Methods of screening for agents that delay a cell cycle and compositions comprising era and an analogue of wild-type era

BACKGROUND OF THE INVENTION
For an Escherichia coli cell to duplicate itself, three important processes must be completed. The cell must first accurately replicate its DNA. The newly formed chromosomes must then be resolved and segregated. An accurately placed septum at the midpoint of the cell must be made to ensure that each newly formed daughter cell receives a full complement of the cellular components. Over the past 30 years, studies investigating the E. coli cell cycle have identified many of the genes involved in these processes. However, identification of the internal signals that regulate these processes has been elusive.
At least 13 proteins are involved in the process of DNA replication (Kornberg and Baker, 1992.) The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety. Initiation begins at a specific site, oriC, when DnaA binds to four dnaA boxes located within oriC (Kornberg and Baker, 1992). After loading of the other proteins (including DNA polymerase III, helicase, and primase) involved in the primosomal complex, replication proceeds bidirectionally until it reaches a site located approximately 180.degree. from oriC where replication terminates (ter). Termination requires two inverted ter sequences and the action of the Tus protein (Kornberg and Baker, 1992).
The era gene was discovered by sequencing downstream of the rnc gene in E. coli (Ahnn et al., 1986). The nomenclature for this gene and protein follows: Era represents the bacterial protein, era represents the bacterial gene, ERA represents the human protein, and ERA represents the human gene. The era gene is named for the initial identification of the gene from E. coli, (for E. coli ras). The era gene is a member of the rnc-era-recO rnc operon (Takiff et al., 1989). The rnc gene encodes the protein RNaseIII, which is an RNA endonuclease that plays a role in the processing of several RNA transcripts in E. coli (Bram et al., 1980; Gegenheimer and Apirion, 1981; King et al., 1986). Examples of RNA molecules processed by RNaseIII include rRNA transcripts, in which RNaseIII provides the initial cleavage in the maturation of precursor 30S rRNA to the functional 23S rRNA and 16S rRNA molecules, and the rnc operon, in which RNaseIII cleaves the transcribed message at a RNA hairpin found in the leader region of the operon (Bram et al., 1980; Gegenheimer and Apirion, 1981; King et al., 1986). This cleavage is important for the autoregulation of the rnc operon (Bardwell et al., 1989). The era gene in E. coli encodes a GTPase that has been proposed to play roles in the cell cycle, cell division, and growth rate (Ahnn et al., 1986; Gollop and March, 1991a; Gollop and March, 1991b). The RecO protein is involved in the RecF recombination pathway (Weinstock, 1987). Of the three genes, only the era gene is required for viability in E. coli (Takiff et al., 1989).
Presently the essential function of Era is unknown. Biochemical and cell biological studies of Era have been performed to attempt to elucidate the function of Era. The Era protein autophosphorylates in a GTP-dependent manner, however the function of the phosphorylated form of the protein in vivo is unknown (Sood et al., 1994). Immunoelectron microcopy to determine where Era localizes on the membrane showed that the protein is found near regions of potential division sites in E. coli (Gollop and March, 1991 a). The Era protein binds to the membrane in a GTP-dependent manner (Lin et al., 1994). The component of the membrane Era binds has not been discovered.
Morphological analysis of a strain in which the expression of era is repressed at low temperatures showed that cells formed long filaments in the absence of the Era protein (Gollop and March, 1991b). Based on these results and the localization of Era to potential division sites Gollop and March proposed that era may be involved in cell division (Gollop and March, 1991b). However, a similar type of strain construction where era is no longer expressed at high temperatures did not cause cells to filament, thus it is unclear whether decreased era expression results in an inhibition of cell division (Lerner and Inouye, 1991).
Proteins that comprise the GTPase superfamily have been discovered in organisms ranging from Escherichia coli to humans (Bourne et al., 1991). These proteins are turned "on" by the binding of GTP and "off" by the hydrolysis of GTP to GDP, thereby acting as a molecular switch (Bourne et al., 1991). The binding of GTP causes a conformational change in the protein that allows interaction with a target molecule (Bourne et al., 1991). GTPases are involved in diverse cellular processes such as signal transduction, protein translocation, and cell cycle regulation (Bourne et al., 1991). Much attention has focused on the small molecular weight GTPase ras and members of the ras subfamily of GTPases due to the identification of ras mutations in human cancer (Marshall, 1985).
Little is known about the functions of bacterial GTPases with respect to their possible role in the regulation of the bacterial cell cycle. However, GTPases are now being shown to play important roles in the cell cycles of bacteria. Recently it has been determined that a key cell division protein in E. coli, FtsZ, is a GTPase and requires GTP to polymerize in vitro (De Boer et al., 1992; RayChaudhuri and Park, 1992; Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994). Also, the Obg protein in Bacillus subtilus is a GTPase that may play an important role in sporulation and DNA replication (Kok et al., 1994; Vidwans et al., 1995).
Era has been shown to be able to bind guanine nucleotides and is able to hydrolyze GTP to GDP (Ahnn et al., 1986; Chen et al., 1990). The protein also autophosphorylates in a GTP dependent manner in vitro (Sood et al., 1994). The function of this phosphorylated form of Era is unknown but it has been suggested that it is the active form of the protein (Sood et al., 1994). Binding of Era to the membrane has been demonstrated biochemically (Lin et al., 1994). Lin et al. have proposed that the regulation of Era binding to the membrane is determined within its GTPase activity (Lin et al., 1994).
The present invention describes the characterization of the sdgE class of suppressors. Both suppressors of this class are mutations affecting era, one of which (sdgE1) is a single point mutation within the era coding sequence. This is the first point mutation isolated within the chromosomal copy of era that affects the function of the Era protein without affecting other genes in the rnc operon. This mutation affects the GTPase activity of Era which reveals a cell cycle defect, at or near cell division.
The mechanism of cell division has been elusive in view of the lack of understanding of the roles of various genes thought to be involved in the process. In addition, the number of potential candidate drugs and agents for treating infectious diseases is vast. Accordingly, the development of a rational drug design, in addition to conventional antibiotic treatment, for treating infectious diseases has not progressed at an acceptable rate. With the continuing emergence of antibiotic-resistant pathogenic bacteria, efforts are needed to identify target genes and alternative methods of treating bacterial infectious diseases. To this end, the present invention satisfies this need by providing a method of screening for agents that delay a cell cycle. Accordingly, the present invention provides for methods of reducing or stopping the growth of infectious organisms and thus decreasing or eliminating infection. In addition, in view of the relation between bacterial era and human era, and by analogy to Ras, which Ras is mutated in several types of human cancers, the present invention also provides for a method of screening for anti-cancer agents.
SUMMARY OF THE INVENTION
The present invention is directed to a method of screening for an agent that delays a cell cycle by comparing the activity of an agent suspected of inhibiting Era with a control assay. The level of GDP resulting from the combination of the agent with Era and GTP is compared to the level of GDP of a control assay. Where GDP is reduced in the agent-Era-GTP containing assay as compared to the control assay, an agent that delays a cell cycle is identified.
In addition, another method of screening for an agent that delays a cell cycle comprises comparing the growth of a cell on medium comprising an agent suspected of delaying the cell cycle. The growth of two different cells is compared, one having a heterologous era nucleic acid sequence, and another having wild-type era nucleic acid sequence. The growth of each cell is observed following growth in the medium for an effective amount of time. Slow growth of the cell having a heterologous era sequence as compared to normal growth of the cell having wild-type era sequence results in identification of an agent that delays a cell cycle is also provided in the present invention.
An analogue of wild-type Era, having proline at amino acid 17 replaced with a basic amino acid is also an embodiment of the present invention. A vector, host cell, cell culture, protein preparation, and composition comprising the analogue are additional embodiments of the present invention.
The present invention provides methods that permit the discovery of agents that block certain cell cycles. As a result, a number of diseases may be treated with the agents discovered by the methods of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and B displays complementation of suppression by pACS3/BstXI (era.sup.+). KY2903 (dnaG2903) is temperature-sensitive at 42.degree. C. The suppression of temperature-sensitivity and slow growth at 25.degree. C. is associated with the suppressor mutations sdgE1 and sdgE15. Plasmid pACS3/BstXI, which only contains the era gene, is able to reverse suppression (yield the original dnaG2903 phenotype) in both sdgE1 and sdgE15.
FIG. 2 (Parts A-C) shows mutations affecting era in sdgE1 and sdgE15. FIG. 2A=Sequence of a portion of the era gene in sdgE1. Note the G-C transversion mutation. FIG. 2B=PCR amplification of the leader region of the rnc operon in wild-type and sdgE15. Lane 1=123 bp ladder. Lane 2=wild-type DNA. Lane3=sdgE15 DNA. FIG. 2C=Summary of the mutations affecting era that suppress dnaGts mutations. A previously identified mutation affecting era, rnc40, is also shown.
FIG. 3 exhibits suppression of the temperature sensitivity of dnaG2903 and parB by rnc40. Cells were grown at 42.degree. C. and 25.degree. C. on LB plates containing either no tetracycline or tetracycline at final concentrations of 10 .mu.g/ml or 20 .mu.g/ml.
FIG. 4 shows doubling times of RABA17 and BSP750.
FIG. 5 shows GTPase activity of wild-type Era and Era1. No protein was added to the NC (negative control lane).
FIG. 6 displays the alignment of the G1 domain, SEQ ID NOS: 24 and 25, of the GTP binding proteins Era and Human H/N/K Ras. The P17R substitution caused by the era1 mutations shown. Mutations at the analogous position in Ras (Glycine-12) have been shown to be associated with several types of human cancers.
FIGS. 7A and B exhibits the effect of pACS74 on the growth of era1 and rnc15. pACS74 contains the era1 mutation cloned into pBR322. Compare the growth of these strains with those carrying the sdgE1 and sdgE15 mutations depicted in FIG. 1.
FIGS. 8A and B show fluorescence phase microscopy results. FIG. 8A shows KY2903 (dnaG2903) and FIG. 8B displays BSP754 (dnaG2903, era1) at 42.degree. C. Cells were incubated 42.degree. C. for 2.5 hours and then prepared for microscopy as described in Materials and Methods. Magnification=2000.times..
FIGS. 9A and B exhibits fluorescence phase microscopy results. FIG. 9A displays RABA17 and FIG. 9B shows BSP750 (era1) grown at 25.degree. C. Cells were diluted 1:1000 in LB broth and grown to an A.sub.600 of 0.3. Magnification=2000.times..
FIGS. 10A and B show suppression of multiple cell cycle alleles by era1.
FIGS. 11A-D show phenotypic analysis of W3110 during recovery from inhibition of protein synthesis with chloramphenicol. Photographs were taken at different time points. FIG. 11A=0 hours, FIG. 11B=2 hours, FIG. 11C=3 hours, and FIG. 11D=4 hours during the resumption of protein synthesis. Magnification=2000.times..
FIGS. 12A-F exhibits phenotypic analysis of BSP750 (era1) during recovery from inhibition of protein synthesis with chloramphenicol. Photographs were taken at different time points. FIG. 12A=0 hours, FIG. 12B=2 hours, FIG. 12C=3 hours, FIG. 12D=4 hours, FIG. 12E=5 hours, and FIG. 12F=6 hours during the resumption of protein synthesis. Magnification=2000.times..
FIGS. 13A-F reveal the percentage of cells containing one, two, or four nucleoids in wild-type and era1 cells. FIG. 13A=W3110, one nucleoid cell. FIG. 13B=era1, one nucleoid cells. FIG. 13C=W3110, two nucleoid cells. FIG. 13D=era1, two nucleoid cells. FIG. 13E=W3110, four nucleoid cells. FIG. 13F=era1, four nucleoid cells.
FIGS. 14A and B display growth of HT120 (rnc40) with various combinations of the rnc and era genes. Each strain depicted above contains the rnc40 mutation. Genes provided by the .lambda. phage are indicated. rnc.sup.+ =wild-type rnc gene. era.sup.+ =wild-type era gene. rnc105 is a loss of function mutation in rnc. rnc*=40 bp BssH2 deletion filled in frame, inactivating the rnc gene.
FIGS. 15A-D show phenotypic analysis of rnc40 containing strains with various combinations of the rnc and era genes. FIG. 15A=BSP848 (rnc40, .lambda. vector control). FIG. 15B=BSP850 (rnc40, .lambda.-rnc.sup.+). FIG. 15C=BSP851 (rnc40, .lambda.-rnc105). FIG. 15D=BSP853 (rnc40, .lambda.-rnc*,era.sup.+). Magnification=2000.times..
FIG. 16 is a summary of the cell cycle mutations tested for suppression by era1. Mutations are listed with the process they are known to affect. Where the era gene is thought to act during the cell cycle is shown. The block (or delay) in the cell cycle caused by era1 is also shown. *=mutation was suppressed by era1.
FIG. 17 reveals heterologous genetic complementation of the tet-dependent E. coli HT 120 defective for the expression of Era by the F. tularensis era contained on the plasmid pLVS120-1. The arrow indicates the direction of transcription from a potential promoter (P) located between aspC and era. Solid lines between aspC and era, and also between era and mdh represent the noncoding sequences between the ORFs. The extent of deletions are indicated by the broken lines.
FIG. 18 displays amino acid sequence alignment of Era from F. tularensis (Ftul), C. burnetii (Cbur), B. subtilis (Bsub), E. coli (Ecol), H. influenzae (Hinf), M. leprae (Mlep), S. mutans (Smut), Synechocystis sp. (Syne), and M. genitalium (Mgen), SEQ ID NOS: 26-34, respectively. The consensus sequence, SEQ ID NO: 35, derived from this alignment is shown in the bottom line. Amino acid residues that are highly conserved are indicated by upper-case letters in the bottom line for the consensus sequence. "A" in the consensus sequence represents an amino acid residue that could be different in the nine Era sequences presented. However, "a" in the nine different Era sequences presented indicates identity with the respective amino acid residue in the consensus sequence. Amino acid residues presented as lower-case letters in the nine Era sequences shown indicate differences from the respective residues in the consensus sequence. Periods indicate gaps in the sequences. Boxes A, B, C, and D are conserved domains identified in addition to the G-1, G-3, and G-4 regions that are known to be common to all GTP-binding proteins (Bourne et al., 1991).
FIG. 19 is a nucleic acid sequence of human ERA, SEQ ID NO: 36.
FIG. 20 is an amino acid sequence of human ERA, SEQ ID NO: 37.

DETAILED DESCRIPTION OF THE INVENTION
In the context of the present invention, "Era protein moiety" is defined to be at least one of Era (bacterial protein) or ERA (human protein). Further, "era gene moiety" is defined to be at least one of era (bacterial gene) or ERA (human gene). These two definitions are solely for convenience.
The present invention is directed to a method of screening for an agent that delays a cell cycle comprising preparing a purified Era, preparing at least one agent suspected of inhibiting Era, combining said purified Era and said agent with GTP to form a Era-agent-GTP combination, measuring GDP resulting from said combination, and comparing GDP resulting therefrom with GDP resulting from a control, wherein GDP resulting from said combination is reduced compared to GDP resulting from said control thereby identifying an agent that delays a cell cycle.
In addition, a method of screening for an agent that delays a cell cycle comprising preparing a cell having a heterologous era nucleic acid sequence, preparing a cell having wild-type era nucleic acid sequence, exposing each of the cells for an effective time to an agent suspected of delaying a cell cycle, and observing the growth of each cell whereby slow growth of cell harboring a heterologous era and normal growth of the cell harboring the wild-type era result in identification of an agent that delays a cell cycle is also provided in the present invention.
In view of conserved sequences of GTPases, the cell cycle that is delayed may be selected from any cell such as any cell that is recognized as foreign to the organism which it infects. Organisms which may be infected include and are not limited to mammals, such as and not limited to humans, and any other organism from mammal to bacteria. A foreign cell may be a microorganism such as and not limited to a bacterium of the Genus Escherichia, Francisella, Salmonella, Coxiella, Streptococcus, Bacillus, Haemophilus, Mycobacterium, Mycoplasm, Pseudomonas, Synechocystis, and the like. In addition to bacterial infections, infection by other pathogens is within the scope of the invention. A foreign cell may also be a cell usually recognized by the organism as self, which self cell is mutated in era such that it no longer expresses the wild-type Era and no longer recognized as self. Examples of foreign cells of this definition include viral and fungal pathogens, cancer cells, neoplastic cells, metastasized cells, and the like. Era, an essential GTPase, is the preferable GTPase useful in the method of the present invention in identifying agents that delay the cell cycle.
Era is a protein essential to a cell cycle. For purposes of the present invention, Era represents a protein in the GTPase superfamily which is involved in the cell cycle of organisms from bacteria to mammals, including humans. While not intending to be bound by any particular theory of operation, it is believed that Era acts at about the time of cell division. "About the time of cell division" refers to at or near the point of cell division. That is, following DNA replication and nucleoid formation, wild-type Era is involved in cell division, cell cycling, and septum formation. Other GTPases have substantially similar activity. For purposes of the present invention, screening may include any one or more of the following characteristics: identifying an agent that inhibits the activity of Era, inhibits GTPase activity, inhibits GDP formation, inhibits GTP binding to Era, inhibits cell division, inhibits septum formation, arrests cell growth, and decreases infection. The word "delay" and variations thereof are used herein synonymously with inhibit, reduce, suppress, retard, slow, and suspend. Further, agents may be identified that completely inhibit cell division such that it is arrested, stopped, or blocked. Further to these definitions, in accordance with the method of screening for an agent that delays a cell cycle using a heterologous era sequence, agents which inhibit any protein, proteins including and not limited to Era, in a cell cycle may be identified. Indeed, membrane binding proteins and GTPase activation by other proteins may be identified as delaying a cell cycle. In accordance with the claimed invention, a cell in which the cell cycle is delayed is one which is unable to display cell division typically seen in that cell type under normal conditions. Accordingly, the present invention includes the identification of agents that substantially inhibit cell division. Substantial inhibition of cell division refers to less than about 1% to about 100% of the cells of a given population are inhibited. Preferably cell division is inhibited about 10%, more preferably about 20%, more preferably about 50%, even more preferably about 75%, even more preferably about 100%.
A gene coding for Era may be obtained from a cDNA or genomic library. Suitable libraries can be obtained from commercial sources such as Clontech, Palo Alto, Calif. Libraries may also be prepared using the following non-limiting examples hamster insulin-secreting tumor (HIT), mouse .alpha.TC-6, a muscle cell library, and rat insulinoma (RIN) cells. Positive clones are then subjected to DNA sequencing to determine the presence of a DNA sequence coding for Era. DNA sequencing may be accomplished using the chain termination method of Sanger et al. 1977. The DNA sequence encoding Era is then inserted into an expression vector for later expression in a host cell.
Briefly, cells are transformed using any of the methods known in the art such as those disclosed by Sambrook, et al., 1989, such as and not limited to calcium chloride transformation, rubidium chloride transformation, or electroporation. Expression vectors and host cells are selected to form an expression system capable of synthesizing Era. As used herein, expression vectors refer to any type of vector that can be manipulated to contain a nucleic acid sequence coding for Era, such as plasmid expression vectors and viral vectors. Plasmid expression vectors comprise a nucleic acid sequence of the invention operably linked with at least one expression control element such as a promoter. In general, plasmid vectors contain replicon and control sequences derived from species compatible with the host cell. To facilitate selection of plasmids containing nucleic acid sequences of the invention, plasmid vectors may also contain a selectable marker such as a gene coding for antibiotic resistance. Suitable examples include the genes coding for ampicillin, tetracycline, chloramphenicol or kanamycin resistance. Vectors including and not limited to baculovirus vectors may be used in the present invention. The vector preferably has a strong promoter that is tightly controlled and inducible. Nonlimiting examples of tightly controlled promoters include temperature sensitive promoters, such as those which increase expression of Era at a particular temperature, and pET (Novagen), which contains a T7 promoter and T7 RNA polymerase under control of a lac promoter. Strains containing T7 promoters include BL21 and HMS174. Host cells suitable for use in the invention include prokaryotic and eukaryotic cells that can be transformed to stably contain and express Era. For example, nucleic acid coding for the protein may be expressed in prokaryotic or eukaryotic host cells, including the most commonly used bacterial host cell for the production of recombinant proteins, E. coli. Other microbial strains may also be used, however, such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, various species of Pseudomonas, or other bacterial strains. Suitable expression vectors, promoters, enhancers, and other expression control elements are known in the art and may be found in Sambrook et al., 1989.
Transformed host cells containing a DNA sequence encoding Era may then be grown in an appropriate medium for the host. The cells are then grown until product accumulation reaches desired levels at which time the cells are then harvested and the protein product purified in accordance with conventional techniques. Era may be purified by any method of protein purification known to skilled artisans such as the techniques set forth herein and by Sambrook, et al., 1989 and Chen, et al., 1990. Briefly, a culture of E. coli is grown at about 37.degree. C. until reaching an A.sub.600 of about 0.4 to about 0.5 at which time the culture is shifted to 40.degree. C. and grown in LB broth, for example, for about two hours to allow for expression of Era.
Cells are then lysed and centrifuged to separate insoluble proteins from soluble proteins. The soluble, Era-containing fraction is then precipitated in ammonium sulfate in fractions of between about 5% to about 50% ammonium sulfate, about 10% to about 40% ammonium sulfate, or about 20% to about 30% ammonium sulfate. The ammonium sulfate precipitated soluble fraction is then separated by size, preferably over a column such as a Sepharose or Sephadex column, a G-25 Sepadex column, for example, will separate proteins of about 25 kD or smaller from the larger sized proteins containing Era, having a molecular weight of about 33 kD. The fraction larger than about 25 kD is then separated by charge, such as over a column, a Q-Sepharose column for example. The Era protein is then stored in 50% glycerol and used for subsequent analysis. Other suitable purification methods include, but are not limited to, SDS PAGE electrophoresis, phenylboronate-agarose, reactive green 19-agarose, concanavalin A sepharose, ion exchange chromatography, affinity chromatography, electrophoresis, dialysis and other methods of purification known in the art.
The agents tested in the method of the present invention include any agent suspected of delaying the certain cell cycle. Agents suspected of inhibiting pathogens such as bacteria or any other foreign cell may be identified by the method of the present invention. The screening method may include a cocktail of a number of agents suspected of delaying a cell cycle, such that a sample of agents containing about five to about fifty suspected agents may be tested together. The agents will be tested for their ability to inhibit the GTPase aactivity of purified Era protein. Depending on the results resulting therefrom, smaller groups of the original agents, such as groups of about two to about twenty (depending on the number of agents in the original screen), or each agent of the cocktail, may then be individually tested. In addition to in vitro testing, individual agents (or a combination of agents) that inhibit Era activity may also be tested for their ability to inhibit the growth of bacteria in vivo. Agents may be added to growth media (such as, for example, LB broth, minimal media, and the like) and the growth of bacteria may be monitored.
Era from E. coli is the preferred Era useful in the methods of the present invention. "For an effective time" refers to a period of time that would permit GTP to form GDP under normal conditions, from about 20 minutes to about 120 minutes, preferably about 60 minutes, more preferably about 30 minutes at about 35.degree. C. to about 45.degree. C., preferably about 37.degree. C.
Era, as purified according to the methods set forth above, is used in the method of the present invention. About one to about 500 picomoles, more preferably about 10 to about 400 picomoles, more preferably about 20 to about 250 picomoles, and most preferably about 40 picomoles of Era is combined with GTP of about 0.1 .mu.moles to about 100 .mu.moles, more preferably about 10 .mu.moles. ATP may be added to inhibit the GTPase activity of ATPases. The amount of agent added to the Era-GTP combination is dependent on a number of factors such as and not limited to the concentration of agent used, synergy between agents used together in a pool of agents, the dosage of agents useful against other diseases.
The methods of measuring GTPase activity, including methods of measuring GDP, resulting from the combination of Era-agent-GTP combination include any quantitative or qualitative measurement, which are readily known to those of skill in the art, and may be and are not limited to spectrophotometry, densitometry, and chromatography. That is, following the combination for an effective time of Era-agent-GTP, in which GTP may be labeled, a small sample, about one .mu.l to about five .mu.ls may be placed on PEI-cellulose for example and thin layer chromatography carried out in 0.5M KH.sub.2 PO.sub.4, 1M NaCl at room temperature. After autoradiography of the chromatogram, the spots corresponding to GTP and GDP are cut out and counted. The amount of GTP hydrolyzed is calculated. Labels include a radioactive label such as .sup.32 p, biotin, fluorescein, and ethidium bromide. Similarly, spectrophotometry and densitometry may be performed by placing a sample in a spectrophotometer or densitometer and reading the value of the sample as compared to a control in the appropriate apparatus.
Other GTPase assays include and are not limited to those that follow. GTPase hydrolysis may be monitored by measuring free phosphate as described by Welch et al., 1994. After the standard reaction using gamma-.sup.32 P GTP, the reaction is quenched with a slurry of activated charcoal in 1 mM kPi buffer (pH 7.5) which selectively removes organics including all nucleotides (GTP, GDP, GMP). The charcoal is pelleted by centrifugation and the amount of free .sup.32 PO.sub.4 is quantified by Cherenkov counting of the supernatant. Alternatively, the rate of hydrolysis may be monitored using the fluorescent GTP analog 3'-mdGTP according to the methods set forth in Methods in Enzymology, 1995 255:95-107 or Moore and Lohman, 1994. Nucleotide binding and hydrolysis is measured using a fluorescence spectrometer. Another alternative is to measure activity by immunoprecipitation of the Era-nucleotide complex as disclosed in Methods in Enzymology, 1995 255:110-125.
Complementation permits the screening of agents having cell cycle delay activity such that a cell having a nonfunctioning or null era nucleic acid sequence may be complemented by a heterologous era sequence. The heterologous era sequence may be incorporated, such as by transformation, into the cell genome or harbored on a plasmid or vector. A cell comprising the heterologous era sequence is grown for an effective time on or in a medium containing the agent suspected of having cell cycle delay activity. era genes from other organisms may be identified by identifying them in genomes from organisms whose DNA has been sequenced. Complementation may be carried out by methods known to those of skill in the art such as according to the methods set forth in the examples below and methods of Sambrook et al., 1989, and Miller, 1992. A nonfunctioning era sequence may be obtained by removing the protein from control of the natural promoter such as by splicing into the genome additional sequences that contain a promoter causing the cell to become dependent thereon. Such an example of a promoter is an antibiotic resistant promoter such as and not limited to tetracycline, ampicillin, kanamycin; arabinose, T7, lactose, .lambda. thermosensitive promoter, and the like. A library is then constructed with vectors appropriate to the size of the sequence to be cloned, the library is then screened. Growth on or in a medium includes plating or growing the cells on plates comprising solid medium or growing the cells in liquid culture medium.
Once heterologous era sequences are identified, they can be used to construct E. coli organisms that depend only on the heterologous era gene for growth. For example, the era gene from another bacterium, such as Helicobacter pylori, can be used to functionally replace the era gene in the chromosome of E. coli. Replacement of the E. coli era gene with the H. pylori era gene can be performed by complementing an era defect with the H. pylori era gene. The resulting strain is the same as a wild-type E. coli strain, except that it has the era gene from H. pylori. These two strains are then grown on culture media LB, minimal media, or the like, that contain agents and the growth of the two strains are compared. Any agent that inhibits the growth of the E. coli strain with the H. pylori era gene but not the wild-type E. coli strain is found to inhibit the H. pylori Era protein. This agent (or combination of agents) is then tested to see if it inhibits the growth of an H. pylori strain. Such an agent would then be a candidate for a treatment against H. pylori.
era is the second gene in the polycistronic rnc operon of E. coli and is flanked upstream by rnc and downstream by recO (Ahnn et al., 1986; Morrison et al., 1989; Takiff et al., 1989). Heterologous genetic complementation may be achieved in bacteria that contains a transposon insertion in the leader sequence of the rnc preventing operon expression from its natural promoter and causing conditional growth of the cell. A genomic library may be constructed, transformed into cells, and selecting for conditional growth, such as for example in the presence of ampicillin (AMP.sup.R may be encoded by a vector). A clone may be obtained that contains an insert that hybridizes to the genomic DNA of the bacterial era on Southern blots.
Heterologous and exogenous, as used herein, denote a nucleic acid sequence that is not obtained from and would not normally form a part of the genetic make-up of the cell to be transformed, in its untransformed state. Accordingly, a heterologous era sequence may be a sequence from another source, which is wild-type for that source. ""For an effective time" refers to a time period that would permit growth of wild-type cells under normal conditions, from about 24 to about 72 hours, preferably about 36 hours, more preferably about 48 hours, at about 35.degree. C. to about 40.degree. C., preferably about 37.degree. C. The preferably era nucleic acid sequence useful in the method set forth above is era from E. coli. Alternatively, depending upon the growth conditions of the cell, an effective time may include about one week to about two weeks, as well.
The era gene encodes a protein in the GTPase superfamily that has guanine-nucleotide binding and GTPase activities. While era is the preferred embodiment of the present invention, GTPases in general are also the subject of the present invention. Indeed, GTPases are known to exist in organisms from bacteria to mammals. The sequence of era (E. coli ras) gene is set forth in Ahnn et al., 1986. Amino acid 17 of mature wild-type Era, proline, is found to be replaced in era1 of the present invention with a basic amino acid, such as and not limited to arginine, lysine, and histidine. The era1 allele is the first single point mutation within the era gene isolated on the chromosome for which the function of era has been analyzed to the knowledge of the inventors. era1 affects the GTPase activity of Era. The Era1 protein expressed by the era gene revealed a decrease in GTPase activity by about four to about five fold and caused a delay in the cell cycle. Thus, Era1 elucidated the role of wild-type Era; GTPase activity resulting from wild-type Era permits normal cell division and/or a normal cell cycle.
The era1 mutation causes the substitution P17R that occurs at a position in the protein of considerable interest in GTPases. Proline-17 of Era corresponds to Gly-12 in Ras; Gly-12 has been found to be mutated in several types of human cancers (Marshall, 1985). Interestingly, proline is the only amino acid that can be placed in position 12 of ras and not cause the protein to become oncogenic (Seeburg et al., 1984). Proline 17 of era was mutated to valine and when the mutation causing this change was present on a multicopy plasmid with the chromosomal allele of era inactivated, the cells became cold-sensitive (Lerner et al., 1992). Analysis of substitutions of Glycine-12 in Ras has demonstrated that mutants at this position (including G12R) are not defective in GTP or GDP binding and that it is the intrinsic GTPase activity of the protein that is inhibited (Finkel et al., 1984). Structural analysis of Ras position 12 mutants led to the hypothesis that cleavage of the gamma phosphate of GTP was inhibited by steric hindrance caused by the bulky side chains of amino acids other than glycine (and presumably proline) at position 12 (Krengel et al., 1990). Because GTP binding domains are well conserved, it is likely that the defect in GTPase activity of Era1 is caused by a similar mechanism. The era sequence is conserved among bacteria as well, see Table 7.
Analysis of the GTPase activity of the Era1 protein showed that Era1 has a 4-5 fold decrease in GTPase activity. Era1 was able to bind GTP and GDP similar to wild-type Era, indicating that the protein was still folded properly. This defect in the GTPase activity may be due to reduced cleavage of the gamma phosphate or to a reduced ability of the protein to cycle between its GTP and GDP bound forms. Analysis of substitutions of Glycine-12 in Ras has demonstrated that mutants at this position (including G12R) are not defective in GTP or GDP binding and that it is the intrinsic GTPase activity of the protein that is inhibited (Finkel et al., 1984). Structural analysis of Ras position 12 mutants led to the hypothesis that cleavage of the gamma phosphate of GTP was inhibited by steric hindrance caused by the bulky side chains of amino acids other than glycine (and presumably proline) at position 12 (Krengel et al., 1990). Because GTP binding domains are well conserved, it is likely that the defect in GTPase activity of Era1 is caused by a similar mechanism.
The purified amino acid sequence may comprise all or part of the sequence of Era1, such that a fragment having substantially the same Era activity is included within the scope of the present invention. Fragments of the amino acid sequence of Era include a fragment comprising codon 17, a fragment comprising codon 21, and a fragment comprising each of codon 17 and codon 21. Mutations which do not substantially alter the activity of the sequence or part of the sequence are embodied by the present invention. Nucleic acids which encode the amino acid sequences of Era1 within in the scope of the present invention include cDNA, RNA, genomic DNA, sequences within these larger sequences, antisense oligonucleotides. Variations in the nucleic acid and polypeptide sequences of the present invention are within the scope of the present invention and include N terminal and C terminal extensions, transcription and translation modifications, and modifications in the cDNA sequence to facilitate and improve transcription and translation efficiency. In addition, mismatches within the sequences identified herein, which achieve the methods of the invention, such that the mismatched sequences are substantially complementary to the Era sequences identified, are also considered within the scope of the present invention. Mismatches which permit substantial complementarity to the Era sequences, such as similarity in residues in hydrophobicity, will be known to those of skill in the art once armed with the present disclosure. In addition, the sequences of the present invention may be natural or synthetic.
In addition to the Era1 protein set forth above, Era homologs, including a human ERA, different from human Ras, and Era from C. elegans, are within the scope of the invention. The human ERA nucleic acid is set forth in FIG. 19, SEQ ID NO: 36, the human ERA amino acid sequence is set forth in FIG. 20, SEQ ID NO: 37. The definition of these Eras include all of the sequence variations in set forth above for bacterial Era, including alternatively spliced sequences and fragments of the sequences which are substantially similar or have substantially the same activity of the sequences of FIGS. 19 and 20. It is the human ERA which is the preferred protein for use in detecting an agent that delays a foreign cell cycle, which agent is useful as an anti-cancer treatment.
The term "purified", when used to describe the state of the protein of the invention, refers to a protein substantially free of other cellular material including proteins not coding for Era or other materials such as those normally associated with nucleic acid in non-recombinant cells, i.e., in its "native state."
The term "purified" or "in purified form" when used to describe the state of Era protein, polypeptide, or amino acid sequence, refers to Era free, to at least some degree, of cellular material or other material normally associated with it in its native state. Preferably the protein has a purity (homogeneity) of at least about 25% to about 100%. More preferably the purity is at least about 50%.
Generally, the protein of the invention may be produced in host cells transformed with an expression vector comprising a nucleic acid sequence encoding Era. The transformed cells are cultured under conditions whereby the nucleic acid sequence coding for the Era is expressed. After a suitable amount of time for the protein to accumulate, the protein is purified from the transformed cells.
A gene coding for Era may be obtained from a cDNA or genomic DNA library. Suitable libraries can be obtained from commercial sources such as Clontech, Palo Alto, Calif. Libraries may also be prepared using the following non-limiting examples hamster insulin-secreting tumor (HIT), mouse .alpha.TC-6, muscle cells, and rat insulinoma (RIN) cells. Positive clones are then subjected to DNA sequencing to determine the presence of a DNA sequence coding for Era. DNA sequencing is accomplished using the chain termination method of Sanger et al. 1977. The DNA sequence encoding Era is then inserted into an expression vector for later expression in a host cell.
Expression vectors and host cells are selected to form an expression system capable of synthesizing Era. Vectors including and not limited to baculovirus vectors may be used in the present invention. Host cells suitable for use in the invention include prokaryotic and eukaryotic cells that can be transformed to stably contain and express Era. For example, nucleic acid coding for the protein may be expressed in prokaryotic or eukaryotic host cells, including the most commonly used bacterial host cell for the production of recombinant proteins, E. coli. Other microbial strains may also be used, however, such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescens, various species of Pseudomonas, or other bacterial strains.
Commonly used eukaryotic systems include yeast, such as Saccharomyces cerevisiae; insect cells, such as Spodoptera frugiperda; chicken cells, such as E3C/O and SL-29; mammalian cells, such as HeLa, Chinese hamster ovary cells (CHO), COS-7 or MDCK cells and the like. The foregoing list is illustrative only and is not intended in any way to limit the types of host cells suitable for expression of the nucleic acid sequences of the invention.
As used herein, expression vectors refer to any type of vector that can be manipulated to contain a nucleic acid sequence coding for Era, such as plasmid expression vectors and viral vectors. The selection of the expression vector is based on compatibility with the desired host cell such that expression of the nucleic acid encoding Era results. Plasmid expression vectors comprise a nucleic acid sequence of the invention operably linked with at least one expression control element such as a promoter. In general, plasmid vectors contain replicon and control sequences derived from species compatible with the host cell. To facilitate selection of plasmids containing nucleic acid sequences of the invention, plasmid vectors may also contain a selectable marker such as a gene coding for antibiotic resistance. Suitable examples include the genes coding for ampicillin, tetracycline, chloramphenicol or kanamycin resistance.
Suitable expression vectors, promoters, enhancers, and other expression control elements are known in the art and may be found in Sambrook et al., 1989.
Transformed host cells containing a DNA sequence encoding Era may then be grown in an appropriate medium for the host. The cells are then grown until product accumulation reaches desired levels at which time the cells are then harvested and the protein product purified in accordance with conventional techniques. Suitable purification methods include, but are not limited to, SDS PAGE electrophoresis, phenylboronate-agarose, reactive green 19-agarose, concanavalin A sepharose, ion exchange chromatography, affinity chromatography, electrophoresis, dialysis and other methods of purification known in the art.
Protein preparations, of purified or unpurified Era produced by host cells, are accordingly produced which comprise Era and other material such as host cell components and/or cell medium, depending on the degree of purification of the protein.
The era gene was sequenced in accordance with standard techniques known to those of skill in the art once armed with the present disclosure, such as Ahnn et al., 1986, and Sambrook, et al., 1989. The method of amplifying nucleic acids may be the polymerase chain reaction (PCR) using a pair of primers wherein at least one primer within the pair is selected from the group consisting of SEQ ID NOS: 9-16.
A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990. PCR is performed using a pair of primers wherein at least one primer within the pair is selected from the group consisting of SEQ ID NOS: 9-16. In addition, other methods known to skilled artisans for amplifying nucleic acids may be used in place of PCR, such as and not limited to LCR disclosed in EPA No. 320,308, Qbeta Replicase, described in PCT Application No. PCT/US87/00880, isothermal amplification methods, disclosed in Walker, G. T., et al., 1992, transcription-based amplification systems (TAS) (Kwoh D., et al., 1989, Gingeras T. R., et al., PCT Application WO 88/10315, including nucleic acid sequence based amplification (NASBA) and 3SR, Davey, C., et al., European Patent Application Publication No. 329,822, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA ("dsDNA") which may be used in accordance with the present invention. Miller, H. I., et al., PCT application WO 89/06700, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" disclosed by Frohman, M. A., 1990) and "one-sided PCR" (Ohara, O., et al., 1989).
Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and which involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA.
Era specific nucleic acids can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having a 3' and 5' sequences of non-era specific DNA and middle sequence of era specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe identified as distinctive products, generate a signal which is released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridization of a probe to an era specific expressed nucleic acid.
Still other amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, may be used in accordance with the present invention. In the former application, "modified" primers are used in a PCR like, template and enzyme dependent synthesis. The primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labelled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence.
Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide (Wu, D. Y. et al., 1989), may also be used in the amplification step of the present invention.
The following examples are illustrative but are not meant to be limiting of the invention.
EXAMPLES
Strains And Genetic Techniques
The strains used in this study are listed in Table 1. P1 transductions using P1vir were performed as described (Miller, 1972). Electroporations were done using the Gene Pulser II apparatus (Bio-Rad) as per manufacturer specifications. Cell were grown in LB broth (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl). Bactoagar (15 g) was added to LB broth to make plates. Antibiotics timentin (50 .mu.g/ml), kanamycin (30 .mu.g/ml) and tetracycline (15 .mu.g/ml) were added when necessary. Restriction enzymes were purchased from New England Biolabs and Boehringer Mannheim. Taq polymerase was purchased from Cetus.
TABLE 1______________________________________Bacterial Strains Strain Relevant Genotype Source______________________________________W3110 prototroph E. coli Genetic Stock Center, Yale University, New Haven, CT RABA17 W3110, TD1-17::.DELTA.Tn10 W3110 transduced by P1 generalized transduction as per Miller, 1989. JV53 parB linked to Cm.sup.r Versalovic (1994). Evolution of the macromolecular synthesis operon and analysis of bacterial primase. Ph. D. Thesis. Baylor College of Medicine, Houston, TX. KY2903 W3110, dnaG2903 Murakami, Y., et al., 1985. RABPARB MG1655, parB linked P1 transduction of JV53 into to Cm.sup.r MG1655 and selecting for Cmr and temperature sensitivity (parB). RAB10 W3110, parB linked Transduction of RABPARB to Cm.sup.r into W3110 with same selection as RABPARB above SDG1 W3110, dnaG2903, sdgE1 RAB2903 (KY2903 (era1), .DELTA.tn10 .DELTA.tn10, proC.sup.-) spontaneous suppressor of RAB2903 SDG15 W3110, dnaG2903, sdgE15 RAB2903 (KY2903 (rnc15), .DELTA.Tn10 .DELTA.tn10, proC.sup.-) spontaneous suppressor of RAB2903 RAB1 W3110, dnaG2903, era1 P1 transduction of W3110 into SDG1, selecting for proC.sup.+, then checked for Tc sensitivity. RAB15 W3110, dnaG2903, rnc15 P1 transduction of W3110 into SDG15, selecting for proC.sup.+, then checked for Tc sensitivity. HT120 W3110, rnc40::.DELTA.Tn10 Takiff et al., 1989 HT31 N4903, TD1-17::.DELTA.Tn10 Takiff et al., 1989 BSP750 W3110, era1, P1 transduction of W3110 TD1-17::.DELTA.Tn10 with BSP754 BSP754 W3110, dnaG2903, era1 P1 transduction of RAB1 TD1-17::.DELTA.Tn10 with HT31 BSP756 W3110, parB, era1 P1 transduction of RAB10 TD1-17::.DELTA.Tn10 with BSP750 BSP751 W3110, rnc15 P1 transduction of W3110 TD1-17::.DELTA.Tn10 with BSP755 BSP755 W3110, dnaG2903, P1 transduction of RAB15 rnc15 TD1-17::.DELTA.Tn10 with HT31 BSP757 W3110, parB, rnc15 P1 transduction of RAB10 TD1-17::.DELTA.Tn10 with BSP550 TAP106 N4956, (kil cI857.DELTA.bio) Patterson, T. A., et al., 1993. N::kan______________________________________
Identification Of era As The Gene Responsible For Suppression In The sdgE Class Of Suppressors
Two suppressors of dnaG2903, sdgE1 and sdgE15 mapped near a marker located at 58.2' on the E. coli chromosome. A shotgun library of the E. coli chromosome was electroporated into SDG1 (sdgE1) in an effort to identify plasmids that could reverse the suppression in this mutant. Transformants were selected based on their ability to form colonies at 25.degree. C. after two days and then tested for growth at 42.degree. C. Four plasmids, pBL301-1, pBL301-2, pBL301-3, and pBL301-4, were able to restore colony formation at 25.degree. C. after two days and reverse the suppression of sdgE1; that is strains harboring these plasmids could no longer grow at 42.degree. C. and could form colonies on plates at 25.degree. C. after 2 days. This demonstrated that each plasmid likely contained the gene responsible for suppression in sdgE1. All four plasmids were electroporated into SDG15 (sdgE15) and in each case suppression was reversed, suggesting that the same gene was responsible for suppression in both strains.
The map locations of pBL301-1 and pBL301-4 were determined by hybridization of the plasmids to the E. coli genome mapping Kohara filter. The Kohara filter contains 476 phage .lambda. clones that contain greater than 99% of the E. coli genome. Both plasmids hybridized to overlapping .lambda. clones 7G4 (434) and 4A12 (435), which contain DNA from the 58.0' region of the E. coli chromosome. Thus two independent methods have localized the gene responsible for suppression in this class to the 58.0' region of the chromosome. Restriction mapping of the pBL301 plasmids was performed to determine which genes were contained in the chimeric pBL plasmids. Digestion of the DNA with BamHI and EcoRI identified two common bands among all four of the plasmids that were approximately 1.4 and 1.6 kb in size. Genes encoded within this 3 kb of DNA include rnc, era, recO, lepB, and pdxJ.
To identify which of these genes was responsible for suppression of dnaG2903, plasmids containing various combinations of genes from the rnc operon were electroporated into strains SDG1 (sdgE1) and SDG15 (sdgE15). The plasmid pDLC142 contains the rnc, era, and recO genes and was able to reverse suppression in both SDG1 and SDG15 (Table 2). The plasmid pACS3, which contains only rnc and era, was also able to reverse suppression. The plasmid pACS21 contains only the rnc gene and was unable to be transformed into either SDG1 and SDG15, suggesting that overexpression of RNaseIII is lethal in both strains. pACS3/BstXI is a plasmid in which the rnc gene has been inactivated, leaving era as the only functional gene on the plasmid. This plasmid was also able to reverse suppression, clearly demonstrating that era was the gene responsible for suppression in the sdgE class of suppressors (see FIG. 1).
TABLE 2______________________________________Effect of genes in the rnc operon on suppression in sdgE1 and sdgE15 Reverse suppression ofPlasmid Genes sdgE1 sdgE15______________________________________pBR322 none No No pDLC142 rnc, era, recO Yes Yes pACS3 rnc, era Yes Yes pACS3/BstXI era Yes Yes pACS21 rnc No.sup.b No.sup.b______________________________________
Sequence Analysis Of The era Gene In sdgE1 And sdgE15
Direct DNA sequencing of PCR products was performed in accordance with the teachings of Sambrook, et al., 1989. Primers used in sequencing the rnc gene were rnc1:
TTGGCGGCATCCATTAATAGCC, SEQ ID NO: 1, nrc2: - GGCACTACGATGAGTTAATGCC, SEQ ID NO: 2, nrc3: - AACCCCATCGTAATTAATCGGC, SEQ ID NO: 3, nrc4: - TTGAGGATTAATTTCTCGACGG, SEQ ID NO: 4, nrc5: - CGTCGTGAGTCAATTCTCGCCG, SEQ ID NO: 5, nrc6: - CACTCAGGCCGACTGACCTGGC, SEQ ID NO: 6, nrc7: - GGTCGTCCATCTGCCGCTGCCG, SEQ ID NO: 7, and nrc8: - CGAGTTGTCTGCGCCTTGCGGG, SEQ ID NO: 8. -Primers used in sequencing the era gene were eral: GGTGGTTGGCACAGGTTCAAGCCG, SEQ ID NO: 9, era2: - ACATCGCCAATAGAGCTGCTCGCC, SEQ ID NO: 10, era3: - CGATCTACGTCGATACACCGGGCC, SEQ ID NO: 11, era4: - TAGATGCTTACGCACGATTGCCGC, SEQ ID NO: 12, era5: - GATGAACTTCCTCGATATCGTGCC, SEQ ID NO: 13, era6: - ATGTCTTTACGCGCTTCAATCCCG, SEQ ID NO: 14, era7: - TGATTCTCGTTGAGCGTGAAGGGC, SEQ ID NO: 15, and era8: - TTGGCAACCAGACGCACGCGCCCC, SEQ ID NO: 16.
The era gene from sdgE1 and sdgE15 bearing strains was sequenced to determine the nucleotide changes within era that are responsible for suppression of dnaG2903. The sdgE1 mutation was found to result from a single transversion point mutation at nucleotide 50 that causes a Pro-to-Arg (P17R) substitution at amino acid 17 (FIG. 2A). This nucleotide substitution destroys a BsiYI site within the era gene. Restriction analysis with BsiYI of a PCR amplified product from genomic DNA of a sdgE1 harboring strain confirmed the presence of the mutation. The P17R mutation occurs in the G1 region of the GTP-binding domain in era. The sdgE1 mutation will now be referred to as era1.
No mutation was found within the era gene in sdgE15; therefore the rnc gene and promoter region of the rnc operon were sequenced. PCR amplification of the promoter region in sdgE15 with primers rnc1 and rnc2 should yield a 370 bp fragment of DNA. However, this fragment was found to be close to 1.1 kb in sdgE15 (FIG. 2B). Partial DNA sequencing of this fragment and subsequent computer analysis identified a 100 % match to the IS1 element of E. coli. IS1 elements are 729 bp in length, accounting for the increased size of the PCR product. The insertion of this element occurred one nucleotide after the stem-loop structure in the leader region of the operon and likely exerts a polar effect on the expression of all three genes. No mutation in the rnc gene was found. As shown above by the growth characteristics of sdgE15 harboring strains, it is most likely that the polar effect on the era gene is responsible for both the suppression of dnaG2903 and slow growth at 25.degree. C. The sdgE15 mutation will now be referred to as rnc15. A summary of the mutations and their location in the rnc operon are shown in FIG. 2C.
Suppression Of dnaG2903 And parB By rnc40
A previously identified mutation known to affect the expression of era was tested for suppression of dnaG2903 and parB. The rnc40 allele is a .DELTA.Tn10 insertion into the leader region of the rnc operon (FIG. 2C). This insertion blocks transcription from the rnc promoter. Expression of the rnc operon was proposed to be driven by the tetracycline-induced promoter of the tetR gene encoded within the .DELTA.Tn10 element (Takiff et al., 1989). Strains containing the rnc40 mutation are unable to form colonies in the absence of tetracycline at low temperatures. This conditional-lethal phenotype can be corrected by plasmids containing the era gene (Takiff et al., 1989).
The rnc40 mutation was transduced using P1vir into dnaG2903 and parB at 30.degree. C. by selecting for tetracycline resistance. Presence of the rnc40 allele was confirmed by absence of colony formation at 25.degree. C. on LB plates containing no tetracycline (FIG. 3). Both dnaG2903 and parB are suppressed at 42.degree. C. by the rnc40 mutation, demonstrating that a second polar mutation affecting era expression can also suppress dnaG.sup.ts FIG. 3. Suppression also occurred in the presence of tetracycline, confirming the previous observation that expression of the tetR promoter does not reach wild-type levels (FIG. 3).
Suppression Of parB By rnc15 And era1
To test if the rnc15 and era1 mutations were allele specific, the rnc15 and era1 mutations were transduced into RAB10 (parB) creating strains BSP757 and BSP756, respectively. In both cases the temperature-sensitivity of parB was suppressed. Attempts to transduce rnc15 and era1 into dnaG3 and dnaG308 were unsuccessful presumably due to the poor growth of these dnaG alleles at the permissive temperatures.
Tests For rnc And recO Phenotypes
The rnc phenotype of strains was tested by plating .lambda. on E. coli as described (Takiff et al., 1989). A forms turbid plaques on wild-type strains but forms clear plaques on rnc.sup.- mutants (Altuvia et al., 1987). Mutations in recO are more sensitive to UV than wild-type cells (Kolodner et al., 1985). To test for a recO.sup.- phenotype, cells were streaked on an LB plate and irradiated with UV. Plates were wrapped in foil and grown for 24 hours at about 37.degree. C. at which time they were checked for growth.
The plasmids pDLC142, pACS3, pACS3/BstXI, and pACS21 (all are pBR322 derivatives) contain various combinations of the rec, era, and recO genes. The pCE31 plasmid contains the era under the control of the pL promoter of .lambda. (Chen et al., 1990). The pCE74 plasmid was constructed by subcloning the era1 mutation from pBP74 on a ClaI-BamHI fragment into pCE31. pACS74 was constructed by cloning the same ClaI-BamHI fragment from pBP74 into pACS3.
Characterization Of rnc And recO Phenotypes In era1 And rnc15
The rnc and recO phenotypes in era1 and rnc15 were determined. The bacteriophage .lambda. normally forms turbid plaques on wild-type E. coli but forms clear plaques on strains that are rnc.sup.- (Altuvia et al., 1987). Mutations in recO cause E. coli to be sensitive to UV light (Kolodner et al., 1985). Table 3 summarizes the results of the rnc and recO phenotypes in both strains. The rnc40 allele, which has been previously shown to have a polar effect on all three genes of the operon, was included as a control. When rnc15 is used as the indicator strain the phage .lambda. plates with clear plaques, indicating a defect in the rnc gene. The rnc15 allele is also UV sensitive, demonstrating that recO is also defective. The era1 allele had no effect on either rnc or recO, demonstrating that era1 is the first mutation affecting era that doesn't affect the other two genes of the rnc operon. The fact that era1 has no effect on the other two genes of the operon shows that this mutation is a good candidate for the analysis of the in vivo function of era.
TABLE 3______________________________________Characterization of rnc and recO phenotypes in era1 and rnc15 Allele RnaseIII Phenotype UV Sensitive Growth at 25.degree. C.______________________________________era+ RNaseIII+ No Yes era1 RNaseIII+ No Slow rnc15 RNaseIII- Yes Slow rnc40 RnaseIII- Yes No______________________________________
Effect Of era1 On Growth Rate
The era1 mutation was linked to a .DELTA.tn10 marker TD1-17 that is located between the glyA gene and the rnc operon and shows 65% co-transduction with the rnc gene (Takiff et al., 1989). The linked era1 mutation was then transduced into the wild-type strain W3110 creating BSP750. Growth rates were determined for RABA17 and BSP750 (era1) at 42.degree. C., 37.degree. C., and 25.degree. C. At all temperatures BSP750 (era1) grew slower than wild-type, demonstrating that era1 cells are defective for growth at all temperatures and are not cold-sensitive (FIG. 4).
GTPase Assays
Wild-type and mutant Era1 protein were purified essentially as previously described with one exception (Chen et al., 1990). Era precipitates in the 20%-30% ammonium sulphate fraction instead of the previously reported 13%-20% fraction.
GTPase assays were performed essentially as described (Chen et al., 1990). Reaction mixture (80 .mu.l): 1.5 .mu.g (40 pmol) of Era protein, 50 mM Tris-HCl, pH 8.0, 5 mM MgCl.sub.2, 1 mM dithiothreitol, 0.1 M NaCl, 0.5 mg/ml bovine serum albumin, 10 mM GTP, 1 mM ATP, 0.1 mCi [.alpha.-.sup.32 P]GTP. Reactions were incubated at 37.degree. C. for 60 minutes. Four ml of the reaction was spotted on PEI-cellulose and thin layer chromatography was performed in 0.5 M KH.sub.2 PO.sub.4, 1 M NaCl at room temperature. Quantitation of GTP and GDP in GTPase assays was performed by exposing the TLC chromatogram to a Molecular Dynamics phosphoimager and quantitating the amount of GTP and GDP using ImageQuant software.
Effect Of Era1 On The GTPase Activity Of Era
In order to determine the effect of the Era1 (P17R) substitution on the GTPase activity of Era, the mutant protein was overexpressed and purified. The pCE31 plasmid is a previously described vector used for overexpressing Era (Chen et al., 1990). A ClaI-BamHI fragment containing the Era1 mutation from pBP74 (containing the nucleotides AGG for amino acid position 17) was cloned into pCE31 creating plasmid pCE74 (era 74). Protein from both plasmids was purified as previously described (Chen et al., 1990).
The ability of Era and Era1 proteins to convert [.alpha.-.sup.32 P]GTP to [.alpha.-.sup.32 P]GDP was determined and it was found that Era1 is defective in GTPase activity (FIG. 5). The amounts of GTP and GDP were quantitated using a phosphoimager and in three independent experiments Era1 was shown to have 4-5 fold less GTPase activity than the wild-type protein. Preliminary equilibrium dialysis assays performed on Era and Era1 suggest that they bind GTP and GDP similarly.
GTPases are believed to be molecular switches, with the GTP bound form thought of as being "on" and the GDP bound form as being "off" (Bourne et al., 1991). Oncogenic mutations in ras have been shown to be in the GTP bound form longer than normal, suggesting that the "on" form of the protein causes the activated state. FIG. 6 shows an alignment of the G1 domain of Era and human Ras. It was hypothesized that if era1, like many ras mutations, is an activating mutation, then overexpression of era1 in trans would not correct the defects of rnc15 and era1 mutations and may make the cells have a more severe phenotype. On the other hand, if the cell senses the GTPase activity of Era or the phosphorylated state of Era, then overexpressing era1 may be able to reverse suppression of dnaG2903 by era1 and rnc15 and/or complement the slow growth of era1 and rnc15.
The era1 mutation was cloned into the plasmid pACS3 by replacing a ClaI-BamHI fragment of pACS3 with the ClaI-BamHI fragment from pBP74 that contains the era1 mutation in era. The resulting plasmid, pACS74, was transformed into four different strains: BSP750 (era1), BSP754 (era1, dnaG2903); BSP751 (rnc15); and BSP755 (rnc15, dnaG2903). The strains containing the plasmid were then tested for their ability to grow at different temperatures (FIG. 7). In BSP754 and BSP755, overexpression of era1 was able to reverse the suppression caused by the chromosomal era1 or rnc15 mutation. In addition, the slow growth was also complemented by overexpression of era1 in all four strains. The pACS74 plasmid was unable to suppress the temperature sensitivity of dnaG2903 or parB. These results suggest that era1 is not an activating mutation and that the cells may sense the total GTPase activity of Era or the phosphorylation state of Era in vivo.
Phenotypic Analysis Of The era1 Mutation By Combination Fluorescence-Phase Contrast Microscopy
The protocol for the preparation of cells for microscopy is a modified version of previously published methods (Hiraga et al., 1989; Grompe et al., 1991). One ml of cells was centrifuged at 7000 rpm (4000G) to pellet the cells. The pellet was then washed with 1 ml of 0.84% NaCl and centrifuged again. The resulting pellet was resuspended in 100 ml of 0.84% NaCl. Ten ml of the cells were then spread on a slide and allowed to dry. A few drops of methanol was added to fix the cells to the slide. After five minutes the slides were dipped in tap water five times and then dried at room temperature. 10 .mu.l of DAPI (5 .mu.g/ml) was added just prior to addition of the cover slip. Cells were visualized at a magnification of 2000.times. with combination fluorescence-phase contrast illumination using a Zeiss Axioskop. Excitation of the DAPI dye was achieved by using a filter unit that allowed tramsmission of emitted light of wavelengths greater than 400 nm. Photographs were taken using Ektachrome 400 color slide film (Kodak).
To determine the extent of suppression of dnaG2903 by era1, cells and nucleoids were visualized by combination fluorescence phase contrast microscopy. Strain KY2903(dnaG2903), which is inviable at 42.degree. C. forms long filaments with centrally located nucleoids at the non-permissive temperature (FIG. 8A). Analysis of strain BSP754 (dnaG2903, era1) demonstrates that era1, in addition to suppressing lethality at 42.degree. C., also corrects the phenotypic defects (filamentation and abnormal chromosome partitioning) associated with the dnaG2903 mutation (FIG. 8B).
To determine if era1 has a cell shape and/or a nucleoid morphology phenotype in a wild-type background, W3110 and BSP750 (era1) were grown in LB broth at 25.degree. C. and their phenotypes were visualized by fluo-phase microscopy. Wild-type strain W3110 consisted of cells containing either one or two nucleoids (FIG. 9A). The era1 mutant, although it had a 2.8 times longer doubling time than W3110, formed slightly longer cells of which 10% contained four segregated nucleoids (FIG. 9B). Based on previous studies of the E. coli cell cycle, wild-type cells theoretically should never contain more than two segregated nucleoids (Cooper and Helmstetter, 1968). These results suggest that era1 cells are defective in the cell cycle.
Suppression Of Multiple Cell Cycle Temperature-Sensitive Mutations By era1
The strains used in this study are listed in Table 4. Construction of specific strains with various alleles was performed by P1 transduction as described in Miller (Miller, 1972). Cells were grown in LB broth (10 g of tryptone, 10 g of NaCl, and 5 g of yeast extract) or on LB plates (LB broth+15 g of bactoagar) unless otherwise indicated. Kanamycin (30 .mu.g/ml) and tetracycline (15 .mu.g/ml) were added when necessary.
TABLE 4______________________________________Bacterial strains Strain Relevant Genotype Source______________________________________W3100 Prototroph as Table 1 KY2903 W3100, dnaG2903 as Table 1 RAB10 W3100, parB linked to Cm.sup.r as Table 1 BSP750 W3110, era1 TD1-17::.DELTA.Tn10 as Table 1 BSP754 W3110, dnaG2903, era1 as Table 1 TD1-17::.DELTA.Tn10 BSP755 W3110, parB, era1 as Table 1 TD1-17::.DELTA.Tn10 BSP610 JFL100, leuTN10; W3110, ftsZ84 ftsz84 mobilized from JFL100tn10 leu by selection for Tetracycline resistance by P1 transduction selecting for Tn10 tetracycline resistance. Leutn 10 moved into JFL100 and the final strain made leu+ by P1 transduction and selection on minimal glucose plates for leucine prototrophy. BSP832 W3110, ftsZ84, era1 P1 transduction of TD1-17:.DELTA.Tn10 BSP610 with BSP750 BSP830 W3110, .DELTA.mukB::kan Niki et al., 1991; Funnel & Cagnier, 1995. BSP831 W3110, .DELTA.mukB::kan, era1 P1 transduction of TD1-17::.DELTA.Tn10 BSP830 with BSP750 FA22 dnaB22 E. coli Stock Center RAB23 dnaB22, era1 TD1-17::.DELTA.Tn10 P1 transduction of FA22 with BSP750 RAB203 W3110, gyrB203 N4177, E. coli Stock Center RAB204 W3110, gyrB203, ear1 P1 transduction of TD1-17::.DELTA.Tn10 N4177 with BSP750 E177 W3110, dnaA177 E. coli Stock Center BSP844 dnaA177, era1 TD1-17::.DELTA.Tn10 P1 transduction of E177 with BSP750 AX727 dnaX2016 E. coli Stock Center RAB37 dnaX2016, era1 TD1-17::.DELTA.Tn10 P1 transduction of AX727 with BSP750 AX621 ftsA1882 E. coli Stock Center RAB621 ftsA1882, era1 TD1-17::.DELTA.Tn10 P1 transduction of AX621 with BSP750 AX655 ftsI2158 E. coli Stock Center JV47 mg1655, parC281, TN10 Versalovic 1994. Evolution of the macromolecular synthesis operon and analysis of bacterial primase. Ph.D. Thesis. Baylor College of Medicine, Houston, TX. BSP854 mg1655, parC281, TN10, era1, Takiff et al., 1989 A17::.DELTA.TN10:kan RAB655 ftsI2158, era1 TD1-17::.DELTA.Tn10 P1 transduction of AX655 with BSP750 HT120 W3110, rnc40::.DELTA.Tn10 set forth below BSP848 W3110, rnc40::.DELTA.Tn10 RS74 set forth below (vector control) BSP849 W3110, rnc40::.DELTA.Tn10 RS473 set forth below (rnc`) BSP850 W3110, rnc40::.DELTA.Tn10 RS463 set forth below (rnc+) BSP851 W3110, rnc40::.DELTA.Tn10 RS462 set forth below (rnc*) BSP853 W3110, rnc40::.DELTA.Tn10 RS559 set forth below (rnc+, era+) BSP853 W3110, rnc40::.DELTA.Tn10 RS560 set forth below (rnc*, era+)______________________________________
BSP830 was prepared by P1 transduction of BSP750 (era1) with BF177 as the donor for mukB::kan allele. Kan.sup.R transductants were screened for temperature sensitivity and found to be resistant up to 39.degree. C. Control constructs into W3110 produced transductants temperature sensitive above 32.degree. C. All procedures were carried out at 30.degree. C. or less. In addition, the BSP848 through 853 strains were constructed in the following manner. Strain HT120 was used to construct strains containing single copy .lambda. prophages expressing the rnc and/or the era genes under control of the native rnc operon promoter. Tetracycline was used continuously throughout manipulations at the concentration of 12.5 .mu.g/ml to insure adequate expression of era. The bacteriophage A constructs listed below were gifts from Dr. Robert Simons (UCLA) and introduced into HT120 using standard techniques (Miller, J., ed., 1992). Detection of strains that contained a single copy of the .lambda. prophage was performed as described by Powell et al.,199. The following .lambda. phages contain various combinations of wild-type and mutant rnc and era genes as follows:
.lambda.RS74=vector, imm21;
.lambda.RS473=.lambda.RS74 rnc.zeta.::lacZ
.lambda.RS463=.lambda.RS74 rnc, era'::lacZ
.lambda.RS462=.lambda.RS74 rnc*, era'::lacZ
.lambda.RS559=.lambda.RS74 rnc, era, recO'::lacZ
.lambda.RS560=.lambda.RS74 rnc*, era, recO'::lacZ
' is a nonfunctional insertion into .lambda. caused by insertion of only a part of the gene; * is the insertion of entire gene into .lambda. which is inactivated by a mutation.
The protocol for the preparation of cells for microscopy is a modified version of previously published methods (Hiraga et al., 1989; Grompe et al., 1991). One ml of cells was centrifuged at 7000 rpm to pellet the cells. The pellet was then washed with 1 ml of 0.84% NaCl and centrifuged again. The resulting pellet was resuspended in 100 .mu.l of 0.84% NaCl. Ten .mu.l of the cells were then spread on a slide and allowed to dry. A few drops of methanol were added to fix the cells to the slide. After five minutes the slides were dipped in tap water five times and then dried at room temperature. Ten .mu.L of DAPI (5 .mu.g/ml) was added just prior to addition of the cover slip. Cells were visualized at a magnification of 2000.times. with combination fluorescence-phase contrast illumination using a Zeiss Axioskop. Excitation of the DAPI dye was achieved by using a filter unit that allowed tramsmission of emitted light of wavelengths greater than 400 nm. Photographs were taken using Ektachrome 400 color slide film (Kodak).
The era1 mutation was isolated as a suppressor of dnaG2903 and has been shown to suppress a second dnaG allele, parB. Phenotypic analysis of a strain carrying the era1 mutation suggested that cells may be blocked just prior to cell division or delayed in the initiation of cell division. This observation raised the possibility that the suppression of the two dnaG alleles by era1 was indirect and might not be specific to dnaG. To test this possibility, several other temperature-sensitive mutations affecting various aspects of the cell cycle were analyzed for suppression by the era1 mutation.
The era1 allele was transduced by P1 into strains carrying temperature-sensitive mutations in genes involved in DNA replication, chromosome partitioning, chromosome segregation, and cell division. These strains were then tested for colony formation at 25.degree. C., 37.degree. C., and 42.degree. C. FIG. 10 shows the results for three mutations tested. dnaG2903 is a temperature-sensitive allele that affects the primase protein and displays its temperature-sensitivity at 42.degree. C. The era1 mutation restores the ability of dnaG2903 to grow at 42.degree. C. The mukB gene was identified in a screen for genes that are involved in chromosome segregation. A null mutation in the mukB gene (.DELTA.mukB) results in strains that can no longer form colonies at temperatures above 30.degree. C. The era1 mutation suppresses the temperature-sensitivity of .DELTA.mukB at 25.degree. C. (compare .DELTA.mukB and .DELTA.mukB, era1 at 37.degree. C.), but not at 42.degree. C. A mutation affecting ftsA (ftsA1882), a gene involved in cell division, was not suppressed by era1 and in fact is actually more temperature-sensitive (compare ftsA1882 and ftsA1882, era1 at 37.degree. C.)
Table 5 lists all of the mutations tested for suppression by era1. dnaB22, dnaG2903, parB, gyrB203, parC281, and .DELTA.mukB were all suppressed by the era1 mutation. The mutations dnaA177, dnaX36,ftsZ84,ftsA1882, and ftsI2158 were not suppressed. In addition, ftsZ84 and ftsA1882 became more temperature-sensitive in the presence of the era1 allele. To be sure that strain background differences had no effect on the suppression by era1 isogenic strains were constructed. The dnaG2903, parB, .DELTA.mukB, dna177, and ftsZ84 mutations were transduced by P1 into W3110 and again tested for suppression by era1. In all cases the strains behaved the same as in their original background. Thus era1 can suppress mutations affecting DNA replication, chromosome partitioning and segregation, but not mutations affecting cell division. This suggests that era1 acts at a point in the cell cycle after nucleoid segregation but prior to or during the cell division process.
TABLE 5______________________________________Summary of suppression of multiple cell cycle temperature-sensitive (Ts) alleles by era1. Growth on M63 media Suppression Growth plus Protein and Function by at glucose at Ts Allele of wild-type allele era1 37.degree. C. 42.degree. C.______________________________________dnaG2903 Primase. DNA Yes Yes NO replication initiation and elongation parB Primase. DNA Yes Yes No replication initiation and elongation dnaA177 DnaA. DNA No Yes No replication initiation dnaB22 Helicase. DNA Yes Yes No replication initiation and elongation dnaX2016 tau and .gamma. subunits of No Yes ND DNA polymerase III gyrB203 Subunited of DNA Yes Yes No gyrase. DNA supercoiling .DELTA.mukB MukB. Chromosome Yes No No Segregation ftsZ84 FtsZ. Septum No Yes Yes formation ftsI2158 PBP3. Septal wall No Yes ND synthesis ftsA1882 FtsA. Unknown No Yes ND function in cell division parC281 Topo.IV subunit. Yes No ND Decatenation of the chromosomes______________________________________ ND = no data.
An alternative possibility for the difference in suppression of the temperature-sensitive alleles is that perhaps the mutations that were not suppressed have a more severe effect on E. coli cell growth than the ones that were suppressed. To test this hypothesis, strains were grown at 37.degree. C. on LB plates to see if they were able to form colonies. The mutations that were not suppressed by era1 were all able to form colonies at 37.degree. C., demonstrating that they are no more defective for colony formation than the alleles that were suppressed (Table 5).
Another possibility for the difference in suppression is that merely slowing down cell growth was responsible for suppression by era1 and that the fts mutations are unable to be suppressed by this mechanism. To test this possibility the strains were plated on M63 minimal media supplemented with glucose, which will slow down the growth of the cells. The temperature-sensitivity of ftsZ84 was suppressed by growth on minimal media. Because ftsZ84 was able to form colonies at 42.degree. C. on minimal media it is unlikely that the mechanism of suppression by era1, as indicated by the absence of a complete correlation between the ability to suppress the temperature-sensitive alleles with era1 or by plating on minimal media, is merely due to a slow down of cell growth.
Analysis of Cells After Recovery From Chloramphenicol inhibition
Overnight cultures of W3110 and BSP750 (era1) were diluted 1:200 in LB broth and grown to an A.sub.600 of 0.3 at 25.degree. C. Chloramphenicol (150 mg/ml) was added to each culture to a final concentration of 300 .mu.g/ml. Cells were incubated for 2 hours in the presence of chloramphenicol. Cultures were filtered through a 0.22 .mu.M filter and washed twice with an equal volume of LB to remove the chloramphenicol. Cells were then resuspended in an equal volume of LB and grown at 25.degree. C. Samples were taken to measure A.sub.600 and for microscopy. Cells were prepared for microscopy as described above. Cultures were sampled for approximately one doubling time (W3110=105 minutes, BSP750 (era1)=300 minutes) after the resumption of growth (initial increase in A.sub.600). Photographs were taken and the number of nucleoids per cell were counted for each time point. A minimum of 120 cells was counted for each time point.
The data presented above and the observation that era1 cells contain four segregated nucleoids suggests that era acts after nucleoid segregation and prior to cell division. However, it is possible that era acts earlier in the cell cycle but the era1 phenotype is not observed until just prior to cell division. In an attempt to demonstrate where Era acts during the cell cycle, era1 cells were inhibited at a specific point in the cell cycle and their phenotypes were observed upon recovery from this inhibition.
When protein synthesis is inhibited in E. coli, cells arrest with a single nucleoid located in the center of the cell, demonstrating that nucleoid segregation requires post-replication protein synthesis (Donachie and Begg, 1989; Hiraga et al., 1990; van Helvoort and Woldringh, 1994). Ongoing rounds of DNA replication are completed and terminated in the absence of protein synthesis but new initiations do not occur. In addition to the completion of already initiated rounds of DNA replication, cells in the process of division do not require de novo protein synthesis. If era1 causes a delay at a stage in the cell cycle between nucleoid segregation and cell division, then it may be possible to see an accumulation of four nucleoid cells immediately after resumption of growth after protein synthesis inhibition. To test this hypothesis, W3110 and BSP750 (era1) were incubated with chloramphenicol (300 .mu.g/ml) for two hours at 25.degree. C. to block cells prior to nucleoid segregation. The chloramphenicol was then washed away and cells were allowed to resume growth. Both W3110 and BSP750 showed a 1.5-2 hour delay in the increase in A.sub.600 during recovery at which time A.sub.600 began to increase in both strains. A delay in the resumption of growth after chloramphenicol treatment has been previously reported (Hiraga et al., 1990). The fact that wild-type and era1 cells both began increasing A.sub.600 at the same time suggests that era is not necessary for translation, unless Era negatively regulates this process.
Samples of cells were taken from the cultures and prepared for microscopy as described above. FIGS. 11A-D shows the phenotypes of wild-type cells during recovery from chloramphenicol. After two hours of chloramphenicol inhibition, almost all of the cells contain a single centrally located nucleoid (FIG. 11A). Coinicident with the increase in A.sub.600 at 1.5-2 hours after removal of chloramphenicol, cells with two nucleoids were observed indicating that the cells had begun to segregate their nucleoids (FIG. 11B). At three and four hours after recovery W3110 consisted of an approximately equal number of one and two nucleoid cells (FIGS. 11C and 11D). These results are consistent with previous reports investigating the segregation of nucleoids during recovery from chloramphenicol inhibition (Hiraga et al., 1990; van Helvoort and Woldringh, 1994).
The recovery of BSP750 (era1) cells following inhibition of protein synthesis is shown in FIGS. 12A-F. As observed in wild-type cells, nearly all of the era1 harboring cells contain a centrally located nucleoid following chloramphenicol treatment (FIG. 12A). However, the era1 cells are on average about 1.5 to about 2 times longer than wild-type cells (compare FIG. 11A with FIG. 12A). At 2 hours after recovery from chloramphenicol treatment cells with two nucleoids were observed but another type of cell was also found that was not present in wild-type cells, those containing four segregated nucleoids (FIG. 12B). These four nucleoid cells were found three, four, and five hours after recovery but were greatly decreased in numbers six hours after recovery (compare FIG. 12C, FIG. 12D, and FIG. 12E, with FIG. 12F).
The number of nucleoids in W3110 and BSP750 (era1) cells was counted at different timepoints after the recovery from chloramphenicol treatment and the results are shown in FIG. 13. In both wild-type and era1 cells 95% of the cells contained a single nucleoid (compare FIG. 13A and FIG. 13B) at 0 hours. The population of wild-type cells gradually accumulated two nucleoid cell types until four hours after recovery at which time there were approximately equal numbers of one and two nucleoid cells (FIG. 13A and FIG. 13C). In era1 cells, four nucleoid cells began to accumulate at two hours after inhibition and increased in number to almost 60% of the culture at 3.5 hours (FIG. 13F). Interestingly, the number of four nucleoid cells decreased from a maximum at 3.5 hours to 7% at 6 hours. Also, cell length decreased during this time period by 40%. These results indicate that the era1 mutation causes a block or a delay in the cell cycle. They also suggest that wild-type era function is required between nucleoid segregation and cell division.
Analysis Of The rnc40 Mutation With Various Combinations Of The rnc And era Genes
Cells were grown in LB with tetracycline (15mg/ml) at 37.degree. C. until an A.sub.600 of 0.6-0.8 at which time they were washed with 10 volumes of LB to and diluted 1:20 in LB. Cells were grown at 25.degree. C. and samples were taken and analyzed by fluorescence-phase contrast microscopy. Cells were prepared for microscopy as described above.
Phenotypic analysis has demonstrated that cells containing the era1 mutation have four segregated nucleoids. However, era1 is not lethal at any temperature and therefore the phenotype observed may be indicative of a partial defect in era. The rnc40 mutation, which is lethal at 25.degree. C., was used to determine the phenotype of a conditional-lethal mutation affecting era.
Because the rnc40 mutation is polar on all of the downstream genes of the rnc operon, .lambda. phage containing different combinations of the rnc and era genes were transduced into HT120 (rnc40). These strains were then tested for growth at 37.degree. C. and 25.degree. C. with and without tetracycline. FIG. 14 shows the results of this test. All strains grow well on tetracycline (15 .mu.g/ml). When a strain harboring the rnc40 mutation as a chromosomal allele receives a recombinant .lambda. phage containing no genes from the rnc operon or only the rnc gene, cells are unable to form colonies at 25.degree. C. on LB plates (FIG. 14). When era is provided in trans on a recombinant .lambda. phage, cells grow well at 25.degree. C., whether or not rnc gene is present. This confirms earlier observations that the cold-sensitivity of rnc40 is due to the polar effect of the .DELTA.Tn10 insertion on the era gene.
The cell morphology of these strains was analyzed by combination fluorescence-phase contrast microscopy. Cells were grown into exponential phase and then shifted to 25.degree. C. for six hours at which time cells were prepared for microscopy. BSP848 (rnc40, .lambda. vector control) formed cells with a single nucleoid present in the middle of the cell, much like when cells are inhibited with chloramphenicol (FIG. 15A). HT120 (rnc40) looks the same as BSP848, indicating that the presence of .lambda. has no effect on the phenotype of the cells. Interestingly, strain BSP850 (rnc40, .lambda. rnc.sup.+), forms four nucleoid cells at the non-permissive temperature (FIG. 15B). BSP851, which is the same as BSP850 except that the rnc gene contains an activated rnc gene, has the same phenotype as BSP848 (FIG. 15C). BSP853 (rnc40, .lambda. rnc*, era.sup.+) which contains era as the only wild-type gene on .lambda., looked similar to wild-type cells (FIG. 15D). These results demonstrate that rnc40 is defective in chromosome partitioning at the non-permissive temperature and that this phenotype is due to a polar effect exerted on both rnc and era. In strains with a single copy of the rnc gene provided in trans to the polar rnc40 chromosomal allele on a .lambda. phage, four nucleoid cells were observed at the non-permissive temperature, suggesting that this era lethal mutation has a similar effect on the cells as the era1 mutation.
Cloning Of The F. tularensis era
era is the second gene in the polycistronic rnc operon of E. coli and is flanked upstream by rnc and downstream by recO (Ahnn et al., 1986; Morrison et al., 1989; Takiff et al., 1989). The indispensable characteristic of E. coli Era was taken advantage of in adapting the strategy of heterologous genetic complementation in an E. coli strain HT120 that contains a mini-Tn10 transposon insertion in the leader sequence of the rnc preventing operon expression from its natural promoter and causing the cell to become conditionally dependent on tetracycline and the tet promoter for growth (Takiff et al., 1992; Takiff et al., 1989). A library was constructed by ligating Bg1II-restricted genomic DNA of the F. tularensis Live Vaccine Strain (LVS) to the BamHI-digested vector pBluescriptII KS.sup.+ (Stratagene, La Jolla, Calif., USA). By transformation of this library into HT120 and selecting for tetracycline-independent growth in the presence of ampicillin (AMP.sup.R being encoded by the vector), a clone, pLVS120-1, was obtained that contained a 4-kb DNA insert. The DNA insert contained in this clone hybridizes to the genomic DNA of F. tularensis LVS on Southern blots.
Nucleotide Sequence Analysis And Heterologous Genetic Complementation Of The F. tularensis era
Results of the nucleotide sequence analysis of the 4-kb DNA insert of PLVS120-1 indicated that it contains a gene homologous to E. coli era; but unlike E. coli (Ahnn, et al. 1986; Chen et al., 1990; Takiff et al., 1989) and Coxiella burnetii (Zuber et al, 1994), rnc and recO do not flank era in F. tularensis (FIG. 17). In fact, pLVS 120-1 failed to complement rnc E. coli in two different--[1] multicopy suppression of capsule synthesis (Zuber et al., 1994) and [2] activation .lambda. N gene expression (Kameyama et al, 1991). Also, the location and organization of the F. tularensis era are remarkably different from those of the gram-positive Streptococcus mutans, where era is located downstream but in the same operon as the gene encoding diacyl glycerol kinase, dgk (Wu et al., 1995; Yamashita et al., 1993). Upstream to the F. tularensis era open reading frame (ORF), a truncated ORF encoding a protein with significant homology to aspartate aminotransferase, aspC, of Bacillus sp. (GenBank Accession No. M59430) was found. There is an intercistronic region of 114 bp between the aspC and era ORFs, containing potential promoter-like sequence. Also, immediately downstream to era, a 200 bp of noncoding sequence followed by a truncated ORF encoding a protein with homologies to malate dehydrogenase (mdh) of Bacillus israeli (GenBank Accession No. X90527) was found. Thus, the era of F. tularensis is organized, with respect to flanking genes, in a unique manner from other bacterial era genes (Table 6). Interestingly, the F. tularensis genes coding for the components of the DnaK molecular chaperone system are organized in a fashion that is characteristic of some gram-positive bacteria (Zuber et al, 1995).
TABLE 6______________________________________Relative location of era in various microorganisms Function or Gene Organism Upstream of era Downstream of era______________________________________E. coli RnaseIII (rnc) RecO (recO) C. burnetii RnaseIII (rnc) RecO (recO) H. influenzae RnaseIII (rnc) RecO (recO) S. typhimurium RnaseIII (rnc) RecO (recO) F. tularensis Aspartate amino- Malate dehydrogenase transferase (aspC) (Mdh) B. subtilis Cytidine deaminase RecO S. mutans Diacyl glycerol- No sequence kinase (dgk) available M. leprae Hypothetical ORF RecO (recO) but spaced 1500-bp downstream of era Synechocystis sp. Sigma factor Histidine kinase (cheA) Cytadherence accessory N. genitalium Hypothetical ORF protein (hmwl)______________________________________
To define the precise location and functional activity of era on the 4-kb DNA insert of pLVS120-1 and to facilitate nucleotide sequencing, unidirectional deletions were generated in vitro using Promega's Erase-a-Base system (Promega, Madison, Wis., USA). Clearly, deletions extending into the era ORF result in the loss of tetracycline-independent growth in HT120 (FIG. 17). The era complementation activity mediated by pLVS120-1 (FIG. 17) appears to be a direct result of its expression from the promoter-like sequence located between the aspC and era ORFs. The 1 ac promoter of the vector is oriented in the antisense direction for era. The deduced amino acid sequence of the F. tularensis era is highly similar to the Era proteins of B. subtilis, C. burnetii, E. coli, Haemophilus influenzae, Mycobacterium leprae, Mycoplasma genitalium, S. mutans and Synechocystis sp. (Table 7). Interestingly, the F. tularensis Era shares a higher percent identity with the Era proteins of the gram-negative E. coli and H. influenzae, and also the gram-variable C. burnetii relative to the Era proteins of the gram-positive S. mutans and B. subtilis. The Era of the more distantly related eubacterial Synechocystis sp. is also well conserved (Table 7; FIG. 18). Because F. tularensis era complements E. coli defective for the expression of Era, era may also be an essential gene in F. tularensis. The era genes of the gram-positive S. mutans (Pillutla et al., 1995) and gram-variable C. burnetii (Zuber et al., 1994) have also been shown to complement for Era function in E. coli, thus suggesting the Era probably responds to the same signal(s) in these bacteria despite their phylogenetic diversity.
TABLE 7______________________________________Relatedness of Era proteins to F. tularensis Accession F. tularensis Eraera of Number Identity (%) Similarity (%)______________________________________E. coli P06616 49.8 70.2 C. burnetii L27436 50.3 68.7 H. influenzae P43728 52.2 70.9 S. mutans P37214 42.6 66.0 B. subtilis P42182 43.4 65.5 M. leprae U00016 34.5 57.9 Synechocystis sp. D63999 36.6 61.3 M. genitalium D43967 23.7 49.8______________________________________
Computer Analysis Of Bacterial Era Protein Sequences
Results of the computer analysis of all the available bacterial Era protein sequences are presented in FIG. 18. From this analysis, four conserved domains have been identified in addition to the known GTP-binding domains G-1, G-3, and G-4 (Bourne et al, 1991). In fact, results suggest that the consensus sequence for G-1 (GRPNVGKS, SEQ ID NO: 17) and G-3 (DTPGL, SEQ ID NO: 18) domains (Bourne et al., 1991) can be extended by four and one aa, respectively (FIG. 18). A point mutation in E. coli era resulting in a change at aa position 21 from K (lysine) to R (arginine) located in G-1 domain (FIG. 18) has been shown to cause loss of complementation activity for bacterial growth (Pillutla et al, 1995). Interestingly, this K21 residue is conserved in all nine Era proteins (FIG. 18). Sood et al. (1994) have identified a tryptic peptide (ISITSR, SEQ ID NO: 19) in E. coli era from residues 33 to 38 as a site for autophosphorylation. Point mutations resulting in changes at amino acid positions S34P and 135F have been reported to cause a loss of Era complementation activity (Pillutla et al., 1995). Results presented in FIG. 18 (see box A) suggest a consensus sequence of IXITSXKXQTTR, SEQ ID NO: 20 for this autophosphorylation site. Changes in amino acid residues at P21 IT or S213P of E. coli Era also cause a loss of functional complementation (Pillutla, 1995). In fact, these residues also comprise a consensus sequence (EXPHSXAXV, SEQ ID NO: 21) indicated as box B (FIG. 18). Of the two alleles mutated in box B, only P211 is present in all nine Era protein sequences available. Another consensus sequence (SQKGIVIGKKG, SEQ ID NO: 22) representing box C in FIG. 18 is yet to be mutagenized to determine it importance. A fourth consensus sequence (LWVKVK, SEQ ID NO: 23) is represented by box D in FIG. 18. Amino acid changes at W278A or K280A of E. coli Era, however, retain complementation activity for bacterial growth (Pillutla et al., 1995). The consensus sequence domains identified in this study (boxes A, B, C, and D of FIG. 2) might prove to be useful to define functionally important regions outside the nucleotide-binding domains. A carboxyl-terminal deletion of 80 amino acid from the F. tularensis Era (FIG. 17) that includes box C and box D domains (FIG. 18) resulted in a loss of functional complementation of E. coli.
Identification of the human ERA homolog of the Escherichia coli era gene.
To identify a human homolog of the Escherichia coli era gene, the carboxy terminal portion of the Era protein was used to search for similar sequences in the Expressed Sequence Tag (EST) database using Baylor College of Medicine search launcher program and the tblastx dbest database. tblastx provides a six frame translation of ESTs of the dbest database. The COOH terminus of Era from E. coli was used such that any ESTs that contain a GTP-binding domain (found in the amino terminus of Era) would not be found, so that Era specific sequences would be identified. The EST R57504 (GenBank ID number) was identified as having sequences that when translated were similar to the carboxy terminus of the E. coli Era protein. The DNA sequence from R57504 was used to search the blastn dbest database for any sequences that were partially identical and thus overlapped with R57504. Blastn provides DNA sequences of the dbest database. This search and subsequent searches identified the following ESTs (identified below by GenBank ID number) that comprise part of the human ERA sequence: R21701, D79048, R19122, H10444, H10445, T87374, H53502, H64871, T79830, N46232, H65468, F19739, H53872, T32992, F02922, F02187, F04605, T34378, N57957, Z43596, F07179, F05941, H11840, F08384, and W98375. The EST H10444 (clone ID#47088), containing a 1.7 kb human genomic DNA insert, was purchased from Genome Systems (St. Louis, Mo.) and the DNA sequence of the insert was determined. A contig of the sequence generated from H10444, along with sequence published in the EST database from clones R21701, D79048, and R57504 identified a 1317 bp sequence that encodes for a protein of 438 amino acids (FIG. 19 and FIG. 20).
An alignment of the carboxy terminal 331 amino acids of human ERA and the full E. coli Era protein demonstrated significant identity and similarity between the two proteins. The human ERA protein shows identity with E. coli Era in all regions that have been determined to be conserved in bacterial Era proteins. An additional region of homology is also observed. Overall identity between the two proteins is 30% (having the same amino acid at a particular position) and they are 53% similar (having functionally similar amino acids at a particular position).
A 1.1 kb fragment (5') of the EST H10444, containing the human ERA sequence, was hybridized to a somatic cell hybrid panel PstI (Oncor, Inc., Gaithersburg, Md.) and was found to specifically hybridize to human chromosome 17. This 1.1 kb fragment of H10444 was then used to probe a human chromosome cosmid library to identify cosmids with which Fluorescence In Situ Hybridization (FISH) was performed. Cosmids identified as having sequence from H10444 were 36d4, 150g3, 38d3, and 78f3. FISH analysis of the cosmids demonstrated that the Human ERA gene maps to the chromosome 17q 1.2-12 region. Subsequent mapping of the mouse ERA gene using the EST H10444 to the syntenic region of chromosome 11 confirmed the mapping data. Northern analysis using a Clonetech human multiple tissue northern (MTN) blot showed that human ERA is expressed in all tissues, most highly in the heart and skeletal muscle.
The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.
Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
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__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 37 - - - - (2) INFORMATION FOR SEQ ID NO: 1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 1: - - TTGGCGGCAT CCATTAATAG CC - # - # 22 - - - - (2) INFORMATION FOR SEQ ID NO: 2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 2: - - GGCACTACGA TGAGTTAATG CC - # - # 22 - - - - (2) INFORMATION FOR SEQ ID NO: 3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 3: - - AACCCCATCG TAATTAATCG GC - # - # 22 - - - - (2) INFORMATION FOR SEQ ID NO: 4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - 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# 8: - - CGAGTTGTCT GCGCCTTGCG GG - # - # 22 - - - - (2) INFORMATION FOR SEQ ID NO: 9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 9: - - GGTGGTTGGC ACAGGTTCAA GCCG - # - # 24 - - - - (2) INFORMATION FOR SEQ ID NO: 10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 10: - - ACATCGCCAA TAGAGCTGCT CGCC - # - # 24 - - - - (2) INFORMATION FOR SEQ ID NO: 11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 11: - - CGATCTACGT CGATACACCG GGCC - # - # 24 - - - - (2) INFORMATION FOR SEQ ID NO: 12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - 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# 16: - - TTGGCAACCA GACGCACGCG CCCC - # - # 24 - - - - (2) INFORMATION FOR SEQ ID NO: 17: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 17: - - Gly Arg Pro Asn Val Gly Lys Ser 5 - - - - (2) INFORMATION FOR SEQ ID NO: 18: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 18: - - Asp Thr Pro Gly Leu 5 - - - - (2) INFORMATION FOR SEQ ID NO: 19: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 19: - - Ile Ser Ile Thr Ser Arg 5 - - - - (2) INFORMATION FOR SEQ ID NO: 20: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 20: - - Ile Xaa Ile Thr Ser Xaa Lys Xaa Gln Thr Th - #r Arg 5 - # - #10 - - - - (2) INFORMATION FOR SEQ ID NO: 21: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 21: - - Glu Xaa Pro His Ser Xaa Ala Xaa Val 5 - - - - (2) INFORMATION FOR SEQ ID NO: 22: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 22: - - Ser Gln Lys Gly Ile Val Ile Gly Lys Lys Gl - #y 5 - # - #10 - - - - (2) INFORMATION FOR SEQ ID NO: 23: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 23: - - Leu Trp Val Lys Val Lys 5 - - - - (2) INFORMATION FOR SEQ ID NO: 24: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 24: - - Phe Ile Ala Ile Val Gly Arg Pro Asn Val Gl - #y Lys Ser Thr 5 - # 10 - - Leu Leu 15 - - - - (2) INFORMATION FOR SEQ ID NO: 25: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 25: - - Lys Ile Val Val Val Gly Gly Gly Gly Val Gl - #y Lys Ser Ala 5 - # - #10 - - Leu Thr 15 - - (2) INFORMATION FOR SEQ ID NO: 26: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 346 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa 1 5 - # 10 - # 15 - - Met Lys Lys Xaa Xaa Cys Xaa Tyr Ile Ser Xa - #a Ile Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Ile Le - #u Lys Tyr Xaa Val Ser 35 - # 40 - # 45 - - Xaa Xaa Xaa Arg Xaa Pro Xaa Xaa Xaa Xaa Hi - #s Gln Xaa Xaa Thr Gly 50 - # 55 - # 60 - - Ile Lys Xaa Leu Xaa Asp Thr Xaa Phe Xaa Ty - #r Xaa Xaa Xaa Xaa Xaa 65 - #70 - #75 - #80 - - Ile Xaa Ile Lys Glu Pro Lys Ala Ile Asn Ly - #s Phe Xaa Xaa Xaa Ala 85 - # 90 - # 95 - - Xaa Thr Thr Met Phe Lys Asp Xaa Xaa Val Xa - #a Leu Xaa Xaa Xaa Xaa 100 - # 105 - # 110 - - Xaa Xaa Xaa Glu Met Gly Lys Trp Thr Glu Le - #u Glu Asp Asn Ile Val 115 - # 120 - # 125 - - Glu Xaa Xaa Lys Xaa Lys His Ser Glu Xaa Xa - #a Ile Xaa Ile Phe Xaa 130 - # 135 - # 140 - - Val Val Xaa Xaa Val Xaa Xaa Xaa Lys Lys Se - #r Leu Glu Ala Ala Met 145 1 - #50 1 - #55 1 -#60 - - Phe Xaa Glu Tyr Ile Lys Glu Lys Leu Xaa Se - #r Xaa Tyr Asp ValXaa 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Tyr Val Xa - #a Xaa Lys Xaa Xaa Xaa 180 - # 185 - # 190 - - Xaa Gln Xaa His Xaa Ile Asn Glu Xaa Glu Se - #r Arg Ile Glu Lys Leu 195 - # 200 - # 205 - - Xaa Xaa Xaa Ser Glu Tyr Phe Phe Tyr Glu Gl - #u Asp Gln Ile Xaa Xaa 210 - # 215 - # 220 - - Arg Ser Ile Lys Xaa Met Val Ala Xaa Ile Xa - #a Xaa Xaa Xaa Ile Met 225 2 - #30 2 - #35 2 -#40 - - Arg Thr Ile Gly Ser Xaa Val Xaa Tyr Gln Il - #e Xaa Xaa Xaa ValGlu 245 - # 250 - # 255 - - Xaa Asp Ser Tyr Lys Val Asp Gln Glu Lys As - #n Ile Xaa Xaa Val Tyr 260 - # 265 - # 270 - - Xaa Tyr Xaa Ser Xaa Leu Xaa Xaa Xaa Asn Xa - #a Xaa Xaa Xaa Xaa Xaa 275 - # 280 - # 285 - - Xaa Xaa Ala Xaa Xaa Ala Lys Xaa Xaa Lys Xa - #a Xaa Thr Asp Ser Xaa 290 - # 295 - # 300 - - Ile Xaa Xaa Xaa Arg Xaa Xaa Xaa Val Xaa Me - #t Gln Xaa Asn Xaa Lys 305 3 - #10 3 - #15 3 -#20 - - Thr His Xaa Xaa Xaa Xaa Ser Gly Xaa Ser Xa - #a Asp Asp Arg AlaXaa 325 - # 330 - # 335 - - Lys Ser Xaa Xaa Xaa Asp Leu Ile Xaa Xaa 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:27: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 346 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Met 1 5 - # 10 - # 15 - - Lys Pro Thr Tyr Cys Xaa Tyr Ala Xaa Xaa Il - #e Xaa Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Xaa Leu Gl - #u Xaa Xaa Xaa Ser Xaa 35 - # 40 - # 45 - - Xaa Xaa Arg Xaa Pro Xaa Xaa Xaa Xaa Tyr Gl - #n Xaa Leu Xaa Gly Val 50 - # 55 - # 60 - - Lys Xaa Phe Lys Asp Ile Xaa Val Xaa Tyr Xa - #a Xaa Xaa Xaa Xaa Xaa 65 - #70 - #75 - #80 - - Leu Xaa Ala Gly Thr Glu Arg Thr Ile Asn Ar - #g Tyr Xaa Xaa Arg Thr 85 - # 90 - # 95 - - Xaa Arg Gly Ala Leu Arg Asp Xaa Xaa Ala Xa - #a Val Xaa Xaa Ile Xaa 100 - # 105 - # 110 - - Xaa Glu Pro His Xaa Xaa Xaa Trp Glu Ser Gl - #n Xaa Ala Trp Val Leu 115 - # 120 - # 125 - - Asp Xaa Xaa Asn Xaa Lys Glu Ile Glu Xaa Xa - #a Thr Xaa Xaa Phe Xaa 130 - # 135 - # 140 - - Val Ile Xaa Xaa Val Xaa Xaa Ile Lys Asn Ar - #g Ala Glu Xaa Xaa Pro 145 1 - #50 1 - #55 1 -#60 - - Leu Xaa Glu Lys Xaa Val Ser Ser Leu Tyr Al - #a Xaa Gln Lys IleXaa 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Leu Xa - #a Xaa Lys Thr Xaa Xaa 180 - # 185 - # 190 - - Xaa Xaa Xaa Asp Gln Xaa Gly Thr Xaa Glu Gl - #n Ala Val His Gln Leu 195 - # 200 - # 205 - - Met Xaa Xaa Ser Pro Xaa Phe Tyr Phe Xaa Pr - #o Glu Gln Val Xaa Xaa 210 - # 215 - # 220 - - Arg Ser Asp Gln Xaa Met Ala Xaa Xaa Ile Xa - #a Xaa Xaa Xaa Leu Met 225 2 - #30 2 - #35 2 -#40 - - Arg Leu Leu Gly Gln Xaa Ile Xaa Tyr Xaa Le - #u Xaa Xaa Xaa ValThr 245 - # 250 - # 255 - - Leu Ile Glu Phe Arg Xaa Xaa Lys Glu Xaa Ly - #s Ile Xaa Xaa Ile Arg 260 - # 265 - # 270 - - Xaa Ser Xaa Val Xaa Trp Xaa Xaa Lys Lys Xa - #a Xaa Xaa Xaa Xaa Xaa 275 - # 280 - # 285 - - Xaa Xaa Xaa Gly Xaa Glu Arg Xaa Xaa Arg Va - #l Xaa Thr Asn Xaa Xaa 290 - # 295 - # 300 - - Leu Xaa Met Xaa Lys Trp Xaa Xaa Phe Xaa Ly - #s Arg Xaa Phe Xaa Gln 305 3 - #10 3 - #15 3 -#20 - - Xaa Xaa Xaa Xaa Xaa Xaa Ser Gly Xaa Ala Xa - #a Asn Glu Arg LeuXaa 325 - # 330 - # 335 - - Arg Glu Xaa Xaa Phe Glu Glu Xaa Xaa Xaa 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:28: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 345 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Met Thr Asn 1 5 - # 10 - # 15 - - Glu Ser Phe Lys Ser Xaa Xaa Xaa Ser Xaa Il - #e Xaa Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Xaa Xaa Xaa Xaa Phe Xaa Xaa Arg Val Ile Xa - #a Xaa Xaa Xaa Ala Xaa 35 - # 40 - # 45 - - Met Xaa Asp Xaa Pro Xaa Xaa Xaa Xaa Asn Ly - #s Val Xaa Gln Gly Val 50 - # 55 - # 60 - - Leu Xaa Thr Xaa Thr Ser Xaa Thr Xaa Phe Il - #e Xaa Xaa Xaa Xaa Ile 65 - #70 - #75 - #80 - - Xaa Lys Pro Lys His Lys Xaa Leu Gly Asp Ph - #e Xaa Met Xaa Val Xaa 85 - # 90 - # 95 - - Gln Asn Thr Leu Lys Glu Xaa Xaa Xaa Xaa Le - #u Xaa Met Ile Asn Ala 100 - # 105 - # 110 - - Glu Xaa Xaa Glu Xaa Xaa Gly Tyr Gly Lys Gl - #y Xaa Glu Phe Ile Ile 115 - # 120 - # 125 - - Glu Xaa Xaa Lys Xaa Gln Thr Met Ser Xaa Xa - #a Thr Xaa Xaa Phe Xaa 130 - # 135 - # 140 - - Ile Val Xaa Xaa Ile Xaa Xaa Ile Xaa His Pr - #o Asp Gln Xaa Xaa Leu 145 1 - #50 1 - #55 1 -#60 - - Leu Xaa Asp Glu Tyr Xaa Arg Lys Arg Tyr Pr - #o Xaa Lys Xaa IleXaa 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Xa - #a Xaa Xaa Xaa Xaa Xaa 180 - # 185 - # 190 - - Leu Glu Xaa Asn Xaa Ile Glu Thr Xaa Leu Al - #a Gln Ile Glu Ala Tyr 195 - # 200 - # 205 - - Xaa Xaa Xaa Xaa Pro Gln Phe Tyr Xaa Xaa Se - #r Asp Gln Val Xaa Xaa 210 - # 215 - # 220 - - His Pro Glu Xaa Xaa Ile Ile Xaa Xaa Leu Xa - #a Xaa Xaa Xaa Val Leu 225 2 - #30 2 - #35 2 -#40 - - His Leu Thr Arg Glu Xaa Ile Xaa Xaa Xaa Il - #e Xaa Xaa Xaa AlaXaa 245 - # 250 - # 255 - - Xaa Glu Xaa Xaa Ser Ile Lys Gly Gln Asp As - #n Gly Ser Xaa Val His 260 - # 265 - # 270 - - Val Ala Xaa Thr Xaa Val Xaa Xaa Xaa Asp Xa - #a Xaa Xaa Xaa Xaa Xaa 275 - # 280 - # 285 - - Xaa Xaa Xaa Xaa Xaa Ser Leu Xaa Xaa Glu Va - #l Xaa Lys Arg Xaa Xaa 290 - # 295 - # 300 - - Ala Xaa Xaa Xaa Ala Xaa Xaa Xaa Leu Xaa Se - #r Arg Xaa Tyr Xaa Glu 305 3 - #10 3 - #15 3 -#20 - - Xaa Xaa Xaa Xaa Gln Lys Asp Xaa Arg Asn Ly - #s Met Ser Gln XaaArg 325 - # 330 - # 335 - - Asp Phe Xaa Phe Lys Glu Asp Glu Tyr 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:29: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 346 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Met Ser 1 5 - # 10 - # 15 - - Ile Asp Lys Ser Tyr Cys Xaa Xaa Ile Xaa Xa - #a Val Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Le - #u Xaa Xaa Xaa Xaa Ser 35 - # 40 - # 45 - - Xaa Xaa Xaa Arg Xaa Ala Xaa Xaa Xaa Xaa Hi - #s Arg Xaa Val Xaa Gly 50 - # 55 - # 60 - - Ile His Xaa Glu Xaa Ala Tyr Xaa Ala Xaa Ty - #r Xaa Xaa Xaa Xaa Xaa 65 - #70 - #75 - #80 - - Leu Xaa Met Glu Glu Lys Arg Ala Ile Asn Ar - #g Leu Xaa Xaa Xaa Ala 85 - # 90 - # 95 - - Xaa Ser Ser Ser Ile Gly Asp Xaa Glu Xaa Va - #l Ile Xaa Xaa Xaa Xaa 100 - # 105 - # 110 - - Xaa Glu Xaa Xaa Gly Thr Arg Xaa Trp Thr Pr - #o Asp Xaa Glu Met Val 115 - # 120 - # 125 - - Leu Asn Xaa Xaa Lys Xaa Arg Xaa Xaa Glu Gl - #y Lys Ala Xaa Xaa Ile 130 - # 135 - # 140 - - Xaa Ala Val Xaa Xaa Val Xaa Asn Val Gln Gl - #u Lys Ala Asp Xaa Xaa 145 1 - #50 1 - #55 1 -#60 - - Pro His Leu Gln Phe Leu Ala Ser Gln Met Xa - #a Asn Xaa Leu AspIle 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Il - #e Xaa Xaa Glu Xaa Xaa 180 - # 185 - # 190 - - Xaa Xaa Thr Xaa Leu Xaa Xaa Asp Thr Ile Al - #a Ala Ile Val Arg Lys 195 - # 200 - # 205 - - His Xaa Xaa Xaa Ala Thr His His Phe Xaa Xa - #a Glu Asp Tyr Ile Xaa 210 - # 215 - # 220 - - Xaa Arg Ser Gln Xaa Xaa Met Ala Xaa Xaa Il - #e Xaa Xaa Xaa Xaa Leu 225 2 - #30 2 - #35 2 -#40 - - Met Arg Phe Leu Gly Ala Xaa Leu Xaa Tyr Xa - #a Val Thr Xaa XaaGlu 245 - # 250 - # 255 - - Xaa Xaa Glu Xaa Xaa Arg Phe Val Ser Asn Xa - #a Arg Gly Gly Tyr Xaa 260 - # 265 - # 270 - - Asp Xaa Asn Gly Leu Xaa Leu Xaa Xaa Xaa Gl - #u Gly Xaa Xaa Lys Met 275 - # 280 - # 285 - - Xaa Xaa Xaa Asn Xaa Xaa Ala Lys Ile Xaa Th - #r Xaa Xaa Ile Glu Xaa 290 - # 295 - # 300 - - Xaa Lys Xaa Met Gln Glu Met Xaa Xaa Phe Gl - #u Ala Pro Xaa His Xaa 305 3 - #10 3 - #15 3 -#20 - - Glu Xaa Xaa Xaa Xaa Xaa Xaa Ser Gly Glu Al - #a Xaa Asp Glu ArgAla 325 - # 330 - # 335 - - Xaa Arg Ser Xaa Xaa Xaa Val Asp Asp Leu 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:30: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 345 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Met Thr Glu Gln 1 5 - # 10 - # 15 - - Phe Asp Lys Thr Tyr Cys Xaa Xaa Ile Xaa Xa - #a Val Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Ile Le - #u Xaa Xaa Xaa Xaa Ser 35 - # 40 - # 45 - - Xaa Xaa Xaa Arg Xaa Ala Xaa Xaa Xaa Xaa Hi - #s Arg Xaa Val Gly Ile 50 - # 55 - # 60 - - Xaa Lys Xaa Glu Xaa Ala Tyr Xaa Glu Xaa Ty - #r Xaa Xaa Xaa Xaa Xaa 65 - #70 - #75 - #80 - - Leu Xaa Ile Glu Glu Lys Arg Ala Ile Asn Ar - #g Leu Xaa Xaa Arg Ala 85 - # 90 - # 95 - - Xaa Ser Ser Ala Ile Gly Asp Xaa Xaa Xaa Xa - #a Ile Xaa Xaa Xaa Xaa 100 - # 105 - # 110 - - Xaa Asp Xaa Xaa Gly Thr His Trp Asn Ala As - #p Xaa Glu Met Val Leu 115 - # 120 - # 125 - - Asn Xaa Xaa Lys Xaa Arg Asn Xaa Xaa Ala Ly - #s Ala Xaa Xaa Val Xaa 130 - # 135 - # 140 - - Ala Ile Xaa Xaa Val Xaa Asn Ile Lys Asn Ly - #s Asp Asp Xaa Xaa Pro 145 1 - #50 1 - #55 1 -#60 - - Phe Xaa Thr Asp Leu Ser Ser Lys Phe Xaa As - #n Xaa Ala His XaaXaa 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Ile Xa - #a Xaa Gln Arg Xaa Xaa 180 - # 185 - # 190 - - Xaa Xaa Xaa Asn Xaa Xaa His Glu Xaa Glu Ly - #s Ile Val Arg Gln Ser 195 - # 200 - # 205 - - Xaa Arg Xaa Xaa Val His His Phe Xaa Xaa Gl - #u Asp Tyr Val Xaa Xaa 210 - # 215 - # 220 - - Arg Ser Gln Xaa Xaa Met Ala Xaa Xaa Ile Xa - #a Xaa Xaa Xaa Leu Met 225 2 - #30 2 - #35 2 -#40 - - Arg Phe Thr Gly Glu Xaa Leu Xaa Tyr Xaa Va - #l Thr Xaa Xaa GluXaa 245 - # 250 - # 255 - - Xaa Glu Xaa Xaa Gln Phe Lys Asn Xaa Arg Gl - #y Thr Tyr Xaa Glu Xaa 260 - # 265 - # 270 - - Asn Gly Leu Xaa Leu Xaa Xaa Xaa Glu Gly Xa - #a Xaa Lys Met Xaa Xaa 275 - # 280 - # 285 - - Xaa Ala Gly Xaa Gln Lys Ile Xaa Thr Xaa Xa - #a Met Glu Xaa Xaa Ala 290 - # 295 - # 300 - - Xaa Met Xaa Arg Xaa Xaa Xaa Phe Asp Asn Ly - #s Xaa His Xaa Glu Xaa 305 3 - #10 3 - #15 3 -#20 - - Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Ala Xaa As - #p Glu Arg Ala XaaArg 325 - # 330 - # 335 - - Ser Xaa Xaa Xaa Met Asp Glu Xaa Xaa 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:31: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 346 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Val Asp 1 5 - # 10 - # 15 - - Met Ala Glu Phe Arg Ser Xaa Xaa Xaa Cys Le - #u Ile Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Thr Xaa Xaa Xaa Xaa Xaa Thr Xaa Ala Xaa Va - #l Xaa Thr Xaa Val Ala 35 - # 40 - # 45 - - Xaa Xaa Xaa Met Xaa Pro Xaa Xaa Xaa Xaa Hi - #s Thr Xaa Arg Gly Ile 50 - # 55 - # 60 - - Val His Arg Glu Xaa Asn Phe Xaa Ile Val Le - #u Xaa Xaa Xaa Xaa Xaa 65 - #70 - #75 - #80 - - Leu Xaa Xaa Arg Pro Arg Thr Leu Leu Gly Ly - #s Arg Leu Xaa Asp Leu 85 - # 90 - # 95 - - Val Arg Asp Thr Tyr Thr Glu Xaa Xaa Xaa Xa - #a Gly Leu Cys Ile Pro 100 - # 105 - # 110 - - Ala Asp Xaa Xaa Glu Ala Thr Xaa Xaa Gly Pr - #o Gly Xaa Arg Trp Ile 115 - # 120 - # 125 - - Val Asn Xaa Xaa Gln Ile Arg Ser Val Ala Pr - #o Lys Thr Ile Leu Val 130 - # 135 - # 140 - - Val Ile Val Thr Xaa Ile Xaa Xaa Val Pro Xa - #a Lys Asp Arg Xaa Ser 145 1 - #50 1 - #55 1 -#60 - - Ala Gln Leu Val Ala Val Ser Asp Leu Val Al - #a Asp Ser Ala XaaXaa 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Va - #l Xaa Xaa Xaa Xaa Xaa 180 - # 185 - # 190 - - Xaa Val Thr Xaa Glu Gln Xaa Asp Val Xaa Il - #e Asp Val Leu Ala Ala 195 - # 200 - # 205 - - Ala Xaa Pro Xaa Pro Ala Tyr Tyr Ser Ala Gl - #y Glu Xaa Leu Xaa Xaa 210 - # 215 - # 220 - - Glu Pro Glu Glu Leu Leu Met Ala Xaa Leu Xa - #a Xaa Xaa Xaa Ala Val 225 2 - #30 2 - #35 2 -#40 - - Leu Glu Gly Val His Asp Xaa Leu Xaa Xaa Xa - #a Leu Xaa Val XaaXaa 245 - # 250 - # 255 - - Ile Asp Glu Val Ser Pro Xaa Xaa Arg Ala Gl - #y Arg Gly Asp Leu Ile 260 - # 265 - # 270 - - Asp Val His Xaa Val Leu Tyr Xaa Xaa Xaa Pr - #o Xaa Xaa Xaa Xaa Xaa 275 - # 280 - # 285 - - Xaa Xaa Xaa Xaa Ser Xaa Ala Arg Xaa Arg Gl - #u Val Xaa Ile Ala Xaa 290 - # 295 - # 300 - - Xaa Arg Gln Xaa Xaa Lys Xaa Xaa Xaa Leu Xa - #a Thr Asn Ile Tyr Xaa 305 3 - #10 3 - #15 3 -#20 - - Asp Xaa His Xaa Asn Xaa Ala Lys Asn Xaa Gl - #n Arg Asn Pro LysGln 325 - # 330 - # 335 - - Xaa Gly Arg Xaa Xaa Phe Xaa Xaa Xaa Xaa 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:32: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 347 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa 1 5 - # 10 - # 15 - - Xaa Met Ser Phe Lys Ser Xaa Xaa Xaa Xaa Xa - #a Leu Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa His Val Me - #t Xaa Xaa Xaa Xaa Ala 35 - # 40 - # 45 - - Xaa Met Xaa Asp Xaa Ala Xaa Xaa Xaa Xaa As - #n Lys Xaa Met Gly Ile 50 - # 55 - # 60 - - Tyr Thr Xaa Asp Lys Glu Xaa Xaa Ile Val Ph - #e Ile Xaa Xaa Xaa Xaa 65 - #70 - #75 - #80 - - Ile Xaa Xaa Lys Pro Lys Thr Ala Leu Gly As - #p Phe Xaa Val Glu Ser 85 - # 90 - # 95 - - Xaa Tyr Ser Thr Leu Arg Glu Xaa Xaa Thr Va - #l Leu Xaa Met Xaa Pro 100 - # 105 - # 110 - - Ala Asp Xaa Xaa Glu Xaa Xaa Lys Arg Gly Ly - #s Gly Xaa Asn Met Ile 115 - # 120 - # 125 - - Ile Glu Xaa Xaa Arg Xaa Lys Xaa Xaa Ala Al - #a Lys Val Xaa Xaa Ile 130 - # 135 - # 140 - - Xaa Val Ile Xaa Xaa Ile Xaa Xaa Val His Xa - #a Pro Asp Gln Xaa Xaa 145 1 - #50 1 - #55 1 -#60 - - Glu Gln Xaa Asp Asp Phe Arg Asn Gln Met Xa - #a Asp Xaa Gln XaaXaa 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Il - #e Xaa Xaa Xaa Xaa Xaa 180 - # 185 - # 190 - - Xaa Leu Gln Xaa Asn Xaa Xaa Ser His Xaa Va - #l Asp Leu Leu Val Asp 195 - # 200 - # 205 - - His Xaa Glu Xaa Xaa Phe Gln Tyr Phe Pro Al - #a Asp Gln Xaa Ile Xaa 210 - # 215 - # 220 - - Xaa His Pro Glu Xaa Xaa Leu Val Xaa Xaa Me - #t Xaa Xaa Xaa Xaa Val 225 2 - #30 2 - #35 2 -#40 - - Leu Leu Leu Thr Arg Glu Xaa Ile Xaa Xaa Xa - #a Val Xaa Val XaaXaa 245 - # 250 - # 255 - - Ile Asp Xaa Xaa Ser Met Xaa Ala Arg Asp Xa - #a Glu Thr His Lys Ile 260 - # 265 - # 270 - - His Xaa Arg Xaa Thr Xaa Met Xaa Xaa Xaa As - #p Xaa Xaa Xaa Xaa Xaa 275 - # 280 - # 285 - - Ile Xaa Xaa Xaa Xaa Xaa Ala Met Xaa Xaa Ly - #s Xaa Xaa Gln Met Xaa 290 - # 295 - # 300 - - Xaa Arg Xaa Xaa Xaa Leu Met Xaa Xaa Leu Xa - #a Asp Lys Xaa Tyr Xaa 305 3 - #10 3 - #15 3 -#20 - - Glu Thr Xaa Xaa Xaa Xaa Xaa Lys Asn Xaa Ar - #g Xaa Lys Lys LeuAsp 325 - # 330 - # 335 - - Xaa Ala Asp Phe Xaa Xaa Asn Lys Lys Glu Ty - #r 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:33: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 347 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: - - Met Asp Ile Pro Asn Thr Thr Ala Thr Ile Al - #a Thr Ile Pro Gln Ala 1 5 - # 10 - # 15 - - Pro Ala Gly Phe Arg Ser Xaa Xaa Xaa Xaa Xa - #a Val Xaa Xaa Xaa Xaa 20 - # 25 - # 30 - - Xaa Xaa Xaa Xaa Xaa Xaa Met Xaa Gln Xaa Va - #l Xaa Xaa Xaa Xaa Ala 35 - # 40 - # 45 - - Xaa Xaa Xaa Pro Val Ala Xaa Xaa Xaa Xaa As - #n Arg Leu Gln Gly Ile 50 - # 55 - # 60 - - Ile Thr Xaa Pro Ser Ser Xaa Xaa Ile Xaa Le - #u Leu Xaa Xaa Xaa Xaa 65 - #70 - #75 - #80 - - Ile Xaa Xaa Lys Pro His His Glu Leu Gly Ar - #g Val Leu Val Xaa Asn 85 - # 90 - # 95 - - Xaa Ile Gln Ala Ile His Ser Xaa Xaa Xaa Va - #l Val Xaa Leu Xaa Xaa 100 - # 105 - # 110 - - Xaa Asp Ser Ser Ala Thr Leu Xaa Xaa Gly Ar - #g Gly Xaa Arg Phe Val 115 - # 120 - # 125 - - Val Asp Leu Xaa Xaa Xaa Gln Lys Thr Asp Gl - #y Xaa Xaa Xaa Xaa Val 130 - # 135 - # 140 - - Val Gly Leu Xaa Xaa Gln Xaa Xaa Xaa Xaa Xa - #a Xaa Gln Gln Pro Pro 145 1 - #50 1 - #55 1 -#60 - - Asp Gln Arg Glu Glu Leu Asn Ala Ser Tyr Gl - #u Thr Leu Thr XaaAsn 165 - # 170 - # 175 - - His Gly Trp Pro Cys Phe Xaa Lys Xaa Xaa Ph - #e Xaa Xaa Xaa Xaa Xaa 180 - # 185 - # 190 - - Xaa Leu Thr Xaa Glu Gly Leu Ser Asn Phe Gl - #n Ser Ala Leu Glu Ala 195 - # 200 - # 205 - - Arg Xaa Asp Pro Xaa Pro Tyr Tyr Tyr Pro Xa - #a Asp Xaa Leu Val Xaa 210 - # 215 - # 220 - - Xaa Gln Pro Glu Xaa Xaa Ile Met Ala Xaa Le - #u Xaa Xaa Xaa Gln Leu 225 2 - #30 2 - #35 2 -#40 - - Leu Leu Leu Thr Arg Gln Xaa Val Xaa Xaa Xa - #a Val Xaa Ile AlaXaa 245 - # 250 - # 255 - - Xaa Xaa Ile Glu Lys Val Xaa Xaa Xaa Glu Xa - #a Thr Pro Glu Arg Thr 260 - # 265 - # 270 - - Asn Val Phe Xaa Ala Xaa Thr Xaa Xaa Xaa Gl - #y Xaa Xaa Xaa Xaa Xaa 275 - # 280 - # 285 - - Ile Xaa Xaa Gln Xaa Xaa Ser Met Xaa Gln Al - #a Xaa Xaa Thr Ala Xaa 290 - # 295 - # 300 - - Xaa Gln Gln Xaa Gln Lys Xaa Xaa Xaa Ile Se - #r Gly Asp Xaa Tyr Xaa 305 3 - #10 3 - #15 3 -#20 - - Lys Xaa Phe Xaa Xaa Xaa Glu Pro Lys Xaa Ar - #g Gln Ser Arg GlnGln 325 - # 330 - # 335 - - Xaa Leu Glu Phe Xaa Xaa Arg Val Glu Glu Xa - #a 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:34: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 346 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa 1 5 - # 10 - # 15 - - Met Lys Val Leu Lys Xaa Xaa Xaa Xaa Gly Va - #l Leu Xaa Pro Thr Xaa 20 - # 25 - # 30 - - Ala Xaa Xaa Xaa Xaa Xaa Ile Xaa Phe Xaa Hi - #s Asn Asp Asp Ser Leu 35 - # 40 - # 45 - - Met Val Xaa Ser Met Asn Asn Xaa Xaa Leu Le - #u Ser Xaa Ser Thr Glu 50 - # 55 - # 60 - - Val Ile Asn Gln Ala Asn Lys Asn Ile Val Ph - #e Ile Xaa Val Xaa Xaa 65 - #70 - #75 - #80 - - Phe Thr Glu Lys Lys His Ser Asn Tyr Glu Xa - #a Leu Ile Thr Xaa Glu 85 - # 90 - # 95 - - Ile Arg Lys Ala Leu Ser Gln Ile Xaa Val Le - #u Leu Leu Xaa Xaa Xaa 100 - # 105 - # 110 - - Xaa Arg Ser Asp Gln Asn Xaa Xaa Xaa Asn Ly - #s Ile Glu Phe Leu Lys 115 - # 120 - # 125 - - Thr Gln Leu Gln Gln Xaa Lys Arg Tyr Gln As - #n Leu Thr Arg Ile Phe 130 - # 135 - # 140 - - Xaa Ile Asn Lys Phe His Gln Xaa Ser Leu Se - #r Glu Val Asn Lys Ala 145 1 - #50 1 - #55 1 -#60 - - Ile Ile Leu Glu Glu Phe Lys Pro Gln Xaa Ly - #s Thr Ile Xaa XaaIle 165 - # 170 - # 175 - - Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Le - #u Xaa Xaa Xaa Xaa Leu 180 - # 185 - # 190 - - Phe Asp Lys Asn Leu Phe Trp Ser Ile Phe Ly - #s Gln Val Glu Leu Arg 195 - # 200 - # 205 - - Tyr Asn Xaa Xaa Xaa Ile Phe Arg Lys Asp Il - #e Asn Phe Ile Asp Ala 210 - # 215 - # 220 - - Asn Asn Asp Asp Xaa Lys Ile Leu Xaa Gly Le - #u Xaa Xaa Gln Ile Ile 225 2 - #30 2 - #35 2 -#40 - - Phe Tyr Cys Lys Asn Xaa Ile Xaa Xaa Xaa Il - #e Xaa Xaa Arg IleGlu 245 - # 250 - # 255 - - Xaa Ile Glu Lys Ser Phe Asn Lys Glu Lys As - #n Leu Leu Lys Ile His 260 - # 265 - # 270 - - Leu Xaa Xaa Val Xaa Ser Xaa Pro Lys Leu Xa - #a Xaa Xaa Lys Xaa Ile 275 - # 280 - # 285 - - Xaa Xaa Xaa Asn Ala Glu Met Ile Xaa Ala Xa - #a Xaa Ile Ala Thr Xaa 290 - # 295 - # 300 - - Lys Lys Leu Xaa Xaa Xaa Asn His Phe Asp Cy - #s Asp Ile Phe Ile Asp 305 3 - #10 3 - #15 3 -#20 - - Ile Phe Xaa Xaa Thr Glu Xaa Xaa Xaa Xaa Xa - #a Xaa Lys Gln LysXaa 325 - # 330 - # 335 - - Pro Val Tyr Ser Phe Leu Ser Lys Xaa Xaa 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO:35: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 348 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa 1 5 - # 10 - # 15 - - Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Val Ala Il - #e Xaa Gly Arg Pro Asn 20 - # 25 - # 30 - - Val Gly Lys Ser Thr Leu Leu Asn Xaa Leu Xa - #a Gly Gln Lys Ile Xaa 35 - # 40 - # 45 - - Ile Thr Ser Xaa Lys Xaa Gln Thr Thr Arg Xa - #a Xaa Ile Xaa Xaa Xaa 50 - # 55 - # 60 - - Xaa Xaa Thr Xaa Gly Xaa Xaa Gln Xaa Ile Xa - #a Val Asp Thr Pro Gly 65 - #70 - #75 - #80 - - Xaa His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Met Asn Lys Xaa 85 - # 90 - # 95 - - Ala Xaa Xaa Xaa Xaa Xaa Xaa Met Asn Lys Xa - #a Ala Xaa Xaa Xaa Xaa 100 - # 105 - # 110 - - Xaa Xaa Val Asp Leu Ile Xaa Phe Val Val Xa - #a Xaa Xaa Xaa Xaa Xaa 115 - # 120 - # 125 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa 130 - # 135 - # 140 - - Leu Xaa Xaa Xaa Xaa Xaa Lys Asp Xaa Xaa Xa - #a Xaa Xaa Xaa Leu Leu 145 1 - #50 1 - #55 1 -#60 - - Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Phe Xaa GluXaa 165 - # 170 - # 175 - - Xaa Xaa Xaa Xaa Xaa Xaa Val Pro Xaa Xaa Xa - #a Ser Ser Ala Xaa Xaa 180 - # 185 - # 190 - - Xaa Xaa Xaa Xaa Gly Xaa Asn Val Xaa Xaa Le - #u Xaa Xaa Xaa Xaa Xaa 195 - # 200 - # 205 - - Xaa Xaa Leu Pro Glu Gly Xaa Xaa Xaa Xaa Xa - #a Pro Xaa Xaa Xaa Xaa 210 - # 215 - # 220 - - Thr Asp Xaa Xaa Xaa Arg Phe Xaa Xaa Ser Gl - #u Xaa Ile Arg Glu Lys 225 2 - #30 2 - #35 2 -#40 - - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Xaa Pro Hi - #s Ser Xaa Ala XaaVal 245 - # 250 - # 255 - - Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Glu Xaa Xaa Xaa Xaa 260 - # 265 - # 270 - - Xaa Xaa Ile Xaa Ala Xaa Ile Xaa Val Glu Ar - #g Xaa Ser Gln Lys Gly 275 - # 280 - # 285 - - Ile Val Ile Gly Lys Lys Gly Xaa Xaa Leu Ly - #s Xaa Ile Gly Xaa Xaa 290 - # 295 - # 300 - - Ala Arg Xaa Asp Ile Glu Xaa Leu Xaa Xaa Xa - #a Gly Xaa Xaa Val Xaa 305 3 - #10 3 - #15 3 -#20 - - Leu Xaa Leu Trp Val Lys Val Lys Xaa Xaa Tr - #p Xaa Asp Xaa XaaXaa 325 - # 330 - # 335 - - Xaa Leu Xaa Xaa Leu Gly Tyr Xaa Xaa Xaa Xa - #a Xaa 340 - # 345 - - - - (2) INFORMATION FOR SEQ ID NO: 36: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1317 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 36: - - GAGGTGGCTG CCCCCAGCTG GCGCGGGGCT AGGCTTGTTC AATCGGCGTT AA -#GAGTCTGG 60 - - CAGGTGGGCC CTCATGTCGC GAGGGAGCGG GTGATCCCTT TTTCCTCACT CT -#TAGGCTTC 120 - - CAACGGAGGT GCGTGTCCTG CGTCGCGGGG TCCGCTTTCT CTGGTCCCCG CT -#TGGCCTCG 180 - - GCTTCTCGCA GTAATGGCCA GGGCTCTGCC CTGGACCACT TCCTCGGATT CT -#CTCAGCCC 240 - - GACAGTTCGG TGACTCCTTG CGTCCCCGCG GTGTCCATGA ACAGAGATGA GC -#AGGATGTC 300 - - CTCTTGGTCC ATCACCCTGA TATGCCTGAG AATTCCCGGG TCCTACGAGT GG -#TCCTCCTG 360 - - GGAGCCCCGA ATGCAGGGAA GTCAACACTC TCCAACCAGC TACTGGGCCG AA -#AGGTGTTC 420 - - CCTGTTTCCA GGAAGGTGCA TACTACTCGC TGCCAAGCTC TGGGGGTCAT CA -#CAGAGAAG 480 - - GAGACCCAGG TGATTCTACT TGACACACCT GGCATTATCA GTCCTGGTAA AC -#AGAAGAGG 540 - - CATCACCTGG AGCTCTCTTT GTTGGAAGAT CCATGGAAGA GCATGGAATC TG -#CTGATCTT 600 - - GTTGTGGTTC TTGTGGATGT CTCAGACAAG TGGACACGGA ACCAGCTCAG CC -#CCCAGTTG 660 - - CTCAGGTGCT TGACCAAGTA CTCCCAGATC CCTAGTGTCC TGGTCATGAA CA -#AGGTAGAT 720 - - TGTTTGAAGC AGAAGTCAGT TCTCCTGGAG CTCACGGCAG CCCTCACTGA AG -#GTGTGGTC 780 - - AATGGCAAAA AGCTCAAGAT GAGGCAGGCC TTCCACTCAC ACCCTGGCAC CC -#ATTGCCCC 840 - - AGCCCAGCAG TTAAGGACCC AAACACACAA TCTGTGGGAA ATCCTCAGAG GA -#TTGGCTGG 900 - - CCCCACTTCA AGGAGATCTT CATGTTGTCA GCCCTAAGCC AGGAGGATGT GA -#AAACACTA 960 - - AAGCAATACC TTCTGACACA GGCCCAGCCA GGGCCCTGGG AGTACCACAG TG -#CAGTCCTC 1020 - - ACTAGCCAGA CACCAGAAGA GATCTGTGCC AACATTATCC GAGAGAAGCT CC -#TAGAACAC 1080 - - CTGCCCCAGG AGGTGCCTTA CAATGTACAG CAGAAGACAG CAGTGTGGGA GG -#AAGGACCA 1140 - - GGTGGGGAGC TGGTTATCCA ACAGAAGCTT CTGGTGCCCA AAGAATCTTA TG -#TGAAACTC 1200 - - CTGATTGGTC CGAAGGGCCA CGTGATCTCC CAGATAGCAC AGGAGGCAGG CC -#ATGACCTC 1260 - - ATGGACATCT TCCTCTGCGA TGTTGACATC CGCCTCTCTG TGAAGCTCCT CA - #AGTGA 1317 - - - - (2) INFORMATION FOR SEQ ID NO: 37: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 438 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 37: - - Glu Val Ala Ala Pro Ser Trp Arg Gly Ala Ar - #g Leu Val Gln Ser Ala 1 5 - # 10 - # 15 - - Leu Arg Val Trp Gln Val Gly Pro His Val Al - #a Arg Glu Arg Val Ile 20 - # 25 - # 30 - - Pro Phe Ser Ser Leu Leu Gly Phe Gln Arg Ar - #g Cys Val Ser Cys Val 35 - # 40 - # 45 - - Ala Gly Ser Ala Phe Ser Gly Pro Arg Leu Al - #a Ser Ala Ser Arg Ser 50 - # 55 - # 60 - - Asn Gly Gln Gly Ser Ala Leu Asp His Phe Le - #u Gly Phe Ser Gln Pro 65 - # 70 - # 75 - #80 - - Asp Ser Ser Val Thr Pro Cys Val Pro Ala Va - #l Ser Met Asn Arg Asp 85 - # 90 - # 95 - - Glu Gln Asp Val Leu Leu Val His His Pro As - #p Met Pro Glu Asn Ser 100 - # 105 - # 110 - - Arg Val Leu Arg Val Val Leu Leu Gly Ala Pr - #o Asn Ala Gly Lys Ser 115 - # 120 - # 125 - - Thr Leu Ser Asn Gln Leu Leu Gly Arg Lys Va - #l Phe Pro Val Ser Arg 130 - # 135 - # 140 - - Lys Val His Thr Thr Arg Cys Gln Ala Leu Gl - #y Val Ile Thr Glu Lys 145 - #150 155 - # 160 - - Glu Thr Gln Val Ile Leu Leu Asp Thr Pro Gl - #y Ile Ile Ser Pro Gly 165 - # 170 - # 175 - - Lys Gln Lys Arg His His Leu Glu Leu Ser Le - #u Leu Glu Asp Pro Trp 180 - # 185 - # 190 - - Lys Ser Met Glu Ser Ala Asp Leu Val Val Va - #l Leu Val Asp Val Ser 195 - # 200 - # 205 - - Asp Lys Trp Thr Arg Asn Gln Leu Ser Pro Gl - #n Leu Leu Arg Cys Leu 210 - # 215 - # 220 - - Thr Lys Tyr Ser Gln Ile Pro Ser Val Leu Va - #l Met Asn Lys Val Asp 225 2 - #30 2 - #35 2 -#40 - - Cys Leu Lys Gln Lys Ser Val Leu Leu Glu Le - #u Thr Ala Ala LeuThr 245 - # 250 - # 255 - - Glu Gly Val Val Asn Gly Lys Lys Leu Lys Me - #t Arg Gln Ala Phe His 260 - # 265 - # 270 - - Ser His Pro Gly Thr His Cys Pro Ser Pro Al - #a Val Lys Asp Pro Asn 275 - # 280 - # 285 - - Thr Gln Ser Val Gly Asn Pro Gln Arg Ile Gl - #y Trp Pro His Phe Lys 290 - # 295 - # 300 - - Glu Ile Phe Met Leu Ser Ala Leu Ser Gln Gl - #u Asp Val Lys Thr Leu 305 3 - #10 3 - #15 3 -#20 - - Lys Gln Tyr Leu Leu Thr Gln Ala Gln Pro Gl - #y Pro Trp Glu TyrHis 325 - # 330 - # 335 - - Ser Ala Val Leu Thr Ser Gln Thr Pro Glu Gl - #u Ile Cys Ala Asn Ile 340 - # 345 - # 350 - - Ile Arg Glu Lys Leu Leu Glu His Leu Pro Gl - #n Glu Val Pro Tyr Asn 355 - # 360 - # 365 - - Val Gln Gln Lys Thr Ala Val Trp Glu Glu Gl - #y Pro Gly Gly Glu Leu 370 - # 375 - # 380 - - Val Ile Gln Gln Lys Leu Leu Val Pro Lys Gl - #u Ser Tyr Val Lys Leu 385 3 - #90 3 - #95 4 -#00 - - Leu Ile Gly Pro Lys Gly His Val Ile Ser Gl - #n Ile Ala Gln GluAla 405 - # 410 - # 415 - - Gly His Asp Leu Met Asp Ile Phe Leu Cys As - #p Val Asp Ile Arg Leu 420 - # 425 - # 430 - - Ser Val Lys Leu Leu Lys 435__________________________________________________________________________

Number	Name	Date
4683195	Mullis et al.	Jul 1987
4683202	Mullis	Jul 1987
4800159	Mullis et al.	Jan 1989

Number	Date	Country
0 320 308 A2	Jun 1989	EPX
0 329 822 A2	Aug 1989	EPX
2 202 328	Sep 1988	GBX
WO 8810315	Dec 1988	WOX
WO 8906700	Jul 1989	WOX
WO 8909284	Oct 1989	WOX
WO 8909283	Oct 1989	WOX

Methods of screening for agents that delay a cell cycle and compositions comprising era and an analogue of wild-type era

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