Compounds and methods for treatment and diagnosis of mycobacterial infections

TECHNICAL FIELD
The present invention relates generally to the detection, treatment and prevention of infectious diseases. In particular, the invention is related to compounds and methods for the treatment of mycobacterial infections including Mycobacterium tuberculosis and Mycobacterium avium. The invention is further related to compounds that function as non-specific immune response amplifiers, and the use of such non-specific immune response amplifiers as adjuvants in vaccination or immunotherapy against infectious disease, and in certain treatments for immune disorders and cancer.
BACKGROUND OF THE INVENTION
Tuberculosis is a chronic, infectious disease, that is caused by infection with Mycobacterium tuberculosis (M. tuberculosis). It is a major disease in developing countries, as well as an increasing problem in developed areas of the world, with about 8 million new cases and 3 million deaths each year. Although the infection may be asymptomatic for a considerable period of time, the disease is most commonly manifested as a chronic inflammation of the lungs, resulting in fever and respiratory symptoms. If left untreated, significant morbidity and death may result.
Although tuberculosis can generally be controlled using extended antibiotic therapy, such treatment is not sufficient to prevent the spread of the disease. Infected individuals may be asymptomatic, but contagious, for some time. In addition, although compliance with the treatment regimen is critical, patient behaviour is difficult to monitor. Some patients do not complete the course of treatment, which can lead to ineffective treatment and the development of drug resistant mycobacteria.
Inhibiting the spread of tuberculosis requires effective vaccination and accurate, early diagnosis of the disease. Currently, vaccination with live bacteria is the most efficient method for inducing protective immunity. The most common mycobacterium employed for this purpose is Bacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacterium bovis. However, the safety and efficacy of BCG is a source of controversy and some countries, such as the United States, do not vaccinate the general public. Diagnosis of M. tuberculosis infection is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, thereby indicating exposure to mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals.
A less well-known mycobacterium that has been used for immunotherapy for tuberculosis, and also leprosy, is Mycobacterium vaccae, which is non-pathogenic in humans. However, there is less information on the efficacy of M. vaccae compared with BCG, and it has not been used widely to vaccinate the general public. M. bovis BCG and M. vaccae are believed to contain antigenic compounds that are recognised by the immune system of individuals exposed to infection with M. tuberculosis.
Several patents and other publications disclose treatment of various conditions by administering mycobacteria, including M. vaccae, or certain mycobacterial fractions. International Patent Publication WO 91/02542 discloses treatment of chronic inflammatory disorders in which a patient demonstrates an abnormally high release of IL-6 and/or TNF or in which the patient's IgG shows an abnormally high proportion of agalactosyl IgG. Among the disorders mentioned in this publication are psoriasis, rheumatoid arthritis, mycobacterial disease, Crohn's disease, primary biliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, Guillain-Barre syndrome, primary diabetes mellitus, and some aspects of graft rejection. The therapeutic agent preferably comprises autoclaved M. vaccae administered by injection in a single dose.
U.S. Pat. No. 4,716,038 discloses diagnosis of, vaccination against and treatment of autoimmune diseases of various types, including arthritic diseases, by administering mycobacteria, including M. vaccae. U.S. Pat. No. 4,724,144 discloses an immunotherapeutic agent comprising antigenic material derived from M. vaccae for treatment of mycobacterial diseases, especially tuberculosis and leprosy, and as an adjuvant to chemotherapy. International Patent Publication WO 91/01751 discloses the use of antigenic and/or immunoregulatory material from M. vaccae as an immunoprophylactic to delay and/or prevent the onset of AIDS. International Patent Publication WO 94/06466 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for therapy of HIV infection, with or without AIDS and with or without associated tuberculosis.
U.S. Pat. No. 5,599,545 discloses the use of mycobacteria, especially whole, inactivated M. vaccae, as an adjuvant for administration with antigens which are not endogenous to M. vaccae. This publication theorises that the beneficial effect as an adjuvant may be due to heat shock protein 65 (hsp 65). International Patent Publication WO 92/08484 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of uveitis. International Patent Publication WO 93/16727 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for the treatment of mental diseases associated with an autoimmune reaction initiated by an infection. International Patent Publication WO 95/26742 discloses the use of antigenic and/or immunoregulatory material derived from M. vaccae for delaying or preventing the growth or spread of tumors.
There remains a need in the art for effective compounds and methods for preventing, treating and detecting tuberculosis.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compounds and methods for the prevention, treatment and diagnosis of mycobacterial infection, together with adjuvants for use in vaccines or immunotherapy of infectious diseases and cancers.
In a first aspect, polypeptides derived from Mycobacterium vaccae are provided comprising an immunogenic portion of an antigen, or a variant of such an antigen. In one embodiment, the antigen includes an amino acid sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 70, 75, 89, 94, 100, 105, 109, 110, 112, 121, 124, 125, 134, 135, 140, 141, 143, 145, 147, 152, 154 156, 158, 160, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192 and 194; and (b) sequences having at least about a 99% probability of being the same as a sequence recited in SEQ ID NOS: 70, 75, 89, 94, 100, 105, 109, 110, 112, 121, 124, 125, 134, 135, 140, 141, 143, 145, 147, 152, 154 156, 158, 160, 165, 166, 170, 172, 174, 177, 178, 181, 182, 184, 186, 187, 192 and 194 as measured by the computer algorithm BLASTP.
In a second aspect, the invention provides polypeptides comprising an immunogenic portion of an M. vaccae antigen wherein the antigen comprises an amino acid sequence encoded by a DNA molecule selected from the group consisting of: (a) sequences recited in SEQ ID NOS: 74, 88, 93, 97, 99, 106-108, 111, 120, 122, 123, 132, 133, 136-138, 142, 144, 146, 151, 153, 155, 157, 159, 161, 162, 163, 164, 169, 171, 173, 175, 176, 179, 180, 183, 185, 191 and 193; (b) complements of the sequences recited in SEQ ID NOS: 74, 88, 93, 97, 99, 106-108, 111, 120, 122, 123, 132, 133, 136-138, 142, 144, 146, 151, 153, 155, 157, 159, 161, 162, 163, 164, 169, 171, 173, 175, 176, 179, 180, 183, 185, 191 and 193; and (c) sequences having at least about a 99% probability of being the same as a sequence of (a) or (b) as measured by the computer algorithm FASTA.
DNA sequences encoding the inventive polypeptides, expression vectors comprising these DNA sequences, and host cells transformed or transfected with such expression vectors are also provided.
In another aspect, the present invention provides fusion proteins comprising a first and a second inventive polypeptide or, alternatively, an inventive polypeptide and a known M. tuberculosis antigen.
Within other aspects, the present invention provides pharmaceutical compositions that comprise at least one of the inventive polypeptides, or a DNA molecule encoding such a polypeptide, and a physiologically acceptable carrier. The invention also provides vaccines comprising at least one of the above polypeptides and a non-specific immune response amplifier, together with vaccines comprising at least one DNA sequence encoding such polypeptides and a non-specific immune response amplifier.
In yet another aspect, methods are provided for inducing protective immunity in a patient, comprising administering to a patient an effective amount of one or more of the above polypeptides together with an immune response amplifier.
In further aspects of this invention, methods and diagnostic kits are provided for detecting tuberculosis in a patient. In a first embodiment, the method comprises contacting dermal cells of a patient with one or more of the above polypeptides and detecting an immune response on the patient's skin. In a second embodiment, the method comprises contacting a biological sample with at least one of the above polypeptides; and detecting in the sample the presence of antibodies that bind to the polypeptide or polypeptides, thereby detecting M. tuberculosis infection in the biological sample. Suitable biological samples include whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid and urine.
Diagnostic kits comprising one or more of the above polypeptides in combination with an apparatus sufficient to contact the polypeptide with the dermal cells of a patient are provided. The present invention also provides diagnostic kits comprising one or more of the inventive polypeptides in combination with a detection reagent.
In yet another aspect, the present invention provides antibodies, both polyclonal and monoclonal, that bind to the polypeptides described above, as well as methods for their use in the detection of M. tuberculosis infection.
The present invention also provides methods for enhancing a non-specific immune response to an antigen. In one embodiment, such methods comprise administering a composition comprising a component selected from the group consisting of: (a) delipidated M. vaccae cells, (b) deglycolipidated M. vaccae cells; (c) delipidated and deglycolipidated M. vaccae cells and (d) M. vaccae culture filtrate. In a second embodiment, such methods comprise administering a polypeptide, the polypeptide comprising an immunogenic portion of an antigen, wherein said antigen includes a sequence selected from the group consisting of: (a) sequences recited in SEQ ID NOS: **114, 117 and 118; and (b) sequences having at least about 97% identity to a sequence recited in SEQ ID NOS: **114, 117 and 118.
In yet a further aspect, compositions comprising a component selected from the group consisting of delipidated M. vaccae cells, deglycolipidated M. vaccae cells, and delipidated and deglycolipidated M. vaccae cells are provided, together with vaccines comprising such components and methods of using such compositions and vaccines to induce protective immunity in a patient.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B illustrate the protective effects of immunizing mice with autoclaved M. vaccae or unfractionated M. vaccae culture filtrates, respectively, prior to infection with live M. tuberculosis H37Rv.
FIGS. 2A and B show components of M. vaccae and M. tuberculosis culture filtrates, respectively, as analysed by 2-dimensional polyacrylamide gel electrophoresis.
FIG. 3 is a comparison of the Antigen 85A protein sequence obtained from M. vaccae with those from M. bovis, M. tuberculosis and M. leprae.
FIG. 4A(i)-(iv) illustrate the non-specific immune amplifying effects of 10 .mu.g, 100 .mu.g and 1 mg autoclaved M. vaccae and 75 .mu.g unfractionated culture filtrates of M. vaccae, respectively.
FIG. 4B(i) and (ii) illustrate the non-specific immune amplifying effects of autoclaved M. vaccae, and delipidated and deglycolipidated M. vaccae, respectively. FIG. 4C(i) illustrates the non-specific immune amplifying effects of whole autoclaved M. vaccae.
FIG. 4C(ii) illustrates the non-specific immune amplifying effects ofsoluble M. vaccae proteins extracted with SDS from delipidated and deglycolipidated M. vaccae.
FIG. 4C(iii) illustrates that the non-specific amplifying effects of the preparation of FIG. 4C(ii) are destroyed by treatment with the proteolytic enzyme Pronase.
FIG. 4D illustrates the non-specific immune amplifying effects of heat-killed M. vaccae (FIG. 4D(i)),whereas a non-specific immune amplifying effect was not seen with heat-killed preparations of M. tuberculosis (FIG. 4D(ii)), M. bovis BCG (FIG. 4D(iii)), M. phlei (FIG. 4D(iv)) and M. smegmatis (FIG. 4D(v)).
FIG. 5 shows the results of polyacrylamide gel electrophoresis analysis of SDS-extracted proteins derived from delipidated and deglycolipidated M. vaccae.
FIG. 6 illustrates the non-specific immune amplifying effects of different molecular weight fractions of SDS-extracted M. vaccae proteins.
FIG. 7 illustrates the non-specific immune amplifying effects of different pI fractions of SDS-extracted M. vaccae proteins.
FIG. 8 illustrates the induction of IL-12 by autoclaved M. vaccae , lyophilized M. vaccae, delipidated and deglycolipidated M. vaccae and M. vaccae glycolipids.
FIGS. 9A, B and C illustrate the stimulation of interferon-gamma production by different concentrations of M. vaccae recombinant proteins, heat-killed M. vaccae, delipidated and deglycolipidated M. vaccae (referred to in the figure as "delipidated M. vaccae"), M. vaccae glycolipids and lipopolysaccharide, in peritoneal macrophages from C57BL/6 mice (FIG. 9A), BALB/C mice (FIG. 9B) or C3H/HeJ mice (FIG. 9C).
FIG. 10 compares the in vitro stimulation of interferon-gamma production in spleen cells from Severe Combined ImmunoDeficient (SCID) mice by different concentrations of heat-killed (autoclaved) M. vaccae, delipidated and deglycolipidated M. vaccae, and M. vaccae glycolipids.

DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is generally directed to compositions and methods for preventing, treating and diagnosing mycobacterial infections, including M. tuberculosis and M. avium infections.
Considerable research efforts have been directed towards elucidating the mechanism of immune response to mycobacterial infection, in particular M. tuberculosis infection. While macrophages have been shown to act as the principal effectors of M. tuberculosis immunity, T cells are the predominant inducers of such immunity. The essential role of T cells in protection against M. tuberculosis infection is illustrated by the frequent occurrence of M. tuberculosis in AIDS patients, due to the depletion of CD4 T cells associated with human immunodeficiency virus (HIV) infection. Mycobacterium-reactive CD4 T cells have been shown to be potent producers of gamma-interferon (IFN-.gamma.), which, in turn, has been shown to trigger the anti-mycobacterial effects of macrophages in mice. While the role of IFN-.gamma. in humans is less clear, studies have shown that 1,25-dihydroxy-vitamin D3, either alone or in combination with IFN-.gamma. or tumor necrosis factor-alpha, activates human macrophages to inhibit M. tuberculosis infection. Furthermore, it is known that IFN-.gamma. stimulates human macrophages to make 1,25-dihydroxy-vitamin D3. Similarly, IL-12 has been shown to play a role in stimulating resistance to M. tuberculosis infection. Another property of CD4.sup.+ T cells and macrophages is their ability to activate CD8.sup.+ cytotoxic T cells which are capable of killing pathogen-infected cells. CD8.sup.+ T cells have been shown to kill macrophages and other cells that harbour M. tuberculosis. For a review of the immunology of M. tuberculosis infection see Chan and Kaufmann in Tuberculosis: Pathogenesis, Protection and Control, Bloom (ed.), ASM Press, Washington, DC, 1994.
The compositions of the present invention include polypeptides that comprise at least one immunogenic portion of aM. vaccae antigen, or a variant thereof. Such polypeptides stimulate T cell proliferation, and/or, interferon gamma secretion from T cells of individuals exposed to M. tuberculosis. In certain embodiments, the inventive polypeptides comprise at least an immunogenic portion of a soluble M. vaccae antigen. A "soluble M. vaccae antigen" is a protein of M. vaccae origin that is present in M. vaccae culture filtrate. As used herein, the term "polypeptide" encompasses amino acid chains of any length, including full length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising an immunogenic portion of one of the above antigens may consist entirely of the immunogenic portion, or may contain additional sequences. The additional sequences may be derived from the native M. vaccae antigen or may be heterologous, and such sequences may (but need not) be immunogenic.
"Immunogenic," as used herein, refers to the ability to elicit an immune response in a patient, such as a human, or in a biological sample. In particular, immunogenic antigens are capable of stimulating cell proliferation, interleukin-12 production or interferon-.gamma. production in biological samples comprising one or more cells selected from the group of T cells, NK cells, B cells and macrophages, where the cells are derived from an M. tuberculosis-immune individual. Polypeptides comprising at least an immunogenic portion of one or more M. vaccae antigens may generally be used to detect tuberculosis or to induce protective immunity against tuberculosis in a patient.
The compositions and methods of this invention also encompass variants of the above polypeptides. As used herein, the term "variant" covers any sequence which exhibits at least about 50%, more preferably at least about 70% and more preferably yet, at least about 90% identity to a sequence of the present invention. Most preferably, a "variant" is any sequence which has at least about a 99% probability of being the same as the inventive sequence. The probability for DNA sequences is measured by the computer algorithm FASTA (version 2.0u4, February 1996; Pearson W. R. et al., Proc. Natl. Acad. Sci., 85:2444-2448, 1988), the probability for translated DNA sequences is measured by the computer algorithm TBLASTX and that for protein sequences is measured by the computer algorithm BLASTP (Altschul, S. F. et al. J. Mol. Biol., 215:403-410, 1990). The term "variants" thus encompasses sequences wherein the probability of finding a match by chance (smallest sum probability), is less than about 1% as measured by any of the above tests.
A polypeptide of the present invention may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.
In general, M. vaccae antigens, and DNA sequences encoding such antigens, may be prepared using any of a variety of procedures. For example, soluble antigens may be isolated from M. vaccae culture filtrate as described below. Antigens may also be produced recombinantly by inserting a DNA sequence that encodes the antigen into an expression vector and expressing the antigen in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner may encode naturally occurring antigens, portions of naturally occurring antigens, or other variants thereof.
DNA sequences encoding M. vaccae antigens may be obtained by screening an appropriate M. vaccae cDNA or genomic DNA library for DNA sequences that hybridize to degenerate oligonucleotides derived from partial amino acid sequences of isolated soluble antigens. Suitable degenerate oligonucleotides may be designed and synthesized, and the screen may be performed as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989. As described below, polymerase chain reaction (PCR) may be employed to isolate a nucleic acid probe from genomic DNA, or a cDNA or genomic DNA library. The library screen may then be performed using the isolated probe.
DNA molecules encoding M. vaccae antigens may also be isolated by screening an appropriate M. vaccae expression library with anti-sera (e.g., rabbit or monkey) raised specifically against M. vaccae antigens.
Regardless of the method of preparation, the antigens described herein have the ability to induce an immunogenic response. More specifically, the antigens have the ability to induce cell proliferation and/or cytokine production (for example, interferon-.gamma. and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from an M. tuberculosis-immune individual. An M. tuberculosis-immune individual is one who is considered to be resistant to the development of tuberculosis by virtue of having mounted an effective T cell response to M. tuberculosis. Such individuals may be identified based on a strongly positive (i.e., greater than about 10 mm diameter induration) intradermal skin test response to tuberculosis proteins (PPD), and an absence of any symptoms of tuberculosis infection.
The selection of cell type for use in evaluating an immunogenic response to an antigen will depend on the desired response. For example, interleukin-12 production is most readily evaluated using preparations containing T cells, NK cells, B cells and macrophages derived from M. tuberculosis-immune individuals may be prepared using methods well known in the art. For example, a preparation of peripheral blood mononuclear cells (PBMCs) may be employed without further separation of component cells. PBMCs may be prepared, for example, using density centrifugation through Ficoll.TM. (Winthrop Laboratories, NY). T cells for use in the assays described herein may be purified directly from PBMCs. Alternatively, an enriched T cell line reactive against mycobacterial proteins, or T cell clones reactive to individual mycobacterial proteins, may be employed. Such T cell clones may be generated by, for example, culturing PBMCs from M. tuberculosis-immune individuals with mycobacterial proteins for a period of 2-4 weeks. This allows expansion of only the mycobacterial protein-specific T cells, resulting in a line composed solely of such cells. These cells may then be cloned and tested with individual proteins, using methods well known in the art, to more accurately define individual T cell specificity. Assays for cell proliferation or cytokine production in T cells, NK cells, B cells or macrophages may be performed, for example, using the procedures described below.
In general, immunogenic antigens are those antigens that stimulate proliferation or cytokine production (i.e., interferon-.gamma. and/or interleukin-12 production) in T cells, NK cells, B cells or macrophages derived from at least about 25% of M. tuberculosis-immune individuals. Among these immunogenic antigens, polypeptides having superior therapeutic properties may be distinguished based on the magnitude of the responses in the above assays and based on the percentage of individuals for which a response is observed. In addition, antigens having superior therapeutic properties will not stimulate cell proliferation or cytokine production in vitro in cells derived from more than about 25% of individuals that are not M. tuberculosis-immune, thereby eliminating responses that are not specifically due to M. tuberculosis-responsive cells. Thus, those antigens that induce a response in a high percentage of T cell, NK cell, B cell or macrophage preparations from M. tuberculosis-immune individuals (with a low incidence of responses in cell preparations from other individuals) have superior therapeutic properties.
Antigens with superior therapeutic properties may also be identified based on their ability to diminish the severity of M. tuberculosis infection, or other mycobacterial infection, in experimental animals, when administered as a vaccine. Suitable vaccine preparations for use in experimental animals are described in detail below.
Antigens having superior diagnostic properties may generally be identified based on the ability to elicit a response in an intradermal skin test performed on an individual with active tuberculosis, but not in a test performed on an individual who is not infected with M. tuberculosis. Skin tests may generally be performed as described below, with a response of at least about 5 mm induration considered positive.
Immunogenic portions of the antigens described herein may be prepared and identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3d ed., Raven Press, 1993, pp. 243-247. Such techniques include screening polypeptide portions of the native antigen for immunogenic properties. The representative proliferation and cytokine production assays described herein may be employed in these screens. An immunogenic portion of a polypeptide is a portion that, within such representative assays, generates an immune response (e.g., cell proliferation, interferon-.gamma. production or interleukin-12 production) that is substantially similar to that generated by the full-length antigen. In other words, an immunogenic portion of an antigen may generate at least about 20%, preferably about 65%, and most preferably about 100%, of the proliferation induced by the full-length antigen in the model proliferation assay described herein. An immunogenic portion may also, or alternatively, stimulate the production of at least about 20%, preferably about 65% and most preferably about 100%, of the interferon-.gamma. and/or interleukin-12 induced by the full length antigen in the model assay described herein.
Portions and other variants of M. vaccae antigens may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems, Inc. (Foster City, Calif.), and may be operated according to the manufacturer's instructions. Variants of a native antigen may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis. Sections of the DNA sequence may also be removed using standard techniques to permit preparation of truncated polypeptides.
In general, regardless of the method of preparation, the polypeptides disclosed herein are prepared in substantially pure form. Preferably, the polypeptides are at least about 80% pure, more preferably at least about 90% pure and most preferably at least about 99% pure. In certain preferred embodiments, described in detail below, the substantially pure polypeptides are incorporated into pharmaceutical compositions or vaccines for use in one or more of the methods disclosed herein.
The present invention also provides fusion proteins comprising a first and a second inventive polypeptide or, alternatively, a polypeptide of the present invention and a known M. tuberculosis antigen, such as the 38 kDa antigen described in Andersen and Hansen, Infect. Immun. 57:2481-2488, 1989, together with variants of such fusion proteins. The fusion proteins of the present invention may also include a linker peptide between the first and second polypeptides.
A DNA sequence encoding a fusion protein of the present invention is constructed using known recombinant DNA techniques to assemble separate DNA sequences encoding the first and second polypeptides into an appropriate expression vector. The 3' end of a DNA sequence encoding the first polypeptide is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide so that the reading frames of the sequences are in phase to permit mRNA translation of the two DNA sequences into a single fusion protein that retains the biological activity of both the first and the second polypeptides.
A peptide linker sequence may be employed to separate the first and the second polypeptides by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci USA 83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may be from 1 to about 50 amino acids in length. Peptide linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
The ligated DNA sequences encoding the fusion proteins are cloned into suitable expression systems using techniques known to those of ordinary skill in the art.
In another aspect, the present invention provides methods for using one or more of the inventive polypeptides or fusion proteins (or DNA molecules encoding such polypeptides or fusion proteins) to induce protective immunity against tuberculosis in a patient. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may be afflicted with a disease, or may be free of detectable disease or infection. In other words, protective immunity may be induced to prevent or treat tuberculosis.
In this aspect, the polypeptide, fusion protein or DNA molecule is generally present within a pharmaceutical composition or a vaccine. Pharmaceutical compositions may comprise one or more polypeptides, each of which may contain one or more of the above sequences (or variants thereof, and a physiologically acceptable carrier. Vaccines may comprise one or more of the above polypeptides and a non-specific immune response amplifier, such as an adjuvant or a liposome, into which the polypeptide is incorporated. Such pharmaceutical compositions and vaccines may also contain other mycobacterial antigens, either, as discussed above, incorporated into a fusion protein or present within a separate polypeptide.
Alternatively, a vaccine of the present invention may contain DNA encoding one or more polypeptides as described above, such that the polypeptide is generated in situ. In such vaccines, the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminator signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerin) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic, or defective, replication competent virus. Techniques for incorporating DNA into such expression systems are well known in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
A DNA vaccine as described above may be administered simultaneously with or sequentially to either a polypeptide of the present invention or a known mycobacterial antigen, such as the 38 kDa antigen described above. For example, administration of DNA encoding a polypeptide of the present invention, may be followed by administration of an antigen in order to enhance the protective immune effect of the vaccine.
Routes and frequency of administration, as well as dosage, will vary from individual to individual and may parallel those currently being used in immunization using BCG. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intradermal, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 3 doses may be administered for a 1-36 week period. Preferably, 3 doses are administered, at intervals of 3-4 months, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of polypeptide or DNA that, when administered as described above, is capable of raising an immune response in a patient sufficient to protect the patient from mycobacterial infection for at least 1-2 years. In general, the amount of polypeptide present in a dose (or produced in situ by the DNA in a dose) ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 .mu.g. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 ml to about 5 ml.
While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
Any of a variety of adjuvants may be employed in the vaccines of this invention to non-specifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a non-specific stimulator of immune responses, such as lipid A, Bordetella pertussis, M. tuberculosis, or, as discussed below, M. vaccae. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Freund's Complete Adjuvant (Difco Laboratories, Detroit, Mich.), and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and Quil A.
In another aspect, this invention provides methods for using one or more of the polypeptides described above to diagnose tuberculosis using a skin test. As used herein, a "skin test" is any assay performed directly on a patient in which a delayed-type hypersensitivity (DTH) reaction (such as swelling, reddening or dermatitis) is measured following intradermal injection of one or more polypeptides as described above. Preferably, the reaction is measured at least 48 hours after injection, more preferably 48-72 hours.
The DTH reaction is a cell-mediated immune response, which is greater in patients that have been exposed previously to the test antigen (i.e., the immunogenic portion of the polypeptide employed, or a variant thereof). The response may be measured visually, using a ruler. In general, a response that is greater than about 0.5 cm in diameter, preferably greater than about 1.0 cm in diameter, is a positive response, indicative of tuberculosis infection.
For use in a skin test, the polypeptides of the present invention are preferably formulated, as pharmaceutical compositions containing a polypeptide and a physiologically acceptable carrier, as described above. Such compositions typically contain one or more of the above polypeptides in an amount ranging from about 1 .mu.g to about 100 .mu.g, preferably from about 10 .mu.g to about 50 .mu.g in a volume of 0.1 ml. Preferably, the carrier employed in such pharmaceutical compositions is a saline solution with appropriate preservatives, such as phenol and/or Tween 80.TM..
In a preferred embodiment, a polypeptide employed in a skin test is of sufficient size such that it remains at the site of injection for the duration of the reaction period. In general, a polypeptide that is at least 9 amino acids in length is sufficient. The polypeptide is also preferably broken down by macrophages or dendritic cells within hours of injection to allow presentation to T-cells. Such polypeptides may contain repeats of one or more of the above sequences or other immunogenic or nonimmunogenic sequences.
In another aspect, methods are provided for detecting mycobacterial infection in a biological sample, using one or more of the above polypeptides, either alone or in combination. In embodiments in which multiple polypeptides are employed, polypeptides other than those specifically described herein, such as the 38 kDa antigen described above, may be included. As used herein, a "biological sample" is any antibody-containing sample obtained from a patient. Preferably, the sample is whole blood, sputum, serum, plasma, saliva, cerebrospinal fluid or urine. More preferably, the sample is a blood, serum or plasma sample obtained from a patient or a blood supply. The polypeptide(s) are used in an assay, as described below, to determine the presence or absence of antibodies to the polypeptide(s) in the sample, relative to a predetermined cut-off value. The presence of such antibodies indicates the presence of mycobacterial infection.
In embodiments in which more than one polypeptide is employed, the polypeptides used are preferably complementary (i.e., one component polypeptide will tend to detect infection in samples where the infection would not be detected by another component polypeptide). Complementary polypeptides may generally be identified by using each polypeptide individually to evaluate serum samples obtained from a series of patients known to be infected with a Mycobacterium. After determining which samples test positive (as described below) with each polypeptide, combinations of two or more polypeptides may be formulated that are capable of detecting infection in most, or all, of the samples tested. For example, approximately 25-30% of sera from tuberculosis-infected individuals are negative for antibodies to any single protein, such as the 38 kDa antigen mentioned above. Complementary polypeptides may, therefore, be used in combination with the 38 kDa antigen to improve sensitivity of a diagnostic test.
A variety of assay formats employing one or more polypeptides to detect antibodies in a sample are well known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In a preferred embodiment, the assay involves the use of polypeptide immobilized on a solid support to bind to and remove the antibody from the sample. The bound antibody may then be detected using a detection reagent that contains a reporter group. Suitable detection reagents include antibodies that bind to the antibody/polypeptide complex and free polypeptide labelled with a reporter group (e.g., in a semi-competitive assay). Alternatively, a competitive assay may be utilized, in which an antibody that binds to the polypeptide is labelled with a reporter group and allowed to bind to the immobilized antigen after incubation of the antigen with the sample. The extent to which components of the sample inhibit the binding of the labelled antibody to the polypeptide is indicative of the reactivity of the sample with the immobilized polypeptide.
The solid support may be any solid material to which the antigen may be attached. Suitable materials are well known in the art. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681.
The polypeptides may be bound to the solid support using a variety of techniques well known in the art. In the context of the present invention, the term "bound" refers to both noncovalent association, such as adsorption, and covalent attachment, which may be a direct linkage between the antigen and functional groups on the support or a linkage by way of a cross-linking agent. Binding by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the polypeptide, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of polypeptide ranging from about 10 ng to about 1 .mu.g, and preferably about 100 ng, is sufficient to bind an adequate amount of antigen.
Covalent attachment of polypeptide to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the polypeptide. For example, the polypeptide may be bound to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the polypeptide (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
In certain embodiments, the assay is an enzyme-linked immunosorbent assay (ELISA). This assay may be performed by first contacting a polypeptide antigen that has been immobilized on a solid support, with the sample, such that antibodies to the polypeptide within the sample are allowed to bind to the immobilized polypeptide. Unbound sample is then removed from the immobilized polypeptide and a detection reagent capable of binding to the immobilized antibody-polypeptide complex is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific detection reagent.
More specifically, once the polypeptide is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20.TM. (Sigma Chemical Co., St. Louis, Mo.) may be employed. The immobilized polypeptide is then incubated with the sample, and antibody is allowed to bind to the antigen. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time, or incubation time, is that period of time that is sufficient to detect the presence of antibody within a M. tuberculosis-infected sample. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound antibody. The time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
Unbound sample may be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20.TM.. Detection reagent may then be added to the solid support. An appropriate detection reagent is any compound that binds to the immobilized antibody-polypeptide complex and that can be detected by any of a variety of means known in the art. Preferably, the detection reagent contains a binding agent (such as, for example, Protein A, Protein G, immunoglobulin, lectin or free antigen) conjugated to a reporter group. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of binding agent to reporter group may be achieved using standard methods known in the art. Common binding agents may also be purchased conjugated to a variety of reporter groups from many commercial sources (e.g., Zymed Laboratories, San Francisco, Calif., and Pierce, Rockford, Ill.).
The detection reagent is incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound antibody. An appropriate amount of time may generally be determined from the manufacturer's instructions or by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
To determine the presence or absence of anti-mycobacterial antibodies in the sample, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value. In one preferred embodiment, the cut-off value is the average mean signal obtained when the immobilized antigen is incubated with samples from an uninfected patient. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, pp. 106-107. In general, signals higher than the predetermined cut-off value are considered to be positive for mycobacterial infection.
The assay may also be performed in a rapid flow-through or strip test format, wherein the antigen is immobilized on a membrane, such as nitrocellulose. In the flow-through test, antibodies within the sample bind to the immobilized polypeptide as the sample passes through the membrane. A detection reagent (e.g., protein A-colloidal gold) then binds to the antibody-polypeptide complex as the solution containing the detection reagent flows through the membrane. The detection of bound detection reagent may then be performed as described above. In the strip test format, one end of the membrane to which polypeptide is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing detection reagent and to the area of immobilized polypeptide. Concentration of detection reagent at the polypeptide indicates the presence of anti-mycobacterial antibodies in the sample. Typically, the concentration of detection reagent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of polypeptide immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of antibodies that would be sufficient to generate a positive signal in an ELISA, as discussed above. Preferably, the amount of polypeptide immobilized on the membrane ranges from about 25 ng to about 1 .mu.g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount (e.g., one drop) of patient serum or blood.
Numerous other assay protocols exist that are suitable for use with the polypeptides of the present invention. The above descriptions are intended to be exemplary only.
The present invention also provides antibodies to the inventive polypeptides. Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising the antigenic polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep and goats). The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.
Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells may then be immortalized by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal, using one of a variety of techniques well known in the art.
Monoclonal antibodies may be isolated from the supernatants of the resulting hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
Antibodies may be used in diagnostic tests to detect the presence of mycobacterial antigens using assays similar to those detailed above and other techniques well known to those of skill in the art, thereby providing a method for detecting mycobacterial infection, such as M. tuberculosis infection, in a patient.
Diagnostic reagents of the present invention may also comprise DNA sequences encoding one or more of the above polypeptides, or one or more portions thereof For example, primers comprising at least 10 contiguous oligonucleotides of the subject DNA sequences may be used in polymerase chain reaction (PCR) based tests. Similarly, probes comprising at least 18 contiguous oligonucleotides of the subject DNA sequences may be used for hybridizing to specific sequences. Techniques for both PCR based tests and hybridization tests are well known in the art. Primers or probes may thus be used to detect M. tuberculosis and other mycobacterial infections in biological samples, preferably sputum, blood, serum, saliva, cerebrospinal fluid or urine. DNA probes or primers comprising oligonucleotide sequences described above may be used alone, in combination with each other, or with previously identified sequences, such as the 38 kDa antigen discussed above.
As discussed above, effective vaccines contain at least two different components. The first is a polypeptide comprising an antigen, which is processed by macrophages and other antigen-presenting cells and displayed for CD4.sup.+ T cells or for CD8.sup.+ T cells. This antigen forms the "specific" target of an immune response. The second component of a vaccine is a non-specific immune response amplifier, such as an adjuvant or a liposome, into which the antigen is incorporated. An adjuvant amplifies immune responses to a structurally unrelated compound or polypeptide. Several adjuvants are prepared from microbes such as Bordetella pertussis, M. tuberculosis and M. bovis BCG. Adjuvants may also contain components designed to protect polypeptide antigens from degradation, such as aluminum hydroxide or mineral oil.
While the antigenic component of a vaccine contains polypeptides that direct the immune attack against a specific pathogen, such as M. tuberculosis, the adjuvant is often capable of broad use in many different vaccine formulations. Certain pathogens, such as M. tuberculosis, as well as certain cancers, are effectively contained by an immune attack directed by T cells, known as cell-mediated immunity. Other pathogens, such as poliovirus, also require antibodies produced by B cells for containment. These different classes of immune attack (T cell or B cell) are controlled by different subpopulations of CD4.sup.+ T cells, commonly referred to as Th1 and Th2 cells. A desirable property of an adjuvant is the ability to selectively amplify the function of either Th1 or Th2 populations of CD4.sup.+ T cells. As shown below in Example 6, M. vaccae and a modified form of autoclaved M. vaccae have been found to have adjuvant properties. As used herein, the term "modified M. vaccae" includes delipidated M. vaccae cells, deglycolipidated M. vaccae cells and M. vaccae cells that have been both delipidated and deglycolipidated (hereinafter referred to as DD-M. vaccae). Furthermore, it has been found that M. vaccae produces compounds which amplify the immune response to M. vaccae antigens, as well as to antigens from other sources. The present invention thus provides methods for enhancing immune responses to an antigen comprising administering killed M. vaccae cells, M. vaccae culture filtrate or modified M. vaccae cells. As detailed below, further studies have demonstrated that this non-specific immune amplifying effect is due, at least in part, to an M. vaccae polypeptide having homology to heat shock protein 65 (GroEL), previously identified in M. tuberculosis.
As described below in Example 10, it has also been found that heat-killed M. vaccae and M. vaccae constituents have cytokine stimulation properties. In particular, it has been found that heat-killed M. vaccae, lyophilised M. vaccae and DD-M. vaccae stimulate the production of interleukin 12 (IL-12) from macrophages. Production of IL-12 from macrophages is known to enhance stimulation of a Th1 immune response.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLE 1
Effect of Immunization of Mice with M. vaccae on Tuberculosis
This example illustrates the effect of immunization with M. vaccae or M. vaccae culture filtrate in mice prior to challenge with live M. tuberculosis.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/l; tryptone, 5 g/l; glucose, 1 g/l) at 37.degree. C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich., USA) with glucose at 37.degree. C. for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10.sup.10 M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120.degree. C. The culture filtrate was passaged through a 0.45 .mu.m filter into sterile bottles.
As shown in FIG. 1A, when mice were immunized with 1 mg, 100 .mu.g or 10 .mu.g of M. vaccae and infected three weeks later with 5.times.10.sup.5 colony forming units (CFU) of live M. tuberculosis H37Rv, significant protection from infection was seen. In this example, spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli determined (expressed as CFU). The reduction in bacilli numbers, when compared to tissue from non-immunized control mice, exceeded 2 logs in liver and lung tissue, and 1 log in spleen tissue. Immunization of mice with heat-killed M. tuberculosis H37Rv had no significant protective effects on mice subsequently infected with live M tuberculosis H37Rv.
FIG. 1B shows that when mice were immunized with 100 .mu.g of M. vaccae culture filtrate, and infected three weeks later with 5.times.10.sup.5 CFU of M. tuberculosis H37Rv, significant protection was also seen. When spleen, liver and lung tissue was harvested from mice three weeks after infection, and live bacilli numbers (CFU) determined, a 1-2 log reduction in numbers, as compared to non-immunized control mice, was observed.
EXAMPLE 2
Purification and Characterization of Polypeptides from M. vaccae Culture Filtrate
This example illustrates the preparation of M. vaccae soluble proteins from culture filtrate. Unless otherwise noted, all percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37.degree. C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37.degree. C. for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 .mu.m filter into sterile bottles.
The culture filtrate was concentrated by lyophilization, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45 .mu.m membrane. The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3 kDa molecular weight cut-off (MWCO) membrane. The pressure was maintained at 50 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 20 1 volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, Calif., USA).
The desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech, Uppsala, Sweden) (16.times.100 mm) equilibrated with 10 mM Tris HCl buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCl from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.
The pool of polypeptides eluting from the ion exchange column was concentrated in a 400 ml Amicon stirred cell which contained a 3 kDa MWCO membrane. The pressure was maintained at 50 psi using nitrogen gas. The polypeptides were repeatedly concentrated by membrane filtration and diluted with 1% glycine until the conductivity of the sample was less than 0.1 mS.
The purified polypeptides were then fractionated by preparative isoelectric focusing in a Rotofor device (Bio-Rad, Hercules, Calif., USA). The pH gradient was established with a mixture of Ampholytes (Pharmacia Biotech) comprising 1.6% pH 3.5-5.0 Ampholytes and 0.4% pH 5.0-7.0 Ampholytes. Acetic acid (0.5 M) was used as the anolyte, and 0.5 M ethanolamine as the catholyte. Isoelectric focusing was carried out at 12W constant power for 6 hours, following the manufacturer's instructions. Twenty fractions were obtained.
Fractions from isoelectric focusing were combined, and the polypeptides were purified on a Vydac C4 column (Separations Group, Hesperia, Calif., USA) 300 Angstrom pore size, 5 micron particle size (10.times.250 mm). The polypeptides were eluted from the column with a linear gradient of acetonitrile (0-80% v/v) in 0.05% (v/v) trifluoroacetic acid (TFA). The flow-rate was 2.0 ml/min and the HPLC eluent was monitored at 220 nm. Fractions containing polypeptides were collected to maximize the purity of the individual samples.
Relatively abundant polypeptide fractions were rechromatographed on a Vydac C4 column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6.times.250 mm). The polypeptides were eluted from the column with a linear gradient from 20-60% (v/v) of acetonitrile in 0.05% (v/v) TFA at a flow-rate of 1.0 ml/min. The column eluent was monitored at 220 nm. Fractions containing the eluted polypeptides were collected to maximise the purity of the individual samples. Approximately 20 polypeptide samples were obtained and they were analysed for purity on a polyacrylamide gel according to the procedure of Laemmli (Laemmli, U. K., Nature 277:680-685, 1970).
The polypeptide fractions which were shown to contain significant contamination were further purified using a Mono Q column (Pharmacia Biotech) 10 micron particle size (5.times.50 mm) or a Vydac Diphenyl column (Separations Group) 300 Angstrom pore size, 5 micron particle size (4.6.times.250 mm). From a Mono Q column, polypeptides were eluted with a linear gradient from 0-0.5 M NaCl in 10 mM Tris HCl pH 8.0. From a Vydac Diphenyl column, polypeptides were eluted with a linear gradient of acetonitrile (20-60% v/v) in 0.1% TFA. The flow-rate was 1.0 ml/min and the column eluent was monitored at 220 nm for both columns. The polypeptide peak fractions were collected and analysed for purity on a 15% polyacrylamide gel as described above.
For sequencing, the polypeptides were individually dried onto Biobrene.TM. (Perlin Elmer/Applied BioSystems Division, Foster City, Calif.)--treated glass fiber filters. The filters with polypeptide were loaded onto a Perkin Elmer/Applied BioSystems Procise 492 protein sequencer and the polypeptides were sequenced from the amino terminal end using traditional Edman chemistry. The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards.
Internal sequences were also determined on some antigens by digesting the antigen with the endoprotease Lys-C, or by chemically cleaving the antigen with cyanogen bromide. Peptides resulting from either of these procedures were separated by reversed-phase HPLC on a Vydac C 18 column using a mobile phase of 0.05% (v/v) trifluoroacetic acid with a gradient of acetonitrile containing 0.05% (v/v) TFA (1%/min). The eluent was monitored at 214 nm. Major internal peptides were identified by their UV absorbance, and their N-terminal sequences were determined as described above.
Using the procedures described above, six soluble M. vaccae antigens, designated GVc-1, GVc-2, GVc-7, GVc-13, GVc-20 and GVc-22, were isolated. Determined N-terminal and internal sequences for GVc-1 are shown in SEQ ID NOS: 1, 2 and 3, respectively; the N-terminal sequence for GVc-2 is shown in SEQ ID NO: 4; internal sequences for GVc-7 are shown in SEQ ID NOS: 5-8; internal sequences for GVc-13 are shown in SEQ ID NOS: 9-11; internal sequence for GVc-20 is shown in SEQ ID NO: 12; and N-terminal and internal sequences for GVc-22 are shown in SEQ ID NO: 56-59, respectively. Each of the internal peptide sequences provided herein begins with an amino acid residue which is assumed to exist in this position in the polypeptide, based on the known cleavage specificity of cyanogen bromide (Met) or Lys-C (Lys).
Three additional polypeptides, designated GVc-16, GVc-18 and GVc-21, were isolated employing a preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) purification step in addition to the preparative isoelectric focusing procedure described above. Specifically, fractions comprising mixtures of polypeptides from the preparative isoelectric focusing purification step previously described were purified by preparative SDS-PAGE on a 15% polyacrylamide gel. The samples were dissolved in reducing sample buffer and applied to the gel. The separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting in 10 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer pH 11 containing 10% (v/v) methanol. The transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant polypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The N-terminal sequences for GVc-16, GVc-18 and GVc-21 are provided in SEQ ID NOS: 13, 14 and 15, respectively.
Additional antigens, designated GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19, were isolated employing a preparative SDS-PAGE purification step in addition to the chromatographic procedures described above. Specifically, fractions comprising a mixture of antigens from the Vydac C4 HPLC purification step previously described were fractionated by preparative SDS-PAGE on a polyacrylamide gel. The samples were dissolved in non-reducing sample buffer and applied to the gel. The separated proteins were transferred to a PVDF membrane by electroblotting in 10 mM CAPS buffer, pH 11 containing 10% (v/v) methanol. The transferred protein bands were identified by staining the PVDF membrane with Coomassie blue. Regions of the PVDF membrane containing the most abundant polypeptide species were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. Protein sequences were determined as described above. The determined N-terminal sequences for GVc-12, GVc-14, GVc-15, GVc-17 and GVc-19 are provided in SEQ ID NOS: 16-20, respectively.
All of the above amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R32) using the GeneAssist system. No significant homologies to the amino acid sequences GVc-2 to GVc-22 were obtained. The amino acid sequence for GVc-1 was found to bear some similarity to sequences previously identified from M. bovis and M. tuberculosis. In particular, GVc-1 was found to have some homology with M. tuberculosis MPT83, a cell surface protein, as well as MPT70. These proteins form part of a protein family (Harboe et al., Scand. J. Immunol. 42:46-51, 1995).
Subsequent studies led to the isolation of DNA sequences for GV-13c, GVc-14 and GVc-22 (SEQ ID NO: 142, 107 and 108, respectively). The corresponding predicted amino acid sequences for GV-13c, GVc-14 and GVc-22 are provided in SEQ ID NO: 143, 109 and 110, respectively. Further studies with GVc-22 suggested that only a part of the gene encoding GVc-22 was cloned. When sub-cloned into the expression vector pET16, no protein expression was obtained. Subsequent screening of the M. vaccae BamHI genomic DNA library with the incomplete gene fragment led to the isolation of the complete gene encoding GVc-22. To distinguish between the full-length clone and the partial GVc-22, the antigen expressed by the full-length gene was called GV-22B. The determined nucleotide sequence of the gene encoding GV-22B and the predicted amino acid sequence are provided in SEQ ID NOS: 144 and 145 respectively.
Amplifications primers AD86 and AD112 (SEQ ID NO: 60 and 61, respectively) were designed from the amino acid sequence of GVc-1 (SEQ ID NO: 1) and the M. tuberculosis MPT70 gene sequence. Using these primers, a 310 bp fragment was amplified from M. vaccae genomic DNA and cloned into EcoRV-digested vector pBluescript II SK.sup.+ (Stratagene). The sequence of the cloned insert is provided in SEQ ID NO: 62. The insert of this clone was used to screen a M. vaccae genomic DNA library constructed in lambda ZAP-Express (Stratagene, La Jolla, Calif.). The clone isolated contained an open reading frame with homology to the M. tuberculosis antigen MPT83 and was re-named GV-1/83. This gene also had homology to the M. bovis antigen MPB83. The determined nucleotide sequence and predicted amino acid sequences are provided in SEQ ID NOS: 146 and 147 respectively.
From the amino acid sequences provided in SEQ ID NOS: 1 and 2, degenerate oligonucleotides EV59 and EV61 (SEQ ID NOS: 148 and 149 respectively) were designed. Using PCR, a 100 bp fragment was amplified, cloned into plasmid pBluescript II SK.sup.+ and sequenced (SEQ ID NO: 150) following standard procedures (Maniatis) The cloned insert was used to screen a M. vaccae genomic DNA library constructed in lambda ZAP-Express. The clone isolated had homology to M. tuberculosis antigen MPT70 and M. bovis antigen MPB70, and was named GV-1/70. The determined nucleotide sequence and predicted amino acid sequence for GV-1/70 are provided in SEQ ID NOS: 151 and 152 respectively.
For expression and purification, the genes encoding GV1/83, GV1/70, GVc-13, GVc-14 and GV-22B were sub-cloned into the expression vector pET16 (Novagen, Madison, Wis.). Expression and purification were done according to the manufacturer's protocol.
The purified polypeptides were screened for the ability to induce T-cell proliferation and IFN-.gamma. in peripheral blood cells from immune human donors. These donors were known to be PPD (purified protein derivative from M. tuberculosis) skin test positive and their T cells were shown to proliferate in response to PPD. Donor PBMCs and crude soluble proteins from M. vaccae culture filtrate were cultured in medium comprising RPMI 1640 supplemented with 10% (v/v) autologous serum, penicillin (60 .mu.g/ml), streptomycin (100 .mu.g/ml), and glutamine (2 mM).
After 3 days, 50 .mu.l of medium was removed from each well for the determination of IFN-.gamma. levels, as described below. The plates were cultured for a further 4 days and then pulsed with 1 .mu.Ci/well of tritiated thymidine for a further 18 hours, harvested and tritium uptake determined using a scintillation counter. Fractions that stimulated proliferation in both replicates two-fold greater than the proliferation observed in cells cultured in medium alone were considered positive.
IFN-.gamma. was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a mouse monoclonal antibody directed to human IFN-.gamma. (Endogen, Wobural, Mass. 1 .mu.g/ml phosphate-buffered saline (PBS) for 4 hours at 4.degree. C. Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS/0.2% Tween 20, and samples diluted 1:2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed, and a biotinylated polyclonal rabbit anti-human IFN-.gamma. serum (Endogen), diluted to 1 .mu.g/ml in PBS, was added to each well. The plates were then incubated for 1 hour at room temperature, washed, and horseradish peroxidase-coupled avidin A (Vector Laboratories, Burlingame, Calif.) was added at a 1:4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and orthophenylenediamine (OPD) substrate added. The reaction was stopped after 10 min with 10% (v/v) HCl. The optical density (OD) was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.
Examples of polypeptides containing sequences that stimulate peripheral blood mononuclear cells (PBMC) T cells to proliferate and produce IFN-.gamma. are shown in Table 1, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
TABLE 1______________________________________Antigen Proliferation IFN-.gamma.______________________________________GVc-1 ++ +/-GVc-2 + ++GVc-7 +/- -GVc-13 + ++GVc-14 ++ +GVc-15 + +GVc-20 + +______________________________________
EXAMPLE 3
Purification and Characterisation of Polypeptides from M. vaccae Culture Filtrate by 2-Dimensional Polyacrylamide Gel Electrophoresis
M. vaccae soluble proteins were isolated from culture filtrate using 2-dimensional polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 at 37.degree. C. M. tuberculosis strain H37Rv (ATCC number 27294) was cultured in sterile Middlebrook 7H9 medium with Tween 80 and oleic acid/albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Mich.). The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium with glucose at 37.degree. C. for one day. The medium was then centrifuged (leaving the bulk of the cells) and filtered through a 0.45 .mu.m filter into sterile bottles. The culture filtrate was concentrated by lyophilisation, and redissolved in MilliQ water. A small amount of insoluble material was removed by filtration through a 0.45 .mu.m membrane filter.
The culture filtrate was desalted by membrane filtration in a 400 ml Amicon stirred cell which contained a 3 kDa MWCO membrane. The pressure was maintained at 60 psi using nitrogen gas. The culture filtrate was repeatedly concentrated by membrane filtration and diluted with water until the conductivity of the sample was less than 1.0 mS. This procedure reduced the 20 1 volume to approximately 50 ml. Protein concentrations were determined by the Bradford protein assay (Bio-Rad, Hercules, Calif., USA).
The desalted culture filtrate was fractionated by ion exchange chromatography on a column of Q-Sepharose (Pharmacia Biotech) (16.times.100 mm) equilibrated with 10 mM TrisHCl buffer pH 8.0. Polypeptides were eluted with a linear gradient of NaCl from 0 to 1.0 M in the above buffer system. The column eluent was monitored at a wavelength of 280 nm.
The pool of polypeptides eluting from the ion exchange column were fractionated by preparative 2D gel electrophoresis. Samples containing 200-500 .mu.g of polypeptide were made 8 M in urea and applied to polyacrylamide isoelectric focusing rod gels (diameter 2 mm, length 150 mm, pH 5-7). After the isoelectric focusing step, the first dimension gels were equilibrated with reducing buffer and applied to second dimension gels (16% polyacrylamide). FIGS. 2A and 2B are the 2-D gel patterns observed with M. vaccae culture filtrate and M. tuberculosis H37Rv culture filtrate, respectively. Polypeptides from the second dimension separation were transferred to PVDF membranes by electroblotting in 10 mM CAPS buffer pH 11 containing 10% (v/v) methanol. The PVDF membranes were stained for protein with Coomassie blue. Regions of PVDF containing polypeptides of interest were cut out and directly introduced into the sample cartridge of the Perkin Elmer/Applied BioSystems Procise 492 protein sequencer. The polypeptides were sequenced from the amino terminal end using traditional Edman chemistry. The amino acid sequence was determined for each polypeptide by comparing the retention time of the PTH amino acid derivative to the appropriate PTH derivative standards. Using these procedures, eleven polypeptides, designated GVs-1, GVs-3, GVs-4, GVs-5, GVs-6, GVs-8, GVs-9, GVs-10, GVs-11, GV-34 and GV-35 were isolated. The determined N-terminal sequences for these polypeptides are shown in SEQ ID NOS: 21-29, 63 and 64, respectively. Using the purification procedure described above, more protein was purified to extend the amino acid sequence previously obtained for GVs-9. The extended amino acid sequence for GVs-9 is provided in SEQ ID NO: 65. Further studies resulted in the isolation of DNA sequences for GVs-9 (SEQ ID NO: 111) and GV-35 (SEQ ID NO: 155. The corresponding predicted amino acid sequences are provided in SEQ ID NO: 112 and 156, respectively. An extended DNA sequence for GVs-9 is provided in SEQ ID NO: 153, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 154.
All of these amino acid sequences were compared to known amino acid sequences in the SwissProt data base (version R32) using the GeneAssist system. No significant homologies were obtained, with the exceptions of GVs-3, GVs-4, GVs-5 and GVs-9. GVs-9 was found to bear some homology to two previously identified M. tuberculosis proteins, namely M. tuberculosis cutinase precursor and an M. tuberculosis hypothetical 22.6 kDa protein. GVs-3, GVs-4 and GVs-5 were found to bear some similarity to the antigen 85A and 85B proteins from M. leprae (SEQ ID NOS: 30 and 31, respectively), M. tuberculosis (SEQ ID NOS: 32 and 33, respectively) and M. bovis (SEQ ID NOS: 34 and 35, respectively), and the antigen 85C proteins from M. leprae (SEQ ID NO: 36) and M. tuberculosis (SEQ ID NO: 37). A comparison of the inventive antigen 85A protein from M. vaccae with those from M. tuberculosis, M. bovis and M. leprae, is presented in FIG. 3.
EXAMPLE 4
DNA Cloning Strategy for the M. vaccae Antigen 85 Series
Probes for antigens 85A, 85B, and 85C were prepared by the polymerase chain reaction (PCR) using degenerate oligonucleotides (SEQ ID NOS: 38 and 39) designed to regions of antigen 85 genomic sequence that are conserved between family members in a given mycobacterial species, and between mycobacterial species. These oligonucleotides were used under reduced stringency conditions to amplify target sequences from M. vaccae genomic DNA. An appropriately-sized 485 bp band was identified, purified, and cloned into T-tailed p Bluescript II SK (Stratagene, La Jolla, Calif.). Twenty-four individual colonies were screened at random for the presence of the antigen 85 PCR product, then sequenced using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the M13-based primers, T3 and T7. Homology searches of the GenBank databases showed that twenty-three clones contained insert with significant homology to published antigen 85 genes from M. tuberculosis and M. bovis. Approximately half were most homologous to antigen 85C gene sequences, with the remainder being more similar to antigen 85B sequences. In addition, these two putative M. vaccae antigen 85 genomic sequences were 80% homologous to one another. Because of this high similarity, the antigen 85C PCR fragment was chosen to screen M. vaccae genomic libraries at low stringency for all three antigen 85 genes.
An M. vaccae genomic library was created in lambda Zap-Express (Stratagene, La Jolla, Calif.) by cloning BamHI partially-digested M. vaccae genomic DNA into similarly-digested X vector, with 3.4.times.10.sup.5 independent plaque-forming units resulting. For screening purposes, twenty-seven thousand plaques from this non-amplified library were plated at low density onto eight 100 cm.sup.2 plates. For each plate, duplicate plaque lifts were taken onto Hybond-N.sup.+ nylon membrane (Amersham International, United Kingdom), and hybridised under reduced-stringency conditions (55.degree. C.) to the radiolabelled antigen 85C PCR product. Autoradiography demonstrated that seventy-nine plaques consistently hybridised to the antigen 85C probe under these conditions. Thirteen positively-hybridising plaques were selected at random for further analysis and removed from the library plates, with each positive clone being used to generate secondary screening plates containing about two hundred plaques. Duplicate lifts of each plate were taken using Hybond-N.sup.+ nylon membrane, and hybridised under the conditions used in primary screening. Multiple positively-hybridising plaques were identified on each of the thirteen plates screened. Two well-isolated positive phage from each secondary plate were picked for further analysis. Using in vitro excision, twenty-six plaques were converted into phagemid, and restriction-mapped. It was possible to group clones into four classes on the basis of this mapping. Sequence data from the 5' and 3' ends of inserts from several representatives of each group was obtained using the Perkin Elmer/Applied Biosystems Model 377 automated sequencer and the T3 and T7 primers. Sequence homologies were determined using FASTA analysis of the GenBank databases with the GeneAssist software package. Two of these sets of clones were found to be homologous to M. bovis and M. tuberculosis antigen 85A genes, each containing either the 5' or 3' ends of the M. vaccae gene (this gene was cleaved during library construction as it contains an internal BamHI site). The remaining clones were found to contain sequences homologous to antigens 85B and 85C from a number of mycobacterial species. To determine the remaining nucleotide sequence for each gene, appropriate subclones were constructed and sequenced. Overlapping sequences were aligned using the DNA Strider software. The determined DNA sequences for M. vaccae antigens 85A, 85B and 85C are shown in SEQ ID NOS: 40-42, respectively, with the predicted amino acid sequences being shown in SEQ ID NOS: 43-45, respectively.
The M. vaccae antigens GVs-3 and GVs-5 were expressed and purified as follows. Amplification primers were designed from the insert sequences of GVs-3 and GVs-5 (SEQ ID NO: 40 and 42, respectively) using sequence data downstream from the putative leader sequence and the 3' end of the clone. The sequences of the primers for GVs-3 are provided in SEQ ID NO: 66 and 67, and the sequences of the primers for GVs-5 are provided in SEQ ID NO: 68 and 69. A XhoI restriction site was added to the primers for GVs-3, and EcoRI and BamHI restriction sites were added to the primers for GVs-5 for cloning convenience. Following amplification from genomic M. vaccae DNA, fragments were cloned into the appropriate site of pProEX HT prokaryotic expression vector (Gibco BRL, Life Technologies, Gaithersburg, Md.) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the recombinant protein was performed according to the manufacturer's protocol.
Expression of a fragment of the M. vaccae antigen GVs-4 (antigen 85B homolog) was performed as follows. The primers AD58 and AD59, described above, were used to amplify a 485 bp fragment from M. vaccae genomic DNA. This fragment was gel-purified using standard techniques and cloned into EcoRV-digested pBluescript containing added dTTP residues. The base sequences of inserts from five clones were determined and found to be identical to each other. These inserts had highest homology to Ag85B from M. tuberculosis. The insert from one of the clones was subcloned into the EcoRI/XhoI sites of pProEX HT prokaryotic expression vector (Gibco BRL), expressed and purified according to the manufacturer's protocol. This clone was renamed GV-4P because only a part of the gene was expressed. The amino acid and DNA sequences for the partial clone GV-4P are provided in SEQ ID NO: 70 and 106, respectively.
Similar to the cloning of GV-4P, the amplification primers AD58 and AD59 were used to amplify a 485 bp fragment from a clone containing GVs-5 (SEQ ID NO: 42). This fragment was cloned into the expression vector pET16 and was called GV-5P. The determined nucleotide sequence and predicted amino acid sequence of GV-5P are provided in SEQ ID NOS: 157 and 158, respectively.
In subsequent studies, using procedures similar to those described above, GVs-3, GV-4P and GVs-5 were re-cloned into the alternative vector pET16 (Novagen, Madison, Wis.).
The ability of purified recombinant GVs-3, GV-4P and GVs-5 to stimulate proliferation of T cells and interferon-.gamma. production in human PBL from PPD-positive, healthy donors, was assayed as described above in Example 2. The results of this assay are shown in Table 2, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, (++) indicates polypeptides having activity greater than four times above background, and ND indicates not determined.
TABLE 2__________________________________________________________________________Donor Donor Donor Donor Donor DonorG97005 G97006 G97007 G97008 G97009 G97010Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma.__________________________________________________________________________GVs-3 ++ + ND ND ++ ++ ++ ++ ++ +/.+-. + ++GV-4P + +/- ND ND + ++ ++ ++ +/- +/- +/- ++GVs-5 ++ ++ ++ ++ ++ ++ + ++ ++ + + ++__________________________________________________________________________
EXAMPLE 5
DNA Cloning Strategy for M. vaccae Antigens
An 84 bp probe for the M. vaccae antigen GVc-7 was amplified using degenerate oligonucleotides designed to the determined amino acid sequence of GVc-7 (SEQ ID NOS: 5-8). This probe was used to screen a M. vaccae genomic DNA library as described in Example 4. The determined nucleotide sequence for GVc-7 is shown in SEQ ID NO: 46 and predicted amino acid sequence in SEQ ID NO: 47. Comparison of these sequences with those in the databank revealed homology to a hypothetical 15.8 kDa membrane protein of M. tuberculosis.
The sequence of SEQ ID NO: 46 was used to design amplification primers (provided in SEQ ID NO: 71 and 72) for expression cloning of the GVc-7 gene using sequence data downstream from the putative leader sequence. A XhoI restriction site was added to the primers for cloning convenience. Following amplification from genomic M. vaccae DNA, fragments were cloned into the XhoI-site of pProEX HT prokaryotic expression vector (Gibco BRL) and submitted for sequencing to confirm the correct reading frame and orientation. Expression and purification of the fusion protein was performed according to the manufacturer's protocol. In subsequent studies, GVc-7 was re-cloned into the vector pET16 (Novagen).
The ability of purified recombinant GVc-7 to stimulate proliferation of T-cells and stimulation of interferon-.gamma. production in human PBL, from PPD-positive, healthy donors, was assayed as described previously in Example 2. The results are shown in Table 3, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
TABLE 3______________________________________Donor Proliferation Interferon-.gamma.______________________________________G97005 ++ +/-G97008 ++ +G97009 + +/-G97010 +/- ++______________________________________
A redundant oligonucleotide probe (SEQ ID NO 73, referred to as MPG15) was designed to the GVs-8 peptide sequence shown in SEQ ID NO: 26 and used to screen a M. vaccae genomic DNA library using standard protocols. A genomic clone containing genes encoding four different antigens was isolated. The determined DNA sequences for GVs-8A (re-named GV-30), GVs-8B (re-named GV-3 1), GVs-8C (re-named GV-32) and GVs-8D, re-named GV-33, are shown in SEQ ID NOS: 48-51, respectively, with the corresponding amino acid sequences being shown in SEQ ID NOS: 52-55, respectively. GV-30 contains regions showing some similarity to known prokaryotic valyl-tRNA synthetases; GV-31 shows some similarity to M. smegmatis aspartate semialdehyde dehydrogenase; and GV-32 shows some similarity to the H. influenza folylpolyglutamate synthase gene. GV-33 contains an open reading frame which shows some similarity to sequences previously identified in M. tuberculosis and M. leprae, but whose function has not been identified.
The determined partial DNA sequence for GV-33 is provided in SEQ ID NO:74 with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 75. Sequence data from the 3' end of the clone showed homology to a previously identified 40.6 kDaa outer membrane protein of M. tuberculosis. Subsequent studies led to the isolation of a full-length DNA sequence for GV-33 (SEQ ID NO: 193). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 194.
The gene encoding GV-33 was amplified from M. vaccae genomic DNA with primers based on the determined nucleotide sequence. This DNA fragment was cloned into EcoRv-digested pBluescript II SK.sup.+ (Stratagene), and then transferred to pET16 expression vector. Recombinant protein was purified following the manufacturer's protocol.
The ability of purified recombinant GV-33 to stimulate proliferation of T-cells and stimulation of interferon-.gamma. production in human PBL was assayed as described previously in Example 2. The results are shown in Table 4, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, and (++) indicates polypeptides having activity greater than four times above background.
TABLE 4______________________________________Stimulatory Activity of PolypeptidesDonor Proliferation Interferon-.gamma.______________________________________G97005 ++ +G97006 ++ ++G97007 - +/-G97008 +/- -G97009 +/- -G97010 +/- ++______________________________________
EXAMPLE 6
Detection of Nonspecific Immune Amplifier from Whole M. vaccae and the Culture Filtrate of M. vaccae
This example illustrates the preparation of whole M. vaccae and M. vaccae culture filtrate and its non-specific immune amplifying or `adjuvant` property.
M. vaccae bacteria was cultured, pelleted and autoclaved as described in Example 1. Culture filtrates of live M. vaccae refer to the supernatant from 24 hour cultures of M. vaccae in 7H9 medium with glucose. A delipidated form of M. vaccae was prepared by sonicating autoclaved M. vaccae for four bursts of 30 seconds on ice using the Virsonic sonicator (Virtis, Disa, USA). The material was then centrifuged (9000 rpm, 20 minutes, JA10 rotor, brake=5). The resulting pellet was suspended in 100 ml of chloroform/methanol (2:1), incubated at room temperature for 1 hour; re-centrifuged, and the chloroform/methanol extraction repeated. The pellet was obtained by centrifugation, dried in vacuo, weighed and resuspended in PBS at 50 mg (dry weight) per ml as delipidated M. vaccae.
Glycolipids were removed from the delipidated M. vaccae preparation by refluxing in 50% v/v ethanol for 2 hours. The insoluble material was collected by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake=5). The extraction with 50% v/v ethanol under reflux was repeated twice more. The insoluble material was collected by centrifugation and washed in PBS. Proteins were extracted by resuspending the pellet in 2% SDS in PBS at 56.degree. C. for 2 hours. The insoluble material was collected by centrifugation and the extraction with 2% SDS/PBS at 56.degree. C. was repeated twice more. The pooled SDS extracts were cooled to 4.degree. C., and precipitated SDS was removed by centrifugation (10,000 rpm, JA20 rotor, 15 mins, brake=5). Proteins were precipitated from the supernatant by adding an equal volume of acetone and incubating at -20.degree. C. for 2 hours. The precipitated proteins were collected by centrifugation, washed in 50% v/v acetone, dried in vacuo, and redissolved in PBS.
M. vaccae culture supernatant (S/N), killed M. vaccae and delipidated M. vaccae were tested for adjuvant activity in the generation of cytotoxic T cell immune response to ovalbumin, a structurally unrelated protein, in the mouse. This anti-ovalbumin-specific cytotoxic response was detected as follows. C57BL/6 mice (2 per group) were immunized by the intraperitoneal injection of 100 .mu.g of ovalbumin with the following test adjuvants: autoclaved M. vaccae; delipidated M vaccae; delipidated M. vaccae with glycolipids also extracted and proteins extracted with SDS; the SDS protein extract treated with Pronase (an enzyme which degrades protein); whole M. vaccae culture filtrate; and heat-killed M. tuberculosis or heat-killed M. bovis BCG, M. phlei or M. smegmatis or M. vaccae culture filtrate. After 10 days, spleen cells were stimulated in vitro for a further 6 days with E.G7 cells which are EL4 cells (a C57BL/6-derived T cell lymphoma) transfected with the ovalbumin gene and thus express ovalbumin. The spleen cells were then assayed for their ability to kill non-specifically EL4 target cells or to kill specifically the E.G7 ovalbumin expressing cells. Killing activity was detected by the release of 51 Chromium with which the EL4 and E.G7 cells have been labelled (100 .mu.Ci per 2.times.10.sup.6), prior to the killing assay. Killing or cytolytic activity is expressed as % specific lysis using the formula: ##EQU1##
It is generally known that ovalbumin-specific cytotoxic cells are generated only in mice immunized with ovalbumin with an adjuvant but not in mice immunized with ovalbumin alone.
The diagrams that make up FIG. 4 show the effect of various M. vaccae derived adjuvant preparations on the generation of cytotoxic T cells to ovalbumin in C57BL/6 mice. As shown in FIG. 4A, cytotoxic cells were generated in mice immunized with (i) 10 .mu.g, (ii) 100 .mu.g or (iii) 1 mg of autoclaved M. vaccae or (iv) 75 .mu.g of M. vaccae culture filtrate. FIG. 4B shows that cytotoxic cells were generated in mice immunized with (i) 1 mg whole autoclaved M. vaccae or (ii) 1 mg delipidated and deglycolipidated (DD-) M. vaccae. As shown in FIG. 4C(i), cytotoxic cells were generated in mice immunized with 1 mg whole autoclaved M. vaccae; FIG. 4C(ii) shows the active material in M. vaccae soluble proteins extracted with SDS from DD-M. vaccae. FIG. 4C(iii) shows that active material in the adjuvant preparation of FIG. 4C(ii) was destroyed by treatment with the proteolytic enzyme Pronase. By way of comparison, 100 .mu.g of the SDS-extracted proteins had significantly stronger immune-enhancing ability (FIG. 4C(ii)) than did 1 mg whole autoclaved M. vaccae (FIG. 4C(i)).
Mice immunized with 1 mg heat-killed M. vaccae (FIG. 4D(i)) generated cytotoxic cells to ovalbumin, but mice immunized separately with 1 mg heat-killed M. tuberculosis (FIG. 4D(ii)), 1 mg M. bovis BCG (FIG. 4D(iii)), 1 mg M. phlei (FIG. 4D(iv)), or 1 mg M. smegmatis (FIG. 4D(v)) failed to generate cytotoxic cells.
These findings demonstrate that heat-killed M. vaccae and DD-M. vaccae have adjuvant properties not seen in other mycobacteria. Furthermore, delipidation and deglycolipidation of M. vaccae removes an NK cell-stimulating activity but does not result in a loss of T-cell stimulating activity.
The SDS-extracted proteins derived from delipidated and deglycolipidated M. vaccae were analysed by polyacrylamide gel electrophoresis. As shown in FIG. 5, three major bands were observed after staining with silver.
In subsequent studies, more of the SDS-extracted proteins described above were prepared by preparative SDS-PAGE on a BioRad Prep Cell (Hercules, Calif.). Fractions corresponding to molecular weight ranges were precipitated by trichloroacetic acid to remove SDS before assaying for adjuvant activity in the anti-ovalbumin-specific cytotoxic response assay in C57BL/6 mice as described above. As seen in FIG. 6, the adjuvant activity was highest in the 60-70 kDa a fraction. The most abundant protein in this size range was purified by SDS-PAGE blotted on to a polyvinylidene difluoride (PVDF) membrane and then sequenced. The sequence of the first ten amino acid residues is provided in SEQ ID NO:76. Comparison of this sequence with those in the gene bank as described above, revealed homology to the heat shock protein 65 (GroEL) gene from M. tuberculosis, indicating that this protein is an M. vaccae member of the GroEL family.
An expression library of M. vaccae genomic DNA in BamH1-lambda ZAP- Express (Stratagene) was screened using sera from cynomolgous monkeys immunised with M. vaccae secreted proteins prepared as described above. Positive plaques were identified using a colorimetric system. These plaques were re-screened until plaques were pure following standard procedures. pBK-CMV phagemid 2-1 containing an insert was excised from the lambda ZAP Express (Stratagene) vector in the presence of ExAssist helper phage following the manufacturer's protocol. The base sequence of the 5' end of the insert of this clone, hereinafter referred to as GV-27, was determined using Sanger sequencing with fluorescent primers on Perkin Elmer/Applied Biosystems Dvision automatic sequencer. The determined nucleotide sequence of the partial M. vaccae GroEL-homologue clone GV-27 is provided in SEQ ID NO: 77 and the predicted amino acid sequence in SEQ ID NO: 78. This clone was found to have homology to M. tuberculosis GroEL. A partial sequence of the 65 kDa heat shock protein of M. vaccae has been published by Kapur et al. (Arch Pathol. Lab. Med. 119 :131-138, 1995). The nucleotide sequence of the Kapur et al. fragment is shown in SEQ ID NO: 79 and the predicted amino acid sequence in SEQ ID NO: 80.
In subsequent studies, an extended (full-length except for the predicted 51 terminal nucleotides) DNA sequence for GV-27 was obtained (SEQ ID NO: 113). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 114. Further studies led to the isolation of a full-length DNA sequence for GV-27 (SEQ ID NO: 159). The corresponding predicted amino acid sequence is provided in SEQ ID NO: 160. GV-27 was found to be 93.7% identical to the M. tuberculosis GroEL at the amino acid level.
Two peptide fragments, comprising the N-terminal sequence (hereinafter referred to as GV-27A) and the carboxy terminal sequence of GV-27 (hereinafter referred to as GV-27B) were prepared using techniques well known in the art. The nucleotide sequences for GV-27A and GV-27B are provided in SEQ ID NO: 115 and 116, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 117 and 118. Subsequent studies led to the isolation of an extended DNA sequence for GV-27B. This sequence is provided in SEQ ID NO: 161, with the corresponding amino acid sequence being provided in SEQ ID NO: 162. The sequence of GV-27A is 95.8% identical to the M. tuberculosis GroEL sequence and contains the shorter M. vaccae sequence of Kapur et al. discussed above. The sequence for GV-27B shows about 92.2% identity to the corresponding region of M tuberculosis HSP65.
Following the same protocol as for the isolation of GV-27, pBK-CMV phagemid 3-1 was isolated. The antigen encoded by this DNA was named GV-29. The determined nucleotide sequences of the 5' and 3' ends of the gene are provided in SEQ ID NOS: 163 and 164, respectively, with the predicted corresponding amino acid sequences being provided in SEQ ID NOS: 165 and 166 respectively. GV-29 showed homology to yeast urea amidolyase. The DNA encoding GV-29 was sub-cloned into the vector pET16 (Novagen, Madison, Wis.) for expression and purification according to standard protocols.
The M. vaccae culture filtrate described above was also fractionated by iso-electric focusing and the fractions assayed for adjuvant activity in the anti-ovalbumin-specific cytotoxic response assay in C57BL/6 mice as described above. As shown in FIG. 7, peak adjuvant activities were demonstrated in fractions corresponding to pI of 4.2-4.32 (fraction nos. 7-9), 4.49-4.57 (fraction nos. 13-17) and 4.81-5.98 (fraction nos. 23-27).
EXAMPLE 7
Autoclaved M. vaccae Generates Cytotoxid CD8 T Cells Against M. Tuberculosis Infected Macrophages
This example illustrates the ability of killed M. vaccae to stimulate cytotoxic CD8 T cells which preferentially kill macrophages that have been infected with M. tuberculosis.
Mice were immunized by the intraperitoneal injection of 500 .mu.g of killed M. vaccae which was prepared as described in Example 1. Two weeks after immunization, the spleen cells of immunized mice were passed through a CD8 T cell enrichment column (R&D Systems, St. Paul, Minn., USA). The spleen cells recovered from the column have been shown to be enriched up to 90% CD8 T cells. These T cells, as well as CD8 T cells from spleens of non-immunized mice, were tested for their ability to kill uninfected macrophages or macrophages which have been infected with M. tuberculosis.
Macrophages were obtained from the peritoneal cavity of mice five days after they have been given 1 ml of 3% thioglycolate intraperitoneally. The macrophages were infected overnight with M. tuberculosis at the ratio of 2 mycobacteria per macrophage. All macrophage preparations were labelled with .sup.51 Chromium at 2 .mu.ci per 10.sup.4 macrophages. The macrophages were then cultured with CD8 T cells overnight (16 hours) at killer to target ratios of 30:1. Specific killing was detected by the release of .sup.51 Chromium and expressed as % specific lysis, calculated as in Example 5.
The production of IFN-.gamma. and its release into medium after 3 days of co-culture of CD8 T cells with macrophages was measured using an enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with a rat monoclonal antibody directed to mouse IFN-.gamma. (Pharmigen, San Diego, Calif., USA) in PBS for 4 hours at 4.degree. C. Wells were blocked with PBS containing 0.2% Tween 20 for 1 hour at room temperature. The plates were then washed four times in PBS containing 0.2% Tween 20, and samples diluted 1:2 in culture medium in the ELISA plates were incubated overnight at room temperature. The plates were again washed, and a biotinylated monoclonal rat anti-mouse IFN-.gamma. antibody (Pharmigen), diluted to 1 .mu.g/ml in PBS, was added to each well. The plates were then incubated for 1 hour at room temperature, washed, and horseradish peroxidase-coupled avidin D (Sigma A-3151) was added at a 1:4,000 dilution in PBS. After a further 1 hour incubation at room temperature, the plates were washed and OPD substrate added. The reaction was stopped after 10 min with 10% (v/v) HCl. The optical density was determined at 490 nm. Fractions that resulted in both replicates giving an OD two-fold greater than the mean OD from cells cultured in medium alone were considered positive.
As shown in Table 5, CD8 T cells from spleens of mice immunized with M. vaccae were cytotoxic for macrophages infected with M tuberculosis and did not lyse uninfected macrophages. The CD8 T cells from non-immunized mice did not lyse macrophages. CD8 T cells from naive or non-immunized mice do produce IFN-.gamma. when cocultured with infected macrophages. The amount of IFN-.gamma. produced in coculture was greater with CD8 T cells derived from M. vaccae immunized mice.
TABLE 5______________________________________EFFECT WITH M. TUBERCULOSIS INFECTEDAND UNINFECTED MACROPHAGES % Specific Lysis of Macrophages IFN-.gamma. (ng/ml)CD8 T cells uninfected infected uninfected infected______________________________________Control 0 0 0.7 24.6M. vaccae Immunized 0 95 2.2 43.8______________________________________
EXAMPLE 8
DNA Cloning Strategy for the M. vaccae Antigens GV-23, GV-24, GV-25, GV-26, GV-38A and GV-38B
M. vaccae (ATCC Number 15483) was grown in sterile Medium 90 at 37.degree. C. for 4 days and harvested by centrifugation. Cells were resuspended in 1 ml Trizol (Gibco BRL, Life Technologies, Gaithersburg, Md.) and RNA extracted according to the standard manufacturer's protocol. M tuberculosis strain H37Rv (ATCC Number 27294) was grown in sterile Middlebrooke 7H9 medium with Tween 80.TM. and oleic acid/albumin/dextrose/catalase additive (Difco Laboratories, Detroit, Mich.) at 37.degree. C. and harvested under appropriate laboratory safety conditions. Cells were resuspended in 1 ml Trizol (Gibco BRL) and RNA extracted according to the manufacturer's standard protocol.
Total M. tuberculosis and M. vaccae RNA was depleted of 16S and 23S ribosomal RNA (rRNA) by hybridisation of the total RNA fraction to oligonucleotides AD10 and AD 11 (SEQ ID NO: 81 and 82) complementary to M. tuberculosis rRNA. These oligonucleotides were designed from mycobacterial 16S rRNA sequences published by Bottger (FEMS Microbiol Lett. 65:171-176, 1989) and from sequences deposited in the databanks. Depletion was done by hybridisation of total RNA to oligonucleotides AD10 and AD11 immobilised on nylon membranes (Hybond N, Amersham International, United Kingdom). Hybridisation was repeated until rRNA bands were not visible on ethidium bromide-stained agarose gels. An oligonucleotide, AD12 (SEQ ID NO: 83), consisting of 20 dATP-residues, was ligated to the 3' ends of the enriched mRNA fraction using RNA ligase. First strand cDNA synthesis was performed following standard protocols, using oligonucleotide AD7 (SEQ ID NO: 84) containing a poly(dT) sequence.
The M. tuberculosis and M. vaccae cDNA was used as template for single-sided-specific PCR (3S-PCR). For this protocol, a degenerate oligonucleotide ADI (SEQ ID NO: 85) was designed based on conserved leader sequences and membrane protein sequences. After 30 cycles of amplification using primer AD1 as 5'-primer and AD7 as 3'-primer, products were separated on a urealpolyacrylamide gel. DNA bands unique to M. vaccae were excised and re-amplified using primers AD1 and AD7. After gel purification, bands were cloned into pGEM-T (Promega) and the base sequence determined.
Searches with the determined nucleotide and predicted amino acid sequences of band 12B21 (SEQ ID NOS: 86 and 87, respectively) showed homology to the pota gene of E. coli encoding the ATP-binding protein of the spermidine/putrescine ABC transporter complex published by Furuchi et al. (Jnl. Biol. Chem. 266: 20928-20933, 1991). The spermidine/putrescine transporter complex of E. coli consists of four genes and is a member of the ABC transporter family. The ABC (ATP-binding Cassette) transporters typically consist of four genes: an ATP-binding gene, a periplasmic, or substrate binding, gene and two transmembrane genes. The transmembrane genes encode proteins each characteristically having six membrane-spanning regions. Homologues (by similarity) of this ABC transporter have been identified in the genomes of Haemophilus influenza (Fleischmann et al. Science 269 :496-512, 1995) and Mycoplasma genitalium (Fraser, et al. Science, 270:397-403, 1995).
An M. vaccae genomic DNA library constructed in BamH1-digested lambda ZAP Express (Stratagene) was probed with the radiolabelled 238 bp band 12B21 following standard protocols. A plaque was purified to purity by repetitive screening and a phagemid containing a 4.5 kb insert was identified by Southern blotting and hybridisation. The nucleotide sequence of the full-length M. vaccae homologue of pota (ATP-binding protein) was identified by subcloning of the 4.5 kb fragment and base sequencing. The gene consisted of 1449 bp including an untranslated 5' region of 320 bp containing putative -10 and -35 promoter elements. The nucleotide and predicted amino acid sequences of the M. vaccae pota homologue are provided in SEQ ID NOS: 88 and 89, respectively.
The nucleotide sequence of the M. vaccae pota gene was used to design primers EV24 and EV25 (SEQ ID NO: 90 and 91) for expression cloning. The amplified DNA fragment was cloned into pProEX HT prokaryotic expression system (Gibco BRL) and expression in an appropriate E. coli host was induced by addition of 0.6 mM isopropylthio-.beta.-galactoside (IPTG). The recombinant protein was named GV-23 and purified from inclusion bodies according to the manufacturer's protocol. In subsequent studies, GV-23 (SEQ ID NO: 88) was re-cloned into the alternative vector pET16 (Novagen).
A 322 bp Sal1-BamH1 subclone at the 3'-end of the 4.5 kb insert described above showed homology to the potd gene, (periplasmic protein), of the spermidine/putrescine ABC transporter complex of E. coli. The nucleotide sequence of this subclone is shown in SEQ ID NO: 92. To identify the gene, the radiolabelled insert of this subclone was used to probe a M. vaccae genomic DNA library constructed in the Sal1-site of lambda Zap Express (Stratagene) following standard protocols. A clone was identified of which 1342 bp showed homology with the potd gene of E. coli. The potd homologue of M. vaccae was identified by subcloning and base sequencing. The determined nucleotide and predicted amino acid sequences are shown in SEQ ID NO: 93 and 94.
For expression cloning, primers EV-26 and EV-27 (SEQ ID NOS: 95-96) were designed from the determined M. vaccae potd homologue. The amplified fragment was cloned into pProEX HT Prokaryotic expression system (Gibco BRL). Expression in an appropriate E. coli host was induced by addition of 0.6 mM IPTG and the recombinant protein named GV-24. The recombinant antigen was purified from inclusion bodies according to the protocol of the supplier. In subsequent studies, GV-24 (SEQ ID NO: 93) was re-cloned into the alternative vector pET16 (Novagen).
To improve the solubility of the purified recombinant antigen, the gene encoding GV-24, but excluding the signal peptide, was re-cloned into the expression vector, employing amplification primers EV101 and EV102 (SEQ ID NOS: 167 and 168). The construct was designated GV-24B. The nucleotide sequence of GV-24B is provided in SEQ ID NO: 169 and the predicted amino acid sequence in SEQ ID NO: 170. This fragment was cloned into pET16 for expression and purification of GV-24B according to the manufacturer's protocols.
The ability of purified recombinant protein GV-23 and GV-24 to stimulate proliferation of T cells and interferon- production in human PBL was determined as described in Example 2. The results of these assays are provided in Table 6, wherein (-) indicates a lack of activity, (+/-) indicates polypeptides having a result less than twice higher than background activity of control media, (+) indicates polypeptides having activity two to four times above background, (++) indicates polypeptides having activity greater than four times above background, and (ND) indicates not determined.
TABLE 6__________________________________________________________________________Donor Donor Donor Donor Donor DonorG97005 G97006 G97007 G97008 G97009 G97010Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma. Prolif IFN-.gamma.__________________________________________________________________________GV-23 ++ ++ ++ ++ + + ++ ++ + - + ++GV-24 ++ + ++ + ND ND + +/- + +/- +/- ++__________________________________________________________________________
Base sequence adjacent to the M. vaccae potd gene-homologue was found to show homology to the potb gene of the spermidine/putrescine ABC transporter complex of E. coli, which is one of two transmembrane proteins in the ABC transporter complex. The M. vaccae potb homologue (referred to as GV-25) was identified through further subcloning and base sequencing. The determined nucleotide and predicted amino acid sequences for GV-25 are shown in SEQ ID NOS: 97 and 98, respectively.
Further subcloning and base sequence analysis of the adjacent 509 bp failed to reveal significant homology to PotC, the second transmembrane protein of E. coli, and suggests that a second transmembrane protein is absent in the M. vaccae homologue of the ABC transporter. An open reading frame with homology to M. tuberculosis acetyl-CoA acetyl transferase, however, was identified starting 530 bp downstream of the transmembrane protein and the translated protein was named GV-26. The determined partial nucleotide sequence and predicted amino acid sequence for GV-26 are shown in SEQ ID NO:99 and 100.
Using a protocol similar to that described above for the isolation of GV-23, the 3S-PCR band 12B28 (SEQ ID NO: 119) was used to screen the M. vaccae genomic library constructed in the BamHI-site of lambda ZAP Express (Stratagene). The clone isolated from the library contained a novel open reading frame and the antigen encoded by this gene was named GV-38A. The determined nucleotide sequence and predicted amino acid sequence of GV-38A are shown in SEQ ID NO: 120 and 121, respectively. Subsequent studies led to the isolation of an extended DNA sequence for GV-38A, provided in SEQ ID NO: 171. The corresponding amino acid sequence is provided in SEQ ID NO: 172. Comparison of these sequences with those in the gene bank, revealed some homology to an unknown M. tuberculosis protein previously identified in cosmid MTCY428.12. (SPTREMBL:P71915).
Upstream of the GV-38A gene, a second novel open reading frame was identified and the antigen encoded by this gene was named GV-38B. The determined 5' and 3' nucleotide sequences for GV-38B are provided in SEQ ID NO: 122 and 123, respectively, with the corresponding predicted amino acid sequences being provided in SEQ ID NO: 124 and 125, respectively. Further studies led to the isolation of the full-length DNA sequence for GV-38B, provided in SEQ ID NO: 173. The corresponding amino acid sequence is provided in SEQ ID NO: 174. This protein was found to show homology to an unknown M. tuberculosis protein identified in cosmid MTCY428.11 (SPTREMBL: P71914).
Both the GV-38A and GV-38B antigens were amplified for expression cloning into pET16 (Novagen). GV-38A was amplified with primers KR11 and KR12 (SEQ ID NO: 126 and 127) and GV-38B with primers KR13 and KR14 (SEQ ID NO: 128 and 129). Protein expression in the host cells BL21(DE3) was induced with 1 mM IPTG, however no protein expression was obtained from these constructs. Hydrophobic regions were identified in the N-termini of antigens GV-38A and GV-38B which may inhibit expression of these constructs. The hydrophobic region present in GV-38A was identified as a possible transmembrane motif with six membrane spanning regions. To express the antigens without the hydrophobic regions, primers KR20 for GV-38A, (SEQ ID NO: 130) and KR21 for GV-38B (SEQ ID NO: 131) were designed. The truncated GV-38A gene was amplified with primers KR20 and KR12, and the truncated GV-38B gene with KR21 and KR14. The determined nucleotide sequences of truncated GV38A and GV-38B are shown in SEQ ID NO: 132 and 133, respectively, with the corresponding predicted amino acid sequences being shown in SEQ ID NO: 134 and 135, respectively. Extended DNA sequences for truncated GV-38A and GV-38B are provided in SEQ ID NO: 175 and 176, respectively, with the corresponding amino acid sequences being provided in SEQ ID NO: 177 and 178, respectively.
EXAMPLE 9
Purification and Characterisation of Polypeptides from M. vaccae Culture Filtrate by Preparative Isoelectric Focusing and Preparative Polyacrylamide Gel Electrophoresis
M. vaccae soluble proteins were isolated from culture filtrate using preparative isoelectric focusing and preparative polyacrylamide gel electrophoresis as described below. Unless otherwise noted, all percentages in the following example are weight per volume.
M. vaccae (ATCC Number 15483) was cultured in 250 1 sterile Medium 90 which had been fractionated by ultrafiltration to remove all proteins of greater than 10 kDa molecular weight. The medium was centrifuged to remove the bacteria, and sterilised by filtration through a 0.45 .mu.m filter. The sterile filtrate was concentrated by ultrafiltration over a 10 kDa molecular weight cut-off membrane.
Proteins were isolated from the concentrated culture filtrate by precipitation with 10% trichloroacetic acid. The precipitated proteins were re-dissolved in 100 mM Tris.HCl pH 8.0. and re-precipitated by the addition of an equal volume of acetone. The acetone precipitate was dissolved in water, and proteins were re-precipitated by the addition of an equal volume of chloroform:methanol 2:1 (v/v). The chloroform:methanol precipitate was dissolved in water, and the solution was freeze-dried.
The freeze-dried protein was dissolved in iso-electric focusing buffer, containing 8 M deionised urea, 2% Triton X-100, 10 mM dithiothreitol and 2% ampholytes (pH 2.5-5.0). The sample was fractionated by preparative iso-electric focusing on a horizontal bed of Ultrodex gel at 8 watts constant power for 16 hours. Proteins were eluted from the gel bed fractions with water and concentrated by precipitation with 10% trichloroacetic acid.
Pools of fractions containing proteins of interest were identified by analytical polyacrylamide gel electrophoresis and fractionated by preparative polyacrylamide gel electrophoresis. Samples were fractionated on 12.5% SDS-PAGE gels, and electroblotted onto nitrocellulose membranes. Proteins were located on the membranes by staining with Ponceau Red, destained with water and eluted from the membranes with 40% acetonitrile/0.1 M ammonium bicarbonate pH 8.9 and then concentrated by lyophilisation.
Eluted proteins were assayed for their ability to induce proliferation and interferon-.gamma. secretion from the peripheral blood lymphocytes of immune donors as detailed in Example 2. Proteins inducing a strong response in these assays were selected for further study.
Selected proteins were further purified by reversed-phase chromatography on a Vydac Protein C4 column, using a trifluoroacetic acid-acetonitrile system. Purified proteins were prepared for protein sequence determination by SDS-polyacrylamide gel electrophoresis, and electroblotted onto PVDF membranes. Protein sequences were determined as in Example 3. The proteins were named GV-40, GV-41, GV-42, GV-43 and GV-44. The determined N-terminal sequences for these polypeptides are shown in SEQ ID NOS: 101-105, respectively. Subsequent studies led to the isolation of a 5', middle fragment and 3' DNA sequence for GV-42 (SEQ ID NO: 136, 137 and 138, respectively). The corresponding predicted amino acid sequences are provided in SEQ ID NO: 139, 140 and 141, respectively.
Following standard DNA amplification and cloning procedures as described in Example 5, the genes encoding GV-41 and GV-42 were cloned. The determined nucleotide sequences are provided in SEQ ID NOS: 179 and 180, respectively, and the predicted amino acid sequences in SEQ ID NOS: 181 and 182. GV-41 had homology to the ribosome recycling factor of M. tuberculosis and M. leprae, and GV-42 had homogy to a M. avium fibronectin attachment protein FAP-A. Within the full-length sequence of GV-42, the amino acid sequence determined for GV-43 (SEQ ID NO:104 ) was identified, indicating that the amino acid sequences for GV-42 and GV-43 were obtained from the same protein.
Murine polyclonal antisera were prepared against GV-40 and GV-44 following standard procedures. These antisera were used to screen a M. vaccae genomic DNA library consisting of randomly sheared DNA fragments. Clones encoding GV-40 and GV-44 were identified and sequenced. The determined nucleotide sequence of the partial gene encoding GV-40 is provided in SEQ ID NO: 183 and the predicted amino acid sequence in SEQ ID NO: 184. The determined nucleotide sequence of the gene encoding GV-44 is provided in SEQ ID NO: 185, and the predicted amino acid sequence in SEQ ID NO: 186. Homology of GV-40 to M. leprae Elongation factor G was found and GV-44 had homology to M. leprae glyceraldehyde-3-phosphate dehydrogenase.
EXAMPLE 10
Immune Modulating Properties of Delipidated and Deglycolipidated M. vaccae and Recombinant Proteins From M. vaccae
This example illustrates the processing of different constituents of M. vaccae and their immune modulating properties.
Heat-killed M. vaccae and M. vaccae culture filtrate
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/l; tryptone, 5 g/l; glucose 1 g/l) at 37.degree. C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich., USA) with glucose at 37.degree. C. for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10 mg/ml, equivalent to 10.sup.10 M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120.degree. C. The culture filtrate was passaged through a 0.45 .mu.m filter into sterile bottles.
Preparation of Delipidated and Deglycolipidated (DD-) M. vaccae and Compositional Analysis
To prepare delipidated M. vaccae, the autoclaved M. vaccae was pelleted by centrifugation, the pellet washed with water and collected again by centrifugation and then freeze-dried. An aliquot of this freeze-dried M. vaccae was set aside and referred to as lyophilised M. vaccae. When used in experiments it was resuspended in PBS to the desired concentration. Freeze-dried M. vaccae was treated with chloroform/methanol (2:1) for 60 mins at room temperature to extract lipids, and the extraction was repeated once. The delipidated residue from chloroform/methanol extraction was further treated with 50% ethanol to remove glycolipids by refluxing for two hours. The 50% ethanol extraction was repeated two times. The pooled 50% ethanol extracts were used as a source of M. vaccae glycolipids (see below). The residue from the 50% ethanol extraction was freeze-dried and weighed. The amount of delipidated and deglycolipidated M. vaccae prepared was equivalent to 11.1% of the starting wet weight of M. vaccae used. For bioassay, the delipidated and deglycolipidated M. vaccae (DD-M. vaccae; referred to as delipidated M. vaccae in FIG. 9), was resuspended in phosphate-buffered saline by sonication, and sterilised by autoclaving.
The compositional analyses of heat-killed M. vaccae and DD-M. vaccae are presented in Table 7. Major changes are seen in the fatty acid composition and amino acid composition of DD-M. vaccae as compared to the insoluble fraction of heat-killed M. vaccae. The data presented in Table 1 show that the insoluble fraction of heat-killed M. vaccae contains 10% w/w of lipid, and the total amino acid content is 2750 nmoles/mg, or approximately 33% w/w. DD-M. vaccae contains 1.3% w/w of lipid and 4250 nmoles/mg amino acids, which is approximately 51% w/w.
TABLE 7______________________________________Compositional analyses of heat-killed M. vaccae and DD-M. vaccaeMONOSACCHARIDE COMPOSITIONsugar alditol M. vaccae DD-M. vaccae______________________________________Inositol 3.2% 1.7%Ribitol* 1.7% 0.4%Arabinitol 22.7% 27.0%Mannitol 8.3% 3.3%Galactitol 11.5% 12.6%Glucitol 52.7% 55.2%______________________________________
______________________________________FATTY ACID COMPOSITIONFatty acid M. vaccae DD-M. vaccae______________________________________C14:0 3.9% 10.0%C16:0 21.1% 7.3%C16:1 14.0% 3.3%C18:0 4.0% 1.5%C18:1* 1.2% 2.7%C18:1w9 20.6% 3.1%C18:1w7 12.5% 5.9%C22:0 12.1% 43.0%C24:1* 6.5% 22.9%______________________________________
The insoluble fraction of heat-killed M. vaccae contains 10% w/w of lipid, and DD-M. vaccae contains 1.3% W/W of lipid.
______________________________________AMINO ACID COMPOSITIONNmoles/mg M. vaccae DD-M. vaccae______________________________________ASP 231 361THR 170 266SER 131 199GLU 319 505PRO 216 262GLY 263 404ALA 416 621CYS* 24 26VAL 172 272MET* 72 94ILE 104 171LEU 209 340TYR 39 75PHE 76 132GlcNH2 5 6HIS 44 77LYS 108 167ARG 147 272______________________________________
The total amino acid content of the insoluble fraction of heat-killed M. vaccae is 2750 nmoles/mg, or approximately 33% w/w. The total amino acid content of DD-M. vaccae is 4250 nmoles/mg, or approximately 51% w/w.
M. vaccae glycolipids
The pooled 50% ethanol extracts described above were dried by rotary evaporation, redissolved in water, and freeze-dried. The amount of glycolipid recovered was 1.2% of the starting wet weight of M. vaccae used. For bioassay, the glycolipids were dissolved in phosphate-buffered saline.
Production of Interleukin-12 from macrophages
Whole heat-killed M. vaccae and DD-M. vaccae were shown to have different cytokine stimulation properties. The stimulation of a Th1 immune response is enhanced by the production of interleukin-12 (IL-12) from macrophages. The ability of different M. vaccae preparations to stimulate IL-12 production was demonstrated as follows.
A group of C57BL/6J mice were injected intraperitoneally with DIFCO thioglycolate and after three days, peritoneal macrophages were collected and placed in cell culture with interferon-gamma for three hours. The culture medium was replaced and various concentrations of whole heat-killed (autoclaved) M. vaccae, lyophilized M. vaccae, DD-M. vaccae (referred to as delipidated-deglycolipidated M. vaccae in FIG. 8) and M. vaccae glycolipids were added. After a further three days at 37.degree. C., the culture supernatants were assayed for the presence of IL-12 produced by macrophages. As shown in FIG. 8, the M. vaccae preparations stimulated the production of IL-12 from macrophages.
By contrast, these same M. vaccae preparations were examined for the ability to stimulate interferon-gamma production from Natural Killer (NK) cells. Spleen cells were prepared from Severe Combined Immunodeficient (SCID) mice. These populations contain 75-80% NK cells. The spleen cells were incubated at 37.degree. C. in culture with different concentrations of heat-killed M. vaccae, DD-M. vaccae, or M. vaccae glycolipids. The data shown in FIG. 10 demonstrates that, while heat-killed M. vaccae and M. vaccae glycolipids stimulate production of interferon-gamma, DD-M. vaccae stimulated relatively less interferon-gamma. The combined data from FIGS. 8 and 10 indicate that, compared with whole heat-killed M. vaccae, DD-M. vaccae is a better stimulator of IL-12 than interferon gamma.
FIGS. 9A, B, and C show data from separate experiments in which groups of C57BL/6 mice (FIG. 9A), BALB/C mice (FIG. 9B) or C3H/HeJ mice (FIG. 9C) were given DIFCO thioglycolate intraperitoneally and, after three days, peritoneal macrophages were collected and placed in culture with interferon-gamma for three hours. The culture medium was replaced and various concentrations of M. vaccae recombinant proteins GVs-3 (GV-3), GV-4P (GV-4P), GVc-7 (GV-7), GV-23, GV-27, heat killed M. vaccae, DD-M. vaccae (referred to as delipidated M. vaccae in FIGS. 9A, B and C), M. vaccae glycolipids or lipopolysaccharide were added. After three days at 37.degree. C., the culture supernatants were assayed for the presence of IL-12 produced by macrophages. As shown in FIGS. 9A, B and C, the recombinant proteins and M. vaccae preparations stimulated the production of IL-12 from macrophages.
EXAMPLE 11
Effect of Intradermal Route of Immunisation with M. vaccae on Tuberculosis in Cynomolgous Monkeys
This example illustrates the effect of immunisation with M. vaccae or M. vaccae culture filtrate intradermally in cynomolgous monkeys prior to challenge with live M. tuberculosis.
M. vaccae (ATCC Number 15483) was cultured in sterile Medium 90 (yeast extract, 2.5 g/l; tryptone, 5 g/l; glucose, 1 g/l) at 37.degree. C. The cells were harvested by centrifugation, and transferred into sterile Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich., USA) with glucose at 37.degree. C. for one day. The medium was then centrifuged to pellet the bacteria, and the culture filtrate removed. The bacterial pellet was resuspended in phosphate buffered saline at a concentration of 10mg/ml, equivalent to 10.sup.10 M. vaccae organisms per ml. The cell suspension was then autoclaved for 15 min at 120.degree. C. The culture filtrate was passaged through a 0.45 .mu.M filter into sterile bottles.
Three groups of cynomolgous monkeys were included in this study, with each group containing 2 monkeys. One group of monkeys were immunised with whole heat-killed M. vaccae; one group were immunised with M. vaccae culture filtrate and a control group received no immunisations. The composition employed for immunisation, amount of immunogen and route of administration for each group of monkeys are provided in Table 7.
TABLE 7______________________________________COMPARISON OF INTRADERMALROUTE OF IMMUNISATION IdentificationGroup Number of Amount of Route ofNumber Monkey Antigen Immunisation______________________________________1 S3101-E 0 --(Controls) 3144-B 0 --2 4080-B 500 .mu.g intradermal(Immunised 3586-B 500 .mu.g intradermalwith heat-killedM. vaccae)3 3564-B 100 .mu.g intradermal(Immunised 3815-B 100 .mu.g intradermalwith culture filtrate)______________________________________
Prior to immunisation, all monkeys were weighed (Wt kgs), body temperature measured (temp), and a blood sample taken for determination of erythrocyte sedimentation rate (ESR mm/hr) and lymphocyte proliferation (LPA) to an in vitro challenge with purified protein (PPD) prepared from Mycobacterium bovis. At day 33 post-immunisation these measurements were repeated. At day 34, all monkeys received a second immunisation using the same amount of M. vaccae. On day 62, body weight, temperature, ESR and LPA to PPD were measured, then all monkeys were infected with 103 colony forming units of the Erdman strain of M. tuberculosis. Twenty eight days following infection, body weight, temperature, ESR and LPA to PPD were measured in all monkeys, and the lungs were X-rayed to determine whether infection with live M. tuberculosis had resulted in the onset of pneumonia As shown in Tables 8A, B and C, the monkeys in the control group showed radiologic evidence of pulmonary tuberculosis by 28 days after infection with M. tuberculosis. Clinical disease was not evident 84 days after infection in monkeys immunised intradermally with two doses of 500 .mu.g of M. vaccae. The onset of clinical disease was delayed in both monkeys immunised intradermally with 100 .mu.g of M. vaccae culture filtrate.
TABLE 8A______________________________________CONTROL MONKEYS LPA LPA Wt. ESR PPD PPDID# Days Kgs Temp. Mm/hr 10 .mu.g 1 .mu.g X-Ray______________________________________S3101E 0 2.17 37.0 0 0.47 1.1 Negative 34 1.88 37.3 ND 0.85 1.4 ND 62 2.02 36.0 ND 1.3 1.5 ND.fwdarw. Time of Infection 28 2.09 38.0 2 1.3 3.7 Positive 56 1.92 37.2 20 5.6 9.1 Positive 84 1.81 37.5 8 4.7 5.6 Positive3144-B 0 2.05 36.7 0 0.87 1.8 Negative 34 1.86 37.6 ND 2.2 1.4 ND 62 1.87 36.5 ND 1.6 1.6 ND.fwdarw. Time of Infection 28 2.10 38.0 0 12 8.7 Positive 56 1.96 37.6 0 29.6 21.1 Positive 84 1.82 37.3 4 45.3 23.4 Positive______________________________________ ND = Not Done
TABLE 8B______________________________________MONKEYS IMMUNISED WITH WHOLE HEAT-KILLED M. VACCAE(500 .mu.g) INTRADERMALLY LPA LPA Wt. ESR PPD PPDID# Days Kgs Temp. Mm/hr 10 .mu.g 1 .mu.g X-Ray______________________________________4080-B 0 2.05 37.1 1 1.1 0.77 Negative 34 1.97 38.0 ND 1.7 1.4 ND 62 2.09 36.7 ND 1.5 1.5 ND.fwdarw. Time of Infection 28 2.15 37.6 0 2.6 2.1 Negative 56 2.17 37.6 0 8.2 7.6 Negative 84 2.25 37.3 0 3.8 2.8 Negative3586-B 0 2.29 37.0 0 1.1 1.4 Negative 34 2.22 38.0 ND 1.9 1.6 ND 62 2.39 36.0 ND 1.3 1.6 ND.fwdarw. Time of Infection 28 2.31 38.2 0 3.2 2.6 Negative 56 2.32 37.2 0 7.8 4.2 Negative 84 2.81 37.4 0 3.4 1.8 Negative______________________________________ ND = Not Done
TABLE 8C______________________________________MONKEYS IMMUNISED WITH CULTURE FILTRATE (100 .mu.g)INTRADERMALLY LPA LPA Wt. ESR PPD PPDID# Days Kgs Temp. Mm/hr 10 .mu.g 1 .mu.g X-Ray______________________________________3564-B 0 2.40 37.2 0 1.4 1.4 Negative 34 2.42 38.1 ND 3.3 2.7 ND 62 2.31 37.1 ND 3.1 3.4 ND.fwdarw. Time of Infection 28 2.41 38.6 13 24 13.6 Negative 56 2.38 38.6 0 12.7 12.0 Negative 84 2.41 38.6 2 21.1 11.8 Positive3815-B 0 2.31 36.3 0 1.0 1.4 Negative 34 2.36 38.2 ND 1.9 2.0 ND 62 2.36 36.4 ND 3.7 2.8 ND.fwdarw. Time of Infection 28 2.45 37.8 0 2.1 3.3 Negative 56 2.28 37.3 4 8.0 5.6 Negative 84 2.32 37.4 0 1.9 2.2 Positive______________________________________ ND = Not Done
EXAMPLE 12
DNA Cloning Strategy for the DD-M. vaccae Antigen GV-45
Proteins were extracted from DD-M. vaccae (500 mg; prepared as described in Example 10) by suspension in 10 ml 2% SDS/PBS and heating to 50.degree. C. for 2 h. The insoluble residue was removed by centrifugation, and proteins precipitated from the supernatant by adding an equal volume of acetone and incubating at -20.degree. C. for 1 hr. The precipitated proteins were collected by centrifugation, dissolved in reducing sample buffer, and fractionated by preparative SDS-polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto PVDF membrane in 10 mM CAPS/0.0 1% SDS pH 11.0, and N-terminal sequences were determined in a gas-phase sequenator.
The amino acid sequence obtained from these experiments was designated GV-45. The determined N-terminal sequence for GV-45 is provided in SEQ ID NO: 187.
From the amino acid sequence of GV-45, degenerate oligonucleotides KR32 and KR33 (SEQ ID NOS: 188 and 189, respectively) were designed. A 100 bp fragment was amplified, cloned into plasmid pBluescript II SK.sup.+ (Stratagene, La Jolla, Calif.) and sequenced (SEQ ID NO: 190) following standard procedures (Maniatis). The cloned insert was used to screen a M. vaccae genomic DNA library constructed in the BamHI-site of lambda ZAP-Express (Stratagene). The isolated clone showed homology to a 35 kDa M. tuberculosis and a 22 kDa M. leprae protein containing bacterial histone-like motifs at the N-terminus and a unique C-terminus consisting of a five amino acid basic repeat. The determined nucleotide sequence for GV-45 is provided in SEQ ID NO: 191, with the corresponding predicted amino acid sequence being provided in SEQ ID NO: 192.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, changes and modifications can be carried out without departing from the scope of the invention which is intended to be limited only by the scope of the appended claims.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 194- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:#Val Gln Gln Val Pro Asply Xaa Ala Ala Tyr# 15- Gly Pro Gly Ser Val Gln Gly Met Ala# 25- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 10 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Xaa Asp Gln Leu Lys Val Asn Asp Asp# 10- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 11 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:#Tyret Xaa Pro Val Pro Val Ala Thr Ala Ala# 10- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 21 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:#Asp His Val Glu Gln Alaro Pro Pro Tyr Val# 15- Lys Phe Gly Asp Leu 20- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 29 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:#Phe Ala Lys Arg Glu Lysla Asp Ala Tyr Ala# 15#Phe Glu Threu Ala Pro Gly Val Pro Xaa Val# 25- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 21 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:#Val Ser Lys Thr Thr Argaa Ala Ile Leu Gln# 15- Gly Gly Gln Ala Ala 20- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 11 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:#Arget Pro Ile Leu Gln Val Ser Gln Thr Gly# 10- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 14 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:#Val Ser Ser Thrro Ile Xaa Leu Gln Leu Gln# 10- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 16 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:#Arg Ile Glu Ala Arg Valln Gly Gly Leu Gly# 15- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 9 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Lys Xaa Gly Leu Ala Asp Leu Ala Pro 1 5- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 14 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 12...12#Residue can be either Glu or Ile- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:#Val Xaa Ala Alala Leu Ala Leu Met Ser Ala# 10- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 11 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:#Thrys Asn Pro Gln Val Ser Asp Glu Leu Xaa# 10- (2) INFORMATION FOR SEQ ID NO:13:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 21 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:#Asp Pro Ala Ala Val Valla Pro Ala Xaa Gly# 15- Ala Ala Met Ser Thr 20- (2) INFORMATION FOR SEQ ID NO:14:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:#Gly Glu Leu Val Asnaa Tyr Leu Gly Gln Pro# 15- (2) INFORMATION FOR SEQ ID NO:15:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 2...2#Residue can be either Gly or Ala (A) NAME/KEY: Other (B) LOCATION: 15...15#Residue can be either Pro or Ala- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:#Ala Pro Gly Ala Xaaro Pro Xaa Gly Pro Pro# 15- (2) INFORMATION FOR SEQ ID NO:16:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:#Val Ser Thr Leu Sersp Leu Gln Gly Pro Leu# 15- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:#Val Val Thr Phe Ala Serer Gly Arg Tyr Thr# 15- Asp Lys Leu Gly Thr Ser Val Ala Ala# 25- (2) INFORMATION FOR SEQ ID NO:18:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 15...15#Residue can be either Ala or Arg (A) NAME/KEY: Other (B) LOCATION: 23...23#Residue can be either Val or Leu- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:#Asp Ser Thr Ala Xaa Xaasp Arg Gly Tyr Val# 15- Ala Ser Pro Pro Thr Leu Xaa Val Val# 25- (2) INFORMATION FOR SEQ ID NO:19:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- Glu Pro Glu Gly Val Ala Pro Pro 1 5- (2) INFORMATION FOR SEQ ID NO:20:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:#Asp Val Ser Ala Tyr Alaro Ala Gly Phe Pro# 15- Ala Val Asp Pro Xaa Xaa Tyr Val Val# 25- (2) INFORMATION FOR SEQ ID NO:21:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:#Val Gln Gln Val Proro Gly Xaa Ala Ala Tyr# 15- (2) INFORMATION FOR SEQ ID NO:22:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:#Leu Met Val Pro Serly Leu Pro Val Glu Tyr# 15- (2) INFORMATION FOR SEQ ID NO:23:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 19 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:#Leu Met Val Pro Ser Proeu Pro Val Glu Tyr# 15- Ser Met Gly- (2) INFORMATION FOR SEQ ID NO:24:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:#Leu Asp Val Phe Serly Leu Pro Val Glu Tyr# 15- (2) INFORMATION FOR SEQ ID NO:25:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 14 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:#Met Val Pro Asnly Leu His Arg Leu Arg Met# 10- (2) INFORMATION FOR SEQ ID NO:26:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 20 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 16...16#Residue can be either Ser or Val (A) NAME/KEY: Other (B) LOCATION: 17...17#Residue can be either Gln or Val- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:#Gln Ala Glu Pro Ala Xaaal Gly Ala Ala Ala# 15- Xaa Arg Ile Asp 20- (2) INFORMATION FOR SEQ ID NO:27:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 14 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 4...4#Residue can be either Tyr or Pro (A) NAME/KEY: Other (B) LOCATION: 8...8#Residue can be either Val or Gly (A) NAME/KEY: Other (B) LOCATION: 9...9#Residue can be either Ile or Tyr- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:#Ala Arg Gly Thraa Asp Ile Glu Xaa Xaa Phe# 10- (2) INFORMATION FOR SEQ ID NO:28:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:#Arg Asp Ala Gly Pheer Val Ser Asp Tyr Ala# 15- (2) INFORMATION FOR SEQ ID NO:29:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 16 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 2...2#Residue can be either Leu or Pro- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:#Thr Val Asp Ala Asp Glnla Xaa Leu Gly Xaa# 15- (2) INFORMATION FOR SEQ ID NO:30:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 330 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:#Val Ala Gly Met Leu Argrg Phe Arg Gly Ala# 15#Leu Leu Ser Ala Leu Ilela Met Gly Val Ala# 30#Phe Ser Arg Pro Gly Leula Pro Ala Glu Ala# 45#Ser Met Gly Arg Asp Ileln Val Pro Ser Pro# 60#Ser Pro Ala Leu Tyr Leusn Gly Gly Ala Asn# 80#Ser Gly Trp Asp Ile Asnla Gln Asp Asp Phe# 95#Gly Ile Ser Val Val Metrp Tyr Tyr Gln Ser# 110#Asp Trp Tyr Ser Pro Alaer Ser Phe Tyr Ser# 125#Trp Glu Thr Phe Leu Thrys Gln Thr Tyr Lys# 140#Lys Gln Ile Lys Pro Thryr Leu Gln Ser Asn#160#Gly Leu Ser Ala Leu Thrly Leu Ser Met Ala# 175#Tyr Val Gly Ser Met Serro Asp Gln Phe Ile# 190#Pro Ser Leu Ile Gly Leuer Asn Ala Met Gly# 205#Ala Asp Met Trp Gly Proly Gly Tyr Lys Ala# 220#Pro Thr Val Asn Val Glyrp Lys Arg Asn Asp#240#Met Tyr Cys Gly Asn Glysn Thr Arg Ile Trp# 255#Pro Ala Lys Leu Leu Gluly Gly Asn Asn Leu# 270#Gln Asp Gly Tyr Asn Alaer Asn Ile Lys Phe# 285#Pro Asp Ser Gly Thr Hisla Val Phe Asn Phe# 300#Asp Met Lys Pro Asp Leuly Glu Gln Leu Asn#320- Gln Gln Tyr Leu Gly Ala Thr Pro Gly Ala# 330- (2) INFORMATION FOR SEQ ID NO:31:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 327 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:#Trp Gly Arg Trp Leu Leuly Lys Ile Arg Ala# 15#Ile Ser Leu Ala Gly Glyhr Leu Pro Ser Leu# 30#Gly Leu Pro Val Glu Tyrla Phe Ser Arg Pro# 45#Thr Ile Lys Val Gln Phelu Ala Met Gly Arg# 60#Tyr Leu Leu Asp Gly Leuly Ser Pro Ala Val# 80#Ile Asn Thr Ser Ala Pheyr Asn Gly Trp Asp# 95#Val Met Pro Val Gly Glyer Gly Leu Ser Val# 110#Pro Ala Cys Gly Lys Alaer Asp Trp Tyr Ser# 125#Leu Thr Ser Glu Leu Proys Trp Glu Thr Phe# 140#Ser Thr Gly Ser Ala Valsn Arg Ser Val Lys#160#Leu Ile Leu Ala Ala Tyrla Gly Ser Ser Ala# 175#Leu Ser Ala Leu Met Asple Tyr Ala Gly Ser# 190#Gly Leu Ala Met Gly Asplu Pro Gln Leu Ile# 205#Gly Pro Pro Asn Asp Prola Ala Asp Met Trp# 220#Ala Gly Lys Leu Val Alasp Pro Ile Leu Gln#240#Asn Gly Thr Pro Ser Glurp Val Tyr Cys Gly# 255#Leu Glu Asn Phe Val Hisal Pro Ala Glu Phe# 270#Asn Gly Ala Gly Gly Hishe Gln Asp Ala Tyr# 285#Thr His Ser Trp Glu Tyreu Asn Ala Asp Gly# 300#Asp Leu Gln Asn Thr Leusn Ala Met Lys Pro#320- Met Ala Val Pro Arg Ser Gly 325- (2) INFORMATION FOR SEQ ID NO:32:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 338 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:#Val Thr Gly Met Ser Argrg Val Arg Gly Ala# 15#Leu Val Ser Gly Leu Valla Val Gly Ala Ala# 30#Ala Phe Ser Arg Pro Glyhr Ala Thr Ala Gly# 45#Pro Ser Met Gly Arg Aspeu Gln Val Pro Ser# 60#Asn Ser Pro Ala Leu Tyrln Ser Gly Gly Ala# 80#Phe Ser Gly Trp Asp Ilerg Ala Gln Asp Asp# 95#Ser Gly Leu Ser Val Vallu Trp Tyr Asp Gln# 110#Ser Asp Trp Tyr Gln Proln Ser Ser Phe Tyr# 125#Lys Trp Glu Thr Phe Leuly Cys Gln Thr Tyr# 140#Asn Arg His Val Lys Proly Trp Leu Gln Ala#160#Ala Ala Ser Ser Ala Leual Gly Leu Ser Met# 175#Val Tyr Ala Gly Ala Metis Pro Gln Gln Phe# 190#Gly Pro Thr Leu Ile Glyro Ser Gln Ala Met# 205#Ala Ser Asp Met Trp Glyla Gly Gly Tyr Lys# 220#Asp Pro Leu Leu Asn Valla Trp Gln Arg Asn#240#Trp Val Tyr Cys Gly Asnsn Asn Thr Arg Val# 255#Leu Pro Ala Lys Phe Leueu Gly Gly Asn Asn# 270#Phe Gln Asp Ala Tyr Asnhr Ser Asn Ile Lys# 285#Phe Pro Asp Ser Gly Thrsn Gly Val Phe Asp# 300#Asn Ala Met Lys Pro Asprp Gly Ala Gln Leu#320#Thr Gly Pro Ala Pro Glnly Ala Thr Pro Asn# 335- Gly Ala- (2) INFORMATION FOR SEQ ID NO:33:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 325 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:#Trp Gly Arg Arg Leu Metrg Lys Ile Arg Ala# 15#Gly Leu Val Gly Leu Alala Val Val Leu Pro# 30#Arg Pro Gly Leu Pro Valla Gly Ala Phe Ser# 45#Gly Arg Asp Ile Lys Valro Ser Pro Ser Met# 60#Ala Val Tyr Leu Leu Asply Asn Asn Ser Pro# 80#Trp Asp Ile Asn Thr Prosp Asp Tyr Asn Gly# 95#Ser Ile Val Met Pro Valyr Gln Ser Gly Leu# 110#Tyr Ser Pro Ala Cys Glyhe Tyr Ser Asp Trp# 125#Thr Phe Leu Thr Ser Gluhr Tyr Lys Trp Glu# 140#Val Lys Pro Thr Gly Serer Ala Asn Arg Ala#160#Ser Ala Met Ile Leu Alaer Met Ala Gly Ser# 175#Gly Ser Leu Ser Ala Leuln Phe Ile Tyr Ala# 190#Leu Ile Gly Leu Ala Metly Met Gly Pro Ser# 205#Met Trp Gly Pro Ser Seryr Lys Ala Ala Asp# 220#Gln Gln Ile Pro Lys Leurg Asn Asp Pro Thr#240#Cys Gly Asn Gly Thr Prorg Leu Trp Val Tyr# 255#Glu Phe Leu Glu Asn Phela Asn Ile Pro Ala# 270#Ala Tyr Asn Ala Ala Glyeu Lys Phe Gln Asp# 285#Asn Gly Thr His Ser Trphe Asn Phe Pro Pro# 300#Lys Gly Asp Leu Gln Serln Leu Asn Ala Met#320- Ser Leu Gly Ala Gly 325- (2) INFORMATION FOR SEQ ID NO:34:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 338 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:#Val Thr Gly Met Ser Argrg Val Arg Gly Ala# 15#Leu Val Ser Gly Leu Valla Val Gly Ala Ala# 30#Ala Phe Ser Arg Pro Glyhr Ala Thr Ala Gly# 45#Pro Ser Met Gly Arg Aspeu Gln Val Pro Ser# 60#Asn Ser Pro Ala Leu Tyrln Ser Gly Gly Ala# 80#Phe Ser Gly Trp Asp Ilerg Ala Gln Asp Asp# 95#Ser Gly Leu Ser Val Vallu Trp Tyr Asp Gln# 110#Ser Asp Trp Tyr Gln Proln Ser Ser Phe Tyr# 125#Lys Trp Glu Thr Phe Leuly Cys Gln Thr Tyr# 140#Asn Arg His Val Lys Proly Trp Leu Gln Ala#160#Ala Ala Ser Ser Ala Leual Gly Leu Ser Met# 175#Val Tyr Ala Gly Ala Metis Pro Gln Gln Phe# 190#Gly Pro Thr Leu Ile Glyro Ser Gln Ala Met# 205#Ala Ser Asp Met Trp Glyla Gly Gly Tyr Lys# 220#Asp Pro Leu Leu Asn Valla Trp Gln Arg Asn#240#Trp Val Tyr Cys Gly Asnsn Asn Thr Arg Val# 255#Leu Pro Ala Lys Phe Leueu Gly Gly Asn Asn# 270#Phe Gln Asp Ala Tyr Asnhr Ser Asn Ile Lys# 285#Phe Pro Asp Ser Gly Thrsn Gly Val Phe Asp# 300#Asn Ala Met Lys Pro Asprp Gly Ala Gln Leu#320#Thr Gly Pro Ala Pro Glnly Ala Thr Pro Asn# 335- Gly Ala- (2) INFORMATION FOR SEQ ID NO:35:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 323 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:#Trp Gly Arg Arg Leu Metrg Lys Ile Arg Ala# 15#Gly Leu Val Gly Leu Alala Val Val Leu Pro# 30#Arg Pro Gly Leu Pro Valla Gly Ala Phe Ser# 45#Gly Arg Asp Ile Lys Valro Ser Pro Ser Met# 60#Ala Val Tyr Leu Leu Asply Asn Asn Ser Pro# 80#Trp Asp Ile Asn Thr Prosp Asp Tyr Asn Gly# 95#Ser Ile Val Met Pro Valyr Gln Ser Gly Leu# 110#Tyr Ser Pro Ala Cys Glyhe Tyr Ser Asp Trp# 125#Thr Leu Leu Thr Ser Gluhr Tyr Lys Trp Glu# 140#Val Lys Pro Thr Gly Serer Ala Asn Arg Ala#160#Ser Ala Met Ile Leu Alaer Met Ala Gly Ser# 175#Gly Ser Leu Ser Ala Leuln Phe Ile Tyr Ala# 190#Gly Leu Ala Met Gly Asply Met Gly Leu Ile# 205#Gly Pro Ser Ser Asp Prola Ala Asp Met Trp# 220#Ile Pro Lys Leu Val Alasp Pro Thr Gln Gln#240#Asn Gly Thr Pro Asn Glurp Val Tyr Cys Gly# 255#Leu Glu Asn Phe Val Argle Pro Ala Glu Phe# 270#Lys Pro Ala Gly Gly Hishe Gln Asp Ala Tyr# 285#Thr His Ser Trp Glu Tyrhe Pro Pro Asn Gly# 300#Asp Leu Gln Ser Ser Leusn Ala Met Lys Gly#320- Gly Ala Gly- (2) INFORMATION FOR SEQ ID NO:36:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 333 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:#Phe Gly Leu Ala Ala Lysln Met Arg Lys Leu# 15#Gly Thr Ala Leu Leu Alahr Ile Ala Val Ile# 30#Ile Ala Val Ala Phe Seral Gly Asp Thr Ala# 45#Val Pro Ser Pro Ser Metal Glu Tyr Leu Gln# 60#Gly Gly Gln His Ala Valle Gln Phe Gln Gly# 80#Asp Tyr Asn Gly Trp Aspeu Arg Ala Gln Glu# 95#His Ser Gly Leu Ser Valhe Glu Glu Tyr Tyr# 110#Tyr Ser Asn Trp Tyr Glnly Gln Ser Ser Phe# 125#Tyr Lys Trp Glu Thr Phely Gln His Tyr Thr# 140#Ala Asn Lys Asn Val Leuro Ser Trp Leu Gln#160#Met Ser Gly Ser Ser Alala Val Gly Leu Ser# 175#Phe Pro Tyr Ala Ala Seryr Tyr Pro Gln Gln# 190#Trp Trp Pro Thr Met Ilesn Pro Ser Glu Gly# 205#Asn Ala Asn Ser Met Trpsp Ser Gly Gly Tyr# 220#Asn Asp Pro Met Val Glnro Ala Trp Lys Arg#240#Ile Trp Val Tyr Cys Glyla Asn Asn Thr Arg# 255#Asn Ile Pro Ala Lys Phelu Leu Gly Gly Asp# 270#Ile Phe Gln Asn Thr Tyreu Ser Thr Asn Glu# 285#Asn Phe Pro Pro Asn Glyrg Asn Gly Val Phe# 300#Leu Val Ala Met Lys Proyr Trp Asn Gln Gln#320#Asn Asn Alaln Gln Ile Leu Asn Gly Ser Asn# 330- (2) INFORMATION FOR SEQ ID NO:37:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 340 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:#Arg Ser Ala Ala Thr Thrln Val Arg Arg Leu# 15#Gly Ala Val Leu Val Tyrla Ile Ala Ala Met# 30#Thr Ala Gly Ala Phe Serhe Gly Gly Pro Ala# 45#Val Pro Ser Ala Ser Metal Glu Tyr Leu Gln# 60#Gly Gly Pro His Ala Valal Gln Phe Gln Gly# 80#Asp Tyr Asn Gly Trp Aspeu Arg Ala Gln Asp# 95#Gln Ser Gly Leu Ser Valhe Glu Glu Tyr Tyr# 110#Tyr Thr Asp Trp Tyr Glnly Gln Ser Ser Phe# 125#Tyr Lys Trp Glu Thr Phely Gln Asn Tyr Thr# 140#Ala Asn Lys Gly Val Serro Ala Trp Leu Gln#160#Met Ser Gly Gly Ser Alala Val Gly Leu Ser# 175#Phe Pro Tyr Ala Ala Seryr Tyr Pro Gln Gln# 190#Trp Trp Pro Thr Leu Ilesn Pro Ser Glu Gly# 205#Asn Ala Asn Ser Met Trpsp Ser Gly Gly Tyr# 220#Asn Asp Pro Met Val Glnro Ala Trp Lys Arg#240#Ile Trp Val Tyr Cys Glyla Asn Asn Thr Arg# 255#Asn Ile Pro Ala Lys Phesp Leu Gly Gly Asp# 270#Thr Phe Arg Asp Thr Tyreu Arg Thr Asn Gln# 285#Asn Phe Pro Pro Asn Glyrg Asn Gly Val Phe# 300#Leu Val Ala Met Lys Alayr Trp Asn Glu Gln#320#Pro Pro Ala Ala Pro Alaeu Asn Gly Ala Thr# 335- Ala Pro Ala Ala 340- (2) INFORMATION FOR SEQ ID NO:38:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:# 20 AACAC- (2) INFORMATION FOR SEQ ID NO:39:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:# 20 TTGGC- (2) INFORMATION FOR SEQ ID NO:40:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1211 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:#TGGGCCTTGG 60GGAGGAT TGACGGTATG AGACTTCTTG ACAGGATTCG#GGTGGGCCTG 120GCGTCGT GGCTGTCGCG ACAGCGATGA TGCCTGCTTT#GGAGTACCTG 180CGACCGC CGGAGCATTC TCCCGGCCAG GTCTGCCGGT#CGGTGGCGAG 240CGTCGAT GGGGCGCGAC ATCAAGATCC AGTTCCAGAG#CAACGGCTGG 300TCTACCT GCTCGACGGC CTGCGTGCGC AGGAGGACTT#GGTGATGCCG 360AGGCTTT CGAGTGGTTC CTCGACAGCG GCATCTCCGT#CAAGGGCCCG 420CCAGCTT CTACACCGAC TGGTACGCCC CCGCCCGTAA#GCTGCAGGCC 480AGTGGGA GACCTTCCTG ACCCAGGAGC TCCCGGGCTG#GGGTTCGGCC 540AGCCGAC CGGCAGCGGC CCTGTCGGTC TGTCGATGGC#GATGTCCGGC 600CGACCTG GCACCCGGAG CAGTTCATCT ACGCGGGCTC#GGGTGACGCC 660CCGAGGG CTGGTGGCCG TTCCTGATCA ACATCTCGAT#AGCGGTTGGA 720CCGACGA CATGTGGGGC AAGACCGAGG GGATCCCAAC#CCGTATCTGG 780CGATGCT GAACATCCCG ACCCTGGTCG CCAACAACAC#CGCCACGTTC 840ACGGCCA GCCCACCGAG CTCGGCGGCG GCGACCTGCC#CGCCGCGGGT 900CCATCCG CACCAACGAG ACCTTCCGCG ACAACTACAT#GTACTGGGGT 960TGTTCAA CTTCCCGGCC AACGGCACGC ACAACTGGGC#GTTGCACGAA 1020CGATGAA GCCTGACCTG CAGGCGCACC TTCTCTGACG#GTGGCCGACA 1080CCGATTG CGGCCGAGGG TTTCGTCGTC CGGGGCTACT#TGCGCTACGA 1140CGCGATG GTGGCTCATC AGGAACGCCG AGGGGGTCAT#CGTCGAGTAC 1200AGCAATC CTTCCTGCCC GACGGAGAGG TCAACATCCA# 1211- (2) INFORMATION FOR SEQ ID NO:41:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 485 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:#TCTCGCGGTG 60TCAACAC CGCCGCCTTC GAGTGGTACG TCGACTCGGG#GGCCTGCGGT 120GCGGGCA GTCCAGCTTC TACAGCGACT GGTACAGCCC#GCCGGCCTAC 180AGACCTA CAAGTGGGAG ACGTTCCTGA CCCAGGAGCT#GTCCATGGCC 240AGGGGGT CGACCCGAAC CGCAACGCGG CCGTCGGTCT#CGCCGGGTCG 300TGACGCT GGCGATCTAC CACCCGCAGC AGTTCCAGTA#CATCTCGATG 360TGAACCC GTCCGAGGGG TGGTGGCCGA TGCTGATCAA#CCCGAGCAGC 420GCTACAA GGCCAACGAC ATGTGGGGTC CACCGAAGGA#CAACACCCCC 480ACGACCC GATGGTCAAC ATCGGCAAGC TGGTGGCCAA# 485- (2) INFORMATION FOR SEQ ID NO:42:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1052 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:#GCGGGGCTCC 60GTGGGTT GTTTGCCGTT ATGAAGTTCA CAGAGAAGTG#GCTGCCCGGA 120TGCACCG GGTGGGCGTT GCCGATATGG CCGCCGTTGC#CGGTCTTCCT 180CCGGGGG TTCGGCAACG GCCGGGGCAT TCTCCCGGCC#CCAGTTCCAG 240ACGTGTT CTCGCCGTCG ATGGGCCGCG ACATCCGGGT#CGACTACAAC 300ATGCGGT CTACCTGCTC GACGGTCTGC GTGCCCAGGA#GTCGACGATC 360ACACCCC TGCGTTCGAG TGGTTCTACG AGTCCGGCTT#TCGGGGCAAC 420GACAGTC CAGCTTCTAC AGCGACTGGT ACCAGCCGTC#GACGTGGCTG 480CCTACAA GTGGGAGACG TTCCTGACCC AGGAGCTGCC#GATGGCGGGC 540GAGTGTC GCGCACCGGC AACGCGTTCG TCGGCCTGTC#CTCGTCGCTG 600CCTACGC GATCCATCAC CCGCAGCAGT TCATCTACGC#GGCGATGAAC 660ACCCGTC CGAGGGCTGG TGGCCGATGC TGATCGGGCT#GGCGTGGAAG 720TCAACGC CGAGAGCATG TGGGGCCCGT CCTCGGACCC#GATCTGGATC 780TGGTCAA CATCAACCAG CTGGTGGCCA ACAACACCCG#GAACCTGATG 840GCACCCC GTCGGAGCTG GACACCGGGA CCCCGGGCCA#TGACAACTAC 900TCGAAGG ATTCACGTTG CGGACCAACA TCGCCTTCCG#CCACAGCTGG 960GCACCAA CGGTGTCTTC AACTTCCCGG CCTCGGGCAC#TCTGGGAGCT 1020AGCAGCT GCAGCAGATG AAGCCCGACA TCCAGCGGGT# 1052 CCACC CACCCCACAC CC- (2) INFORMATION FOR SEQ ID NO:43:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 326 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:#Trp Ala Arg Arg Phe Glyrg Ile Arg Gly Pro# 15#Ala Leu Val Gly Leu Alahr Ala Met Met Pro# 30#Arg Pro Gly Leu Pro Valla Gly Ala Phe Ser# 45#Gly Arg Asp Ile Lys Ilero Ser Pro Ser Met# 60#Ala Leu Tyr Leu Leu Asply Glu Asn Ser Pro# 80#Trp Asp Ile Asn Thr Glnlu Asp Phe Asn Gly# 95#Ser Val Val Met Pro Valeu Asp Ser Gly Ile# 110#Tyr Ala Pro Ala Arg Asnhe Tyr Thr Asp Trp# 125#Thr Phe Leu Thr Gln Gluhr Tyr Lys Trp Glu# 140#Val Lys Pro Thr Gly Serln Ala Asn Arg Ala#160#Ala Ala Leu Asn Leu Alaer Met Ala Gly Ser# 175#Gly Ser Met Ser Gly Pheln Phe Ile Tyr Ala# 190#Leu Ile Asn Ile Ser Metly Trp Trp Pro Phe# 205#Met Trp Gly Lys Thr Gluhe Lys Ala Asp Asp# 220#Asp Pro Met Leu Asn Ileal Gly Gln Arg Asn#240#Trp Val Tyr Cys Gly Asnsn Asn Thr Arg Ile# 255#Leu Pro Ala Thr Phe Leueu Gly Gly Gly Asp# 270#Phe Arg Asp Asn Tyr Ilerg Thr Asn Glu Thr# 285#Phe Pro Ala Asn Gly Thrsn Gly Val Phe Asn# 300#Gln Ala Met Lys Pro Asprp Gly Arg Glu Leu#320- Leu Gln Ala His Leu Leu 325- (2) INFORMATION FOR SEQ ID NO:44:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 161 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:#Glu Trp Tyr Val Asp Sersn Thr Ala Ala Phe# 15#Gln Ser Ser Phe Tyr Seret Pro Val Gly Gly# 30#Gly Cys Gln Thr Tyr Lysla Cys Gly Lys Ala# 45#Ala Tyr Leu Ala Ala Asnhr Gln Glu Leu Pro# 60#Val Gly Leu Ser Met Alasn Arg Asn Ala Ala# 80#His Pro Gln Gln Phe Glnhr Leu Ala Ile Tyr# 95#Pro Ser Glu Gly Trp Trper Gly Tyr Leu Asn# 110#Ala Gly Gly Tyr Lys Alale Ser Met Gly Asp# 125#Ser Ser Ala Trp Lys Argro Pro Lys Asp Pro# 140#Val Ala Asn Asn Thr Prosn Ile Gly Lys Leu#160- Leu- (2) INFORMATION FOR SEQ ID NO:45:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 334 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:#Ala Lys Ala Ala Met Hisys Trp Arg Gly Ser# 15#Ala Leu Pro Gly Leu Ilesp Met Ala Ala Val# 30#Ala Phe Ser Arg Pro Glyer Ala Thr Ala Gly# 45#Pro Ser Met Gly Arg Aspeu Asp Val Phe Ser# 60#His Ala Val Tyr Leu Leuln Gly Gly Gly Thr# 80#Gly Trp Asp Ile Asn Thrln Asp Asp Tyr Asn# 95#Leu Ser Thr Ile Met Prohe Tyr Glu Ser Gly# 110#Trp Tyr Gln Pro Ser Arger Phe Tyr Ser Asp# 125#Glu Thr Phe Leu Thr Glnyr Thr Tyr Lys Trp# 140#Gly Val Ser Arg Thr Glyeu Glu Ala Asn Arg#160#Ser Ala Ala Leu Thr Tyreu Ser Met Ala Gly# 175#Ala Ser Ser Leu Ser Glyln Gln Phe Ile Tyr# 190#Met Leu Ile Gly Leu Alalu Gly Trp Trp Pro# 205#Ser Met Trp Gly Pro Serly Phe Asn Ala Glu# 220#Met Val Asn Ile Asn Glnys Arg Asn Asp Pro#240#Tyr Cys Gly Thr Gly Thrhr Arg Ile Trp Ile# 255#Gln Asn Leu Met Ala Alahr Gly Thr Pro Gly# 270#Asn Ile Ala Phe Arg Asphe Thr Leu Arg Thr# 285#Val Phe Asn Phe Pro Alaly Gly Thr Asn Gly# 300#Gln Gln Leu Gln Gln Metrp Gly Tyr Trp Gly#320#Gln Ala Thr Alale Gln Arg Val Leu Gly Ala# 330- (2) INFORMATION FOR SEQ ID NO:46:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 795 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:#GGGTAACGAT 60GCCATCT CTTGGGTCCT GGGTCGGGAG GCCATGTTCT#AGGAGCGGCG 120GCGATGT GACCAACATG CGAACAGCGA CAACGAAGCT#GGCGAACGCC 180CATTGGT GGCCGCCACG GGGATGGTCA GCGCGGCGAC#GTTCGACCTG 240AGGTCCG TTACACGCTC ACCTCGGCCG GCGCTTACGA#GTATGCGTTC 300CGCAGCC GCCGAGCATG CAGGCGTTCA ACGCCGACGC#AACCACGATG 360AGGTCAG CCTCGCCCCG GGTGTGCCGT GGGTCTTCGA#GCAGGCCGCC 420GGGCGAT CCTTCAGGTC AGCAGCACCA CCCGCGGTGG#GCACGACGAC 480GCGACAT CGCCGTCGAT GGCCAGGAGG TGCTCAGCCA#AGTCCGGCCA 540GGTGCCA GCTCGGTCAG TGGTGAGTCA CCTCGCCGAG#CGGGTCAGCG 600CGGCTCG CGGTGCAGCA CCCCGAGGCG CTGGGTCGCG#GGGTAGACCA 660GCTGGCC CCGCGCGGCC CCTCGGCGAG GATCTGCTCC#CCCGGGACAC 720TAACTCC AGACCCTTGG TCTGCGTGGG TGCCACCGCG#ACGAAATCGT 780CACCACG CTGGTGCCCT CCCGGTCCGC CTCCGCACGC# 795- (2) INFORMATION FOR SEQ ID NO:47:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 142 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:#Ala Leu Gly Ala Ala Alahr Lys Leu Gly Ala# 15#Ala Thr Ala Asn Ala Glnly Met Val Ser Ala# 30#Ser Ala Gly Ala Tyr Glurg Tyr Thr Leu Thr# 45#Pro Ser Met Gln Ala Pheeu Thr Thr Gln Pro# 60#Glu Lys Val Ser Leu Alala Phe Ala Lys Arg# 80#Met Ala Asp Pro Asn Trpal Phe Glu Thr Thr# 95#Gly Gly Gln Ala Ala Proer Ser Thr Thr Arg# 110#Gln Glu Val Leu Ser Glnle Ala Val Asp Gly# 125#Leu Gly Gln Trpro Tyr Asn Val Arg Cys Gln# 140- (2) INFORMATION FOR SEQ ID NO:48:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 300 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:#CTGCGCTTGC 60CGGTTTT CATCGATGCC GCACACAACC CCGGTGGGCC#GGTGATGGGG 120ACGAGTT CGACTTCCGG TATCTCGTCG GCGTCGTCTC#CGGTCTCGCA 180ACGGGAT CCGCCAGGAC CCGGGCGTGC CGGACGGGCG#CCAGATCGCC 240GCGACAA CCTTCGAAAG GGTGCGGCGC TCAACACGAT#CCGATAATCG 300CCCAGTT GTAAGTGTTC CGCCGAAATT GCATTCCACG- (2) INFORMATION FOR SEQ ID NO:49:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 563 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:#CGGCTACGAG 60GCTCAAG AGTCCGCGCC GAGGTGGATG TGACGCTGGA#CGACTGGTAT 120GCGAGGC GCTGTACCAC TTCGCCTGGG ACGAGTTCTG#CGTGTTGGCC 180AAGTGCA ACTGGGTGAA GGTTTCTCGC ACACCACGGC#CACCGAGGTG 240TGCTGCT CAAGCTTCTG CACCCGGTCA TGCCGTTCGT#TGTGGAGTCA 300TGACCGG GCGGGCCGGC GCGAGCGAAC GTCTGGGAAA#TGCCGCACAA 360ACTGGCC CACGCCCACC GGATACGCGC TGGATCAGGC#CGATCAGGGT 420CCCAGAA GTTGATCACC GAGGTGCGCC GGTTCCGCAG#GGGTCTGGAC 480AGCGGGT GCCTGCCCGG TTGTCCGGCA TCGACACCGC#AGGGCTTCAC 540CGGTGCG CGCGCTGGCC TGGCTTGACC GAGGGTGATG# 563CGAGG TGC- (2) INFORMATION FOR SEQ ID NO:50:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 434 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:#CGCGGCTTTC 60GGATGAG CAAGTTCGAA GTCGTCACCG GGATGGCGTT#CTGGGACGCG 120TCGACGT CGCCGTCGTC GAGGTCGGGC TCGGTGGTCG#CCACACCGAC 180ACGCACC GGTCGCGGTC ATCACCCCGA TCGGGGTGGA#TCACCCGCCA 240CGATCGC CGAGATCGCC GGGGAGAAGG CCGGAAATCA#TCCCGAGGCC 300TGCCGAC CGACACCGTC GCCGTGCTGG CGCGGCAGGT#CGAGGATTCG 360TGGCCCA GGCGGTGCGC TCGGATGCGG CTGTAGCGCG#TTGCAGGGGC 420TGGGCCG TCAGGTCGCC ATCGGCGGCA GCTGCTCCGG# 434- (2) INFORMATION FOR SEQ ID NO:51:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 438 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:#TGCTGATCGA 60GCGCCGG CGGCGGCCAG CTGGTACGGC CATTCCAGCG#CGCCCTCACG 120CGCGTGC TGGCCGACCC GGTGTGGAGC AACAGATGTT#TTCCCGCCGT 180CAGCGCA TGCACGACGT CCCGGTGCCG CTGGAGGCGC#CCATCGTCGC 240ATCGCCA ACGACCACTA CGACCACCTC GACATCGACA#CACACCTGCG 300CAGCGGG CCCCGTTCGT GGTGCCGTTG GGCATCGGCG#CCCACCGCAT 360CCCGAGG CGCGGATCGT CGAGTTGGAC TGGCACGAAG#TGTTCTCCCG 420CTGGTCT GCACCCCCGC CCGGCACTTC TCCGGCCGGT# 438 GGC- (2) INFORMATION FOR SEQ ID NO:52:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 87 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:#Ala His Asn Pro Gly Glyal Phe Ile Asp Ala# 15#Phe Asp Phe Arg Tyr Leurg Leu Arg Asp Glu# 30#Asp Val Asp Gly Ile Argal Met Gly Asp Lys# 45#Leu Ala Leu Phe Val Serro Asp Gly Arg Gly# 60#Asn Thr Ile Gln Ile Alays Gly Ala Ala Leu# 80- Glu Leu Leu Ala Ala Gln Leu 85- (2) INFORMATION FOR SEQ ID NO:53:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 175 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:#Glu Val Asp Val Thr Leuer Arg Val Arg Ala# 15#Ala Leu Tyr His Phe Alaer Arg Ala Cys Glu# 30#Leu Ala Lys Val Gln Leusp Trp Tyr Val Glu# 45#Leu Ala Thr Val Leu Aspis Thr Thr Ala Val# 60#Pro Phe Val Thr Glu Valeu His Pro Val Met# 80#Ala Ser Glu Arg Leu Glyhr Gly Arg Ala Gly# 95#Pro Thr Pro Thr Gly Tyral Val Ala Asp Trp# 110#Ala Asp Thr Gln Lys Leula Ala Gln Arg Ile# 125#Gln Gly Leu Ala Asp Argrg Phe Arg Ser Asp# 140#Asp Thr Ala Gly Leu Asprg Leu Ser Gly Ile#160#Trp Leu Asp Arg Glyla Val Arg Ala Leu Ala# 175- (2) INFORMATION FOR SEQ ID NO:54:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 144 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:#Val Val Thr Gly Met Alasn Ser Lys Phe Glu# 15#Val Ala Val Val Glu Valsp Ala Pro Ile Asp# 30#Val Val Asn Ala Pro Valrp Asp Ala Thr Asn# 45#Thr Asp Tyr Leu Gly Asple Gly Val Asp His# 60#Gly Asn His His Pro Prola Gly Glu Lys Ala# 80#Ala Val Leu Ala Arg Glnro Thr Asp Thr Val# 95#Gln Ala Val Arg Ser Asplu Val Leu Leu Ala# 110#Ala Val Leu Gly Arg Glnlu Asp Ser Glu Cys# 125#Arg Gly Ser Val Ala Serer Cys Ser Gly Cys# 140- (2) INFORMATION FOR SEQ ID NO:55:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 145 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:#Trp Tyr Gly His Ser Serro Ala Ala Ala Ser# 15#Leu Ala Asp Pro Val Trpsp Gly Tyr Arg Val# 30#Gly Pro Gln Arg Met Hisro Ser Arg Ala Val# 45#Ala Val Asp Ala Val Valeu Glu Ala Leu Pro# 60#Ile Asp Thr Ile Val Alayr Asp His Leu Asp# 80#Val Pro Leu Gly Ile Glyrg Ala Pro Phe Val# 95#Ala Arg Ile Val Glu Leurp Gly Val Pro Glu# 110#Leu Thr Leu Val Cys Thris Arg Ile Asp Asp# 125#Ser Arg Asp Ser Thr Leuer Gly Arg Leu Phe# 140- Trp 145- (2) INFORMATION FOR SEQ ID NO:56:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 10 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...1#Residue can be either Gly, Ile, Leu or Val (A) NAME/KEY: Other (B) LOCATION: 2...2#Residue can be either Ile, Leu, Gly or Ala- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:- Xaa Xaa Ala Pro Xaa Gly Asp Ala Xaa Arg# 10- (2) INFORMATION FOR SEQ ID NO:57:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 7...7#Residue can be either Ile or Leu- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:- Pro Glu Ala Glu Ala Asn Xaa Arg 1 5- (2) INFORMATION FOR SEQ ID NO:58:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 11 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 4...4#Residue can be either Gln or Gly (A) NAME/KEY: Other (B) LOCATION: 5...5#Residue cn be either Gly or Gln- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:#Arghr Ala Asn Xaa Xaa Glu Tyr Tyr Asp Asn# 10- (2) INFORMATION FOR SEQ ID NO:59:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 34 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:#Leu Arg Gly Tyr Phe Thrlu Ala Glu Ala Asn# 15#Gly Ile Leu Ala Pro Ileyr Tyr Asp Leu Arg# 30- Gly Asp- (2) INFORMATION FOR SEQ ID NO:60:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:# 20 TGCGC- (2) INFORMATION FOR SEQ ID NO:61:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:# 20 TGGTA- (2) INFORMATION FOR SEQ ID NO:62:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 313 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:#CGGCCTCGGT 60GGCTGCG CGGAATACGC GGCAGCCAAT CCCACTGGGC#AGTTGACAAC 120CAGGACC CGGTCGCGGT GGCGGCCTCG AACAATCCGG#GACACCCTCA 180ACTGTCG GGCCAGCTCA ATCCGCAAGT AAACCTGGTG#CTGCCGGCAT 240CACGGTG TTCGCACCGA CCAACGCGGC ATTTAGCAAG#ACCTACCACG 300GCTCAAG ACCAATTCGT CACTGCTGAC CAGCATCCTG# 313- (2) INFORMATION FOR SEQ ID NO:63:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 18 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:#Glu Arg Leu His Thr Leueu Pro Xaa Tyr Asn# 15- Xaa Gln- (2) INFORMATION FOR SEQ ID NO:64:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 25 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:#Gly Gln Gly Arg Thr Leueu Ser Leu Val Asp# 15- Thr Val Gln Gln Xaa Asp Thr Phe Leu# 25- (2) INFORMATION FOR SEQ ID NO:65:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:#Ala Arg Gly Thr Gly Alale Glu Val Glu Phe# 15- Glu Pro Gly Leu Xaa Xaa Val Xaa Asp Ala# 25- (2) INFORMATION FOR SEQ ID NO:66:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 32 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:# 32 TCCCG GCCAGGTCTG CC- (2) INFORMATION FOR SEQ ID NO:67:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 32 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:# 32 CTCTT CCACGCGGAC GT- (2) INFORMATION FOR SEQ ID NO:68:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 30 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:# 30 CCGGC CCGGTCTTCC- (2) INFORMATION FOR SEQ ID NO:69:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 26 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:# 26 GTGGC CTGAGC- (2) INFORMATION FOR SEQ ID NO:70:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 161 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:#Glu Trp Tyr Val Asp Sersn Thr Ala Ala Phe# 15#Gln Ser Ser Phe Tyr Seret Pro Val Gly Gly# 30#Gly Cys Gln Thr Tyr Lysla Cys Gly Lys Ala# 45#Ala Tyr Leu Ala Ala Asnhr Gln Glu Leu Pro# 60#Val Gly Leu Ser Met Alasn Arg Asn Ala Ala# 80#His Pro Gln Gln Phe Glnhr Leu Ala Ile Tyr# 95#Pro Ser Glu Gly Trp Trper Gly Tyr Leu Asn# 110#Ala Gly Gly Tyr Lys Alale Ser Met Gly Asp# 125#Ser Ser Ala Trp Lys Argrg Thr Glu Asp Pro# 140#Val Ala Asn Asn Thr Prosn Ile Gly Lys Leu#160- Leu- (2) INFORMATION FOR SEQ ID NO:71:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 33 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:# 33 GCCCA GGAAGGGCAC CAG- (2) INFORMATION FOR SEQ ID NO:72:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 32 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:# 32 CTCAC CACTGACCGA GC- (2) INFORMATION FOR SEQ ID NO:73:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:# 20 GARCC- (2) INFORMATION FOR SEQ ID NO:74:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 825 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:#CGTGCTGATC 60CCGCGCC GGCGGCGGCC AGCTGGTACG GCCATTCCAG#TTCGCCCTCA 120ACCGCGT GCTGGCCGAC CCGGTGTGGA GCAACAGATG#GCTTCCCGCC 180CGCAGCG CATGCACGAC GTCCCGGTGC CGCTGGAGGC#CACCATCGTC 240TGATCAG CCACGACCAC TACGACCACC TCGACATCGA#CGCACACCTG 300CCCAGCG GGCCCCGTTC GTGGTGCCGT TGGGCATCGG#AGCCCACCGC 360TCCCCGA GGCGCGGATC GTCGAGTTGG ACTGGCACGA#GTTGTTCTCC 420CGCTGGT CTGCACCCCC GCCCGGCACT TCTCCGGACG#GGCGTTCTTC 480TGTGGGC GTCGTGGGTG GTCACCGGCT CGTCGCACAA#CGGTCCGTTC 540GATACAC GAAGAGCTTC GCCGAGATCG GCGACGAGTA#CCACATGAAC 600TGCCGAT CGGGGCCTAC CATCCCGCGT TCGCCGACAT#CCTGATGGTG 660TGCGCGC CCATCTGGAC CTGACCGAGG TGGACAACAG#CGCCGAACGC 720CGACATT CCGCCTCGCC CCGCATCCGT GGTCCGAGCC#CGGTCAGCGG 780CCGACGC CGAGCGGGTA CGCCTGACCG TGCCGATTCC# 825GTT CGACCCGTGG TGGCGGTTCT GAACC- (2) INFORMATION FOR SEQ ID NO:75:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 273 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:#Ser Trp Tyr Gly His Serla Pro Ala Ala Ala# 15#Val Leu Ala Asp Pro Valal Asp Gly Tyr Arg# 30#Val Gly Pro Gln Arg Meter Pro Ser Arg Ala# 45#Pro Ala Val Asp Ala Valro Leu Glu Ala Leu# 60#Asp Ile Asp Thr Ile Valis Tyr Asp His Leu# 80#Val Val Pro Leu Gly Ileln Arg Ala Pro Phe# 95#Glu Ala Arg Ile Val Gluys Trp Gly Val Pro# 110#Asp Leu Thr Leu Val Cysla His Arg Ile Asp# 125#Phe Ser Arg Asp Ser Thrhe Ser Gly Arg Leu# 140#Ser His Lys Ala Phe Pheal Val Thr Gly Ser#160#Ala Glu Ile Gly Asp Gluyr Thr Lys Ser Phe# 175#Ile Gly Ala Tyr His Proeu Thr Leu Leu Pro# 190#Glu Ala Val Arg Ala Hisis Met Asn Pro Glu# 205#Met Val Pro Ile His Trpal Asp Asn Ser Leu# 220#Ser Glu Pro Ala Glu Argla Pro His Pro Trp#240#Arg Leu Thr Val Pro Ilesp Ala Glu Arg Val# 255#Phe Asp Pro Trp Trp Argsp Pro Glu Ser Thr# 270- Phe- (2) INFORMATION FOR SEQ ID NO:76:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 10 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:- Ala Lys Thr Ile Ala Tyr Asp Glu Glu Ala# 10- (2) INFORMATION FOR SEQ ID NO:77:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 337 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:#GCTCCCGCTG 60TGCTGGT CAGCTCCAAG GTGTCGACCG TCAAGGATCT#CGTCGAGGGC 120TCCAGGC CGGCAAGCCG CTGCTGATCA TCGCCGAGGA#CGTCGCCGTC 180CGCTGGT GGTCAACAAG ATCCGCGGCA CCTTCAAGTC#CATCCTCACC 240TCGGTGA CCGCCGCAAG GCGATGCTGC AGGACATGGC#CGTCTCGCTG 300TCAGCGA AAGAGTCGGG CTGTCCCTGG AGACCGCCGA# 337 AAGGT CGTCGTCACC AAGGACA- (2) INFORMATION FOR SEQ ID NO:78:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 112 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:#Val Ser Thr Val Lys Aspeu Val Ser Ser Lys# 15#Ala Gly Lys Pro Leu Leulu Lys Val Ile Gln# 30#Leu Ser Thr Leu Val Valal Glu Gly Glu Ala# 45#Ala Val Lys Ala Pro Glyhr Phe Lys Ser Val# 60#Asp Met Ala Ile Leu Thrys Ala Met Leu Gln# 80#Leu Ser Leu Glu Thr Alaer Glu Arg Val Gly# 95#Val Val Val Thr Lys Asply Gln Ala Arg Lys# 110- (2) INFORMATION FOR SEQ ID NO:79:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 360 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:#CGACGACGTC 60TCGGCGC TGAGCTGGTC AAAGAGGTCG CCAAGAAGAC#CGAAGGCCTG 120CCACCAC CGCCACCGTG CTCGCTCAGG CTCTGGTTCG#GAAGGCTGTC 180CCGGCGC CAACCCGCTC GGCCTCAAGC GTGGCATCGA#GGAGCAGATT 240AGTCGCT GCTGAAGTCG GCCAAGGAGG TCGAGACCAA#CGCCGAGGCC 300CGATCTC CGCCGGCGAC ACCCAGATCG GCGAGCTCAT#CTTCGGCCTG 360GCAACGA GGGTGTCATC ACCGTCGAGG AGTCGAACAC- (2) INFORMATION FOR SEQ ID NO:80:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 120 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:#Lys Glu Val Ala Lys Lysly Ala Glu Leu Val# 15#Thr Ala Thr Val Leu Alaly Asp Gly Thr Thr# 30#Val Ala Ala Gly Ala Asnlu Gly Leu Arg Asn# 45#Ala Val Glu Ala Val Thrrg Gly Ile Glu Lys# 60#Glu Thr Lys Glu Gln Ileer Ala Lys Glu Val# 80#Thr Gln Ile Gly Glu Leule Ser Ala Gly Asp# 95#Glu Gly Val Ile Thr Valsp Lys Val Gly Asn# 110- Glu Glu Ser Asn Thr Phe Gly Leu# 120- (2) INFORMATION FOR SEQ ID NO:81:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 43 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:# 43 CGAAA GCGTGGGGAG CGAACAGGAT TAG- (2) INFORMATION FOR SEQ ID NO:82:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 43 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:# 43 CTACC TTAGGACCGT CATAGTTACG GGC- (2) INFORMATION FOR SEQ ID NO:83:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:# 20 AAAAA- (2) INFORMATION FOR SEQ ID NO:84:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 31 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:# 31 CTTTT TTTTTTTTTT T- (2) INFORMATION FOR SEQ ID NO:85:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 31 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:# 31 CATGC TSCTSCTSCT S- (2) INFORMATION FOR SEQ ID NO:86:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 238 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:#GTTCGAGCTC 60TCGGAGC GCTCGACCTG AAGCTGCGCC ACGTCATGCA#CCAGGAAGAG 120GGGAGGT CGGGATCACG TTCATCTACG TGACCCACGA#ACAGATCGGC 180GTGACCG CATCGCGGTG ATGAACGCCG GCAACGTCGA#CATCGAAT 238TCTACGA CCGTCCCGCG ACGGTGTTCG TCGCCAGCTT- (2) INFORMATION FOR SEQ ID NO:87:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 79 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:#Lys Leu Arg His Val Metly Ala Leu Asp Leu# 15#Val Gly Ile Thr Phe Ilerg Ile Gln Arg Glu# 30#Thr Met Ser Asp Arg Ileln Glu Glu Ala Leu# 45#Ile Gly Ser Pro Thr Gluly Asn Val Glu Gln# 60#Ala Ser Phe Ile Gluro Ala Thr Val Phe Val# 75- (2) INFORMATION FOR SEQ ID NO:88:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1518 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:#CAGAACCGTG 60TGTTACA ATACCCCACC AGTTCCTCGA AGTAAACGAA#TGGGGTCCGG 120AAAATAT TCACAGCGAC GAAGCCCGGC CGATGCCTGA#GTGACGAAGG 180CGCTTTC CTGCGCGGAT TCTATTGTCG AGTCCGGGGT#TCAGATCCGC 240AATGTAA ATTCGTTGCG GAATCACTTG CATAGGTCCG#GACCGGCACA 300ACAGCCA CGACGGCTGT CCCCGAGGAG GACCTGCCCT#GCCCAAGGGC 360CAGAACC TGCAGAACAG ACGGCGGATT CCGCGGCACC#GGCCGTCGCG 420AGATCGA CCATGTCACG AAGCGCTTCG GCGACTACCT#GTCCGGGTGT 480CCATCGC GCCCGGGGAG TTCTTCTCCA TGCTCGGCCC#AGGGGCGATC 540CGTTGCG CATGATCGCG GGATTCGAGA CCCCGACTGA#CAACACGGTG 600CCGACGT GTCGAGGACC CCACCCAACA AGCGCAACGT#GTACGGCCCG 660CGCTGTT CCCGCACATG ACGGTCTGGG ACAACGTCGC#GCTGGAGATC 720TCGGCAA AGGCGAGGTC CGCAAGCGCG TCGACGAGCT#GCAGCAGCAG 780AATTTGC CGAGCGCAGG CCCGCCCAGC TGTCCGGCGG#CGATGAACCG 840CCCGGGC ACTGGTGAAC TACCCCAGCG CGCTGCTGCT#GCGCATCCAG 900ACCTGAA GCTGCGCCAC GTCATGCAGT TCGAGCTCAA#GCTCACGATG 960TCACGTT CATCTACGTG ACCCACGACC AGGAAGAGGC#CCCGACCGAG 1020CGGTGAT GAACGCCGGC AACGTCGAAC AGATCGGCAG#CAACCTCTGG 1080CCGCGAC GGTGTTCGTC GCCAGCTTCA TCGGACAGGC#TCTCGGCTCG 1140CCGGCCG CTCCAACCGC GATTACGTCG AGATCGACGT#CACCCTGATG 1200GCCCGGG CGAGACCACG ATCGAGCCCG GCGGGCACGC#CGGTGACGTC 1260GCATCCG GGTCACCCCG GGCTCCCAGG ACGCGCCGAC#GCGGCTCTCG 1320CCACCGT CACCGACCTG ACCTTCCAAG GTCCGGTGGT#GGATCTGCCG 1380ACGACTC GACCGTGATC GCCCACGTCG GCCCCGAGCA#CCTGGTGCTT 1440GCGACGA CGTGTACGTC AGCTGGGCAC CGGAAGCCTC#CTCCTGAGTC 1500TCCCCAC CACCGAGGAC CTCGAAGAGA TGCTCGACGA#1518 CGA- (2) INFORMATION FOR SEQ ID NO:89:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 376 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:#Phe Gly Asp Tyr Leu Alais Val Thr Lys Arg# 15#Gly Glu Phe Phe Ser Methe Ser Ile Ala Pro# 30#Thr Leu Arg Met Ile Alays Gly Lys Thr Thr# 45#Arg Leu Glu Gly Ala Asphr Glu Gly Ala Ile# 60#Val Asn Thr Val Phe Glnro Asn Lys Arg Asn# 80#Trp Asp Asn Val Ala Tyrro His Met Thr Val# 95#Glu Val Arg Lys Arg Valys Leu Gly Lys Gly# 110#Glu Phe Ala Glu Arg Argle Val Arg Leu Thr# 125#Arg Val Ala Leu Ala Argly Gly Gln Gln Gln# 140#Leu Asp Glu Pro Leu Glyro Ser Ala Leu Leu#160#Gln Phe Glu Leu Lys Argeu Arg His Val Met# 175#Tyr Val Thr His Asp Glnly Ile Thr Phe Ile# 190#Ala Val Met Asn Ala Glyet Ser Asp Arg Ile# 205#Ile Tyr Asp Arg Pro Alaly Ser Pro Thr Glu# 220#Ala Asn Leu Trp Ala Glyer Phe Ile Gly Gln#240#Val Glu Ile Asp Val Leuer Asn Arg Asp Tyr# 255#Thr Thr Ile Glu Pro Glyla Arg Pro Gly Glu# 270#Arg Ile Arg Val Thr Proet Val Arg Pro Glu# 285#Ala Cys Val Arg Ala Thrro Thr Gly Asp Val# 300#Val Arg Leu Ser Leu Alahe Gln Gly Pro Val#320#Val Gly Pro Glu Gln Asphr Val Ile Ala His# 335#Tyr Val Ser Trp Ala Proro Gly Asp Asp Val# 350#Ile Pro Thr Thr Glu Aspeu Pro Gly Asp Asp# 365- Leu Glu Glu Met Leu Asp Asp Ser# 375- (2) INFORMATION FOR SEQ ID NO:90:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 33 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:# 33 ATCGA GATCGACCAT GTC- (2) INFORMATION FOR SEQ ID NO:91:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 31 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:# 31 CGGGA AGCGTGACTC A- (2) INFORMATION FOR SEQ ID NO:92:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 323 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:#CAAGGAGCCG 60AAGACTT CAACGACAAC GAGCAGTGGT TCGCCAAGGT#CATGGCCGCG 120AGGACAT AGGCGCCGAC CTGGTGATCC CCACCGAGTT#CAATCGCAAG 180TGGGATG GCTCAATGAG ATCAGCGAAG CCGGCGTGCC#CACCGCGCCG 240ACCTGTT GGACTCGAGC ATCGACGAGG GCCGCAAGTT#CGATATCCGC 300TGGTCGG TCTCGCCTAC AACAAGGCAG CCACCGGACG# 323TGGGA TCC- (2) INFORMATION FOR SEQ ID NO:93:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1341 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:#ACATCGATCC 60CCTGGAG CCGACGAAAG GCACCCGCAC ATGTCCCGTG#TCGGCGGTGG 120CGAATGA CCGCACGCCG CACCTTGCGT CGCCGCTTCA#GCGGGTCCGA 180GCGGGCC TGACCCTCGG TTCGTCGTTC CTGGCGGCGT#CCCTGCGCGT 240AGCACCA CGTCACAGGA CAGCGGCCCC GCCAGCGGCG#CCGCCTCGGG 300CTCTATA TGGCCGACGG TTTCATCGCA GCGTTCCAGA#CCAAGGTCAA 360TACAAAG AAGACTTCAA CGACAACGAG CAGTGGTTCG#CCGAGTTCAT 420CGCAAGC AGGACATAGG CGCCGACCTG GTGATCCCCA#GCGTGCCCAA 480AAGGGCC TGGGATGGCT CAATGAGATC AGCGAAGCCG#GCAAGTTCAC 540CGTCAGG ACCTGTTGGA CTCGAGCATC GACGAGGGCC#CCGGACGCGA 600ACCGGCA TGGTCGGTCT CGCCTACAAC AAGGCAGCCA#GTCTGTTCTC 660GACGACC TCTGGGATCC CGCGTTCAAG GGCCGCGTCA#CGGAGAATCC 720GGCCTCG GCATGATCAT GCTCTCGCAG GGCAACTCGC#ACAGGGGGTC 780ATTCAGC AGGCGGTCGA TCTGGTCCGC GAACAGAACG#ACATCGCCAT 840CACCGGC AACGACTACG CCGACGACCT GGCCGCAGAA#ATCTGCAGTT 900TCCGGTG ACGTCGTGCA GCTGCAGGCG GACAACCCCG#CGTACACCAC 960TCCGGCG GCGACTGGTT CGTCGACACG ATGGTGATCC#CCAACTACGC 1020GCCGCCG AGGCGTGGAT CGACTACATC TACGACCGAG#ACGAACTCGC 1080TTCACCC AGTTCGTGCC CGCACTCTCG GACATGACCG#AGGTGCAGGC 1140GCATCGG CGGAGAACCC GCTGATCAAC CCGTCGGCCG#ACACTGCGTA 1200TGGGCGG CACTGACCGA CGAGCAGACG CAGGAGTTCA#TAAATGGCCC 1260GGCGGCT GACGCGGTGG TAGTGCCGAT GCGAGGGGCA#CGGACAAGGT 1320AGCATAA ATGGCCGGTG TCGCCACCAG CAGCCGTCAG# 1341TCC T- (2) INFORMATION FOR SEQ ID NO:94:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 393 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:#Ala Arg Met Thr Ala Argsp Pro His Leu Leu# 15#Gly Ala Ala Ala Ala Alarg Phe Ile Gly Gly# 30#Ala Cys Gly Ser Asp Serer Ser Phe Leu Ala# 45#Gly Pro Ala Ser Gly Alahr Ser Gln Asp Ser# 60#Ala Asp Gly Phe Ile Alarp Pro Leu Tyr Met# 80#Asp Tyr Lys Glu Asp Pheer Gly Ile Thr Val# 95#Lys Glu Pro Leu Ser Argrp Phe Ala Lys Val# 110#Pro Thr Glu Phe Met Alala Asp Leu Val Ile# 125#Glu Ile Ser Glu Ala Glyeu Gly Trp Leu Asn# 140#Leu Leu Asp Ser Ser Ilesn Leu Arg Gln Asp#160#Met Thr Gly Met Val Glyhe Thr Ala Pro Tyr# 175#Asp Ile Arg Thr Ile Aspla Ala Thr Gly Arg# 190#Val Ser Leu Phe Ser Aspla Phe Lys Gly Arg# 205#Ser Gln Gly Asn Ser Proly Met Ile Met Leu# 220#Ala Val Asp Leu Val Arglu Ser Ile Gln Gln#240#Leu His Arg Gln Arg Leuly Ser Asp Pro Ser# 255#Ile Ala Gln Ala Tyr Serrg Arg Asn Ile Ala# 270#Pro Asp Leu Gln Phe Ileeu Gln Ala Asp Asn# 285#Asp Thr Met Val Ile Proly Asp Trp Phe Val# 300#Ala Trp Ile Asp Tyr Ileln Lys Ala Ala Glu#320#Ala Phe Thr Gln Phe Valyr Ala Lys Leu Val# 335#Ala Lys Val Asp Pro Alaet Thr Asp Glu Leu# 350#Ala Glu Val Gln Ala Asneu Ile Asn Pro Ser# 365#Gln Thr Gln Glu Phe Asnla Leu Thr Asp Glu# 380- Thr Ala Tyr Ala Ala Val Thr Gly Gly#390- (2) INFORMATION FOR SEQ ID NO:95:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:# 22ATCC CC- (2) INFORMATION FOR SEQ ID NO:96:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:#21 GCGTC A- (2) INFORMATION FOR SEQ ID NO:97:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 861 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:#CTGGCACGCA 60TATCTCG CGATCTTCTT CCGTGGTGCC GTTCTTCTCG#GCCTGGGACT 120GACCGGC GGCTCGGTGT TCATGCCGAC GCTGACGTTC#TCGTTCGGCT 180CGACGCG TTCACGATGT ACCACGAGCA GATCTTCCGC#TACGTCATCG 240CACGGTG CTGTGCCTGT TGCTGGCGTT CCCGCTGGCC#CCGTTCTTCG 300CCGGTTC AAGAACCTGA TCCTGGGGCT GGTGATCCTG#GGCTGGGTGG 360CCGCACC ATTGCGTGGA AGACGATCCT GGCCGACGAA#TCCACCAGCT 420CGCCATC GGGCTGCTGC CTGACGAGGG CCGGCTGCTG#CCGCTGTACG 480CGGTCTG ACCTACAACT GGATCATCTT CATGATCCTG#TACTCGTCGG 540GATCGAC CCGCGTCTGC TGGAGGCCTC CCAGGACCTC#CTGGCCGGGA 600CGGCAAG GTGATCCTGC CGATGGCGAT GCCCGGGGTG#CTCGGCAGTA 660CATCCCG GCCGTCGGCG ACTTCATCAA CGCCGACTAT#AAGGACTATC 720GATCGGC AACGTGATCC AGAAGCAGTT CCTGGTCGTC#GTGCTCCTCT 780GCTGAGT CTGGGGCTGA TGTTGCTGAT CCTGATCGGC#CCGCACTGGC 840GGGTTCG GAGGATCTGG TATGACCACC CAGGCAGGCG# 861ATC C- (2) INFORMATION FOR SEQ ID NO:98:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 259 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:#Ser Leu Ser Glu Thr Glyer Leu Ala Arg Thr# 15#Ala Trp Asp Phe Gly Asnro Thr Leu Thr Phe# 30#Gln Ile Phe Arg Ser Phehr Met Tyr His Glu# 45#Leu Leu Leu Ala Phe Prola Thr Val Leu Cys# 60#Arg Phe Lys Asn Leu Ilela Phe Lys Ala Gly# 80#Thr Phe Leu Ile Arg Threu Pro Phe Phe Val# 95#Trp Val Val Thr Ala Leueu Ala Asp Glu Gly# 110#Arg Leu Leu Ser Thr Sereu Pro Asp Glu Gly# 125#Trp Ile Ile Phe Met Ilely Leu Thr Tyr Asn# 140#Asp Pro Arg Leu Leu Gluer Leu Glu Lys Ile#160#Arg Ser Phe Gly Lys Valyr Ser Ser Ala Pro# 175#Ala Gly Ser Met Leu Valet Pro Gly Val Leu# 190#Ala Asp Tyr Leu Gly Serly Asp Phe Ile Asn# 205#Gln Lys Gln Phe Leu Valle Gly Asn Val Ile# 220#Ser Leu Gly Leu Met Leula Ala Ala Ala Leu#240#Arg Ala Leu Gly Ser Glual Leu Leu Tyr Thr# 255- Asp Leu Val- (2) INFORMATION FOR SEQ ID NO:99:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 277 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:#CAAGGACTTC 60GAGCCCG TACGCCGGTA GGCAAACTCA TGGGTTCGCT#CTTCCCCGGC 120TCGGTGC CGTGGCGATC AAGGGCGCCC TGGAGAAAGC#CTCCGCCGGC 180CTCGTCT CGTCGAGTAC GTGATCATGG GCCAAGTGCT#GGACGTCGCC 240CCGCCCG CCAGGCCGCC GTCGCCGCCG GCATCCCGTG# 277 AAGAT GTGCCTGTCG GGCATCG- (2) INFORMATION FOR SEQ ID NO:100:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 92 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:#Gly Lys Leu Met Gly Serla Arg Thr Pro Val# 15#Ala Val Ala Ile Lys Glyly Ser Asp Leu Gly# 30#Asp Pro Ala Arg Leu Valhe Pro Gly Val Asp# 45#Ala Gly Ala Gly Gln Metly Gln Val Leu Ser# 60#Ile Pro Trp Asp Val Alala Val Ala Ala Gly# 80#Gly Ileeu Thr Ile Asn Lys Met Cys Leu Ser# 90- (2) INFORMATION FOR SEQ ID NO:101:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 12 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (ix) FEATURE: (A) NAME/KEY: Other (B) LOCATION: 1...1#Residue can be either Glu or Pro (A) NAME/KEY: Other (B) LOCATION: 2...2#Residue can be either Pro or Glu- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:#Arg Xaaaa Ala Asp Arg Gly Xaa Ser Lys Tyr# 10- (2) INFORMATION FOR SEQ ID NO:102:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 24 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:#Glu Lys Met Glu Lys Alaeu Phe Asp Ala Glu# 15- Val Ser Val Ala Arg Asp Ser Ala 20- (2) INFORMATION FOR SEQ ID NO:103:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 23 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:#Leu Ser Glu Phe Xaa Alala Thr Ser Gly Thr# 15- Xaa Lys Gly Val Thr Met Glu 20- (2) INFORMATION FOR SEQ ID NO:104:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:#Ala Asp Arg Val Glysp Ala Phe Ala Val Leu# 15- (2) INFORMATION FOR SEQ ID NO:105:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 9 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:- Xaa Ile Arg Val Gly Val Asn Gly Phe 1 5- (2) INFORMATION FOR SEQ ID NO:106:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 485 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:#TCTCGCGGTG 60TCAACAC CGCCGCCTTC GAGTGGTACG TCGACTCGGG#GGCCTGCGGT 120GCGGGCA GTCCAGCTTC TACAGCGACT GGTACAGCCC#GCCGGCCTAC 180AGACCTA CAAGTGGGAG ACGTTCCTGA CCCAGGAGCT#GTCCATGGCC 240AGGGGGT CGACCCGAAC CGCAACGCGG CCGTCGGTCT#CGCCGGGTCG 300TGACGCT GGCGATCTAC CACCCGCAGC AGTTCCAGTA#CATCTCGATG 360TGAACCC GTCCGAGGGG TGGTGGCCGA TGCTGATCAA#CCCGAGCAGC 420GCTACAA GGCCAACGAC ATGTGGGGTC GCACCGAGGA#CAACACCCCC 480ACGACCC GATGGTCAAC ATCGGCAAGC TGGTCGCCAA# 485- (2) INFORMATION FOR SEQ ID NO:107:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 501 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:#GGCCGCGTGC 60GTGCGCG CAGTGCGCTT GCGTCCGTGA CCTTCGTCGC#CTACACGGTG 120GCACCGC ACTGGCGGCG ACGCCGGACT GGAGCGGGCG#AGAACCCGAC 180CCGACAA ACTCGGCACG AGTGTGGCCG CCCGCCAGCC#CACCGCGTCC 240ACACCTT CAGCACGTCC TGTGTGGGCA CCTGCGTGGC#CTGGGACGGC 300CGTCGAA CCCGACGATT CCGCAGCCCG CGCGCTACAC#CGACGTCCCG 360TCAACTA CAACTGGCAG TGGGAGTGCT TCCGCGGCGC#CGGGTCGATG 420CCGCGCG TTCGCTGGTG TTCTACGCCC CGACCGCCGA#GATCATGCCG 480GCACCGA NATCCTGGAN GGCCTCTGCA AGGGCACCGT# 501GTA G- (2) INFORMATION FOR SEQ ID NO:108:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 180 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:#CGCCAACCCG 60GGCCCGA GGCCGAGGCG AACCTGCGGG GCTACTTCAC#GCGCAACTGC 120ACCTGCG GGGCATCCTC GCCCCGATCG GTGACGCGCA#GGCCGGCTGA 180TGCCGGT AGAGCTGCAG ACGGCCTACG ACACGTTCAT- (2) INFORMATION FOR SEQ ID NO:109:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 166 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:#Ala Ser Val Thr Phe Valla Arg Ser Ala Leu# 15#Ala Leu Ala Ala Thr Proly Ala Glu Gly Thr# 30#Phe Ala Ser Asp Lys Leuyr Thr Val Val Thr# 45#Pro Asp Phe Ser Gly Glnla Arg Gln Pro Glu# 60#Cys Val Ala Thr Ala Serer Cys Val Gly Thr# 80#Pro Gln Pro Ala Arg Tyrer Asn Pro Thr Ile# 95#Tyr Asn Trp Gln Trp Gluln Trp Val Phe Asn# 110#Tyr Ala Ala Ala Arg Sersp Val Pro Arg Glu# 125#Ser Met Phe Gly Thr Trpro Thr Ala Asp Gly# 140#Gly Thr Val Ile Met Prosp Gly Leu Cys Lys#160- Val Ala Ala Tyr Pro Ala 165- (2) INFORMATION FOR SEQ ID NO:110:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 74 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:#Ala Val Thr Ala Ala Metro Gly Ala Asn Gln# 15#Leu Arg Gly Tyr Phe Thrlu Ala Glu Ala Asn# 30#Gly Ile Leu Ala Pro Ileyr Tyr Asp Leu Arg# 45#Val Leu Pro Val Glu Leusn Cys Asn Ile Thr# 60- Gln Thr Ala Tyr Asp Thr Phe Met Ala Gly# 70- (2) INFORMATION FOR SEQ ID NO:111:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 503 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:#GGCCGCGTTA 60GTGTTCT GGGCAGTGTC GGTGCAGCAG TCGCGGTTTC#CATCGAGGTG 120TTTCGAT ACCGACCGCC TCAGCGGATC CGTGTCCGGA#TGCGTTCGTC 180GGACCGG TGCGGAACCC GGCCTCGGGT GGGTCGGTGA#GAACTACCCG 240CCAAGGT CGGTGAGCAG TCGGTGGGCA CCTACGCGGT#CGGGGCGGGT 300TTCGACA AATCGGCGCC CATGGGCGCG GCCGACGCAT#TGTCGCANGG 360GACAACT GCCCGGACAC CAAGCTTGTC CTGGGCGGCA#TCACCCCCAC 420GACCTGA TCACCGTCGA TCCGCGACCG CTGGGCCGGT#GAAATCCGTT 480CGCGTCG CCGACCACGT GGCCGCCGTT GTGGTCTTCG# 503TGGCG GTC- (2) INFORMATION FOR SEQ ID NO:112:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 167 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:#Gly Ala Ala Val Ala Valal Leu Gly Ser Val# 15#Ile Pro Thr Ala Ser Alaln Thr Gly Val Ser# 30#Ala Arg Gly Thr Gly Alale Glu Val Ile Phe# 45#Phe Val Asn Ala Leu Argrp Val Gly Asp Ala# 60#Tyr Ala Val Asn Tyr Proln Ser Val Gly Thr# 80#Met Gly Ala Ala Asp Alasp Lys Ser Ala Pro# 95#Cys Pro Asp Thr Lys Leurp Met Ala Asp Asn# 110#Val Ile Asp Leu Ile Threr Xaa Gly Ala Gly# 125#Pro Thr Pro Met Pro Proeu Gly Arg Phe Thr# 140#Val Phe Gly Asn Pro Leual Ala Ala Val Val#160- Arg Asp Ile Arg Gly Gly Gly 165- (2) INFORMATION FOR SEQ ID NO:113:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1569 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:#GGGCCTCAAC 60TTGCGTA TGACGAAGAG GCCCGCCGTG GCCTCGAGCG#CGTGCTGGAG 120CCGTAAA GGTGACGTTG GGCCCGAAGG GTCGCAACGT#GGAGATCGAG 180CCCCCAC GATCACCAAC GATGGTGTGT CCATCGCCAA#CAAGAAGACC 240ACGAGAA GATCGGCGCT GAGCTGGTCA AAGAGGTCGC#TCTGGTTCGC 300GCGACGG CACCACCACC GCCACCGTGC TCGCTCAGGC#TGGCATCGAG 360ACGTCGC AGCCGGCGCC AACCCGCTCG GCCTCAAGCG#CGAGACCAAG 420CTGTCAC CCAGTCGCTG CTGAAGTCGG CCAAGGAGGT#CGAGCTCATC 480CCACCGC GGCGATTTCC GCCGGCGACA CCCAGATCGG#GTCGAACACC 540ACAAGGT CGGCAACGAG GGTGTCATCA CCGTCGAGGA#CATCTCGGGT 600TCGAGCT CACCGAGGGT ATGCGCTTCG ACAAGGGCTA#CATCCTGCTG 660ACGCCGA GCGCCAGGAA GCCGTCCTGG AGGATCCCTA#GGTCATCCAG 720TGTCGAC CGTCAAGGAT CTGCTCCCGC TGCTGGAGAA#GTCCACGCTG 780TGCTGAT CATCGCCGAG GACGTCGAGG GCGAGGCCCT#GGGCTTCGGT 840TCCGCGG CACCTTCAAG TCCGTCGCCG TCAAGGCTCC#GGTCGTCAGC 900CGATGCT GCAGGACATG GCCATCCTCA CCGGTGGTCA#GGCCCGCAAG 960TGTCCCT GGAGACCGCC GACGTCTCGC TGCTGGGCCA#CGATGCCATC 1020AGGACGA GACCACCATC GTCGAGGGCT CGGGCGATTC#CTACGACCGC 1080CTCAGAT CCGCGCCGAG ATCGAGAACA GCGACTCCGA#CAAGGCCGGA 1140AGCGCCT GGCCAAGCTG GCCGGCGGTG TTGCGGTGAT#CGTCCGCAAC 1200TGGAGCT CAAGGAGCGC AAGCACCGCA TCGAGGACGC#GCTGCAGTCG 1260TCGAAGA GGGCATCGTC GCCGGTGGCG GCGTGGCTCT#CAACATCGTC 1320ACGACCT CGGCCTGACG GGCGACGAGG CCACCGGTGC#GGAGCCCGGC 1380CGGCTCC GCTCAAGCAG ATCGCCTTCA ACGGCGGCCT#CGCGACCGGT 1440AGGTGTC CAACCTGCCC GCGGGTCACG GCCTCAACGC#CCGCTCGGCG 1500TGCTCAA GGCCGGCGTC GCCGACCCGG TGAAGGTCAC#CGTCGCCGAC 1560CGTCCAT CGCGGCTCTG TTCCTCACCA CCGAGGCCGT# 1569- (2) INFORMATION FOR SEQ ID NO:114:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 523 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:#Ala Arg Arg Gly Leu Glula Tyr Asp Glu Glu# 15#Lys Val Thr Leu Gly Proeu Ala Asp Ala Val# 30#Trp Gly Ala Pro Thr Ileal Leu Glu Lys Lys# 45#Ile Glu Leu Glu Asp Proer Ile Ala Lys Glu# 60#Glu Val Ala Lys Lys Thrla Glu Leu Val Lys# 80#Ala Thr Val Leu Ala Glnsp Gly Thr Thr Thr# 95#Ala Ala Gly Ala Asn Proly Leu Arg Asn Val# 110#Val Glu Ala Val Thr Glnly Ile Glu Lys Ala# 125#Thr Lys Glu Gln Ile Serla Lys Glu Val Glu# 140#Gln Ile Gly Glu Leu Ileer Ala Gly Asp Thr#160#Gly Val Ile Thr Val Gluys Val Gly Asn Glu# 175#Leu Thr Glu Gly Met Argly Leu Gln Leu Glu# 190#Val Thr Asp Ala Glu Argle Ser Gly Tyr Phe# 205#Leu Leu Val Ser Ser Lyslu Asp Pro Tyr Ile# 220#Leu Glu Lys Val Ile Glnsp Leu Leu Pro Leu#240#Asp Val Glu Gly Glu Alaeu Ile Ile Ala Glu# 255#Gly Thr Phe Lys Ser Valal Asn Lys Ile Arg# 270#Arg Lys Ala Met Leu Glnly Phe Gly Asp Arg# 285#Val Ser Glu Arg Val Glyhr Gly Gly Gln Val# 300#Leu Gly Gln Ala Arg Lysla Asp Val Ser Leu#320#Val Glu Gly Ser Gly Aspsp Glu Thr Thr Ile# 335#Ile Arg Ala Glu Ile Gluly Arg Val Ala Gln# 350#Leu Gln Glu Arg Leu Alayr Asp Arg Glu Lys# 365#Ala Gly Ala Ala Thr Glual Ala Val Ile Lys# 380#Glu Asp Ala Val Arg Asnrg Lys His Arg Ile#400#Ala Gly Gly Gly Val Alalu Glu Gly Ile Val# 415#Leu Gly Leu Thr Gly Aspro Ala Leu Asp Asp# 430#Ala Leu Ser Ala Pro Leusn Ile Val Arg Val# 445#Pro Gly Val Val Ala Glusn Gly Gly Leu Glu# 460#Leu Asn Ala Ala Thr Glyro Ala Gly His Gly#480#Ala Asp Pro Val Lys Valeu Lys Ala Gly Val# 495#Ile Ala Ala Leu Phe Leuln Asn Ala Ala Ser# 510#Gluhr Thr Glu Ala Val Val Ala Asp Lys Pro# 520- (2) INFORMATION FOR SEQ ID NO:115:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 647 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic RNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:#GGGCCTCAAC 60TTGCGTA TGACGAAGAG GCCCGCCGTG GCCTCGAGCG#CGTGCTGGAG 120CCGTAAA GGTGACGTTG GGCCCGAAGG GTCGCAACGT#GGAGATCGAG 180CCCCCAC GATCACCAAC GATGGTGTGT CCATCGCCAA#CAAGAAGACC 240ACGAGAA GATCGGCGCT GAGCTGGTCA AAGAGGTCGC#TCTGGTTCGC 300GCGACGG CACCACCACC GCCACCGTGC TCGCTCAGGC#TGGCATCGAG 360ACGTCGC AGCCGGCGCC AACCCGCTCG GCCTCAAGCG#CGAGACCAAG 420CTGTCAC CCAGTCGCTG CTGAAGTCGG CCAAGGAGGT#CGAGCTCATC 480CCACCGC GGCGATTTCC GCCGGCGACA CCCAGATCGG#GTCGAACACC 540ACAAGGT CGGCAACGAG GGTGTCATCA CCGTCGAGGA#CATCTCGGGT 600TCGAGCT CACCGAGGGT ATGCGCTTCG ACAAGGGCTA# 647GCCGA GCGCCAGGAA GCCGTCCTGG AGGATCC- (2) INFORMATION FOR SEQ ID NO:116:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 927 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:#GCTCCCGCTG 60TGCTGGT CAGCTCCAAG GTGTCGACCG TCAAGGATCT#CGTCGAGGGC 120TCCAGGC CGGCAAGCCG CTGCTGATCA TCGCCGAGGA#CGTCGCCGTC 180CGCTGGT GGTCAACAAG ATCCGCGGCA CCTTCAAGTC#CATCCTCACC 240TCGGTGA CCGCCGCAAG GCGATGCTGC AGGACATGGC#CGTCTCGCTG 300TCAGCGA AAGAGTCGGG CTGTCCCTGG AGACCGCCGA#CGAGGGCTCG 360GCAAGGT CGTCGTCACC AAGGACGAGA CCACCATCGT#CGAGAACAGC 420CCATCGC CGGCCGGGTG GCTCAGATCC GCGCCGAGAT#CGGCGGTGTT 480ACCGCGA GAAGCTGCAG GAGCGCCTGG CCAAGCTGGC#GCACCGCATC 540CCGGAGC TGCCACCGAG GTGGAGCTCA AGGAGCGCAA#CGGTGGCGGC 600GCAACGC GAAGGCTGCC GTCGAAGAGG GCATCGTCGC#CGACGAGGCC 660AGTCGGC TCCTGCGCTG GACGACCTCG GCCTGACGGG#CGCCTTCAAC 720TCGTCCG CGTGGCGCTG TCGGCTCCGC TCAAGCAGAT#GGGTCACGGC 780CCGGCGT CGTTGCCGAG AAGGTGTCCA ACCTGCCCGC#CGACCCGGTG 840CCGGTGA GTACGAGGAC CTGCTCAAGG CCGGCGTCGC#CCTCACCACC 900CGGCGCT GCAGAACGCG GCGTCCATCG CGGCTCTGTT# 927 GACAA GCCGGAG- (2) INFORMATION FOR SEQ ID NO:117:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 215 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:#Ala Arg Arg Gly Leu Glula Tyr Asp Glu Glu# 15#Lys Val Thr Leu Gly Proeu Ala Asp Ala Val# 30#Trp Gly Ala Pro Thr Ileal Leu Glu Lys Lys# 45#Ile Glu Leu Glu Asp Proer Ile Ala Lys Glu# 60#Glu Val Ala Lys Lys Thrla Glu Leu Val Lys# 80#Ala Thr Val Leu Ala Glnsp Gly Thr Thr Thr# 95#Ala Ala Gly Ala Asn Proly Leu Arg Asn Val# 110#Val Glu Ala Val Thr Glnly Ile Glu Lys Ala# 125#Thr Lys Glu Gln Ile Serla Lys Glu Val Glu# 140#Gln Ile Gly Glu Leu Ileer Ala Gly Asp Thr#160#Gly Val Ile Thr Val Gluys Val Gly Asn Glu# 175#Leu Thr Glu Gly Met Argly Leu Gln Leu Glu# 190#Val Thr Asp Ala Glu Argle Ser Gly Tyr Phe# 205- Gln Glu Ala Val Leu Glu Asp# 215- (2) INFORMATION FOR SEQ ID NO:118:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 309 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:#Val Ser Thr Val Lys Aspeu Val Ser Ser Lys# 15#Ala Gly Lys Pro Leu Leulu Lys Val Ile Gln# 30#Leu Ser Thr Leu Val Valal Glu Gly Glu Ala# 45#Ala Val Lys Ala Pro Glyhr Phe Lys Ser Val# 60#Asp Met Ala Ile Leu Thrys Ala Met Leu Gln# 80#Leu Ser Leu Glu Thr Alaer Glu Arg Val Gly# 95#Val Val Val Thr Lys Asply Gln Ala Arg Lys# 110#Ser Asp Ala Ile Ala Glylu Gly Ser Gly Asp# 125#Asn Ser Asp Ser Asp Tyrrg Ala Glu Ile Glu# 140#Lys Leu Ala Gly Gly Valln Glu Arg Leu Ala#160#Val Glu Leu Lys Glu Argly Ala Ala Thr Glu# 175#Ala Lys Ala Ala Val Glusp Ala Val Arg Asn# 190#Leu Leu Gln Ser Ala Proly Gly Gly Val Ala# 205#Glu Ala Thr Gly Ala Asnly Leu Thr Gly Asp# 220#Lys Gln Ile Ala Phe Asneu Ser Ala Pro Leu#240#Lys Val Ser Asn Leu Proly Val Val Ala Glu# 255#Glu Tyr Glu Asp Leu Leusn Ala Ala Thr Gly# 270#Thr Arg Ser Ala Leu Glnsp Pro Val Lys Val# 285#Thr Thr Glu Ala Val Valla Ala Leu Phe Leu# 300- Ala Asp Lys Pro Glu 305- (2) INFORMATION FOR SEQ ID NO:119:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 162 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:#CGCCACCCTG 60CGGAGAT CTCCGACGAC GCCACGTCGG TACGGTTGGT#CATCCAGGGC 120TGTTGAC GTTGGTGCTG TCCGGGCTCA ACGCCACCCT# 162 TGGCG CAGGCGGATT CCGTCGATCT TC- (2) INFORMATION FOR SEQ ID NO:120:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1366 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:#CGGTCGGGTT 60CTGAACT CGACCTGGTT GGCCTGGGCC GTCGCGGTCG#GCGGCAGCGC 120GTCGTGC TGACCGAGGT GCACAACGCG TTGCGTCGGC#CGTTGCTGCT 180GTGCAAC TCCTGCGTAC CTACATCCTG CCGCTGGGCG#TGGTCGCCAC 240GCGATGG AGATCTCCGA CGACGCCACG TCGGTACGGT#CCCTCATCCA 300GTGTTGT TGACGTTGGT GCTGTCCGGG CTCAACGCCA#ACGTCGCGCG 360GACAGCT GGCGCAGGCG GATTCCGTCG ATCTTCCTCG#GCGCGAACGT 420GCGGTCG GTATCACCGT GATCATGGCC TATGTCTGGG#CTCTGCAGAA 480ACCGCAC TGGGCGTCAC TTCCATCGTT CTTGGCCTGG#TCCGGCTCGG 540ATCATCT CGGGTCTGCT GCTGCTGTTC GAGCAACCGT#GCGTGGTGGA 600GTCCCCA CCGCGGCGGG CCGGCCGTCC GCCCACGGCC#TGCCCAACGC 660GCAACAC ATATCGACAC CGGCGGCAAC CTGCTGGTAA#ACCGGCTGAC 720GCGTCGT TCACCAATTA CAGCCGGCCC GTGGGAGAGC#TGCTGTCGTC 780TTCAACG CCGCGGACAC CCCCGATGAT GTCTGCGAGA#TCTATCTCGG 840CTGCCCG AACTGCGCAC CGACGGACAG ATCGCCACGC#ACTCGGTCAG 900GAGAAGT CGATCCCGTT GCACACACCC GCGGTGGACG#GCCTNAACGG 960CGATGGG TCTGGTACGC CGCGCGCCGG CAGGAACTTC#CTGTGGCGTC 1020TTCGACA CGCCGGAACG GATCGCCTCG GCCATGCGGG#GTCTGGTCCG 1080GCAGACG ACGAACAGCA GGAGATCGCC GACGTGGTGC#TGAGGTTCAT 1140GAACGCC TCCAGCAGCC GGGTCAGGTA CCGACCGGGA#TCCCGGCGCG 1200GTGAGTC TGTCCGTGAT CGATCAGGAC GGCGACGTGA#CGGTACTGGC 1260GGCGACT TCCTGGGGCA GACCACGCTG ACGCGGGAAC#AGATCGAGCG 1320CTGGAGG AAGTCACCGT GCTGGAGATG GCCCGTGACG# 1366CCGA TCCTGCTGCA CGTGATCGGG GCCGTG- (2) INFORMATION FOR SEQ ID NO:121:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 455 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:#Ala Trp Ala Val Ala Valsn Ser Thr Trp Leu# 15#Leu Thr Glu Val His Asnal Leu Leu Val Val# 30#Arg Pro Val Gln Leu Leuly Ser Ala Leu Ala# 45#Leu Leu Leu Leu Val Glnro Leu Gly Ala Leu# 60#Val Arg Leu Val Ala Thrsp Asp Ala Thr Ser# 80#Leu Ser Gly Leu Asn Alaeu Leu Thr Leu Val# 95#Trp Arg Arg Arg Ile Prola Pro Glu Asp Ser# 110#Leu Ile Ala Val Gly Ileal Ala Arg Phe Ala# 125#Asn Val Gly Gly Leu Pheyr Val Trp Gly Ala# 140#Gly Leu Ala Leu Gln Asnhr Ser Ile Val Leu#160#Leu Leu Phe Glu Gln Prole Ser Gly Leu Leu# 175#Thr Ala Ala Gly Arg Prorp Ile Thr Val Pro# 190#Trp Arg Ala Thr His Ileal Val Glu Val Asn# 205#Asn Ala Glu Leu Ala Glyeu Leu Val Met Pro# 220#Gly Glu His Arg Leu Thryr Ser Arg Pro Val#240#Pro Asp Asp Val Cys Glusn Ala Ala Asp Thr# 255#Glu Leu Arg Thr Asp Glyla Ala Ser Leu Pro# 270#Glu Tyr Glu Lys Ser Ileyr Leu Gly Ala Ala# 285#Val Arg Ser Thr Tyr Leula Val Asp Asp Ser# 300#Glu Leu Arg Xaa Asn Glyla Ala Arg Arg Gln#320#Ile Ala Ser Ala Met Argsp Thr Pro Glu Arg# 335#Asp Glu Gln Gln Glu Ileeu Arg Leu Ala Asp# 350#Asn Gly Glu Arg Leu Glneu Val Arg Tyr Gly# 365#Phe Ile Val Asp Gly Argro Thr Gly Met Arg# 380#Asp Val Ile Pro Ala Argle Asp Gln Asp Gly#400#Thr Thr Leu Thr Arg Glusp Phe Leu Gly Gln# 415#Glu Val Thr Val Leu Glula His Ala Leu Glu# 430#His Arg Lys Pro Ile Leule Glu Arg Leu Val# 445- Leu His Val Ile Gly Ala Val# 455- (2) INFORMATION FOR SEQ ID NO:122:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 898 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:#AAGACGCGGG 60CCTGGAA TGCGCGAACG TCTGAACACC CGACGCGAAA#GCTGCTTCTG 120TGTCGCG GATGAGCATC CAGTCCAAGT TGCTGCTGAT#ACGGTCCTCG 180CGGCTGC GGTGGTCGGT TTCATCGGCT ATCAGTCCGG#GCGCGGGTTG 240TGTTCGA CCGCCTCACC GACATCCGCG AGTCGCAGTC#CAGCACTGCC 300CGGACCT GAAGAACTCG ATGGTGATTT ACTCGCGCGG#GACGATCAAT 360GCGCGTT CAGCGACGGT TTCCGTCAGC TCGGCGATGC#CACCACCCTC 420CGTCATT GCGCCGTTAC TACGACCGGA CGTTCGCCAA#CCCCCAGCGC 480ACCGCGT CGACGTCCGC GCGCTCATCC CGAAATCCAA#CGCGTTCGAC 540TCTATAC CCCGCCGTTT CAGAACTGGG AGAAGGCGAT#GTTCTTCCGC 600GCAGCGC CTGGTCGGCC GCCAATGCCA GATTCAACGA#GGGCAACGTG 660GCTTCAA CTTCGAGGAT CTGATGCTGC TCGACCTCGA#CCCCTATCGC 720ACAAGGG GCCGGATCTC GGGACAAACA TCGTCAACGG#CGACTATGTC 780CGGAAGC CTACGAGAAG GCGGTCGCGT CGAACTCGAT#GTTCCTGTCC 840TCGGGTG GTACCTGCCT GCCGAGGAAC CGACCGCCTG#CGGAATTC 898AGGACCG AGTCGACGGT GTGATGGCGG TCCAGTTCCC- (2) INFORMATION FOR SEQ ID NO:123:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1259 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:#ACCGGTGAGA 60GGCGCGG GGACAGTGGC GTGACACCGG GATGGGAGAC#CGCGAGAACC 120ACCGGAC AATCTGATGC GCTCGGACTC CCGGCTGTTC#GCCGACGAAT 180GGCCGAC GTCGTCGAGG GGGGAACCCC GCCGGAGGTC#GTCGAGGAGG 240CGGCACC ACGCTGGTGC AGCCGGTGAC CACCCGCTCC#GAGGCGTTAC 300CACCGGG ACGACGATCG AGGACGACTA TCTCGGCCAC#AAGATCGACA 360GGTGGAC CTGCCGGGAC TGCACTGGGT GATCGTGGCC#TCGACGGTGA 420CGCCCCG GTGGCGCAGT TCACCAGGAC CCTGGTGCTG#GTCCGTCCGA 480CGTGTCG CTGGCGGCCA TGCTGCTGGC GCGGTTGTTC#CTCGCTCTGC 540GGCCGGC GCCCAGCAGA TCAGCGGCGG TGACTACCGC#ATGAGTCGCA 600TGACGAA TTCGGCGATC TGACAACAGC TTTCAACGAC#CGGCTGATGC 660GGACGAG CTGCTCGGCG AGGAGCGCGC CGAGAACCAA#ACGATCGCCC 720CGAACCG GTGATGCAGC GCTACCTCGA CGGGGAGGAG#GAGTTGTCGC 780CGTCACG GTGATCTTCG CCGACATGAT GGGCCTCGAC#CAGTTCGACG 840CGAGGAA CTGATGGTGG TGGTCAACGA CCTGACCCGC#TACCTGGCCA 900TCTCGGG GTCGACCACG TGCGGACGCT GCACGACGGG#TTCGCGATCG 960CGTGCCG CGGCTGGACA ACGTCCGGCG CACGGTCAAT#CGGCTCCGCG 1020CATCGAC CGGCACGCCG CCGAGTCCGG GCACGACCTG#TTGGCGTACG 1080CGGGTCG GCGGCCAGCG GGCTGGTGGG GCGGTCCACG#CCCCAGCCCG 1140GGCGGTC GATGTCGCCT ACCAGGTGCA GCGCGGCTCC#TTCGTCGCCG 1200CTCGCGG GTGCACGAGG TCATGCAGGA AACTCTCGAC#GGCCACCCG 1259CGGCGAG CGCGGCGTCG AGACGGTCTG GCGGTTGCAG- (2) INFORMATION FOR SEQ ID NO:124:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 299 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:#Ser Glu His Pro Thr Argrp Asn Ala Arg Thr# 15#Arg Met Ser Ile Gln Seryr His Leu Leu Ser# 30#Ile Leu Ser Ala Ala Valeu Leu Leu Thr Ser# 45#Ser Ser Leu Arg Ala Seryr Gln Ser Gly Arg# 60#Ser Gln Ser Arg Gly Leuhr Asp Ile Arg Glu# 80#Met Val Ile Tyr Ser Argsp Leu Lys Asn Ser# 95#Phe Ser Asp Gly Phe Arglu Ala Ile Gly Ala# 110#Gln Ala Ala Ser Leu Arghr Ile Asn Thr Gly# 125#Thr Leu Asp Asp Ser Glyhr Phe Ala Asn Thr# 140#Lys Ser Asn Pro Gln Argrg Ala Leu Ile Pro#160#Gln Asn Trp Glu Lys Alayr Thr Pro Pro Phe# 175#Ala Trp Ser Ala Ala Asnla Arg Asp Gly Ser# 190#Val His Arg Phe Asn Phehe Phe Arg Glu Ile# 205#Asn Val Val Tyr Ser Alaeu Asp Leu Glu Gly# 220#Val Asn Gly Pro Tyr Argeu Gly Thr Asn Ile#240#Ala Val Ala Ser Asn Serlu Ala Tyr Glu Lys# 255#Trp Tyr Leu Pro Ala Glual Thr Asp Phe Gly# 270#Gly Leu Lys Asp Arg Valhe Leu Ser Pro Val# 285#Ilesp Gly Val Met Ala Val Gln Phe Pro Gly# 295- (2) INFORMATION FOR SEQ ID NO:125:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 419 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:#Asp Thr Gly Met Gly Asprg Gly Gln Trp Arg# 15#Asn Leu Met Arg Ser Aspeu Val Gly Pro Asp# 30#Phe Leu Ala Asp Val Vallu Asn Arg Glu Lys# 45#Glu Ser Val Asp Arg Argro Glu Val Ala Asp# 60#Arg Ser Val Glu Glu Alaln Pro Val Thr Thr# 80#Asp Asp Tyr Leu Gly Hisly Thr Thr Ile Glu# 95#Leu Pro Gly Leu His Trpyr Ser Pro Val Asp# 110#Ala Phe Ala Pro Val Alale Asp Thr Asp Glu# 125#Val Ile Ile Ile Phe Glyeu Val Leu Ser Thr# 140#Leu Phe Val Arg Pro Ileet Leu Leu Ala Arg#160#Ser Gly Gly Asp Tyr Argly Ala Gln Gln Ile# 175#Phe Gly Asp Leu Thr Threu Ser Arg Asp Glu# 190#Ile Lys Asp Glu Leu Leuer Arg Asn Leu Ser# 205#Met Leu Ser Leu Met Prolu Asn Gln Arg Leu# 220#Glu Glu Thr Ile Ala Glnrg Tyr Leu Asp Gly#240#Asp Met Met Gly Leu Asphr Val Ile Phe Ala# 255#Leu Met Val Val Val Asneu Thr Ser Glu Glu# 270#Glu Ser Leu Gly Val Asphe Asp Ala Ala Ala# 285#Ala Ser Cys Gly Leu Glyis Asp Gly Tyr Leu# 300#Val Asn Phe Ala Ile Glusn Val Arg Arg Thr#320#Glu Ser Gly His Asp Leusp Arg His Ala Ala# 335#Ala Ala Ser Gly Leu Valle Asp Thr Gly Ser# 350#Gly Ser Ala Val Asp Valla Tyr Asp Met Trp# 365#Pro Gly Ile Tyr Val Thrrg Gly Ser Pro Gln# 380#Leu Asp Phe Val Ala Alaal Met Gln Glu Thr#400#Thr Val Trp Arg Leu Glnlu Arg Gly Val Glu# 415- Gly His Pro- (2) INFORMATION FOR SEQ ID NO:126:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 27 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:# 27 AGCGT GCTGAAC- (2) INFORMATION FOR SEQ ID NO:127:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 26 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:# 26 CCGAT CACGTG- (2) INFORMATION FOR SEQ ID NO:128:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 33 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:# 33 TTTCT GCCCTGGAAT GCG- (2) INFORMATION FOR SEQ ID NO:129:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 32 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:# 32 GGCCC TGCAACCGCC AG- (2) INFORMATION FOR SEQ ID NO:130:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 27 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:# 27 CCGTT CCGGCTC- (2) INFORMATION FOR SEQ ID NO:131:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 27 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Other- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:# 27 CAGTC CGGACGG- (2) INFORMATION FOR SEQ ID NO:132:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 844 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:#CCGGCCGTCC 60GGCTCGG CGACTGGATC ACCGTCCCCA CCGCGGCGGG#CGGCGGCAAC 120TGGTGGA AGTCAACTGG CGTGCAACAC ATATCGACAC#CAGCCGGCCC 180CCAACGC CGAACTCGCC GGCGCGTCGT TCACCAATTA#CCCCGATGAT 240GGCTGAC CGTCGTCACC ACCTTCAACG CCGCGGACAC#CGACGGACAG 300TGTCGTC GGTCGCGGCG TCGCTGCCCG AACTGCGCAC#GCACACACCC 360ATCTCGG TGCGGCCGAA TACGAGAAGT CGATCCCGTT#CGCGCGCCGG 420CGGTCAG GAGCACGTAC CTGCGATGGG TCTGGTACGC#TCGCCTCGGC 480TAACGGC GTCGCCGACG ATTCGACACG CCGGAACGGA#AGATCGCCGA 540GCGTCCA CACTGCGCTT GGCAGACGAC GAACAGCAGG#GTCAGGTACC 600GTCCGTT ACGGCAACGG GGAACGCCTC CAGCAGCCGG#ATCAGGACGG 660TTCATCG TAGACGGCAG GGTGAGTCTG TCCGTGATCG#CCACGCTGAC 720GCGCGGG TGCTCGAGCG TGGCGACTTC CTGGGGCAGA#TGGAGATGGC 780CTGGCGA CCGCGCACGC GCTGGAGGAA GTCACCGTGC#TGATCGGGGC 840GAGCGCC TGGTGCACCG AAAGCCGATC CTGCTGCACG# 844- (2) INFORMATION FOR SEQ ID NO:133:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 742 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:#CACCGACATC 60GACGGTC CTCGCTGCGC GCATCGGTGT TCGACCGCCT#CTCGATGGTG 120CGCGCGG GTTGGAGAAT CAGTTCGCGG ACCTGAAGAA#CGGTTTCCGT 180GCAGCAC TGCCACGGAG GCGATCGGCG CGTTCAGCGA#TTACTACGAC 240CGACGAT CAATACCGGG CAGGCGGCGT CATTGCGCCG#CCGCGCGCTC 300ACACCAC CCTCGACGAC AGCGGAAACC GCGTCGACGT#GTTTCAGAAC 360ACCCCCA GCGCTATCTG CAGGCGCTCT ATACCCCGCC#GGCCGCCAAT 420TCGCGTT CGACGACGCG CGCGACGGCA GCGCCTGGTC#GGATCTGATG 480AGTTCTT CCGCGAGATC GTGCACCGCT TCAACTTCGA#TCTCGGGACA 540AGGGCAA CGTGGTGTAC TCCGCCTACA AGGGGCCGGA#GAAGGCGGTC 600GCCCCTA TCGCAACCGG GAACTGTCGG AAGCCTACGA#GCCTGCCGAG 660TCGACTA TGTCGGTGTC ACCGACTTCG GGTGGTACCT#CGGTGTGATG 720GGTTCCT GTCCCCGGTC GGGTTGAAGG ACCGAGTCGA# 742GAAT TC- (2) INFORMATION FOR SEQ ID NO:134:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 282 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:#Thr Val Pro Thr Ala Alaeu Gly Asp Trp Ile# 15#Glu Val Asn Trp Arg Alais Gly Arg Val Val# 30#Val Met Pro Asn Ala Gluly Gly Asn Leu Leu# 45#Arg Pro Val Gly Glu Hishe Thr Asn Tyr Ser# 60#Ala Asp Thr Pro Asp Asphr Thr Phe Asn Ala# 80#Ser Leu Pro Glu Leu Arger Ser Val Ala Ala# 95#Gly Ala Ala Glu Tyr Glula Thr Leu Tyr Leu# 110#Asp Asp Ser Val Arg Seris Thr Pro Ala Val# 125#Arg Arg Gln Glu Leu Argal Trp Tyr Ala Ala# 140#Pro Glu Arg Ile Ala Sersp Xaa Phe Asp Thr#160#Leu Ala Asp Asp Glu Glnla Ser Thr Leu Arg# 175#Arg Tyr Gly Asn Gly Glual Val Arg Leu Val# 190#Gly Met Arg Phe Ile Vally Gln Val Pro Thr# 205#Gln Asp Gly Asp Val Ileeu Ser Val Ile Asp# 220#Leu Gly Gln Thr Thr Leulu Arg Gly Asp Phe#240#Ala Leu Glu Glu Val Threu Ala Thr Ala His# 255#Arg Leu Val His Arg Lysrg Asp Glu Ile Glu# 270- Pro Ile Leu Leu His Val Ile Gly Ala Val# 280- (2) INFORMATION FOR SEQ ID NO:135:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 247 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:#Ala Ser Val Phe Asp Argrg Ser Ser Leu Arg# 15#Gly Leu Glu Asn Gln Phelu Ser Gln Ser Arg# 30#Ser Arg Gly Ser Thr Alaer Met Val Ile Tyr# 45#Phe Arg Gln Leu Gly Aspla Phe Ser Asp Gly# 60#Leu Arg Arg Tyr Tyr Asply Gln Ala Ala Ser# 80#Ser Gly Asn Arg Val Asphr Thr Leu Asp Asp# 95#Gln Arg Tyr Leu Gln Alaro Lys Ser Asn Pro# 110#Lys Ala Ile Ala Phe Asphe Gln Asn Trp Glu# 125#Ala Asn Ala Arg Phe Asner Ala Trp Ser Ala# 140#Asn Phe Glu Asp Leu Metle Val His Arg Phe#160#Ser Ala Tyr Lys Gly Proly Asn Val Val Tyr# 175#Tyr Arg Asn Arg Glu Leule Val Asn Gly Pro# 190#Asn Ser Ile Asp Tyr Valys Ala Val Ala Ser# 205#Ala Glu Glu Pro Thr Alaly Trp Tyr Leu Pro# 220#Arg Val Asp Gly Val Metal Gly Leu Lys Asp#240- Ala Val Gln Phe Pro Gly Ile 245- (2) INFORMATION FOR SEQ ID NO:136:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 45 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:#45 CGNCC CTGGCGGGTT CTGGCATGTG GCATC- (2) INFORMATION FOR SEQ ID NO:137:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 340 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:#CGCGCCGGCC 60CCGCCGC GGTGCCCGCC GGGGTGAGCG CCCCGGCGGT#CACGCTCAGC 120CCCGCCC GGTGTCCACG ATCGCGCCGG CGACCTCGGG#CTTCCGCGCC 180CCAAGGG CGTCACGATG GAGCCGCAGT CCAGCCGCGA#GAACGTGCCG 240TGCCGAA GCCGCGGGGC TGGGAGCACA TCCCGGACCC#GACAAACGCC 300TGCTGGC CGACCGGGTC AGNGGTAAAG GTCAGNAGTC# 340 AAACA CGTAGGCGAG TTCGACGGCA- (2) INFORMATION FOR SEQ ID NO:138:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 235 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: Genomic DNA- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:#ACGCGATTGT 60GTNGAAC AGGTCGTTGC CGAAGCCGCG GAGGCCACCG#CTGCACCCGG 120GTCAGCG TTCCGGGTCC GGGTCCGGCC GCACCGCCAC#TCGCACCACC 180CCGCCCG CCCCCGGCGC CCCGGCGCTG CCGCTGGCCG#TGCAG 235GTTCCCG CCGTGGCGCC CGCGCCACAG CTGCTGGGAC- (2) INFORMATION FOR SEQ ID NO:139:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:#Leu Ala Cys Gly Ilela Arg Pro Trp Arg Val# 15- (2) INFORMATION FOR SEQ ID NO:140:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 113 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:#Gly Val Ser Ala Pro Alala Ala Val Pro Ala# 15#Pro Val Ser Thr Ile Alala Met Pro Ala Arg# 30#Phe Ala Ala Lys Gly Valhr Leu Ser Glu Phe# 45#Arg Ala Leu Asn Ile Valer Ser Arg Asp Phe# 60#Pro Asp Pro Asn Val Proly Trp Glu His Ile# 80#Gly Gly Lys Gly Gln Xaaeu Ala Asp Arg Val# 95#His Val Gly Glu Phe Aspal Val Val Asp Lys# 110- Gly- (2) INFORMATION FOR SEQ ID NO:141:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 73 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:#Ala Ala Asp Ala Thr Glulu Gln Val Val Ala# 15#Pro Gly Pro Gly Pro Alahe Lys Val Ser Val# 30#Val Pro Pro Ala Pro Glyro Gly Ala Pro Gly# 45#Pro Pro Ala Pro Ala Valeu Ala Val Ala Pro# 60- Pro Ala Val Ala Pro Ala Pro Gln Leu# 70- (2) INFORMATION FOR SEQ ID NO:142:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 273 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:#CAGCGGATAC 60AGGGGGG TCTCGGCCGC ATCGAGGCCC GGGTGGCCGA#CATCGACCAG 120CCAAGGG CTACTTCCCG CTGAGCTTCA CCGTCGCCGG#CGTGGCCACC 180TGACCGC CAACGTCACC GCGGCGGCCC CGACGGGCGC#CAAGCAGTCC 240TCATCGC CGGGCCGAGC CCGACCGGAT GGCAGCTGTC# 273 TCCGC GGTCATCGCC GCA- (2) INFORMATION FOR SEQ ID NO:143:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 91 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:#Ile Glu Ala Arg Val Alaly Gly Leu Gly Arg# 15#Gly Tyr Phe Pro Leu Sersn Ala Ala Ala Lys# 30#Pro Ile Val Thr Ala Asnle Asp Gln Asn Gly# 45#Ala Thr Gln Pro Leu Thrro Thr Gly Ala Val# 60#Gln Leu Ser Lys Gln Serer Pro Thr Gly Trp# 80#Alala Leu Ala Leu Met Ser Ala Val Ile Ala# 90- (2) INFORMATION FOR SEQ ID NO:144:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 554 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:#AACGAGTTAC 60GAGAATG TAACGTTCGA CCGGAGAACG CCGTCGGCAC#GTAACCGATC 120AGATCTC GGTTACCTTG GATTTCAGGC GGGGGAAGCA#CGCAAGCCGC 180ACCCAAA CAACATGAAA TTCACTGGAA TGACCGTGCG#GCAACCGTGG 240CGTCGGG GCGGCATGTC TGTTCGGCGG CGTGGCCGCG#ACCGGCACCG 300GGGCGCC CAGCCGGCCG AGTGCAACGC CAGCTCACTC#GCCAACCAGG 360CGGTCAG GCGCGTCAGT ACCTAGACAC CCACCCGGGC#CGGGGCTACT 420GATGAAC CAGCCGCGGC CCGAGGCCGA GGCGAACCTG#ATCGGTGACG 480GGCGGAG TACTACGACC TGCGGGGCAT CCTCGCCCCG#TACGACACGT 540CAACATC ACCGTGCTGC CGGTAGAGCT GCAGACGGCC# 554- (2) INFORMATION FOR SEQ ID NO:145:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 136 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:#Ser Arg Arg Ala Leu Alaet Thr Val Arg Ala# 15#Val Ala Ala Ala Thr Valys Leu Phe Gly Gly# 30#Glu Cys Asn Ala Ser Serly Ala Gln Pro Ala# 45#Gln Ala Arg Gln Tyr Leuer Ser Val Thr Gly# 60#Thr Ala Ala Met Asn Glnla Asn Gln Ala Val# 80#Gly Tyr Phe Thr Ala Asnlu Ala Asn Leu Arg# 95#Leu Ala Pro Ile Gly Aspsp Leu Arg Gly Ile# 110#Pro Val Glu Leu Gln Thrsn Ile Thr Val Leu# 125- Ala Tyr Asp Thr Phe Met Ala Gly# 135- (2) INFORMATION FOR SEQ ID NO:146:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 808 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:#CCGCAGCTGG 60GNGTGTG ACGGTAGACG TTCCGACCAA TCCAACGACG#GGATTCAGGA 120GCCAATT CAGTGCGGGC AACGGTGTCC GTCCACGAAG#TGGCCATCCT 180CGCCGGA AGTCAGCCGC AGTGGCGGGA ATCGCTGCGG#CCAGCAGCAC 240TGTTCGA GTGAGGACGG TGGGAGCACG GCCTCGTCGG#CGGCCCCTTC 300ATGGAGT CCGCGACCGA CGAGATGACC ACGTCGTCGG#AGCAGGTCCC 360GCCAACC TGATCGGCTC CGGCTGCGCG GCCTACGCCG#CGGCGTCGAA 420TCGGTGG CCGGGATGGC AGCCGATCCG GTGACGGTGG#CGCAGGTCAA 480CAGACGC TGTCCCAGGC GCTGTCCGGC CAGCTCAATC#ACGACGCGTT 540CTCGACG GCGGTGAGTT CACCGTGTTC GCGCCGACCG#TGCTGACCAA 600CCGGCCA CGCTGGAGAC CCTCAAGACG GACTCCGACA#TCGGCGAGCA 660CACGTCG TGCCCGGCCA GGCCGCGCCC GATCAGGTGG#TCAAGGTCAA 720GGGGCGC CGGTCACGGT GTCCGGGATG GCCGACCAGC#ATCTGATCGA 780GTGTGCG GTGGGGTGCA GACCGCCAAC GCGACGGTGT# 808 GCCGG CAGCGTAG- (2) INFORMATION FOR SEQ ID NO:147:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 228 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:#Val Ala Gly Ile Ala Alarg Lys Ser Ala Ala# 15#Ser Glu Asp Gly Gly Serla Ala Ala Cys Ser# 30#Ser Ala Met Glu Ser Alaer Ser Thr Ala Ser# 45#Pro Ser Ala Asp Pro Alahr Ser Ser Ala Ala# 60#Tyr Ala Glu Gln Val Proer Gly Cys Ala Ala# 80#Ala Asp Pro Val Thr Valal Ala Gly Met Ala# 95#Leu Ser Gln Ala Leu Serro Met Leu Gln Thr# 110#Asp Thr Leu Asp Gly Glyln Val Asn Leu Val# 125#Ala Phe Ala Lys Ile Aspla Pro Thr Asp Asp# 140#Ser Asp Met Leu Thr Asnhr Leu Lys Thr Asp#160#Ala Ala Pro Asp Gln Valal Val Pro Gly Gln# 175#Pro Val Thr Val Ser Glyhr Val Glu Gly Ala# 190#Ser Val Val Cys Gly Glyys Val Asn Asp Ala# 205#Ile Asp Thr Val Leu Metla Thr Val Tyr Leu# 220- Pro Pro Ala Ala 225- (2) INFORMATION FOR SEQ ID NO:148:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 22 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:# 22NTGY GC- (2) INFORMATION FOR SEQ ID NO:149:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:#21 NACNG G- (2) INFORMATION FOR SEQ ID NO:150:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 102 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:#GCCGGGATCG 60CCGGCTG TGCGGCCTAC GTGCAACAGG TGCCGGACGG# 102 AGCTC GCCCGTAGCG ACCGCCGCGT AT- (2) INFORMATION FOR SEQ ID NO:151:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 683 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:#TCATGAACAT 60AACCGCC GATCATCCAC TGCAGGAAGG AATCTCACGA#GTCTGTCGCT 120CTTGCCG GAGCGGGTTT CGCGATGACC GCCGCCGTCG#CCTACGTGCA 180AGCGCCG CAGCCGCGCC GGTCGGACCG GGGTGTGCGG#TGGCCACCGC 240GGGCCGG GATCGGTGCA GGGCATGGCG AGCTCGCCGG#AGCTCAACCC 300CCGCTGC TCACCACGCT CTCGCAGGCG ATCTCGGGTC#CGCCGACCAA 360GTCGACA CGTTCAACGG CGGCCAGTTC ACCGTGTTCG#ATTCCGACCT 420AAGATCG ATCCGGCCAC GCTGGAGACC CTCAAGACCG#ATCAGGTGGT 480CTCACCT ACCACGTCGT GCCCGGCCAG GCCGCGCCCG#CCGACCAGCT 540ACGGTGG AGGGGGCGCC GGTCACGGTG TCCGGGATGG#CGACGGTGTA 600GCGTCGG TGGTGTGCGG TGGGGTGCAG ACCGCCAACG#CAGAAGAGGG 660GTGCTGA TGCCGCCGGC AGCGTAGCCG GGCGGCACCA# 683CTCCC CCG- (2) INFORMATION FOR SEQ ID NO:152:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 231 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:#Arg Arg Ser Ser Thr Alaro Pro Ala Asn Asn# 15#Met Lys Thr Leu Ala Glyle Met Asn Ile Ser# 30#Leu Ser Leu Gly Thr Alahr Ala Ala Val Gly# 45#Gly Cys Ala Ala Tyr Valla Pro Val Gly Pro# 60#Gln Gly Met Ala Ser Serly Pro Gly Ser Val# 80#Leu Leu Thr Thr Leu Serla Ala Asp Asn Pro# 95#Val Asn Leu Val Asp Thrln Leu Asn Pro Asn# 110#Pro Thr Asn Asp Ala Phehe Thr Val Phe Ala# 125#Leu Lys Thr Asp Ser Aspla Thr Leu Glu Thr# 140#Val Pro Gly Gln Ala Alaeu Thr Tyr His Val#160#Val Glu Gly Ala Pro Vally Glu His Val Thr# 175#Val Asn Asp Ala Ser Valla Asp Gln Leu Lys# 190#Thr Val Tyr Leu Ile Aspln Thr Ala Asn Ala# 205#Gly Thr Thr Glu Glu Glyro Ala Ala Pro Gly# 220- Pro Pro His Pro Ala Ser Pro#230- (2) INFORMATION FOR SEQ ID NO:153:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1125 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:#GGCCGCGTTA 60GTGTTCT GGGCAGTGTC GGTGCAGCAG TCGCGGTTTC#CATCGAGGTG 120TTTCGAT ACCGACCGCC TCAGCGGATC CGTGTCCGGA#TGCGTTCGTC 180GGACCGG TGCGGAACCC GGCCTCGGGT GGGTCGGTGA#GAACTACCCG 240CCAAGGT CGGTGAGCAG TCGGTGGGCA CCTACGCGGT#CGGGGCGGGT 300TTCGACA AATCGGCGCC CATGGGCGCG GCCGACGCAT#TGTCGCANGG 360GACAACT GCCCGGACAC CAAGCTTGTC CTGGGCGGCA#TCACCCCCAC 420GACCTGA TCACCGTCGA TCCGCGACCG CTGGGCCGGT#GAAATCCGTT 480CGCGTCG CCGACCACGT GGCCGCCGTT GTGGTCTTCG#GGCCGAAGTC 540GGTGGCG GTCCGCTGCC GCAGATGAGC GGCACCTACG#TGCCGGCCCA 600GCGCTCG ACGATCCGTT CTGCTCGCCC GGCTTCAACC#GCCTGGAACC 660GACAACG GCATGGTGGA GGAAGCCGCG AACTTCGCCC#CGCGGGGCGA 720GAGCTGC CCGAGGCGCC CTACCTGCAC CTGTTCGTCC#TCACCGCATC 780GACGCCG GACCGCTGCG CGAAGGCGAC GCAGTGCGTT#AGATGCATGC 840GTGACCG CCACCGCGCC CGCGGAGATC CTCGTCTGGG#CTGCTCGCCG 900GCATAAG CGAATAGGAG TCCTGCTGGC CGGCGCAGCA#AGCACAACCG 960ACCTGGA CCCGGGCCGT CGGCGGCACC GGCCCCGACG#CCTCTGGCCG 1020TCCCGGA CTCGTCCCGG TGACCGTCGC GGTCGACGAA#CTGTCGGTGT 1080CCAGCCC CGGGAGGCCC TGGTGCCGCA GGGTTGGACG# 1125CCG CGGCTGGCCG CGTGGGCCCC GGACG- (2) INFORMATION FOR SEQ ID NO:154:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 748 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:#Gly Ala Ala Val Ala Valal Leu Gly Ser Val# 15#Ile Pro Thr Ala Ser Alaln Thr Gly Val Ser# 30#Ala Arg Gly Thr Gly Alale Glu Val Ile Phe# 45#Phe Val Asn Ala Leu Argrp Val Gly Asp Ala# 60#Tyr Ala Val Asn Tyr Proln Ser Val Gly Thr# 80#Met Gly Ala Ala Asp Alasp Lys Ser Ala Pro# 95#Cys Pro Asp Thr Lys Leurp Met Ala Asp Asn# 110#Val Ile Asp Leu Ile Threr Xaa Gly Ala Gly# 125#Pro Thr Pro Met Pro Proeu Gly Arg Phe Thr# 140#Val Phe Gly Asn Pro Leual Ala Ala Val Val#160#Glu Pro Arg Gly Leu Asnly Gly Pro Arg Leu# 175#His Arg Thr Tyr Arg Glyrg Gly Leu Tyr Thr# 190#Ile Leu Glu Ala Ser Proyr Ser Ser Glu Arg# 205#Ala Ser Pro Ala Ser Prola Leu Ala Leu Glu# 220#Arg Pro Arg Gly Leu Tyrys Tyr Ser Ser Glu#240#Ala Leu Ala His Ile Sersn Leu Glu Pro Arg# 255#Leu Ala Ala Ser Pro Alala Thr Tyr Arg Ala# 270#Leu Gly Leu Gly Leu Alaet Glu Thr Val Ala# 285#Glu Ala Leu Ala Ala Argla Ser Asn Pro His# 300#Gly Leu Asn Ser Glu Argro Arg Gly Leu Tyr#320#Leu Ala Leu Ala Pro Argeu Glu Pro Arg Gly# 335#Pro His Glu Val Ala Leuis Ile Ser Leu Glu# 350#Val Ala Leu Thr His Argly Leu Tyr Gly Leu# 365#Gly Leu Tyr Pro Arg Leuer Pro Ala Leu Ala# 380#Ser Pro Ala Leu Ala Valeu Gly Leu Tyr Ala#400#Arg Ala Leu Ala Ser Gluro His Glu Thr His# 415#Ala Arg Gly Val Ala Leueu Tyr Gly Leu Asn# 430#Leu Ala Pro Arg Ala Leula Thr His Arg Ala# 445#Leu Thr Arg Pro Gly Leulu Leu Glu Val Ala# 460#Leu Tyr Leu Glu Gly Leuer Ala Leu Ala Gly#480#Ala Ser Asn Ala Arg Glyeu Ala Ala Leu Ala# 495#Tyr Ala Arg Gly Ala Argla Leu Ala Gly Leu# 510#Ala Arg Gly Ala Arg Glyis Arg Ala Leu Ala# 525#Arg Gly Thr His Arg Threr Ile Leu Glu Ala# 540#Ala Val Ala Leu Gly Leula Arg Gly Ala Leu#560#Pro Arg Ala Ser Pro Glyis Arg Gly Leu Tyr# 575#Gly Leu Ala Arg Gly Threr Asn Ala Arg Gly# 590#Gly Thr His Arg Ala Arger Glu Arg Ala Arg# 605#Arg Gly Ala Arg Gly Glyyr Ala Ser Pro Ala# 620#Arg Ser Glu Arg Gly Leula Arg Gly Thr His#640#Val Ala Leu Ala Arg Glyrg Gly Ala Leu Ala# 655#Gly Leu Tyr Pro Arg Glyro Arg Gly Leu Tyr# 670#Tyr Leu Glu Ala Ser Prola Leu Ala Gly Leu# 685#Ala Leu Gly Leu Tyr Alaeu Gly Leu Tyr Val# 700#Ala Leu Ala Ala Leu Alala Arg Gly Pro Arg#720#Ala Leu Gly Leu Tyr Proyr Ala Arg Gly Val# 735#Leu Tyrly Leu Tyr Ala Arg Gly Pro Arg Gly# 745- (2) INFORMATION FOR SEQ ID NO:155:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1012 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:#GCATCCATCG 60ATTCGGG ATGCTACAAA TCCGCCGGCC CGATATGGTC#GCTGAGCCTG 120CCGCACT GGCACCATCT CATGCAGGTC TGGACAATGA#CTTCCTCAAC 180AGGGCCC GGAACGACTG ACCATTCAGC AGTGGGACAC#GGGCAAGGCG 240TGGACCG CAACCGGTTG ACCCGGGAGT GGTTCCACTC#GCTGGGCTAC 300CCGGTGA AGGTGCCGAC GAGTTCGAGG GCACGCTGGA#CACCCCGAAC 360CGTGGTC GCTGGGCGTG GGCATCAACT TCAGCTACAC#CGGTGATTCC 420GTTACGG CCTCAACTTC GCCGACCCGC TGCTGGGCTT#CAACGGCCCC 480CGCTGTT CCCGGGTGTC TCGATCACGG CGGACCTGGG#TTCCGTGGTG 540TCGCGAC CTTCTCCGTG GACGTGGCCG GCCCCGGTGG#GCGTCCGTTC 600ACGGCAC GGTCACCGGT GCTGCCGGTG GTGTGCTGCT#CTGCTGAAAC 660CGTCGAC CGGCGACAGC GTCACCACCT ACGGCGCACC#CTTCACGCTG 720ATCACGA TGGAGGCCCC CCGGCGTCAA CCGGGGCCCG#TGCGCGCGCG 780CGAGGTT CGATCGAAGT GGCCGACTGC GGCAAACGCC#ACTCCACCTC 840GACGCAG GGTCTGGTGG TAGTCGAATG TCATCCTGTG#GGCACGTACG 900CGACGGC CGGGGTTCCG GTGTGTGGGC GCCGGCCTTG#CCGGACGGCC 960TCGTGAT GTGACGAGCG TCGCAGTGTT TGCCGGCAAC#AG 1012GCATCCG TCCAGCGAAC CCGGGGGATC CAAAGAATTC- (2) INFORMATION FOR SEQ ID NO:156:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 336 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:#Ser Ala Gly Pro Ile Trper Gly Cys Tyr Lys# 15#Leu Ala Pro Ser His Alaeu Cys Ser Pro Ala# 30#His Gly Gln Gly Pro Glueu Ser Leu Gly Val# 45#Leu Asn Gly Val Phe Proln Trp Asp Thr Phe# 60#Phe His Ser Gly Lys Alaeu Thr Arg Glu Trp# 80#Glu Phe Glu Gly Thr Leuly Glu Gly Ala Asp# 95#Ser Leu Gly Val Gly Ileal Gly Phe Pro Trp# 110#Tyr Asp Gly Tyr Gly Leuhr Pro Asn Ile Thr# 125#Asp Ser Ile Val Thr Proeu Leu Gly Phe Gly# 140#Asp Leu Gly Asn Gly Proal Ser Ile Thr Ala#160#Asp Val Ala Gly Pro Glyla Thr Phe Ser Val# 175#Thr Val Thr Gly Ala Alaer Asn Ala His Gly# 190#Leu Ile Ser Ser Thr Glyrg Pro Phe Ala Arg# 205#Lys His Glu Leu Thr Thryr Gly Ala Pro Leu# 220#Gly Pro Leu His Ala Glyro Gly Val Asn Arg#240#Pro Thr Ala Ala Asn Alaal Arg Ser Lys Trp# 255#Gly Leu Val Val Val Gluer Ser Leu Thr Gln# 270#Arg Arg Asp Gly Arg Glyro Pro His Arg Pro# 285#Tyr Gly Gly Asp Arg Argro Ala Leu Gly Thr# 300#Gly Asn Pro Asp Gly Proal Ala Val Phe Ala#320#Gly Gly Ser Lys Glu Phero Ser Ser Glu Pro# 335- (2) INFORMATION FOR SEQ ID NO:157:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 480 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:#CTTGTCGACG 60TCAACAC CCCTGCGTTC GAGTGGTTCT ACGAGTCCGG#GTCTCGGGGC 120GCGGACA GTCCAGCTTC TACAGCGACT GGTACCAGCC#GCCGACGTGG 180ACACCTA CAAGTGGGAG ACGTTCCTGA CCCAGGAGCT#GTCGATGGCG 240GCGGAGT GTCGCGCACC GGCAACGCGT TCGTCGGCCT#CGCCTCGTCG 300TGACCTA CGCGATCCAT CACCCGCAGC AGTTCATCTA#GCTGGCGATG 360TGAACCC GTCCGAGGGC TGGTGGCCGA TGCTGATCGG#CCCGGCGTGG 420GCTTCAA CGCCGAGAGC ATGTGGGGCC CGTCCTCGGA#CCGGATCTGG 480CGATGGT CAACATCAAC CAGCTGGTGG CCAACAACAC- (2) INFORMATION FOR SEQ ID NO:158:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 161 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:#Glu Trp Phe Tyr Glu Sersn Thr Pro Ala Phe# 15#Gln Ser Ser Phe Tyr Seret Pro Val Gly Gly# 30#Gln Asn Tyr Thr Tyr Lyser Arg Gly Asn Gly# 45#Thr Trp Leu Glu Ala Asnhr Gln Glu Leu Pro# 60#Val Gly Leu Ser Met Alahr Gly Asn Ala Phe# 80#His Pro Gln Gln Phe Ilehr Tyr Ala Ile His# 95#Pro Ser Glu Gly Trp Trper Gly Phe Leu Asn# 110#Ala Gly Gly Phe Asn Alaeu Ala Met Asn Asp# 125#Ala Trp Lys Arg Asn Aspro Ser Ser Asp Pro# 140#Asn Asn Thr Arg Ile Trpsn Gln Leu Val Ala#160- Ile- (2) INFORMATION FOR SEQ ID NO:159:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1626 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:#GGGCCTCAAC 60TTGCGTA TGACGAAGAG GCCCGCCGTG GCCTCGAGCG#CGTGCTGGAG 120CCGTAAA GGTGACGTTG GGCCCGAAGG GTCGCAACGT#GGAGATCGAG 180CCCCCAC GATCACCAAC GATGGTGTGT CCATCGCCAA#CAAGAAGACC 240ACGAGAA GATCGGCGCT GAGCTGGTCA AAGAGGTCGC#TCTGGTTCGC 300GCGACGG CACCACCACC GCCACCGTGC TCGCTCAGGC#TGGCATCGAG 360ACGTCGC AGCCGGCGCC AACCCGCTCG GCCTCAAGCG#CGAGACCAAG 420CTGTCAC CCAGTCGCTG CTGAAGTCGG CCAAGGAGGT#CGAGCTCATC 480CCACCGC GGCGATTTCC GCCGGCGACA CCCAGATCGG#GTCGAACACC 540ACAAGGT CGGCAACGAG GGTGTCATCA CCGTCGAGGA#CATCTCGGGT 600TCGAGCT CACCGAGGGT ATGCGCTTCG ACAAGGGCTA#CATCCTGCTG 660ACGCCGA GCGCCAGGAA GCCGTCCTGG AGGATCCCTA#GGTCATCCAG 720TGTCGAC CGTCAAGGAT CTGCTCCCGC TGCTGGAGAA#GTCCACGCTG 780TGCTGAT CATCGCCGAG GACGTCGAGG GCGAGGCCCT#GGGCTTCGGT 840TCCGCGG CACCTTCAAG TCCGTCGCCG TCAAGGCTCC#GGTCGTCAGC 900CGATGCT GCAGGACATG GCCATCCTCA CCGGTGGTCA#GGCCCGCAAG 960TGTCCCT GGAGACCGCC GACGTCTCGC TGCTGGGCCA#CGATGCCATC 1020AGGACGA GACCACCATC GTCGAGGGCT CGGGCGATTC#CTACGACCGC 1080CTCAGAT CCGCGCCGAG ATCGAGAACA GCGACTCCGA#CAAGGCCGGA 1140AGCGCCT GGCCAAGCTG GCCGGCGGTG TTGCGGTGAT#CGTCCGCAAC 1200TGGAGCT CAAGGAGCGC AAGCACCGCA TCGAGGACGC#GCTGCAGTCG 1260TCGAAGA GGGCATCGTC GCCGGTGGCG GCGTGGCTCT#CAACATCGTC 1320ACGACCT CGGCCTGACG GGCGACGAGG CCACCGGTGC#GGAGCCCGGC 1380CGGCTCC GCTCAAGCAG ATCGCCTTCA ACGGCGGCCT#CGCGACCGGT 1440AGGTGTC CAACCTGCCC GCGGGTCACG GCCTCAACGC#CCGCTCGGCG 1500TGCTCAA GGCCGGCGTC GCCGACCCGG TGAAGGTCAC#CGTCGCCGAC 1560CGTCCAT CGCGGCTCTG TTCCTCACCA CCGAGGCCGT#CGGTATGGAC 1620CGTCCGC ACCCGCGGGC GACCCGACCG GTGGCATGGG# 1626- (2) INFORMATION FOR SEQ ID NO:160:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 541 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:#Ala Arg Arg Gly Leu Glula Tyr Asp Glu Glu# 15#Lys Val Thr Leu Gly Proeu Ala Asp Ala Val# 30#Trp Gly Ala Pro Thr Ileal Leu Glu Lys Lys# 45#Ile Glu Leu Glu Asp Proer Ile Ala Lys Glu# 60#Glu Val Ala Lys Lys Thrla Glu Leu Val Lys# 80#Ala Thr Val Leu Ala Glnsp Gly Thr Thr Thr# 95#Ala Ala Gly Ala Asn Proly Leu Arg Asn Val# 110#Val Glu Ala Val Thr Glnly Ile Glu Lys Ala# 125#Thr Lys Glu Gln Ile Serla Lys Glu Val Glu# 140#Gln Ile Gly Glu Leu Ileer Ala Gly Asp Thr#160#Gly Val Ile Thr Val Gluys Val Gly Asn Glu# 175#Leu Thr Glu Gly Met Argly Leu Gln Leu Glu# 190#Val Thr Asp Ala Glu Argle Ser Gly Tyr Phe# 205#Leu Leu Val Ser Ser Lyslu Asp Pro Tyr Ile# 220#Leu Glu Lys Val Ile Glnsp Leu Leu Pro Leu#240#Asp Val Glu Gly Glu Alaeu Ile Ile Ala Glu# 255#Gly Thr Phe Lys Ser Valal Asn Lys Ile Arg# 270#Arg Lys Ala Met Leu Glnly Phe Gly Asp Arg# 285#Val Ser Glu Arg Val Glyhr Gly Gly Gln Val# 300#Leu Gly Gln Ala Arg Lysla Asp Val Ser Leu#320#Val Glu Gly Ser Gly Aspsp Glu Thr Thr Ile# 335#Ile Arg Ala Glu Ile Gluly Arg Val Ala Gln# 350#Leu Gln Glu Arg Leu Alayr Asp Arg Glu Lys# 365#Ala Gly Ala Ala Thr Glual Ala Val Ile Lys# 380#Glu Asp Ala Val Arg Asnrg Lys His Arg Ile#400#Ala Gly Gly Gly Val Alalu Glu Gly Ile Val# 415#Leu Gly Leu Thr Gly Aspro Ala Leu Asp Asp# 430#Ala Leu Ser Ala Pro Leusn Ile Val Arg Val# 445#Pro Gly Val Val Ala Glusn Gly Gly Leu Glu# 460#Leu Asn Ala Ala Thr Glyro Ala Gly His Gly#480#Ala Asp Pro Val Lys Valeu Lys Ala Gly Val# 495#Ile Ala Ala Leu Phe Leuln Asn Ala Ala Ser# 510#Glu Lys Ala Ser Ala Proal Ala Asp Lys Pro# 525#Met Asp Phesp Pro Thr Gly Gly Met Gly Gly# 540- (2) INFORMATION FOR SEQ ID NO:161:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 985 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:#TGCTCCCGCT 60CTGCTGG TCAGCTCCAA GGTGTCGACC GTCAAGGATC#ACGTCGAGGG 120ATCCAGG CCGGCAAGCC GCTGCTGATC ATCGCCGAGG#CCGTCGCCGT 180ACGCTGG TGGTCAACAA GATCCGCGGC ACCTTCAAGT#CCATCCTCAC 240TTCGGTG ACCGCCGCAA GGCGATGCTG CAGGACATGG#ACGTCTCGCT 300GTCAGCG AAAGAGTCGG GCTGTCCCTG GAGACCGCCG#TCGAGGGCTC 360CGCAAGG TCGTCGTCAC CAAGGACGAG ACCACCATCG#TCGAGAACAG 420GCCATCG CCGGCCGGGT GGCTCAGATC CGCGCCGAGA#CCGGCGGTGT 480GACCGCG AGAAGCTGCA GGAGCGCCTG GCCAAGCTGG#AGCACCGCAT 540GCCGGAG CTGCCACCGA GGTGGAGCTC AAGGAGCGCA#CCGGTGGCGG 600CGCAACG CGAAGGCTGC CGTCGAAGAG GGCATCGTCG#GCGACGAGGC 660CAGTCGG CTCCTGCGCT GGACGACCTC GGCCTGACGG#TCGCCTTCAA 720ATCGTCC GCGTGGCGCT GTCGGCTCCG CTCAAGCAGA#CGGGTCACGG 780CCCGGCG TCGTTGCCGA GAAGGTGTCC AACCTGCCCG#CCGACCCGGT 840ACCGGTG AGTACGAGGA CCTGCTCAAG GCCGGCGTCG#TCCTCACCAC 900TCGGCGC TGCAGAACGC GGCGTCCATC GCGGCTCTGT#ACCCGACCGG 960GCCGACA AGCCGGAGAA GGCGTCCGCA CCCGCGGGCG# 985 GGACT TCTAA- (2) INFORMATION FOR SEQ ID NO:162:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 327 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:#Val Ser Thr Val Lys Aspeu Val Ser Ser Lys# 15#Ala Gly Lys Pro Leu Leulu Lys Val Ile Gln# 30#Leu Ser Thr Leu Val Valal Glu Gly Glu Ala# 45#Ala Val Lys Ala Pro Glyhr Phe Lys Ser Val# 60#Asp Met Ala Ile Leu Thrys Ala Met Leu Gln# 80#Leu Ser Leu Glu Thr Alaer Glu Arg Val Gly# 95#Val Val Val Thr Lys Asply Gln Ala Arg Lys# 110#Ser Asp Ala Ile Ala Glylu Gly Ser Gly Asp# 125#Asn Ser Asp Ser Asp Tyrrg Ala Glu Ile Glu# 140#Lys Leu Ala Gly Gly Valln Glu Arg Leu Ala#160#Val Glu Leu Lys Glu Argly Ala Ala Thr Glu# 175#Ala Lys Ala Ala Val Glusp Ala Val Arg Asn# 190#Leu Leu Gln Ser Ala Proly Gly Gly Val Ala# 205#Glu Ala Thr Gly Ala Asnly Leu Thr Gly Asp# 220#Lys Gln Ile Ala Phe Asneu Ser Ala Pro Leu#240#Lys Val Ser Asn Leu Proly Val Val Ala Glu# 255#Glu Tyr Glu Asp Leu Leusn Ala Ala Thr Gly# 270#Thr Arg Ser Ala Leu Glnsp Pro Val Lys Val# 285#Thr Thr Glu Ala Val Valla Ala Leu Phe Leu# 300#Ala Gly Asp Pro Thr Glyys Ala Ser Ala Pro#320- Gly Met Gly Gly Met Asp Phe 325- (2) INFORMATION FOR SEQ ID NO:163:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 403 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:#GGACGGCCGA 60CGGCTGG TGACGACCAA GTACAACCCG GCCCGCACCT#GCCCCGGCGG 120ATCGGCG GCGCGTACCT GTGCATCTAC GGGATGGAGG#CGGCGCCGTT 180GGCCGCA CCACCCAGGT GTGGAGTCGT TACCGCCACA#ATCCGGTGTC 240CCCTGGC TGCTGCGGTT TTTCGACCGA ATTTCGTGGT#CGGTCGACAT 300CTGGAAT TGCGAGCCGA CATGGCCGCA GGCCGGGGCT#ACGCCGACGA 360TTCTCCC TCGCCGAGCA CGAACGGTTC CTGGCCGACA#403 TTCCC GGCAGGCGGC CGCGTTCTCC GCC- (2) INFORMATION FOR SEQ ID NO:164:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 336 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:#GTCGAAGGCC 60CGGCCGC CGGCGAGTTC GACCGCGCCG AGAAAGCCGC#GACGCTCCGT 120CCGGGGA CCTGGTGCTC TACGACGGTG CGAGCGGGTC#GCCGGACAGC 180GTGGAAG GTCGACGTCG CCGTCGGTGA CCGGGTGGTG#GCCGACGGGG 240GGAGGCG ATGAAGATGG AGACCGTGCT GCGCGCCCCG#CCACTGGTCG 300CCTGGTC TCCGCTGGGC ATCTCGTCGA TCCCGGCACC# 336 TGCGC GCATGAGCGC CGTCGA- (2) INFORMATION FOR SEQ ID NO:165:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 134 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:#Tyr Asn Pro Ala Arg Threu Val Thr Thr Lys# 15#Gly Ala Tyr Leu Cys Ileer Val Gly Ile Gly# 30#Phe Val Gly Arg Thr Thrro Gly Gly Tyr Gln# 45#Pro Phe Glu Pro Gly Seryr Arg His Thr Ala# 60#Ser Trp Tyr Pro Val Serhe Phe Asp Arg Ile# 80#Met Ala Ala Gly Arg Glylu Leu Arg Ala Asp# 95#Leu Ala Glu His Glu Argsp Gly Val Phe Ser# 110#Ala Phe Arg Ser Arg Glnla Asp Asp Ile Ala# 125- Ala Ala Ala Phe Ser Ala 130- (2) INFORMATION FOR SEQ ID NO:166:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 108 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:#Asp Arg Ala Glu Lys Alala Ala Gly Glu Phe# 15#Asp Leu Val Leu Tyr Aspsp Ala Asp Thr Gly# 30#Ser Ser Val Trp Lys Valsp Ala Pro Phe Ala# 45#Gly Gln Pro Leu Leu Alasp Arg Val Val Ala# 60#Arg Ala Pro Ala Asp Glyet Glu Thr Val Leu# 80#His Leu Val Asp Pro Glyeu Val Ser Ala Gly# 95#Arg Alaro Leu Val Val Val Gly Thr Gly Val# 105- (2) INFORMATION FOR SEQ ID NO:167:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 31 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:# 31 CAGTG GGACCTCGAG C- (2) INFORMATION FOR SEQ ID NO:168:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 27 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:# 27 CGTCA GCCGCCG- (2) INFORMATION FOR SEQ ID NO:169:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1111 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:#GCGGCGCCCT 60ACCTCGA GCACCACGTC ACAGGACAGC GGCCCCGCCA#TCCAGACCGC 120TGGCCGC TCTATATGGC CGACGGTTTC ATCGCAGCGT#GGTTCGCCAA 180GTCGACT ACAAAGAAGA CTTCAACGAC AACGAGCAGT#TCCCCACCGA 240TTGTCGC GCAAGCAGGA CATAGGCGCC GACCTGGTGA#AAGCCGGCGT 300CGCGTCA AGGGCCTGGG ATGGCTCAAT GAGATCAGCG#AGGGCCGCAA 360AATCTGC GTCAGGACCT GTTGGACTCG AGCATCGACG#CAGCCACCGG 420TACATGA CCGGCATGGT CGGTCTCGCC TACAACAAGG#GCGTCAGTCT 480ACCATCG ACGACCTCTG GGATCCCGCG TTCAAGGGCC#ACTCGCCGGA 540CAGGACG GCCTCGGCAT GATCATGCTC TCGCAGGGCA#AGAACGACAG 600GAGTCCA TTCAGCAGGC GGTCGATCTG GTCCGCGAAC#GCAGAAACAT 660TCGCTTC ACCGGCAACG ACTACGCCGA CGACCTGGCC#ACCCCGATCT 720GCGTACT CCGGTGACGT CGTGCAGCTG CAGGCGGACA#TGATCCCGTA 780CCCGAAT CCGGCGGCGA CTGGTTCGTC GACACGATGG#ACCGAGCCAA 840CAGAAGG CCGCCGAGGC GTGGATCGAC TACATCTACG#TGACCGACGA 900GTCGCGT TCACCCAGTT CGTGCCCGCA CTCTCGGACA#CGGCCGAGGT 960GATCCTG CATCGGCGGA GAACCCGCTG ATCAACCCGT#AGTTCAACAC 1020AAGTCGT GGGCGGCACT GACCGACGAG CAGACGCAGG#GGGGCATAAA 1080GTCACCG GCGGCTGACG CGGTGGTAGT GCCGATGCGA# 1111 GAGGA GCATAAATGG C- (2) INFORMATION FOR SEQ ID NO:170:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 348 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:#Gln Asp Ser Gly Pro Alaer Ser Thr Thr Ser# 15#Leu Tyr Met Ala Asp Glyal Ser Asn Trp Pro# 30#Ile Thr Val Asp Tyr Lysln Thr Ala Ser Gly# 45#Ala Lys Val Lys Glu Prosn Glu Gln Trp Phe# 60#Leu Val Ile Pro Thr Glusp Ile Gly Ala Asp# 80#Trp Leu Asn Glu Ile Seral Lys Gly Leu Gly# 95#Arg Gln Asp Leu Leu Aspsn Arg Lys Asn Leu# 110#Ala Pro Tyr Met Thr Glyly Arg Lys Phe Thr# 125#Thr Gly Arg Asp Ile Argyr Asn Lys Ala Ala# 140#Lys Gly Arg Val Ser Leurp Asp Pro Ala Phe#160#Ile Met Leu Ser Gln Glysp Gly Leu Gly Met# 175#Ile Gln Gln Ala Val Aspro Thr Thr Glu Ser# 190#Ile Arg Arg Phe Thr Glysn Asp Arg Gly Gln# 205#Asn Ile Ala Ile Ala Glnsp Leu Ala Ala Gly# 220#Ala Asp Asn Pro Asp Leual Val Gln Leu Gln#240#Trp Phe Val Asp Thr Metlu Ser Gly Gly Asp# 255#Ala Ala Glu Ala Trp Ilehr Gln Asn Gln Lys# 270#Lys Leu Val Ala Phe Thrrg Ala Asn Tyr Ala# 285#Asp Glu Leu Ala Lys Valeu Ser Asp Met Thr# 300#Asn Pro Ser Ala Glu Vallu Asn Pro Leu Ile#320#Thr Asp Glu Gln Thr Glner Trp Ala Ala Leu# 335#Gly Glyhe Asn Thr Ala Tyr Ala Ala Val Thr# 345- (2) INFORMATION FOR SEQ ID NO:171:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1420 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:#CGGTCGGGTT 60CTGAACT CGACCTGGTT GGCCTGGGCC GTCGCGGTCG#GCGGCAGCGC 120GTCGTGC TGACCGAGGT GCACAACGCG TTGCGTCGGC#CGTTGCTGCT 180GTGCAAC TCCTGCGTAC CTACATCCTG CCGCTGGGCG#TGGTCGCCAC 240GCGATGG AGATCTCCGA CGACGCCACG TCGGTACGGT#CCCTCATCCA 300GTGTTGT TGACGTTGGT GCTGTCCGGG CTCAACGCCA#ACGTCGCGCG 360GACAGCT GGCGCAGGCG GATTCCGTCG ATCTTCCTCG#GCGCGAACGT 420GCGGTCG GTATCACCGT GATCATGGCC TATGTCTGGG#CTCTGCAGAA 480ACCGCAC TGGGCGTCAC TTCCATCGTT CTTGGCCTGG#TCCGGCTCGG 540ATCATCT CGGGTCTGCT GCTGCTGTTC GAGCAACCGT#GCGTGGTGGA 600GTCCCCA CCGCGGCGGG CCGGCCGTCC GCCCACGGCC#TGCCCAACGC 660GCAACAC ATATCGACAC CGGCGGCAAC CTGCTGGTAA#ACCGGCTGAC 720GCGTCGT TCACCAATTA CAGCCGGCCC GTGGGAGAGC#TGCTGTCGTC 780TTCAACG CCGCGGACAC CCCCGATGAT GTCTGCGAGA#TCTATCTCGG 840CTGCCCG AACTGCGCAC CGACGGACAG ATCGCCACGC#ACTCGGTCAG 900GAGAAGT CGATCCCGTT GCACACACCC GCGGTGGACG#GCCTNAACGG 960CGATGGG TCTGGTACGC CGCGCGCCGG CAGGAACTTC#CTGTGGCGTC 1020TTCGACA CGCCGGAACG GATCGCCTCG GCCATGCGGG#GTCTGGTCCG 1080GCAGACG ACGAACAGCA GGAGATCGCC GACGTGGTGC#TGAGGTTCAT 1140GAACGCC TCCAGCAGCC GGGTCAGGTA CCGACCGGGA#TCCCGGCGCG 1200GTGAGTC TGTCCGTGAT CGATCAGGAC GGCGACGTGA#CGGTACTGGC 1260GGCGACT TCCTGGGGCA GACCACGCTG ACGCGGGAAC#AGATCGAGCG 1320CTGGAGG AAGTCACCGT GCTGGAGATG GCCCGTGACG#CCGACCGGCG 1380AAGCCGA TCCTGCTGCA CGTGATCGGG GCCGTGATCG# 1420 GTTGA TGGCGGACTC GCAGGACTGA- (2) INFORMATION FOR SEQ ID NO:172:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 471 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:#Ala Trp Ala Val Ala Valsn Ser Thr Trp Leu# 15#Leu Thr Glu Val His Asnal Leu Leu Val Val# 30#Arg Pro Val Gln Leu Leuly Ser Ala Leu Ala# 45#Leu Leu Leu Leu Val Glnro Leu Gly Ala Leu# 60#Val Arg Leu Val Ala Thrsp Asp Ala Thr Ser# 80#Leu Ser Gly Leu Asn Alaeu Leu Thr Leu Val# 95#Trp Arg Arg Arg Ile Prola Pro Glu Asp Ser# 110#Leu Ile Ala Val Gly Ileal Ala Arg Phe Ala# 125#Asn Val Gly Gly Leu Pheyr Val Trp Gly Ala# 140#Gly Leu Ala Leu Gln Asnhr Ser Ile Val Leu#160#Leu Leu Phe Glu Gln Prole Ser Gly Leu Leu# 175#Thr Ala Ala Gly Arg Prorp Ile Thr Val Pro# 190#Trp Arg Ala Thr His Ileal Val Glu Val Asn# 205#Asn Ala Glu Leu Ala Glyeu Leu Val Met Pro# 220#Gly Glu His Arg Leu Thryr Ser Arg Pro Val#240#Pro Asp Asp Val Cys Glusn Ala Ala Asp Thr# 255#Glu Leu Arg Thr Asp Glyla Ala Ser Leu Pro# 270#Glu Tyr Glu Lys Ser Ileyr Leu Gly Ala Ala# 285#Val Arg Ser Thr Tyr Leula Val Asp Asp Ser# 300#Glu Leu Arg Xaa Asn Glyla Ala Arg Arg Gln#320#Ile Ala Ser Ala Met Argsp Thr Pro Glu Arg# 335#Asp Glu Gln Gln Glu Ileeu Arg Leu Ala Asp# 350#Asn Gly Glu Arg Leu Glneu Val Arg Tyr Gly# 365#Phe Ile Val Asp Gly Argro Thr Gly Met Arg# 380#Asp Val Ile Pro Ala Argle Asp Gln Asp Gly#400#Thr Thr Leu Thr Arg Glusp Phe Leu Gly Gln# 415#Glu Val Thr Val Leu Glula His Ala Leu Glu# 430#His Arg Lys Pro Ile Leule Glu Arg Leu Val# 445#Arg Arg Ala His Glu Leula Val Ile Ala Asp# 460- Arg Leu Met Asp Ser Gln Asp#470- (2) INFORMATION FOR SEQ ID NO:173:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2172 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:#AAAAAGACGC 60TGCCCTG GAATGCGCGA ACGTCTGAAC ACCCGACGCG#GATGCTGCTT 120TCCTGTC GCGGATGAGC ATCCAGTCCA AGTTGCTGCT#CGGACGGTCC 180TCTCGGC TGCGGTGGTC GGTTTCATCG GCTATCAGTC#GTCGCGCGGG 240CGGTGTT CGACCGCCTC ACCGACATCC GCGAGTCGCA#CGGCAGCACT 300TCGCGGA CCTGAAGAAC TCGATGGTGA TTTACTCGCG#TGCGACGATC 360TCGGCGC GTTCAGCGAC GGTTTCCGTC AGCTCGGCGA#CAACACCACC 420CGGCGTC ATTGCGCCGT TACTACGACC GGACGTTCGC#CAACCCCCAG 480GAAACCG CGTCGACGTC CGCGCGCTCA TCCCGAAATC#GATCGCGTTC 540CGCTCTA TACCCCGCCG TTTCAGAACT GGGAGAAGGC#CGAGTTCTTC 600ACGGCAG CGCCTGGTCG GCCGCCAATG CCAGATTCAA#CGAGGGCAAC 660ACCGCTT CAACTTCGAG GATCTGATGC TGCTCGACCT#CGGCCCCTAT 720CCTACAA GGGGCCGGAT CTCGGGACAA ACATCGTCAA#GATCGACTAT 780TGTCGGA AGCCTACGAG AAGGCGGTCG CGTCGAACTC#CTGGTTCCTG 840ACTTCGG GTGGTACCTG CCTGCCGAGG AACCGACCGC#CCCGATCGCG 900TGAAGGA CCGAGTCGAC GGTGTGATGG CGGTCCAGTT#GGGAGACACC 960TGATGAC GGCGCGGGGA CAGTGGCGTG ACACCGGGAT#GCTGTTCCGC 1020TGGTCGG ACCGGACAAT CTGATGCGCT CGGACTCCCG#GGAGGTCGCC 1080AGTTCCT GGCCGACGTC GTCGAGGGGG GAACCCCGCC#CCGCTCCGTC 1140ACCGCCG CGGCACCACG CTGGTGCAGC CGGTGACCAC#CGGCCACGAG 1200GCGGCAA CACCGGGACG ACGATCGAGG ACGACTATCT#CGTGGCCAAG 1260ACTCACC GGTGGACCTG CCGGGACTGC ACTGGGTGAT#GGTGCTGTCG 1320AGGCGTT CGCCCCGGTG GCGCAGTTCA CCAGGACCCT#GTTGTTCGTC 1380TCTTCGG CGTGTCGCTG GCGGCCATGC TGCTGGCGCG#CTACCGCCTC 1440GGTTGCA GGCCGGCGCC CAGCAGATCA GCGGCGGTGA#CAACGACATG 1500TGTCTCG TGACGAATTC GGCGATCTGA CAACAGCTTT#GAACCAACGG 1560CGATCAA GGACGAGCTG CTCGGCGAGG AGCGCGCCGA#GGAGGAGACG 1620TGATGCC CGAACCGGTG ATGCAGCGCT ACCTCGACGG#CCTCGACGAG 1680ACAAGAA CGTCACGGTG ATCTTCGCCG ACATGATGGG#GACCCGCCAG 1740TGACCTC CGAGGAACTG ATGGTGGTGG TCAACGACCT#CGACGGGTAC 1800CCGAGAG TCTCGGGGTC GACCACGTGC GGACGCTGCA#GGTCAATTTC 1860GGTTAGG CGTGCCGCGG CTGGACAACG TCCGGCGCAC#CGACCTGCGG 1920ACCGCAT CATCGACCGG CACGCCGCCG AGTCCGGGCA#GTCCACGTTG 1980TCGACAC CGGGTCGGCG GCCAGCGGGC TGGTGGGGCG#CGGCTCCCCC 2040GGGGTTC GGCGGTCGAT GTCGCTAACC AGGTGCAGCG#TCTCGACTTC 2100ACGTCAC CTCGCGGGTG CACGAGGTCA TGCAGGAAAC#GTTGCAGGGC 2160AGGTCGT CGGCGAGCGC GGCGTCGAGA CGGTCTGGCG# 2172- (2) INFORMATION FOR SEQ ID NO:174:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 722 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:#Ser Glu His Pro Thr Argrp Asn Ala Arg Thr# 15#Arg Met Ser Ile Gln Seryr His Leu Leu Ser# 30#Ile Leu Ser Ala Ala Valeu Leu Leu Thr Ser# 45#Ser Ser Leu Arg Ala Seryr Gln Ser Gly Arg# 60#Ser Gln Ser Arg Gly Leuhr Asp Ile Arg Glu# 80#Met Val Ile Tyr Ser Argsp Leu Lys Asn Ser# 95#Phe Ser Asp Gly Phe Arglu Ala Ile Gly Ala# 110#Gln Ala Ala Ser Leu Arghr Ile Asn Thr Gly# 125#Thr Leu Asp Asp Ser Glyhr Phe Ala Asn Thr# 140#Lys Ser Asn Pro Gln Argrg Ala Leu Ile Pro#160#Gln Asn Trp Glu Lys Alayr Thr Pro Pro Phe# 175#Ala Trp Ser Ala Ala Asnla Arg Asp Gly Ser# 190#Val His Arg Phe Asn Phehe Phe Arg Glu Ile# 205#Asn Val Val Tyr Ser Alaeu Asp Leu Glu Gly# 220#Val Asn Gly Pro Tyr Argeu Gly Thr Asn Ile#240#Ala Val Ala Ser Asn Serlu Ala Tyr Glu Lys# 255#Trp Tyr Leu Pro Ala Glual Thr Asp Phe Gly# 270#Gly Leu Lys Asp Arg Valhe Leu Ser Pro Val# 285#Ala Arg Ile Asn Glu Leual Gln Phe Pro Ile# 300#Gly Met Gly Asp Thr Glyln Trp Arg Asp Thr#320#Met Arg Ser Asp Ser Argly Pro Asp Asn Leu# 335#Ala Asp Val Val Glu Glyrg Glu Lys Phe Leu# 350#Val Asp Arg Arg Gly Thral Ala Asp Glu Ser# 365#Val Glu Glu Ala Gln Argal Thr Thr Arg Ser# 380#Tyr Leu Gly His Glu Alahr Ile Glu Asp Asp#400#Gly Leu His Trp Val Ilero Val Asp Leu Pro# 415#Ala Pro Val Ala Gln Phehr Asp Glu Ala Phe# 430#Ile Ile Phe Gly Val Sereu Ser Thr Val Ile# 445#Val Arg Pro Ile Arg Argeu Ala Arg Leu Phe# 460#Gly Asp Tyr Arg Leu Alaln Gln Ile Ser Gly#480#Asp Leu Thr Thr Ala Pherg Asp Glu Phe Gly# 495#Asp Glu Leu Leu Gly Glusn Leu Ser Ile Lys# 510#Ser Leu Met Pro Glu Proln Arg Leu Met Leu# 525#Thr Ile Ala Gln Asp Hiseu Asp Gly Glu Glu# 540#Met Gly Leu Asp Glu Leule Phe Ala Asp Met#560#Val Val Val Asn Asp Leuer Glu Glu Leu Met# 575#Leu Gly Val Asp His Valla Ala Ala Glu Ser# 590#Cys Gly Leu Gly Val Proly Tyr Leu Ala Ser# 605#Phe Ala Ile Glu Met Asprg Arg Thr Val Asn# 620#Gly His Asp Leu Arg Leuis Ala Ala Glu Ser#640#Ser Gly Leu Val Gly Arghr Gly Ser Ala Ala# 655#Ala Val Asp Val Ala Asnsp Met Trp Gly Ser# 670#Ile Tyr Val Thr Ser Arger Pro Gln Pro Gly# 685#Phe Val Ala Ala Gly Gluln Glu Thr Leu Asp# 700#Trp Arg Leu Gln Gly Hisly Val Glu Thr Val#720- Arg Arg- (2) INFORMATION FOR SEQ ID NO:175:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 898 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:#CCGGCCGTCC 60GGCTCGG CGACTGGATC ACCGTCCCCA CCGCGGCGGG#CGGCGGCAAC 120TGGTGGA AGTCAACTGG CGTGCAACAC ATATCGACAC#CAGCCGGCCC 180CCAACGC CGAACTCGCC GGCGCGTCGT TCACCAATTA#CCCCGATGAT 240GGCTGAC CGTCGTCACC ACCTTCAACG CCGCGGACAC#CGACGGACAG 300TGTCGTC GGTCGCGGCG TCGCTGCCCG AACTGCGCAC#GCACACACCC 360ATCTCGG TGCGGCCGAA TACGAGAAGT CGATCCCGTT#CGCGCGCCGG 420CGGTCAG GAGCACGTAC CTGCGATGGG TCTGGTACGC#TCGCCTCGGC 480TAACGGC GTCGCCGACG ATTCGACACG CCGGAACGGA#AGATCGCCGA 540GCGTCCA CACTGCGCTT GGCAGACGAC GAACAGCAGG#GTCAGGTACC 600GTCCGTT ACGGCAACGG GGAACGCCTC CAGCAGCCGG#ATCAGGACGG 660TTCATCG TAGACGGCAG GGTGAGTCTG TCCGTGATCG#CCACGCTGAC 720GCGCGGG TGCTCGAGCG TGGCGACTTC CTGGGGCAGA#TGGAGATGGC 780CTGGCGA CCGCGCACGC GCTGGAGGAA GTCACCGTGC#TGATCGGGGC 840GAGCGCC TGGTGCACCG AAAGCCGATC CTGCTGCACG#AGGACTGA 898CGGCGCG CGCACGAACT TCGGTTGATG GCGGACTCGC- (2) INFORMATION FOR SEQ ID NO:176:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2013 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:#CACCGACATC 60GACGGTC CTCGCTGCGC GCATCGGTGT TCGACCGCCT#CTCGATGGTG 120CGCGCGG GTTGGAGAAT CAGTTCGCGG ACCTGAAGAA#CGGTTTCCGT 180GCAGCAC TGCCACGGAG GCGATCGGCG CGTTCAGCGA#TTACTACGAC 240CGACGAT CAATACCGGG CAGGCGGCGT CATTGCGCCG#CCGCGCGCTC 300ACACCAC CCTCGACGAC AGCGGAAACC GCGTCGACGT#GTTTCAGAAC 360ACCCCCA GCGCTATCTG CAGGCGCTCT ATACCCCGCC#GGCCGCCAAT 420TCGCGTT CGACGACGCG CGCGACGGCA GCGCCTGGTC#GGATCTGATG 480AGTTCTT CCGCGAGATC GTGCACCGCT TCAACTTCGA#TCTCGGGACA 540AGGGCAA CGTGGTGTAC TCCGCCTACA AGGGGCCGGA#GAAGGCGGTC 600GCCCCTA TCGCAACCGG GAACTGTCGG AAGCCTACGA#GCCTGCCGAG 660TCGACTA TGTCGGTGTC ACCGACTTCG GGTGGTACCT#CGGTGTGATG 720GGTTCCT GTCCCCGGTC GGGTTGAAGG ACCGAGTCGA#ACAGTGGCGT 780CGATCGC GCGGATCAAC GAATTGATGA CGGCGCGGGG#TCTGATGCGC 840GAGACAC CGGTGAGACC ATCCTGGTCG GACCGGACAA#CGTCGAGGGG 900TGTTCCG CGAGAACCGG GAGAAGTTCC TGGCCGACGT#GCTGGTGCAG 960AGGTCGC CGACGAATCG GTTGACCGCC GCGGCACCAC#GACGATCGAG 1020GCTCCGT CGAGGAGGCC CAACGCGGCA ACACCGGGAC#GCCGGGACTG 1080GCCACGA GGCGTTACAG GCGTACTCAC CGGTGGACCT#GGCGCAGTTC 1140TGGCCAA GATCGACACC GACGAGGCGT TCGCCCCGGT#GGCGGCCATG 1200TGCTGTC GACGGTGATC ATCATCTTCG GCGTGTCGCT#CCAGCAGATC 1260TGTTCGT CCGTCCGATC CGGCGGTTGC AGGCCGGCGC#CGGCGATCTG 1320ACCGCCT CGCTCTGCCG GTGTTGTCTC GTGACGAATT#GCTCGGCGAG 1380ACGACAT GAGTCGCAAT CTGTCGATCA AGGACGAGCT#GATGCAGCGC 1440ACCAACG GCTGATGCTG TCCCTGATGC CCGAACCGGT#GATCTTCGCC 1500AGGAGAC GATCGCCCAG GACCACAAGA ACGTCACGGT#GATGGTGGTG 1560TCGACGA GTTGTCGCGC ATGTTGACCT CCGAGGAACT#CGACCACGTG 1620CCCGCCA GTTCGACGCC GCCGCCGAGA GTCTCGGGGT#GCTGGACAAC 1680ACGGGTA CCTGGCCAGC TGCGGGTTAG GCGTGCCGCG#GCACGCCGCC 1740TCAATTT CGCGATCGAA ATGGACCGCA TCATCGACCG#GGCCAGCGGG 1800ACCTGCG GCTCCGCGCG GGCATCGACA CCGGGTCGGC#TGTCGCTAAC 1860CCACGTT GGCGTACGAC ATGTGGGGTT CGGCGGTCGA#GCACGAGGTC 1920GCTCCCC CCAGCCCGGC ATCTACGTCA CCTCGCGGGT#CGGCGTCGAG 1980TCGACTT CGTCGCCGCC GGGGAGGTCG TCGGCGAGCG# 2013 CAGGG CCACCGGCGA TGA- (2) INFORMATION FOR SEQ ID NO:177:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 297 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:#Thr Val Pro Thr Ala Alaeu Gly Asp Trp Ile# 15#Glu Val Asn Trp Arg Alais Gly Arg Val Val# 30#Val Met Pro Asn Ala Gluly Gly Asn Leu Leu# 45#Arg Pro Val Gly Glu Hishe Thr Asn Tyr Ser# 60#Ala Asp Thr Pro Asp Asphr Thr Phe Asn Ala# 80#Ser Leu Pro Glu Leu Arger Ser Val Ala Ala# 95#Gly Ala Ala Glu Tyr Glula Thr Leu Tyr Leu# 110#Asp Asp Ser Val Arg Seris Thr Pro Ala Val# 125#Arg Arg Gln Glu Leu Argal Trp Tyr Ala Ala# 140#Pro Glu Arg Ile Ala Sersp Xaa Phe Asp Thr#160#Leu Ala Asp Asp Glu Glnla Ser Thr Leu Arg# 175#Arg Tyr Gly Asn Gly Glual Val Arg Leu Val# 190#Gly Met Arg Phe Ile Vally Gln Val Pro Thr# 205#Gln Asp Gly Asp Val Ileeu Ser Val Ile Asp# 220#Leu Gly Gln Thr Thr Leulu Arg Gly Asp Phe#240#Ala Leu Glu Glu Val Threu Ala Thr Ala His# 255#Arg Leu Val His Arg Lysrg Asp Glu Ile Glu# 270#Ala Asp Arg Arg Ala Hisal Ile Gly Ala Val# 285- Glu Leu Arg Leu Met Asp Ser Gln Asp# 295- (2) INFORMATION FOR SEQ ID NO:178:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 670 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:#Ala Ser Val Phe Asp Argrg Ser Ser Leu Arg# 15#Gly Leu Glu Asn Gln Phelu Ser Gln Ser Arg# 30#Ser Arg Gly Ser Thr Alaer Met Val Ile Tyr# 45#Phe Arg Gln Leu Gly Aspla Phe Ser Asp Gly# 60#Leu Arg Arg Tyr Tyr Asply Gln Ala Ala Ser# 80#Ser Gly Asn Arg Val Asphr Thr Leu Asp Asp# 95#Gln Arg Tyr Leu Gln Alaro Lys Ser Asn Pro# 110#Lys Ala Ile Ala Phe Asphe Gln Asn Trp Glu# 125#Ala Asn Ala Arg Phe Asner Ala Trp Ser Ala# 140#Asn Phe Glu Asp Leu Metle Val His Arg Phe#160#Ser Ala Tyr Lys Gly Proly Asn Val Val Tyr# 175#Tyr Arg Asn Arg Glu Leule Val Asn Gly Pro# 190#Asn Ser Ile Asp Tyr Valys Ala Val Ala Ser# 205#Ala Glu Glu Pro Thr Alaly Trp Tyr Leu Pro# 220#Arg Val Asp Gly Val Metal Gly Leu Lys Asp#240#Glu Leu Met Thr Ala Argle Ala Arg Ile Asn# 255#Thr Gly Glu Thr Ile Leuhr Gly Met Gly Asp# 270#Ser Arg Leu Phe Arg Glueu Met Arg Ser Asp# 285#Glu Gly Gly Thr Pro Proeu Ala Asp Val Val# 300#Gly Thr Thr Leu Val Glner Val Asp Arg Arg#320#Gln Arg Gly Asn Thr Glyer Val Glu Glu Ala# 335#Glu Ala Leu Gln Ala Tyrsp Tyr Leu Gly His# 350#Val Ile Val Ala Lys Ilero Gly Leu His Trp# 365#Gln Phe Thr Arg Thr Leuhe Ala Pro Val Ala# 380#Val Ser Leu Ala Ala Metle Ile Ile Phe Gly#400#Arg Arg Leu Gln Ala Glyhe Val Arg Pro Ile# 415#Leu Ala Leu Pro Val Leuly Gly Asp Tyr Arg# 430#Ala Phe Asn Asp Met Serly Asp Leu Thr Thr# 445#Gly Glu Glu Arg Ala Gluys Asp Glu Leu Leu# 460#Glu Pro Val Met Gln Argeu Ser Leu Met Pro#480#Asp His Lys Asn Val Thrlu Thr Ile Ala Gln# 495#Glu Leu Ser Arg Met Leuet Met Gly Leu Asp# 510#Asp Leu Thr Arg Gln Pheet Val Val Val Asn# 525#His Val Arg Thr Leu Hiser Leu Gly Val Asp# 540#Val Pro Arg Leu Asp Asner Cys Gly Leu Gly#560#Met Asp Arg Ile Ile Aspsn Phe Ala Ile Glu# 575#Arg Leu Arg Ala Gly Ileer Gly His Asp Leu# 590#Gly Arg Ser Thr Leu Alala Ser Gly Leu Val# 605#Ala Asn Gln Val Gln Arger Ala Val Asp Val# 620#Ser Arg Val His Glu Vally Ile Tyr Val Thr#640#Gly Glu Val Val Gly Glusp Phe Val Ala Ala# 655#Gly His Arg Arglu Thr Val Trp Arg Leu Gln# 670- (2) INFORMATION FOR SEQ ID NO:179:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 520 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:#CTCGGTGGCA 60CCCTCTT CCATGCCGAG GAGAAGATGG AGAAGGCCGT#CAACCGGATC 120CGTCGAT TCGTACCGGC CGCGCGAACC CCGGCATGTT#CAACGTGCCC 180ACGGCGC CTCCACCCCG ATCACGCAGC TGTCCAGCAT#CATCGAGGAT 240TGGTGAT CAAGCCCTAC GAGGCGAGCC AGCTGCGCCT#CATCCGGGTG 300CCGACCT CGGCGTCAAT CCGACCAACG ACGGCAACAT#CAAGGCCAAG 360TCACCGA GGAGCGCCGC CGCGACCTGG TCAAGCAGGC#CACCTTTCGC 420AGGTGTC GGTGCGCAAC ATCCGTCGCA ACGATATGAA#GATCGCAGGT 480GGCTGCC GACGCCACCG CCGTCGTAGA AGCGACAGAG# 520 GCCTT CTGTGGCGGG CCGACACCAC- (2) INFORMATION FOR SEQ ID NO:180:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1071 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:#TCTGGCAGGT 60TGCACTC TATGAGCGAA ATCGCCCGTC CCTGGCGGGT#CACCACGGCG 120GCGCCGC GGGTATCGCC GGGGTGCTGA GCATCGCGGT#GACGCAAACC 180GCCTCCC GCAGCCCCCG CTGCCCGCCC CTGCCACAGT#GACGCCTGCC 240CCAACGC CGCGCCACAA CTCATCCCGC GCCCCGGTGT#GCCGGCCCCC 300CCGCGGT GCCCGCCGGG GTGAGCGCCC CGGCGGTCGC#GCTCAGCGAG 360GCCCGGT GTCCACGATC GCCCCGGCCA CCTCGGGCAC#CCGCGCCCTC 420AGGGCGT CACGATGGAG CCGCAGTCCA GCCGCGACTT#CGTGCCGGAC 480CGAAGCC GCGGGGCTGG GAGCACATCC CGGACCCGAA#GAACGCCCAG 540TGGCCGA CCGGGTCGGC GGCAACGGCC TGTACTCGTC#CCACGGCTTC 600AACTCGT CGGCGAGTTC GACCCCAAGG AAGCGATCAG#CGACTTCGGC 660AGCTGCC GGCGTGGCGT TCCACCGACG CGTCGCTGGC#GCTGAACACG 720CGCTGAT CGAGGGCACC TACCGCGAGA ACAACATGAA#GCTGTCGGTG 780TCATTGC CACCGCGGGG CCCGACCACT ACCTGGTGTC#GATTGTCAAC 840AACAGGC CGTGGCCGAA GCCGCGGAGG CCACCGACGC#ACCCGGTGCC 900GCGTTCC GGGTCCGGGT CCGGCCGCAC CGCCACCTGC#ACCACCCCCG 960CCGCCCC CGGCGCCCCG GCGCTGCCGC TGGCCGTCGC#GGGATAGACG 1020CCGCCGT GGCGCCCGCG CCACAGCTGC TGGGACTGCA#T 1071GGCGAAG CCTGGCGCCC GGGGGACGAC GGCCCCTTTC- (2) INFORMATION FOR SEQ ID NO:181:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 152 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:#Glu Lys Met Glu Lys Alaeu Phe His Ala Glu# 15#Ile Arg Thr Gly Arg Alasp Asp Leu Ala Ser# 30#Asp Tyr Tyr Gly Ala Sersn Arg Ile Asn Ile# 45#Val Pro Glu Ala Arg Meteu Ser Ser Ile Asn# 60#Leu Arg Leu Ile Glu Aspyr Glu Ala Ser Gln# 80#Pro Thr Asn Asp Gly Asnsp Leu Gly Val Asn# 95#Glu Glu Arg Arg Arg Asple Pro Gln Leu Thr# 110#Asp Ala Lys Val Ser Valys Ala Lys Gly Glu# 125#Phe Arg Ile Ala Pro Valsn Asp Met Asn Thr# 140- Arg Leu Pro Thr Pro Pro Pro Ser#150- (2) INFORMATION FOR SEQ ID NO:182:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 331 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:#Leu Ala Gly Gly Ile Glyrg Pro Trp Arg Val# 15#Ser Ile Ala Val Thr Thrle Ala Gly Val Leu# 30#Pro Leu Pro Ala Pro Alaly Leu Pro Gln Pro# 45#Asn Ala Ala Pro Gln Leual Thr Val Ala Pro# 60#Gly Gly Ala Ala Ala Valal Thr Pro Ala Thr# 80#Pro Ala Pro Ala Leu Prola Pro Ala Val Ala# 95#Thr Ser Gly Thr Leu Serhr Ile Ala Pro Ala# 110#Glu Pro Gln Ser Ser Argys Gly Val Thr Met# 125#Lys Pro Arg Gly Trp Glusn Ile Val Leu Pro# 140#Phe Ala Val Leu Ala Aspsn Val Pro Asp Ala#160#Asn Ala Gln Val Val Vally Leu Tyr Ser Ser# 175#Glu Ala Ile Ser His Glylu Phe Asp Pro Lys# 190#Arg Ser Thr Asp Ala Serys Leu Pro Ala Trp# 205#Leu Ile Glu Gly Thr Tyrly Met Pro Ser Ser# 220#Arg Arg His Val Ile Alays Leu Asn Thr Ser#240#Leu Ser Val Thr Thr Seris Tyr Leu Val Ser# 255#Ala Thr Asp Ala Ile Valla Glu Ala Ala Glu# 270#Gly Pro Ala Ala Pro Proer Val Pro Gly Pro# 285#Ala Pro Gly Ala Pro Alaro Gly Val Pro Pro# 300#Pro Ala Val Pro Ala Valla Pro Pro Pro Ala#320#Glyla Pro Ala Pro Gln Leu Leu Gly Leu Gln# 330- (2) INFORMATION FOR SEQ ID NO:183:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 207 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:#CATCCCGTCG 60AGAACAA GGTCACGGGC GGCCGCATCC CGCGCGAGTA#CCCGCTGGTT 120CGCAGGA CGCCATGCAG TACGGCGTGC TGGCCGGCTA#GGAAATGGCA 180CGCTGCT CGACGGTGCC TACCACGAAG TCGACTCGTC# 207 TCCCA GGTCATA- (2) INFORMATION FOR SEQ ID NO:184:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 69 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:#Gly Arg Ile Pro Arg Glusn Lys Val Thr Gly# 15#Asp Ala Met Gln Tyr Glysp Ala Gly Ala Gln# 30#Lys Leu Thr Leu Leu Aspro Leu Val Asn Val# 45#Met Ala Phe Lys Val Alaal Asp Ser Ser Glu# 60- Gly Ser Gln Val Ile 65- (2) INFORMATION FOR SEQ ID NO:185:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 898 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185:#GACGGCACAC 60GGCGTGA GGCCAACCAC TAGGCTGGTC ACCAGTAGTC#TGTGAACGGC 120TGAGGAC AGAGGAGACA CCCGTGACGA TCCGTGTTGG#CGAAGGCAAG 180GACGCAA CTTCTTCCGC GCGCTGGACG CGCAGAAGGC#GCTGGCGCAC 240AGATCGT CGCGGTCAAC GACCTCACCG ACAACGCCAC#CGAAGGCGAG 300ACTCGAT CCTGGGCCGG CTGCCCTACG ACGTGAGCCT#AGGCCCGGCG 360TCGGCAG CACCAAGATC AAGGCGCTCG AGGTCAAGGA#CATCTTCACC 420GCGACCT GGGCGTCGAC GTCGTCGTCG AGTCCACCGG#CATCTCCGCG 480CCCAGGG CCACCTCGAC GCGGGCGCCA AGAAGGTCAT#GTACGACGGC 540AGGACAT CACCATCGTG CTCGGCGTCA ACGACGACAA#GCTGGCGAAG 600TCTCCAA CGCGTCGTGC ACCACGAACT GCCTCGGCCC#CGCCTACACC 660AGTTCGG CATCGTCAAG GGCCTGNTGA CCACCATCCA#CGCCGCCGCG 720TGCAGGA CGGCCCGCAC AAGGATCTGC GCCGGGCCCG#GCTGCCCGAG 780CGACCTC CACCGGTGCC GCCAAGGCCA TCGGACTGGT#CTCGGTCACC 840TCGACGG CTACGCGCTG CGGGTGCCGA TCCCCACCGG#CGCGATGA 898AGCTGGG CAAGTCGGCC ACCGTGGACG AGATCAACGC- (2) INFORMATION FOR SEQ ID NO:186:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 268 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:#Gly Arg Ile Gly Arg Asnly Val Asn Gly Phe# 15#Glu Gly Lys Asn Lys Aspsp Ala Gln Lys Ala# 30#Asp Asn Ala Thr Leu Alaal Asn Asp Leu Thr# 45#Arg Leu Pro Tyr Asp Valsp Ser Ile Leu Gly# 60#Gly Ser Thr Lys Ile Lyssp Thr Ile Val Val# 80#Leu Pro Trp Gly Asp Leulu Gly Pro Ala Ala# 95#Ile Phe Thr Lys Arg Aspal Glu Ser Thr Gly# 110#Lys Lys Val Ile Ile Sereu Asp Ala Gly Ala# 125#Val Leu Gly Val Asn Asplu Asp Ile Thr Ile# 140#Ser Asn Ala Ser Cys Threr Gln Asn Ile Ile#160#Ile Asn Asp Glu Phe Glyro Leu Ala Lys Val# 175#Ala Tyr Thr Xaa Val Glnaa Thr Thr Ile His# 190#Arg Arg Ala Arg Ala Alaro His Lys Asp Leu# 205#Ala Ala Lys Ala Ile Glyro Thr Ser Thr Gly# 220#Asp Gly Tyr Ala Leu Argeu Lys Gly Lys Leu#240#Leu Thr Ala Glu Leu Glyly Ser Val Thr Asp# 255#Ala Meter Ala Thr Val Asp Glu Ile Asn Ala# 265- (2) INFORMATION FOR SEQ ID NO:187:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 41 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:#Thr Glu Lys Leu Gly Sereu Ile Asp Val Leu# 15#Asn Val Val Asp Thr Ilehr Ala Ala Val Glu# 30- Val Ala Ala Val Pro Lys Xaa Val Val# 40- (2) INFORMATION FOR SEQ ID NO:188:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 26 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:# 26 CTSAT YGAYGT- (2) INFORMATION FOR SEQ ID NO:189:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 20 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:# 20 TTYTC- (2) INFORMATION FOR SEQ ID NO:190:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 84 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:190:#GACTGCGGCA 60TACTCAC TGAGAAGCTG GGCTCGGATT GTCGGCAAGC# 84CACAC CATA- (2) INFORMATION FOR SEQ ID NO:191:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 337 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:191:#GACTGCGGCG 60TACTCAC TGAGAAGCTG GGCTCGGATT GTCGGCAAGC#CGTCACCATC 120TCGACAC CATCGTGCGC GCCGTGCACA AGGGTGAGAG#CAATCCGCGC 180TTTTCGA GCAGCGTCGT CGCGCAGCAC GCGTGGCACG#CGGCGCTCAG 240TGAAGGT CAAGCCCACC TCAGTCCCGG CATTCCGTCC#GGTCAAGCGC 300TCTCTGG CGCACAGAAG CTTCCGGCCG AGGGTCCGGC# 337 AGCAC CGCCCGCAAG GCAGCCA- (2) INFORMATION FOR SEQ ID NO:192:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 111 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:#Gly Ser Asp Arg Gln Alaeu Thr Glu Lys Leu# 15#Ile Val Arg Ala Val Hissn Val Val Asp Thr# 30#Gly Val Phe Glu Gln Arghr Ile Thr Gly Phe# 45#Arg Thr Gly Glu Thr Valal Ala Arg Asn Pro# 60#Arg Pro Gly Ala Gln Pheer Val Pro Ala Phe# 80#Pro Ala Glu Gly Pro Alaly Ala Gln Lys Leu# 95#Ala Arg Lys Ala Alaal Thr Ala Thr Ser Thr# 110- (2) INFORMATION FOR SEQ ID NO:193:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1164 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193:#GGTGCGGGCG 60GAGAAGC GCCCGCCCCG GTTCACGGGC GCCTGATCAT#GCTGCGCGCC 120GCTTCGG GACGGCCTCA CTGCTGGCCG GCGGGTTCGT#GGTGGCGCGC 180CTGCCGC CCTCGGCGCG ACTCCGGGCG AGGTCGCGCC#GGGCATCACG 240ACCGCGA CGGCAAGTTC GTCAACCTGG AGCCCCCGTC#ATCCCAGGGC 300TGCAGCG GATGCTGTTG CGCGATCTGG CCAACGCCGC#TCCCGCGCCG 360CGATCCC GCTGGCCGAG CCGCCGAAGG GGGATCCCAC#CTACCGCGTG 420GGTACGG CCATTCCAGC GTGCTGATCG AGGTCGACGG#ACCGCAGCGC 480TGTGGAG CAACAGATGT TCGCCCTCAC GGGCGGTCGG#GGTGATCAGC 540CGGTGCC GCTGGAGGCG CTTCCCGCCG TGGACGCGGT#CACCCAGCGG 600ACCACCT CGACATCGAC ACCATCGTCG CGTTGGCGCA#CGTCCCCGAG 660TGCCGTT GGGCATCGGC GCACACCTGC GCAAGTGGGG#GACGCTGGTC 720AGTTGGA CTGGCACGAA GCCCACCGCA TAGACGACCT#GCTGTGGGCG 780GGCACTT CTCCGGACGG TTGTTCTCCC GCGACTCGAC#CGGATACACG 840CCGGCTC GTCGCACAAG GCGTTCTTCG GTGGCGACAC#GCTGCCGATC 900AGATCGG CGACGAGTAC GGTCCGTTCG ATCTGACCCT#GGTGCGCGCC 960CCGCGTT CGCCGACATC CACATGAACC CCGAGGAGGC#GGCGACATTC 1020CCGAGGT GGACAACAGC CTGATGGTGC CCATCCACTG#TGCCGACGCC 1080ATCCGTG GTCCGAGCCC GCCGAACGCC TGCTGACCGC#GTCGACGTTC 1140TGACCGT GCCGATTCCC GGTCAGCGGG TGGACCCGGA# 1164TTCTG AACC- (2) INFORMATION FOR SEQ ID NO:194:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 370 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:#Gly Thr Ala Ser Leu Leueu Arg Tyr Gly Phe# 15#Gly Thr Pro Ala Ala Leueu Arg Ala Leu Gln# 30#Ala Arg Arg Ser Pro Asnlu Val Ala Pro Val# 45#Pro Pro Ser Gly Ile Thrhe Val Asn Leu Glu# 60#Arg Asp Leu Ala Asn Alaln Arg Met Leu Leu# 80#Pro Leu Ala Glu Pro Proro Pro Gly Pro Ile# 95#Ala Ser Trp Tyr Gly Hisro Ala Pro Ala Ala# 110#Arg Val Leu Ala Asp Prolu Val Asp Gly Tyr# 125#Ala Val Gly Pro Gln Argys Ser Pro Ser Arg# 140#Leu Pro Ala Val Asp Alaal Pro Leu Glu Ala#160#Leu Asp Ile Asp Thr Ilesp His Tyr Asp His# 175#Phe Val Val Pro Leu Glyhr Gln Arg Ala Pro# 190#Pro Glu Ala Arg Ile Valrg Lys Trp Gly Val# 205#Asp Asp Leu Thr Leu Vallu Ala His Arg Ile# 220#Leu Phe Ser Arg Asp Seris Phe Ser Gly Arg#240#Ser Ser His Lys Ala Pherp Val Val Thr Gly# 255#Phe Ala Glu Ile Gly Asply Tyr Thr Lys Ser# 270#Pro Ile Gly Ala Tyr Hissp Leu Thr Leu Leu# 285#Glu Glu Ala Val Arg Alale His Met Asn Pro# 300#Leu Met Val Pro Ile Hislu Val Asp Asn Ser#320#Trp Ser Glu Pro Ala Glueu Ala Pro His Pro# 335#Val Arg Leu Thr Val Prola Asp Ala Glu Arg# 350#Thr Phe Asp Pro Trp Trpal Asp Pro Glu Ser# 365- Arg Phe 370__________________________________________________________________________

Number	Name	Date
3956481	Jolles et al.	May 1976
4036953	Adam et al.	Jul 1977
4716038	Stanford et al.	Dec 1987
4724144	Rook et al.	Feb 1988
5599545	Stanford et al.	Feb 1997
5833996	Stanford et al.	Nov 1998

	Number	Date	Country
Parent	873970	Jun 1997
Parent	705347	Aug 1996

Compounds and methods for treatment and diagnosis of mycobacterial infections

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

REFERENCE TO RELATED APPLICATIONS

US Referenced Citations (6)

Foreign Referenced Citations (1)

Continuation in Parts (2)