRNA synthetases.
Enzyme Assay
Upon the isolation of an aaRS gene from helicobacter (as described herein), the gene can be incorporated into an expression system for production of the aaRS or a fusion protein, followed by isolation and testing of the enzyme in vitro. The isolated or purified helicobacter aaRSs can also be used in further structural studies that allow for the design of antibiotics which specifically target one or more aaRSs of helicobacter, while not affecting or minimally affecting host or mammalian (e.g., human) aaRSs. Because the amino acid sequences of the tRNA synthetases have diverged over evolution, significant differences exist between the structure of the enzymes from mammals (e.g., human, bovine) and mammalian pathogens, and the design or selection of inhibitors can exploit the structural differences between the pathogen aaRS and the host (e.g., a mammalian host, such a human) aaRS to yield specific inhibitors of the pathogen aaRS, which may further have antimicrobial activity.
Furthermore, isolated, active helicobacter aaRSs can be used in an in vitro method of screening for inhibitors of aminoacyl-tRNA synthetase activity in which the inhibitory effect of a compound is assessed by monitoring aaRS activity according to standard techniques. For example, inhibitors of the activity of isolated, recombinant H. pylori IleRS, MetRS, LeuRS, ValRS, LysRS or SerRS can be identified by the method. In one embodiment, the isolated aaRS enzyme is maintained under conditions suitable for aminoacyl-adenylate formation, the enzyme is contacted with a compound to be tested, and formation of the aminoacyl-adenylate is monitored by standard assay. A reduction in the activity measured in the presence of compound, as compared with the activity in the absence of compound, is indicative of inhibition of aminoacyl-tRNA synthetase activity by the compound. For example, the extent of isoleucyl-adenylate formation catalyzed by urified IleRS can be measured using an ATP-pyrophosphate exchange assay in the presence and in the absence of a candidate inhibitor (Calendar, R. and P. Berg, Biochemistry, 5:1690-1695 (1966)). In this reaction, the enzymatic synthesis of ATP from AMP and pyrophosphate in the absence of tRNA is monitored. A candidate inhibitor can be added to a suitable reaction mixture (e.g., 100 mM TrisCl, pH 7.5 / 5 mM MgCl.sub.2 / 10 mM 2-mercaptoethanol/ 10 mM KF / 2 mM ATP / 2 mM �.sup.32 p! pyrophosphate / 1 mM isoleucine), and the mixture is incubated at 25.degree. C. IleRS (to a final concentration of .about.10 nM) is added to initiate the reaction. Aliquots of the reaction are removed at various times and quenched in 7% (vol/vol) cold perchloric acid, followed by the addition of 3% (wt/vol) charcoal suspended in 0.5% HCl. The ATP adsorbed to charcoal is filtered onto glass fiber pads (Schleicher & Schuell), and formation of �.sup.32 P!ATP is quantified by liquid scintillation counting in Hydrofluor (National Diagnostics, Manville, N.J.). The enzyme activity measured in the presence of the compound is compared with the activity in the absence of the compound to assess inhibition. Alternatively, a candidate inhibitor can be preincubated with enzyme under suitable conditions. Preincubation in the absence of substrate provides a more sensitive assay for the detection of inhibition (e.g., detects slow binding inhibitors). For example, the compound can be added to a mixture containing .about.10 nM isoleucyl-tRNA synthetase in 100 mM TrisCl, pH 7.5 / 5 mM MgCl.sub.2 / 10 mM 2-mercaptoethanol/ 10 mM KF, and preincubated at 25.degree. C. for 20 minutes. To initiate the reaction, ATP, �.sup.32 P! pyrophosphate and isoleucine are added to final concentrations of 2 mM, 2 mM and 1 mM, respectively. The reaction is monitored as described above, and the activity measured in the presence of compound is compared with the activity in the absence of compound to assess inhibition.
In another embodiment, formation of the aminoacylated tRNA is monitored in a standard aminoacylation assay. Inhibitors identified by enzymatic assay can be further assessed for antimicrobial activity using tester strains as described herein, or using other suitable assays. For example, the extent of aminoacylation of tRNA with isoleucine catalyzed by IleRS (e.g., a GST fusion) can be measured by monitoring the incorporation of �.sup.3 H! isoleucine into trichloroacetic acid-precipitable �.sup.3 H! isoleucyl-tRNA in the presence of a candidate inhibitor, as compared with activity in the absence inhibitor. Appropriately diluted IleRS (.about.0.4 nM) can be preincubated for 20 minutes at 25.degree. C. in, for example, 50 mM HEPES, pH 7.5 / 0.1 mg/ml BSA (bovine serum albumin) / 10 mM MgCl.sub.2 / 10 mM 2-mercaptoethanol / 20 mM KCl / 1-20% DMSO (preferably about 1%) in the presence or absence of a compound to be tested. The preincubation mixture can be supplemented with ATP, �.sup.3 H!isoleucine and tRNA to final concentrations of, for example, 4 mM ATP/ 20 .mu.M �.sup.3 H!isoleucine (0.6 .mu.Ci), and 90 .mu.M crude tRNA or 2 .mu.M specific tRNA.sup.Ile. The reaction can be maintained at 25.degree. C., and aliquots are removed at specific times, and applied to filter paper discs (3 MM, Whatman) that have been presoaked with 5% (wt/vol) trichloroacetic acid. Filters are washed for three 10-minute periods in 5% trichloroacetic acid, rinsed in 95% ethanol and 100% ether, and the incorporation of .sup.3 H-isoleucine into tRNA (formation of .sup.3 H-Ile-tRNA) is measured in Betafluor by liquid scintillation counting. The aminoacylation assay can also be performed without preincubation under suitable conditions (e.g., using .about.0.4 nM IleRS in a reaction mixture containing 50 mM HEPES, pH 7.5 / 0.1 mg/ml BSA (bovine serum albumin) / 10 mM MgCl.sub.2 / 10 mM 2-mercaptoethanol / 20 mM KCl / 1-20% DMSO / 4 mM ATP/ 20 .mu.M �.sup.3 H!isoleucine (0.6 .mu.Ci), and 90 .mu.M crude tRNA or 2 .mu.M specific tRNA.sup.Ile) in the presence or absence of test compound. An IC.sub.50 value (the concentration of inhibitor causing 50% inhibition of enzyme activity) for a known amount of active IleRS can be determined.
Binding Assay
Isolated, recombinant aaRS or a portion thereof, and suitable fusion proteins can be used in a method to select and identify compounds which bind specifically to the aaRS, such as H. pylori isoleucyl-, methionyl-, leucyl-, valyl-, lysyl- or seryl-tRNA synthetase, and which are potential inhibitors of aaRS activity. Compounds selected by the method can be further assessed for their inhibitory effect on aaRS activity and for antimicrobial activity.
In one embodiment, an isolated or purified H. pylori aaRS can be immobilized on a suitable affinity matrix by standard techniques, such as chemical cross-linking, or via an antibody raised against the isolated or purified aaRS, and bound to a solid support. The matrix can be packed in a column or other suitable container and is contacted with one or more compounds (e.g., a mixture) to be tested under conditions suitable for binding of compound to the aaRS. For example, a solution containing compounds can be made to flow through the matrix. The matrix can be washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds. Compounds which remain bound can be released by a suitable elution buffer. For example, a change in the ionic strength or pH of the elution buffer can lead to a release of compounds. Alternatively, the elution buffer can comprise a release component or components designed to disrupt binding of compounds (e.g., one or more substrates or substrate analogs which can disrupt binding of compound to the aaRS, such as lysine, ATP, tRNA.sup.Lys for a lysyl-tRNA synthethase, or other suitable molecules which competitively inhibit binding).
Fusion proteins comprising all of, or a portion of, the aaRS linked to a second moiety not occurring in the helicobacter aaRS as found in nature (see above), can be prepared for use in another embodiment of the method. Suitable fusion proteins for this purpose include those in which the second moiety comprises an affinity ligand (e.g., an enzyme, antigen, epitope). The fusion proteins can be produced by the insertion of an aaRS gene or portion thereof into a suitable expression vector, which encodes an affinity ligand (e.g., pGEX-4T-2 and pET-15b, encoding glutathione S-transferase and His-Tag affinity ligands, respectively). The expression vector is introduced into a suitable host cell for expression. Host cells are lysed and the lysate, containing fusion protein, can be bound to a suitable affinity matrix by contacting the lysate with an affinity matrix under conditions sufficient for binding of the affinity ligand portion of the fusion protein to the affinity matrix.
In one aspect of this embodiment, the fusion protein is immobilized on a suitable affinity matrix under conditions sufficient to bind the affinity ligand portion of the fusion protein to the matrix, and is contacted with one or more compounds (e.g., a mixture) to be tested, under conditions suitable for binding of compounds to the aaRS portion of the bound fusion protein. Next, the affinity matrix with bound fusion protein is washed with a suitable wash buffer to remove unbound compounds and non-specifically bound compounds. Compounds which remain bound can be released by contacting the affinity matrix with fusion protein bound thereto with a suitable elution buffer (a compound elution buffer). Wash buffer is formulated to permit binding of the fusion protein to the affinity matrix, without significantly disrupting binding of specifically bound compounds. In this aspect, compound elution buffer is formulated to permit retention of the fusion protein by the affinity matrix, but is formulated to interfere with binding of the compound(s) tested to the aaRS portion of the fusion protein. For example, a change in the ionic strength or pH of the elution buffer can lead to release of compounds, or the elution buffer can comprise a release component or components designed to disrupt binding of compounds to the aaRS portion of the fusion protein (e.g., one or more substrates or substrate analogs which can disrupt binding of compounds to the aaRS portion of the fusion protein, such as lysine, ATP, or tRNA.sup.Lys for LysRS, or other suitable molecules which competitively inhibit binding).
Immobilization can be performed prior to, simultaneous with, or after contacting the fusion protein with compound, as appropriate. Various permutations of the method are possible, depending upon factors such as the compounds tested, the affinity matrix-ligand pair selected, and elution buffer formulation. For example, after the wash step, fusion protein with compound bound thereto can be eluted from the affinity matrix with a suitable elution buffer (a matrix elution buffer, such as glutathione for a GST fusion). Where the fusion protein comprises a cleavable linker, such as a thrombin cleavage site, cleavage from the affinity ligand can release a portion of the fusion with compound bound thereto. Bound compound can then be released from the fusion protein or its cleavage product by an appropriate method, such as extraction.
To enrich for specific binding to the aaRS portion of the fusion protein, compounds can be pre-treated, for example with affinity matrix alone, with affinity ligand or a portion thereof (e.g., the portion present in the fusion protein), either alone or bound to matrix, under conditions suitable for binding of compound to the aaRS portion of the bound fusion protein.
One or more compounds can be tested simultaneously according to the method. Where a mixture of compounds is tested, the compounds selected by the foregoing processes can be separated (as appropriate) and identified by suitable methods (e.g., PCR, sequencing, chromatography). Large combinatorial libraries of compounds (e.g., organic compounds, peptides, nucleic acids) produced by combinatorial chemical synthesis or other methods can be tested (see e.g., Ohlmeyer, M. H. J. et al., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating to tagged compounds; see also Rebek et al., Process for Creating Molecular Diversity, U.S. Ser. No. 08/180,215, filed Jan. 12, 1994, relating to compounds without tags; see also, Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D. et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No. 4,833,092). Where compounds selected from a combinatorial library by the present method carry unique tags, identification of individual compounds by chromatographic methods is possible. Where compounds do not carry tags, chromatographic separation, followed by mass spectrophotometry to ascertain structure, can be used to identify individual compounds selected by the method, for example.
Random sequence RNA and DNA libraries (see Ellington, A. D. et al., Nature 346: 818-822 (1990); Bock, L. C. et al., Nature 355: 584-566 (1992); and Szostak, J. W., Trends in Biochem. Sci. 17:89-93 (March, 1992)) can also be screened according to the present method to select RNA or DNA molecules which bind to a helicobacter aaRS. Such molecules can be further assessed for antimicrobial effect upon introduction into a cell (e.g., by expression in the case of an RNA molecule selected by the method).
Tester Strains
Nucleic acids of the present invention can also be used in constructing tester strains for in vivo assays of the effect on the activity of the helicobacter enzyme of a substance which is added to tester strain cells. A tester strain comprises a host cell having a defect in a gene encoding an endogenous aaRS, and a heterologous aaRS gene which complements the defect in the host cell gene. Thus, complementation of a particular defective host cell aaRS gene by a heterologous aaRS gene is a threshold requirement for a tester strain. Because the aaRS genes are essential, the heterologous gene can be introduced into the host cell simultaneously with inactivation of the host cell gene to preserve viability. Alternatively, the heterologous gene can be introduced into the host cell before inactivation or loss of the host cell gene. In this case, to test for complementation, the host cell is then subjected to some change in conditions (e.g., a change in temperature, growth medium, selection conditions) which causes inactivation or loss of either the host aaRS gene or gene product, or both.
If the heterologous gene complements the inactivated host cell gene, such a cell can be used to determine whether a substance that is introduced into the cells for testing, can interact specifically with the heterologous tRNA synthetase (or a component in the pathway of the expression of the heterologous tRNA synthetase gene) to cause loss of function of the tested heterologous tRNA synthetase in those host cells. Thus, such cells are "tester strains". Successful cross-species complementation has been described already, for example, for yeast seryl-tRNA synthetase and for yeast isoleucyl-tRNA synthetase in E. coli (Weygand-Durasevic, I., et al., Eur. J. Biochem 214:869-877 (1993); Racher, K. I., et al., J. Biol. Chem. 266:17158-17164 (1991)).
In tester cells to be used in an assay for chemical substances that can inhibit the function of a specific aaRS, the gene for the aminoacyl-tRNA synthetase can, for example, physically replace the host cell aaRS gene or can be present in addition to a host aaRS gene that does not produce a functional product, and the heterologous gene whose gene product is to be tested complements the host gene. A substance to be tested is administered to the tester cells, and the viability or growth of such cells can be compared with that of cells of a suitable control.
As a tester strain comprises a host cell comprising a heterologous aaRS gene (i.e., one from a heterologous species), a suitable host cell is heterologous with respect to the species from which the gene to be tested is isolated. For instance, suitable host cells to test Helicobacter pylori genes can be host cells of a species other than H. pylori. Examples of species which are suitable for use as hosts for the construction of tester strains are E. coli, S. cerevisiae, and B. subtilis. These species are especially amenable to genetic manipulation because of their history of extensive study.
Suitable host cells having a genotype useful for the construction of a tester strain can be constructed or selected using known methods. For example, both in E. coli and in S. cerevisiae, a first plasmid which contains a functional copy of a host chromosomal aaRS gene (which is to be inactivated later), and some selectable marker gene, can be constructed and introduced into cells. Then, an inactivating mutation can be caused in the chromosomal copy of the aaRS gene.
This can be accomplished, for instance, by causing or selecting for a double crossover event which creates a deletion and insertion. This can be done by introducing into the cells double-stranded DNA having regions of homology to the DNA flanking the target aaRS gene, and having between these regions a gene encoding a selectable marker, either on a suitable vector or as a DNA fragment, as appropriate (Jasin et al., U.S. Pat. No. 4,713,337; Schimmel, P., U.S. Pat. No. 4,963,487; Toth, M. J. and Schimmel, P., J. Biol. Chem. 261:6643-6646 (1986); Rothstein, R., Methods in Enzymology 194:281-301 (1991)). Such an approach simultaneously inserts a selectable marker and results in a deletion of the endogenous gene between the flanking sequences provided. Where needed to maintain viability, a compatible maintenance plasmid is provided encoding an endogenous or complementing aaRS.
A test plasmid which is compatible with the maintenance plasmid, and which contains the aaRS gene to be tested for complementation, can be introduced into the host cells. If the first plasmid has been constructed to have a mechanism to allow for inhibition of its replication (for example, a temperature sensitive replicon) or to have a mechanism by which cells containing the first plasmid can be selected against (by, for example, the use of 5-fluoroorotic acid to select against S. cerevisiae cells which have a first plasmid containing the URA3 gene), cells which survive by virtue of having a complementing aaRS gene on the second plasmid can be selected (Sikorsky, R. S. and Boeke, J. D., Methods in Enzymology 194:302-318 (1991)).
Causing or selecting for a double crossover event which creates a deletion and insertion can be used in itself as a one-step method of constructing a tester strain in which a native aaRS gene is replaced by the corresponding foreign gene whose gene product is to be tested. Endogenous recombination mechanisms have been used to advantage previously in E. coli, B. subtilis, and S. cerevisiae, among other organisms. This method depends on the ability of the heterologous gene to be tested to complement the native corresponding aaRS gene. This can be done by introducing into the cells double-stranded DNA having regions of homology to the DNA flanking the target native aaRS gene, and having between these regions a gene encoding a selectable marker as well as the heterologous aaRS gene intended to replace the native aaRS gene. The survival of cells expressing the selectable marker is indicative of expression of the introduced heterologous aaRS gene and complementation of the defect in the endogenous synthetase.
For example, a tester strain useful for testing the effect of a compound on the function of LeuRS expressed by an inserted H. pylori gene, can be constructed in a one-step method in a suitable host cell. Optional positive and negative controls for this cross-species transformation can be used to show that the resulting strain depends on the LeuRS gene from H. pylori for growth and that this recombination event is not lethal. For example, B. subtilis cells made competent for transformation (Dubnau, D. and Davidoff-Abelson, R., J. Mol. Biol. 56:206-221 (1971)) can be transformed with a suitable construct, such as a linearized plasmid containing an insert. Generally, the construct includes a selectable marker gene for antibiotic resistance, or other suitable selectable marker. In one embodiment, a linearized plasmid which contains the H. pylori LeuRS gene and an antibiotic resistance gene, situated between sequences homologous to the flanking sequences of the endogenous LeuRS gene of the host cells, is used to transform the host cell. For a positive control, the linearized plasmid can be constructed in a similar fashion, except that the native B. subtilis LeuRS gene replaces the H. pylori gene, such that a normal B. subtilis LeuRS gene is located adjacent to the antibiotic resistance marker in the insert. As a negative control, the insert can be designed to contain only the flanking sequences and the antibiotic resistance marker, for example. Antibiotic resistant transformants are not expected upon transformation with the negative control construct, as homologous recombination with the construct results in deletion of the endogenous LeuRS gene. Successful construction of a tester strain can also be confirmed by Southern analysis.
B. subtilis strains, which can be used as hosts for gene replacement (Dubnau, D. and Davidoff-Abelson, R., J. Mol. Biol. 56:209-221 (1971)), contain a cryptic tyrosyl-tRNA synthetase gene (tyrz) in addition to the tyrs gene, which can maintain viability (Glaser, P. et al., DNA Sequ. and Mapping, 1:251-61 (1990); Henken, T.M et al., J. Bacteriol., 174:1299-1306 (1992)). A suitable tester strain for TyrRS can be constructed in B. subtilis by, for example, simultaneous inactivation of both of the host genes, or by sequential inactivation. For instance, inactivation of one host gene by a suitable method, such as by insertion of a selectable marker, can be followed by a one-step gene replacement of the remaining host gene with a heterologous helicobacter TyrRS gene and a second selectable marker.
The yeast S. cerevisiae offers additional possibilities for genetic manipulations to create tester strains, relative to bacteria. Yeast integrating plasmids, which lack a yeast origin of replication, can be used for making alterations in the host chromosome (Sikorski, R. S. and Heiter, P., Genetics, 122:19-27 (1989); Gietz, R. D. and Sugino, A., Gene, 74:527-534 (1988)). In another embodiment, one-step gene disruptions can be performed in diploid cells using a DNA fragment comprising a copy of an aaRS gene (optionally containing a deletion in the aaRS gene) having an insertion of a selectable marker in the aaRS gene. A suitable fragment can be introduced into a diploid cell to disrupt a chromosomal copy of the yeast gene. Successful integration of the disrupted aaRS gene can be confirmed by Southern blotting and by tetrad analysis of the sporulated diploid cells. The diploid cells heterozygous for the disrupted aaRS gene provide a diploid host strain which can be transformed with a plasmid containing the heterologous aaRS gene. These cells can be sporulated and the haploid spores analyzed for rescue of the defective chromosomal aaRS by the heterologous aaRS gene.
Alternatively, those diploid cells that are found to contain one copy of the disrupted chromosomal aaRS gene, as well as one functional copy, can be transformed with a maintenance plasmid which contains a gene which complements the disruption, such as the corresponding wild type yeast aaRS gene, and which provides for a mechanism to select against survival of the cells containing this plasmid. These cells can then be made to sporulate to obtain a haploid null strain containing the disrupted chromosomal aaRS gene and the wild type gene on the maintenance plasmid. This haploid host strain can then be transformed with a test plasmid which expresses a heterologous aaRS gene, and the maintenance plasmid can be selected against by growing this strain under appropriate conditions.
Construction of a tester strain may start with the isolation of a mutant host strain which produces e.g., an inactive tRNA synthetase specific for a particular amino acid, a tRNA synthetase which is conditionally inactivatible, or no tRNA synthetase at all specific for that amino acid. A number of E. coli and S. cerevisiae strains have been described that can be used for constructing tester strains. Some of these strains are described below for purposes illustration. The procedures used to isolate and/or construct these E. coli and S. cerevisiae strains, or similar procedures, can be used or dapted to make additional mutant strains in E. coli, S. cerevisiae or in other host organisms.
A number of E. coli strains have been characterized in which an aaRS gene has been inactivated by some method, in whole or in part, yielding an observable phenotypic defect which can be detectably complemented. For example, null strains in which the gene encoding MetRS has been inactivated, and a mutant strain of E. coli in which the gene encoding MetRS has been conditionally inactivated, have been described (see Kim, et al., Proc. Natl. Acad. Sci. USA 90:10046-10050 (1993), describing a metG null strain of E. coli carrying a maintenance plasmid, MN9261/pRMS615); and Barker, D. G. et al. Eur. J. Biochem. 127:449-457 (1982) and Starzyk, R. M. et al., Biochemistry, 28:8479-8484 (1989), regarding a mutant strain having a methionine auxotrophy because the K.sub.m for methionine of the enzyme encoded by the chromosomal metG allele is elevated).
E. coli strain IQ843/pRMS711 and its derivative IQ844/pRMS711 contain a chromosomal deletion of the ileS gene (.DELTA.ileS203::kan), and are propagated by expression of wild type IleRS at 30.degree. C. from a temperature-sensitive maintenance plasmid designated pRMS711, which encodes the wild type ileS gene and a gene which confers chloramphenicol resistance. pRMS711 cannot replicate at 42.degree. C., thus, at the non-permissive temperature, the maintenance plasmid is lost. Following the introduction of a test construct into these strains, the growth of chloramphenicol sensitive colonies at a non-permissive temperature (e.g., 42.degree. C.) is indicative of complementation of the chromosomal ileS deletion by the introduced construct (Shiba, K. and P. Schimmel, Proc. Natl. Acad. Sci. USA, 89:1880-1884 (1992); Shiba, K. and P. Schimmel, Proc. Natl. Acad. Sci. USA, 89:9964-9968 (1992); Shiba, K. and P. Schimmel, J. Biol. Chem., 267:22703-22706 (1992)).
An E. coli strain has been constructed in which both genes encoding a lysyl-tRNA synthetase, lyss (Kawakami, K. et al., Mol. Gen. Genet. 219:333-340 (1989)) and lysU (Leveque, F. et al. Nucleic Acids Res. 18:305-312 (1990); Clark, R. L. and Neidhardt, F. C., J. Bacteriol. 172:3237-3243 (1990)), have been mutated. This strain carries a temperature sensitive maintainence plasmid that is lost at 42.degree. C. (Chen, J. et al., J. Bacteriol. 176:2699-2705 (1994)).
Temperature sensitive alleles are examples of genes encoding conditionally inactivatable tRNA synthetases. For example, temperature-sensitive alleles of the genes encoding cytoplasmic IleRS (ils1-1) and MetRS (mes1-1) have been described in S. cerevisiae (Hartwell, L. H., and McLaughlin, C.S., J. Bacteriol. 96:1664-1671 (1968); McLaughlin, C.S., and Hartwell, L. H. Genetics 61:557-566 (1969)), and are available from the Yeast Genetic Stock Center (University of California-Berkeley; catalog nos. 341 and 19:3:4, respectively). Temperature sensitive strains of E. coli having a defect in the tyrs gene encoding TyrRS (see, e.g., Bedouelle, H. and G. Winter, Nature 320:371-373 (1986)) and temperature-sensitive serS (encoding SerRS) strains of E. coli have been described (Low, B., et al., J. Bacterial. 108:742-750 (1971); Clarke, S. J. et al., J. Bacterial. 113:1096-1103 (1973); and Hartlein, M. et al., Nucl. Acids Res., 15(3):1005-1017 (1987)).
Temperature-sensitive alas strains of E. coli have also been described (Buckel, P. et al, J. Bacteriol. 108:1008-1016 (1971); Lee, A. L. and Beckwith, J., J. Bacterial. 166:878-883 (1986)), in addition to a number of strains with well-characterized alas deletions and complementing alas alleles on plasmids (Jasin, M., et al., Cell 36:1089-1095 (1984); Jasin, M. and Schimmel, P., J. Bacteriol. 159:783-786 (1984)). Such strains can be used as host cells for the construction of E. coli tester strains for alanyl-tRNA synthetase genes of helicobacter.
E. coli strains having a defect, such as a null mutation, in an aminoacyl-tRNA synthetase gene can be constructed using a cloned E. coli aaRS gene. Each aminoacyl-tRNA synthetase from E. coli has been cloned (see Meinnel, T. et al., 1995, "Aminoacyl-tRNA synthetases: Occurrence, structure and function", In: tRNA: Structure, Biosynthesis and Function, Soll, D. and U. RajBhandary, Eds., (American Society for Microbiology: Washington, D.C.), Chapter 14, pp. 251-292, the teachings of which are incorporated herein by reference). For example, the E. coli tyrosyl-tRNA synthetase gene (Barker, D. G., Eur. J. Biochem., 125:357-360 (1982); Barker, D. G. et al., FEBS Letters, 150:419-423 (1982)), isoleucyl-tRNA synthetase gene (Webster, T. et al., Science 226:1315-1317 (1984); see also, EMBL/GenBank Accession No. D10483), and seryl-tRNA synthetase gene have been cloned and sequenced (Hartlein, M. et al., Nucl. Acids Res., 15(3):1005-1017 (1987)). The cloned genes can also be used as maintenance plasmids in a suitable host cell or can be incorporated into a suitable construct for use as a maintenance plasmid.
For construction of a tester strain in S. cerevisiae, a plasmid such as the one reported by P. Walter et al. (Proc. Natl. Acad. Sci. USA 80:2437-2441, 1983), which contains the wild type cytoplasmic methionyl-tRNA synthetase gene of S. cerevisiae, MES1, can be used to construct mesl strains, and for the construction of maintenance plasmids to create cytoplasmic tester strains for a MetRS (see also Fasiolo, F. et al., J. Biol. Chem. 260:15571-15576 (1985)). The ILSI gene encoding cytoplasmic isoleucyl-tRNA synthetase (Englisch, U., et al., Biol. Chem. Hoppe-Seyler 368:971-979 (1987)), and the KRS1 gene encoding cytoplasmic lysyl-tRNA synthetase (Mirande, M. et al., Biochemie 68:1001-1007 (1986); Mirande, M. and Waller, J.-P., J. Biol. Chem. 263:18443-18451 (1988)) of S. cerevisiae have been cloned and sequenced. The KRS1 gene was shown to be essential by the construction of a disrupted allele of KRS1 (Martinez, R. et al., Mol. Gen. Genet. 227:149-154 (1991)). The yeast VAS1 gene encodes both mitochondrial and cytoplasmic ValRSs (Chatton, B. et al., J. Biol. Chem., 263(1) :52-57 (1988)). Leucyl- and seryl-tRNA synthetase genes from yeast cytoplasm, among others, have also been cloned and sequenced and can be used in the construction of tester strains (see e.g., Weygand-Durasevic, I. et al., Nucl. Acids Res., 15(5):1887-1904 (1987) regarding S. cerevisiae serS; see also Meinnel, T. et al., 1995, "Aminoacyl-tRNA synthetases: Occurrence, structure and function", In: tRNA: Structure, Biosynthesis and Function, Soll, D. and U. RajBhandary, Eds., (American Society for Microbiology: Washington, D.C.), Chapter 14, pp. 251-292 and references cited therein).
Mitochondrial mutant strains, such as an msml-1 strain or disruption strain QBY43 (aW303.DELTA.MSM1) (MATa ade2-1 his3-11, 15 leu2-3,112 ura3-1 trpl-1 msm1::HIS3; see Tzagoloff, A., et al., Eur. J. Biochem. 179:365-371 (1989)), can be used for the construction of tester strains comprising a helicobacter methionyl-tRNA synthetase (see e.g., Example 10 for construction of a tester strain in a QBY43 derivative). Strains having a defect in another mitochondrial aminoacyl-tRNA synthetase can be constructed using a cloned mitochondrial aaRS gene, and used to make tester strains (see Meinnel, T. et al., 1995, "Aminoacyl-tRNA synthetases: Occurrence, structure and function", In: tRNA: Structure, Biosynthesis and Function, Soll, D. and U. RajBhandary, Eds., (American Society for Microbiology: Washington, D.C.), Chapter 14, pp. 251-292 and ATCC Catalog of Recombinant DNA Materials, American Type Culture Collection, Rockville, M.D., regarding mitochondrial aaRS genes; a sequence designated as mitochondrial IleRS has been recently reported (GenBank Accession No. L38957, locus YSCMSI1)). The sequence and disruption of the S. cerevisiae mitochondrial leucyl-tRNA synthetase gene (MSL1) has been reported (Tzagoloff, A. et al., J. Biol Chem., 263:850-856 (1988)). An S. cerevisiae strain has been constructed which carries a disruption of MSY1, the gene encoding mitochondrial tyrosyl-tRNA synthetase. Plasmids carrying MSY1 which rescue this defect, also have been constructed (Hill, J. and A. Tzagoloff, Columbia University; see Edwards, H. and P. Schimmel, Cell 51:643-649 (1987)). The construction of a tester strain using the gene encoding S. cerevisiae mitochondrial lysyl-tRNA synthetase (Gatti, D. L. and A. Tzagoloff, J. Mol. Biol. 218:557-568 (1991); GenBank Accession No. X57360) is described in Example 9.
In S. cerevisiae, to construct a maintenance plasmid or a test plasmid carrying a heterologous gene, a suitable vector, such as a yeast centromere plasmid (CEN; single-copy) or 2 .mu. vector (high copy) can be used. A heterologous gene to be tested can also be incorporated into the chromosome, using an integrating plasmid, for example. Examples of convenient yeast vectors for cloning include vectors such as those in the pRS series (integrating, CEN, or 2 .mu.plasmids differing in the selectable marker (HIS3, TRP1, LEU2, URA3); see Christianson, T. W., et al., Gene, 110:119-122 (1992) regarding 2 .mu. vectors; see Sikorski, R. S. and Hieter, P. Genetics, 122:19-27 (1989) regarding integrating and CEN plasmids which are available from Stratagene, La Jolla)) and shuttle vectors (integrating, CEN or 2 .mu. vectors) which contain the multiple cloning site of pUC19 (Gietz, R. D. and Sugino, A., Gene, 74:527-534 (1988)). Examples of expression vectors include pEG (Mitchell, D. A. et al., Yeast, 9:715-723 (1993)) and pDAD1 and pDAD2, which contain a GAL1 promoter (Davis, L. I. and Fink, G. R., Cell 61:965-978 (1990)).
A variety of promoters are suitable for expression. Available yeast vectors offer a choice of promoters. In one embodiment, the inducible GAL1 promoter is used. In another embodiment, the constitutive ADH1 promoter (alcohol dehyrogenase; Bennetzen, J. L. and Hall, B. D., J. Biol. Chem., 257:3026-3031 (1982)) can be used to express an inserted gene on glucose-containing media. An example of a vector suitable for expression of a heterologous aaRS gene in yeast is pQB169 (Example 11).
For illustration, a yeast tester strain can be constructed as follows. An S. cerevisiae strain with convenient markers, such as FY83 (MATa/MAT.alpha. lys2-128.delta./lys2-128.delta. leu2.DELTA./leu2.DELTA.l ura3-52/ura3-52 trp1 .DELTA.63/trp1 .DELTA.63) can be used as a host cell.
A clone encoding yeast cytoplasmic seryl-tRNA synthetase has been isolated (Weygand-Durasevic, I. et al., Nucl. Acids Research, 15(5):1887-1904 (1987); GenBank/EMBL Data Bank, Accession No. X04884). A nucleic acid encoding yeast cytoplasmic SerRS can be used to create a null allele of the yeast cytoplasmic SerRS gene. For example, a deletion/insertion allele can be constructed by excising the SerRS open reading frame, including the promoter region and 3' flanking region or portions thereof from a cloned gene, and replacing the excised sequence with a selectable marker (e.g., TRP1). This serrs::TRP1 fragment can be used to transform the diploid strain FY83, and Trp+ transformants can be selected (Rothstein, J., Methods in Enzymol. 101:202-211 (1983)). Standard genetic procedures can be employed to identify the appropriate integrant created by this one-step gene disruption (a diploid having the genotype MATa/MAT.alpha. lys2-128.delta./lys2-128.delta. leu2 .DELTA.1/leu2.DELTA.1 ura3-52/ura3-52 trp1 .DELTA.63/trp1 .DELTA.63 serrs::TRP1/TyrRS); Rose, M. D. et al. Methods in Yeast Genetics, 1990, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
To construct a maintenance plasmid, a fragment from containing the SerRS coding region, its promoter and some of the 3' untranslated region (e.g., a region approximately equivalent to that deleted in the construction of the null allele above) can be excised and introduced into a vector such as YCplac33, a CEN plasmid containing a URA3 selectable marker (Gietz, R. D. and Sugino, A., Gene 74:527-534 (1988)). The resulting plasmid can be used to transform the serrs::TRP1/SerRS diploid described above, and Ura+ transformants which contain the maintenance plasmid can be selected. The resulting diploid can be sporulated and a haploid Trp+Ura+ spore (a SerRS null strain), corresponding to a serrs: :TRP1 strain dependent upon the URA3-SerRS maintenance plasmid can be isolated.
To construct a test plasmid (a plasmid bearing a heterologous tRNA synthetase gene to be tested for its ability to complement the defect in the endogenous yeast gene), a heterologous aaRS gene to be tested can be inserted into a suitable vector for expression. For instance, the multicopy vector pQB169 described in Example 11 can be used. A fragment containing the H. pylori SerRS gene can be inserted into pQB169, using one or more suitable restriction sites in the multiple cloning site, for example. Alternatively, to test whether a relatively reduced level of expression of the heterologous tRNA synthetase gene permits complementation, a fragment containing the H. pylori SerRS gene can be inserted into a CEN plasmid such as pQB172 for expression (Example 11). Preferably, the heterologous gene is inserted into the vector so that its ATG start codon is the first ATG within 50 to 100 bp of the transcription start site of the ADH promoter of the vector.
Because these plasmids bear the LEU2 selectable marker, they can be used to transform a null strain, such as the Trp+Ura+Leu- strain described, and Leu+transformants containing the test plasmid can be selected. Leu+Ura+Trp+ transformants (containing a serrs::TRP1 allele, a URA3 maintenance plasmid, and the LEU2 test plasmid) can be tested for growth on media containing 5-fluoroorotic acid (5-FOA). 5-FOA is toxic to URA3 cells, and causes loss of the URA3 maintenance plasmid (Boeke, J. et al., Mol. Gen. Genet., 197:345-346 (1984)). Accordingly, growth of cells on media containing 5-FOA is indicative of complementation of the lethal deletion in the aaRS gene on the chromosome (serrs::TRP1) by the heterologous TyrRS gene on the test plasmid. Cells that are unable to grow on 5-FOA are dependent upon the maintenance plasmid for viability, and therefore, are indicative of insufficient activity to complement the lethal deletion in the aaRS gene. Where complementation is observed, the strain can be used to test for inhibitors of the product of the heterologous gene encoded by the test plasmid.
In another embodiment, a eucaryotic host cell is used to construct a mitochondrial tester strain. For example, in yeast, each of the mitochondrial tRNA synthetases is essential for growth on non-fermentable carbon sources (e.g., glycerol). Thus, complementation tests can be conducted in mitochondrial tester strains. As the genes encoding mitochondrial aminoacyl-tRNA synthetases are typically nuclear-encoded, the procedures described above can be modified to construct mitochondrial tester strains having a defect in a mitochondrial aminoacyl-tRNA synthetase. Modification is necessitated by the fact that yeast strains with a defect in mitochondrial protein synthesis, such as a defective aminoacyl-tRNA synthetase, lose their mitochondrial DNA, rapidly becoming rho-. As a result, these strains are unable to grow on non-fermentable carbon sources even if a complementing gene is introduced into the strain. Therefore, in a haploid strain having a defect in, for example, the yeast mitochondrial leucyl-tRNA synthetase gene (e.g., a gene disruption with a cosegregating selectable marker constructed as indicated above; see also Tzagoloff, A. et al., J. Biol. Chem. 263(2): 850-856 (1988)), the haploid strain can be crossed with a rho+ strain having a wild-type mitochondrial leucyl-tRNA synthetase gene to restore the mitochondrial DNA. The resulting rho.sup.+ diploid can then be transformed with a plasmid which encodes the wild-type yeast mitochondrial leucyl-tRNA synthetase (i.e., a maintenance plasmid) and a second selectable marker. Following sporulation, progeny spores which carry the defective mitochondrial LeuRS, identified by the presence of the cosegregating selectable marker, and the maintenance plasmid, identified by the presence of the second selectable marker, and which are rho.sup.+, can be isolated (e.g., by tetrad analysis). Strains constructed in this manner would be suitable for complementation assays using the helicobacter aminoacyl-tRNA synthetases.
For instance, a plasmid encoding a helicobacter leucyl-tRNA synthetase gene can be introduced into such a strain on a second plasmid having a third selectable marker. As indicated above, the maintenance plasmid can be selected against (e.g., where the selectable marker is URA3, selection on 5-fluoroorotic acid leads to loss of the maintenance plasmid), and complementation by the helicobacter gene can be monitored on a non-fermentable carbon source.
In another embodiment, a mitochondrial aminoacyl-tRNA synthetase gene disruption with a cosegregating selectable marker can be constructed in a diploid rho.sup.+ strain (see e.g., Edwards, H. and P. Schimmel, Cell, 51:643-649 (1987)). A plasmid encoding a helicobacter aminoacyl-tRNA synthetase gene is introduced on a plasmid having a second selectable marker. Sporulation of a resulting diploid yields two progeny spores carrying the yeast mitochondrial aminoacyl-tRNA synthetase gene disruption, identified by the presence of a cosegregating selectable marker, and two progeny spores carrying the corresponding wild-type gene. The presence of the plasmid can be monitored by the presence of the second selectable marker. Complementation by the helicobacter gene on the introduced plasmid is indicated by growth on non-fermentable carbon sources of spores carrying the disrupted aminoacyl-tRNA synthetase gene.
In the case of a mitochondrial tester strain, the helicobacter aminoacyl-tRNA synthetase can be imported into mitochondria to achieve complementation of the mitochondrial defect. When it is necessary to achieve import or desirable to improve the efficiency of import of the aminoacyl-tRNA synthetase in the host cell, a gene fusion can be constructed using a sequence encoding a mitochondrial targeting sequence which functions in the host cell. For example, a mitochondrial targeting sequence can be introduced at the amino-terminal end of the helicobacter aminoacyl-tRNA synthetase. In one embodiment in yeast, the helicobacter aaRS gene or a sufficient portion thereof is introduced into a vector in which it is placed under the control of the minimal alcohol dehydrogenase promoter and is fused to the yeast cytochrome oxidase IV targeting signal derived from plasmid pMC4 (Bibus et al., J. Biol. Chem., 263:13097 (1988)). Expression of the construct yields a fusion protein with an N-terminally located cytochrome oxidase IV targeting signal joined to the helicobacter aaRS protein.
If the construction methods described here are not successful initially, one or more natural or synthetic helicobacter or other (e.g., procaryotic, such as a bacterial, or eukaryotic, such as a mammalian or fungal) tRNA gene(s) can be introduced into the host cell to provide one or more cognate tRNAs for the helicobacter aaRS. The tRNA genes of a number of species have been cloned and sequenced (Steinberg, S., et al., "Compilation of tRNA sequences and sequences of tRNA genes", Nucleic Acids Res. 21:3011-3015 (1993)). A method for constructing a strain of Streptomyces lividans in which an essential tRNA gene has been inactivated in the chromosome, and the gene is instead maintained on a plasmid, has been described (Cohen, S. N., WO 94/08033 (1994)).
Use of Tester Strains
To assess the inhibitory effect of a substance on a tester strain, the cells are maintained under conditions suitable for complementation of the host cell defect, under which complementation of the host cell defect is dependent upon the test gene (i.e., assay conditions). A substance to be tested is administered to the tester cells, and the viability or growth of the tester cells can be compared with that of cells of one or more suitable controls. A variety of control experiments can be designed to assess the inhibitory effect of a substance and/or the specificity of inhibition. The following examples are provided for purposes of illustration.
A preliminary test for inhibitory effect may be conducted where desired. For example, a substance to be tested can be administered to tester cells maintained under assay conditions, and the viability or growth of the tester cells in the presence of the substance can be compared with that of tester cells maintained under the same conditions in the absence of the substance. If it is determined that the substance inhibits growth of the tester cells, a further assessment of the specificity of inhibition by the substance can be conducted as described below.
Alternatively, the inhibitory effect of a substance on tester cell growth and the specificity of inhibition can be determined without conducting the preliminary test for inhibitory activity. The following examples, in which the various cell types are in each case exposed to drug, are provided for purposes of illustration only.
To determine the specificity of inhibition, the viability or growth of the tester cells can be compared with that of cells of one or more suitable control strains maintained under the same conditions. In particular, tester strains and control strains are maintained under assay conditions, and exposed to the substance to be tested.
Strains which are similar to the tester strain, but lack the heterologous aminoacyl-tRNA synthetase gene present in the tester strain (i.e., the "test gene"), can serve as control strains. These control strains comprise a "control gene" which is an aminoacyl-tRNA synthetase gene other than the heterologous aaRS gene present in the tester strain (i.e., an aaRS gene from a different species, such as a procaryotic or eukaryotic species). The control gene can be a cytoplasmic or mitochondrial aaRS gene, and it encodes an aaRS specific for the same amino acid as the aaRS encoded by the test gene. Viability or growth of the control strain is dependent upon the control gene under the conditions of the assay.
In one embodiment, a cell which is a cell of the same species as the host cell used to construct the tester strain, and which further comprises a control aaRS gene, is selected as a control. For example, the control gene can be a wild-type aaRS gene from the control strain species which encodes an aaRS specific for the same amino acid as the aaRS encoded by the test gene. Such a cell can be used when, for example, the substance or compound to be tested does not significantly affect growth of the control strain under the assay conditions. For example, where an E. coli host is used to construct a tester strain having an H. pylori aaRS gene, an E. coli strain having a wild-type E. coli control gene can be used as a control strain. As another example, if a yeast host cell having a defect in a mitochondrial aaRS gene is used to construct the tester strain, a yeast strain comprising the wild type mitochondrial gene can be used as a control strain.
In another embodiment, the control strain can be a strain distinct from the tester strain, which is constructed in a manner which generally parallels that of the tester strain comprising the test gene, such that complementation of the host cell defect, which is also present in the control strain, is dependent upon the control gene under the assay conditions. In this embodiment, the control strain preferably comprises a host cell of the same species as the host cell used to construct the tester strain, and is closely related in genotype to the tester strain. These preferred control strains comprise a "control gene", which, as indicated above, is an aaRS gene other than the test gene (i.e., an aaRS gene from a different species, such as a heterologous procaryotic or eukaryotic species). Furthermore, the control gene, which can be cytoplasmic or mitochondrial, encodes an aaRS specific for the same amino acid as the test gene. Preferably, the control gene is selected from a species which is a host for the pathogen from which the test gene is derived, permitting the identification of specific inhibitors which selectively inhibit the pathogen aaRS (e.g., human control gene for an H. pylori test gene). Alternatively, because the eukaryotic aminoacyl-tRNA synthetases are generally more closely related to each other than to procaryotic aminoacyl-tRNA synthetases, a control gene from another eukaryote (e.g., a different mammalian species) can be used in lieu of one selected from the host species (e.g., a rat or mouse control gene for an H. pylori test gene).
For example, a strain isogenic with a tester strain, except for the substitution of a human control gene, can serve as a control strain. Such a control strain can be constructed using the same methods and the same host cell used to construct the tester strain, with the exception that a human control gene is introduced into the host cell in lieu of the heterologous helicobacter aaRS gene present in the tester.
Under the conditions of this assay, growth or viability of the control strain is dependent upon the control aaRS gene, which complements the host cell aaRS defect in the control strain. Specific inhibition by a substance can be determined by comparing the viability or growth of the tester strain and control strain in the presence of the substance.
In some cases, further controls may be desired to assess specific inhibition. For this purpose, one or more additional "comparison control" strains are used for purposes of comparison. These additional controls can be used to assess the relative effects of a substance upon growth of the tester and control strains in the presence of the substance.
Strains useful for this purpose include, for example, strains of the same species as the host cell used to construct the tester strain, which contain a wild type version of the aaRS gene which is inactivated in the tester strain. In one embodiment, where an E. coli host is used to construct a tester strain comprising an H. pylori test gene, an E. coli strain comprising a wild-type E. coli aaRS gene can be used as a comparison control strain. In another embodiment, "parental-type" cells (e.g., parent host cells or a similar strain) are used as comparison controls. For example, the parent host cells of the tester strain can serve as a comparison control strain for the tester strain. Where the tester strain and the control strain have the same parent, a single strain can be used as the comparison control strain for both tester and control strains.
For example, a parent host cell from which the tester and control strains were both constructed (e.g., by inactivation and replacement of the wild type host aaRS gene) can be used as a comparison control strain. This comparison control strain contains a wild type version of the aaRS gene which is inactivated in the tester and control strains, and the viability or growth of this comparison control strain is dependent upon the wild type aaRS under the conditions of the assay. Specific inhibition of the heterologous helicobacter aaRS encoded by the test gene (or a step in the expression of the helicobacter gene) is indicated if, after administering the substance to the tester strain, growth of the tester strain is reduced as compared with an appropriate comparison control strain, and growth of the control strain is not reduced, or is relatively less reduced, as compared with its appropriate comparison control strain.
Testing for Antibiotic Resistance to tRNA Synthetase Inhibitors
Mutation of a drug target can reduce the effectiveness of antimicrobial or antibiotic agents, and can confer drug resistance. Thus, mutation of a target aminoacyl-tRNA synthetase, such as a H. pylori IleRS, MetRS, LeuRS, ValRS, LysRS or SerRS, could reduce the effectiveness of an inhibitor of aaRS activity. To test for mutations that confer resistance to an inhibitor (e.g., an inhibitor of aaRS activity, including such an inhibitor having antimicrobial activity) a variety of approaches can be used. Mutant helicobacter aaRS genes can be obtained, for example, by isolation of a mutant gene, or by preparing an individual mutant gene or an expression library of mutant helicobacter aaRS genes, such as a library of mutants of an H. pylori LysRS gene. The mutant gene or gene library can be introduced into suitable host cells for screening for resistance to a compound.
An isolated tRNA synthetase gene, such as an H. pylori aaRS gene, can be mutagenized by any suitable method including, but not limited to, cassette mutagenesis, PCR mutagenesis (e.g., the fidelity of PCR replication can be reduced to induce mutation by varying Mg.sup.2+) concentration, increasing the number of amplification cycles, altering temperatures for annealing and elongation, to yield random mutants), or chemical mutagenesis (e.g., nitrosoguanidine, ethylmethane sulfonate (EMS), hydroxylamine) of the entire gene or a portion thereof. The mutagenesis products can be used to construct an expression library of mutant genes (e.g., by inserting the gene into an expression vector, or replacing a portion of an expression vector comprising the wild-type gene with mutant fragments) which is introduced into a host cell.
In one embodiment, if the inhibitor is known to inhibit the host cell (e.g., E. coli, yeast, Bacillus subtilis) aminoacyl-tRNA synthetase specific for the same amino acid, the mutant genes can be introduced into the wild-type host and the resulting cells can be exposed to drug to assess resistance.
In another embodiment, the procedures described above relating to tester strains are used in the method to identify mutants resistant to inhibitor. Introduction of the heterologous mutant aaRS gene(s) (i.e., mutant test gene(s)) into a host cell is carried out as described above for the production of tester strains. For example, the library can be introduced into a host cell having a defect in the endogenous gene encoding MetRS. The metG null strain of E. coli designated MN9261/pRMS615 is an example of the type of strain that can be constructed and used as a host for the introduction of mutant helicobacter aaRS gene(s) (in that case, MetRS genes; see Kim et al., Proc. Natl. Acad. Sci. USA 90:10046-10050 (1993), describing a strain which carries a null allele of metG, and a temperature sensitive maintenance plasmid, carring a wild type metG allele (encoding E. coli MetRS) and having a temperature sensitive replicon which causes loss of the maintenance plasmid at the non-permissive temperature).
Active, drug-resistant mutants are then identified by a selection process in which cells containing mutant genes encoding active aaRS are identified, and the effect of an inhibitor upon aaRS activity is assessed. Cells are maintained under conditions suitable for expression of the mutated gene, and cells containing an active mutant aaRS (e.g., an active recombinant H. pylori aaRS) are identified by complementation of the host cell defect. Where complementation occurs, each resulting transformant is, in essence, a tester strain comprising a mutant test gene. Cells containing active mutant aaRS as determined by complementation of the host cell defect are then exposed to inhibitor, and the effect of inhibitor on cell growth or viability is assessed to determine whether the active mutant aaRS further confers resistance to inhibitor.
In the case of the metG null strain, complementation by the helicobacter gene is indicated by growth at the non-permissive temperature at which the maintenance plasmid is lost. Cells which survive loss of the maintenance plasmid due to the presence of the complementing mutant gene are then challenged with inhibitor to assess resistance. Resistance can be assessed by comparison to a suitable control by methods analogous to those described above for determining inhibition and/or the specificity of inhibition of a substance in tester cells. For example, the relative effects of an inhibitor upon a tester strain comprising the mutant test gene and upon a tester strain differing only in that it contains the test gene lacking the mutation, can be assessed by comparing the viability or growth of cells which are dependent upon either the test gene or mutant test gene for growth under conditions suitable for complementation of the host cell defect. For instance, the effect of inhibitor on the protein encoded by the test gene lacking the mutation can be determined by comparing the growth of cells containing the test gene in the presence of drug to the growth of such cells in the absence of drug, and the effect of inhibitor on the protein encoded by a mutant test gene can be determined by comparing growth of cells containing the mutant test gene in the presence of drug to the growth of such cells in the absence of drug. A decrease in the inhibitory effect on growth of cells carrying the mutant test gene as compared to the inhibitory effect against cells carrying the test gene lacking the mutation is indicative of resistance.
Cells containing a complementing mutant test gene which further confers resistance to an inhibitor can be used to identify derivatives of the inhibitor with improved antimicrobial effect, which circumvent resistance. Such cells can also be used to identify additional inhibitors having inhibitory activity against the active mutant aaRS encoded by the mutant test gene.
In another embodiment, a naturally occurring mutant helicobacter aaRS gene which confers resistance to an inhibitor upon a helicobacter cell, can be isolated from the helicobacter organism using nucleic acids of the present invention as probes. The cloned gene can then be introduced into a host cell as described for the production of tester strains. Tester cells comprising the mutant test gene which confers resistance, and which complements the host defect, can be used as described herein to identify additional inhibitors having reduced susceptibility to the resistance mutation or derivatives of the inhibitor with improved inhibitory activity.
Vectors carrying mutant genes which confer resistance to inhibitor can be recovered and the insert analyzed to locate and identify the mutation by standard techniques, such as DNA sequence analysis, to yield additional information regarding the nature of mutations capable of conferring resistance to selected inhibitors. Mutant proteins can also be expressed and purified for further characterization by in vitro kinetic and binding assays.
Applications in Biochemistry
The helicobacter aminoacyl-tRNA synthetase or stable subdomains of the protein can be used in a method to separate the amino acid that the enzyme specifically recognizes from a mixture of the amino acid and other compounds such as other amino acids, or to specifically isolate L-amino acid from D-amino acid. The tRNA synthetase can be chemically attached to a solid support material packed in a column or other suitable container. Alternatively, a fusion protein, such as a GST-tRNA synthetase fusion or a His tag-tRNA synthetase fusion (having a histidine hexamer tail), can permit attachment to a suitable solid support which binds the GST portion or His tag portion of the fusion protein, respectively. For example, a mixture of lysine and other compounds can be loaded onto a column under conditions in which lysine binds to lysyl-tRNA synthetase, while other compounds present in the mixture flow through the column. In a later step, lysine can be released from lysyl-tRNA synthetase by changing the conditions in the column, such as washing with a solution of high ionic strength to elute L-lysine, for example.
In a similar manner, the aminoacyl-tRNA synthetase can be used in a method to isolate tRNA that is specifically recognized by the tRNA synthetase.
The helicobacter aminoacyl-tRNA synthetase can be used in the quantitative determination of an amino acid (e.g., lysine) by its conversion to the corresponding aminoacyl-hydroxamate (e.g., lysyl-hydroxamate). An example of an appropriate assay is illustrated by the following series of reactions.
lysine+ATP.fwdarw.lysine-AMP+PP.sub.i
(in the presence of excess pyrophosphatase and ATP at pH 7.5, where pyrophosphatase catalyzes the conversion of the product inorganic pyrophospate (PP.sub.i) to inorganic orthophospate (P.sub.i); ATP is adenosine triphospate; AMP is adenosine monophosphate)
lysine-AMP+NH.sub.2 OH.fwdarw.lysine-NHOH+AMP (at pH 7.5)
lysine-NHOH+FeCl.sub.3 .fwdarw.colored complex (at acidic pH)
The resulting colored complex can be quantitated by spectrophotometric measurements of absorbance at 540 nm, and compared with a standard curve made using known concentrations of lysine. This assay is based on the reactions described by Stulberg and Novelli, Methods in Enzymology 5:703-707 (1962).
The helicobacter aminoacyl-tRNA synthetases can also be used for the quantitative determination of ATP. In the presence of excess amino acid such as lysine, and in the presence of pyrophosphatase to convert the product PP.sub.i to P.sub.i, the ATP is quantitatively converted to AMP and inorganic pyrophosphate by the lysyl-tRNA synthetase. For example,
lysine+ATP.fwdarw.lysine-AMP+PP.sub.i (in the presence of LysRS)
PP.sub.i +H.sub.2 O.fwdarw.2P.sub.i (in the presence of pyrophosphatase)
P.sub.i can be quantitated by reaction with molybdate, measuring the absorbance at 580 nm and comparing to a standard curve made using known quantities of orthophosphate.
EXEMPLIFICATION
The present invention will now be illustrated by the following Examples, which are not intended to be limiting in any way.
Example 1
PCR amplification of DNA fragments of aminoacyl-tRNA synthetase genes from H. pylori genomic DNA
PCR was used to obtain the DNA fragments of aminoacyl-tRNA synthetase (aaRS) genes from H. pylori strain NCTC 11638 (National Collection of Type Cultures (NCTC), Central Public Health Laboratory, 61 Colindale Avenue, London, NW9 5HT, United Kingdom). The PCR primers were designed by aligning polypeptide sequences of corresponding aaRS from different species using the PILEUP program (Needleman and Wunsch, J. Mol. Diol., 48:443-453 (1970)). Table 1 lists all the primers that have been used for amplification of the H. pylori aminoacyl-tRNA synthetases. A convenient EcoRI recognition sequence was introduced at the 5' end of several primers (extra sequence indicated by boldface in Table 1).
The PCR reactions were done in 50 .mu.l volume with 10 mM Tris HCl (pH 8.3 at room temperature), 50 mM KCl, 2.5 mM MgCl.sub.2, 200 .mu.M of each dNTP (pH 7.0), 10 ng of H. pylori genomic DNA (NCTC 11638, provided by Douglas Berg; genomic DNA was prepared as described in Bukanov, N. O. and D. E. Berg, Mol. Microbiol. 11(3): 509-523 (1994)), 100 pmole each of the primers, and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer). The reactions were first incubated at 95.degree. C. for 2 minutes, followed by 30 cycles of 95.degree. C. (1 minute), 50.degree. C. (1 minute), and 72.degree. C. (2 minutes). The thermocycle reaction were extended for 8 minutes at 72.degree. C.
The following combinations of primers were successfully used to amplify the partial sequences of H. pylori aaRS genes. For Ile, 3 combinations of primers produced PCR products: (a) primers KIYO-16 and KIYO-36 generated a DNA fragment of about 400 bp; (b) primers KIYO-37 and KIYO-20 generated a PCR product of 1.3 kb; and (c) primers KIYO-16, -17, and -20 generated a DNA fragment of about 1.5 kb. In the latter case, forward primers KIYO-16 and KIYO-17 were designed from the same region of the IleRS genes, but encode peptides having a different amino acid sequence bias. KIYO-16 and -17 were used simultaneously in a reaction in which KIYO-20 served as the reverse primer.
For Lys, the combination of KIYO-138, 139, and 140 produced a DNA fragment of about 250 bp. Primers KIYO-139 and KIYO-140 were designed from the same region of the LysRS genes, and were used simultaneously in a reaction in which they served as reverse primers and KIYO-138 served as the forward primer.
For Leu, primers KIYO-21 and KIYO-22 produced a DNA fragment of about 300 bp.
For Met, two primer mixtures yielded PCR products: (a) KIYO-12 and KIYO-15 produced a 550 bp DNA fragment; and (b) KIYO-12, -13, -14, and -15 produced a 550 bp fragment, with KIYO-12 and -13 serving as forward primers and KIYO-14 and -15 served as reverse primers.
For Ser, the combination of SerF266, SerR364, and SerR407 produced a DNA fragment of about 500 bp. Reverse primers SerR364 and SerR407 were designed from different regions of SerRS. Use of reverse primer SerR407 and forward primer SerF266 yielded the PCR product, as determined by matching the size of the resulting PCR fragment with the expected sizes.
For Val, primers Val2A and Val6 generated a DNA fragment of 1.2 kb.
In each case, the sizes of the amplified DNA fragments were in good agreement with the predicted sizes of the fragments.
TABLE 1__________________________________________________________________________Degenerate PCR Primers for Amplification of H. pylori Aminoacyl-tRNAsynthetasesaaRS Primer SEQ ID NO: Primer Sequence (5' .fwdarw. 3')__________________________________________________________________________Met KIYO-12 13 GCG AAT TCT WYC TIA CIG GIA CIG AYG ARC AYG G KIYO-13 14 GCG AAT TCT TYA TIT GYG GIA CIG AYG ARY AYG G KIYO-14 15 GCG AAT TCR TAR TTI ATI AGI GCR TCR AWC CAI ACR TA KIYO-15 16 GCG AAT TCR TAI CCR ATI GKI GCR TCI ARC CAI ACR TAIle KIYO-16 17 GCG AAT TCG GIT GGG AYA CIC AYG GIS TIC C KIYO-17 18 GCG AAT TCG GIT GGG AYT GYC AYG GIC TIC C KIYO-18 19 GCG AAT TCG ICA RCG ITA YTG GGG IRT ICC IAT KIYO-19 20 GCG AAT TCG IAA YCG ITW YTG GGG IAC ICC IMT KIYO-20 21 GCG AAT TCR AAC CAI CCI CGI GTY TGR TCI WWI CCY TC KIYO-36 22 GGI ARI GTC CAI GGI GTI GTI GTC CA KIYO-37 23 TWY ATG GAR TCI ACI TGG TGG GYI TTI AAR CAVal Val2A 24 GAY CAY GCI GGI ATH GCI ACN CA Val4 25 RTC RTG IGC IGG IGT DAT YTT Val6 26 RAA CCA IGT RTC IAR IAC RTCLeu KIYO-21 27 GCG AAT TCC IAT IGG ITG GGA YGC ITT YGG ICT ICC KIYO-22 28 GCG AAT TCA CYT GYT CRT TIG CIA GIA CIG T KIYO-34 29 CCI MTI GGI TWY CAY TGY ACI GGI MTI CC KIYO-35 30 GCR TCR TAR TAI GGR TTI GCR TCI GTI GTI ACR AALys KIYO-138 31 TTY MTI GAR GTI GAR ACI CCI ATG ATG KIYO-139 32 TAR AAY TCI ATI GTI GTR AAY TCI GGR TTR TG KIYO-140 33 TAC MAY TCI AKC ATI GTR AAY TCI GGR TTR TGSer KIYO-141 34 AAR AAR TAY GAY CTI GAR GCI TGG TTY CC KIYO-142 35 AAR ACI TAY GAY CTI GAR GTI TGG ATH CC KIYO-143 36 CCY TTY TCI GTY TGR TAR TTY TC KIYO-144 37 CCR TCY TCI GTY TGR TAR TTY TC SerF266 38 SCI TGT TTT MGI TCW GAA GCI GG SerF356 39 TAT MGI GAA ATT TCW TGT TCW AAT SerR364 40 TAA WGA ACA WGA AAT TTC ICK ATA SerR407 41 CCA TCH KST TGT TGA TRA TTT TC__________________________________________________________________________
Example 2
Cloning and characterization of the PCR products
The PCR products were isolated on a 1.2% agarose gel and purified by GeneClean (Bio 101). One third to one fifth of the purified DNA fragments were ligated to 50 ng of pT7Blue (R) T-vector (Novagen). The ligated plasmids were transformed into E. coli DH5.alpha. cells (competent cells purchased from Gibco/BRL), and the transformants were plated on LB-agar plates containing 100 .mu.g/ml ampicillin, 30 .mu.g/ml X-gal, and 0.1 mM IPTG. The white colonies were rapidly screened by using direct colony PCR with T7 and U19 primers that hybridize to the vector sequences flanking the cloning site (Novagen). The plasmids containing the inserts with expected sizes were isolated, and the sequences of the inserts were determined by dideoxy-sequenase sequencing (USB) with the T7 and U19 primers. By querying the sequences against the Non-redundant Protein Data Bases of the BLAST Network Service at the National Center for Biotechnology Information (NCBI), the following clones containing the partial sequences of the corresponding aaRS genes were identified: 2B-3, Ile; 9B-5, Lys; 10B-2, Leu; 13B-3, Met; 19-3, Ser; Val-4, Val.
Example 3
Screening of an ordered cosmid H. Pylori genomic library
Materials
The solutions shown in Table 2 below were used during screening of the H. pylori genomic library.
TABLE 2______________________________________Screening SolutionsSolution Composition______________________________________Solution I 50 mM Tris-Cl, pH 8.0/50 mM EDTA, pH 8.0/ 25% sucrose/1.5 mg/ml lysozymeSolution II 0.5 N NaOH/0.2% Triton X-100Solution III 0.5 N NaOHSolution IV 1 M Tris-Cl, pH 7.5Solution V 0.15 M NaCl/0.1 M Tris-Cl, pH 7.5______________________________________
Library Screening
The cloned PCR fragments of H. pylori aaRS genes were used as the probes to screen an ordered cosmid genomic library of H. pylori (strain NCTC 11638) (Bukanov, N. O. and D. E. Berg, "Ordered cosmid library and high-resolution physical-genetic map of Helicobacter pylori strain NCTC11638", Mol. Microbiol. 11(3): 509-523 (1994)). The library was provided by Douglas Berg in Washington University at St. Louis. It is composed of 68 independent clones and covers 95% of the genomic sequence.
One microliter of bacterial culture of each of the 68 library clones was spotted directly onto a piece of GeneScreen hybridization nylon membrane (Dupont/NEN), which was placed on top of a LB-agar plate containing 50 .mu.g/ml of kanamycin. The plate was then incubated at 37.degree. C. for 16 hours to allow the bacterial cells to grow. The GeneScreen membrane with the bacterial cells was chilled at 4.degree. C. for 1 hour. To lyse the cells and denature the DNA in situ, the prechilled nylon membrane was treated with the solutions listed in Table 2 as follows. First, the membrane was placed right-side-up on a piece of 3MM paper (Whatman), which was saturated with solution I and incubated for 1 minute at room temperature. The membrane was then transferred to a piece of fresh, dry 3MM paper and allowed to stand for 1 minute. These steps were repeated a total of four times for solution I. The same procedure was used to treat the membrane with each of solutions II through V in turn. After treatment with solution V, the membrane was air dried for 30 minutes, baked in a vacuum oven at 80.degree. C. for 2 hours, and washed three times in 300 ml prewashing buffer (3X SSC /0.1% SDS). The first two washes were for 2 hours at 65.degree. C. and the last one was overnight at 65.degree. C. The prewashed filter was then incubated in 10 ml of Southern prehybridization solution (6X SSC / 0.5% SDS / 5X Denhardt's solution / 100 .mu.g/ml sheared and denatured calf thymus DNA) at 65.degree. C. for at least 2 hours before being probed with the partial DNA fragments of the H. pylori aaRS genes generated by PCR.
The polymerase chain reaction was used to generate probes for hybridization with the H. pylori genomic library. The templates for the PCR reactions were the pT7Blue (R) T-vector clones designated 2B-3, 9B-5, 10B-2, 13B-3, 19-3, Val-4. A typical reaction was carried out in 50 .mu.l containing 10 mM Tris HCl (pH 8.3 at room temperature), 50 mM KCl, 2.5 mM MgCl.sub.2, 20 pmole each of T7 and U19 primers, .about.10 ng template, 200 .mu.M each dATP, dGTP, dTTP, 3 .mu.M dCTP, 50 .mu.Ci �.alpha..sup.32 P! dCTP (3000 Ci/mmole, Dupont/NEN), and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer). For the Ile probe, 100 .mu.M of cold dCTP was used. For the Lys and Leu probes, 35 .mu.Ci of �.alpha.-.sup.32 P!dCTP was used. The reactions were first incubated at 95.degree. C. for 2 minutes followed by 30 cycles of 95.degree. C. (30 seconds), 55.degree. C. (30 seconds), 72.degree. C. (2 minutes), and extended for an additional 8 minutes at 72.degree. C. after the thermocycles. Under these conditions, probes with a specific activity calculated to be about 1.times.10.sup.9 cpm/.mu.g DNA were obtained.
The .sup.32 P-labelled probes were then purified by Sephadex G50 spin columns (Boehringer Mannheim), denatured by boiling for 5 minutes, and added to the hybridization nylon membranes in 10 ml of hybridization solution (6X SSC / 10 mM EDTA / 0.5% SDS / 5X Denhardt's solution / 100 .mu.g/ml sheared and denatured calf thymus DNA). The hybridizations were at 65.degree. C. for 16-24 hours. The membranes were then typically washed three times: the first two washes were with 500 ml of 2X SSC, 0.5% SDS solution at room temperature for 15 minutes, and the third wash was with 500 ml of 0.2X SSC / 0.5% SDS at 65.degree. C. for one hour. Hybridizations were then analyzed by autoradiography.
The PCR fragment amplified from Clone 2B-3, which contains a 1.5 kb fragment of IleRS gene, hybridized to the cosmid library clone 51. (Cosmid library clone numbers (51, 67, 54, 55, 29-31, 48, 49, and 40-42) correspond to the clones of the same number in FIG. 2 of Bukanov, N. O. and D. E. Berg, Mol. Microbiol. 11(3): 509-523 (1994).) The PCR fragment amplified from Clone 9B-5, which contains a 250 bp fragment of LysRS gene, hybridized to the cosmid library clone 67. The PCR fragment amplified from Clone 10B-2, which contains a 300 bp fragment of LeuRS gene, hybridized to cosmid library clones 54 and 55. The PCR fragment amplified from Clone 13B-3, which contains a 550 bp fragment of MetRS gene, hybridized to cosmid library clones 29, 30, 31. The PCR fragment amplified from Clone 19-3, which contains a 500 bp fragment of SerRS gene, hybridized to the cosmid library clones 48 and 49. The PCR fragment amplified from Clone Val-4, which contains a 1.2 kb fragment of ValRS gene, hybridized to the cosmid library clones 40, 41, and 42.
Example 4
Sequencing of H. pylori aaRS genes
The partial sequences of the PCR fragments were used to design the initial internal sequencing primers. Promega fmol DNA Sequencing System was used to sequence the H. pylori aaRS genes, using the cosmid DNAs of the positive clones as the templates. The thermal cycle sequencing were carried out according to the Technical Manual of the fmol DNA Sequencing System (Promega) with 5'-.sup.33 P labelled primers. The cosmid clones chosen for sequencing were: Ile, clone 51; Lys, clone 67; Leu, clone 55; Met, clone 30; Ser, clone 49; Val, clone 41.
The DNA sequence generated with each primer was processed with the DNASTAR program (DNASTAR, Inc.), and its homology to the existing aaRS genes in the database was traced using the BLAST program (Basic Local Alignment Search Tool; Gish et al., Nature Genetics 3:266-272 (1993); Altschul et al., J. Mol. Biol. 215:403-410 (1990)). The individual sequences were assembled using the DNA Sequence Management Program with the DNASTAR package to generate full-length genes. The initiation codon of each gene was identified by comparison with known corresponding aaRS sequences in the data bank using the Multiple Sequence Alignment program from the DNASTAR package, by the existence of a putative ribosomal binding site upstream of the initiation codon, and in most cases, by the fact that they are the first methionine codons in the corresponding open reading frames.
The nucleotide sequence determined for the H. pylori isoleucyl-tRNA synthetase gene is shown in SEQ ID NO:1. The open reading frame (ORF) is 2760 base pairs and encodes a polypeptide of 920 amino acids (SEQ ID NO:2), with translation starting at the GTG at position 149 in SEQ ID NO:1. GTG initiation codons (GUG in the mRNA) have been observed in H. pylori (see Labigne et al., J. Bacteriol., 173: 1920-1931 (1991), describing the UreD gene), as well as in other organisms. Where a codon other than AUG has been observed, invariably, the amino acid used for initiation has been determined to be methionine (Varshney, U. and U. L. RajBhandary, Proc. Natl. Acad. Sci. USA, 87:1586-1590 (1990)). Accordingly, SEQ ID NO:1 shows a methionine at the N-terminus of the encoded protein. The deduced amino acid sequence of IleRS contains a .sup.65 HLGH.sup.68 motif, which resembles the HIGH sequence, and a .sup.610 KMSKS.sup.614 motif. These two sequence motifs are characteristic of all class I aaRSs. The H. pylori IleRS amino acid sequence was compared with the IleRS sequences available in the data bank by using the Multiple Sequence Alignment program from the DNASTAR package. Percent similarity and percent divergence among these sequences were determined using the Clustal method with PAM250 residue weight table. The percent similarity between the predicted 920-amino acid sequence of H. pylori IleRS and proteins identified as IleRSs from C. jejuni and Staphylococcus aureus was found to be 65.5% and 54.0%, respectively. Other sequences were even less related.
The nucleotide sequence determined for the H. pylori methionyl-tRNA synthetase gene is shown in SEQ ID NO:3. The ORF is 1944 base pairs, encoding a polypeptide of 648 amino acids (SEQ ID NO:4), with translation starting from the ATG at position 102 in SEQ ID NO:3. The deduced MetRS sequence contains class-defining motifs at position 18 (.sup.18 HIGH.sup.21) and at position 301 (.sup.301 KMSKS.sup.305) SEQ ID NO:3. The H. pylori MetRS sequence was compared with the MetRS sequences available in the data bank by using the Multiple Sequence Alignment program from the DNASTAR package. Percent similarity and percent divergence among these sequences were determined using the Clustal method with PAM250 residue weight table. The percent similarity between the predicted 648-amino acid sequence of H. pylori MetRS and proteins identified as MetRSs from B. stearothermophilus and T. thermophilus was found to be 36.2% and 34.6%, respectively. Other sequences were even less related.
The nucleotide sequence determined for the H. pylori leucyl-tRNA synthetase gene is shown in SEQ ID NO:5. The ORF is 2418 base pairs, encoding a polypeptide of 806 amino acids (SEQ ID NO:6) with translation starting from the ATG at position 153 in SEQ ID NO:5. The putative leucyl-tRNA synthetase also contains class I motifs (.sup.45 HMGH.sup.48 and .sup.572 KMSKS.sup.576, SEQ ID NO:5. The H. pylori LeuRS sequence was compared with the LeuRS sequences available in the data bank by using the Multiple Sequence Alignment program from the DNASTAR package. Percent similarity and percent divergence among these sequences were determined using the Clustal method with PAM250 residue weight table. The percent similarity between the predicted 806-amino acid sequence of H. pylori LeuRS and proteins identified as LeuRSs from E. coli and Bacillus subtilis was found to be 39.3% and 34.2%, respectively. Other sequences were even less related.
The nucleotide sequence determined for the H. pylori valyl-tRNA synthetase gene is shown in SEQ ID NO:7. The ORF is 2616 base pairs, encoding a polypeptide of 872 amino acids (SEQ ID NO:8), with translation starting from the ATG at position 219 in SEQ ID NO:7. In the deduced protein sequence, the class-defining HIGH and KMSKS motifs are located between positions 56-59 and 531-535, respectively SEQ ID NO:8. The H. pylori ValRS sequence was compared with the ValRS sequences available in the data bank by using the multiple sequence alignment program from the DNASTAR package. Percent similarity and percent divergence among these sequences were determined using the Clustal method with PAM250 residue weight table. The percent similarity between the predicted 872-amino acid sequence of H. pylori ValRS and proteins identified as ValRSs from B. stearothermophilus and E. coli was found to be 36.3% and 33.4%, respectively. Other sequences were even less related.
The nucleotide sequence determined for the H. pylori lysyl-tRNA synthetase gene is shown in SEQ ID NO:9. The ORF is 1503 base pairs, encoding a polypeptide of 501 amino acids (SEQ ID NO:10), with translation starting from the ATG at position 121 in SEQ ID NO:9. The three class-defining sequence motifs, which are highly conserved among the class II aminoacyl-tRNA synthetases, are located between positions 191-200, 251-268, and 460-473, respectively in the deduced amino acid sequence of H. pylori LysRS. The H. pylori LysRS sequence was compared with the LysRS sequences available in the data bank by using the multiple sequence alignment program from the DNASTAR package. Percent similarity and percent divergence among these sequences were determined using the Clustal method with PAM250 residue weight table. The percent similarity between the predicted 501-amino acid sequence of H. pylori LysRS and proteins identified as C. jejuni LysRS, the E. coli lysS gene product, and the E. coli lysU gene product was found to be 57.3%, 47.6%, and 46.4% respectively. Other sequences were even less related.
The nucleotide sequence determined for the H. pylori seryl-tRNA synthetase gene is shown in SEQ ID NO:11. The ORF is 1245 base pairs, encoding a polypeptide of 415 amino acids (SEQ ID NO:12), with translation starting from the ATG at position 80 in SEQ ID NO:11. As in the H. pylori LysRS, three class II aaRS sequence motifs can also be identified in the deduced amino acid sequence of H. pylori SerRS. They are located between positions 190-199, 261-285, and 382-394, respectively . The H. pylori SerRS sequence was compared with the SerRS sequences available in the data bank by using the Multiple Sequence Alignment program from the DNASTAR package. Percent similarity and percent divergence among these sequences were determined using the Clustal method with PAM250 residue weight table. The percent similarity between the predicted 415-amino acid sequence of H. pylori SerRS and proteins identified as SerRSs from E. coli and Coxiella burnetii was found to be 44.5% and 42.1%, respectively. Other sequences were even less related.
Example 5
Expression of GST-H. pylori Ile and Met tRNA synthetase fusion proteins
The DNA fragments comprising the ORFs of Ile and Met tRNA synthetases were recovered by PCR amplification using the following PCR primers:
______________________________________Ile 5' primer 5'-CGTGGATCCGTGAAAGAATACAAAGACAC-3'(SEQ ID NO:42):Ile 3' primer 5'-CCCAGTCGACTTATCATCGCTCTTTTAAAACC-3'(SEQ ID NO:43):Met 5' primer 5'-CGTGGATCCATGCAAAAATCACTGATCAC-3'(SEQ ID NO:44):Met 3' primer 5'-CCCAGTCGACTTAGCTGATCAAACTTCCTGC-3'(SEQ ID NO:45):______________________________________
PCR reactions were carried out in 50 .mu.l with 10 mM Tris HCl (pH 8.3 at room temperature), 50 mM KCl, 1 .mu.l of of cosmid DNA (clone 51 for IleRS and clone 30 for MetRS), 20 pmole each of the 5' and 3' primers, 0.2 mM each of dNTPs, 1.5 mM MgCl.sub.2, and 5 units of AmpliTaq DNA polymerase (Perkin-Elmer). The reactions were first denatured for 2 minutes at 95.degree. C., followed by 30 cycles of 95.degree. C. (30 seconds), 55.degree. C. (30 seconds), and 72.degree. C. (2 minutes). The thermal cycles were extended at 72.degree. C. for 8 minutes.
The amplified DNA fragments were purified by using Gene Clean method (Bio 101), and then digested with BamHI/SalI restriction endonucleases (New England Biolabs, Inc.) followed by gel purification. The purified DNA fragments were then cloned into the BamHI/SalI sites of pGEX-4T-2 E. coli expression vector (Pharmacia), yielding the plasmids pGEXHPIRS-1 (for GST-IleRS fusion) and pGEXHPMRS-1 (for GST-MetRS fusion), respectively.
The H. pylori IleRS and MetRS expression constructs were used to transform E. coli DH5.alpha. cells. To test the expression of the recombinant GST (glutathione-S-transferase) fusion proteins, the transformed cells were grown 5 hours at 37.degree. C. in 3 ml of LB broth with 50 .mu.g/ml of ampicillin until the A600 of the bacterial cultures reached 0.6 to 1. Protein expression was induced by the addition of IPTG to 1 mM. After 2.5 hours induction of protein expression at 37.degree. C., the bacterial cells were recovered by centrifugation, and lysed in 1X phosphate-buffer-saline (1X PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2 HPO.sub.4, 1.8 mM KH.sub.2 PO.sub.4, pH7.3) by sonication. The soluble proteins of the crude cell extracts were obtained by centrifugation of the cell lysates and fractionation by electrophoresis on a 10% SDS-polyacrylamide gel. Protein was visualized by staining with Coomassie Blue.
In the case of IleRS, a protein with apparent molecular weight of about 130 kDa was expressed without induction, and a higher level of expression was achieved upon the IPTG induction. For MetRS, a protein of about 97 kDa was expressed to detectable levels upon induction with IPTG. The MetRS protein was not visible in the absence of IPTG induction. Both of the recombinant proteins can be purified using glutathione-agarose affinity chromatography as described below.
Example 6
Purification and Aminoacylation Activities of the H. pylori GST-Ile and GST-Met tRNA Synthetases
20 ml overnight LB cultures (with 50 .mu.g/ml of ampicilline) of E. coli harboring the pGEXHPIRS-1 or pGEXHPMRS-1 plasmid were used to inoculate fresh 2 liter cultures of LB (containing 50 .mu.g/ml of ampicillin). The cells were grown at 37.degree. C. for 3.5 hours to reach OD600 to 0.9-1.0 before IPTG was added to final concentration of 1 mM to induce the expression of recombinant proteins. After 3 hours of IPTG induction of expression, the bacterial cells were pelleted by centrifugation in Beckman JA10 rotor for 20 minutes at 6000 rpm and stored at -80.degree. C. until protein purification.
To purify the proteins, the cells were resuspended in 30 ml of 1X PBS with 5 mM DTT and 2 mg/ml lysozyme. Non-ionic detergent NP-40 was added to the cell suspension to final concentration of 0.1% prior to the cell lysis by French Press. The cell lysates were centrifuged at 12,000 g for 10 min at 4.degree. C. and the supernatants were recovered and loaded onto 15 ml Glutathione-agarose (SIGMA Chemical Co., St. Louis, Mo.) affinity columns equilibrated with 1X PBS/ 5 mM DTT at 4.degree. C.
After the samples were loaded, each column was washed with 150 ml of 1X PBS with 5 mM DTT at 4.degree. C. The GST-aaRS fusion protein bound specifically to the glutathione on the column was eluted with 30 ml of 10 mM glutathione, 50 mM Tris-HCl (pH7.9) solution, at 25.degree. C. The eluted fusion protein was concentrated by using Centricon (Amicon) to final volume of 2 ml. The purified proteins were analyzed on a 10% SDS-polyacrylamide gel. The proteins were stored at -80.degree. C. with 50% glycerol and 5 mM DTT.
The purified GST fusions of H. pylori isoleucyl- and methionyl-tRNA synthetase were tested for their corresponding charging activities. Charging (aminoacylation) assays were based on the procedure of Shepard et al. (Proc. Natl. Acad. Sci. USA, 89: 9964-68 (1992)). Aminoacylation reactions with IleRS were carried out in 50 mM HEPES, pH7.5, 10 mM 2-mercaptoethanol, 4 mM ATP, 10 mM MgCl.sub.2, 20 .mu.M �.sup.3 H!-isoleucine (80 .mu.Ci/ml), 180 .mu.M crude E. coli total tRNA (SIGMA), and 1 .mu.l of the purified GST-H. pylori IleRS protein. Aminoacylation reactions with MetRS were carried out in 20 mM HEPES, pH 7.5, 0.1 mM EDTA, 150 mM NH.sub.4 Cl, 100 .mu.g/ml BSA, 2 mM ATP, 4 .mu.M tRNA.sup.fMet (Boehringer-Mannheim), 4 mM MgCl.sub.2, and 20 .mu.M �.sup.35 S!-methionine (40 uCi/ml) and 1 .mu.l of the purified GST-H. pylori MetRS protein.
Reactions were incubated at 25.degree. C. For each time point, an aliquot of the reaction mixture was removed and spotted onto Whatman 3MM paper filter disks. The filter disks were immediately placed in cold 5% trichloroacetic acid (TCA). After 3 washes in 5% TCA (20 minutes each), the filter disks were washed once with 95% ethanol for 5 minutes, briefly washed once with ether, dried under a heat lamp, and subjected to scintillation counting.
As positive controls, purified E. coli IleRS and MetRS were assayed in the same way as the purified recombinant proteins. E. coli IleRS was purified as previously described (Shepard et al., Proc. Natl. Acad. Sci. USA, 89:9964-68 (1992)). About 10 ng of the purified enzyme was used in the assay. E. coli MetRS was also purified using a published protocol (Burbaum, J. J. and P. Schimmel, Biochemistry, 30: 319-324 (1991)). About 2 .mu.g of the purified MetRS was used in the assay. Reactions without aaRS or without tRNAs were carried out under the same conditions, as negative controls. The results are depicted in FIGS. 1 and 2.
Example 7
Expression of H. pylori leucyl-, valyl-, seryl-, and lysyl-tRNA synthetases
H. pylori leucyl-, valyl-, seryl-, and lysyl-tRNA synthetases were also expressed as GST (glutathione-S-transferase) fusion proteins. For each synthetase, a DNA fragment comprising the open reading frame was recovered by PCR amplification using the following pairs of PCR primers for each synthetase:
______________________________________Leu 5' 5'-GCGCGGATCCATGGATTTTATCAATATAGAAA-3'primer (SEQID NO:46):Leu 3' 5'-ACGCGTCGACTTATGCGATAACAAAATTAACG-3'primer (SEQID NO:47):Val 5' 5'-GCGCGGATCCATGAAACAAGAACCCACCACCT-3'primer (SEQID NO:48):Val 3' 5'-ACGCGTCGACTTATGGTTGTTTTAACAAATC-3'primer (SEQID NO:49):Ser 5' 5'-GCGCGGATCCATGATTGATAGAAAACTTTTAT-3'primer (SEQID NO:50):Ser 3' 5'-ACGCGTCGACTTAAAGGTATTTTTCTAACGC-3'primer (SEQID NO:51):Lys 5' 5'-GCGCGGATCCATGTTTTCTAACCAATACATC-3'primer (SEQID NO:52):Lys 3' 5'-ACGCGTCGACTTATTCTCCACTCTCTCCACA-3'primer (SEQID NO:53):______________________________________
For the Ser primers, the reaction was performed essentially as described above for the Ile and Met GST-fusion proteins (see Example 5) using cosmid DNA clone 49 as template.
For Lys, Leu and Val primers, individual reactions were carried out in a 50 .mu.l volume with 1X Vent DNA polymerase buffer (New England Biolabs), 20 pmole of each of the 5' and 3' primers, 0.1 mM each of dNTPs, and 2 units of Vent DNA polymerase (New England Biolabs). One .mu.l (.about.5-20 ng) of cosmid DNA was used as template in each reaction (templates: Lys, clone 67; Leu, clone 55; Val, clone 41). The reactions were first denatured for 2 minutes at 95.degree. C., followed by 30 cycles of 95.degree. C. (30 seconds), 55.degree. C. (30 seconds), and 72.degree. C. (2 minutes). The thermal cycles were extended at 72.degree. C. for 8 minutes.
The amplified DNA fragments were purified by using Gene Clean (Bio 101), and then digested with BamHI and SalI restriction endonucleases (New England Biolabs, Inc.) followed by gel purification. The purified DNA fragments were then cloned into expression vector pGEX-4T-2 (Pharmacia) which had been linearized with BamHI and SalI.
The resulting GST fusion expression constructs for leucyl-, valyl-, seryl-, and lysyl-tRNA synthetases were designated pGEXHPLEU-3, pGEXHPVAL-1, pGEXHPSER-2, and pGEXHPLYS-1, respectively. Transformants of E. coli DH5-.alpha. and E. coli JM109 were obtained for each construct.
The Lys 3' primer (SEQ ID NO:53) used for amplification of the H. pylori LysRS gene contained a 2 nucleotide insertion (CT, indicated in bold above). As a result of this insertion, the GST-LysRS fusion protein encoded by pGEXHPLYS-1 consists of an N-terminal GST portion fused to amino acids 1-498 of LysRS, and an extra C-terminal 12 amino acids fused to Glu.sup.498 (i.e., -Arg-Val-Glu-Asn-Lys-Ser-Thr-Arg-Ala-Ala-Ala-Ser) (SEQ ID NO:67), which were introduced by frameshift and readthrough into the vector (pGEX-4T-2; amino acids in bold are encoded by the vector).
GST-SerRS and GST-LysRS fusion proteins were expressed in JM109 cells and purified essentially as described for the GST-IleRS and GST-MetRS fusion proteins (see Example 6). GST-LeuRS and GST-ValRS fusion proteins were also expressed to a high level upon IPTG induction.
Example 8
Aminoacylation Activities of the H. pylori GST-Ser and GST-Lys tRNA Synthetases
Aminoacylation reactions with SerRS or LysRS were carried out in 50 mM HEPES, pH 7.5, 8 mM KF, 16 mM 2-mercaptoethanol, 4.4 mM ATP, 10 mM MgCl.sub.2, 18 .mu.M Ser (39 .mu.Ci �.sup.3 H!-serine/ml) or 20 .mu.M Lys (220 .mu.Ci �.sup.3 H!-lysine/ml), and 90 .mu.M E. coli total tRNA (Boehringer-Mannheim). About 0.3 .mu.g of purified GST-SerRS or 0.3 .mu.g of purified GST-LysRS was added to the corresponding charging reaction.
Purified E. coli SerRS and LysRS were assayed under the same conditions as the corresponding recombinant GST-fusion protein. E. coli SerRS and LysRS were partially purified on a DEAE column, and 10 .mu.l of the partially pure preparation was used in an aminoacylation reaction. As negative controls, reactions without tRNA were carried out under the same conditions. The results are depicted in FIGS. 3 and 4.
Example 9
Functional Complementation of a Mitochondrial Lysyl-tRNA Synthetase Null Strain by H. pylori and Human Lysyl-tRNA Synthetase Genes in Saccharomyces cerevisiae
Construction of Expression Vectors for Mitochondrial Targeting
pQB111 and pQB136
The presequence from the cytochrome oxidase IV was used in the mitochondrial import vectors pQB111 and pQB136. This sequence has been used to allow import of several heterologous proteins in the mitochondria (Hurt, E. C., et al., EMBO J. 3:3149-3156 (1984); Pinkham, J., et al., Mol. and Cell. Biol. 14:4643-4652, (1994)).
In order to construct pQB111, an SphI-XbaI fragment bearing the ADH1 promoter and 22 of the 25 amino acids of COXIV (cytochrome oxidase IV) presequence were excised from plasmid pMC4 (obtained from J. Pinkham, University of Massachusetts, Amherst) (Bibus, C. R., et al., J. Biol. Chem. 263:13097-13102 (1988); Hurt, E. C., et al., J. Biol. Chem. 262:1420-1424 (1987)) and cloned into the SphI and XbaI sites of YEplac195 (also referred to as pQB42) (Sugino, A., and Gietz, R. D. Gene 74:527-534 (1988)) to form pQB111.
Plasmid pQB136 is a derivative of pQB111 which allows construction of GST (glutathione-S-transferase) fusion proteins targeted to mitochondria. PCR was used to amplify the GST gene from pGEX-4T-2 (Pharmacia) using the following primers:
5'-GCGCTCTAGATATCTGCTTATGTCCCCTATACTAGGTTATTGG-3' (SEQ ID NO:54),
and
5'-GGGGTACCTCACGATGCGGCCGCTCGAG-3' (SEQ ID NO:55).
(The ATG underlined in the 5'-primer is the start site of GST; the bases in boldface specify amino acids 22-25 of the COXIV presequence.) The 5' primer introduced an XbaI site (underlined), which when fused to the XbaI site in plasmid pQB111, restores the entire (25 amino acid residue) presequence of COXIV. The 3' primer introduced a KpnI site (underlined) downstream of the GST stop codon. The PCR product was cleaved with XbaI and KpnI and inserted into the XbaI and KpnI sites of pQB111 to yield pQB136.
pQB161
Plasmid pQB152, which encodes a GST-MSM1 protein fusion, was constructed by PCR amplification of the wild type MSM1 gene (mitochondrial methionyl-tRNA synthetase) from plasmid pQB104 (pG72/T1) (Tzagoloff, A., et al., Eur. J. Biochem. 179:365-371 (1989)), using the following primers:
5'-CCGCTCGAGCGATGCAATGTCGATCAATTGTGC-3' (SEQ ID NO:56)
and
5'-GGGGTACCCCTTTTTCATGACCTCATATTCG-3' (SEQ ID NO:57).
The PCR product was cleaved with XhoI and KpnI and cloned into the XhoI and KpnI sites of pQB136 to yield pQB152. In subsequent studies pQB152, encoding a GST-MSM1 fusion, was observed to complement msml-1 and msml::HIS3 strains on YEPG medium.
To construct pQB161, a KpnI-HindIII fragment containing the GST-MSM1 fusion gene of pQB152 was excised and cloned into the KpnI and HindIII sites of pQB41 (also referred to as YEplac181; Gietz and Sugino, Gene, 74: 527-534 (1988)), yielding pQB161. The backbone of pQB161 is similar to pQB111 in that it contains an ADH promoter and encodes the first 22 amino acids of COXIV; however, the plasmid bears a LEU2 selectable marker in lieu of the URA3 marker.
pQB218 (MSK1, URA3)
Plasmid pQB106 (also referred to as pG11/T6), which carries the yeast MSK1 gene (GenBank Accession No. X57360), has already been described (Gatti, D.L. and A. Tzagoloff, J. Mol. Biol., 218: 557-568 (1991)). Genomic clone pG11/T6 (ATCC.RTM. No. 77080) is available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852-1776. A 3 kb XbaI fragment comprising the MSK1 gene was excised from pQB106, and inserted into the 2 .mu. URA3 vector YEplac195 (also referred to as pQB42) (Sugino, A., and Gietz, R. D. Gene 74:527-534 (1988)) to yield plasmid pQB218 (pC.sup.3 473). pQB218, which encodes the yeast MSK1 gene, and carries the URA3 selectable marker, can be used as a maintenance plasmid.
pQB211 (H. pylori LysRS gene, LEU2)
In order to clone the lysyl-tRNA synthetase gene from H. pylori into a mitochondrial targeting vector, the gene was first amplified using the following primers:
______________________________________5'hplys (SEQ ID NO:58):5'-CAGACGTCTAGATATCTGCTTATGTTTTCTAACCAATAC-3'3'hplys (SEQ ID NO:59):5'-ACCGCTCGAGCGGTTATTCTCCACTCTCCACATT-3'______________________________________
PCR conditions were as follows: 94.degree., 1'; 60.degree., 1'; 72.degree., 2' for 35 cycles. Reactions contained 50 .mu.l 1X Taq polymerase buffer, 0.2 mM of each dNTP, 2 .mu.M of each primer, and 0.5 .mu.l of Taq polymerase (2.5 units of AmpliTaq polymerase (Perkin-Elmer)). The template used for PCR amplification was cosmid clone #67 (see above). The 1.5 kb PCR product amplified with 5'hplys and 3'hplys primers was excised from 1% agarose gel, purified using a Gene CleanII Kit (Bio 101), and ligated to pT7Blue(R) vector (T-vector; Novagen) to generate plasmid pQB209 (pC.sup.3 464). The insert of pQB209 as excised with XbaI and KpnI and cloned into the corresponding sites of the vector backbone of plasmid pQB161 (which had been cleaved with XbaI and KpnI to release the GST-MSM1 fusion sequences) to generate plasmid pQB211 (pC.sup.3 466).
pQB210 (Human LysRS gene, LEU2)
The procedure for constructing a vector for expression and mitochondrial targeting of the human lysyl-tRNA synthetase gene was identical to that described above for H. pylori LysRS with the following differences. In order to clone the human lysyl-tRNA synthetase gene into the mitochondrial targeting vector, the gene was first amplified using the following primers: ##STR1## (Primer 3'hulys introduces a silent mutation in the codon just before the stop codon (bold); the primer was designed to inactivate an XbaI site at this location). The template for amplification using the 5'hulys and 3'hulys primers was plasmid pM115 which contains a cDNA encoding human LysRS (K. Shiba; see also GenBank, National Center for Biotechnology Information (NCBI Seq ID: 505107), Accession No. D31890). A 1.8 kb PCR product was amplified, isolated as described above, and ligated to pT7Blue(R) vector (T-vector; Novagen) to generate plasmid pQB208 (pC.sup.3 463). The insert of pQB208 was excised with XbaI and KpnI and cloned into the corresponding sites of the vector backbone of plasmid pQB161 (which had been cleaved with XbaI and KpnI) to generate plasmid pQB210 (pC.sup.3 465).
Strains
Standard methods for yeast propagation and transformation were used (Ausubel, F. M., et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, Inc., (1993); Rose et al., 1990, Methods in Yeast Genetics, (Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.).
QBY4
Strain QBY4, also referred to as EY722, has the following genotype: MAT.alpha. ade2-1 his3-11,15 leu2-3,112 ura3-1 trpl-l canl-100 Gal+)
QBY274
Strain QBY274, a kar1.DELTA.15 derivative of QBY4, was made by integrative transformation of W303 strain QBY4 with pMR1593. pMR1593 is a derivative of YIp5, which contains the kar1.DELTA.15 allele, and the URA3 and .beta.-lactamase genes as selectable markers (Mark Rose, Princeton University, N.J.; J. Cell. Biol., 117:1277-1287 (1992); see GenBank Accession No. M15683 (YSCKAR1) for KAR1 sequence). To direct integration into the KAR1 locus by integrative transformation, ten .mu.g of pMR1593 were linearized with BglII and used to transform QBY4. Ura+tranformants were selected, and passaged twice on synthetic complete medium containing 5-fluoroorotic acid (1 g/liter of 5-FOA) to select for the replacement of the chromosomal copy of KAR1 by the kar1.DELTA.15 allele and loss of the YIp5-derived vector. Chromosomal DNA was prepared from 10 independent isolates of the resulting strain. The chromosomal DNA was digested with NsiI and subjected to Southern analysis using a 600 bp fragment from plasmid pMR1593 as a probe. The results of the Southern analysis confirmed the presence of a deletion at the KAR1 locus. Strains containing the kar1.DELTA.15 allele were also tested for their deficiency in karyogamy by mating assay.
QBY47
The mitochondrial lysyl-tRNA synthetase deficient strain used was the disruption strain QBY47 (W303.gradient.MSK1) (MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 trpl-1 msk1::HIS3) (Gatti, D. L. and A. Tzagoloff, J. Mol. Biol., 218: 557-568 (1991)).
QBY47 (pQB218)
Strain QBY47 (pQB218) was made by transforming QBY47 with pQB218. For complementation studies, a rho.sup.+ derivative was constructed by cytoduction. In particular, 5.times.10.sup.6 cells from logarithmic phase cultures of each of QBY47(pQB218) and QBY274 were mixed and spread onto a nitrocellulose filter laid on top of a YPD agar plate. The plate was incubated at 30.degree. C. for 5 hours. Cytoductants were micromanipulated on YPD agar and allowed to form colonies; the colonies were later purified on SC-glycerol media lacking histidine and uracil to select for rho+ derivatives of QBY47(pQB218). Selection on 5-FOA plates following transformation was used to replace pQB218 (MSK+ URA3+) with the test plasmids, which were based on a LEU2-marked 2.mu. vector.
QBY171
Strain QBY171 has the following genotype: MAT.alpha. mal rho+ and is available from the American Type Culture Collection (Accession No. ATCC 24658; Genetics, 49: 39 (1964)). The strain serves as a wildtype control.
Complementation of a Yeast Mitochondrial Gene Defect
Strains of S. cerevisiae having mutations (e.g., point mutations) in nuclear PET genes (petite or pet mutants), whose expression is required for the morphogenesis of respiratory-competent mitochondria, cannot grow on non-fermentable carbon sources such as glycerol media. However, because S. cerevisiae is a facultative anaerobe, such strains are capable of growing on fermentable carbon sources such as glucose, in the absence of mitochondrial function. On rich media such as glucose, these "petite" strains exhibit the small colony phenotype for which they are named. The majority of mitochondrial proteins, including the mitochondrial aminoacyl-tRNA synthetases, are nuclear encoded, synthesized in the cytoplasm and imported into mitochondria. Petite mutants of S. cerevisiae having defects in genes encoding a mitochondrial aminoacyl-tRNA synthetases have been identified (see e.g., Tzagoloff, A. and A. M. Myers, Ann. Rev. Biochem. 55:249-285 (1986); Tzagoloff, A. and C. L. Diekmann, Microbiol. Rev. 54(9):211-225 (1990); Myers, A. M., et al., EMBO J. 4(8) :2087-2092 (1985)).
Although pet strains having mutations in nuclear genes encoding components of the mitochondrial translational apparatus, such as mitochondrial aminoacyl-tRNA synthetase genes, can grow on glucose, these strains tend to lose their mitochondrial DNA at high frequency, converting to rho- or rho.sup.0 strains, with large deletions in their mitochondrial DNA (rho-) or no mitochondrial DNA (rho.sup.0) (Tzagoloff, A. and A. M. Myers, Ann. Rev. Biochem., 55:249-285 (1986); Myers, A. M., et al., EMBO J., 4(8):2087-2092 (1985)).
For complementation studies, functional mitochondria were introduced into strain QBY47, a strain having a mutation in the nuclear gene encoding mitochondrial lysyl-tRNA synthetase. Initial attempts to introduce functional mitochondria by mating strain QBY47 with the wild type strain QBY4 were unsuccessful. Although His.sup.+ rho.sup.+ diploids were isolated, when sporulated, these strains gave rise to inviable spores. Accordingly, functional mitochondria were introduced by cytoduction. First, a set of kar1.DELTA.15 strains was constructed in the W303 background, including strain QBY274 used in this study. Strain QBY47 (pQB218) rho-, which carries the disruption allele (msk1::HIS3) and maintenance plasmid pQB218, was mated with QBY274 (QBY4 kar1.DELTA.15) to introduce mitochondria by cytoduction as described above.
Complementation
Strain QBY47 (pQB218) rho+ was transformed with plasmid pQB211. Leu+ transformants were selected on Sc-Leu plates (synthetic complete medium, minus leucine). Selection on 5-FOA plates (synthetic complete medium containing 1 g/liter 5-fluoroorotic acid) was used to induce loss of the maintenance plasmid pQB218 (which carries the MSK1 and URA3 genes). 5-FOA resistant colonies were grown on a variety of plates to confirm their phenotype (Ura-Leu+His+), including YPD, SD, SC, (SC minus adenine), (SC minus leucine), (SC minus tryptophan), (SC minus uracil), (SC minus histidine), 5-FOA plates, and mating type lawns. 5-FOA resistant single colonies carrying plasmid pQB211 were then tested for complementation as indicated by growth on glycerol media (YEPG). The growth on YEPG plates (rich medium, supplemented with glycerol) of five of the 5-FOA resistant colonies was examined. Growth was weak compared to strain QBY47 (pQB218) rho+ or the general wild type strain QBY171. Growth on glycerol was shown to be plasmid-dependent, since cured Leu- strains (5 colonies tested) were no longer able to grow on YEPG plates. These results indicate that the H. pylori aaRS can substitute for the function of the host cell aminoacyl-tRNA synthetase. The transformants are examples of tester strains containing an H. pylori LysRS gene.
In the case of human LysRS complementation, plasmid pQB210 was isolated from complementing strains and its identity was verified by restriction digestion with EcoRI or EcoRV, using the original plamsid and pQB183 as controls. Attempts to recover pQB211 plasmid (hp lysRS) from complementing strains failed to yield E. coli transformants; detection of pQB211 by more sensitive methods such as PCR will further confirm these results.
The color of Ade- strains on YPD medium is often a good reflection of how well a certain plasmid can complement a mitochondrial tRNA synthetase defect. In the case of complementation by the H. pylori LysRS gene carried by pQB211, and the human LysRS gene carried by pQB210, the strains had a white appearance, suggesting weak complementation.
Example 10
Complementation Studies with H. pylori Isoleucyl- and Methionyl-tRNA Synthetase Genes in E. coli and Saccharomyces cervisiae
E. coli Strains
E. coli strain MI1 carries a chromosomal point mutation in the ileS gene, conferring an isoleucine auxotrophy (Iaccarino, M. and Berg, P., J. Bacteriol. 105:527-537 (1971); Treiber, G. and Iaccarino, M., J. Bacteriol. 107:828-832 (1971); and Schmidt, E. S. and P. Schimmel, Science, 264: 265-268 (1994)).
E. coli strain IQ844/pRMS711 is a derivative of IQ843/pRMS711 (Shiba, K. and P. Schimmel, Proc. Natl. Acad. Sci. USA, 89:1880-1884 (1992); Shiba, K. and P. Schimmel, Proc. Natl. Acad. Sci. USA, 89:9964-9968 (1992); Shiba, K. and P. Schimmel, J. Biol. Chem., 267:22703-22706 (1992)). The IQ843/pRMS711 and IQ844/pRMS711 strains each contain a chromosomal deletion of the ileS gene (.DELTA.ileS203::kan), and are propagated by expression of wild type IleRS at 30.degree. C. from a temperature-sensitive maintenance plasmid designated pRMS711, which encodes the wild type ileS gene and a gene which confers chloramphenicol resistance. pRMS711 cannot replicate at 42.degree. C.; thus, at the non-permissive temperature, the maintenance plasmid is lost. Following the introduction of a test construct into these strains, the growth of chloramphenicol sensitive colonies at 42.degree. C. is indicative of complementation of the chromosomal ileS deletion by the introduced construct.
E. coli strain MN9261/pRMS615 (Kim, S., et al., Proc. Natl. Acad. Sci. USA 90:10046-10050 (1993)) carries a chromosomal null allele of metG. Strain MN9261/pRMS615 also contains a temperature sensitive maintenance plasmid (pRMS615) which carries a wild type metG allele (encoding E. coli MetRS), and has a temperature sensitive replicon which causes loss of the maintenance plasmid at the non-permissive temperature (e.g., 42.degree. C.).
GST-H. pylori IleRS fusion gene complements the ileS defect in E. coli Strain MI1
E. coli strain MI1, which carries a chromosomal point mutation in the ileS gene (conferring an isoleucine auxotrophy), was used in complementation tests. MI1 cells were transformed with pGEXHPIRS-1 and were tested for viability on M9 medium (Maniatis et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.), containing 0.008% arginine and tryptophan, 100 .mu.g/ml ampicillin, and 50 .mu.g/ml IPTG at 37.degree. C. for two days. MI1 cells transformed with the expression vector pGEX4T-2 (Pharmacia) served as a negative control, and MI1 cells transformed with a plasmid containing E. coli IleRS gene served as a positive control. Under the conditions set forth above, complementation of the ileS mutation of E. coli MI1 by the H. pylori IleRS fusion gene carried on pGEXHPIRS-1 was clearly observed.
The fusion constructs, which direct the expression of (a) a GST-IleRS fusion protein (pGEXHPIRS-1) or (b) a GST-MetRS fusion protein (pGEXHPMRS-1) were also introduced into E. coli strains having a null mutation in the corresponding E. coli host aaRS gene. In particular, E. coli strain IQ844/pRMS711 was transformed with pGEXHPIRS-1, and E. coli strain MN9261/pRMS615 was transformed with pGEXHPMRS-1. Transformants were tested at 42.degree. C. to induce loss of the respective maintenance plasmids. Under the conditions of the assay, complementation was not observed. In contrast, E. coli strain IQ844/pRMS711 transformed with pKS21 (a control containing the E. coli ileS gene, encoding IleRS; Shiba, K. and P. Schimmel, Proc. Natl. Acad. Sci. USA, 89:1880-1884 (1992)) and E. coli strain MN9261/pRMS615 transformed with pJB104 (a control containing the E. coli MetG gene, encoding MetRS; Kim, S. and P. Schimmel, J. Biol. Chem., 267: 15563-67 (1992)) were able to grow at the non-permissive temperature (420). Loss of the maintenance plasmid was verified by assessing antibiotic resistance. The nucleotide sequences of the IleRS and MetRS portions of the fusion constructs were confirmed by DNA sequencing. Adjustment of growth conditions may permit complementation.
S. cerevisiae cytoplasmic IleRS
A construct designed to express an H. pylori IleRS having the amino acid sequence shown in SEQ ID NO:2 was tested for complementation of a null allele of the S. cerevisiae cytoplasmic IleRS gene (ILS1) in strain QBY187. Strain QBY187 was constructed by transformation of a diploid (QBY182) (ILS1/ils1.DELTA.::TRP1) with an ILS1 maintenance plasmid (pQB89; see Example 11) and Ura+transformants were selected. The transformant was sporulated, tetrads were dissected, a haploid Trp+Ura+spore was identified, and was designated QBY187 (MAT.alpha. leu2.DELTA.1 lys2-128.delta. ura3-52 trp1.DELTA.63 ils1.DELTA.::TRP1/pQB89). QBY187 was transformed with pQBHPIRS, which contains the H. pylori IleRS gene between the PstI/SalI sites of E. coli/S. cerevisiae shuttle vector pQB169 (see Example 11, below). To provide positive and negative controls, QBY187 cells were transformed with either plasmid pQB197 (which contains the wild type yeast ILS1 gene and a LEU2 selectable marker) or with vector pQB169, respectively. Leu+transformants of QBY187 containing pQBHPIRS, pQB197 or pQB169 were selected, and tested for viability at 30.degree. C. on plates containing synthetic complete (SC) medium and 5-FOA. 5-FOA leads to loss of the maintenance plasmid pQB89, which carries a URA3 gene. Under the conditions used, complementation of the ils1.DELTA.::TRP1 defect of strain QBY187 by pQBHPIRS was not observed. Similarly, no complementation was observed in the vector control. In contrast, QBY187 cells harboring pQB197, which carries the wild type yeast ILS1 gene, were able to grow under these conditions.
S. cerevisiae mitochondrial IleRS
A haploid yeast strain having a disruption (a HIS3 insertion) in the gene encoding the mitochondrial isoleucyl-tRNA synthetase (msi1::HIS3) was constructed. This strain was transformed with pQB240, a URA3 maintenance (CEN) plasmid in which the MSI1 gene is expressed from its natural promoter. A rho.sup.+ derivative carrying the maintenance plasmid was derived by cytoduction �strain QBY343 (W303 msi1::HIS3 (pQB240) rho.sup.+)!. The resulting strain was used to test two different constructs.
One construct (pQB242) was designed to express an H. pylori IleRS having the COXIV presequence fused to SEQ ID NO:2 ("native") (using an ATG initiation codon in place of the GTG initiation codon for expression). A second construct (pQB249) was designed to express a GST-H. pylori IleRS fusion protein, again using the COXIV presequence for mitochondrial targeting. pQB249 was constructed by excising the BamHI-SalI fragment containing a GST-H. pylori IleRS fusion protein from pGEXHPIRS-1 (see above), and ligating the fragment to the BamHI-SalI backbone of pQB153-GST (also referred to as pQB248)(see below).
Strain QBY343 was transformed with pQB242, pQB249, or a positive control construct. Leu+ transformants were isolated, and purified on 5-FOA plates to select for loss of the maintenance plasmid pQB240. Complementation of the petite phenotype was assessed on YEPG plates (2 days, 30.degree. C.). Neither pQB242 nor pQB249 was observed to complement the msi1::HIS3 defect under conditions where control constructs (e.g., the yeast MSI1 gene) showed complementation. Expression of heterologous protein in yeast was confirmed in the case of the GST-fusion by visualization of the expressed fusion protein using anti-GST antibodies on a Western blot.
An H. pylori MetRS gene complements an msm1::HIS3 defect in S. cerevisiae
Construction of pQB153
Plasmid pQB150 is a derivative of pQB111 (Example 9), which contains a 1.7 kb BamHI fragment encoding the methionyl-tRNA synthetase gene from M. tuberculosis in the BamHI site of pQB111. pQB150 was digested with HindIII and KpnI, and the HindIII-KpnI fragment containing the methionyl-tRNA synthetase gene was inserted into the HindIII and KpnI sites of YEplac181 (also referred to as pQB41) (Sugino, A. and Gietz, R. D., Gene, 74: 527-534 (1988)) to form pQB154. pQB154 was digested with BamHI, releasing the methionyl-tRNA synthetase gene fragment. The vector portion was purified and religated to yield pQB153. pQB153 encodes the first 22 amino acids of COXIV under the control of an ADH promoter and bears a LEU2 selectable marker.
Construction of pQB153-GST (also referred to as pQB248)
Plasmid pQB153 was digested with XbaI and KpnI and the vector backbone was isolated. The GST coding region from plasmid pQB136 (see Example 9) was released by digestion with XbaI and KpnI, and the fragment was ligated to the XbaI-KpnI backbone of plasmid pQB153. The resulting construct is designated pQB153-GST or pQB248.
pQB153-GST is suitable for expressing GST-aaRS fusion proteins and mitochondrial targeting of the encoded fusion protein. The construct carries LEU2 as a selectable marker.
Construction of test plasmid pQB224 (pC.sup.3 479)
The H. pylori MetRS gene was cloned into the mitochondrial vector pQB153 (2.mu., LEU2, ADH-COXIV) using XbaI and SalI sites (underlined in the primer sequences shown below) introduced during PCR amplification. The following primers were used:
______________________________________5'-HP Met (SEQ ID NO:66): 5'-CAGACGTCTAGATATCTGCTTATGCAAAAATCACTGATCA-3'Met 3' primer (SEQ ID NO:45): 5'-CCCAGTCGACTTAGCTGATCAAACTTCCTGC-3'______________________________________
PCR conditions were as follows: 94.degree. C., 60"; 55.degree. C., 60"; 72.degree. C., 120", for 35 cycles. Reactions contained 50 .mu.l 1X Taq polymerase buffer, 0.2 mM of each dNTP, 2 .mu.M of each primer, and 0.5 .mu.l of Taq polymerase (2.5 units of AmpliTaq polymerase (Perkin-Elmer)). Approximately 20 ng of cosmid clone #30 (see Examples 3 and 4) was used as the template for amplification. The PCR product was cleaved with XbaI and SalI, and the fragment containing the amplified gene was isolated and inserted into pQB153 which had been cleaved with XbaI and SalI. The resulting plasmid, maintained in E. coli DH5.alpha., is designated pQB224 or pC.sup.3 479.
Construction of maintenance plasmid pQB141
Plasmid pQB104 (pQB104 is referred to as pG72/T1 in Tzagoloff, A., et al., Eur. J. Biochem. 179:365-371 (1989); American Type Culture Collection, Rockville, Md., ATCC.RTM. Accession Nos. 37663, 66319) was digested with KpnI and PstI to obtain a 2.5 kb KpnI-PstI fragment containing the MSM1 gene promoter and entire MSM1 coding region. The 2.5 kb fragment was cloned into the KpnI and PstI sites of plasmid YCplac33 (Sugino, A., and Gietz, R. D., Gene 74:527-534 (1988)), which carries a URA3 selectable marker, to yield pQB141.
Strain QBY43:
Strain QBY43 (aW303.DELTA.MSM1) has the following genotype: MATa ade2-1 his3-11, 15 leu2-3,112 ura3-1 trp1-1 msm1::HIS3 (Tzagoloff, A., et al., Eur. J. Biochem. 179:365-371 (1989)).
Strain QBY43 (pQB141):
Strain QBY43 (pQB141) was made by transformation of QBY43 with plasmid pQB141.
Strain QBY281:
Strain QBY281 (QBY43 (pQB141) rho+) was made by cytoduction from QBY43 (pQB141). 5.times.10.sup.6 cells from logarithmic phase cultures of each of QBY43(pQB141) and QBY274 (see Example 9) were mixed and spread onto a nitrocellulose filter laid on top of a YPD agar plate. The plate was incubated at 30.degree. C. for 5 hours. Cytoductants were micromanipulated on YPD agar and allowed to form colonies; the colonies were later purified on SC-glycerol media lacking histidine and uracil to select for rho+ derivatives of QBY43 (pQB141).
Strain QBY51 and QBY52:
The rho.sup.0 strains QBY51 and QBY52 were obtained by growing QBY19 (MATa ade6 lys1) and QBY20 (MAT.alpha. ade6 lys1) in YEPD (yeast extract, peptone, dextrose medium) containing 10 .mu.g/ml of ethidium bromide (Sigma) to induce loss of mitochondrial DNA.
Complementation
Strain QBY281 was used as a recipient for transformation of plasmid pQB224. This strain was also transformed with individual positive control constructs containing (a) the wild type mitochondrial gene MSM1, (b) the E. coli metG gene, or (c) an M. tuberculosis MetRS gene, or with a negative control construct (vector pQB153-GST). The test plasmid and each of the control constructs contains a LEU2 selectable marker.
Leu+transformants containing the test plasmid and each of the control plasmids were selected on synthetic complete medium lacking leucine (SC - Leu). Leu+transformants were purified on plates containing 5-FOA, to select for loss of the maintenance plasmid pQB141, which carries the URA3 selectable marker. Single colonies selected on 5-FOA plates were resuspended in water in 32-well plates, and cells were transferred to a series of plates for analysis using an inoculating manifold (by "frogging"). In particular, transformants were transferred to the following types of plates to verify auxotrophies and to assess complementation (by growth on glycerol medium (YEPG)): YEPD; YEPG; SC; SD;. SC minus adenine; SC minus histidine; SC minus leucine; SC minus tryptophan; SC minus uracil, and 5-FOA plates. Subsequently, the 5-FOA plates were replica-plated to YEPG, and to mating lawns of rho.degree. tester strains QBY51 and QBY52, which were then replica plated onto YEPG and SD. Growth on these plates verified mating type and the rho state of the transformants. Strains which form a diploid (as indicated by growth on SD medium) when crossed to QBY52 are MATa. Growth of a diploid on YEPG indicates that the transformant crossed to the rho.degree. tester is rho.sup.+.
Four Leu.sup.+ colonies of QBY281 transformed with pQB224 and selected on 5-FOA plates were tested for their ability to complement on YEPG plates. After 2 days at 30.degree. C. complementation was observed for all four colonies. All four Pet.sup.+ transformants were Ura-His+Leu+, confirming loss of the maintenance plasmid (which carries URA3), the presence of the disruption allele (msm1::HIS3), and the presence of the test plasmid, pQB224. Thus, these transformants are examples of tester strains containing an H. pylori MetRS gene. Growth of the four colonies was indistinguishable from that of Ura-His+Leu+ positive control strains (harboring the wild type mitochondrial gene MSM1, the E. coli metG gene, or an M. tuberculosis MetRS gene on a plasmid). In contrast, a strain transformed with the vector alone (pQB153-GST) was unable to grow on glycerol. These results indicate that an H. pylori MetRS gene can complement the defect in the yeast mitochondrial MSM1 gene of strain QBY281.
EXAMPLE 11
Construction of pQB169 and pQB172
Plasmid pMC4 carries the ADH promoter of S. cerevisiae, and downstream of the promoter, the coding sequence for the cytochrome oxidase IV mitochondrial targeting peptide (Pinkham, J. et al., Mol. Cell. Biol., 14:4643-4652, (1994); Hurt, E. C. et al., J. Biol. Chem., 262:1420-1424 (1987); Hurt, E. C., et al., EMBO J., 3:3149-3156 (1984)). Derivatives of plasmid pMC4 can be constructed which lack or interrupt the sequence encoding the mitochondrial targeting sequence (e.g., by insertion of a gene between the promoter and targeting sequence), permitting cytoplasmic expression. Alternatively, the ADH promoter of pMC4 can be excised and inserted into another suitable vector. pQB169 and pQB172, which were constructed for the expression of heterologous genes in yeast cytoplasm, are examples of vectors constructed in this manner.
pQB169 contains the constitutive ADH promoter, a polylinker and the ILS1 transcriptional terminator. A 450 bp fragment containing the constitutive ADH promoter (PADH) with its transcriptional start sites (but not a translational start site (i.e., ATG)) was amplified by PCR using plasmid pMC4 as template. Primers were designed to incorporate a HindIII site at the 5' end (primer JK-1, SEQ ID NO:62) of the fragment and a PstI site at the 3' end (primer JK-2, SEQ ID NO:63):
__________________________________________________________________________ HindIIIJK-1: 5'-CCA AGA AGC TTG AAG TAA TAA TAG GCG CAT GC-3' (SEQ ID NO:62) Pst IJK-2: 5'-CGT ACT GCA GGA TTG TAT GCT TGG TAT AGC-3' (SEQ ID NO:63)__________________________________________________________________________
The resulting PCR product was cleaved with HindIII and PstI, and the HindIII-PstI fragment containing PADH was subcloned into the HindIII and PstI sites of vector YEplac181 (Gietz and Sugino, Gene, 74: 527-534 (1988)), a 2.mu. LEU2 yeast shuttle vector, to yield intermediate plasmid pQB147.
For efficient transcription termination, a 270 bp terminator fragment (tILS1), containing conserved transcription termination signals (Zaret and Sherman, Cell, 28: 563-573 (1982)) was generated by PCR, using plasmid pQB89 as template. pQB89 is a derivative of YCplac33 (a URA3, CEN4 plasmid; Geitz and Sugino, Gene, 74:527-534 (1988))). pQB89 was constructed by subcloning a 5.2 kb BamHI fragment obtained from a X clone PM4967 (ATCC Accession No. 70323) containing a yeast genomic fragment which includes the ILS1 gene (yeast cytoplasmic isoleucyl-tRNA synthetase gene; Englisch et al., Biol. Chem. Hoppe-Seyler, 368: 971-979 (1987)) into YCplac33.
The 270 bp tILS1 PCR fragment was engineered to have an EcoRI site at the 5' end (JK-5, SEQ ID NO:64), and a NarI site at the 3' end (JK-6, SEQ ID NO:65), and contains the 3' untranslated region of ILS1, including bases 3519-3846 of the ILS1 gene. The primers used to prepare this fragment were:
__________________________________________________________________________EcoRIJK-5: 5'-GGA ATT CTG AAA ACA ACT CAT ATA AAT ACG-3' (SEQ ID NO:64) NarIJK-6: 5'-GAG GCG CCC TCT TAT CAA TCC CCT CCT CAA CC-3' (SEQ ID NO:65)__________________________________________________________________________
The resulting PCR product was cleaved with EcoRI and NarI. pQB147 was cleaved with EcoRI and NarI, and the EcoRI-NarI tILS1 fragment was subcloned into the EcoRI and NarI sites of the vector to yield expression vector pQB169. Transformants of E. coli DH5.alpha. containing pQB169 were obtained. Transcription of a gene inserted into this vector can be initiated from pADH, and translation can be initiated at the first ATG of the insert.
To make a single-copy (CEN) version of this vector, the expression cassette (pADH-polylinker-tILS1) of pQB169 was excised with HindIII and NarI, and was subcloned into the HindIII and NarI sites of HindIII-NarI cut YCplac111 (Gietz and Sugino, Gene, 74:527-534 (1988)) to yield pQB172. Transformants of E. coli DH5.alpha. containing pQB172 were obtained.
Equivalents
Those skilled in the art will be able to recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 67(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2998 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 149..2908(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CGATGCGATTAGCTTGCATTATTTAAAAGGGATAGAAGAATACACGATTTTACAAATCCC60CACTTTAAAAAATGTGCCGCGAAAAGACACGCACCTTTATATCGCTCCTAAAACAAAAGA120ATAAAGATAAAAGATTAAGGAACAATCAGTGAAAGAATACAAAGACACCCTA172MetLysGluTyrLysAspThrLeu15AACTTAAACACAACCACCTTTTCTATGAAAGGGAATTTGAGCGTTAAT220AsnLeuAsnThrThrThrPheSerMetLysGlyAsnLeuSerValAsn101520GAGCCTAAAACTTACGCAAAATGGCAAGAGCAACAAGCGTTCAAACGC268GluProLysThrTyrAlaLysTrpGlnGluGlnGlnAlaPheLysArg25303540ATGCAAGCTAGGAAAGATAACCATGGGGATTTCACCTTGCATGACGGG316MetGlnAlaArgLysAspAsnHisGlyAspPheThrLeuHisAspGly455055CCGCCTTATGCGAACGGGCATTTGCATTTAGGGCATGCCTTAAATAAA364ProProTyrAlaAsnGlyHisLeuHisLeuGlyHisAlaLeuAsnLys606570ATTTTAAAAGACATTGTTATTAAAAGGGAATATTTTAAAGGGAAGAAA412IleLeuLysAspIleValIleLysArgGluTyrPheLysGlyLysLys758085ATCTATTACACGCCCGGTTGGGATTGCCATGGCTTGCCCATTGAGCAG460IleTyrTyrThrProGlyTrpAspCysHisGlyLeuProIleGluGln9095100CAAATTTTAGAGCGATTAGAAAAAGAAAAAACGAGCCTAGAAAACCCC508GlnIleLeuGluArgLeuGluLysGluLysThrSerLeuGluAsnPro105110115120ACGCTGTTTAGAGAAAAGTGCCGAGATCATGCGAAGAAATTTTTAGAA556ThrLeuPheArgGluLysCysArgAspHisAlaLysLysPheLeuGlu125130135ATCCAAAAGAATGAATTTTTGCAATTAGGCGTTTTGGGGGATTTTGAA604IleGlnLysAsnGluPheLeuGlnLeuGlyValLeuGlyAspPheGlu140145150GATCCTTATAAAACCATGGATTTTAAATTTGAAGCGAGCATTTATAGG652AspProTyrLysThrMetAspPheLysPheGluAlaSerIleTyrArg155160165GCCTTAGTGGAAGTGGCTAAAAAAGGGCTTTTGAAAGAGCGCCATAAG700AlaLeuValGluValAlaLysLysGlyLeuLeuLysGluArgHisLys170175180CCTATTTATTGGAGTTATGCATGCGAGAGCGCTTTAGCGGAAGCTGAA748ProIleTyrTrpSerTyrAlaCysGluSerAlaLeuAlaGluAlaGlu185190195200GTGGAATACAAGATGAAAAAATCGCCCTCCATTTTCGTGGCGTTTGAT796ValGluTyrLysMetLysLysSerProSerIlePheValAlaPheAsp205210215TTGAAAAAAGAGAGTTTAGAAAAATTAAAAGTCAAAAAAGCGAGCTTG844LeuLysLysGluSerLeuGluLysLeuLysValLysLysAlaSerLeu220225230GTGATTTGGACGACCACGCCCTGGACTTTGTATGCGAATGAAGCGATC892ValIleTrpThrThrThrProTrpThrLeuTyrAlaAsnGluAlaIle235240245GCTTTGAAAAAGGACGCTGTTTATGTGCTCACCCAAAAAGGCTATTTA940AlaLeuLysLysAspAlaValTyrValLeuThrGlnLysGlyTyrLeu250255260GTCGCTAAAGCCTTGCATGAAAAATTAGCCGCTTTAGGGGTGGTGGAT988ValAlaLysAlaLeuHisGluLysLeuAlaAlaLeuGlyValValAsp265270275280AGTGAGATCACGCATGAATTTAACGCTAATGATTTAGAATACTTGAAG1036SerGluIleThrHisGluPheAsnAlaAsnAspLeuGluTyrLeuLys285290295GCCACCAATCCTTTAAACCAAAGAGATTCCCTAATCACTCTAGGAGAG1084AlaThrAsnProLeuAsnGlnArgAspSerLeuIleThrLeuGlyGlu300305310CATGTCGGTTTAGAAGATGGCACAGGAGCCGTGCATACCGCACCTGGG1132HisValGlyLeuGluAspGlyThrGlyAlaValHisThrAlaProGly315320325CATGGTGAAGAGGACTATTATTTAGGCTTAAAATACAATTTAGAAGTG1180HisGlyGluGluAspTyrTyrLeuGlyLeuLysTyrAsnLeuGluVal330335340TTAATGTCCGTAGATGAGAAAGGTTGCTATGATGAGGGCATTATCCAT1228LeuMetSerValAspGluLysGlyCysTyrAspGluGlyIleIleHis345350355360AACCAACTATTAGATGAAAGCTATCTGGGCGAGCATGTTTTTAAGGCT1276AsnGlnLeuLeuAspGluSerTyrLeuGlyGluHisValPheLysAla365370375CAAAAACGCATTATAGAGCAATTGGGCGATTCTTTATTGCTGGAGCAA1324GlnLysArgIleIleGluGlnLeuGlyAspSerLeuLeuLeuGluGln380385390GAGATTGAGCATTCCTACCCGCATTGCTGGAGGACGCACAAGCCTGTG1372GluIleGluHisSerTyrProHisCysTrpArgThrHisLysProVal395400405ATTTACAGAGCGACCACGCAATGGTTTATTTTAATGGATGAGCCTTTT1420IleTyrArgAlaThrThrGlnTrpPheIleLeuMetAspGluProPhe410415420ATTCAAAATGATGGTTCTCAAAAAACCTTAAGAGAAGTGGCTTTGAGT1468IleGlnAsnAspGlySerGlnLysThrLeuArgGluValAlaLeuSer425430435440GCGATTGAAAAGGTGGAATTTGTGCCAAGCAACGGGAAAAACCGCCTA1516AlaIleGluLysValGluPheValProSerAsnGlyLysAsnArgLeu445450455AAAACCATGATAGAAAACCGCCCTGATTGGTGCTTGAGCCGGCAAAGA1564LysThrMetIleGluAsnArgProAspTrpCysLeuSerArgGlnArg460465470AAATGGGGCGTGCCACTGGCCTTTTTCATAGACAAACGTACGAATAAG1612LysTrpGlyValProLeuAlaPhePheIleAspLysArgThrAsnLys475480485CCTTGTTTTGAAAGCGAAGTTTTAGAGCATGTGGCGAATCTTTTTGAG1660ProCysPheGluSerGluValLeuGluHisValAlaAsnLeuPheGlu490495500AAAAAAGGTTGTGATGTGTGGTGGGAGTCTAGCGTAAAAGATTTATTA1708LysLysGlyCysAspValTrpTrpGluSerSerValLysAspLeuLeu505510515520CCCCCTAGCTATCAAGAGGACGCCAAGCATTACGAAAAAATCATGCAC1756ProProSerTyrGlnGluAspAlaLysHisTyrGluLysIleMetHis525530535ATTTTAGATGTGTGGTTTGATAGTGGTAGCACCTTTAAGGCGGTTTTA1804IleLeuAspValTrpPheAspSerGlySerThrPheLysAlaValLeu540545550GAAGACTATCATGGAGAAAAAGGGCAAAGCCCTAGTGATGTGGTTTTA1852GluAspTyrHisGlyGluLysGlyGlnSerProSerAspValValLeu555560565GAAGGGAGCGATCAGCATAGGGGGTGGTTTCAAAGTTCGCTTCTAATC1900GluGlySerAspGlnHisArgGlyTrpPheGlnSerSerLeuLeuIle570575580GGTTGTGTTTTAAACAACCAAGCCCCTTTTAAAAAGGTCATTACGCAT1948GlyCysValLeuAsnAsnGlnAlaProPheLysLysValIleThrHis585590595600GGCTTTATCGTCGATGAAAAGGGCGAGAAAATGAGTAAATCTAAGGGC1996GlyPheIleValAspGluLysGlyGluLysMetSerLysSerLysGly605610615AATGTGGTGTCTTTGGACAACTTACTCAAAAAGCATGGGAGCGATGTG2044AsnValValSerLeuAspAsnLeuLeuLysLysHisGlySerAspVal620625630GTGCGTTTGTGGGTAGCGTTTAATGACTATCAAAACGATTTGAGGGTC2092ValArgLeuTrpValAlaPheAsnAspTyrGlnAsnAspLeuArgVal635640645TCTCAAACCTTCTTCATTCAAACAGAACAGCATTATAAGAAATTCCGC2140SerGlnThrPhePheIleGlnThrGluGlnHisTyrLysLysPheArg650655660AACACCCTTAAATTCTTACTCGCCAATTTTAGCGATATGGATCTTAAG2188AsnThrLeuLysPheLeuLeuAlaAsnPheSerAspMetAspLeuLys665670675680AATTTAGAACGATCCCATGACTTCAGCCCTTTAGATCATTTTATATTA2236AsnLeuGluArgSerHisAspPheSerProLeuAspHisPheIleLeu685690695GAGGCTTTAGAAACAACAAGCGTTGGAGTCAATAGCGCGTTTGAAGAG2284GluAlaLeuGluThrThrSerValGlyValAsnSerAlaPheGluGlu700705710CATGATTTTGTGAAGGGCTTGAATATTTTAATGGCGTTTGTTACCAAT2332HisAspPheValLysGlyLeuAsnIleLeuMetAlaPheValThrAsn715720725GAATTGAGTGGGATTTATTTAGACGCTTGTAAGGATAGCTTGTATTGC2380GluLeuSerGlyIleTyrLeuAspAlaCysLysAspSerLeuTyrCys730735740GATAGCAAAAACAATGAAAAACGCCAAGCCATTCAAATGGTCTTACTC2428AspSerLysAsnAsnGluLysArgGlnAlaIleGlnMetValLeuLeu745750755760GCTGTGGCTAGTAAGTTGTGCTACTTTTTAGCCCCGATTTTAACGCAC2476AlaValAlaSerLysLeuCysTyrPheLeuAlaProIleLeuThrHis765770775ACGATTGAAGAGGTTTTAGAGCATAGTCAGGTGCTGTGCGCGTTTTTA2524ThrIleGluGluValLeuGluHisSerGlnValLeuCysAlaPheLeu780785790CAAGCCAAAGATGTGTTTGATTTAAAAGACATTAGCGTTTCAGAAAAA2572GlnAlaLysAspValPheAspLeuLysAspIleSerValSerGluLys795800805CTCCACCTTAAAGAGTTTAAAAAACCAGAAAATTTTGAAGCCGTTTTA2620LeuHisLeuLysGluPheLysLysProGluAsnPheGluAlaValLeu810815820GCCTTGCGTTCTGCCTTTAATGAAGAGTTAGACCGATTGAAAAAAGAA2668AlaLeuArgSerAlaPheAsnGluGluLeuAspArgLeuLysLysGlu825830835840GGCGTCATTAAAAATTCGTTGGAGTGCGCTATTGAAGTAAAAGAAAAA2716GlyValIleLysAsnSerLeuGluCysAlaIleGluValLysGluLys845850855GCGTTGCGTGAAAATTTGATAGAAGAATTGCTGATGGTGAGCTTTGTG2764AlaLeuArgGluAsnLeuIleGluGluLeuLeuMetValSerPheVal860865870GGGGTTGCAAAAGAAAAATTAAGCGAAACGCCAGCATTCACGCTCTTT2812GlyValAlaLysGluLysLeuSerGluThrProAlaPheThrLeuPhe875880885AAAGCCCCCTTTTATAAATGCCCTAGGTGTTGGCGTTTTAAAAGCGAA2860LysAlaProPheTyrLysCysProArgCysTrpArgPheLysSerGlu890895900TTAGAAAACACCCCTTGCAAGCGCTGCGAAGAGGTTTTAAAAGAGCGA2908LeuGluAsnThrProCysLysArgCysGluGluValLeuLysGluArg905910915920TGATAAAAGGATAGGGCTTTTGGAAACTTTACAAACCCATAGAGTTTTACAAGCCCTAAT2968CGGCCATTTCACCCCATTTTTAGAAAGTGG2998(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 920 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetLysGluTyrLysAspThrLeuAsnLeuAsnThrThrThrPheSer151015MetLysGlyAsnLeuSerValAsnGluProLysThrTyrAlaLysTrp202530GlnGluGlnGlnAlaPheLysArgMetGlnAlaArgLysAspAsnHis354045GlyAspPheThrLeuHisAspGlyProProTyrAlaAsnGlyHisLeu505560HisLeuGlyHisAlaLeuAsnLysIleLeuLysAspIleValIleLys65707580ArgGluTyrPheLysGlyLysLysIleTyrTyrThrProGlyTrpAsp859095CysHisGlyLeuProIleGluGlnGlnIleLeuGluArgLeuGluLys100105110GluLysThrSerLeuGluAsnProThrLeuPheArgGluLysCysArg115120125AspHisAlaLysLysPheLeuGluIleGlnLysAsnGluPheLeuGln130135140LeuGlyValLeuGlyAspPheGluAspProTyrLysThrMetAspPhe145150155160LysPheGluAlaSerIleTyrArgAlaLeuValGluValAlaLysLys165170175GlyLeuLeuLysGluArgHisLysProIleTyrTrpSerTyrAlaCys180185190GluSerAlaLeuAlaGluAlaGluValGluTyrLysMetLysLysSer195200205ProSerIlePheValAlaPheAspLeuLysLysGluSerLeuGluLys210215220LeuLysValLysLysAlaSerLeuValIleTrpThrThrThrProTrp225230235240ThrLeuTyrAlaAsnGluAlaIleAlaLeuLysLysAspAlaValTyr245250255ValLeuThrGlnLysGlyTyrLeuValAlaLysAlaLeuHisGluLys260265270LeuAlaAlaLeuGlyValValAspSerGluIleThrHisGluPheAsn275280285AlaAsnAspLeuGluTyrLeuLysAlaThrAsnProLeuAsnGlnArg290295300AspSerLeuIleThrLeuGlyGluHisValGlyLeuGluAspGlyThr305310315320GlyAlaValHisThrAlaProGlyHisGlyGluGluAspTyrTyrLeu325330335GlyLeuLysTyrAsnLeuGluValLeuMetSerValAspGluLysGly340345350CysTyrAspGluGlyIleIleHisAsnGlnLeuLeuAspGluSerTyr355360365LeuGlyGluHisValPheLysAlaGlnLysArgIleIleGluGlnLeu370375380GlyAspSerLeuLeuLeuGluGlnGluIleGluHisSerTyrProHis385390395400CysTrpArgThrHisLysProValIleTyrArgAlaThrThrGlnTrp405410415PheIleLeuMetAspGluProPheIleGlnAsnAspGlySerGlnLys420425430ThrLeuArgGluValAlaLeuSerAlaIleGluLysValGluPheVal435440445ProSerAsnGlyLysAsnArgLeuLysThrMetIleGluAsnArgPro450455460AspTrpCysLeuSerArgGlnArgLysTrpGlyValProLeuAlaPhe465470475480PheIleAspLysArgThrAsnLysProCysPheGluSerGluValLeu485490495GluHisValAlaAsnLeuPheGluLysLysGlyCysAspValTrpTrp500505510GluSerSerValLysAspLeuLeuProProSerTyrGlnGluAspAla515520525LysHisTyrGluLysIleMetHisIleLeuAspValTrpPheAspSer530535540GlySerThrPheLysAlaValLeuGluAspTyrHisGlyGluLysGly545550555560GlnSerProSerAspValValLeuGluGlySerAspGlnHisArgGly565570575TrpPheGlnSerSerLeuLeuIleGlyCysValLeuAsnAsnGlnAla580585590ProPheLysLysValIleThrHisGlyPheIleValAspGluLysGly595600605GluLysMetSerLysSerLysGlyAsnValValSerLeuAspAsnLeu610615620LeuLysLysHisGlySerAspValValArgLeuTrpValAlaPheAsn625630635640AspTyrGlnAsnAspLeuArgValSerGlnThrPhePheIleGlnThr645650655GluGlnHisTyrLysLysPheArgAsnThrLeuLysPheLeuLeuAla660665670AsnPheSerAspMetAspLeuLysAsnLeuGluArgSerHisAspPhe675680685SerProLeuAspHisPheIleLeuGluAlaLeuGluThrThrSerVal690695700GlyValAsnSerAlaPheGluGluHisAspPheValLysGlyLeuAsn705710715720IleLeuMetAlaPheValThrAsnGluLeuSerGlyIleTyrLeuAsp725730735AlaCysLysAspSerLeuTyrCysAspSerLysAsnAsnGluLysArg740745750GlnAlaIleGlnMetValLeuLeuAlaValAlaSerLysLeuCysTyr755760765PheLeuAlaProIleLeuThrHisThrIleGluGluValLeuGluHis770775780SerGlnValLeuCysAlaPheLeuGlnAlaLysAspValPheAspLeu785790795800LysAspIleSerValSerGluLysLeuHisLeuLysGluPheLysLys805810815ProGluAsnPheGluAlaValLeuAlaLeuArgSerAlaPheAsnGlu820825830GluLeuAspArgLeuLysLysGluGlyValIleLysAsnSerLeuGlu835840845CysAlaIleGluValLysGluLysAlaLeuArgGluAsnLeuIleGlu850855860GluLeuLeuMetValSerPheValGlyValAlaLysGluLysLeuSer865870875880GluThrProAlaPheThrLeuPheLysAlaProPheTyrLysCysPro885890895ArgCysTrpArgPheLysSerGluLeuGluAsnThrProCysLysArg900905910CysGluGluValLeuLysGluArg915920(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2157 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 102..2045(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:ACCCCACAAATAAGCATAAATTCAACCTCTATTCTCTAAATTTTTAAAAGCATGTTAAAA60TTAAGAGATTTAAACAAATTTAAGGTAACACGATTATAAAGATGCAAAAATCA113MetGlnLysSerCTGATCACAACCCCCATTTATTATGTGAATGATATTCCCCATATTGGC161LeuIleThrThrProIleTyrTyrValAsnAspIleProHisIleGly5101520CATGCTTATACGACTTTGATTGCGGACACTTTAAAGAAGTATTACACG209HisAlaTyrThrThrLeuIleAlaAspThrLeuLysLysTyrTyrThr253035CTTCAAGGCGAAGAAGTCTTTTTTTTAACCGGCACCGATGAGCATGGG257LeuGlnGlyGluGluValPhePheLeuThrGlyThrAspGluHisGly404550CAAAAGATCGAACAAAGCGCGAGATTGAGAAATCAAAGCCCTAAAGCT305GlnLysIleGluGlnSerAlaArgLeuArgAsnGlnSerProLysAla556065TACGCCGATAGCATTAGCGCGATTTTTAAAGACCAGTGGGATTTTTTC353TyrAlaAspSerIleSerAlaIlePheLysAspGlnTrpAspPhePhe707580AATTTGGATTATGATGGTTTTATCCGCACCACAGACAGCGAGCATCAA401AsnLeuAspTyrAspGlyPheIleArgThrThrAspSerGluHisGln859095100AAATGCGTGCAAAACGCCTTTGAAATCATGTTTGAAAAAGGGGATATT449LysCysValGlnAsnAlaPheGluIleMetPheGluLysGlyAspIle105110115TATAAAGGCGCTTATAGTGGGTATTATTGCGTGAGCTGTGAGAGTTAT497TyrLysGlyAlaTyrSerGlyTyrTyrCysValSerCysGluSerTyr120125130TGCGCGATTTCTAAAGCGGACAATACGAGTGATAAAGTCCTATGCCCT545CysAlaIleSerLysAlaAspAsnThrSerAspLysValLeuCysPro135140145GATTGCTTGAGGGAGACTGCGCTTTTAGAAGAAGAGAGTTATTTTTTT593AspCysLeuArgGluThrAlaLeuLeuGluGluGluSerTyrPhePhe150155160AAATTGAGCGCGTATGAGAAGCCTTTATTGGAGTTTTACGCCAAAAAC641LysLeuSerAlaTyrGluLysProLeuLeuGluPheTyrAlaLysAsn165170175180CCTGAAGCGATTTTGCCTATTTATCGTAAAAATGAGGTAACTTCTTTT689ProGluAlaIleLeuProIleTyrArgLysAsnGluValThrSerPhe185190195ATTGAGCAGGGTTTATTGGATCTGTCTATCACGCGCACGAGCTTTGAA737IleGluGlnGlyLeuLeuAspLeuSerIleThrArgThrSerPheGlu200205210TGGGGCATTCCTTTGCCTAAAAAAATGAACGATCCTAAACATGTGGTG785TrpGlyIleProLeuProLysLysMetAsnAspProLysHisValVal215220225TATGTTTGGCTAGACGCTTTATTGAATTATGCGAGCGCGTTAGGGTAT833TyrValTrpLeuAspAlaLeuLeuAsnTyrAlaSerAlaLeuGlyTyr230235240TTGAATGGTTTAGACAATAAAATGGCGCATTTTGAATGCGCTAGGCAT881LeuAsnGlyLeuAspAsnLysMetAlaHisPheGluCysAlaArgHis245250255260ATTGTGGGTAAGGATATTTTACGCTTCCATGCCATTTATTGGCCGGCT929IleValGlyLysAspIleLeuArgPheHisAlaIleTyrTrpProAla265270275TTTTTGATGAGTTTGAATTTGCCCCTATTCAAACAGCTTTGCGTGCAT977PheLeuMetSerLeuAsnLeuProLeuPheLysGlnLeuCysValHis280285290GGGTGGTGGACGATAGAGGGCGTGAAAATGAGTAAGAGCTTGGGTAAT1025GlyTrpTrpThrIleGluGlyValLysMetSerLysSerLeuGlyAsn295300305GTTTTAGACGCTCAAAAGCTCGCTATGGAGTATGGGATTGAAGAATTA1073ValLeuAspAlaGlnLysLeuAlaMetGluTyrGlyIleGluGluLeu310315320CGCTATTTTTTATTGCGTGAGGTGCCTTTTGGGCAAGATGGGGATTTT1121ArgTyrPheLeuLeuArgGluValProPheGlyGlnAspGlyAspPhe325330335340TCTAAAAAAGCGTTAATAGAAAGGATCAACGCGAATTTGAACAACGAT1169SerLysLysAlaLeuIleGluArgIleAsnAlaAsnLeuAsnAsnAsp345350355TTGGGGAATTTGTTGAATCGCTTGCTAGGCATGGCTAAAAAATATTTC1217LeuGlyAsnLeuLeuAsnArgLeuLeuGlyMetAlaLysLysTyrPhe360365370AATTATTCTCTAAAAAGCGCCAAAATCACCGCGTATTATTCTAAAGAG1265AsnTyrSerLeuLysSerAlaLysIleThrAlaTyrTyrSerLysGlu375380385CTAGAAAAAGCGCATCAAATCTTAGATAACGCTAATTCTTTTGTGCCT1313LeuGluLysAlaHisGlnIleLeuAspAsnAlaAsnSerPheValPro390395400AAAATGCAATTGCATAAAGCTTTAGAGGAATTGTTTAACGTTTATGAT1361LysMetGlnLeuHisLysAlaLeuGluGluLeuPheAsnValTyrAsp405410415420TTTTTAAACAAACTCATCGCTAAAGAAGAGCCGTGGGTTTTGCACAAA1409PheLeuAsnLysLeuIleAlaLysGluGluProTrpValLeuHisLys425430435AACAACGAATCAGAAAAACTAGAAGCCTTGTTGAGTTTGATCGCAAAC1457AsnAsnGluSerGluLysLeuGluAlaLeuLeuSerLeuIleAlaAsn440445450GCGCTTTTGCAATCAAGCTTTTTGCTCTATGCGTTCATGCCAAAGAGT1505AlaLeuLeuGlnSerSerPheLeuLeuTyrAlaPheMetProLysSer455460465GCTGTGAAATTAGCGAGCGCTTTCAACACAGAAATCACGCCCAATAAT1553AlaValLysLeuAlaSerAlaPheAsnThrGluIleThrProAsnAsn470475480TACGAACGCTTTTTTAAGGCCAAAAAATTACAAGATATGGTTTTACAA1601TyrGluArgPhePheLysAlaLysLysLeuGlnAspMetValLeuGln485490495500GACACCGAGCCTTTATTTTGTAAAATGGAGAAAATTGAAAAGACTGAC1649AspThrGluProLeuPheCysLysMetGluLysIleGluLysThrAsp505510515AAAAGGGAAAAAGAAGTCCCACCAGAAAAAGCAGAAAAAAAAGAAAAA1697LysArgGluLysGluValProProGluLysAlaGluLysLysGluLys520525530GAAAAAGCCCCACCAAAACAAGAAAACTATATCGGCATTGAGGATTTC1745GluLysAlaProProLysGlnGluAsnTyrIleGlyIleGluAspPhe535540545AAAAAAGTAGAAATTAAAGTAGGGCTTATCAAAGAAGCTCAAAGGATT1793LysLysValGluIleLysValGlyLeuIleLysGluAlaGlnArgIle550555560GAAAAATCCAATAAATTATTGCGCTTAAAAGTGGATTTAGGCGAAAAT1841GluLysSerAsnLysLeuLeuArgLeuLysValAspLeuGlyGluAsn565570575580CGTTTGAGGCAGATCATCTCAGGGATCGCTTTGGATTATGAGCCTGAA1889ArgLeuArgGlnIleIleSerGlyIleAlaLeuAspTyrGluProGlu585590595AGTTTGGTGGGGCAAATGGTGTGCGTGGTGGCTAATTTAAAGCCTGCA1937SerLeuValGlyGlnMetValCysValValAlaAsnLeuLysProAla600605610AAGCTTATGGGCGAAATGAGTGAGGGCATGATTTTAGCGGTGCGAGAT1985LysLeuMetGlyGluMetSerGluGlyMetIleLeuAlaValArgAsp615620625AGCGATAATCTGGCTTTAATCAGCCCTACCAAAGAAAAAATCGCAGGA2033SerAspAsnLeuAlaLeuIleSerProThrLysGluLysIleAlaGly630635640AGTTTGATCAGCTAAATGCGATTAGGGGTGAATGAAGCCGTAGAATTGAGTT2085SerLeuIleSer645TGGGCGAATTGCAAAACACGCCCTCAATCAGCTATTTCAATTCCATTGTCTTGTCTTTAA2145ACAAAGTCAAAA2157(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 648 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:MetGlnLysSerLeuIleThrThrProIleTyrTyrValAsnAspIle151015ProHisIleGlyHisAlaTyrThrThrLeuIleAlaAspThrLeuLys202530LysTyrTyrThrLeuGlnGlyGluGluValPhePheLeuThrGlyThr354045AspGluHisGlyGlnLysIleGluGlnSerAlaArgLeuArgAsnGln505560SerProLysAlaTyrAlaAspSerIleSerAlaIlePheLysAspGln65707580TrpAspPhePheAsnLeuAspTyrAspGlyPheIleArgThrThrAsp859095SerGluHisGlnLysCysValGlnAsnAlaPheGluIleMetPheGlu100105110LysGlyAspIleTyrLysGlyAlaTyrSerGlyTyrTyrCysValSer115120125CysGluSerTyrCysAlaIleSerLysAlaAspAsnThrSerAspLys130135140ValLeuCysProAspCysLeuArgGluThrAlaLeuLeuGluGluGlu145150155160SerTyrPhePheLysLeuSerAlaTyrGluLysProLeuLeuGluPhe165170175TyrAlaLysAsnProGluAlaIleLeuProIleTyrArgLysAsnGlu180185190ValThrSerPheIleGluGlnGlyLeuLeuAspLeuSerIleThrArg195200205ThrSerPheGluTrpGlyIleProLeuProLysLysMetAsnAspPro210215220LysHisValValTyrValTrpLeuAspAlaLeuLeuAsnTyrAlaSer225230235240AlaLeuGlyTyrLeuAsnGlyLeuAspAsnLysMetAlaHisPheGlu245250255CysAlaArgHisIleValGlyLysAspIleLeuArgPheHisAlaIle260265270TyrTrpProAlaPheLeuMetSerLeuAsnLeuProLeuPheLysGln275280285LeuCysValHisGlyTrpTrpThrIleGluGlyValLysMetSerLys290295300SerLeuGlyAsnValLeuAspAlaGlnLysLeuAlaMetGluTyrGly305310315320IleGluGluLeuArgTyrPheLeuLeuArgGluValProPheGlyGln325330335AspGlyAspPheSerLysLysAlaLeuIleGluArgIleAsnAlaAsn340345350LeuAsnAsnAspLeuGlyAsnLeuLeuAsnArgLeuLeuGlyMetAla355360365LysLysTyrPheAsnTyrSerLeuLysSerAlaLysIleThrAlaTyr370375380TyrSerLysGluLeuGluLysAlaHisGlnIleLeuAspAsnAlaAsn385390395400SerPheValProLysMetGlnLeuHisLysAlaLeuGluGluLeuPhe405410415AsnValTyrAspPheLeuAsnLysLeuIleAlaLysGluGluProTrp420425430ValLeuHisLysAsnAsnGluSerGluLysLeuGluAlaLeuLeuSer435440445LeuIleAlaAsnAlaLeuLeuGlnSerSerPheLeuLeuTyrAlaPhe450455460MetProLysSerAlaValLysLeuAlaSerAlaPheAsnThrGluIle465470475480ThrProAsnAsnTyrGluArgPhePheLysAlaLysLysLeuGlnAsp485490495MetValLeuGlnAspThrGluProLeuPheCysLysMetGluLysIle500505510GluLysThrAspLysArgGluLysGluValProProGluLysAlaGlu515520525LysLysGluLysGluLysAlaProProLysGlnGluAsnTyrIleGly530535540IleGluAspPheLysLysValGluIleLysValGlyLeuIleLysGlu545550555560AlaGlnArgIleGluLysSerAsnLysLeuLeuArgLeuLysValAsp565570575LeuGlyGluAsnArgLeuArgGlnIleIleSerGlyIleAlaLeuAsp580585590TyrGluProGluSerLeuValGlyGlnMetValCysValValAlaAsn595600605LeuLysProAlaLysLeuMetGlyGluMetSerGluGlyMetIleLeu610615620AlaValArgAspSerAspAsnLeuAlaLeuIleSerProThrLysGlu625630635640LysIleAlaGlySerLeuIleSer645(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2662 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 153..2570(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:CCTCATACCGGCGTTTGTGTTTTTACAGATTTTAAATAATTTGGTTACAGCTTACATGCT60CATGATTGGGGCTTTGATTAGTAACGCTTTCAGCCTCATCTTTTTGTTGATTGAAAGCGT120TGTAACGAGCGAAACGGATTAAGGGGTAGTTGATGGATTTTATCAATATAGAA173MetAspPheIleAsnIleGlu15AAAAAATGGCAAGAATTTTGGTGGAAAAATAAGAGTTTTGAGCCTAAA221LysLysTrpGlnGluPheTrpTrpLysAsnLysSerPheGluProLys101520GACGATTTTAACCTCCCTAAAAAATACATTCTGAGCATGCTGCCTTAT269AspAspPheAsnLeuProLysLysTyrIleLeuSerMetLeuProTyr253035CCTAGTGGGGAAATCCATATGGGGCATGTGCGCAATTACACCATTGGC317ProSerGlyGluIleHisMetGlyHisValArgAsnTyrThrIleGly40455055GATGCTTTGGCGCGTTATTATCGTTTGCACCATTATAATGTGTTACAC365AspAlaLeuAlaArgTyrTyrArgLeuHisHisTyrAsnValLeuHis606570CCTATGGGGTTTGATTCTTTTGGGATGCCTGCAGAAAATGCGGCCATT413ProMetGlyPheAspSerPheGlyMetProAlaGluAsnAlaAlaIle758085AAGCATGGTATCCACCCTAAAACCTGGACTTATGAAAACATTGAAGCC461LysHisGlyIleHisProLysThrTrpThrTyrGluAsnIleGluAla9095100ATGCAAAAAGAGTTTGAAGCTTTAGGGTTTTCTTTTTCTAAAAACAGG509MetGlnLysGluPheGluAlaLeuGlyPheSerPheSerLysAsnArg105110115GAATTTGCCACTTCAGATCCGGATTACACGAAATTTGAGCAGCAATTT557GluPheAlaThrSerAspProAspTyrThrLysPheGluGlnGlnPhe120125130135TTCATTGATTTGTGGGAAAAAGGGTTAATTTATCGCAAGAAAGCCATG605PheIleAspLeuTrpGluLysGlyLeuIleTyrArgLysLysAlaMet140145150CTCAATTGGTGCCCTAACGACAAGACCGTTTTAGCTAATGAGCAAGTC653LeuAsnTrpCysProAsnAspLysThrValLeuAlaAsnGluGlnVal155160165ATCGATGGGAGGTGTTGGCGTTGCGATACGGAAGTTGTTCAAAAAGAA701IleAspGlyArgCysTrpArgCysAspThrGluValValGlnLysGlu170175180CTCTATCAATATTATTTGAAGATCACAAACTACGCTGAAGAATTACTA749LeuTyrGlnTyrTyrLeuLysIleThrAsnTyrAlaGluGluLeuLeu185190195AAAGACTTAGAAACTTTAGAAAATCATTGGCCTTCTCAAGTCCTAACC797LysAspLeuGluThrLeuGluAsnHisTrpProSerGlnValLeuThr200205210215ATGCAAAAAAACTGGATAGGAAAATCTATCGGGTTGCAATTTGGTTTT845MetGlnLysAsnTrpIleGlyLysSerIleGlyLeuGlnPheGlyPhe220225230AAAATCGCTGATGAGTGCTTGAAGGCTTGCAACGGCATTCAAGAAATT893LysIleAlaAspGluCysLeuLysAlaCysAsnGlyIleGlnGluIle235240245GAAGTTTTTACCACAAGAGCGGACACCATTTATGGCGTCACTTACATC941GluValPheThrThrArgAlaAspThrIleTyrGlyValThrTyrIle250255260GCCATCGCCCCAGAACACCCTTTAGTAGAGCATGCCATTAAGCGAGTG989AlaIleAlaProGluHisProLeuValGluHisAlaIleLysArgVal265270275AGCCAAGAAGATTCAAAGATCATTAAAGCGATTTTAAACACGACTCAA1037SerGlnGluAspSerLysIleIleLysAlaIleLeuAsnThrThrGln280285290295AGAGAAAGAGCTTTAGAGAAAAAAGGGGCGTTTTTAGGCGTTTATGCT1085ArgGluArgAlaLeuGluLysLysGlyAlaPheLeuGlyValTyrAla300305310ATCCACCCTTTAACAAAGCAAAAAATCCCTGTTTGGGTGGCTAATTTC1133IleHisProLeuThrLysGlnLysIleProValTrpValAlaAsnPhe315320325GCCTTAGCTAATTATGGCTCTGGGGCGTTAATGGGCGTGCCAGCCTGC1181AlaLeuAlaAsnTyrGlySerGlyAlaLeuMetGlyValProAlaCys330335340GATGAAAGGGATTTTGAATTCGCCAATCTTTATCATATCCCTATTAAA1229AspGluArgAspPheGluPheAlaAsnLeuTyrHisIleProIleLys345350355GTGATCACTCAAAGCCCTCAAAATTTGCCCCACACCAAAGAAGAGGTT1277ValIleThrGlnSerProGlnAsnLeuProHisThrLysGluGluVal360365370375TTAAAAAATAGCGGGGAGTGGAGCGATCTTTCTAGCTCAGTGGCCAGA1325LeuLysAsnSerGlyGluTrpSerAspLeuSerSerSerValAlaArg380385390GAGCAAATCATCGCTTATTTTGAAAAAGAAAACCTCGGTAAAAGGGTG1373GluGlnIleIleAlaTyrPheGluLysGluAsnLeuGlyLysArgVal395400405ATCAACTACCGTTTGCAAGATTGGGGAGTGAGCCGTCAAAGGTATTGG1421IleAsnTyrArgLeuGlnAspTrpGlyValSerArgGlnArgTyrTrp410415420GGAGCGCCCATTCCTATGATTCATTGCAACAATTGCGGGATTGTTCCT1469GlyAlaProIleProMetIleHisCysAsnAsnCysGlyIleValPro425430435GAAACCCAACTGCCGGTAACTTTACCCGAAGACATTGTGATTGATGGG1517GluThrGlnLeuProValThrLeuProGluAspIleValIleAspGly440445450455GAGGGCAATCCGTTAGAAAAGCATGTGAGTTGGAAATTCGCTCAATGC1565GluGlyAsnProLeuGluLysHisValSerTrpLysPheAlaGlnCys460465470CCCAAATGCCACAAAGACGCTTTAAGAGAAACAGACACCATGGATACT1613ProLysCysHisLysAspAlaLeuArgGluThrAspThrMetAspThr475480485TTCATCCAGTCTAGTTGGTATTTCTTGCGCTACACCACCCCCAAAAAT1661PheIleGlnSerSerTrpTyrPheLeuArgTyrThrThrProLysAsn490495500CAGCGTGAAAATCAAGCGTTTGATCAAAATTACTTGAAGTATTTCATG1709GlnArgGluAsnGlnAlaPheAspGlnAsnTyrLeuLysTyrPheMet505510515CCAGTGGATACTTATATTGGCGGCATTGAACATGCGATTTTGCACTTG1757ProValAspThrTyrIleGlyGlyIleGluHisAlaIleLeuHisLeu520525530535TTATACGCGCGCTTTTTCACTAAGGCTTTAAGGGATTTGGGCTATATT1805LeuTyrAlaArgPhePheThrLysAlaLeuArgAspLeuGlyTyrIle540545550CATTTAGATGAGCCTTTCAAACAGCTTATCACTCAAGGCATGGTCTTA1853HisLeuAspGluProPheLysGlnLeuIleThrGlnGlyMetValLeu555560565AAAAATGGCACTAAGATGAGCAAATCTAAAGGTAATGTCGTTAGCCCT1901LysAsnGlyThrLysMetSerLysSerLysGlyAsnValValSerPro570575580AAAGAAATACTCAAAAAATACGGGGCCGATGCCGCAAGGCTTTTTATC1949LysGluIleLeuLysLysTyrGlyAlaAspAlaAlaArgLeuPheIle585590595CTTTTTGCTGCCCCACCGGCTAAAGAATTAGAATGGAATGACAGCGCT1997LeuPheAlaAlaProProAlaLysGluLeuGluTrpAsnAspSerAla600605610615TTAGAAGGTGCGCACCGGTTTATCAAGCGCTTATACGATAAAGCGAGT2045LeuGluGlyAlaHisArgPheIleLysArgLeuTyrAspLysAlaSer620625630GCCATTACCCCTACCACTTCTAAGCCTGAATTTAAAGAAGTCAGCCTG2093AlaIleThrProThrThrSerLysProGluPheLysGluValSerLeu635640645AATGAAGCGCAAAAACTAGCTCGTAAAAAAGTCTATGAAGCGTTAAAA2141AsnGluAlaGlnLysLeuAlaArgLysLysValTyrGluAlaLeuLys650655660AAATCGCATGAAATTTTCAATAAGGCTGAAAGCGCTTACGCGTTTAAC2189LysSerHisGluIlePheAsnLysAlaGluSerAlaTyrAlaPheAsn665670675ACTTTGATCGCAAGTTGCATGGAGGCTTTAAACGCTTTGAGTGCGCAA2237ThrLeuIleAlaSerCysMetGluAlaLeuAsnAlaLeuSerAlaGln680685690695ACTAATGAGCAAATTTTATGCGAAGGTTATTTTGTGTTGTTGCAAATT2285ThrAsnGluGlnIleLeuCysGluGlyTyrPheValLeuLeuGlnIle700705710TTAGAGCCTATTATCCCGCACACCGCATGGGAGTTGAGTGAGAGGCTT2333LeuGluProIleIleProHisThrAlaTrpGluLeuSerGluArgLeu715720725TTTAAAAGAGAGAATTTTAAGCCTATAGCGATCGATGAAAGCGCTTTG2381PheLysArgGluAsnPheLysProIleAlaIleAspGluSerAlaLeu730735740ATGGAAGACTTTATGACTTTAGGGCTTACCATTAATGGCAAAAGGCGC2429MetGluAspPheMetThrLeuGlyLeuThrIleAsnGlyLysArgArg745750755GCGGAATTGAAAGTCAATATTAACGCCAGTAAAGAAGAAATTATTGTT2477AlaGluLeuLysValAsnIleAsnAlaSerLysGluGluIleIleVal760765770775TTGGCTAAAAAAGAATTAGAGAAATATTTAGAAAAGGCGAGCGTTAAA2525LeuAlaLysLysGluLeuGluLysTyrLeuGluLysAlaSerValLys780785790AAAGAAATTTATGTGCCTAATAAGCTCGTTAATTTTGTTATCGCA2570LysGluIleTyrValProAsnLysLeuValAsnPheValIleAla795800805TGAAAGCTTTACTTTTTTTTATTTTGTTGCTTTTGTTCAAGGGCTGTGGGTATAAGCCCA2630TCGCCGCTTACGCTCAAAACGCTTTAGGCGAT2662(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 806 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetAspPheIleAsnIleGluLysLysTrpGlnGluPheTrpTrpLys151015AsnLysSerPheGluProLysAspAspPheAsnLeuProLysLysTyr202530IleLeuSerMetLeuProTyrProSerGlyGluIleHisMetGlyHis354045ValArgAsnTyrThrIleGlyAspAlaLeuAlaArgTyrTyrArgLeu505560HisHisTyrAsnValLeuHisProMetGlyPheAspSerPheGlyMet65707580ProAlaGluAsnAlaAlaIleLysHisGlyIleHisProLysThrTrp859095ThrTyrGluAsnIleGluAlaMetGlnLysGluPheGluAlaLeuGly100105110PheSerPheSerLysAsnArgGluPheAlaThrSerAspProAspTyr115120125ThrLysPheGluGlnGlnPhePheIleAspLeuTrpGluLysGlyLeu130135140IleTyrArgLysLysAlaMetLeuAsnTrpCysProAsnAspLysThr145150155160ValLeuAlaAsnGluGlnValIleAspGlyArgCysTrpArgCysAsp165170175ThrGluValValGlnLysGluLeuTyrGlnTyrTyrLeuLysIleThr180185190AsnTyrAlaGluGluLeuLeuLysAspLeuGluThrLeuGluAsnHis195200205TrpProSerGlnValLeuThrMetGlnLysAsnTrpIleGlyLysSer210215220IleGlyLeuGlnPheGlyPheLysIleAlaAspGluCysLeuLysAla225230235240CysAsnGlyIleGlnGluIleGluValPheThrThrArgAlaAspThr245250255IleTyrGlyValThrTyrIleAlaIleAlaProGluHisProLeuVal260265270GluHisAlaIleLysArgValSerGlnGluAspSerLysIleIleLys275280285AlaIleLeuAsnThrThrGlnArgGluArgAlaLeuGluLysLysGly290295300AlaPheLeuGlyValTyrAlaIleHisProLeuThrLysGlnLysIle305310315320ProValTrpValAlaAsnPheAlaLeuAlaAsnTyrGlySerGlyAla325330335LeuMetGlyValProAlaCysAspGluArgAspPheGluPheAlaAsn340345350LeuTyrHisIleProIleLysValIleThrGlnSerProGlnAsnLeu355360365ProHisThrLysGluGluValLeuLysAsnSerGlyGluTrpSerAsp370375380LeuSerSerSerValAlaArgGluGlnIleIleAlaTyrPheGluLys385390395400GluAsnLeuGlyLysArgValIleAsnTyrArgLeuGlnAspTrpGly405410415ValSerArgGlnArgTyrTrpGlyAlaProIleProMetIleHisCys420425430AsnAsnCysGlyIleValProGluThrGlnLeuProValThrLeuPro435440445GluAspIleValIleAspGlyGluGlyAsnProLeuGluLysHisVal450455460SerTrpLysPheAlaGlnCysProLysCysHisLysAspAlaLeuArg465470475480GluThrAspThrMetAspThrPheIleGlnSerSerTrpTyrPheLeu485490495ArgTyrThrThrProLysAsnGlnArgGluAsnGlnAlaPheAspGln500505510AsnTyrLeuLysTyrPheMetProValAspThrTyrIleGlyGlyIle515520525GluHisAlaIleLeuHisLeuLeuTyrAlaArgPhePheThrLysAla530535540LeuArgAspLeuGlyTyrIleHisLeuAspGluProPheLysGlnLeu545550555560IleThrGlnGlyMetValLeuLysAsnGlyThrLysMetSerLysSer565570575LysGlyAsnValValSerProLysGluIleLeuLysLysTyrGlyAla580585590AspAlaAlaArgLeuPheIleLeuPheAlaAlaProProAlaLysGlu595600605LeuGluTrpAsnAspSerAlaLeuGluGlyAlaHisArgPheIleLys610615620ArgLeuTyrAspLysAlaSerAlaIleThrProThrThrSerLysPro625630635640GluPheLysGluValSerLeuAsnGluAlaGlnLysLeuAlaArgLys645650655LysValTyrGluAlaLeuLysLysSerHisGluIlePheAsnLysAla660665670GluSerAlaTyrAlaPheAsnThrLeuIleAlaSerCysMetGluAla675680685LeuAsnAlaLeuSerAlaGlnThrAsnGluGlnIleLeuCysGluGly690695700TyrPheValLeuLeuGlnIleLeuGluProIleIleProHisThrAla705710715720TrpGluLeuSerGluArgLeuPheLysArgGluAsnPheLysProIle725730735AlaIleAspGluSerAlaLeuMetGluAspPheMetThrLeuGlyLeu740745750ThrIleAsnGlyLysArgArgAlaGluLeuLysValAsnIleAsnAla755760765SerLysGluGluIleIleValLeuAlaLysLysGluLeuGluLysTyr770775780LeuGluLysAlaSerValLysLysGluIleTyrValProAsnLysLeu785790795800ValAsnPheValIleAla805(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2973 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 219..2834(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:TGAATAAGGAATCAATTTTTTCCAAACGCACTTCATTGATATGCTCAAATCCTAAAATAG60GGGCTTTTAAAAAGTAATTCACTGGACTATCCTTTTTAATTTAAATGCTTTGTTGTATAA120TTTTGAAAAAATACGCAACAATTAAAAGCGTAAAAGCAAAAGGCGTTCCAAATTCAACCA180TTTTTGAAAGAACGCTCTATTTTAGGATAATAATAATAATGAAACAAGAACCC233MetLysGlnGluPro15ACCACCTACCAACCAGAAGAGATAGAAAAAAAGATTTATGAAATTTGC281ThrThrTyrGlnProGluGluIleGluLysLysIleTyrGluIleCys101520TCTCATAGGGGGTATTTTGAAATTGATGGCAATGAAGCAATCCAAGAA329SerHisArgGlyTyrPheGluIleAspGlyAsnGluAlaIleGlnGlu253035AAAAACAAACGATTTTGCTTGATGATGCCCCCTCCTAATGTGACCGGT377LysAsnLysArgPheCysLeuMetMetProProProAsnValThrGly404550ATCTTACACATAGGGCATGCTTTGACTTTAAGCTTGCAAGATATTTTA425IleLeuHisIleGlyHisAlaLeuThrLeuSerLeuGlnAspIleLeu556065GCGCGCTATAAGCGCATGGACGGGTATAAGACTTTGTATCAGCCCGGA473AlaArgTyrLysArgMetAspGlyTyrLysThrLeuTyrGlnProGly70758085TTGGATCACGCCGGCATTGCGACGCAAAATGTCGTGGAAAAGCAACTT521LeuAspHisAlaGlyIleAlaThrGlnAsnValValGluLysGlnLeu9095100TTAAATCAAGGGATTAAAAAAGAAGATTTAGGGCGTGAAGCGTTCGTT569LeuAsnGlnGlyIleLysLysGluAspLeuGlyArgGluAlaPheVal105110115CAAAAAGTGTGGGAGTGGAAAGAAAAGAGTGGGGGAGCGATTTTAGAG617GlnLysValTrpGluTrpLysGluLysSerGlyGlyAlaIleLeuGlu120125130CAAATGAAGCGTTTAGGCGTGAGCGCGGCCTTTTCTAGGACTCGTTTC665GlnMetLysArgLeuGlyValSerAlaAlaPheSerArgThrArgPhe135140145ACGATGGATAAGGGCTTGCAAAGAGCGGTTAAATTGGCGTTTTTGAAA713ThrMetAspLysGlyLeuGlnArgAlaValLysLeuAlaPheLeuLys150155160165TGGTATGAAAAAGGTCTCATCGTTCAAGATAATTACATGGTGAATTGG761TrpTyrGluLysGlyLeuIleValGlnAspAsnTyrMetValAsnTrp170175180TGCACTAAAGATGGGGCATTGAGCGATATTGAAGTGGAGTATGAAGAG809CysThrLysAspGlyAlaLeuSerAspIleGluValGluTyrGluGlu185190195CGTAAGGGGGCGTTGTATTATATTAGATATTATTTAGAAAATCAAAAA857ArgLysGlyAlaLeuTyrTyrIleArgTyrTyrLeuGluAsnGlnLys200205210GATTATTTAGTGGTGGCCACCACACGCCCTGAAACTTTGTTTGGCGAT905AspTyrLeuValValAlaThrThrArgProGluThrLeuPheGlyAsp215220225AGCGCGCTTATGGTCAATCCTAATGATGAAAGATACAGGCATTTAGTG953SerAlaLeuMetValAsnProAsnAspGluArgTyrArgHisLeuVal230235240245GGGCAAAAAGCGGTCTTGCCTTTAATCAATCGCACAATCCCTATTATC1001GlyGlnLysAlaValLeuProLeuIleAsnArgThrIleProIleIle250255260GCTGATGAACATGTGGAAATGGAGTTTGGCACAGGGTGTGTGAAAGTT1049AlaAspGluHisValGluMetGluPheGlyThrGlyCysValLysVal265270275ACCCCTGGGCATGATTTTAACGATTACGAAGTGGGCAAACGCCACCAT1097ThrProGlyHisAspPheAsnAspTyrGluValGlyLysArgHisHis280285290TTGGAGGCGATTAAAATCTTTGATGAAAAAGGGATTTTAAACGCGCAT1145LeuGluAlaIleLysIlePheAspGluLysGlyIleLeuAsnAlaHis295300305TGTGGGGAGTTTGAAAATTTAGAGCGATTAGAAGCTAGAGATAAGGTC1193CysGlyGluPheGluAsnLeuGluArgLeuGluAlaArgAspLysVal310315320325GTAGAAAGATTAAAAGAAAACGCCTTATTGGAAAAAATAGAAGAGCAC1241ValGluArgLeuLysGluAsnAlaLeuLeuGluLysIleGluGluHis330335340ACGCATCAAGTGGGGCATTGCTATCGTTGTCATAATGTGGTAGAGCCT1289ThrHisGlnValGlyHisCysTyrArgCysHisAsnValValGluPro345350355TATGTGTCTAAGCAATGGTTTGTCAAGCCTGAAATCGCTCAAAGTTCT1337TyrValSerLysGlnTrpPheValLysProGluIleAlaGlnSerSer360365370ATTGAAAAAATCCAACAAGGTTTAGCACGATTCTACCCTTCTAATTGG1385IleGluLysIleGlnGlnGlyLeuAlaArgPheTyrProSerAsnTrp375380385ATCAATAATTATAACGCTTGGATGAGGGAATTACGCCCTTGGTGTATC1433IleAsnAsnTyrAsnAlaTrpMetArgGluLeuArgProTrpCysIle390395400405AGCAGGCAATTGTTTTGGGGGCATCAAATACCGGTATTTACTTGTGAA1481SerArgGlnLeuPheTrpGlyHisGlnIleProValPheThrCysGlu410415420AATAACCACCAGTTTGTAAGCCTAGACACCCCCTTAAGTTGCCCTACT1529AsnAsnHisGlnPheValSerLeuAspThrProLeuSerCysProThr425430435TGCAAGAGTGAAATACTAGAGCAAGATAAAGATGTGCTAGACACATGG1577CysLysSerGluIleLeuGluGlnAspLysAspValLeuAspThrTrp440445450TTTAGTTCAGGGCTATGGGCGTTTTCCACTCTAGGGTGGGGGCAAGAA1625PheSerSerGlyLeuTrpAlaPheSerThrLeuGlyTrpGlyGlnGlu455460465AAAAGCGGTTTGTTTAATGAAAGCGATTTGAAAGATTTCTACCCTAAC1673LysSerGlyLeuPheAsnGluSerAspLeuLysAspPheTyrProAsn470475480485ACAACGCTCATTACCGGGTTTGACATCCTCTTTTTTTGGGTGGCCAGA1721ThrThrLeuIleThrGlyPheAspIleLeuPhePheTrpValAlaArg490495500ATGCTCTTTTGCAGCGAATCGCTTTTAGGCGAATTGCCTTTTAAAGAT1769MetLeuPheCysSerGluSerLeuLeuGlyGluLeuProPheLysAsp505510515ATTTACTTGCACGCCTTGGTTAGGGATGAAAAGGGTGAAAAAATGAGC1817IleTyrLeuHisAlaLeuValArgAspGluLysGlyGluLysMetSer520525530AAATCTAAGGGTAATGTGATCGATCCTTTAGAGATGATAGAAAAATAC1865LysSerLysGlyAsnValIleAspProLeuGluMetIleGluLysTyr535540545GGCGCGGATAGTTTGCGTTTCACTTTAGCCAATTTGTGCGCTACGGGC1913GlyAlaAspSerLeuArgPheThrLeuAlaAsnLeuCysAlaThrGly550555560565AGGGACATTAAGCTTTCCACTACGCATTTAGAAAACAACAAGAATTTC1961ArgAspIleLysLeuSerThrThrHisLeuGluAsnAsnLysAsnPhe570575580GCCAACAAGATTTTCAATGCGGTGAGTTATTTGAAACTCAAACAAGAA2009AlaAsnLysIlePheAsnAlaValSerTyrLeuLysLeuLysGlnGlu585590595GCTTTCAAAGACAGGGAGCGTTTGAACGAATACCAAACGCCTTTGGGG2057AlaPheLysAspArgGluArgLeuAsnGluTyrGlnThrProLeuGly600605610CGTTATGCGAAATCGCGCCTAAATTCAGCGACTAAAGAGGCGCGTAAC2105ArgTyrAlaLysSerArgLeuAsnSerAlaThrLysGluAlaArgAsn615620625GCTTTGGATAACTACCGCTTTAATGATGCGACGACTTTGTTATACCGC2153AlaLeuAspAsnTyrArgPheAsnAspAlaThrThrLeuLeuTyrArg630635640645TTTTTGTGGGGGGAATTTTGCGATTGGTTCATTGAATTTTCTAAAGTG2201PheLeuTrpGlyGluPheCysAspTrpPheIleGluPheSerLysVal650655660GAAAATGGAGCGATAGACGAGTTAGGGAGCGTGTTAAAAGAGGCTTTA2249GluAsnGlyAlaIleAspGluLeuGlySerValLeuLysGluAlaLeu665670675AAACTCTTGCACCCTTTCATGCCCTTTATCAGCGAGTCTTTATACCAC2297LysLeuLeuHisProPheMetProPheIleSerGluSerLeuTyrHis680685690AAGCTCAGTAACACGGAACTAGAAAACACTGAATCTATCATGGTCATG2345LysLeuSerAsnThrGluLeuGluAsnThrGluSerIleMetValMet695700705CCTTACCCTAAAGATTTGGCACAAGATGAAAAACTAGAGCATGAATTT2393ProTyrProLysAspLeuAlaGlnAspGluLysLeuGluHisGluPhe710715720725GAAGTGATCAAAGATTGCATTGTGTCTTTAAGGCGTTTGAAAATCATG2441GluValIleLysAspCysIleValSerLeuArgArgLeuLysIleMet730735740CTAGAAACCCCACCGATTGTTTTAAAGGAAGCGAGCGTGGGATTAAGG2489LeuGluThrProProIleValLeuLysGluAlaSerValGlyLeuArg745750755GAAAAAATAGAAAACACAGAGCGTTTGCAAACTTATGCTCAAAAATTA2537GluLysIleGluAsnThrGluArgLeuGlnThrTyrAlaGlnLysLeu760765770GCGAGGTTAGAAAAAGTCAGCGTGATAACTTATAAGCCTTTAAAAAGC2585AlaArgLeuGluLysValSerValIleThrTyrLysProLeuLysSer775780785GTGAGCGATGTGGGGGAATTTTGCCAAACTTATGCAAATTTAGAAAAT2633ValSerAspValGlyGluPheCysGlnThrTyrAlaAsnLeuGluAsn790795800805CTTGATTTAAGCCCGCTCATTGCTCGTTTAAAAAAGCAGCTAGAAAAA2681LeuAspLeuSerProLeuIleAlaArgLeuLysLysGlnLeuGluLys810815820CTGGAAAAAGAAAAATTAAAACTCAATTTGCACAATGAAAATTTTGTT2729LeuGluLysGluLysLeuLysLeuAsnLeuHisAsnGluAsnPheVal825830835AAAAACGCACCTAAAAGCGTGCTAGAAAAAGCCAAAGAGAGTTTAAAA2777LysAsnAlaProLysSerValLeuGluLysAlaLysGluSerLeuLys840845850ACGCTTTTAGAAAAAGAAAGTAAAATTAAGCAAGAATTGGATTTGTTA2825ThrLeuLeuGluLysGluSerLysIleLysGlnGluLeuAspLeuLeu855860865AAACAACCATAATAAAAGGATAGAAAATGTTTCAAGCATTAAGCGATGG2874LysGlnPro870GTTTAAAAACGCGCTCAATAAAATCCGCTTTCAAGACGATGAAAAAGCGCTAGACAGAGC2934GTTAGATGAATTGAAAAAAACGCTTTTAAAAAACGATGT2973(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 872 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:MetLysGlnGluProThrThrTyrGlnProGluGluIleGluLysLys151015IleTyrGluIleCysSerHisArgGlyTyrPheGluIleAspGlyAsn202530GluAlaIleGlnGluLysAsnLysArgPheCysLeuMetMetProPro354045ProAsnValThrGlyIleLeuHisIleGlyHisAlaLeuThrLeuSer505560LeuGlnAspIleLeuAlaArgTyrLysArgMetAspGlyTyrLysThr65707580LeuTyrGlnProGlyLeuAspHisAlaGlyIleAlaThrGlnAsnVal859095ValGluLysGlnLeuLeuAsnGlnGlyIleLysLysGluAspLeuGly100105110ArgGluAlaPheValGlnLysValTrpGluTrpLysGluLysSerGly115120125GlyAlaIleLeuGluGlnMetLysArgLeuGlyValSerAlaAlaPhe130135140SerArgThrArgPheThrMetAspLysGlyLeuGlnArgAlaValLys145150155160LeuAlaPheLeuLysTrpTyrGluLysGlyLeuIleValGlnAspAsn165170175TyrMetValAsnTrpCysThrLysAspGlyAlaLeuSerAspIleGlu180185190ValGluTyrGluGluArgLysGlyAlaLeuTyrTyrIleArgTyrTyr195200205LeuGluAsnGlnLysAspTyrLeuValValAlaThrThrArgProGlu210215220ThrLeuPheGlyAspSerAlaLeuMetValAsnProAsnAspGluArg225230235240TyrArgHisLeuValGlyGlnLysAlaValLeuProLeuIleAsnArg245250255ThrIleProIleIleAlaAspGluHisValGluMetGluPheGlyThr260265270GlyCysValLysValThrProGlyHisAspPheAsnAspTyrGluVal275280285GlyLysArgHisHisLeuGluAlaIleLysIlePheAspGluLysGly290295300IleLeuAsnAlaHisCysGlyGluPheGluAsnLeuGluArgLeuGlu305310315320AlaArgAspLysValValGluArgLeuLysGluAsnAlaLeuLeuGlu325330335LysIleGluGluHisThrHisGlnValGlyHisCysTyrArgCysHis340345350AsnValValGluProTyrValSerLysGlnTrpPheValLysProGlu355360365IleAlaGlnSerSerIleGluLysIleGlnGlnGlyLeuAlaArgPhe370375380TyrProSerAsnTrpIleAsnAsnTyrAsnAlaTrpMetArgGluLeu385390395400ArgProTrpCysIleSerArgGlnLeuPheTrpGlyHisGlnIlePro405410415ValPheThrCysGluAsnAsnHisGlnPheValSerLeuAspThrPro420425430LeuSerCysProThrCysLysSerGluIleLeuGluGlnAspLysAsp435440445ValLeuAspThrTrpPheSerSerGlyLeuTrpAlaPheSerThrLeu450455460GlyTrpGlyGlnGluLysSerGlyLeuPheAsnGluSerAspLeuLys465470475480AspPheTyrProAsnThrThrLeuIleThrGlyPheAspIleLeuPhe485490495PheTrpValAlaArgMetLeuPheCysSerGluSerLeuLeuGlyGlu500505510LeuProPheLysAspIleTyrLeuHisAlaLeuValArgAspGluLys515520525GlyGluLysMetSerLysSerLysGlyAsnValIleAspProLeuGlu530535540MetIleGluLysTyrGlyAlaAspSerLeuArgPheThrLeuAlaAsn545550555560LeuCysAlaThrGlyArgAspIleLysLeuSerThrThrHisLeuGlu565570575AsnAsnLysAsnPheAlaAsnLysIlePheAsnAlaValSerTyrLeu580585590LysLeuLysGlnGluAlaPheLysAspArgGluArgLeuAsnGluTyr595600605GlnThrProLeuGlyArgTyrAlaLysSerArgLeuAsnSerAlaThr610615620LysGluAlaArgAsnAlaLeuAspAsnTyrArgPheAsnAspAlaThr625630635640ThrLeuLeuTyrArgPheLeuTrpGlyGluPheCysAspTrpPheIle645650655GluPheSerLysValGluAsnGlyAlaIleAspGluLeuGlySerVal660665670LeuLysGluAlaLeuLysLeuLeuHisProPheMetProPheIleSer675680685GluSerLeuTyrHisLysLeuSerAsnThrGluLeuGluAsnThrGlu690695700SerIleMetValMetProTyrProLysAspLeuAlaGlnAspGluLys705710715720LeuGluHisGluPheGluValIleLysAspCysIleValSerLeuArg725730735ArgLeuLysIleMetLeuGluThrProProIleValLeuLysGluAla740745750SerValGlyLeuArgGluLysIleGluAsnThrGluArgLeuGlnThr755760765TyrAlaGlnLysLeuAlaArgLeuGluLysValSerValIleThrTyr770775780LysProLeuLysSerValSerAspValGlyGluPheCysGlnThrTyr785790795800AlaAsnLeuGluAsnLeuAspLeuSerProLeuIleAlaArgLeuLys805810815LysGlnLeuGluLysLeuGluLysGluLysLeuLysLeuAsnLeuHis820825830AsnGluAsnPheValLysAsnAlaProLysSerValLeuGluLysAla835840845LysGluSerLeuLysThrLeuLeuGluLysGluSerLysIleLysGln850855860GluLeuAspLeuLeuLysGlnPro865870(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1692 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 121..1623(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:GGCGTGGAATCTTTAAAAGAAAAGGCTAAAGACTTGCCTAAAAACATGTTAGATCCAAAA60GCTAACCAAACCCCACCAAACCCTACCCCATCTAATAAAGAACCCCTATAAAGGCATCAC120ATGTTTTCTAACCAATACATCCAACAACGCATCCATAAAGCCAATAGC168MetPheSerAsnGlnTyrIleGlnGlnArgIleHisLysAlaAsnSer151015TTGAGGGAAGAAGGGAAAAACCCTTATCAAAATGGCTTGAAACGAAGC216LeuArgGluGluGlyLysAsnProTyrGlnAsnGlyLeuLysArgSer202530CTCACCAACGCCGCTTTTTTAGAAAAATACGCTTATATTAAGGATTTA264LeuThrAsnAlaAlaPheLeuGluLysTyrAlaTyrIleLysAspLeu354045GAAGAGCCTAAAGACAAAGAAAAATGCGAGAGTGTTGTAGGGAGAGTC312GluGluProLysAspLysGluLysCysGluSerValValGlyArgVal505560AAGCTTTTGCGTTTAATGGGTAAGGCTTGTTTTATTAAAATTGAAGAT360LysLeuLeuArgLeuMetGlyLysAlaCysPheIleLysIleGluAsp65707580GAAAGCGCGATTTTGCAAGCCTATGTTTCGCAAAATGAATTGAATGAT408GluSerAlaIleLeuGlnAlaTyrValSerGlnAsnGluLeuAsnAsp859095GAATTTAAAAGCCTGAAAAAGCATTTAGAAGTGGGCGATATTGTGTTG456GluPheLysSerLeuLysLysHisLeuGluValGlyAspIleValLeu100105110GTGAAAGGTTTCCCTTTTGCTACCAAAACCGGTGAATTAAGCGTTCAT504ValLysGlyPheProPheAlaThrLysThrGlyGluLeuSerValHis115120125GCCCTAGAATTTCATATTTTAAGCAAAACCATTGTGCCTTTACCTGAA552AlaLeuGluPheHisIleLeuSerLysThrIleValProLeuProGlu130135140AAGTTTCATGGATTAAGCGATATAGAATTGCGTTACCGCCAGCGCTAC600LysPheHisGlyLeuSerAspIleGluLeuArgTyrArgGlnArgTyr145150155160TTGGATTTGATCGTCAATCCTAGCGTTAAAGATGTGTTCAAAAAACGC648LeuAspLeuIleValAsnProSerValLysAspValPheLysLysArg165170175AGTTTGATTGTTTCTAGCGTGCGGAAATTCTTTGAAATGGCAGGGTTT696SerLeuIleValSerSerValArgLysPhePheGluMetAlaGlyPhe180185190TTAGAAGTGGAAACCCCCATGATGCACCCCATTCCTGGCGGGGCGAAC744LeuGluValGluThrProMetMetHisProIleProGlyGlyAlaAsn195200205GCAAGGCCTTTTATCACTTACCATAACGCTTTGGAGGTGGAGAGGTAT792AlaArgProPheIleThrTyrHisAsnAlaLeuGluValGluArgTyr210215220TTAAGAATCGCCCCAGAATTATACCTCAAACGCTTGATTGTAGGGGGG840LeuArgIleAlaProGluLeuTyrLeuLysArgLeuIleValGlyGly225230235240TTTGAAGCGGTGTTTGAAATCAATCGTAATTTCAGGAATGAAGGCATG888PheGluAlaValPheGluIleAsnArgAsnPheArgAsnGluGlyMet245250255GATCACAGCCATAACCCCGAATTCACGATGATTGAGTTTTATTGGGCG936AspHisSerHisAsnProGluPheThrMetIleGluPheTyrTrpAla260265270TATCACACTTATGAAGATTTGATTGAACTCAGTAAGAGGCTGTTTGAC984TyrHisThrTyrGluAspLeuIleGluLeuSerLysArgLeuPheAsp275280285TACTTGCTAAAGACTTTGAACTTACCTTCAAAAATCATTTATAACGAT1032TyrLeuLeuLysThrLeuAsnLeuProSerLysIleIleTyrAsnAsp290295300ATGGAAGTGGATTTCAACCAAACGAGCGTGATTTCCTATTTGGACGCT1080MetGluValAspPheAsnGlnThrSerValIleSerTyrLeuAspAla305310315320TTAGAAACGATAGGGGGCATTAGTAAGGGTATTTTAGAAAAAGAAGAC1128LeuGluThrIleGlyGlyIleSerLysGlyIleLeuGluLysGluAsp325330335AGGCTTTTGGCTTATTTGTTAGAGCAAGGCATCAAAGTAGAGCCCAAT1176ArgLeuLeuAlaTyrLeuLeuGluGlnGlyIleLysValGluProAsn340345350CTCACTTATGGCAAGTTGCTCGCTGAAGCGTTTGATCATTTTGTAGAG1224LeuThrTyrGlyLysLeuLeuAlaGluAlaPheAspHisPheValGlu355360365CATCAACTCATTAACCCCACTTTTGTAACCCAATACCCTATTGAGATT1272HisGlnLeuIleAsnProThrPheValThrGlnTyrProIleGluIle370375380AGCCCTTTAGCCAGACGCAACGATAGTAACCCTAATATTGCTGACAGG1320SerProLeuAlaArgArgAsnAspSerAsnProAsnIleAlaAspArg385390395400TTTGAATTGTTCATTGCAGGAAAAGAAATCGCTAATGGCTTTAGCGAG1368PheGluLeuPheIleAlaGlyLysGluIleAlaAsnGlyPheSerGlu405410415TTGAACGACCCTTTAGATCAATTAGAACGCTTTAAAAATCAAGTGGCT1416LeuAsnAspProLeuAspGlnLeuGluArgPheLysAsnGlnValAla420425430GAAAAAGAAAAAGGCGATGAAGAAGCCCAATACATGGATGAAGATTAC1464GluLysGluLysGlyAspGluGluAlaGlnTyrMetAspGluAspTyr435440445GTGTGGGCCCTAGCCCATGGAATGCCCCCCACTGCAGGGCAAGGCATA1512ValTrpAlaLeuAlaHisGlyMetProProThrAlaGlyGlnGlyIle450455460GGCATTGACCGATTAGTGATGTTACTCACTGGAGCTAAAAGCATTAAA1560GlyIleAspArgLeuValMetLeuLeuThrGlyAlaLysSerIleLys465470475480GATGTGATTTTATTCCCAGCGATGCGTCCTGTTAAAAACGATTTTAAT1608AspValIleLeuPheProAlaMetArgProValLysAsnAspPheAsn485490495GTGGAGAGTGGAGAATAATGGCGTATTTTTTAGAACAAACGGATAGTGAAATTTT1663ValGluSerGlyGlu500TGAATTGATCTTTGAAGAATATAAGCGGC1692(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 501 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:MetPheSerAsnGlnTyrIleGlnGlnArgIleHisLysAlaAsnSer151015LeuArgGluGluGlyLysAsnProTyrGlnAsnGlyLeuLysArgSer202530LeuThrAsnAlaAlaPheLeuGluLysTyrAlaTyrIleLysAspLeu354045GluGluProLysAspLysGluLysCysGluSerValValGlyArgVal505560LysLeuLeuArgLeuMetGlyLysAlaCysPheIleLysIleGluAsp65707580GluSerAlaIleLeuGlnAlaTyrValSerGlnAsnGluLeuAsnAsp859095GluPheLysSerLeuLysLysHisLeuGluValGlyAspIleValLeu100105110ValLysGlyPheProPheAlaThrLysThrGlyGluLeuSerValHis115120125AlaLeuGluPheHisIleLeuSerLysThrIleValProLeuProGlu130135140LysPheHisGlyLeuSerAspIleGluLeuArgTyrArgGlnArgTyr145150155160LeuAspLeuIleValAsnProSerValLysAspValPheLysLysArg165170175SerLeuIleValSerSerValArgLysPhePheGluMetAlaGlyPhe180185190LeuGluValGluThrProMetMetHisProIleProGlyGlyAlaAsn195200205AlaArgProPheIleThrTyrHisAsnAlaLeuGluValGluArgTyr210215220LeuArgIleAlaProGluLeuTyrLeuLysArgLeuIleValGlyGly225230235240PheGluAlaValPheGluIleAsnArgAsnPheArgAsnGluGlyMet245250255AspHisSerHisAsnProGluPheThrMetIleGluPheTyrTrpAla260265270TyrHisThrTyrGluAspLeuIleGluLeuSerLysArgLeuPheAsp275280285TyrLeuLeuLysThrLeuAsnLeuProSerLysIleIleTyrAsnAsp290295300MetGluValAspPheAsnGlnThrSerValIleSerTyrLeuAspAla305310315320LeuGluThrIleGlyGlyIleSerLysGlyIleLeuGluLysGluAsp325330335ArgLeuLeuAlaTyrLeuLeuGluGlnGlyIleLysValGluProAsn340345350LeuThrTyrGlyLysLeuLeuAlaGluAlaPheAspHisPheValGlu355360365HisGlnLeuIleAsnProThrPheValThrGlnTyrProIleGluIle370375380SerProLeuAlaArgArgAsnAspSerAsnProAsnIleAlaAspArg385390395400PheGluLeuPheIleAlaGlyLysGluIleAlaAsnGlyPheSerGlu405410415LeuAsnAspProLeuAspGlnLeuGluArgPheLysAsnGlnValAla420425430GluLysGluLysGlyAspGluGluAlaGlnTyrMetAspGluAspTyr435440445ValTrpAlaLeuAlaHisGlyMetProProThrAlaGlyGlnGlyIle450455460GlyIleAspArgLeuValMetLeuLeuThrGlyAlaLysSerIleLys465470475480AspValIleLeuPheProAlaMetArgProValLysAsnAspPheAsn485490495ValGluSerGlyGlu500(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1431 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 80..1324(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:TTTGAACGCTCAAATAGATAAAAATGAAATTGATGAATGGGCTAAATTGTGGCATTTTAG60AACGATTAAAGAGGGTTGAATGATTGATAGAAAACTTTTATTGCAAGATTTT112MetIleAspArgLysLeuLeuLeuGlnAspPhe1510GACAAGGTGGCTCTTTCTTTAAAAAAGCGTAATCATGCGATGGATGAT160AspLysValAlaLeuSerLeuLysLysArgAsnHisAlaMetAspAsp152025GGATTGGAGCGTTTGCGCGAAGTCATCACGCGTTATAAAAAGCAACTC208GlyLeuGluArgLeuArgGluValIleThrArgTyrLysLysGlnLeu303540ATTGAATTGGAAGGCTTGCAAGCCTTTCAAAACAAGGTTTCTAAAGAA256IleGluLeuGluGlyLeuGlnAlaPheGlnAsnLysValSerLysGlu455055TTTGGTATCAAAATGGCTCAAAAAGTGGATACAAGCGATCTCAAAAAA304PheGlyIleLysMetAlaGlnLysValAspThrSerAspLeuLysLys60657075GAGCTAGAAAGCAATAAAATCAAATTGAATGAGCTTTCTAAAAGCGTG352GluLeuGluSerAsnLysIleLysLeuAsnGluLeuSerLysSerVal808590GGCGAATTGGAGCAACAAATTGATTTGAAGCTTTCCATAATCCCTAAT400GlyGluLeuGluGlnGlnIleAspLeuLysLeuSerIleIleProAsn95100105CTAGTGGATGAAAAAACCCCTTTAGGCGCAAATGAAGAAGACAACATA448LeuValAspGluLysThrProLeuGlyAlaAsnGluGluAspAsnIle110115120GAAATTAAAAAAATCTTAACCCCAAGGGTTTTTACTTTCAAACCCAAA496GluIleLysLysIleLeuThrProArgValPheThrPheLysProLys125130135GAGCATTTTGAGCTCGCTCAACAAAACGGCTGGATTGATTTTGAAGGC544GluHisPheGluLeuAlaGlnGlnAsnGlyTrpIleAspPheGluGly140145150155GGCGTGAAACTCGCCAAAAGCCGTTTTTCGGTCATTAGGGGTTTTGGG592GlyValLysLeuAlaLysSerArgPheSerValIleArgGlyPheGly160165170GCTAAAATTTATCGCGCGCTCATTCATTTAATGCTGGATTTTAATGAA640AlaLysIleTyrArgAlaLeuIleHisLeuMetLeuAspPheAsnGlu175180185AAAAATGGCTTTGAAATCATCTACACGCCGGCGTTAGTGAATGAAAAA688LysAsnGlyPheGluIleIleTyrThrProAlaLeuValAsnGluLys190195200ATGCTTTTTGGGACCGGGCAATTACCCAAATTCAAAGAAGATATTTTC736MetLeuPheGlyThrGlyGlnLeuProLysPheLysGluAspIlePhe205210215AAAATAGAAAATGAAAATTTGTATCTGATTCCCACCGCTGAGGTAACG784LysIleGluAsnGluAsnLeuTyrLeuIleProThrAlaGluValThr220225230235CTCACCAATCTATACAACGACACCATTATTAGCGTTGAAAACCTCCCC832LeuThrAsnLeuTyrAsnAspThrIleIleSerValGluAsnLeuPro240245250ATTAAAATGACCGCGCACACGCCTTGTTTCAGGAGCGAAGCGGGGAGC880IleLysMetThrAlaHisThrProCysPheArgSerGluAlaGlySer255260265GCGGGCAAGGACACAAGGGGGATGATAAGACAGCACCAATTTGATAAA928AlaGlyLysAspThrArgGlyMetIleArgGlnHisGlnPheAspLys270275280GTAGAATTAGTGGCTATCACGCACCCTAAAGAAAGCGATGTTATGCAA976ValGluLeuValAlaIleThrHisProLysGluSerAspValMetGln285290295GAGCATATGCTAGAGAGCGCGAGCGAGATCTTAAAGGCTTTGGAATTA1024GluHisMetLeuGluSerAlaSerGluIleLeuLysAlaLeuGluLeu300305310315CCGCACCGGTTCGTGCAATTGTGCAGCGCGGATTTAGGCTTTAGTGCG1072ProHisArgPheValGlnLeuCysSerAlaAspLeuGlyPheSerAla320325330AGCAACACGATAGACATTGAAGTGTGGCTGCCCGGGCAAAATTGCTAC1120SerAsnThrIleAspIleGluValTrpLeuProGlyGlnAsnCysTyr335340345CGAGAAATCAGCTCCGTGTCTAACACGAGGGATTTCCAGGCCAGGCGT1168ArgGluIleSerSerValSerAsnThrArgAspPheGlnAlaArgArg350355360GCCAAAATCCGCTTCAAAGAAAATCAAAAAAACCAATTAGCGCACACC1216AlaLysIleArgPheLysGluAsnGlnLysAsnGlnLeuAlaHisThr365370375TTAAACGGCTCTTCTTTAGCGGTAGGCAGGACGATGGTCGCTTTAATG1264LeuAsnGlySerSerLeuAlaValGlyArgThrMetValAlaLeuMet380385390395GAAAACCACCAGCAAGCGGATGGAAACATCCACATTCCTAAGGCGTTA1312GluAsnHisGlnGlnAlaAspGlyAsnIleHisIleProLysAlaLeu400405410GAAAAATACCTTTAAGGCTAGTTCGTGCTGAATGAAGAGCAAAATTCATTAG1364GluLysTyrLeu415AAGAAAAAGGGGGCGAAAACAAAAACGAAAAAGAAACCCCCTAAAAGGCATTCATTCTAA1424AATCCCC1431(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 415 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:MetIleAspArgLysLeuLeuLeuGlnAspPheAspLysValAlaLeu151015SerLeuLysLysArgAsnHisAlaMetAspAspGlyLeuGluArgLeu202530ArgGluValIleThrArgTyrLysLysGlnLeuIleGluLeuGluGly354045LeuGlnAlaPheGlnAsnLysValSerLysGluPheGlyIleLysMet505560AlaGlnLysValAspThrSerAspLeuLysLysGluLeuGluSerAsn65707580LysIleLysLeuAsnGluLeuSerLysSerValGlyGluLeuGluGln859095GlnIleAspLeuLysLeuSerIleIleProAsnLeuValAspGluLys100105110ThrProLeuGlyAlaAsnGluGluAspAsnIleGluIleLysLysIle115120125LeuThrProArgValPheThrPheLysProLysGluHisPheGluLeu130135140AlaGlnGlnAsnGlyTrpIleAspPheGluGlyGlyValLysLeuAla145150155160LysSerArgPheSerValIleArgGlyPheGlyAlaLysIleTyrArg165170175AlaLeuIleHisLeuMetLeuAspPheAsnGluLysAsnGlyPheGlu180185190IleIleTyrThrProAlaLeuValAsnGluLysMetLeuPheGlyThr195200205GlyGlnLeuProLysPheLysGluAspIlePheLysIleGluAsnGlu210215220AsnLeuTyrLeuIleProThrAlaGluValThrLeuThrAsnLeuTyr225230235240AsnAspThrIleIleSerValGluAsnLeuProIleLysMetThrAla245250255HisThrProCysPheArgSerGluAlaGlySerAlaGlyLysAspThr260265270ArgGlyMetIleArgGlnHisGlnPheAspLysValGluLeuValAla275280285IleThrHisProLysGluSerAspValMetGlnGluHisMetLeuGlu290295300SerAlaSerGluIleLeuLysAlaLeuGluLeuProHisArgPheVal305310315320GlnLeuCysSerAlaAspLeuGlyPheSerAlaSerAsnThrIleAsp325330335IleGluValTrpLeuProGlyGlnAsnCysTyrArgGluIleSerSer340345350ValSerAsnThrArgAspPheGlnAlaArgArgAlaLysIleArgPhe355360365LysGluAsnGlnLysAsnGlnLeuAlaHisThrLeuAsnGlySerSer370375380LeuAlaValGlyArgThrMetValAlaLeuMetGluAsnHisGlnGln385390395400AlaAspGlyAsnIleHisIleProLysAlaLeuGluLysTyrLeu405410415(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 14(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 17(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 20(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 23(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:GCGAATTCTWYCTNACNGGNACNGAYGARCAYGG34(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 14(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 20(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 23(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:GCGAATTCTTYATNTGYGGNACNGAYGARYAYGG34(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 18(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 33(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:GCGAATTCRTARTTNATNAGNGCRTCRAWCCANACRTA38(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 18(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 27(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 33(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:GCGAATTCRTANCCRATNGKNGCRTCNARCCANACRTA38(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 11(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 20(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 26(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 29(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GCGAATTCGGNTGGGAYACNCAYGGNSTNCC31(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 11(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 26(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 29(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GCGAATTCGGNTGGGAYTGYCAYGGNCTNCC31(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 10(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 16(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 25(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 28(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 31(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:GCGAATTCGNCARCGNTAYTGGGGNRTNCCNAT33(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 10(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 16(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 25(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 28(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 31(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:GCGAATTCGNAAYCGNTWYTGGGGNACNCCNMT33(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 18(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 30(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 33(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:GCGAATTCRAACCANCCNCGNGTYTGRTCNWWNCCYTC38(2) INFORMATION FOR SEQ ID NO:22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 26 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 3(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 6(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 18(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:GGNARNGTCCANGGNGTNGTNGTCCA26(2) INFORMATION FOR SEQ ID NO:23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 24(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 27(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:TWYATGGARTCNACNTGGTGGGYNTTNAARCA32(2) INFORMATION FOR SEQ ID NO:24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 9(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 18(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:GAYCAYGCNGGNATHGCNACNCA23(2) INFORMATION FOR SEQ ID NO:25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 7(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 10(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 13(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:RTCRTGNGCNGGNGTDATYTT21(2) INFORMATION FOR SEQ ID NO:26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 7(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 13(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 16(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:RAACCANGTRTCNARNACRTC21(2) INFORMATION FOR SEQ ID NO:27:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 36 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 10(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 13(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 16(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 25(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 31(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 34(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:GCGAATTCCNATNGGNTGGGAYGCNTTYGGNCTNCC36(2) INFORMATION FOR SEQ ID NO:28:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 20(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 23(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 26(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 29(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:GCGAATTCACYTGYTCRTTNGCNAGNACNGT31(2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 3(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 6(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 9(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 24(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 27(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:CCNMTNGGNTWYCAYTGYACNGGNMTNCC29(2) INFORMATION FOR SEQ ID NO:30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 35 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 18(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 24(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 27(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 30(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:GCRTCRTARTANGGRTTNGCRTCNGTNGTNACRAA35(2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 27 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 6(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 18(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:TTYMTNGARGTNGARACNCCNATGATG27(2) INFORMATION FOR SEQ ID NO:32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 9(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 24(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:TARAAYTCNATNGTNGTRAAYTCNGGRTTRTG32(2) INFORMATION FOR SEQ ID NO:33:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 9(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 24(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:TACMAYTCNAKCATNGTRAAYTCNGGRTTRTG32(2) INFORMATION FOR SEQ ID NO:34:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:AARAARTAYGAYCTNGARGCNTGGTTYCC29(2) INFORMATION FOR SEQ ID NO:35:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 6(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 15(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:AARACNTAYGAYCTNGARGTNTGGATHCC29(2) INFORMATION FOR SEQ ID NO:36:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 9(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:CCYTTYTCNGTYTGRTARTTYTC23(2) INFORMATION FOR SEQ ID NO:37:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 9(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:CCRTCYTCNGTYTGRTARTTYTC23(2) INFORMATION FOR SEQ ID NO:38:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 3(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 12(D) OTHER INFORMATION: /mod.sub.-- base=i(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 21(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:SCNTGTTTTMGNTCWGAAGCNGG23(2) INFORMATION FOR SEQ ID NO:39:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 6(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:TATMGNGAAATTTCWTGTTCWAAT24(2) INFORMATION FOR SEQ ID NO:40:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 24 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 19(D) OTHER INFORMATION: /mod.sub.-- base=i(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:TAAWGAACAWGAAATTTCNAKATA24(2) INFORMATION FOR SEQ ID NO:41:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:CCATCHKSTTGTTGATRATTTTC23(2) INFORMATION FOR SEQ ID NO:42:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:CGTGGATCCGTGAAAGAATACAAAGACAC29(2) INFORMATION FOR SEQ ID NO:43:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:CCCAGTCGACTTATCATCGCTCTTTTAAAACC32(2) INFORMATION FOR SEQ ID NO:44:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:CGTGGATCCATGCAAAAATCACTGATCAC29(2) INFORMATION FOR SEQ ID NO:45:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:CCCAGTCGACTTAGCTGATCAAACTTCCTGC31(2) INFORMATION FOR SEQ ID NO:46:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:GCGCGGATCCATGGATTTTATCAATATAGAAA32(2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:ACGCGTCGACTTATGCGATAACAAAATTAACG32(2) INFORMATION FOR SEQ ID NO:48:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:GCGCGGATCCATGAAACAAGAACCCACCACCT32(2) INFORMATION FOR SEQ ID NO:49:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:ACGCGTCGACTTATGGTTGTTTTAACAAATC31(2) INFORMATION FOR SEQ ID NO:50:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:GCGCGGATCCATGATTGATAGAAAACTTTTAT32(2) INFORMATION FOR SEQ ID NO:51:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:ACGCGTCGACTTAAAGGTATTTTTCTAACGC31(2) INFORMATION FOR SEQ ID NO:52:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:GCGCGGATCCATGTTTTCTAACCAATACATC31(2) INFORMATION FOR SEQ ID NO:53:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:ACGCGTCGACTTATTCTCCACTCTCTCCACA31(2) INFORMATION FOR SEQ ID NO:54:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 43 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:GCGCTCTAGATATCTGCTTATGTCCCCTATACTAGGTTATTGG43(2) INFORMATION FOR SEQ ID NO:55:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 28 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:GGGGTACCTCACGATGCGGCCGCTCGAG28(2) INFORMATION FOR SEQ ID NO:56:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:CCGCTCGAGCGATGCAATGTCGATCAATTGTGC33(2) INFORMATION FOR SEQ ID NO:57:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 31 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:GGGGTACCCCTTTTTCATGACCTCATATTCG31(2) INFORMATION FOR SEQ ID NO:58:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:CAGACGTCTAGATATCTGCTTATGTTTTCTAACCAATAC39(2) INFORMATION FOR SEQ ID NO:59:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 34 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:ACCGCTCGAGCGGTTATTCTCCACTCTCCACATT34(2) INFORMATION FOR SEQ ID NO:60:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 39 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:CAGACGTCTAGATATCTGCTTATGGCGGCCGTGCAGGCG39(2) INFORMATION FOR SEQ ID NO:61:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 33 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:TCTAGCTCGAGCTACACAGAAGTGCCAACTGTT33(2) INFORMATION FOR SEQ ID NO:62:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:CCAAGAAGCTTGAAGTAATAATAGGCGCATGC32(2) INFORMATION FOR SEQ ID NO:63:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:CGTACTGCAGGATTGTATGCTTGGTATAGC30(2) INFORMATION FOR SEQ ID NO:64:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 30 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:GGAATTCTGAAAACAACTCATATAAATACG30(2) INFORMATION FOR SEQ ID NO:65:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 32 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:GAGGCGCCCTCTTATCAATCCCCTCCTCAACC32(2) INFORMATION FOR SEQ ID NO:66:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 40 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:CAGACGTCTAGATATCTGCTTATGCAAAAATCACTGATCA40(2) INFORMATION FOR SEQ ID NO:67:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: Not Relevant(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:ArgValGluAsnLysSerThrArgAlaAlaAlaSer1510__________________________________________________________________________

Number	Name	Date
4713337	Jasin et al.	Dec 1987
4788148	Nilsson et al.	Nov 1988
4952501	Jasin et al.	Aug 1990
4963487	Schimmel et al.	Oct 1990
5370995	Henneck et al.	Dec 1994
5561054	Kron et al.	Oct 1996
5688655	Housey	Nov 1997

Helicobacter aminoacyl-tRNA synthetase proteins, nucleic acids and strains comprising same

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

US Referenced Citations (7)

Foreign Referenced Citations (1)

Non-Patent Literature Citations (51)

Entry
Langenberg et al. "Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis" J. Clinical Microbiol. 24(3):414-417, Sep. 1986.
Taylor et al. "Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level." J. Bacteriol. 174(21):6800-6806, Nov. 1992.
Webster's II New Riveside Dictionary The Riverside Publishing Co., p. 1232, 1994.
Meinnel, T., et al., "Aminoacyl-tRNA Synthetases: Occurence, Structure, and Function." In tRNA: Structure, Biosynthesis, and Function, Soll, D. and RajBhandary, U., eds. (Washington, DC: American Society for Microbiology), pp. 251-300 (1995).
von der Haar, F. et al, "Target Directed Drug Synthesis: The Aminoacyl-tRNA Synthetase as Possible Targets," Angew. Chem. Int. Ed. Engl., 20(3):217-223 (1981).
Walter, R. D. and Kuhlow, F., "Parasite-Specific Interaction of N-�4-(4' Nitroanilino)-Phenyl!-S-(.beta.-Carboxyethyl)-Dithiocarbamic Acid-Ester with Arginyl-tRNA-Synthetase from Dirofilaria immitis," Trop. Med. Parasit., 36:230-232 (1985).
Hughes, J., et al., "Inhibition of Isoleucyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli by Pseudomonic Acid," Biochem. J., 176:305-318 (1978).
Hughes, J. and Mellows, G., "Interaction of Pseudomonic Acid A with Escherichia coli B Isoleucyl-tRNA Synthetase," Biochem J., 191:209-219 (1980).
Racher, K.I., et al., "Expression and Characterization of a Recombinant Yeast Isoleucyl-tRNA Synthetase," J. Biol. Chem., 266(26):17158-17164 (1991).
Chalker, A.F., et al., "Analysis and Toxic Overexpression in Escherichia coli of a Staphylococcal Gene Encoding Isoleucyl-tRNA Synthetase," Gene, 141:103-108 (1994).
Jasin, M. and Schimmel, P., "Deletion of an Essential Gene in Eschericia coli by Site-Specific Recombinant with Linear DNA Fragments," J. Bacteriol., 159(2):783-786 (1984).
Shiba, K. and Shimmel, P., "Functional Assembly of a Randomly Cleaved Protein," Proc. Natl. Acad. Sci. USA, 89:1880-1884 (1992).
Shepard, A., et al., "RNA Binding Determinant in Some Class I tRNA Synthetases Identified by Alignment-Guided Mutagenesis," Proc. Natl. Acad. Sci. USA, 89:9964-9968 (1992).
Kim, S., et al., "Diversified Sequences of Peptide Epitope for Same-RNA Recognition," Proc. Natl. Acad. Sci. USA, 90:10046-10050 (1993).
Edwards, H., et al., "An E. coli Aminoacyl-tRNA Synthetase Can Substitute for Yeast Mitochondrial Enzyme Function In Vivo," Cell, 51:643-649 (1987).
Edwards, H. and Schimmel, P., "A Bacterial Amber Suppressor in Saccharomyces cerevisiae Is Selectively Recognized by a Bacterial Aminoacyl-tRNA Synthetase," Mol. Cell. Biol., 10(4):1633-1641 (1990).
Weygand-Durasevic, I., et al., "Yeast Seryl-tRNA Synthetase Expressed in Escherichia coli Recognizes Bacterial Serine-Specific tRNAs in vivo," Eur. J. Biochem., 214:869-877 (1993).
Jones, M. D., et al., "Natural Variation of Tyrosyl-tRNA Sythetase and Comparison with Engineered Mutants," Biochemistry, 25:1887-1891 (1986).
Henkin, T. M., et al., "Analysis of the Bacillus subtilis tyrS Gene: Conservation of a Regulatory Sequence in Multiple tRNA Synthetase Genes," J. Bacteriol., 174(4):1299-1306 (1992).
Salazar, O., et al., "Thiobacillus ferrooxidans Tyrosyl-tRNA Synthetase Functions In Vivo in Escherichia coli," J. Bacteriol., 176(14):4409-4415 (1994).
Iaccarino, M. and Berg, P., "Isoleucine Auxotrophy as a Consequence of a Mutationally Altered Isoleucyl-Transfer Ribonucleic Acid Synthetase," J. Bacteriol., 105:527-537 (1970).
Archibold, E.R., and Williams, L.S., "Regulation fo Methionyl-Transfer Ribonucleic Acid Synthetase Formation in Eschericia coli and Salmonella typhimurium," J. Bacteriol., 114(3):1007-1013 (1973).
Dardel, F., et al., "Molecular Cloning and Primary Structure of the Eschericia coli Methionyl-tRNA Synthetase Gene," J. Bacteriol., 160(3):1115-1122 (1984)
Low, B., et al., "Isolation and Partial Characterization of Temperature-Sensitive Eschericia coli Mutants with Altered Leucyl- and Seryl- Transfer Ribonucleic Acid Synthetases," J. Bacteriol., 108(2):742-750 (1971).
Clarke, S. J., et al., "Isolation and Characterization of a Regulatory Mutant of an Aminoacyl-Transfer Ribonucleic Acid Synthetase in Escherichia coli K-12," J. Bacteriol., 113(3):1096-1103 (1973).
Schlesinger, S., and Nester, E. W., "Mutants of Escherichia coli with an Altered Tryosyl-Transfer Ribonucleic Acid Synthetase," J. Bacteriol., 100(1):167-175 (1969).
Gatti, D.L., and Tzagoloff, A., "Structure and Evolution of a Group of Related Aminoacyl-tRNA Synthetases," J. Mol. Biol., 218:557-568 (1991).
Tzagoloff, A., et al., "Characterization of MSM1, the Structural Gene for Yeast Mitochondiral Methionyl-tRNA Synthetase," Eur. J. Biochem., 179:365-371 (1989).
Webster, T., et al., "Specific Sequence Homology and Three-Dimensional Structural of an Aminoacyl Transfer RNA Synthetase," Science, 226:1315-1317 (1984).
Jenal, U., et al., "Isoleucyl-tRNA Synthetase of MEthanobacterium thermautotrophicum Marburg," J. Biol. Chem., 266(16):10570-10577 (1991).
Shiba, K., et al., "Human cytoplasmic isoleucyl-tRNA synthetase: Selective divergence of the anticondon-binding domain and acquisition of a new structural unit," Proc. Natl. Acad. Sci. USA, 91:7435-7439 (1994).
Shiba, K., et al., "Isolation of Higher Eukaryote Aminoacyl-tRNA Synthetase Genes by an Alignment-Guided Cross-Species PCR: Application to Human Isoleucine tRNA Synthetase," �From Programme and Abstracts, p. F.46!, 15th International tRNA Workshop, SocieteFrancaise de Biochimie et Biologie Moleculaire, Cap d'Agde, France, May 30-Jun. 4 (1993), Abstract No. 364.
Printout of a computer record of parts of a poster presented at Cap d'Agde, France, May 30-Jun. 4, 1993, 15th International tRNA Workshop, Societe Francaise de Biochimie et Biologie Molecularie.
Hong, Y., et al., Data Submission, Isoleucyl-tRNA Synthetase, Campylobacter jejuni, GenBank Accession No. U15295 (1994).
Nureki, O., et al., "Methionyl-tRNA Synthetase from an Extreme Thermophile, Thermus thermopilus HB8," J. Biol. Chem., 266 (5):3268-3277 (1991).
Mechulam, Y., et al., "Methionyl-tRNA Synthetase from Bacillus stearothermophilus: Structural and Functional Indentities with the Escherichia coli Enzyme," Nucleic Acids Research, 19(13):3673-3681 (1991).
Hartlein, M. and Madern, D., "Molecular Cloning and Nucleotide Sequence of the Gene for Escherichia coli Leucyl-tRNA Synthetase," Nucleic Acids Research, 15(24): 10199-10210 (1987).
Vander Horn, P. B., and Zahler, S. A., "Cloning and Nucleotide Sequence of the Leucyl-tRNA Sythetase Gene fo Bacillus subtilis," J. Bacteriol., 174(12):3928-3935 (1992).
Borgford, T.J., et al., "The Valyl-tRNA Synthetase from Bacilus stearothermophilus Has Considerable Sequence Homology with the Isoleucyl-tRNA Synthetase from Escherichia coli", Biochemistry, 26: 2480-2486 (1987).
Hartlein, M., et al., "Nucleotide sequence of Escherichia coli valyl-tRNA synthetase gene valS", Nucleic Acids Research, 15(21): 9081-9082 (1987).