WO1998027819A1 - dapE GENE OF HELICOBACTER PYLORI AND dapE- MUTANT STRAINS OF $i(HELICOBACTER PYLORI) - Google Patents

dapE GENE OF HELICOBACTER PYLORI AND dapE- MUTANT STRAINS OF $i(HELICOBACTER PYLORI) Download PDF

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WO1998027819A1
WO1998027819A1 PCT/US1997/024147 US9724147W WO9827819A1 WO 1998027819 A1 WO1998027819 A1 WO 1998027819A1 US 9724147 W US9724147 W US 9724147W WO 9827819 A1 WO9827819 A1 WO 9827819A1
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Martin J. Blaser
Mikio Karita
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Vanderbilt University
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Abstract

The invention provides the dapE gene of Helicobacter pylori and H. pylori dapE- mutants and to methods of using the mutants to express foreign genes and immunize against foreign agents. The dapE gene can consist of the nucleotide sequence defined in SEQ ID NO:1. Nucleic acids of the gidA gene and ORF2 or H. pylori are provided. Examples of these nucleic acids can be found in SEQ ID NO:3 and SEQ ID NO:5, respectively. Having provided these nucleic acids, hybridizing nucleic acids in accord with the description of hybridizing nucleic acids of dapE are also provided.

Description

dapE GENE OF HELICOEΛCTER PYLORI AND dapE" MUTANT STRAINS OF HELICOBACTER PYLORI
This work was supported by NIHR01DK 50837. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
Field of the Invention.
The invention pertains to the dapE gene of Heli cobacter pyl ori and H. pylori dapK mutants and to methods of using the mutants to express foreign genes and immunize against foreign agents.
Background Art
Heli cobacter pyl ori are gram negative enteric bacteria that colonize the human gastric mucosa and cause gastritis and peptic ulcer disease (6,11,15) and are implicated in malignant neoplasms of the stomach
(5,26,30,37). Thus, there exists a need for a method of treating and preventing H. pyl ori infection.
The present invention meets these needs by providing the dapE gene of H. pyl ori and conditionally lethal mutants of H. pyl ori which can be used to express foreign proteins and to immunize against H. pyl ori infection.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows physical maps of recombinant pBluescript plasmids containing gidA, dapE, and orf2. pAKl contains the 3' region of orf2 plus approximately 4kb downstream. pAK2 contains a 5kb EcoRI fragment that includes gidA, dapE , and orf2. Boxes and arrows beneath the plasmids represent the location of the genes and the presumed direction of translation, and km represents a cassette encoding kanamycin-resistance . Arrowheads with numbers represent sites of oligonucleotide primers used in PCR. Restriction endonuclease cleavage sites: Ba ( BamHI ) , Be (Bell), N ( del), E ( EcoRI ) , and H (JJindlll) .
Figure 2 shows the nucleotide and deduced amino acid sequences of the region on the H. pyl ori chromosome including gidA, and dapE, and orf2. SEQ ID NO: 7 defines the nucleotide sequence shown in Figure 2. The sequence of gidA, dapE and orf2, and 579bp upstream and 595bp downstream are shown. The 1866bp gidA commences at nucleotide 580 and ends at nucleotide 2445. The 1167bp dapE commences at nucleotide 2456 and ends at nucleotide 3622. The 753bp orf2 commences at nucleotide 3703 and ends at nucleotide 4455. The deduced amino acid sequence is shown beneath the nucleotides. A potential ribosome- binding site (Shine-Dalgarno sequence) and putative promoter elements (-35 and -10 sequences) are indicated. An open reading frame, tentatively called ORF1, which is deduced to be translated in the opposite orientation from gi dA begins at nucleotide 483. An open reading frame, tentatively called ORF3, which is deduced to be translated in the opposite orientation from orf2 ends at nucleotide 4655.
Figure 3 shows alignment of the deduced amino acid sequences of the gi dA (Panel A) and dapE (Panel B) products in E. coli and H. infl uenzae and the respective H. pyl ori homologs. To optimize the alignments, gaps were introduced when necessary. The vertical lines between residues indicate identity whereas two dots represents a conservative substitution.
DETAILED DESCRIPTION OF THE INVENTION
Nucleic Acids
An isolated dapE gene of Heli cobacter pyl ori is provided. "Isolated" means a nucleic acid is separated from at least some of other components of the naturally occurring organism, for example, the cell structural components and/or other genes. The isolation of the nucleic acids can therefore be accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids (24). It is not contemplated that the isolated nucleic acids are necessarily totally free of non-nucleic acid components, but that the isolated nucleic acids are isolated to a degree of purification to be useful in a clinical, diagnostic, experimental, or other procedure such as gel electrophoresis, Southern or dot blot hybridization, or PCR. A skilled artisan in the field will readily appreciate that there are a multitude of procedures which may be used to isolate the nucleic acids prior to their use in other procedures. These include, but are not limited to, lysis of the cell followed by gel filtration or anion exchange chromatography, binding DNA to silica in the form of glass beads, filters or diatoms in the presence of high concentration of chaotropic salts, or ethanol precipitation of the nucleic acids.
The nucleic acids of the present invention can include positive and negative strand RNA as well as DNA and is meant to include genomic and subgenomic nucleic acids found in the naturally occurring organism. The nucleic acids contemplated by the present invention include double stranded and single stranded DNA of the genome, complementary positive stranded cRNA and mRNA, and complementary cDNA produced therefrom and any nucleic acid which can selectively or specifically hybridize to the isolated nucleic acids provided herein.
The dapE gene can consist of the nucleotide sequence defined in SEQ ID NO:l. Other examples of the dapE gene of H. pyl ori can be found in any H. pyl ori isolate. Although there may be small differences (e.g., point mutations) among the dapE genes of H. pylori strains, these differences, if any, do not prevent the isolation and sequencing or other uses of this gene from other H. pyl ori strains. Thus, primers from the present dapE sequence can be used to amplify dapE from any sample in which it occurs, and oligonucleotide segments of the exemplified dapE gene can be used to probe a sample for the presence of the H. pyl ori dapE ox, more generally, H. pyl ori . It may be preferable to use slightly longer primers than the standard primers of 17 nucleotides, for example, primers of approximately 25 nucleotides.
The dapE gene can be distinguished from other nucleic acids, because of its conserved genomic location. Particularly, dapE is flanked upstream by gidA and downstream by orf2. This conserved location also makes obtaining dapE from other H. pylori strains routine and predictable. For example, primers that hybridize with the highly conserved gi dA and orf2 can be used to amplify dapE from any sample in which it occurs. Similarly, a primer that hybridizes with one or the other of the highly conserved gidA and orf2 can be paired with a primer from the exemplified dapE to amplify dapE (or a segment of it) from any sample in which it occurs. Additionally, since the position of this gene in the H. pylori genome is known, it can be mutated in any strain, according to the methods taught herein.
DapE-encoding nucleic acids can be isolated from H. pyl ori ) using any of the routine techniques. For example, a genomic DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest using one of the present dapE nucleic acids as a probe. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA) . Furthermore, genomic DNA can be isolated from an H. pyl ori strain and screened using one of the present dapE nucleic acids as a probe. Once isolated, the dapE nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid. General methods are set forth in Sambrook et al . (24).
A H. pyl ori- speci fic nucleic acid fragment of the dapE gene is provided. For example, the fragment can consist of the nucleotide sequence of the 1.1 kb dapE- specific fragment, further described in the Examples. Other examples can be obtained routinely using the methods taught herein and in the art.
A nucleic acid that encodes a naturally occurring DapE protein of Heli cobacter pyl ori and hybridizes with the nucleic acid of SEQ ID N0:1 under the stringency conditions of about 16 hrs at about 65°C, about 5x SSC, about 0.1% SDS, about 2x Denhardt ' s solution, about 150 g/ml salmon sperm DNA with washing at about 65°C, 30 min, 2x, in about 0. lx SSPE/0.1% SDS is provided. Alternative hybridization conditions include 68°C for about 16 hours in buffer containing about 6X SSC, 0.5% sodium dodecyl sulfate, about 5X Denhardt ' s solution and about lOOμg salmon sperm DNA, with washing at about 60 °C in about 0.5X SSC (Tummuru, M.K.R., T. Cover, and M.J. Blaser (24).
A nucleic acid probe that hybridizes with the nucleic acid of SEQ ID NO:l under either of the above described stringency conditions can be used to identify dapE in other strains of H. pyl ori .
A nucleic acid primer that hybridizes with the nucleic acid of SEQ ID NO:l under the polymerase chain reaction conditions of 35 cycles of 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min, with a terminal extension at 72°C for 10 min. These conditions can be used with the relevant primers to identify dapE, including interstrain variants. Examples of these primers are described in the Examples .
The selectively hybridizing nucleic acids of the invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99% complementarity with the segment and strand of the sequence to which it hybridizes. The nucleic acids can be at least 15, 18, 20, 25, 50, 100, 150, 200, 300, 500, 750, 1000, 2000, 3000 or 4000 nucleotides in length. Thus, the nucleic acid can be an alternative coding sequence for the H. pylori dapE, or can be used as a probe or primer for detecting the presence H pylori . If used as primers, the invention provides compositions including at least two nucleic acids which selectively hybridize with different regions so as to amplify a desired region. Depending on the length of the probe or primer, it can range between 70% complementary bases and full complementarity with the segment to which it hybridizes. For example, for the purpose of diagnosing the presence of H. pylori , the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes ( H. pylori DNA from a sample) should be at least enough to exclude hybridization with a nucleic acid from related bacterium. Thus, a nucleic acid that selectively hybridizes with a H. pylori DapE coding sequence will not selectively hybridize under stringent conditions with a nucleic acid for a DapE of another species, and vice versa. The degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid. It should also be clear that a selectively hybridizing nucleic acid will not hybridize with nucleic acids encoding unrelated proteins.
Nucleic acids of the gidA gene and ORF2 of H. pyl ori are provided. Examples of these nucleic acids can be found in SEQ ID NO: 3 and SEQ ID NO: 5, respectively.
Having provided these nucleic acids, hybridizing nucleic acids in accord with the description of hybridizing nucleic acids of dapE are also provided. The nucleic acids of the invention ( dapE, gidA and ORF2, and their fragments) can also be in a composition such as a genetic construct, which includes other nucleic acids such as origins of replication, promoters, other protein coding sequences, etc. The composition can also, or alternatively, contain compounds, such as non-nucleic acid marker molecules attached to the nucleic acids.
Vectors The DapE-encodmg nucleic acid and selectively hybridizing nucleic acids of the invention can be in a vector suitable for expressing the nucleic acid. The nucleic acid in a vector can be in a host suitable for expressing the nucleic acid.
A plasmid comprising a nucleic acid encoding a functional DapE protein of H. pylori is provided. The plasmid can further comprise a nucleic acid encoding a non-H. pyl ori (foreign) protein. The foreign protein encoded can be immunogenic, antigenic, or enzymatic proteins of bacteria, virus, fungus or parasite. For example the protein can be from Salmonella ( Salmonella enteπ tidi s) , Shigell a species, Yersima, enterotoxigenic and enterohemorrhagic E. coli , Mycobacteri um tuberculosis, Streptococcus pyogenes, Borda tella pertussi s, Bacill us anthraci s, P. fal ciparum, human immunodeficiency virus, respiratory syncytial virus, influenza virus, histoplasma capsula tum or other infectious organisms.
Other foreign proteins include sperm antigens for use to immunize against sperm as a form of birth control. Also contemplated are lmmunomodulating proteins to treat against inflammatory diseases and cytotoxic proteins to treat against malignancies. Likewise, the foreign protein need not be an antigen, but can instead be protein of the host. In this embodiment the H. pylori mutant strain is used to express a host protein in vi vo to provide or augment an activity of the host protein. Thus, the H. pyl ori dapET mutant can include an insulin gene for use in a method of delivering insulin to diabetics or it can include a gene for TNF for modulating or augmenting the host response to stress. In each case the amount of DAP provided to the host can be used to control the amount of the protein that is expressed. As noted elsewhere in the application, the H. pyl ori dapK strain can also have other introduced attenuating mutations or it can be a naturally occurring vacuolating toxin" strain.
The plasmids of the present invention can be any of the well know plasmids used in bacteria. For example, the plasmid can include a 1.5 kb Hindlll fragment cloned into the polylinker of a pUC vector. One such vector has a kanamycin resistance cassette inserted into this hybrid clone and is called pHPl .
Another shuttle vector that can be used as an example of the type of construct that would be useful, was that described by Schmitt et al . (Mol. Gen. Genetics. 1995.
248:563-472). In the case described, a cryptic H. pylori plasmid, pHel was ligated with an E. coli replicon to prepare an E. coli-H. pylori shuttle vector. Schmitt et al . cloned the H. pyl ori recA gene into the shuttle vector to create pDH38 which was introduced into an H. pylori strain by natural transformation. This vector was shown to complement the recA deficiency on a parental strain. Vectors other than plasmids that can be used in H. pylori include transducing bacteriophage .
Mutant H. pylori A purified mutant strain of H. pylori that does not express a functional DapE protein is provided. The mutant can either not express DapE or express a non-functioning
DapE. Such a mutant is also referred to herein as a dapE' mutant. Mutations to the dapE gene on the H. pylori chromosome that result in a dapE' mutant can be made by insertion, deletion or internal modification.
In one example, the mutant H. pylori strain is obtained by making an insertion substitution mutation in the coding sequence for the DapE as described in the Examples. Since the present invention provides the nucleic acid encoding DapE, other methods of mutating the coding sequence of DapE can be used to obtain other mutant strains as contemplated herein.
One example of the dapE~ mutant strain is deposited with the American Type Culture Collection, 12301 Parklawn Drive Rockville, MD 20852 on December 6, 1996 under accession no. ATCC 55897. This example was made according to one method of making such mutants taught in the Examples .
Additional mutants can be prepared, for example, by inserting a nucleic acid in the dapE gene or deleting a portion of the dapE gene so as to render the gene nonfunctional or protein produced in such low amounts that the organism dies in the absence of DapE supplementation. Furthermore, by providing the nucleotide sequence for the nucleic acid encoding the DapE, the present invention permits the making of specific point mutations having the desired effect. The deletion, insertion or substitution mutations can be made in either the regulatory or coding region to prevent transcription or translation or to render the transcribed and translated product nonfunctional .
One such approach to the construction of a deletion or insertion mutant is via the Donnenberg method (Donnenberg and Kaper Infect . Immun . 4310-4317, 1991) . A deletion in dapE is created by deleting a restriction fragment and religating the clone. This mutant is cloned into suicide vector pILL570. The sacB gene of Bacillus subtilis is also cloned into the suicide vector to provide a conditionally lethal phenotype. This construct is transformed into H. pyl ori by electroporation, and transformants selected by spectinomycin resistance. The merodiploid strain which contains the suicide vector and the mutated version of the dapE gene are exposed to sucrose to directly select for organisms that have undergone a second recombination, resulting in the loss of the vector. These and other well known methods of making mutations can be applied to the nucleic acids provided herein to obtain other desired mutations. Included are insertional mutagenesis as described in reference 8, as well as linker-scanning mutagenesis (46) and site-directed mutagenesis (47) .
Non-isogenic mutants are also within the scope of the invention. For example, a live attenuated H. pylori that is also a dapE" mutant according to the present invention, is provided. A dapE~recA~ mutant strain is constructed, for example, by insertion mutation of both the dapE and recA genes, according to the methods taught herein for dapE and in U.S. Patent No. 5,434,253, issued on July 18, 1995 for recA. A dapE'recA-cagA- mutant strain is constructed, for example, by insertion mutation of all three genes, according to the methods taught herein, in U.S. Patent No. 5,434,253 and in U.S. Application Serial No. 08/053,614, which describes the generation of a cagA (referred to therein as tagA) mutant. A dapE'recA'vacA' mutant strain is constructed, for example, by insertion mutation of all three genes, according to the methods taught herein. A dapE' recA cagA~vacA~ mutant strain is constructed, for example, by insertion mutation of all four genes, according to the methods taught herein and the above cited patents and patent applications. Furthermore, a mutation in dapE combined with any one or more of the above or other mutations can be made. Any of the well known methods of mutating a gene can be used in the present invention to generate H. pylori mutant strains. The strains can be tested as provided for immunogenicity, conditional lethality, vacuolating activity, etc.
The dapE~ mutant strain can also have in its chromosome a nucleic acid encoding a foreign protein. Briefly, this can be accomplished by inserting SacB in the chromosome as above, then using a suicide vector that has the foreign gene replacing dapE and flanked by gidA and orf2. The suicide vector is then transformed into H. pyl ori using natural transformation conditions such as described in U.S. Patent No. 5,434,253, and in U.S. Application Serial Nos. 08/053,614, 08/215,928 and
08/200,232. After transformation the H. pyl ori is then grown on sucrose-containing plates. This selects for replacement of the sacB insert by the foreign gene. The foreign protein encoded can be as described above.
The dapE~ mutant strain can also contain a plasmid comprising a nucleic acid encoding a foreign protein. The foreign protein encoded can be as described above.
The dapE~ mutant H. pyl ori can be transformed with, and thus, contain a plasmid comprising a nucleic acid encoding a functional DapE protein. This can be accomplished using natural transformation such as is described in U.S. Patent No. 5,434,253, and in U.S. Application Serial Nos. 08/053,614, 08/215,928 and 08/200,232. Under the appropriate conditions the dapE~ mutant H. pylori can express a functioning dapE protein due to trans complementation by the dapE gene on the plasmid. These are the typical growth conditions for H. pyl ori , such as are described in the example.
The mutant H. pyl ori containing a plasmid comprising a nucleic acid encoding a functional DapE protein can include in its chromosome a foreign nucleic acid encoding a foreign protein. A method of inserting a foreign gene in the H. pylori chromosome is described above. The mutant H. pyl ori containing a plasmid comprising a nucleic acid encoding a functional DapE protein wherein the plasmid further comprises a nucleic acid encoding a foreign protein is provided.
Having provided the present conditionally lethal dapE~ mutant and method of generating such a conditionally lethal mutation in the genome of H. pylori , the present invention leads predictably to other conditionally lethal mutations in H. pyl ori . Such mutants include those with mutations in genes essential for cell wall synthesis or particular biochemical pathways for which the product can be used to complement the mutation.
The dapE~ mutant strain containing a foreign protein in either its chromosome or in a plasmid can be used as an expression system for expressing the foreign protein.
Foreign Gene Expression Method
The present invention provides methods to deliver foreign antigens via the present dapET mutant H. pyl ori strain engineered to also contain nucleic acids either in the chromosome or on a plasmid (e.g., an H. pylori shuttle plasmid) which express the foreign antigen of interest.
A method is provided for maintaining the expression of a foreign antigen in Heli cobacter pyl ori , comprising: a) transforming a mutant Heli cobacter pyl ori that does not produce of functioning DapE protein with a plasmid comprising a nucleic acid encoding a functional DapE protein and comprising a nucleic acid encoding the foreign protein; and b) maintaining the mutant Heli cobacter pylori from step a under conditions that permit expression of the foreign protein. Furthermore, by maintaining the H. pylori from step a in medium without added L-DAP, only the H. pyl ori that contain the plasmid will survive.
Screening for H. pylori mutants
The present dapET mutant can be used in a method of screening for and selecting H. pylori mutants. Because of the attributes of the dapET mutant, mutants can be selected without the use of antibiotic resistance. This makes the mutants safer for use in humans, because there is no risk of introducing an organism that is resistant to antibiotics, and thus, not removable by antibiotic treatment. Briefly, dapE is deleted from the chromosome or insertionally mutated and the stain is grown in L-DAP- containing medium. A shuttle vector (plasmid) is constructed that encodes DapE to complement the dapE mutation in the chromosome and encodes a foreign protein. The plasmid is transformed into the dapET H. pyl ori , which are grown on medium without L-DAP. This selects for maintenance of the plasmid.
It should be noted that in the methods and compositions described, the foreign antigen gene can either be in the chromosome replacing dapE or elsewhere in the chromosome. The foreign gene can be on the plasmid that encodes DapE, so that dapET strains can be used as hosts for any number (multiple copies) or type (cocktail) of gene that may be on any number of plasmids. These strains are expected to survive well since functional DapE is provided. They can be attenuated elsewhere, for example in recA or in vacA so that they are less toxic.
Immunization Methods
An immunogenic amount of the dapET mutant H. pylori in a pharmaceutically acceptable carrier can be used as a vaccine .
A method is provided for immunizing a subject against infection with Heli cobacter pyl ori , comprising: a) administering to the subject the dapE~ mutant strain of H. pylori ; b) supplementing the subject's diet with diaminopimelic acid (DAP) in the form of L-DAP or meso DAP (a mixture of L-DAP and D-DAP) to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the strain; and c) ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject. The immunization methods described herein comprise administering to the subject an immunogenic amount of mutant H. pylori in a pharmaceutically acceptable carrier for the mutant. The length of time to which the subject is exposed to the mutant strain, i.e., the length of time L-DAP supplementation is provided, will typically be from a few days (2 or 3) up to a few weeks (2 or 3) . The exact time course may vary from individual to individual and can be verified by tests for the presence of an immune response such as indicated by the presence of antibodies against the H. pylori in a tissue sample (e.g., gastric juice, blood, plasma, urine and saliva) from the subject.
A method of immunizing a subject against bacterial infection is provided, comprising: a) administering to the subject the dapE' mutant strain having in its chromosome a nucleic acid encoding a foreign protein (e.g., an immunogen of the bacterium) ; b) supplementing the subject's diet with L-DAP to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the strain; and c) ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject. The immunization methods described herein comprise administering to the subject an immunogenic amount of mutant H. pylori in a pharmaceutically acceptable carrier for the mutant. The length of time to which the subject is exposed to the mutant strain is as described above. The exact time course will vary from individual to individual and can be verified by tests for the presence of an immune response such as is measured by assays for antibodies against the foreign protein in a tissue sample (e.g., gastric juice, blood, plasma, urine and saliva) from the subject.
A method of immunizing a subject against a bacterial infection, comprising: a) administering to the subject the dapE~ mutant strain containing a plasmid comprising a nucleic acid encoding a foreign protein; b) supplementing the subject's diet with diaminopimelic acid to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the strain; and c) ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject.
In the above methods wherein the foreign antigen encoding gene is on the dap£-containing plasmid, the host strain can be attenuated in other genes (e.g., recA, vacA, etc. )
The present immunization methods are useful in immunizing against infection with bacteria, fungi, protozoa and viruses. Thus, the foreign protein encoded can be an antigen (immunogen) of the bacterium, virus, fungus or protozoan against which immunization is being made. The foreign protein encoded can be immunogenic, antigenic, or enzymatic proteins of Salmonella ( Salmonella enteri tidi s) , Shi gella, Yersinia, enterotoxigenic and enterohemorrhagic E. coli , M. tubercul osi s, Streptococcus pyogenes, P. fal ciparum, human immunodeficiency virus, respiratory syncytial virus, influenza virus or other infectious organisms. Other foreign proteins include sperm antigens for use to immunize against sperm as a form of birth control or immunomodulating proteins or cytotoxic proteins as described above. The immunization methods for these microorganisms also comprise administering to the subject an immunogenic amount of mutant H. pyl ori containing the gene for a foreign immunogen in a pharmaceutically acceptable carrier for the mutant. The length of time to which the subject is exposed to the mutant strain, i.e., the length of time L-DAP supplementation is provided, will typically be from a few days (2 or 3) up to a few weeks (2 or 3) . The exact time course may vary from individual to individual and can be verified by tests for the presence an of immune response (e.g., by the presence of antibodies) against the foreign protein in a tissue sample (gastric juice, blood, plasma, urine and saliva) from the subject. Clearly, if the subject produces antibodies against the microorganism, it is understood that the antibodies are against the recombinant protein produced by the altered H. pyl ori strain .
Determining immunogenicity and immunogenic amounts The isolated mutant strains of the invention can be tested to determine their immunogenicity. Briefly, various concentrations of a putative immunogen are prepared and administered to an animal and the immunological response (e.g., the production of antibodies or cell mediated immunity) of an animal to each concentration is determined. The amounts of antigen administered depend on the subject, e.g. a human, mouse or gerbil, the condition of the subject, the size of the subject, etc. Thereafter, an animal so inoculated with the strain can be exposed to the bacterium to test the potential vaccine effect of the specific immunogenic protein or fragment.
For example, well-established models include gnotobiotic piglets and mice. The dapET mutant strain is first fed to the piglets or mice and maintained by DapE supplementation. After a suitable interval, the supplementation is stopped and the clearance of the vaccine strain is evaluated. Next, this piglet or mouse is challenged with a wild-type H. pyl ori strain or other microorganism and the presence or absence of infection is ascertained (48, 49) . This same system can also be used to determine the amounts of mutant required to have a protective effect.
Once immunogenicity is established as described above, immunogenic amounts of the antigen can be determined using standard procedures. Briefly, various concentrations of the dapET mutant are prepared, administered to an animal and the immunological response (e.g., the production of antibodies) of an animal to each concentration is determined.
Pharmaceutically acceptable carrier
The pharmaceutically acceptable carrier in the vaccine of the instant invention can comprise saline or other suitable carriers (Arnon, R. (Ed.) Syntheti c Vaccines 1:83-92, CRC Press, Inc., Boca Raton, Florida, 1987) . An adjuvant can also be a part of the carrier of the vaccine, in which case it can be selected by standard criteria based on the antigen used, the mode of administration and the subject (Arnon, R. (Ed.), 1987). Methods of administration can be by oral or sublingual means, or by injection, depending on the particular vaccine used and the subject to whom it is administered.
It can be appreciated from the above that the vaccine and immunization method can be used as a prophylactic or a therapeutic modality, for example, by inducing a therapeutic immune response. Thus, the invention provides methods of preventing or treating H. pyl ori infection and the associated diseases by administering the vaccine to a subject. Because the dapE~ mutant can contain and express many different foreign immunogens, the invention also provides methods of treating or preventing infections with other organisms.
DapE , GidA and ORF2 proteins
A purified DapE protein of Heli cobacter pyl ori is provided. The DapE protein can consist of the amino acid sequence defined in SEQ ID NO: 2. A H. pyl ori-speci fic fragment of the DapE protein can be routinely obtained in accord with teaching herein and in the art, and is contemplated.
Similarly, the GidA protein and the protein encoded by ORF2 are provided in SEQ ID NOS : 4 and 6. EXAMPLES
Characterization of Heli cobacter pylori dapE and construction of a conditionally lethal dapE~ mutant
Bacterial strains, plasmids and growth conditions.
H. pyl ori strain 60190 was used for the molecular cloning studies, and 21 well characterized clinical H. pyl ori strains from the Vanderbilt University Heli cobacter/ Campyl obacter culture collection were used to determine the conservation of the cloned genes. Stock cultures were maintained at -70°C in Brucella Broth (BBL Microbiology Systems, Cockysville, MD) supplemented with 15% glycerol. E. coli DH5 , XL-lblue, and Dam" strains were used for transformation, and pBluescript (Stratagene, La Jolla, CA, USA) was used as a cloning vector. E. coli strains were routinely cultured in Luria-Bertani (LB) medium with shaking at 37°C, and the clinical H. pylori isolates were cultured on Trypticase soy agar plates containing 5% sheep blood in a microaerobic atmosphere, as described (13). For transformation of H. pyl ori (14), strains were grown at 37°C in a microaerobic atmosphere on Brucella agar plates containing 5% Fetal Calf Serum (FCS) and 30μg /ml kanamycin and supplements of 0 to 2mM DAP (a racemic mixture of all three DAP isomers, Sigma Chemical Co, St. Louis, MO) or 1 mM lysine (Sigma).
Genetic techniques and nucleotide sequence analysis.
Chromosomal DNA was prepared as described previously (40) . All other standard molecular genetic techniques including Southern and colony hybridizations were performed, as described (24,39). For molecular cloning, positive plaques were purified from a bank of approximately 5 kb random chromosomal fragments of H. pyl ori 60190 using ZapII and recombinant DNA was prepared as described (40) . Restriction enzyme cleavage maps were generated, and a 5kb fragment was subcloned into pBluescript to create pAK2 (Figure 1). Another 5kb fragment carrying a portion of the H. pylori genome overlapping only the orf2 region of pAK2 was subcloned into pBluescript to create pAKl (Figure 1). The nucleotide sequence was determined unambiguously on both strands using double-stranded DNA templates using an automated DNA sequencer (Perkin Elmer, Model ABI377, Foster City, CA) with the ThermoSequenase dye primer reaction kit (Amersham, Arlington Heights, IL) . Oligonucleotide primers were synthesized at the Vanderbilt Cancer Center DNA core facility with an ABI 392 DNA synthesizer sequencer (Perkin Elmer) . Nucleotide sequences were compiled and analyzed using programs in the GCG Package (16) . Amplifications were conducted in a Perkin-Elmer Thermal Cycler. PCR conditions used in this study were 35 cycles of 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min, with a terminal extension at 72°C for 10 min, and the primers used in this study are listed in Table 1.
Table 1 PCR Primers used in this study
CO
Figure imgf000025_0001
Figure imgf000026_0001
Tosition in sequence shown in Figure 2 except for km and vacA primers. Position refer to those in the cited publications.
-p-
Construction of recombinant plasmids with insertion of kanamycin-resistance cassettes into targeted genes.
A C. coli km gene (22) was ligated into the unique Bell site of pAK2 within the gidA ORF to create pAK2 : gi dA : km (pME36) (Figure 1). An E. coli km cassette from pUC4K (38) was inserted into the unique Ndel site within the dapE ORF to create pAK2 : dapE : km (pMAK36) (Figure 1) . orf2 contained no unique sites for km insertion, but 3 Hin i I I sites were present within 107bp. Therefore, to create orf2 :km, the orf2 ORF from pAKl was PCR-amplified and subcloned the amplified fragment into pT7Blue (Novagen, Madison, WI) to create pAK7. The 430 bp insert was subcloned into pCR-Script Cam SK (+), (Stratagene, La Jolla, CA) , a pBluescript derivative encoding chloramphenicol resistance, to create pAK8. After Hindi ! I digestion of pAK8, the km cassette from pUC4K was inserted into orf2 to create pAK8 : or f2 :km (pAKQ) (Figure 1) .
Construction of H. pylori dapE and orf2 mutants.
The constructs, pAK2 : gi dA : km (pME36) , pAK2 : dapE:km (pMAK36), or pAK8 : orf2 :km (pAKQ) , all of which are unable to replicate in H. pyl ori , were introduced into H. pylori 60190 by natural transformation; pCTB8: m containing vacA : km was used as a positive control (14) . The transformants were selected on Brucella broth agar plates containing 5% FCS and 30 μg /ml kanamycin. In certain experiments, plates were supplemented with ImM DAP to determine the conditions necessary for dapE~ mutant viability. To determine the minimum concentration needed for growth of the dapE' mutant, strains were grown on media supplemented with 0 to 2.0 mM DAP or 1.0 itiM lysine. To provide genetic evidence in the transformed strain of dapE disruption by the km insertion, DNA isolated from both the H. pylori mutant strain 60190 pAK2 : dapE : km and wild-type strain 60190 was digested with BamHI and hybridized to dapE and km probes. The authenticity of the mutant strain also was verified by PCR, using primers based on dapE and km (Figure 1 and Table 1) . The authenticity of the orf2~ mutants also was verified by Southern hybridization and PCR using parallel methods.
Evidence of homologous recombination between pAK2 : gri A: Jan and H. pylori strain 60190 chromosomal DNA.
No viable gidA mutants were obtained, even with selection on media supplemented with DAP or lysine. To provide genetic evidence that double cross-over events had occurred during the pre-selective growth phase, allowing for the insertion of km in the H. pyl ori chromosome within gidA, PCR was performed with a forward primer specific for km (primer 10 in Figure 1 and Table 1) and a reverse primer (primer 8 in Figure 1 and Table 1) specific for a region of the H. pyl ori chromosome present in pAKl that is beyond the fragment cloned in pAK2. DNA isolated from wild type strain 60190 was examined after overnight incubation with pAK2 : gi dA : km . As negative controls, DNA from wild type strain 60190 and a mixture of DNA from wild type strain 60190 and pAK2 : gidA: km were used. As a positive control, the forward km primer (primer 10 in Figure 1 and Table 1) and a confirmed vacA reverse primer (primer 17 in Table 1) (14) were tested on DNA isolated from wild type strain 60190 after overnight incubation with pCTB8 : vacA ikm . RNA isolation, reverse transcriptase-polymerase chain reaction (RT-PCR) , and slot-blot analysis.
To determine whether gidA, dapE, and orf2 are co- transcribed, wild type and mutant H. pyl ori strains were cultured for 24h on Brucella agar plates containing 5% FCS supplemented with ImM DAP, cells were harvested, and RNA was recovered for RT-PCR by two rounds of hot-phenol extraction, as described previously (40) . cDNA was synthesized from Iμq of DNAse-treated total RNA by priming with lμg of random hexamer (Pharmacia, Inc., Piscataway,
NJ) , ImM of each dNTP, 20 units of RNAse inhibitor and AMV reverse transcriptase (Promega, Madison, WI) in a final volume of 20ul at 42°C for 15 min. PCR reactions were performed as described above.
Agarose gel electrophoresis was performed on specific RT-PCR amplified products from H. pylori wild-type and mutant strains using primers within gidA, dapE, or orf2 as follows: Lanes 1, 4, 7 - wild type strain 60190; lanes 2, 5, 8 - dapE~ mutant strain (60190E"); lanes 3, 6, 9 - orf2' mutant strain (60190-2") . PCR was performed using primers 13 and 14 and DNA (lanes 1-3), cDNA (lanes 4- 6) or RNA (lanes 7-9) as templates. PCR was performed using primers 15 and 16 and DNA (lanes 1-3), cDNA (lanes 4- 6) or RNA (lanes 7-9) as templates.
To provide further evidence that orf2 is co- transcribed with dapE , slot-blot RNA analysis of mRNA transcripts of gidA, dapE, and orf2 was used. DNAse- treated RNA samples (12μg) from wild type( T) H. pylori strain 60190, or its dapE' or orf2~ mutants were transferred to nylon membranes, and hybridized with equal amounts (50,000 cpm) of radiolabelled cagA, gidA, dapE, or orf2 probes. Hybridization used probes specific for gidA (1.9kb PCR-amplified gidA-specific fragment), dapE (l.lkb PCR-amplified dap£-specific fragment), orf2 (0.7kb PCR- amplified or~2-specific fragment) , or cagA as positive control (0.5kb PCR-amplified cagA-specific fragment). The amount of radiolabel (50,000 cpm) was standardized for each probe, and experiments were performed as previously described (32 ) .
Isolation of H. pylori dapE.
A 5 kb EcoRI genomic fragment from H. pyl ori strain 60190 was cloned into pBluescript to create pAK2 (Figure 1), and the nucleotide sequence of this fragment was determined (Figure 2) . Analysis of translation of the 5050 bp nucleotide sequence in all possible reading frames revealed five complete or partial open reading frames (ORFs) . The three complete ORFs, consisting of 1866, 1167, and 753 nucleotides, were oriented in the same direction and opposite to the partial ORFs present at the ends of the fragment (Figures 1 and 2) . The first complete ORF begins with GTG as the initiation codon, and encodes a 621 amino acid polypeptide, yielding a predicted product with a 69,665 Da molecular mass. The second ORF begins with an ATG codon, 10 bp after the termination of the first ORF and encodes a 388 amino acid polypeptide, yielding a predicted 42,822 Da product. The third ORF begins with ATG 80 bp after the termination of the second ORF, and encodes a 250 amino acid polypeptide with a predicted molecular mass of 27,585 Da. Potential ribosome binding sites begin 6 or 7 bp upstream of each ORF.
Upstream of the translational start of the first ORF is the sequence TATTTT, which resembles the consensus σ70 -10 sequence (33), and is 19 bp downstream of the sequence TTGGCA that shares 5 of 6 bases with the corresponding -35 consensus sequence (33) . Nucleotides 4456 to 4654 following the third ORF exhibit the sequence of a putative three-hairpin stem-loop structure (ΔG= -40.2) that could permit a strong mRNA transcriptional terminator. The single putative promoter and transcription terminator and the close location and orientation of the ORFs suggest that they may represent an operon.
Analysis of the deduced products of the ORFs.
The translated amino acid sequence for genes in pAK2 was compared with databases using the FASTA, FASTDB, and BLAST network services of the National Center for Biotechnology Information. The deduced product from the first complete ORF showed significant homology throughout the translated amino acid sequence (48.3% identity and 66.5% similarity) with the glucose inhibited division protein, encoded by gidA in E. coli (9,36,43), H. infl uenzae (47.1% identity and 67.5% similarity) (18) (Figure 3A) , Pseudomonas putida (28) (47.9% identity and 68.9% similarity), and Bacill us subtili s (27,28) (46.1% identity and 64.4% similarity). The putative product of the second ORF showed significant homology (37.9 % identity and 61.0% similarity) with N-Succinyl-L- diaminopimelic acid desuccinylase (encoded by dapE) of E. coli (7,45) (Figure 3B) and H. infl uenzae (18) (39.1% identity and 58.8% similarity) (Figure 3B) . There was no substantial overall homology between the products of the other complete or the two incomplete ORFs and other known sequences. These genes are tentatively identified as orfl , orf2, and orf3, as shown in Figure 1. Conservation of gidA , dapE, and orf2 among H. pylori strains .
To determine whether other H. pyl ori strains possess sequences homologous to gidA, dapE, or orf2, 21 strains (10 cagA+ and 11 cagA "strains) were studied by colony hybridization, using PCR-amplified gidA, dapE, and orf2 specific fragments. A positive signal was obtained from each of these strains, indicating that these genes are conserved in H. pyl ori , despite other genotypic variations.
Characterization of a dapE mutant.
To create a dapE mutant, H. pyl ori strain 60190 was transformed with pMAK36 ( dapE:km) , and plated transformants on kanamycin-containing medium including 1 mM DAP. Southern and PCR analysis of the kanamycin- resistant transformants indicated that the km cassette was stably incorporated into the single chromosomal dapE gene creating a dapE mutant. However, in repeated experiments, transformation of H. pyl ori with pMAK36 ( dapE km) on plates lacking DAP did not yield any transformants (Table 2) . Similarly, the dapE" mutants obtained on DAP- containing plates were unable to grow when re-plated on TSA agar or Brucella agar with 5% FCS without the addition of DAP. Transformation of H. pylori with pCTB8 : vacA:km yielded a similar number of transformants whether or not DAP was present in the selective media and served as a positive control. The minimum DAP concentration required for survival of the dapE~ mutant was found to be 0.2mM (Table 3) . The dapE' mutant was unable to grow on media supplemented with lysine only (Table 2), emphasizing the specific DAP requirement of H. pyl ori for growth and/or survival . Table 2. Growth of wild type and mutant H. pylori strains on brucella agar in the presence or absence of DAP
Figure imgf000033_0001
aBrucella broth with 1.5% agar supplemented with 5% FCS and 30ug/ml kanamycin additionally supplemented with 1 mM DAP in medium "Additionally supplemented with 1 mM lysine in medium
Table 3. Minimum concentration of DAP required for growth of the dapE- H.pylori mutant
Figure imgf000033_0002
aDAP concentration in Brucella broth with 1.5% agar supplemented with 5% FCS and 30ug/ml kanamycin b3 mutant strains were tested c3 mutant strains were tested Characterization of an H. pylori mutant lacking orf2.
The orf2 ORF begins only 80 bp downstream from dapE and lacks its own consensus promoter, suggesting that these genes could be co-transcribed and their products could be functionally related. To test this hypothesis, orf2 was disrupted by insertional mutagenesis, and demonstrated the authenticity of H. pyl ori mutant 60190 orf2 :km by Southern hybridization and PCR. However, the orf2 ' mutant was found to grow well with or without exogenous DAP in the growth medium (Table 2 ) , demonstrating that orf2 is not required in the metabolic pathway leading to DAP formation.
Evidence that mutation of gidA is lethal in H. pylori . The dapE open reading frame is separated by only 10 bp from gidA, suggesting co-transcription and a functional relationship between these two genes. To determine whether the gidA product in H. pyl ori is associated with dapE synthesis, an attempt was made to insertionally inactivate gidA. However, efforts to inactivate gidA by transforming H. pyl ori strain 60190 with pAK2 : gi dA : km were unsuccessful. No transformants were observed even on media supplemented with DAP (or lysine) , while parallel transformations that led to the inactivation of dapE or vacA yielded more than 100 transformants for each. Since these data suggested that interruption of gidA was lethal for H. pylori , PCR was performed to determine whether insertion of the kanamycin cassette within gidA had occurred to transiently create 60190 gidA km, but this organism was not viable. As a positive control, wild type strain 60190 was incubated in parallel with pCTB8 : vacA: km to create an insertion in vacA. Agarose gel electrophoresis was performed on of PCR- amplified products from DNA isolated from wild type strain 60190 after overnight incubation and transformation with or without specified plasmid. All PCRs used a forward primer specific for km (primer 10 in Figure 1 and Table 1) . The reverse primer was either specific for a region of the H. pyl ori chromosome not included in the fragment cloned in pAK2 (primer 8 in Figure 1 and Table 1) or was specific for vacA (primer 17 in Table 1) as a control. Lane 1: Template isolated from DNA strain 60190 after overnight incubation with pAK2 : gidA :km, and primers 10 and 8. Lane 2: Template DNA isolated from strain 60190, and primers 10 and 8. Lane 3: Template is a mixture of DNA from strain 60190 and pAK2 : gidA .'km, and primers 10 and 8. Lane 4: Template: DNA from strain 60190 after overnight incubation with pCTB8 : vacA .'km, and primers 10 and 17.
When the forward km primer (primer 10 in Figure 1 and Table 1) and reverse vacA primer (primer 17 in Table 1) were used, a 3.1 kb band was amplified, as expected.
Using a forward km primer (primer 10) and a reverse primer (primer 8 in Figure 1 and Table 1) that is not present in pAK2 (Figure 1), a 4.2 kb band was amplified in DNA isolated from wild type strain 60190 that had been incubated overnight with pAK2 : gidA: km. No band was present in DNA from wild type strain 60190 alone, or if 60190 DNA was mixed with pAK2 : gidA: km in the absence of H. pyl ori cells.
These results indicate that homologous recombination had occurred between the chromosomal DNA of the wild type strain 60190 and pAK2 : gidA : km leading to km insertion within gidA, and provided further evidence that this transformation event was lethal to H. pylori .
RT-PCR and slot-blot analysis. To ascertain whether gidA, dapE, and orf2 are co- transcribed, RNA was extracted for analysis from wild type H. pyl ori strain 60190 and its dapET and orf2~ mutants. Reverse transcriptase-PCR (RT-PCR) of cDNA template was performed with a pair of primers bridging the gidA and dapE ORFs (primers 13 and 14, Table 1) .
A 0.35kb product was detected for each strain, as expected. In RT-PCR using primers bridging the dapE-orf2 ORFs (primers 15 and 16), no product was detected m the dapE mutant, as expected, but both the wild type strain and the orf2 mutant showed a product of the expected size (0.4kb). Negative-control PCR using RNA as template showed no products. As expected, the positive control cagA probe hybridized with equal intensity to the wild type strain and its dapE and orf2 mutants. The gidA probe hybridized to RNA with similar intensity for the wild type strain and its dapET and orf2 mutants. The dapE probe hybridized equally well to wild type and orf2~ mutant RNA, and less well to its dapE mutant. The orf2 probe hybridized well to RNA m the wild type strain, but only weakly to RNA from the dapE and orf2 mutants (Figure 6) . The results of both sets of experiments indicate that orf2 can be co-transcribed with dapE.
The results in this study suggest that the ability of H. pyl ori to synthesize DAP is based only on the succmylase pathway, or that its synthesis via the dehydrogenase and/or acetylase pathways is too low to allow for survival.
The gidA, dapE, and orf2 ORFs are closely spaced and oppositely oriented to the flanking genes (Figure 1), suggesting that they form an operon. A sequence bearing strong homology to the σ70 promoter is present 5' to gidA, but no promoter-like elements were observed upstream of dapE and orf2. The presence of a strong putative transcriptional terminator downstream of orf2 also is consistent with the notion that these 3 genes form an operon, and RT-PCR and slot blot data indicate that dapE and orf2 may be co-transcribed. The presence of another putative transcriptional terminator, an 80-nucleotide palindromic sequence (DG= -2.9), beginning at nucleotide 3623 to 3702 in the intergenic region between dapE and orf2, may, under certain conditions, allow for transcription of gidA and dapE without orf2.
The dapE ORF is separated by only 10 bp from gidA. In E. coli , gidA lies near the origin of replication ( oriC) (29); inactivation of gidA by transposon insertion reduces the E. coli growth rate by 20% and causes filamentation of cells in media containing glucose (41,42) . However, in H. pylori , the arrangement of gidA and dapE in the same operon suggests that the products of these two genes could be functionally related. However, the inability of DAP or lysine supplementation to permit gidA mutants to survive suggests that its critical activity does not involve the DAP/lysine pathway.
The presence of orf2 80 bp downstream from dapE, with no unique promoter sequence, suggests that these two genes may be co-transcribed and that their protein products may be functionally related. RT-PCR and slot-blot results support this hypothesis, since orf2 RNA was not transcribed in the dapE ' mutant. That the orf2 ~ mutant strain grew normally without exogenous DAP indicates that the orf2 product is not required for DAP biosynthesis.
The observations made in this study suggest that the enzymes involved in DAP biosynthesis represent targets for the development of novel agents against H. pylori
(3,4,20,21) . DAP biosynthetic genes also may be used to stabilize shuttle plasmids for use in H. pyl ori in the absence of antibiotic markers (25) . If H. pyl ori strains carrying mutations in DAP biosynthesis genes can be constructed, plasmids carrying the respective complementing gene could be maintained. Such plasmids may lead to the ability to stably maintain recombinant DNA in humans for the expression of H. pylori or heterologous antigens, and may provide tools in the investigation of H. pyl ori pathogenesis as well as for the development of new anti-iϊ. pylori agents.
These results also suggest that co-administration of H. pylori dapET mutant strains with a DAP supplement may serve as an immunization strategy. After sufficient time for evoking an immune response directed at this H. pyl ori strain, cessation of DAP supplementation would lead to its death. Ideally, optimal timing of supplementation could result in the establishment of long-term immunity to H. pyl ori or to heterologous antigens delivered by this superb mucosal colonizer. Throughout this application various publications are referenced by numbers within parentheses. Full citations for these publications are as follows. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
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19. Galan, J. E., K. Nakayama, and R. Curtiss,III. 1990. Cloning and characterization of the asd gene of Salmonella typhimuri um : use in stable maintenance of recombinant plasmids in Salmonella vaccine strains. Gene 94:29-35.
20. Galardy, R. E. and Z. P. Kortylewicz. 1984. Inhibition of Carboxypeptidase A by aldehyde and ketone substrate analogues. Biochemistry 23:2083-2087.
21. Gelb, M. H., J. P. Svaren, and R. H. Abeles . 1985. Fluoro ketone inhibitors of hydrolytic enzymes. Biochemistry 24:1813-1817.
22. Labigne-Roussel , A., J. Harel , and L. Tompkins. 1987. Gene transfer from Escheri chia coli to Campylobacter species. Development of shuttle vectors for genetic analysis of Campyl obacter jej uni . J. Bacteriol. 169:5320-5323.
23. Lin, Y., R. Myhrman, M. L. Schrag, and M. H. Gelb. 1987. Bacterial N-succinyl-L-diaminopimelic acid desuccinylase . J. Biol. Chem. 1622-1627.
24. Maniatis, T. , E. F. Fritsch, and J. Sambrook . 1989. Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
25. Nakayama, K. , S. M. Kelley, and R. Curtiss,III. 1988. Construction of an asd expression-cloning vector: stable maintenance and high level expression of cloned genes in a salmonella vaccine strain. Bio. Technol. 6:693-697.
26. Nomura, A., G. N. Ste mermann, P. Chyou, I. K to, G. I. Perez-Perez, and M. J. Blaser. 1991. Heli cobacter pyl ori infection and gastric carcinoma in a population of Japanese-Americans in Hawaii. N. Engl . J. Med. 325:1132-1136.
27. Ogasawara, N., S. Nakai , and H. Yoshiokawa. 1994. Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res 1:1-14. 28. Ogasawara, N. and H. Yoshikawa. 1992. Genes and their organization in the replication origin region of the bacterial chromosome. Mol. Microbiol. 6:629-634.
29. Ogawa, T. and T. Okazaki . 1994. Cell cycle-dependent transcription from the gid and mi oC promoters of Escheri chia coli . J. Bacteriol. 176:1609-1615.
30. Parsonnet, J. , G. D. Friedman, D. P. Vandersteen, Y. Chang, J. H. Vogelman, N. Orentreich, and R. K. Sibley. 1991. Heli cobacter pylori infection and the risk of gastric carcinoma. N. Engl . J Med 325:1127-1131.
31. Patte, J. C. 1996. Biosynthesis of Threonine and lysine, p. 532-535. In F. C. Neidhardt (ed.), Escherichia coli and Salmonella , cellular and molecular biology. ASM Press, Washington, DC.
32. Peek, R. M. , Jr., S. A. Thompson, J. C. Atherton, M. J. Blaser, and G. G. Miller. 1996. Expression of a novel ulcer-associated H. pyl ori gene, i ceA, after contact with gastric epithelium. Gastroenterol . (In Press)
33. Rosenberg, M. and D. Court. 1979. Regulatory sequences involved in the promotion and termination of RNA transcription. ( ) 13:319-353.
34. Schrumpf, B. , A. Schwarzer, J. Kalinowski, A. Puhler, L. Eggeling, and H. Sahm. 1991. A functionally split pathway for lysine synthesis in Corynebacterium glutamicum. J. Bacteriol. 173:4510-4516.
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43. Walker, J. E., N. J. Gay, M. Saraste, and A. N. Eberle. 1984. DNA sequence around the Escherichia coli unc operon. Completion of the sequence of a 17 kilobase segment containing a snA, ori C, unc , glmS, and phoS . Biochem. J. 224:799-815. 44. Weinberger, S. and C. Gilvarg. 1970. Bacterial distribution of the use of succinyl and acetyl blocking groups in diaminopimelic acid biosynthesis. J. Bacteriol. 101:323-324.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(l) APPLICANT: Vanderbilt University 305 Kirkland Hall Nashville, Tennessee 37240
(ii) TITLE OF THE INVENTION: dapE Gene of Helicobacter pylori and dapE- Mutant Strains of H. pylori
(ill) NUMBER OF SEQUENCES: 24
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: NEEDLE & ROSENBERG, P.C.
(B) STREET: 127 Peachtree Street, N.E., Suite 1200
(C) CITY: Atlanta
(D) STATE: GA
(E) COUNTRY: USA
(F) ZIP: 30303-1811
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(V ) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 23-DEC-1997
(C) CLASSIFICATION:
(VII) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/033,824
(B) FILING DATE: 23-DEC-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Spratt, Gwendolyn DD
(B) REGISTRATION NUMBER: 36,016
(C) REFERENCE/DOCKET NUMBER: 22000.0058/P
(lx) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 404 688 0770
(B) TELEFAX: 404 688 9880
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:l:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1866 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (IX) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 1...1863 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
GTG GTA AAA GAA AGT GAT ATT TTA GTG ATT GGT GGG GGG CAT GCG GGC 48 Met Val Lys Glu Ser Asp He Leu Val He Gly Gly Gly His Ala Gly 1 5 10 15
ATT GAA GCG AGC TTG ATT GCA GCC AAA ATG GGG GCT AGG GTG CAT TTA 96 He Glu Ala Ser Leu He Ala Ala Lys Met Gly Ala Arg Val His Leu 20 25 30
ATC ACC ATG CTC ATA GAC ACG ATC GGT TTA GCG AGC TGT AAC CCG GCG 144 He Thr Met Leu He Asp Thr He Gly Leu Ala Ser Cys Asn Pro Ala 35 40 45
ATT GGG GGC TTG GGT AAA GGG CAT TTG ACT AAA GAA GTG GAT GTT TTA 192 He Gly Gly Leu Gly Lys Gly His Leu Thr Lys Glu Val Asp Val Leu 50 55 60
GGG GGG GCT ATG GGG ATT ATT ACA GAT CAT AGC GGT TTG CAA TAT CGT 240 Gly Gly Ala Met Gly He He Thr Asp H s Ser Gly Leu Gin Tyr Arg 65 70 75 80
GTG TTA AAC GCT TCT AAA GGG CCG GCG GTT AGG GGG ACT AGA GCG CAA 288 Val Leu Asn Ala Ser Lys Gly Pro Ala Val Arg Gly Thr Arg Ala Gin 85 90 95
ATT GAT ATG GAT ACT TAC CGC ATT TTT GCA AGA AAT CTT GTT TTA AAC 336 He Asp Met Asp Thr Tyr Arg He Phe Ala Arg Asn Leu Val Leu Asn 100 105 110
ACC CCT AAT TTG AGC GTC TCT CAA GAA ATG ACC GAA AGT TTA ATC CTT 384 Thr Pro Asn Leu Ser Val Ser Gin Glu Met Thr Glu Ser Leu He Leu 115 120 125
GAA AAC GAT GAG GTA GTG GGC GTA ACC ACG AAC ATT AAT AAC ACT TAC 432 Glu Asn Asp Glu Val Val Gly Val Thr Thr Asn He Asn Asn Thr Tyr 130 135 140
AGA GCT AAA AAA GTG ATC ATC ACC ACA GGC ACT TTT TTA AAA GGG GTG 480 Arg Ala Lys Lys Val He He Thr Thr Gly Thr Phe Leu Lys Gly Val 145 150 155 160
GTG CAT ATT GGC GAG CAC CAA AAC CAA AAC GGG CGT TTT GGG GAA AAC 528 Val His He Gly Glu His Gin Asn Gin Asn Gly Arg Phe Gly Glu Asn 165 170 175
GCT TCC AAT TCT TTA GCC TTG AAT TTA AGG GAG CTT GGC TTT AAG GTG 576 Ala Ser Asn Ser Leu Ala Leu Asn Leu Arg Glu Leu Gly Phe Lys Val 180 185 190
GAG AGG TTA AAA ACC GGC ACT TGC CCA AGA GTG GCC GGC AAT AGC ATT 624 Glu Arg Leu Lys Thr Gly Thr Cys Pro Arg Val Ala Gly Asn Ser He 195 200 205
GAT TTT GAA GGC TTA GAA GAG CAT TTT GGG GAT GCA AAC CCT CCC TAT 672 Asp Phe Glu Gly Leu Glu Glu H s Phe Gly Asp Ala Asn Pro Pro Tyr 210 215 220 TTC AGC TAT AAA ACC AAA GAT TTT AAC CCC ACC CAA CTC TCT TGT TTC 720
Phe Ser Tyr Lys Thr Lys Asp Phe Asn Pro Thr Gin Leu Ser Cys Phe
225 230 235 240
ATC ACT TAC ACT AAC CCC ATT ACC CAC CAA ATC ATT AGG GAT AAT TTC 768
He Thr Tyr Thr Asn Pro He Thr H s Gin He He Arg Asp Asn Phe 245 250 255
CAC CGA GCT CCC CTT TTT AGC GGT CAA ATT GAA GGC ATA GGC CCA AGG 816
His Arg Ala Pro Leu Phe Ser Gly Gin He Glu Gly He Gly Pro Arg 260 265 270
TAT TGC CCT AGC ATT GAA GAT AAA ATC AAC CGC TTT AGT GAA AAA GAA 864
Tyr Cys Pro Ser He Glu Asp Lys He Asn Arg Phe Ser Glu Lys Glu 275 280 285
CGC CAC CAG CTG TTT TTA GAG CCT CAA ACC ATT CAT AAA AAC GAA TAT 912
Arg His Gin Leu Phe Leu Glu Pro Gin Thr He His Lys Asn Glu Tyr 290 295 300
TAT ATC AAC GGC TTA AGC ACC TCT TTG CCC CTA GAT GTG CAA GAA AAG 960
Tyr He Asn Gly Leu Ser Thr Ser Leu Pro Leu Asp Val Gin Glu Lys
305 310 315 320
GTC ATT CAT TCT ATC AAA GGC TTA GAA AAC GCC CTC ATC ACG CGC TAT 1008
Val He His Ser He Lys Gly Leu Glu Asn Ala Leu He Thr Arg Tyr 325 330 335
GGC TAT GCG ATA GAG TAT GAT TTC ATC CAG CCT ACA GAA TTA ACC CAC 1056
Gly Tyr Ala He Glu Tyr Asp Phe He Gin Pro Thr Glu Leu Thr His 340 345 350
GCT TTA GAA ACC AAA AAA ATC AAA GGG CTT TAT TTG GCC GGG CAA ATC 1104
Ala Leu Glu Thr Lys Lys He Lys Gly Leu Tyr Leu Ala Gly Gin He 355 360 365
AAT GGG ACT ACC GGC TAT GAA GAA GCG GCG GAT CAA GGG CTT ATG GCT 1152
Asn Gly Thr Thr Gly Tyr Glu Glu Ala Ala Asp Gin Gly Leu Met Ala 370 375 380
GGG ATT AAT GCG GTA TTA GCC TTA AAG AAT CAA GCC CCC TTT ATT TTA 1200
Gly He Asn Ala Val Leu Ala Leu Lys Asn Gin Ala Pro Phe He Leu
385 390 395 400
AAG CGC AAT GAA GCT TAT ATT GGC GTT TTG ATT GAT GAT TTG GTT ACT 1248
Lys Arg Asn Glu Ala Tyr He Gly Val Leu He Asp Asp Leu Val Thr 405 410 415
AAA GGC ACG AAT GAG CCT TAC AGA ATG TTT ACT AGC CGA GCC GAA TAC 1296
Lys Gly Thr Asn Glu Pro Tyr Arg Met Phe Thr Ser Arg Ala Glu Tyr 420 425 430
CGC TTG CTT TTA AGA GAG GAC AAC ACG CTT TTT AGG TTG GGC GAA CAT 1344
Arg Leu Leu Leu Arg Glu Asp Asn Thr Leu Phe Arg Leu Gly Glu His 435 440 445
GCC TAT CGT TTA GGG CTT ATG GAA CAG GAT TTT TAT AAG GAA TTA AAA 1392
Ala Tyr Arg Leu Gly Leu Met Glu Gin Asp Phe Tyr Lys Glu Leu Lys 450 455 460 AAA GAT AAA CAA GAG ATA CAA GAC AAT CTC AAA CGC CTT AAA GAA TGC 1440
Lys Asp Lys Gin Glu He Gin Asp Asn Leu Lys Arg Leu Lys Glu Cys
465 470 475 480
GTC CTT ACC CCT AGT AAA AAA TTG TTA AAA CGC TTG AAC GAA TTA GAC 1488
Val Leu Thr Pro Ser Lys Lys Leu Leu Lys Arg Leu Asn Glu Leu Asp 485 490 495
GAA AAC CCT ATC AAT GAC AAG GTT AAT GGC GTT AGT TTG TTA GCA CGC 1536
Glu Asn Pro He Asn Asp Lys Val Asn Gly Val Ser Leu Leu Ala Arg 500 505 510
GAT AGT TTT AAT GCA GAA AAA ATG CGC TCC TTT TTC AGC TTT TTA GCC 1584
Asp Ser Phe Asn Ala Glu Lys Met Arg Ser Phe Phe Ser Phe Leu Ala 515 520 525
CCC TTG AAC GAG CGG GTT TTA GAG CAG ATT AAA ATT GAA TGC AAA TAT 1632
Pro Leu Asn Glu Arg Val Leu Glu Gin He Lys He Glu Cys Lys Tyr 530 535 540
AAT ATT TAT ATT GAA AAG CAA CAC GAA AAT ATC GCT AAA ATG GAT AGC 1680
Asn He Tyr He Glu Lys Gin His Glu Asn He Ala Lys Met Asp Ser
545 550 555 560
ATG CTC AAA GTT TCT ATC CCT AAA GGT TTT GTG TTT AAA GGC ATT CCA 1728
Met Leu Lys Val Ser He Pro Lys Gly Phe Val Phe Lys Gly He Pro 565 570 575
GGC TTA AGC TTA GAA GCG GTA GAA AAA TTA GAA AAA TTC CGC CCC AAA 1776
Gly Leu Ser Leu Glu Ala Val Glu Lys Leu Glu Lys Phe Arg Pro Lys 580 585 590
AGC CTT TTT GAA GCC TCA GAA ATC AGC GGG ATC ACT CCA GCG AAT TTA 1824
Ser Leu Phe Glu Ala Ser Glu He Ser Gly He Thr Pro Ala Asn Leu 595 600 605
GAC GTT TTG CAT TTA TAC ATC CAT TTG CGA AAA AAC TCT TAA 1866
Asp Val Leu His Leu Tyr He His Leu Arg Lys Asn Ser 610 615 620
(2) INFORMATION FOR SEQ ID NO: 2:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 621 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Val Lys Glu Ser Asp He Leu Val He Gly Gly Gly His Ala Gly
1 5 10 15
He Glu Ala Ser Leu He Ala Ala Lys Met Gly Ala Arg Val His Leu
20 25 30
He Thr Met Leu He Asp Thr He Gly Leu Ala Ser Cys Asn Pro Ala 35 40 45 He Gly Gly Leu Gly Lys Gly H s Leu Thr Lys Glu Val Asp Val Leu
50 55 60
Gly Gly Ala Met Gly He He Thr Asp His Ser Gly Leu Gin Tyr Arg 65 70 75 80
Val Leu Asn Ala Ser Lys Gly Pro Ala Val Arg Gly Thr Arg Ala Gin
85 90 95
He Asp Met Asp Thr Tyr Arg He Phe Ala Arg Asn Leu Val Leu Asn
100 105 110
Thr Pro Asn Leu Ser Val Ser Gin Glu Met Thr Glu Ser Leu He Leu
115 120 125
Glu Asn Asp Glu Val Val Gly Val Thr Thr Asn He Asn Asn Thr Tyr
130 135 140
Arg Ala Lys Lys Val He He Thr Thr Gly Thr Phe Leu Lys Gly Val 145 150 155 160
Val His He Gly Glu His Gin Asn Gin Asn Gly Arg Phe Gly Glu Asn
165 170 175
Ala Ser Asn Ser Leu Ala Leu Asn Leu Arg Glu Leu Gly Phe Lys Val
180 185 190
Glu Arg Leu Lys Thr Gly Thr Cys Pro Arg Val Ala Gly Asn Ser He
195 200 205
Asp Phe Glu Gly Leu Glu Glu His Phe Gly Asp Ala Asn Pro Pro Tyr
210 215 220
Phe Ser Tyr Lys Thr Lys Asp Phe Asn Pro Thr Gin Leu Ser Cys Phe 225 230 235 240
He Thr Tyr Thr Asn Pro He Thr His Gin He He Arg Asp Asn Phe
245 250 255
His Arg Ala Pro Leu Phe Ser Gly Gin He Glu Gly He Gly Pro Arg
260 265 270
Tyr Cys Pro Ser He Glu Asp Lys He Asn Arg Phe Ser Glu Lys Glu
275 280 285
Arg His Gin Leu Phe Leu Glu Pro Gin Thr He His Lys Asn Glu Tyr
290 295 300
Tyr He Asn Gly Leu Ser Thr Ser Leu Pro Leu Asp Val Gin Glu Lys 305 310 315 320
Val He His Ser He Lys Gly Leu Glu Asn Ala Leu He Thr Arg Tyr
325 330 335
Gly Tyr Ala He Glu Tyr Asp Phe He Gin Pro Thr Glu Leu Thr H s
340 345 350
Ala Leu Glu Thr Lys Lys He Lys Gly Leu Tyr Leu Ala Gly Gin He
355 360 365
Asn Gly Thr Thr Gly Tyr Glu Glu Ala Ala Asp Gin Gly Leu Met Ala
370 375 380
Gly He Asn Ala Val Leu Ala Leu Lys Asn Gin Ala Pro Phe He Leu 385 390 395 400
Lys Arg Asn Glu Ala Tyr He Gly Val Leu He Asp Asp Leu Val Thr
405 410 415
Lys Gly Thr Asn Glu Pro Tyr Arg Met Phe Thr Ser Arg Ala Glu Tyr
420 425 430
Arg Leu Leu Leu Arg Glu Asp Asn Thr Leu Phe Arg Leu Gly Glu His
435 440 445
Ala Tyr Arg Leu Gly Leu Met Glu Gin Asp Phe Tyr Lys Glu Leu Lys
450 455 460
Lys Asp Lys Gin Glu He Gin Asp Asn Leu Lys Arg Leu Lys Glu Cys 465 470 475 480
Val Leu Thr Pro Ser Lys Lys Leu Leu Lys Arg Leu Asn Glu Leu Asp
485 490 495
Glu Asn Pro He Asn Asp Lys Val Asn Gly Val Ser Leu Leu Ala Arg
500 505 510
Asp Ser Phe Asn Ala Glu Lys Met Arg Ser Phe Phe Ser Phe Leu Ala 515 520 525 Pro Leu Asn Glu Arg Val Leu Glu Gin He Lys He Glu Cys Lys Tyr
530 535 540
Asn He Tyr He Glu Lys Gin H s Glu Asn He Ala Lys Met Asp Ser
545 550 555 560
Met Leu Lys Val Ser He Pro Lys Gly Phe Val Phe Lys Gly He Pro
565 570 575
Gly Leu Ser Leu Glu Ala Val Glu Lys Leu Glu Lys Phe Arg Pro Lys
580 585 590
Ser Leu Phe Glu Ala Ser Glu He Ser Gly He Thr Pro Ala Asn Leu
595 600 605
Asp Val Leu His Leu Tyr He H s Leu Arg Lys Asn Ser
610 615 620
(2) INFORMATION FOR SEQ ID NO: 3:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1167 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (IX) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...1164 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ATG AAC GCT TTA GAA ATC ACC CAA AAG CTC ATC AGC TAC CCC ACC ATT 48 Met Asn Ala Leu Glu He Thr Gin Lys Leu He Ser Tyr Pro Thr He 1 5 10 15
ACG CCC AAA GAA TGC GGT ATT TTT GAA TAC ATT AAA TCG CTT TTT CCT 96 Thr Pro Lys Glu Cys Gly He Phe Glu Tyr He Lys Ser Leu Phe Pro 20 25 30
GCT TTT AAA ACA CTA GAG TGT GGA GAA AAT GGC GTG AAA AAC CTT TTT 144 Ala Phe Lys Thr Leu Glu Cys Gly Glu Asn Gly Val Lys Asn Leu Phe 35 40 45
TTA TAC CGC ATT TTT AAC CCC CCC AAA GAG CAT GCA GAA AAA GAA CAT 192 Leu Tyr Arg He Phe Asn Pro Pro Lys Glu His Ala Glu Lys Glu His 50 55 60
GCA AAA GAA AAG CAT GCA AAA GAA AAT GTT AAG CCC TTG CAT TTT TCT 240 Ala Lys Glu Lys His Ala Lys Glu Asn Val Lys Pro Leu His Phe Ser 65 70 75 80
TTT GCA GGG CAT ATT GAT GTC GTG CCT CCT GGA GAT AAT TGG CAA AGC 288 Phe Ala Gly His He Asp Val Val Pro Pro Gly Asp Asn Trp Gin Ser 85 90 95
GAT CCC TTT AAA CCC ATC ATT AAA GAG GGG TTT TTA TAC GGC CGT GGG 336 Asp Pro Phe Lys Pro He He Lys Glu Gly Phe Leu Tyr Gly Arg Gly 100 105 110 GCG CAA GAC ATG AAA GGG GGC GTG GGG GCG TTT TTG AGC GCG AGT TTA 384 Ala Gin Asp Met Lys Gly Gly Val Gly Ala Phe Leu Ser Ala Ser Leu 115 120 125
AAT TTT AAC CCT AAA ACC CCT TTT TTG CTT TCT ATT TTA CTC ACG AGC 432 Asn Phe Asn Pro Lys Thr Pro Phe Leu Leu Ser He Leu Leu Thr Ser 130 135 140
GAT GAA GAA GGG CCA GGG ATT TTT GGC ACA AAA CTC ATG CTA GAA AAA 480 Asp Glu Glu Gly Pro Gly He Phe Gly Thr Lys Leu Met Leu Glu Lys 145 150 155 160
CTC AAA GAA AAA GAT TTA TTG CCC CAT ATG GCG ATT GTG GCT GAA CCC 528 Leu Lys Glu Lys Asp Leu Leu Pro His Met Ala He Val Ala Glu Pro 165 170 175
ACT TGC GAA AAA GTC TTA GGC GAT AGC ATC AAA ATT GGT CGA AGA GGT 576 Thr Cys Glu Lys Val Leu Gly Asp Ser He Lys He Gly Arg Arg Gly 180 185 190
TCC ATT AAT GGC AGA CTC ATT TTA AAA GGC GTT CAA GGG CAT GTG GCT 624 Ser He Asn Gly Arg Leu He Leu Lys Gly Val Gin Gly His Val Ala 195 200 205
TAC CCA CAA AAA TGC CAA AAC CCC ATT GAT ACG CTC GCT TCT GTT TTG 672 Tyr Pro Gin Lys Cys Gin Asn Pro He Asp Thr Leu Ala Ser Val Leu 210 215 220
CCT TCA ATT TCA GGA GTC CAT TTA GAC GAT GGC GAT GAA TAT TTT GAC 720 Pro Ser He Ser Gly Val His Leu Asp Asp Gly Asp Glu Tyr Phe Asp 225 230 235 240
CCT TCA AAA TTG GTT GTC ACC AAC TTG CAT GCA GGG TTA GGG GCT AAT 768 Pro Ser Lys Leu Val Val Thr Asn Leu His Ala Gly Leu Gly Ala Asn 245 250 255
AAT GTG ACT CCA GGG AGC GTA GAA ATT ACC TTT AAT GCG CGC CAT TCT 816 Asn Val Thr Pro Gly Ser Val Glu He Thr Phe Asn Ala Arg H s Ser 260 265 270
TTA AAA ACC ACC AAA GAG AGT TTG AAA GAA TAT TTA GAA AAA GTT TTA 864 Leu Lys Thr Thr Lys Glu Ser Leu Lys Glu Tyr Leu Glu Lys Val Leu 275 280 285
AAA GAT TTG CCT CAC ACT TTA GAA TTA GAG TCA AGC AGT TCG CCT TTC 912 Lys Asp Leu Pro His Thr Leu Glu Leu Glu Ser Ser Ser Ser Pro Phe 290 295 300
ATC ACG GCT TCT CAT TCA AAG CTT ACC AGC GTT TTA AAA GAA AAT ATT 960 He Thr Ala Ser His Ser Lys Leu Thr Ser Val Leu Lys Glu Asn He 305 310 315 320
TTA AAA ACA TGC CGC ACC ACC CCC CTT TTA AAC ACC AAA GGC GGC ACG 1008 Leu Lys Thr Cys Arg Thr Thr Pro Leu Leu Asn Thr Lys Gly Gly Thr 325 330 335
AGC GAT GCG CGA TTT TTT AGC GCT CAT GGT ATA GAA GTG GTG GAG TTT 1056 Ser Asp Ala Arg Phe Phe Ser Ala His Gly He Glu Val Val Glu Phe 340 345 350 GGC GTT ATT AAT GAC AGG ATC CAT GCC ATT GAT GAA AGG GTG AGC TTG 1104 Gly Val He Asn Asp Arg He His Ala He Asp Glu Arg Val Ser Leu 355 360 365
AAA GAA TTA GAG CTT TTA GAA AAA GTG TTT TTG GGG GTT TTA GAG GGC 1152 Lys Glu Leu Glu Leu Leu Glu Lys Val Phe Leu Gly Val Leu Glu Gly 370 375 380
TTG AGT GAG GCA TAA 1167
Leu Ser Glu Ala
385
(2) INFORMATION FOR SEQ ID NO: 4:
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 388 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(x ) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Asn Ala Leu Glu He Thr Gin Lys Leu He Ser Tyr Pro Thr He
1 5 10 15
Thr Pro Lys Glu Cys Gly He Phe Glu Tyr He Lys Ser Leu Phe Pro
20 25 30
Ala Phe Lys Thr Leu Glu Cys Gly Glu Asn Gly Val Lys Asn Leu Phe
35 40 45
Leu Tyr Arg He Phe Asn Pro Pro Lys Glu His Ala Glu Lys Glu His
50 55 60
Ala Lys Glu Lys His Ala Lys Glu Asn Val Lys Pro Leu His Phe Ser 65 70 75 80
Phe Ala Gly His He Asp Val Val Pro Pro Gly Asp Asn Trp Gin Ser
85 90 95
Asp Pro Phe Lys Pro He He Lys Glu Gly Phe Leu Tyr Gly Arg Gly
100 105 110
Ala Gin Asp Met Lys Gly Gly Val Gly Ala Phe Leu Ser Ala Ser Leu
115 120 125
Asn Phe Asn Pro Lys Thr Pro Phe Leu Leu Ser He Leu Leu Thr Ser
130 135 140
Asp Glu Glu Gly Pro Gly He Phe Gly Thr Lys Leu Met Leu Glu Lys 145 150 155 160
Leu Lys Glu Lys Asp Leu Leu Pro His Met Ala He Val Ala Glu Pro
165 170 175
Thr Cys Glu Lys Val Leu Gly Asp Ser He Lys He Gly Arg Arg Gly
180 185 190
Ser He Asn Gly Arg Leu He Leu Lys Gly Val Gin Gly His Val Ala
195 200 205
Tyr Pro Gin Lys Cys Gin Asn Pro He Asp Thr Leu Ala Ser Val Leu
210 215 220
Pro Ser He Ser Gly Val His Leu Asp Asp Gly Asp Glu Tyr Phe Asp 225 230 235 240
Pro Ser Lys Leu Val Val Thr Asn Leu His Ala Gly Leu Gly Ala Asn
245 250 255
Asn Val Thr Pro Gly Ser Val Glu He Thr Phe Asn Ala Arg His Ser 260 265 270 Leu Lys Thr Thr Lys Glu Ser Leu Lys Glu Tyr Leu Glu Lys Val Leu
275 280 285
Lys Asp Leu Pro His Thr Leu Glu Leu Glu Ser Ser Ser Ser Pro Phe
290 295 300
He Thr Ala Ser His Ser Lys Leu Thr Ser Val Leu Lys Glu Asn He 305 310 315 320
Leu Lys Thr Cys Arg Thr Thr Pro Leu Leu Asn Thr Lys Gly Gly Thr
325 330 335
Ser Asp Ala Arg Phe Phe Ser Ala His Gly He Glu Val Val Glu Phe
340 345 350
Gly Val He Asn Asp Arg He His Ala He Asp Glu Arg Val Ser Leu
355 360 365
Lys Glu Leu Glu Leu Leu Glu Lys Val Phe Leu Gly Val Leu Glu Gly
370 375 380
Leu Ser Glu Ala 385
(2) INFORMATION FOR SEQ ID NO : 5 :
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 753 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (lx) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 1...750 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ATG CTA GGA AGC GTT AAA AAA ACC TTT TTT TGG GTC TTG TGT TTG GGC 48 Met Leu Gly Ser Val Lys Lys Thr Phe Phe Trp Val Leu Cys Leu Gly 1 5 10 15
GCG TTG TGT TTA AGA GGG TTA ATG GCA GAG CCA GAC GCT AAA GAG CTT 96 Ala Leu Cys Leu Arg Gly Leu Met Ala Glu Pro Asp Ala Lys Glu Leu 20 25 30
GTT AAT TTA GGC ATA GAG AGC GCG AAG AAG CAA GAT TTC GCT CAA GCT 144 Val Asn Leu Gly He Glu Ser Ala Lys Lys Gin Asp Phe Ala Gin Ala 35 40 45
AAA ACG CAT TTT GAA AAA GCT TGT GAG TTA AAA AAT GGC TTT GGG TGT 192 Lys Thr His Phe Glu Lys Ala Cys Glu Leu Lys Asn Gly Phe Gly Cys 50 55 60
GTT TTT TTA GGG GCG TTC TAT GAA GAA GGG AAA GGA GTG GGA AAA GAC 240 Val Phe Leu Gly Ala Phe Tyr Glu Glu Gly Lys Gly Val Gly Lys Asp 65 70 75 80
TTG AAA AAA GCC ATC CAG TTT TAC ACT AAA AGT TGT GAA TTA AAT GAT 288 Leu Lys Lys Ala He Gin Phe Tyr Thr Lys Ser Cys Glu Leu Asn Asp 85 90 95 GGT TAT GGG TGC AAC CTG CTA GGA AAT TTA TAC TAT AAC GGA CAA GGC 336
Gly Tyr Gly Cys Asn Leu Leu Gly Asn Leu Tyr Tyr Asn Gly Gin Gly 100 105 110
GTA TCT AAA GAC GCT AAA AAA GCC TCA CAA TAC TAC TCT AAA GCT TGC 384
Val Ser Lys Asp Ala Lys Lys Ala Ser Gin Tyr Tyr Ser Lys Ala Cys 115 120 125
GAC TTA AAC CAT GCT GAA GGG TGT ATG GTA TTA GGA AGC TTA CAC CAT 432
Asp Leu Asn His Ala Glu Gly Cys Met Val Leu Gly Ser Leu His His 130 135 140
TAT GGC GTA GGC ACG CCT AAG GAT TTA AGA AAG GCT CTT GAT TTG TAT 480
Tyr Gly Val Gly Thr Pro Lys Asp Leu Arg Lys Ala Leu Asp Leu Tyr
145 150 155 160
GAA AAA GCT TGC GAT TTA AAA GAC AGC CCA GGG TGT ATT AAT GCA GGA 528
Glu Lys Ala Cys Asp Leu Lys Asp Ser Pro Gly Cys He Asn Ala Gly 165 170 175
TAT ATA TAT AGT GTA ACA AAG AAT TTT AAG GAG GCT ATC GTT CGT TAT 576
Tyr He Tyr Ser Val Thr Lys Asn Phe Lys Glu Ala He Val Arg Tyr 180 185 190
TCT CAA GCA TGC GAG TTG AAC GAT GGT AGG GGG TGT TAT AAT TTA GGG 624
Ser Gin Ala Cys Glu Leu Asn Asp Gly Arg Gly Cys Tyr Asn Leu Gly 195 200 205
GTT ATG CAA TAC AAC GCT CAA GGC ACA GCA AAA GAC GAA AAG CAA GCG 672
Val Met Gin Tyr Asn Ala Gin Gly Thr Ala Lys Asp Glu Lys Gin Ala 210 215 220
GTA GAA AAC TTT AAA AAA GGT TGC AAA TCA GGC GTT AAA GAA GCA TGC 720
Val Glu Asn Phe Lys Lys Gly Cys Lys Ser Gly Val Lys Glu Ala Cys
225 230 235 240
GAC GCT CTC AAG GAA TTG AAA ATA GAA CTT TAG 753
Asp Ala Leu Lys Glu Leu Lys He Glu Leu 245 250
(2) INFORMATION FOR SEQ ID NO : 6 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 250 ammo acids
(B) TYPE: ammo ac d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Leu Gly Ser Val Lys Lys Thr Phe Phe Trp Val Leu Cys Leu Gly
1 5 10 15
Ala Leu Cys Leu Arg Gly Leu Met Ala Glu Pro Asp Ala Lys Glu Leu
20 25 30
Val Asn Leu Gly He Glu Ser Ala Lys Lys Gin Asp Phe Ala Gin Ala 35 40 45 Lys Thr His Phe Glu Lys Ala Cys Glu Leu Lys Asn Gly Phe Gly Cys
50 55 60
Val Phe Leu Gly Ala Phe Tyr Glu Glu Gly Lys Gly Val Gly Lys Asp 65 70 75 80
Leu Lys Lys Ala He Gin Phe Tyr Thr Lys Ser Cys Glu Leu Asn Asp
85 90 95
Gly Tyr Gly Cys Asn Leu Leu Gly Asn Leu Tyr Tyr Asn Gly Gin Gly
100 105 110
Val Ser Lys Asp Ala Lys Lys Ala Ser Gin Tyr Tyr Ser Lys Ala Cys
115 120 125
Asp Leu Asn His Ala Glu Gly Cys Met Val Leu Gly Ser Leu His His
130 135 140
Tyr Gly Val Gly Thr Pro Lys Asp Leu Arg Lys Ala Leu Asp Leu Tyr 145 150 155 160
Glu Lys Ala Cys Asp Leu Lys Asp Ser Pro Gly Cys He Asn Ala Gly
165 170 175
Tyr He Tyr Ser Val Thr Lys Asn Phe Lys Glu Ala He Val Arg Tyr
180 185 190
Ser Gin Ala Cys Glu Leu Asn Asp Gly Arg Gly Cys Tyr Asn Leu Gly
195 200 205
Val Met Gin Tyr Asn Ala Gin Gly Thr Ala Lys Asp Glu Lys Gin Ala
210 215 220
Val Glu Asn Phe Lys Lys Gly Cys Lys Ser Gly Val Lys Glu Ala Cys 225 230 235 240
Asp Ala Leu Lys Glu Leu Lys He Glu Leu 245 250
(2) INFORMATION FOR SEQ ID NO : 7 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5049 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
AATTCCCTAT CATGAAACCT AAAATCAATC TCCTAGGGCT TGTGCCTACT AGTAAATAAT 60
GCTTAAAGCG ATGCGCGTGT GCAAATTCCA TTTTTGCATG CTTAAAGCTA AAATAAACCC 120
TCCCATAAAA AGAAAGATAA TCGGCGATGC GTAAGAAGAG CTGACGCTAG CGAATTGATC 180
CACGCTAAAG ACGCTAAAAA GCACCAAAGG TAAAAGCGCG GTTGCAGGCA GGTCAATGGT 240
TTCTGTCATC CACCATATCC CCATTAAAAC AGCCACCCCA GCCACAACAG GCATCGCCTT 300
ATAATTTAAG GAATTAAGCT TGGGGATTTC TTCTACAATA TGAGGCAGTT GAGAATTGAG 360
CGCATAACAG ATAATAAGCG CGATTAACAC TCCTCCTATT AACCCCAACA AGTGCACGAT 420
CTTAGTGCTT TTATCATCGG TGCGCGTATC GGTATGCGTA TTGGCATGCG AATGATTTTC 480
CATTTTATTT TACCCTTTAA AATTACTAAC CTCCATGCTA CAATAAAACG TTTTCAAAAC 540
TAAGATTTTA GAAAAATCAT ATCAAAACAG GAAAAAGAGT GGTAAAAGAA AGTGATATTT 600
TAGTGATTGG TGGGGGGCAT GCGGGCATTG AAGCGAGCTT GATTGCAGCC AAAATGGGGG 660
CTAGGGTGCA TTTAATCACC ATGCTCATAG ACACGATCGG TTTAGCGAGC TGTAACCCGG 720
CGATTGGGGG CTTGGGTAAA GGGCATTTGA CTAAAGAAGT GGATGTTTTA GGGGGGGCTA 780
TGGGGATTAT TACAGATCAT AGCGGTTTGC AATATCGTGT GTTAAACGCT TCTAAAGGGC 840
CGGCGGTTAG GGGGACTAGA GCGCAAATTG ATATGGATAC TTACCGCATT TTTGCAAGAA 900
ATCTTGTTTT AAACACCCCT AATTTGAGCG TCTCTCAAGA AATGACCGAA AGTTTAATCC 960
TTGAAAACGA TGAGGTAGTG GGCGTAACCA CGAACATTAA TAACACTTAC AGAGCTAAAA 1020
AAGTGATCAT CACCACAGGC ACTTTTTTAA AAGGGGTGGT GCATATTGGC GAGCACCAAA 1080
ACCAAAACGG GCGTTTTGGG GAAAACGCTT CCAATTCTTT AGCCTTGAAT TTAAGGGAGC 1140
TTGGCTTTAA GGTGGAGAGG TTAAAAACCG GCACTTGCCC AAGAGTGGCC GGCAATAGCA 1200
TTGATTTTGA AGGCTTAGAA GAGCATTTTG GGGATGCAAA CCCTCCCTAT TTCAGCTATA 1260
AAACCAAAGA TTTTAACCCC ACCCAACTCT CTTGTTTCAT CACTTACACT AACCCCATTA 1320 CCCACCAAAT CATTAGGGAT AATTTCCACC GAGCTCCCCT TTTTAGCGGT CAAATTGAAG 1380
GCATAGGCCC AAGGTATTGC CCTAGCATTG AAGATAAAAT CAACCGCTTT AGTGAAAAAG 1440
AACGCCACCA GCTGTTTTTA GAGCCTCAAA CCATTCATAA AAACGAATAT TATATCAACG 1500
GCTTAAGCAC CTCTTTGCCC CTAGATGTGC AAGAAAAGGT CATTCATTCT ATCAAAGGCT 1560
TAGAAAACGC CCTCATCACG CGCTATGGCT ATGCGATAGA GTATGATTTC ATCCAGCCTA 1620
CAGAATTAAC CCACGCTTTA GAAACCAAAA AAATCAAAGG GCTTTATTTG GCCGGGCAAA 1680
TCAATGGGAC TACCGGCTAT GAAGAAGCGG CGGATCAAGG GCTTATGGCT GGGATTAATG 1740
CGGTATTAGC CTTAAAGAAT CAAGCCCCCT TTATTTTAAA GCGCAATGAA GCTTATATTG 1800
GCGTTTTGAT TGATGATTTG GTTACTAAAG GCACGAATGA GCCTTACAGA ATGTTTACTA 1860
GCCGAGCCGA ATACCGCTTG CTTTTAAGAG AGGACAACAC GCTTTTTAGG TTGGGCGAAC 1920
ATGCCTATCG TTTAGGGCTT ATGGAACAGG ATTTTTATAA GGAATTAAAA AAAGATAAAC 1980
AAGAGATACA AGACAATCTC AAACGCCTTA AAGAATGCGT CCTTACCCCT AGTAAAAAAT 2040
TGTTAAAACG CTTGAACGAA TTAGACGAAA ACCCTATCAA TGACAAGGTT AATGGCGTTA 2100
GTTTGTTAGC ACGCGATAGT TTTAATGCAG AAAAAATGCG CTCCTTTTTC AGCTTTTTAG 2160
CCCCCTTGAA CGAGCGGGTT TTAGAGCAGA TTAAAATTGA ATGCAAATAT AATATTTATA 2220
TTGAAAAGCA ACACGAAAAT ATCGCTAAAA TGGATAGCAT GCTCAAAGTT TCTATCCCTA 2280
AAGGTTTTGT GTTTAAAGGC ATTCCAGGCT TAAGCTTAGA AGCGGTAGAA AAATTAGAAA 2340
AATTCCGCCC CAAAAGCCTT TTTGAAGCCT CAGAAATCAG CGGGATCACT CCAGCGAATT 2400
TAGACGTTTT GCATTTATAC ATCCATTTGC GAAAAAACTC TTAAAGGATT TTTAATGAAC 2460
GCTTTAGAAA TCACCCAAAA GCTCATCAGC TACCCCACCA TTACGCCCAA AGAATGCGGT 2520
ATTTTTGAAT ACATTAAATC GCTTTTTCCT GCTTTTAAAA CACTAGAGTG TGGAGAAAAT 2580
GGCGTGAAAA ACCTTTTTTT ATACCGCATT TTTAACCCCC CCAAAGAGCA TGCAGAAAAA 2640
GAACATGCAA AAGAAAAGCA TGCAAAAGAA AATGTTAAGC CCTTGCATTT TTCTTTTGCA 2700
GGGCATATTG ATGTCGTGCC TCCTGGAGAT AATTGGCAAA GCGATCCCTT TAAACCCATC 2760
ATTAAAGAGG GGTTTTTATA CGGCCGTGGG GCGCAAGACA TGAAAGGGGG CGTGGGGGCG 2820
TTTTTGAGCG CGAGTTTAAA TTTTAACCCT AAAACCCCTT TTTTGCTTTC TATTTTACTC 2880
ACGAGCGATG AAGAAGGGCC AGGGATTTTT GGCACAAAAC TCATGCTAGA AAAACTCAAA 2940
GAAAAAGATT TATTGCCCCA TATGGCGATT GTGGCTGAAC CCACTTGCGA AAAAGTCTTA 3000
GGCGATAGCA TCAAAATTGG TCGAAGAGGT TCCATTAATG GCAGACTCAT TTTAAAAGGC 3060
GTTCAAGGGC ATGTGGCTTA CCCACAAAAA TGCCAAAACC CCATTGATAC GCTCGCTTCT 3120
GTTTTGCCTT CAATTTCAGG AGTCCATTTA GACGATGGCG ATGAATATTT TGACCCTTCA 3180
AAATTGGTTG TCACCAACTT GCATGCAGGG TTAGGGGCTA ATAATGTGAC TCCAGGGAGC 3240
GTAGAAATTA CCTTTAATGC GCGCCATTCT TTAAAAACCA CCAAAGAGAG TTTGAAAGAA 3300
TATTTAGAAA AAGTTTTAAA AGATTTGCCT CACACTTTAG AATTAGAGTC AAGCAGTTCG 3360
CCTTTCATCA CGGCTTCTCA TTCAAAGCTT ACCAGCGTTT TAAAAGAAAA TATTTTAAAA 3420
ACATGCCGCA CCACCCCCCT TTTAAACACC AAAGGCGGCA CGAGCGATGC GCGATTTTTT 3480
AGCGCTCATG GTATAGAAGT GGTGGAGTTT GGCGTTATTA ATGACAGGAT CCATGCCATT 3540
GATGAAAGGG TGAGCTTGAA AGAATTAGAG CTTTTAGAAA AAGTGTTTTT GGGGGTTTTA 3600
GAGGGCTTGA GTGAGGCATA AAATAAATAA ACATTAAGTA AGGCTTATCA ATATTTGATT 3660
ACAATTATAA AGGGTTACAT TTTTTTAATA GGAGATATAC CATGCTAGGA AGCGTTAAAA 3720
AAACCTTTTT TTGGGTCTTG TGTTTGGGCG CGTTGTGTTT AAGAGGGTTA ATGGCAGAGC 3780
CAGACGCTAA AGAGCTTGTT AATTTAGGCA TAGAGAGCGC GAAGAAGCAA GATTTCGCTC 3840
AAGCTAAAAC GCATTTTGAA AAAGCTTGTG AGTTAAAAAA TGGCTTTGGG TGTGTTTTTT 3900
TAGGGGCGTT CTATGAAGAA GGGAAAGGAG TGGGAAAAGA CTTGAAAAAA GCCATCCAGT 3960
TTTACACTAA AAGTTGTGAA TTAAATGATG GTTATGGGTG CAACCTGCTA GGAAATTTAT 4020
ACTATAACGG ACAAGGCGTA TCTAAAGACG CTAAAAAAGC CTCACAATAC TACTCTAAAG 4080
CTTGCGACTT AAACCATGCT GAAGGGTGTA TGGTATTAGG AAGCTTACAC CATTATGGCG 4140
TAGGCACGCC TAAGGATTTA AGAAAGGCTC TTGATTTGTA TGAAAAAGCT TGCGATTTAA 4200
AAGACAGCCC AGGGTGTATT AATGCAGGAT ATATATATAG TGTAACAAAG AATTTTAAGG 4260
AGGCTATCGT TCGTTATTCT CAAGCATGCG AGTTGAACGA TGGTAGGGGG TGTTATAATT 4320
TAGGGGTTAT GCAATACAAC GCTCAAGGCA CAGCAAAAGA CGAAAAGCAA GCGGTAGAAA 4380
ACTTTAAAAA AGGTTGCAAA TCAGGCGTTA AAGAAGCATG CGACGCTCTC AAGGAATTGA 4440
AAATAGAACT TTAGTTTCAA TAAAGTTAAG CCAAACGCCG TGTTTAGCTG GCTTCTACGC 4500
TTTTTAATAT CTTAATGAAA GCATAAACCC TACAAACTAA TCTTTTAATC ATAATAAGGG 4560
TTTTATATCG CACCCATTCA TTGCCGTTTT TAGATTGGCG CTTGAAAGGT TTAAAGCAAG 4620
TTTGTTCAAA CCCTTAAAAA GGGTTTTTAA CCCCTACAAC GCTTTCAATA GCACGCTATT 4680
TAGGCGTTCG GTAAAACTTT TAGCGTCTTT TAAAGCCCCT TTTTCTAAAA GCTTCGCCCC 4740
ATCATAAAGC AACCAGATAA AAGCGTTCAA CTGCTCTTTA TCTTCGCATT TTAAGAGTTT 4800
TTGGAAAATC GCATGGTTAG GGTTTAATTC TAGCGTTTTC TTGCTTTCAG GCACGCTTTG 4860
ACCCATTTGA CGCATAAAAT TAGCCATCAT CGCATTTTGG TCATCGCCTA TTAAAGCCAC 4920
CGCTGAAGTG AGATGACTGG AAAGCTCTAC GCCTTTAATC TCATCTTTAA GATTTTCTTC 4980 AAACGCTTTC ATTAAATCTT TAAACTGATC TTTTATCTCA TCAAGGATTT CTTCCAAACC 5040 AAGGGTTAA 5049
(2) INFORMATION FOR SEQ ID NO: 8:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CAGGAAAAAG AG GGTAA 18
(2) INFORMATION FOR SEQ ID NO : 9 :
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TTAAGAGTTT TTTCGCAA 18
(2) INFORMATION FOR SEQ ID NO: 10:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: AAGGATATTT AATGAACG 18
(2) INFORMATION FOR SEQ ID NO: 11:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x ) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GTTTATTTAT TTTATGCCTC A 21
(2) INFORMATION FOR SEQ ID NO: 12:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: TAATTTAGGC ATAGAGAGC 19
(2) INFORMATION FOR SEQ ID NO: 13:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TATAACGGAC AAGGCGTATC T 21
(2) INFORMATION FOR SEQ ID NO: 14:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(x ) SEQUENCE DESCRIPTION: SEQ ID NO:14: GTTCTATTTT CAATTCCTTG AGAG 24
(2) INFORMATION FOR SEQ ID NO: 15:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GCGTGAATGA ATACGATA 18
(2) INFORMATION FOR SEQ ID NO: 16:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CTCCCACCAG CTTATATACC TTAG 24
(2) INFORMATION FOR SEQ ID NO: 17: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CTGGGGATCA AGCCTGATTG G 21
(2) INFORMATION FOR SEQ ID NO: 18:
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GACCGTTCCG TGGCAAAGCA 20
(2) INFORMATION FOR SEQ ID NO: 19:
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CTTGTGCAAT GTAACATCAG AG 22
(2) INFORMATION FOR SEQ ID NO: 20:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GCATTCCAGG CTTAAGCT 18
(2) INFORMATION FOR SEQ ID NO: 21:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic ac d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: TGCATGTTCT TTTTCTGCAT 20
(2) INFORMATION FOR SEQ ID NO: 22:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:22: GAGTTTGGCG TTATTAAT 18
(2) INFORMATION FOR SEQ ID NO: 23:
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:23: GCTTTTTCAA AATGCGT 17
(2) INFORMATION FOR SEQ ID NO: 24:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: AAGCTTGATC ACTCC 15

Claims

What is claimed is:
1. An isolated dapE gene of Heli cobacter pyl ori .
2 . The dapE gene of claim 1, consisting of the nucleotide sequence defined in SEQ ID NO:l.
3. A Heli cobacter pylori-specific nucleic acid fragment of the gene of claim 2.
4. A nucleic acid that encodes a naturally occurring DapE protein of Helicobacter pylori and hybridizes with the nucleic acid of SEQ ID NO:l under the stringency conditions of about 16 hrs at about 65°C, about 5x SSC, about 0.1% SDS, about 2x Denhardt ' s solution, about 150 μg/ml salmon sperm DNA with washing at about 65°C, 30 min, 2x, in about O.lx SSPE/0.1% SDS.
5. A nucleic acid probe that hybridizes with the nucleic acid of SEQ ID NO:l under the stringency conditions of about 16 hrs at about 65°C, about 5x SSC, about 0.1% SDS, about 2x Denhardt ' s solution, about 150 μg/ml salmon sperm DNA with washing at about 65°C, 30 min, 2x, in about O.lx SSPE/0.1% SDS.
6. A nucleic acid primer that hybridizes with the nucleic acid of SEQ ID NO:l under the stringency conditions of 35 cycles of 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min, with a terminal extension at 72°C for 10 min.
7. A purified mutant strain of Helicobacter pyl ori that does not express a functional DapE protein.
8. The mutant strain of claim 7 deposited with the American Type Culture Collection under accession no. ATCC 55897.
9. The mutant Heli cobacter pylori of claim 7, containing a plasmid comprising a nucleic acid encoding a functional DapE protein.
10. The mutant strain of claim 7, having in its chromosome a nucleic acid encoding a foreign protein.
11. The mutant strain of claim 10, wherein the foreign protein encoded is an immunogen of an infectious bacterium, virus, yeast, fungus or parasite selected from the group consisting of Salmonella enteri tidis, Shigella species, Yersinia, enterotoxigenic and enterohemorrhagic E. coli , Mycobacteri um tuberculosis, Streptococcus pyogenes, Bordatella pertussi s, Bacill us anthraci s, P. fal ciparum, human immunodeficiency virus, respiratory syncytial virus, influenza virus, histoplasma capsula tum .
12. The mutant strain of claim 10, wherein the foreign protein encoded is a sperm immunogen.
13. The mutant Helicobacter pyl ori of claim 10, containing a plasmid comprising a nucleic acid encoding a functional DapE protein.
14. The mutant strain of claim 7, having a plasmid comprising a nucleic acid encoding a foreign protein.
15. The mutant strain of claim 14, wherein the foreign protein encoded is an immunogen of an infectious bacterium, virus, yeast, fungus or parasite selected from the group consisting of Salmonella enteri tidis, Shigella species, Yersinia, enterotoxigenic and enterohemorrhagic E. coli , Mycobacteri um tubercul osi s, Streptococcus pyogenes, Borda tella pertussis, Bacill us anthracis, P. fal ciparum, human immunodeficiency virus, respiratory syncytial virus, influenza virus, histoplasma capsulatum .
16. The mutant Heli cobacter pylori of claim 14, containing a plasmid comprising a nucleic acid encoding a functional DapE protein.
17. A method of maintaining long term expression of a foreign antigen in Heli cobacter pylori , comprising: a. transforming a mutant Heli cobacter pyl ori of claim 7 with a plasmid comprising a nucleic acid encoding a functional DapE protein comprising a nucleic acid encoding the foreign protein; and b. maintaining the mutant Heli cobacter pylori from step a under conditions that permit expression of the foreign protein.
18. A method of maintaining the expression of a foreign protein in Helicobacter pylori , comprising: a. transforming a mutant Heli cobacter pylori of claim 7 having in its chromosome a nucleic acid encoding the foreign protein with a plasmid comprising a nucleic acid encoding a functional DapE protein; b. maintaining the mutant Heli cobacter pylori from step a under conditions that permit expression of the foreign protein.
19. A method of immunizing a subject against infection with Helicobacter pylori , comprising: a. administering to the subject the mutant strain of claim 7; b. supplementing the subject's diet with diaminopimelic acid to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the strain; and c. ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject.
20. A method of immunizing a subject against infection with a bacteria, virus, fungus or parasite, comprising: a) administering to the subject the mutant strain of claim 11; b) supplementing the subject's diet with diaminopimelic acid to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the foreign; and c) ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject.
21. A method of immunizing a subject against infection with a bacteria, virus, fungus or parasite, comprising: a) administering to the subject the mutant strain of claim 15; b) supplementing the subject's diet with diaminopimelic acid to maintain the mutant strain in the subject at least long enough for the subject to mount an immune response to the foreign protein; and c) ceasing the supplementation of the subject's diet with diaminopimelic acid to kill the mutant strain in the immunized subject.
PCT/US1997/024147 1996-12-23 1997-12-23 dapE GENE OF HELICOBACTER PYLORI AND dapE- MUTANT STRAINS OF $i(HELICOBACTER PYLORI) WO1998027819A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU59576/98A AU5957698A (en) 1996-12-23 1997-12-23 (dape) gene of (helicobacter pylori) and (dape)- mutant strains of (helicobacter pylori)
US09/331,568 US6570004B1 (en) 1996-12-23 1997-12-23 dapE gene on Helicobacter pylori and dapE− mutant strains of Helicobacter pylori

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3382496P 1996-12-23 1996-12-23
US60/033,824 1996-12-23

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09/331,568 A-371-Of-International US6570004B1 (en) 1996-12-23 1997-12-23 dapE gene on Helicobacter pylori and dapE− mutant strains of Helicobacter pylori
US10/447,013 Division US20030204068A1 (en) 1996-12-23 2003-05-27 dapE gene of helicobacter pylori and dapE- mutant strains of helicobacter pylori

Publications (1)

Publication Number Publication Date
WO1998027819A1 true WO1998027819A1 (en) 1998-07-02

Family

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WO2006015445A1 (en) * 2004-08-13 2006-02-16 Marshall Barry J Bacterial delivery system
JP2008509168A (en) * 2004-08-13 2008-03-27 マーシャル，バリー，ジェー． Bacteria delivery system
AU2005270660B2 (en) * 2004-08-13 2010-11-11 Barry J. Marshall Bacterial delivery system
US7968324B2 (en) * 2004-08-13 2011-06-28 Barry J Marshall Helicobacter system and uses thereof
US8420374B2 (en) 2004-08-13 2013-04-16 Ondek Pty. Ltd. Helicobacter system and uses thereof
JP2010508828A (en) * 2006-11-10 2010-03-25 マーシャル，バリー，ジェー． Method and apparatus for delivering peptides into the gastric mucosa

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