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WO1998018927A1 - Candida albicans proteins associated with virulence and hyphal formation and uses thereof - Google Patents

Candida albicans proteins associated with virulence and hyphal formation and uses thereof Download PDF

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WO1998018927A1
WO1998018927A1 PCT/CA1997/000809 CA9700809W WO9818927A1 WO 1998018927 A1 WO1998018927 A1 WO 1998018927A1 CA 9700809 W CA9700809 W CA 9700809W WO 9818927 A1 WO9818927 A1 WO 9818927A1
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Ekkehard Leberer
David Y. Thomas
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National Research Council Of Canada
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Priority to CA002269633A priority Critical patent/CA2269633A1/en
Priority to AU48585/97A priority patent/AU4858597A/en
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Priority to US10/093,524 priority patent/US20030166886A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56961—Plant cells or fungi

Definitions

  • the invention relates to Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, associated with virulence and hyphal formation and uses thereof, such as to design screening tests for inhibi- tors for the treatment of pathogenic fungi infections and/or inflammation conditions, (b) Description of Prior Art
  • Candida albicans is the major fungal pathogen in humans, causing various forms of candidiasis. The incidence of infections is increasing in immunocom- promised patients. This fungus is diploid with no sexual cycle and is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of a mycelium displaying hyphae with branches and lateral buds. In view of the observation that hyphae seem to adhere to and invade host tissues more readily than does the yeast form, the switch from the yeast to the filamentous form probably contributes to the virulence of this organism (for a review see Fidel, P. L. & Sobel, J. D. (1994) Trends Microbiol . 2, 202-205). The molecular mechanisms by which morphological switching is regulated are poorly understood.
  • bakers yeast Saccharomyces cerevisiae is also a dimorphic organism capable of switching under certain nutritional conditions from a budding yeast to a filamentous form. Under the control of nutritional signals, diploid cells switch to pseudo- hyphal growth (Gimeno, C. J. et al . (1992) Cell 68, 1077-1090), and haploid cells to invasive growth (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985) .
  • S. cerevisiae The similarities between the dimorphic switching of S. cerevisiae and C. albicans suggest that these morphological pathways may be regulated by similar mechanisms in both organisms.
  • morphological transitions are controlled by signaling components that are also involved in the mating response of haploid cells (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al . (1993) Science 262, 1741-1744).
  • the switch to pseudohyphal growth requires a transcription factor encoded by the STE12 gene, and a mitogen-activated protein (MAP) kinase cascade including Ste7 ⁇ (a homolog of MAP kinase kinase or MEK), Stellp (a MEK kinase homolog) and Ste20p (a MEK kinase kinase) (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al . (1993) Science 262, 1741-1744).
  • the MAP kinases involved in this response are as yet unknown (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al . (1993) Science 262, 1741-1744).
  • Ste20p serine/threonine protein kinases are thought to be involved in triggering morphogenetic processes in response to external signals in organisms ranging from yeast to mammalian cells.
  • Two of these kinases, Ste20p and Cla4p, are well characterized in S. cerevisiae (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824; Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830).
  • Ste20p is required for pheromone signal transduction (Leberer, E. et al . (1992) EMBO J.
  • Cst20p a C. albicans homolog of the Ste20p protein kinase
  • Cst20p a C. albicans homolog of the Ste20p protein kinase
  • CaCla4p a C. albicans homolog of the Cla4p protein kinase
  • CaCla4p a C. albicans homolog of the Cla4p protein kinase
  • One aim of the present invention is to provide Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, and their uses thereof.
  • One aim of the present invention is to provide the nucleotide and amino acid sequences of CaCla4p, Cst20p, CaCdc42p and CaBemlp.
  • Another aim of the present invention is to provide screening tests for inhibitors of CaCla4p, Cst20p, CaCdc42p and CaBemlp or of their interactions.
  • fungi when used herein is intended to mean any fungi, pathogenic or not, which show hyphal induction using kinases, such as C.
  • albicans Saccharo- myces cerevisiae, Aspergillus, Ustilago maydis, and all the species of the fungal genera Aspergillus, Blastomy- ces, Candida, Cladosporiu , Coccidioides , Cryptococcus, Epidermophyton, Exophilia, Fonsecaea, Histoplasma, Madurella, Malassezia, Microsporum, Paracoccidioides, Penicillium, Phaeoannellomyces , Phialophora, Scedospo- rium, Sporothrix, Torulopsis, Trichophyton, Trichospo- ron, Ustilago, Wangiella, Xylohypha, among others.
  • an in vi tro screening test for compounds to inhibit the biological activity of at least one protein selected from the group consisting of CaCla4p, Cst20p, Cdc42p and Bemlp, which comprises: a) at least one of the proteins; and b) means to monitor the biological activity of at least one protein; thereby compounds are tested for their inhibiting potential.
  • the inhibition of the interactions between CaCla4p and CaCdc42p is determined. In accordance with another embodiment of the present invention, the inhibition of the interactions between Cst20p and CaCdc42p is determined.
  • FIG. IA to ID illustrate photomicrographs which show that C. albicans CST20 gene complements defects in pseudohyphal growth of ste20/ste20 S. cerevisiae diploid cells.
  • Figs. 2A to 2C show the morphology of S. cere- visiae MAT ⁇ cells (strain YEL306-1A) deleted for STE20 and CLA4, and transformed with plasmids expressing CLA4 (Fig. 2A), STE20 (Fig. 2B) and C. albicans CST20 (Fig. 2C).
  • Figs. 3A to 3C show the nucleotide (SEQ ID NO: 5) and predicted amino acid sequences of CST20 (SEQ ID NO : 6 ) .
  • Figs. 5A to 5J show colonies of C. albicans cells grown for 5 days at 37 °C on solid "Spider" medium containing mannitol.
  • Wild type strain SC5314 A
  • ura3/ura3 cst20 ⁇ /cst20 ⁇ : : URA3 strain CDH22
  • B ura3/ura3 cst20 ⁇ /cst20 ⁇ : : CST20 : : URA3 strain CDH36 (obtained by reintegration of CST20 into strain CDH25 by homologous recombination using linearized plasmid PDH190)
  • C ura3/ura3 cst20 ⁇ /cst20 ⁇ strain CDH25 transformed with plasmids pYPBl-ADHpt
  • E pYPBl- ADH ⁇ t-HST7
  • E ura3/ura3 hst l ⁇ /hst l ⁇ strain CDH12 transformed with plasmids pVEC
  • Figs. 7A to 7B illustrate the nucleotide (SEQ ID NO: 7) and predicted amino acid (SEQ ID NO: 8) sequences of CaCLA4.
  • Fig. 8A illustrates the deletion of CaCLA4 in C. albicans .
  • Fig. 8C illustrates the Northern blot analysis with the CaCLA4 fragment as a probe.
  • PCR with the divergent oligodeoxynucleotides OEL109 and OELllO was used to delete the coding sequence of CaCLA4.
  • Fig. 10 illustrates the staining of mouse kidney sections with periodic acid Schiff ' s stain 48 h after infection with C. albicans strains SC5314 and CLJ1.
  • Fig. 11 illustrates the nucleotide (SEQ ID NO: 9) and predicted amino acid (SEQ ID NO: 10) sequences of CaCdc42p.
  • Figs. 12A to 12B illustrate the nucleotide (SEQ ID NO: 11) and predicted amino acid (SEQ ID NO: 12) sequences of CaBemlp.
  • albicans one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans .
  • C. albicans cells into kidneys after infection into mice and completely suppressed virulence in the mouse model.
  • hyphal formation of C. albicans mediated by the CaCla4p protein kinase may contribute to the pathogenicity of this dimorphic fungus.
  • the yeast form of C. albicans was cultured at 30°C in YPD medium. Hyphal growth was induced at 37 °C on solid "Spider” media (Liu, H. et al. (1994) Science 266, 1723-1726) containing 1% (w/v) nutrient broth, 0.2% (w/v) K 2 HP0 4 , 2% (w/v) agar and 1% (w/v) of the indicated sugars (pH 7.2 after autoclaving) . Cells were grown in liquid "Spider” media at 30 °C to stationary phase, and then incubated for 5 days at 37 °C on solid "Spider” media at a density of about 200 cells per 80 mm plates.
  • the CST20 gene was isolated from a genomic C. albicans library constructed in plasmid YEp352 from genomic DNA of the clinical isolate W01 (Boone, C. et al. (1991) J. Bacteriol . 173, 6859-6864).
  • a plasmid carrying an amino-terminally truncated version of CST20 missing the first 918 nucleotides of coding sequence was isolated by screening for suppressors of defects in basal FUSl : :HIS3 expression and mating in S. cerevisiae strain YEL64 which was disrupted in STE20.
  • the S. cerevisiae MAT ⁇ strain YEL257-1A-2 deleted for STE20 and CLA4 and carrying plasmid pDH129 with CLA4 under control of the GAL1 promoter was transformed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al . (1991) J. Bacte- riol . 173, 6859-6864). Transformants were grown on selective medium in 4% galactose and then replica- plated to selective medium containing 2% glucose to select for plasmids that were able to support growth in the absence of Cla4p and Ste20p.
  • a plasmid that contained CST20- lanking sequences from nucleotides 989 to 1,674, and 3,423 to 4,134 joined with BamHI sites was then created by PCR using the divergent oligodeoxynucleotide primers 0DH68 (5 1 - CGGGATCCAGACCAACCACTCGAACTACT-3' (SEQ ID N0:1) and ODH69 ( 5 ' -CGGGATCCGAAGGTGAACCACCATATTTG-3 ' ( SEQ ID N0:2); newly introduced BamHI sites are underlined) and plasmid pDH119 as a template.
  • a plasmid that contained CaCLA4 flanking sequences joined with Bglll sites was then created by PCR using the divergent oligodeoxynucleotide primers OEL109 (5 1 - GAAGATCTTGTAATCAATGTTCCCGTGGA-3' (SEQ ID NO : 3 ) and OELllO ( 5 ' -GAAGATCTCATCGTGATATTAAATCCGAT-3 ' ( SEQ ID NO:4); newly introduced Bglll sites are underlined) and plasmid pDH205 as template.
  • the amplified DNA was cleaved with Bglll and ligated with a 4 kb BamHI-Bglll fragment of a hisG-URA3-hisG cassette derived from plasmid pCUB-6 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to yield plasmid pDH210.
  • This plasmid was linearized with PstI and SacI and transformed into the Ura " C. albicans strain CAI4 (Fonzi, W. A. & Irwin, M. Y.
  • the C. albicans integration plasmid pDH190 was constructed by subcloning a Kpnl to PstI fragment of CST20 into pBS-cC7RA3 (pBluescript KS( + ) into which the C. albicans URA3 gene was cloned between the NotI and Xbal sites of the polylinker). The integration plasmid was then linearized with Nsil and transformed into C. albicans to target integration into the Nsil site of the CST20 ⁇ : :hisG fusion gene. Integrations were selected on Ura- medium and confirmed by Southern blot analysis.
  • the C. albicans CST20 expression plasmid pDH188 was constructed by subcloning a SacI to PstI fragment of CST20 into plasmid pVEC carrying a C. albicans autonomously replicating sequence and URA3 as selectable marker.
  • the C. albicans plasmid pVEC-CaCLA4 was constructed by subcloning the Kpnl to SacI insert of YEp 352-CaCLA4 into plasmid pVEC.
  • mice were sacrificed by cervical dislocation 48 hours after injection and kidneys were homogenized in 5 ml phosphate buffered saline, serially diluted and plated on YNG medium (0.67% yeast nitrogen base, 1% glucose, pH 7.0). Histological examination of kidney sections was done with periodic acid Schiff * s stain.
  • a C. albicans homolog of the S. cerevisiae STE20 gene was cloned by functional complementation of the pheromone signaling defect of S. cerevisiae cells that were deleted for the STE20 gene.
  • the mating defect of the STE20 deleted S. cerevisiae strain YEL20 was fully complemented by introduction of the centromeric plasmid pRL53 carrying full length CST20 (mating efficiency was
  • the diploid strain YEL306 heterozygous for ste20 ⁇ : : TRP1 /STE20 cla4 ⁇ : : LEU2/CLA4 was transformed with plasmid pRS316 carrying either no insert, CLA4 (pRL21), CST20 (pRL53) or STE20 (pSTE20-5), and then sporulated and dissected.
  • No viable haploid ste20 ⁇ cla4 ⁇ spores were obtained from transformants with the plasmid without insert, but were obtained from transformants with plasmids carrying CLA4 (Fig. 2A), STE20 (Fig. 2B) or CST20 (Fig. 2C).
  • the open reading frame of CST20 is capable of encoding a protein of 1,229 amino acids with a pre- dieted molecular weight of 133 kDa and a domain structure characteristic of the Ste20p/p65- PAK family of protein kinases (Fig. 3). Numerals at the left margin indicate nucleotide and amino acid positions (Fig. 3). Nucleotide 1 corresponds to the first nucleotide of the initiation codon and amino acid 1 to the first residue of the deduced protein. The putative p21 binding domain has been shadowed, and the kinase domain has been boxed.
  • the catalytic domain present in the carboxyl terminal half of the protein has sequence identities of 76 and 56%, respectively, with S. cerevisiae Ste20p (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824) and Cla4p (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817- 1830).
  • the amino terminal, non-catalytic region con- tains a sequence from amino acid residues 473 to 531 with 68% identity to the p21 binding domain of Ste20p that has been shown to bind the small GTPase Cdc42p.
  • This region contains the sequence motif ISxPxxxxHxxH thought to be important for the interaction of the p21 binding domain with the GTP-bound forms of Cdc42Hs and Racl (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817- 1830).
  • the remaining non-catalytic sequences are less conserved.
  • Unique sequences not present in Ste20p and the other members of the family are found at the amino terminus and between the p21 binding and catalytic domains .
  • a CST20 transcript of 4.9 kb in size was detected in Northern blots. This transcript was present at similar levels in yeast cells grown in YPD at room temperature and germ tubes induced by a temperature shift to 37°C. Isolation and characterization of CaCLA4
  • a C. albicans homolog of the S. cerevisiae CLA4 gene was cloned by functional complementation of the growth defect of S. cerevisiae cells that were deleted for the STE20 and CLA4 genes.
  • the open reading frame of the CaCLA4 gene is capable of encoding a protein of 971 amino acids with a predicted molecular weight of 107 kDa and a domain structure characteristic of the Ste20p family of protein kinases (Fig. 7).
  • the catalytic domain present in the carboxyl terminal half of the protein has sequence identities of 74, 63 and 64%, respectively, with S. cerevisiae Cla4p, S. cerevisiae Ste20p and an uncharac- terized open reading frame present in the S. cerevisiae genome, 65% with the C. albicans Ste20p homolog Cst20p, and 61% with rat p ⁇ - ⁇ (Fig. 7).
  • the amino terminal, noncatalytic region contains a sequence from amino acid residues 69 to 180 with similarity to pleckstrin homology (PH) domains and a sequence from amino acid residues 229 to 292 with 63% identity to the Cdc42p binding domain of S. cerevisiae Cla4p that has been shown to bind the small GTPase Cdc42p (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830). The remaining noncatalytic sequences are less conserved. Chromosomal deletion of CST20
  • Homologous recombination was used in a multistep procedure to partially delete CST20 in a URA ⁇ C. albi - cans strain (Fig. 4A) .
  • PCR with the divergent oligodeoxynucleotides ODH68 and ODH69 was used to partially delete the coding sequence of CST20.
  • a hisG-URA3-hisG cassette was then inserted. The deletion was confirmed by Southern blot analyses (Fig. 4B).
  • the genomic DNA samples digested with Xhol were from following strains: Lane #1, CAI4 ( ura3/ura3 CST20/CST20 ) ; lane 2, CDH15 ⁇ ura3/ura3 CST20/cst20 ⁇ : :hisG-URA3-hisG) ; lane 3, CDH18 ( ura3/ura3 CST20/cst20 ⁇ : :hisG) ; lane 4, CDH22 ( ura3/ura3 cst20 ⁇ : :hisG-URA3-hisG/cst20 ⁇ : :hisG) ; lane 5, CDH25 ⁇ ura3/ura3 cst20 ⁇ : :hisG/cst20 ⁇ : :hisG) .
  • Northern blots showed that the CST20 transcript was absent in the corresponding homozygous deletion strains.
  • Mycelial formation was drastically reduced when the media contained galactose, mannose or raffinose.
  • the mutant strains regained the ability to form hyphae when wild type CST20 was reintroduced by transformation with the CST20 expression plasmid pDH188 or reintegrated into the genome by targeted homologous recombination (Fig. 5C).
  • the CST20 transcript was detected in these strains by Northern blot analysis. Mutant strains formed hyphae when colonies were grown on "Spider" media containing either glucose or N- acetyl glucosamine. Normal hyphae formation was also observed on rice agar and on agar containing Lee ' s medium or 10% serum.
  • Fig. 8A shows the restriction endonu- clease map of CaCLA4.
  • the coding sequence is indicated by the arrow.
  • PCR with the divergent oligodeoxynucleotides OEL109 and OELllO was used to delete the coding sequence of CaCLA4.
  • a hisG-URA3-hisG cassette was then inserted and a two-step procedure was used to delete both alleles of CaCLA4 by homologous recombination.
  • the endonuclease restriction sites are as follows: B, BamHI; Bg, Bglll; E, EcdRl ; H, HindiII; P, PstI; S, Sad ; X, Xbal .
  • the deletions were confirmed by Southern blot analyses (Fig. 8B). Southern blot analysis with a 1.1 kb CaCLA4 fragment from Pstl-Xbal as a probe.
  • the genomic DNA samples digested with EcoRI were from following strains: Lanes: 1, CAI4 ( ura3/ura3 CaCLA4/CaCLA4 ) ; 2, CDH77 ⁇ ura3/ura3 CaCLA4/cacla4 ⁇ : :hisG-URA3-hisG) ; 3, CDH88 ( ura3/ura3 CaCLA4/cacla4 ⁇ : :hisG) ; 4, CLJ1 ⁇ ura3/ura3 cacla4 ⁇ : :hisG-URA3- hisG/cacla4 ⁇ : :hisG) ; and 5, CLJ5 ⁇ ura3/ura3 cacla4 ⁇ : :hisG/cacla4 ⁇ : :hisG) .
  • mice were injected intravenously with wild type and mutant strains and monitored for survival and for fun- gal invasion into kidneys.
  • the Ura- strain CAI4 was not pathogenic (Figs. 6A and B).
  • infection with Ura + wild type cells resulted in rapid mortality with a rate that was dependent on the dose of injected cells (1 x 10 ⁇ cells in Fig. 6A, and 1 x 10 ⁇ cells in Fig. 6B).
  • Survival was significantly prolonged, however, in mice infected with Ura + cells deleted for both alleles of CST20 ( cst20 ⁇ /cst20 ⁇ : : URA3 ) .
  • a C. albicans homolog of the CaCDC42 gene was cloned by functional complementation of the temperature-sensitive growth defect of S. cerevisiae cells carrying the cdc42-l ⁇ s mutation.
  • the growth defect was fully complemented by plasmid YEp352-CaCDC42.
  • the open reading frame of the CaCDC42 gene is capable of encoding a protein of 191 amino acids with homology to the Rho-family of small G-proteins (Fig. 11). The highest homology is found with Cdc42p from S. cerevisiae .
  • a C. albicans homolog of the CaBEMl gene was cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM1 gene.
  • Ste20p fulfills multiple functions during mating (Leberer, E. et al . (1992) EMBO J. 11, 4815-4824), pseudohyphae formation (Liu, H., Styles, C. & Fink, G. R. (1993) Science 262, 1741- 1744), invasive growth (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985) and cytokinesis (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830).
  • CST20 expression in S. cerevisiae fully complements these functions.
  • Cst20p has the potential to fulfill similar functions in C. albicans .
  • C. albicans The yeast-to-hyphal transition of C. albicans is a morphological change that can be triggered by a wide variety of factors. Carbohydrates, amino acids, salts, and serum have been described as inducers of germ tube formation, as have pH changes, temperature increases and starvation, but no single environmental factor could be defined as uniquely significant in stimulating the morphological switch. Hence C. albicans appears capable of responding to many divergent environmental signals. Disruption of both CPH1 alleles, which encode a homolog of the S. cerevisiae Stel2p transcription factor (Liu, H. et al .
  • null mutants of CST20 are reduced in virulence (Figs. 6A and 6B), that CaCla4p and Cst20p, and proteins such as CaCdc42p and CaBemlp interacting with these protein kinases, may be valid targets for the development of antifungal agents.
  • CaCla4p and CaCdc42p will be used to test compounds inhibiting their interactions.
  • CaCla4p may be solid phase bound and CaBemlp will be in suspension free to interact with CaCla4p.
  • a labeled antibody specific to CaBemlp will be added to the assay to determine the presence of CaBemlp bound to CaCla4p.
  • the compounds tested to inhibit the CaBemlp- CaCla4p interactions, should when tested positive, cause only a minute quantity of CaBemlp to bind to CaCla4p interactions.
  • the analogous in vi tro assay will be used to test compounds that inhibit the interaction between Cst20p and CaBemlp.
  • the CaCDC42 gene will be fused to the DNA binding domain of GAL4
  • the CaCLA4 gene will be fused to the activation domain of GAL4. Interaction of the two proteins will cause green fluo- rescence. Whereas inhibitors of the interaction will suppress fluorescence.
  • Non-specific inhibitors of the two-hybrid interaction system will be excluded by performing a parallel screen with unrelated fusion proteins known to inter- act. Compounds of general toxicity or inhibitors of the human homologs will also be excluded in this system because those compounds will not allow growth of the cells and therefore reduce the fluorescent readout in both parallel screens.
  • a two-hybrid yeast strain carrying the GAL4-GFP fusion gene is constructed. This strain will be deleted for the CLA4 gene using the TRP1 marker as described (Leberer E. et al . (1997) Embo J. 16, 83-97). The STE20 gene will be replaced by the human PAK gene as described above.
  • an integrating plasmid will be constructed carrying the HsCDC42 gene fused to a URA3 blaster gene and CDC42 flanking sequences. After linearization, the construct will be transformed into the PAK containing two-hybrid strain, and integrants will be selected on -ura medium. The URA3 gene will then be looped out on FOA medium. The various gene disruptions and gene replacements will be verified by Southern blot analyses .
  • the two-hybrid vectors carrying the CaCDC42 gene fused to the GAL4-DNA binding domain and the CaCLA4 gene fused to the transcriptional activation domain of GAL4 will be constructed by standard procedures.
  • sequences of either one of the genes CaCLA4, CST20, CaCDC42 and CaBEMl may be used to derive probes for the detection of C. albicans using PCR techniques or hybridization assays.
  • CaCDC42 and CaBEMl may be used to identify and clone homologues from other fungi.
  • the STE20 gene will be replaced in a supersensi- tive sstl yeast strain by the human PAK gene using homologous recombination.
  • an integrating plasmid will be constructed carrying the PAK gene fused to a URA3 blaster gene and STE20 flanking sequences. The construct will be linearized and transformed into yeast, and integrants will be selected on - ura medium. The URA3 gene will then be looped out on FOA medium to gain back the ura3 marker. Correct inte- gration of the PAK gene will be confirmed by Southern blot analysis.
  • Fluorescence resonance energy transfer as probe for protein-protein interactions
  • the engineering of different GFP mutants with altered fluorescence characteristics allows the use of fluorescence resonance energy transfer (FRET) to probe protein-protein interactions (Heim and Tsien (1996) Curr. Biol. 6, 178-182).
  • FRET fluorescence resonance energy transfer
  • the FRET phenomenon consists in a fluorescence transfer between a donor and a receptor fluorochrome. If excitation and emission wave- lengths are compatible, the FRET is easily measurable.
  • the main parameter of the reaction is the distance between donor and receptor, which must be in the range of nanometers. This is precisely the kind of values in protein-protein interactions.
  • the CaCDC42 gene will be fused to a GFP mutant that acts as donor, and the CaCLA4 gene will be fused to a mutant that acts as receptor.
  • the yeast strain used as an expression sys- tem will be humanized as described in Example VII. Inhibitors of the interaction are expected to reduce energy transfer, and this reduction can be easily measured spectroscopically .
  • the interaction of unrelated proteins known to interact will be used as a reference to exclude non-specific inhibitors of the assay system. Compounds inhibiting the interaction of the human homologs or of general toxicity will be excluded by inhibition of growth and therefore reduced fluorescence in both screens .
  • the CaCDC42 gene will be fused to the gene encoding the GFP Y66H mutant as donor, and the CaCLA4 gene will be fused to the gene encoding the GFP 3657 mutant as receptor (Heim and Tsien (1996), Curr. Biol. 6, 178-182).
  • the constructs will then be transformed into the humanized yeast strain described in Example VII, and the FRET phenomenon will be analyzed in yeast cultures using fluorescence spectroscopy.
  • the conditions for the assay will be worked out and optimized. We will adapt the assay conditions to the scale used in microtiter plates for automated screening.
  • MOLECULE TYPE cDNA
  • MOLECULE TYPE cDNA
  • cDNA cDNA
  • MOLECULE TYPE Genomic RNA
  • FEATURE :
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • AGT TCT AAT AGT CTT GGC GTA ACA GCA AAT CAA ACC AAA CCT ATT CAA 614 Ser Ser Asn Ser Leu Gly Val Thr Ala Asn Gin Thr Lys Pro He Gin 50 55 60
  • ACT CAT AAA GTA CAC GTG GGA TTT GAT CCT GCC AGT GGT AAT TTT ACT 1190 Thr His Lys Val His Val Gly Phe Asp Pro Ala Ser Gly Asn Phe Thr 240 245 250
  • AAC AAT TAC TCA TCA ACC AAA AAC AAT GTC CAA GAG GCA AAT TTA CAA 1430 Asn Asn Tyr Ser Ser Thr Lys Asn Asn Val Gin Glu Ala Asn Leu Gin 320 325 330
  • CAA CAT AAA AAT ATT GTT AAT TTT TTG GAT TCT TAT TTA ATT GGT GAT 2678 Gin His Lys Asn He Val Asn Phe Leu Asp Ser Tyr Leu He Gly Asp 735 740 745
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • MOLECULE TYPE Genomic DNA
  • FEATURE
  • AAA AAT CAA ACT GCC AAA TAT CAA GCT TCA ACA ATC CCC CTT GGT TCA 1064 Lys Asn Gin Thr Ala Lys Tyr Gin Ala Ser Thr He Pro Leu Gly Ser 245 250 255
  • AAA TTA CGA AGA GAA AAA TTG GAT TAT TAT TTA TCA AAT TTA ATT GCA 1448 Lys Leu Arg Arg Glu Lys Leu Asp Tyr Tyr Leu Ser Asn Leu He Ala 375 380 385
  • TTT TCT AAA CCA ATA AGT CAA AAA TCA AAT TCT CAT CAA GAT AGA TTA 1592 Phe Ser Lys Pro He Ser Gin Lys Ser Asn Ser His Gin Asp Arg Leu 420 425 430

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Abstract

The present invention relates to Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, associated with virulence and hyphal formation and uses thereof, such as to design screening tests for inhibitors for the treatment of pathogenic fungi infections and/or inflammation conditions. The invention also relates to an in vitro screening test for compounds to inhibit the biological activity of at least one protein selected from the group consisting of CaCla4p, Cst20p, CaCdc42p and CaBemlp, which comprises: a) at least one of said proteins; and b) means to monitor the biological activity of said at least one protein; thereby compounds are tested for their inhibiting potential.

Description

CANDIDA ALBICANS PROTEINS ASSOCIATED WITH VIRULENCE AND HYPHAL FORMATION AND USES THEREOF
BACKGROUND OF THE INVENTION (a) Field of the Invention
The invention relates to Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, associated with virulence and hyphal formation and uses thereof, such as to design screening tests for inhibi- tors for the treatment of pathogenic fungi infections and/or inflammation conditions, (b) Description of Prior Art
Candida albicans is the major fungal pathogen in humans, causing various forms of candidiasis. The incidence of infections is increasing in immunocom- promised patients. This fungus is diploid with no sexual cycle and is capable of a morphological transition from a unicellular budding yeast to a filamentous form. Extensive filamentous growth leads to the formation of a mycelium displaying hyphae with branches and lateral buds. In view of the observation that hyphae seem to adhere to and invade host tissues more readily than does the yeast form, the switch from the yeast to the filamentous form probably contributes to the virulence of this organism (for a review see Fidel, P. L. & Sobel, J. D. (1994) Trends Microbiol . 2, 202-205). The molecular mechanisms by which morphological switching is regulated are poorly understood.
Like C. albicans, bakers yeast Saccharomyces cerevisiae is also a dimorphic organism capable of switching under certain nutritional conditions from a budding yeast to a filamentous form. Under the control of nutritional signals, diploid cells switch to pseudo- hyphal growth (Gimeno, C. J. et al . (1992) Cell 68, 1077-1090), and haploid cells to invasive growth (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985) .
The similarities between the dimorphic switching of S. cerevisiae and C. albicans suggest that these morphological pathways may be regulated by similar mechanisms in both organisms. In S. cerevisiae, morphological transitions are controlled by signaling components that are also involved in the mating response of haploid cells (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al . (1993) Science 262, 1741-1744). The switch to pseudohyphal growth requires a transcription factor encoded by the STE12 gene, and a mitogen-activated protein (MAP) kinase cascade including Ste7ρ (a homolog of MAP kinase kinase or MEK), Stellp (a MEK kinase homolog) and Ste20p (a MEK kinase kinase) (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al . (1993) Science 262, 1741-1744). The MAP kinases involved in this response are as yet unknown (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al . (1993) Science 262, 1741-1744).
Members of the Ste20p family of serine/threonine protein kinases are thought to be involved in triggering morphogenetic processes in response to external signals in organisms ranging from yeast to mammalian cells. Two of these kinases, Ste20p and Cla4p, are well characterized in S. cerevisiae (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824; Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830). Ste20p is required for pheromone signal transduction (Leberer, E. et al . (1992) EMBO J. 11, 4815-4824) and for filamentous growth in response to nitrogen starvation (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985; Liu, H. et al. (1993) Science 262, 1741-1744), and shares an essential function with Cla4p during budding (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). Ste20p and Cla4p interact with the small G-protein Cdc42p, and this interaction is required for viability of S. cerevisiae cells. Ste20p also interacts with the SH3 domain protein Bemlp, and this interaction plays a role in morphogenetic processes (Leeuw, T. et al. (1995) Science 270, 1210-1213).
Here we show that Cst20p, a C. albicans homolog of the Ste20p protein kinase, is required for hyphal growth of C. albicans under certain in vi tro conditions. We also show in a mouse model for systemic candidiasis that Cst20p plays a role in virulence, as judged from significantly prolonged survival of mice infected with CST20 deleted cells. Our results suggest that Cst20p acts in a regulatory pathway which is involved in hyphal growth of C. albicans .
We also demonstrate that CaCla4p, a C. albicans homolog of the Cla4p protein kinase, is required for hyphal formation in vi tro in response to serum, and in vivo in a mouse model for systemic candidiasis. We also show that CaCla4p is required for efficient colonization of kidneys with C. albicans cells after infection of mice and essential for virulence in the mouse model .
SUMMARY OF THE INVENTION
One aim of the present invention is to provide Candida albicans proteins, such as CaCla4p, Cst20p, CaCdc42p and CaBemlp, and their uses thereof. One aim of the present invention is to provide the nucleotide and amino acid sequences of CaCla4p, Cst20p, CaCdc42p and CaBemlp.
Another aim of the present invention is to provide screening tests for inhibitors of CaCla4p, Cst20p, CaCdc42p and CaBemlp or of their interactions. The term "fungi" when used herein is intended to mean any fungi, pathogenic or not, which show hyphal induction using kinases, such as C. albicans, Saccharo- myces cerevisiae, Aspergillus, Ustilago maydis, and all the species of the fungal genera Aspergillus, Blastomy- ces, Candida, Cladosporiu , Coccidioides , Cryptococcus, Epidermophyton, Exophilia, Fonsecaea, Histoplasma, Madurella, Malassezia, Microsporum, Paracoccidioides, Penicillium, Phaeoannellomyces , Phialophora, Scedospo- rium, Sporothrix, Torulopsis, Trichophyton, Trichospo- ron, Ustilago, Wangiella, Xylohypha, among others.
In accordance with the present invention there is provided an in vi tro screening test for compounds to inhibit the biological activity of at least one protein selected from the group consisting of CaCla4p, Cst20p, Cdc42p and Bemlp, which comprises: a) at least one of the proteins; and b) means to monitor the biological activity of at least one protein; thereby compounds are tested for their inhibiting potential.
In accordance with another embodiment of the present invention, the inhibition of the interactions between CaCla4p and CaCdc42p is determined. In accordance with another embodiment of the present invention, the inhibition of the interactions between Cst20p and CaCdc42p is determined.
BRIEF DESCRIPTION OF THE DRAWINGS Figs. IA to ID illustrate photomicrographs which show that C. albicans CST20 gene complements defects in pseudohyphal growth of ste20/ste20 S. cerevisiae diploid cells.
Figs. 2A to 2C show the morphology of S. cere- visiae MATα cells (strain YEL306-1A) deleted for STE20 and CLA4, and transformed with plasmids expressing CLA4 (Fig. 2A), STE20 (Fig. 2B) and C. albicans CST20 (Fig. 2C).
Figs. 3A to 3C show the nucleotide (SEQ ID NO: 5) and predicted amino acid sequences of CST20 (SEQ ID NO : 6 ) .
Fig. 4A is the deletion of CST20 in C. albicans . Fig. 4B is the Southern blot analysis with a CST20 fragment from EcoRI to Xbal as a probe.
Figs. 5A to 5J show colonies of C. albicans cells grown for 5 days at 37 °C on solid "Spider" medium containing mannitol. Wild type strain SC5314 (A), ura3/ura3 cst20Δ/cst20Δ : : URA3 strain CDH22 (B), ura3/ura3 cst20Δ/cst20Δ : : CST20 : : URA3 strain CDH36 (obtained by reintegration of CST20 into strain CDH25 by homologous recombination using linearized plasmid PDH190) (C), ura3/ura3 cst20Δ/cst20Δ strain CDH25 transformed with plasmids pYPBl-ADHpt (D) and pYPBl- ADHρt-HST7 (E), ura3/ura3 hst lΔ/hst lΔ strain CDH12 transformed with plasmids pVEC (F), pVEC-HST7 (G) , pYPBl-ADHpt (H), and pYPBl-ADHpt-HST7 (I), and ura3/ura3 cphl/cphl strain CDH72 [ ura3/ura3 derivative of strain JK19] transformed with pYPBl-ADHpt-HST7 ( J) . Photomicrographs of representative colonies were taken with a 2χ lens (bar=2mm) . Figs. 6A to 6C illustrate virulence assays. Survival curves of mice (n=10 for each C. albicans strain at each inoculation dose) infected with 1 x 10^ (A) and 1 x 105 (B) cells of C. albicans strains SC5314 (wild type), CAI4 ( ura3/ura3 ) , CDH22 ( ura3/ura3 cst20Δ/cst20Δ : : URA3 ) (C) Staining of mouse kidney sections with periodic acid Schiff ' s stain 48 hours after infection with cst20Δ/cst20Δ : : URA3 mutant strain CDH22 (a). Some hyphal cells are indicated with arrows (bar=0.1 mm). Figs. 7A to 7B illustrate the nucleotide (SEQ ID NO: 7) and predicted amino acid (SEQ ID NO: 8) sequences of CaCLA4.
Fig. 8A illustrates the deletion of CaCLA4 in C. albicans .
Fig. 8B illustrates the Southern blot analysis with the CaCLA4 fragment from PstI to Xbal as a probe.
Fig. 8C illustrates the Northern blot analysis with the CaCLA4 fragment as a probe. PCR with the divergent oligodeoxynucleotides OEL109 and OELllO was used to delete the coding sequence of CaCLA4. A hisG-
URA3-hisG cassette was then inserted, and homologous recombination was used in a two-step procedure to replace both CaCLA4 alleles. Fig. 9 illustrates virulence assays. Survival curves of mice (n=15 for each C. albicans strain) infected with 1 x 10^ cells of C. albicans strains
SC5314 (wild-type), CDH77 ( CaCLA4/cacla4Δ ) , CLJ1
{ cacla4Δ/cacla4Δ ) and CLJ5 { CaCla4Δ/cacla4Δ ) trans- formed with the control plasmid pVEC and plasmid pVEC-
CaCLA4 carrying the CaCLA4 gene.
Fig. 10 illustrates the staining of mouse kidney sections with periodic acid Schiff ' s stain 48 h after infection with C. albicans strains SC5314 and CLJ1. Fig. 11 illustrates the nucleotide (SEQ ID NO: 9) and predicted amino acid (SEQ ID NO: 10) sequences of CaCdc42p.
Figs. 12A to 12B illustrate the nucleotide (SEQ ID NO: 11) and predicted amino acid (SEQ ID NO: 12) sequences of CaBemlp.
DETAILED DESCRIPTION OF THE INVENTION
The CST20 gene of Candida albi cans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65-PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid "Spider" media. However, hyphal development was not impaired in some other media. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans .
The CaCLA4 gene of C. albicans was cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the STE20 and CLA4 genes. CaCLA4 encodes a homolog of the Ste20p family of ser- ine/threonine protein kinases with pleckstrin homology and Cdc42p binding domains in the amino-terminal non- catalytic region. Deletion of both alleles of CaCLA4 in C. albicans caused defects in hyphal formation in vi tro in synthetic liquid and solid media, and in vivo in a mouse model for systemic candidiasis. The deletions reduced the invasion of C. albicans cells into kidneys after infection into mice and completely suppressed virulence in the mouse model. Thus, hyphal formation of C. albicans mediated by the CaCla4p protein kinase may contribute to the pathogenicity of this dimorphic fungus.
The CaBEMl and CaCDC42 genes of C. albi cans were cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM1 and CDC42 genes, respectively. CaBEMl encodes an SH3 domain protein with homology to Bemlp, and CaCDC42 encodes a small G-protein with homology to members of the Rho-family of G-proteins. MATERIALS AND METHODS Yeast manipulations
The yeast form of C. albicans was cultured at 30°C in YPD medium. Hyphal growth was induced at 37 °C on solid "Spider" media (Liu, H. et al. (1994) Science 266, 1723-1726) containing 1% (w/v) nutrient broth, 0.2% (w/v) K2HP04, 2% (w/v) agar and 1% (w/v) of the indicated sugars (pH 7.2 after autoclaving) . Cells were grown in liquid "Spider" media at 30 °C to stationary phase, and then incubated for 5 days at 37 °C on solid "Spider" media at a density of about 200 cells per 80 mm plates. All media were supplemented with uridine (25 μg/ml) for the growth of Ura" strains. Germ tube formation was induced at 37 °C in either 10% fetal bovine serum (GIBCO/BRL) on liquid "Spider" media containing the indicated sugars at an inoculation density of 10^ cells per ml.
Yeast manipulations were performed according to standard procedures. Isolation of CST20
The CST20 gene was isolated from a genomic C. albicans library constructed in plasmid YEp352 from genomic DNA of the clinical isolate W01 (Boone, C. et al. (1991) J. Bacteriol . 173, 6859-6864). A plasmid carrying an amino-terminally truncated version of CST20 missing the first 918 nucleotides of coding sequence was isolated by screening for suppressors of defects in basal FUSl : :HIS3 expression and mating in S. cerevisiae strain YEL64 which was disrupted in STE20. A fragment from nucleotides 958 to 1,252 of CST20 was amplified by the polymerase chain reaction (PCR) and used as a probe to isolate a full length clone by colony hybridization to the C. albicans genomic library transformed into E. coli strain MC1061. Both DNA strands were sequenced by the dideoxy chain termination method. The full length clone was subcloned between the SacI and Hindlll sites of the S. cerevisiae centromere plasmid pRS316 to yield plasmid pRL53. Isolation of CaCLA4
The S. cerevisiae MATα strain YEL257-1A-2 deleted for STE20 and CLA4 and carrying plasmid pDH129 with CLA4 under control of the GAL1 promoter was transformed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al . (1991) J. Bacte- riol . 173, 6859-6864). Transformants were grown on selective medium in 4% galactose and then replica- plated to selective medium containing 2% glucose to select for plasmids that were able to support growth in the absence of Cla4p and Ste20p. By screening 1,600 transformants, we isolated plasmid YEp352-CaCLA4 carrying an insert of 5.6 kb with an open reading frame of 2,913 bp capable of encoding a homolog of Cla4p. Sub- cloning indicated that this open reading frame was responsible for complementation. Both DNA strands were sequenced by the dideoxy chain termination method. Molecular cloning of CaCDC42
The S . cerevisiae MATα strain DJTD2-16A carrying the cdc42-l ts mutation was transformed with the genomic C. albicans library constructed in the S. cerevisiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al. (1991) J. Bacteriol . 173, 6859-6864). Transformants were grown on selective medium at room temperature. Colonies were then replica-plated to selective medium and grown at 34 °C. By screening 2,000 transformants, we isolated plasmid YEp352-CaCDC42 carrying an open reading frame of 573 bp capable of encoding a homolog of Cdc42p. Both DNA strands were sequenced by the dideoxy chain termination method. Sub- cloning of various restriction endonuclease fragments indicated that the open reading frame was responsible for complementation of the temperature-sensitive growth defect caused by the cdc42-2ts mutation. Molecular cloning of CaBEMl
The S. cerevisiae MATα strain YEL220-1A deleted for BEM1 and carrying plasmid pGAL-BEMl with BEM1 under control of the GAL1 promoter was transformed with the genomic C. albicans library constructed in the S. cere- visiae vector YEp352 carrying URA3 as selectable marker (Boone, C. et al. (1991) J. Bacteriol . 173, 6859-6864). Transformants were grown on selective medium in 4% galactose and then replica-plated to selective medium containing 2% glucose to select for plasmids that were capable of supporting growth of Bemlp-depleted cells. We isolated plasmid YEp352-CaBEMl carrying an open reading frame of 1,905 bp fulfilling this criterion and capable of encoding a homolog of Bemlp. Both DNA strands were sequenced by the dideoxy chain termination method, and subcloning of various restriction endonuclease fragments indicated that this open reading frame was responsible for complementation. Construction of C. albicans strains and plasmids
To construct a CST20 null mutant, an EcoRI to Sad fragment from nucleotide positions 989 to 4,134 of CST20 was subcloned into the Bluescript KS(+) vector (Stratagene) to yield plasmid pDH119. A plasmid that contained CST20- lanking sequences from nucleotides 989 to 1,674, and 3,423 to 4,134 joined with BamHI sites, was then created by PCR using the divergent oligodeoxynucleotide primers 0DH68 (51- CGGGATCCAGACCAACCACTCGAACTACT-3' (SEQ ID N0:1) and ODH69 ( 5 ' -CGGGATCCGAAGGTGAACCACCATATTTG-3 ' ( SEQ ID N0:2); newly introduced BamHI sites are underlined) and plasmid pDH119 as a template. The amplified DNA was cleaved with BamHI and ligated with a 4 kb BamHI to BgJlI fragment of a hisG-URA3-hisG cassette derived from plasmid pCUB-6 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to yield plasmid pDH183. This plasmid was linearized with Xhol and Sad and transformed into the Ura" C. albicans strain CAI4 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to partially replace the coding region of one of the chromosomal CST20 alleles with the hisG-URA3-hisG cassette by homologous recombination. Ura+ transformants were selected on Ura- medium, and integration of the cassette into the CST20 locus was verified by Southern blot analysis. Spontaneous Ura" derivatives of two of the heterozygous disruptants were selected on medium containing 5-fluoroorotic acid. These clones were screened by Southern blot hybridization to identify those which had lost the URA3 gene by intrachromosomal recombination mediated by the hisG repeats. This procedure was then repeated to delete the remaining func- tional allele of CST20.
A similar procedure was employed to delete the CaCST20 gene. A 4.6 kb Xbal fragment of YEp352-CaCLA4 was subcloned into the pBluescript KS(+) vector (Stratagene) to yield plasmid pDH205. A plasmid that contained CaCLA4 flanking sequences joined with Bglll sites was then created by PCR using the divergent oligodeoxynucleotide primers OEL109 (51- GAAGATCTTGTAATCAATGTTCCCGTGGA-3' (SEQ ID NO : 3 ) and OELllO ( 5 ' -GAAGATCTCATCGTGATATTAAATCCGAT-3 ' ( SEQ ID NO:4); newly introduced Bglll sites are underlined) and plasmid pDH205 as template. The amplified DNA was cleaved with Bglll and ligated with a 4 kb BamHI-Bglll fragment of a hisG-URA3-hisG cassette derived from plasmid pCUB-6 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to yield plasmid pDH210. This plasmid was linearized with PstI and SacI and transformed into the Ura" C. albicans strain CAI4 (Fonzi, W. A. & Irwin, M. Y. (1993) Genetics 134, 717-728) to replace the coding region of one of the chromosomal CaCLA4 alleles with the hisG-URA3-hisG cassette by homologous recombination. Ura+ transformants were selected on Ura- medium, and integration of the cassette into the CaCLA4 locus was verified by Southern blot analysis. Spontaneous Ura- derivatives were then selected on medium containing 5-fluoroorotic acid. These clones were screened by Southern blot hybridization to identify those which had lost the URA3 gene by intrachromosomal recombination mediated by the hisG repeats . This procedure was then repeated to delete the remaining functional allele of CaCLA4.
To reintegrate CST20 into the genome of mutant strains, the C. albicans integration plasmid pDH190 was constructed by subcloning a Kpnl to PstI fragment of CST20 into pBS-cC7RA3 (pBluescript KS( + ) into which the C. albicans URA3 gene was cloned between the NotI and Xbal sites of the polylinker). The integration plasmid was then linearized with Nsil and transformed into C. albicans to target integration into the Nsil site of the CST20Δ: :hisG fusion gene. Integrations were selected on Ura- medium and confirmed by Southern blot analysis.
The C. albicans CST20 expression plasmid pDH188 was constructed by subcloning a SacI to PstI fragment of CST20 into plasmid pVEC carrying a C. albicans autonomously replicating sequence and URA3 as selectable marker. The C. albicans plasmid pVEC-CaCLA4 was constructed by subcloning the Kpnl to SacI insert of YEp 352-CaCLA4 into plasmid pVEC. Northern blot analyses
Northern blots of total and poly (A)+ RNA from C. albicans cells were performed as described (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824). Signals were quantified by 2-D radioimaging. Animal experiments
Eight week-old, male CFW-1 mice (Halan-Winkel- mann, Paderborn, Germany) were inoculated with 1 x 10^ or 1 x 10^ cells by intravenous injection. Survival curves were calculated according to the Kaplan-Meier method using the PRISM™ program (GraphPad Software Inc., San Diego) and compared using the log-rank test. A P value <0.05 was considered significant.
To quantify colony-forming C. albicans units in kidneys, mice were sacrificed by cervical dislocation 48 hours after injection and kidneys were homogenized in 5 ml phosphate buffered saline, serially diluted and plated on YNG medium (0.67% yeast nitrogen base, 1% glucose, pH 7.0). Histological examination of kidney sections was done with periodic acid Schiff * s stain.
RESULTS
Isolation and characterization of CST20
A C. albicans homolog of the S. cerevisiae STE20 gene was cloned by functional complementation of the pheromone signaling defect of S. cerevisiae cells that were deleted for the STE20 gene. The mating defect of the STE20 deleted S. cerevisiae strain YEL20 was fully complemented by introduction of the centromeric plasmid pRL53 carrying full length CST20 (mating efficiency was
81±9% in cells expressing CST20, compared with 85±8% in cells expressing STE20; n=3). Similarly, defects in growth arrest and morphological changes in response to pheromone were completely cured by transformation with the CST20 plasmid. As shown in Fig. 1, nitrogen deficiency-induced pseudohyphae formation, which is blocked by disruption of STE20 in diploid cells (Liu, H., Styles, C. & Fink, G. R. (1993) Science 262, 1741-1744), was restored by introduction of the CST20 plasmid. Colonies of the diploid STE20 wild type strain L5266 (4) (Fig. IA) and the isogenic ste20/ste20 strain HLY492 (4) transformed with either the control plasmid pRS316 (Fig. IB), the CST20 plasmid pRL53 (Fig. IC), or the STE20 plasmid pSTE20-5 (9) (Fig. ID) were grown on nitrogen starvation medium (2) for 5 days at 30°C. Photomicrographs were taken with a 4x objective (bar=lmm) .
As illustrated in Fig. 2, the cytokinesis defect caused by deletion of CLA4, encoding an S. cerevisiae isoform of Ste20p (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830), was not complemented by CST20 (Fig. 2). However, the lethality caused by deletion of both STE20 and CLA4 { Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830), could be rescued by CST20 (Fig. 2). The diploid strain YEL306 heterozygous for ste20Δ : : TRP1 /STE20 cla4Δ : : LEU2/CLA4 was transformed with plasmid pRS316 carrying either no insert, CLA4 (pRL21), CST20 (pRL53) or STE20 (pSTE20-5), and then sporulated and dissected. No viable haploid ste20Δ cla4Δ spores were obtained from transformants with the plasmid without insert, but were obtained from transformants with plasmids carrying CLA4 (Fig. 2A), STE20 (Fig. 2B) or CST20 (Fig. 2C).
Cells were grown to mid-exponential phase in YPD medium at 30 °C. No viable ste20Δ cla4Δ segregants were obtained in medium containing 5-fluoro-orotic acid suggesting that the plasmids were essential for viability. Neither STE20 nor CST20 were able to suppress the morphological defect of cla4Δ cells. Photomicrographs were taken by phase contrast with a 40x objective (bar=30 μm) .
The open reading frame of CST20 is capable of encoding a protein of 1,229 amino acids with a pre- dieted molecular weight of 133 kDa and a domain structure characteristic of the Ste20p/p65-PAK family of protein kinases (Fig. 3). Numerals at the left margin indicate nucleotide and amino acid positions (Fig. 3). Nucleotide 1 corresponds to the first nucleotide of the initiation codon and amino acid 1 to the first residue of the deduced protein. The putative p21 binding domain has been shadowed, and the kinase domain has been boxed.
The catalytic domain present in the carboxyl terminal half of the protein has sequence identities of 76 and 56%, respectively, with S. cerevisiae Ste20p (Leberer, E. et al. (1992) EMBO J. 11, 4815-4824) and Cla4p (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817- 1830). The amino terminal, non-catalytic region con- tains a sequence from amino acid residues 473 to 531 with 68% identity to the p21 binding domain of Ste20p that has been shown to bind the small GTPase Cdc42p. This region contains the sequence motif ISxPxxxxHxxH thought to be important for the interaction of the p21 binding domain with the GTP-bound forms of Cdc42Hs and Racl (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817- 1830). The remaining non-catalytic sequences are less conserved. Unique sequences not present in Ste20p and the other members of the family are found at the amino terminus and between the p21 binding and catalytic domains .
A CST20 transcript of 4.9 kb in size was detected in Northern blots. This transcript was present at similar levels in yeast cells grown in YPD at room temperature and germ tubes induced by a temperature shift to 37°C. Isolation and characterization of CaCLA4
A C. albicans homolog of the S. cerevisiae CLA4 gene was cloned by functional complementation of the growth defect of S. cerevisiae cells that were deleted for the STE20 and CLA4 genes.
The open reading frame of the CaCLA4 gene is capable of encoding a protein of 971 amino acids with a predicted molecular weight of 107 kDa and a domain structure characteristic of the Ste20p family of protein kinases (Fig. 7). The catalytic domain present in the carboxyl terminal half of the protein has sequence identities of 74, 63 and 64%, respectively, with S. cerevisiae Cla4p, S. cerevisiae Ste20p and an uncharac- terized open reading frame present in the S. cerevisiae genome, 65% with the C. albicans Ste20p homolog Cst20p, and 61% with rat pδδ-^ (Fig. 7). The amino terminal, noncatalytic region contains a sequence from amino acid residues 69 to 180 with similarity to pleckstrin homology (PH) domains and a sequence from amino acid residues 229 to 292 with 63% identity to the Cdc42p binding domain of S. cerevisiae Cla4p that has been shown to bind the small GTPase Cdc42p (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830). The remaining noncatalytic sequences are less conserved. Chromosomal deletion of CST20
Homologous recombination was used in a multistep procedure to partially delete CST20 in a URA~ C. albi - cans strain (Fig. 4A) . PCR with the divergent oligodeoxynucleotides ODH68 and ODH69 was used to partially delete the coding sequence of CST20. A hisG-URA3-hisG cassette was then inserted. The deletion was confirmed by Southern blot analyses (Fig. 4B). The genomic DNA samples digested with Xhol were from following strains: Lane #1, CAI4 ( ura3/ura3 CST20/CST20 ) ; lane 2, CDH15 { ura3/ura3 CST20/cst20Δ : :hisG-URA3-hisG) ; lane 3, CDH18 ( ura3/ura3 CST20/cst20Δ : :hisG) ; lane 4, CDH22 ( ura3/ura3 cst20Δ : :hisG-URA3-hisG/cst20Δ : :hisG) ; lane 5, CDH25 { ura3/ura3 cst20Δ : :hisG/cst20Δ : :hisG) . Northern blots showed that the CST20 transcript was absent in the corresponding homozygous deletion strains.
The lateral outgrowth of hyphae from colonies grown on solid "Spider" media containing mannitol or sorbitol was completely blocked by deletion of CST20 (Fig. 5B).
Mycelial formation was drastically reduced when the media contained galactose, mannose or raffinose. The mutant strains regained the ability to form hyphae when wild type CST20 was reintroduced by transformation with the CST20 expression plasmid pDH188 or reintegrated into the genome by targeted homologous recombination (Fig. 5C). The CST20 transcript was detected in these strains by Northern blot analysis. Mutant strains formed hyphae when colonies were grown on "Spider" media containing either glucose or N- acetyl glucosamine. Normal hyphae formation was also observed on rice agar and on agar containing Lee ' s medium or 10% serum. The frequency of germ-tube for a- tion in either liquid Lee's medium, 10% serum or liquid "Spider" media containing any of the sugars tested above, were also normal. These results indicate that Cst20p is not required for hyphae formation under all conditions but are involved in the lateral formation of mycelia on some solid surfaces. Chromosomal deletion of CaCLA4
Homologous recombination was used in a multistep procedure to delete both alleles of CaCLA4 in C. albi cans (Fig. 8A). Fig. 8A shows the restriction endonu- clease map of CaCLA4. The coding sequence is indicated by the arrow. PCR with the divergent oligodeoxynucleotides OEL109 and OELllO was used to delete the coding sequence of CaCLA4. A hisG-URA3-hisG cassette was then inserted and a two-step procedure was used to delete both alleles of CaCLA4 by homologous recombination. The endonuclease restriction sites are as follows: B, BamHI; Bg, Bglll; E, EcdRl ; H, HindiII; P, PstI; S, Sad ; X, Xbal . The deletions were confirmed by Southern blot analyses (Fig. 8B). Southern blot analysis with a 1.1 kb CaCLA4 fragment from Pstl-Xbal as a probe. The genomic DNA samples digested with EcoRI were from following strains: Lanes: 1, CAI4 ( ura3/ura3 CaCLA4/CaCLA4 ) ; 2, CDH77 { ura3/ura3 CaCLA4/cacla4Δ : :hisG-URA3-hisG) ; 3, CDH88 ( ura3/ura3 CaCLA4/cacla4Δ : :hisG) ; 4, CLJ1 { ura3/ura3 cacla4Δ : :hisG-URA3- hisG/cacla4Δ : :hisG) ; and 5, CLJ5 { ura3/ura3 cacla4Δ : :hisG/cacla4Δ : :hisG) . Northern blots showed that the CaCLA4 transcript with a size of 4.1 kb was reduced to about 40% in heterozygous CaCLA4/cacla4Δ cells and was absent in homozygous cacla4Δ/cacla4Δ deletion cells (Fig. 8C). The transcript was present at about wildtype levels when the CaCLA4 gene was retransformed into the homozygous deletion cells by using an autonomously replicating plasmid carrying the CaCLA4 gene (Fig. 8C). Northern blot analysis of poly(A)+ RNA isolated from following strains grown in the yeast form in YPD at 30°C: Lanes: 1, SC5314 (wild-type); 2, CDH88; 3, CLJ5 transformed with pVEC; 4, CLJ5 transformed with pVEC- CaCLA4. The blot was probed with fragments specific for CaCLA4 (upper panel) or CaACTl (lower panel) and quantified by radioimaging. Numbers at the bottom of the figure depict the relative amounts of CaCLA4 transcript in relation to the amounts of CaACTl transcript (mean values of two independent experiments). We found that viability of C. albicans cells was not affected by deleting either one or both alleles of CaCLA4. Mutant cells showed the same growth behavior as wild-type cells, independently whether the cells were grown under conditions favoring either the yeast or filamentous forms. However, deletion of both CaCLA4 alleles generated defects in cellular morphology producing a heterogeneous population of aberrantly shaped cells that were frequently multibudded and multinucle- ated. This phenotype indicates a defect in cytokinesis resembling the phenotype of S. cerevisiae cells deleted for CLA4 (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830) .
Deletion of both CaCLA4 alleles caused defects in hyphal formation in all media and under all conditions that we investigated. When morphological switching was induced in liquid media by either serum, N-acetyl glucosamine, proline, pH increase, temperature shift, or Lee's medium, wild-type cells and cells deleted for only one or both alleles of CaCLA4 produced germ tubes after about 30 minutes. In wild-type cells and cells deleted for only one allele of CaCLA4 , these germ tubes elongated and grew into long hyphae after prolonged incubation. Cells deleted for both alleles of CaCLA4 failed to produce hyphae, however. Instead, these cells produced multiple short protrusions giving rise to an aberrant morphology.
On solid media containing either serum, rice agar or mannitol, the normal formation of mycelia was completely suppressed by deletion of both CaCLA4 alleles. This phenotype was reversed by introducing the CaCLA4 gene on a plasmid, and deletion of only one allele had no effect. Virulence studies
To determine the role of Cst20p for virulence, mice were injected intravenously with wild type and mutant strains and monitored for survival and for fun- gal invasion into kidneys. We found that the Ura- strain CAI4 was not pathogenic (Figs. 6A and B). However, infection with Ura+ wild type cells resulted in rapid mortality with a rate that was dependent on the dose of injected cells (1 x 10^ cells in Fig. 6A, and 1 x 10^ cells in Fig. 6B). Survival was significantly prolonged, however, in mice infected with Ura+ cells deleted for both alleles of CST20 ( cst20Δ/cst20Δ : : URA3 ) . This effect, which was reproducible and statistically significant, was observed at high (Fig. 6A) or low (Fig. 6B) doses of infection (with P values of 0.027 and 0.001, respectively) and correlated with colony-forming units per kidney (1.5 x 10^ for wild type cells and 7 x 105 for cst20Δ/cst20Δ : : URA3 mutant cells) after 48 hours of infection with 1 x 10^ cells. These effects on virulence could be reversed by reintroducing CST20 into the strain deleted for both CST20 alleles, and were not observed in Ura+ cells deleted for only one CST20 allele. A histological examination revealed that cells deleted for both alleles of CST20, were able to form hyphae in infected kidneys (Fig. 6C).
To investigate whether CaCla4p is required for virulence, mice were injected intravenously with wildtype and mutant C. albicans strains and monitored for survival and for fungal invasion into kidneys. Infec- tions with CaCLA4 wild-type cells (strain SC5314) resulted in rapid mortality (Fig. 9). No difference in the mortality rate was observed after infection with cells deleted for only one allele of CaCLA4 (strain CDH77). All mice survived, however, after infection with cells deleted for both alleles of CaCLA4 (strain CLJ1 and CLJδpVECl). This effect correlated with a reduction in the amount of colony-forming units per kidney of infected animals and was reversed by transformation of the cells with a plasmid carrying the CaCLA4 gene (strain CLJ5CaCLA4) (Fig. 9). A histologi- cal examination revealed that kidneys from mice injected with either wild-type cells or cells deleted for one allele of CaCLA4 were heavily infected with C. albicans cells that produced hyphae densely penetrating the animal tissue (Fig. 10, left panel), whereas kidneys from mice injected with cells deleted for both CaCLA4 alleles contained small foci of aberrantly shaped cells that frequently carried multiple protrusions (Fig. 10, right panel). The morphologies of these cells were similar to those induced by serum under in vi tro conditions. Thus, the function of CaCla4p is required for morphological switching of C. albicans under in vi tro and in vivo conditions and for virulence. Molecular cloning of the CaCDC42 and CaBEMl genes
A C. albicans homolog of the CaCDC42 gene was cloned by functional complementation of the temperature-sensitive growth defect of S. cerevisiae cells carrying the cdc42-l ^s mutation. The growth defect was fully complemented by plasmid YEp352-CaCDC42. The open reading frame of the CaCDC42 gene is capable of encoding a protein of 191 amino acids with homology to the Rho-family of small G-proteins (Fig. 11). The highest homology is found with Cdc42p from S. cerevisiae . A C. albicans homolog of the CaBEMl gene was cloned by functional complementation of the growth defect of S. cerevisiae cells deleted for the BEM1 gene. This defect was fully complemented by plasmid YEp352-CaBEMl carrying the CaBEMl gene. The open read- ing frame of the CaBEMl gene is capable of encoding a protein of 635 amino acids with a domain structure characteristic of Bemlp (Fig. 12). CaBemlp contains two conserved SH3 domains which are most homologous to the SH3 domains of Bemlp, and also has homology to Bemlp outside of the SH3 domains. Discussion
In S. cerevisiae, Ste20p fulfills multiple functions during mating (Leberer, E. et al . (1992) EMBO J. 11, 4815-4824), pseudohyphae formation (Liu, H., Styles, C. & Fink, G. R. (1993) Science 262, 1741- 1744), invasive growth (Roberts, R. L. & Fink, G. R. (1994) Genes Dev. 8, 2974-2985) and cytokinesis (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830). CST20 expression in S. cerevisiae fully complements these functions. Thus, Cst20p has the potential to fulfill similar functions in C. albicans .
The yeast-to-hyphal transition of C. albicans is a morphological change that can be triggered by a wide variety of factors. Carbohydrates, amino acids, salts, and serum have been described as inducers of germ tube formation, as have pH changes, temperature increases and starvation, but no single environmental factor could be defined as uniquely significant in stimulating the morphological switch. Hence C. albicans appears capable of responding to many divergent environmental signals. Disruption of both CPH1 alleles, which encode a homolog of the S. cerevisiae Stel2p transcription factor (Liu, H. et al . (1994) Science 266, 1723-1726), suppressed the lateral formation of mycelia from colo- nies grown on solid "Spider" medium, but did not block hyphal development in other media. We have shown that C. albicans mutant cells deleted for CST20 display a similar phenotype, and that the effect of these mutations on hyphal development is dependent on the carbon source in which the cells were grown. These observations are consistent with the idea that several signaling pathways can trigger morphogenesis in C. albicans . Furthermore, the behavior of C. albicans mutant strains deleted for either CPH1 or CST20 indicates that these pathways might operate independently to activate hyphal development under differing environmental conditions. C. albicans encounters a variety of different microenvironments during the development of superficial and systemic infections. Hence, the existence of parallel morphogenetic signaling pathways might provide a distinct advantage to this pathogen.
Our results indicate that the pathway controlled by Cst20p is not essential for virulence in a mouse model of systemic infections. It is not inconceivable that this pathway plays a role in other forms of infections, for example in the development of superficial infections of the mucosal epithelia (thrush). An as yet undefined role of Cst20p in pathogenicity outside of the Cst20 signaling pathway is suggested, however, by prolonged survival of mice infected with cst20 deleted cells. It is unlikely that this effect is caused by defects in hyphal formation since a his- tological examination of infected kidneys revealed that the CST20 deleted cells are not restricted in their capacity to form hyphae.
In S. cerevisiae, Cla4p plays a role in cytokinesis and shares with Ste20p an essential function for polarized growth during budding (Cvrckova, F. et al. (1995) Genes Dev. 9, 1817-1830). Cla4p binds the Rho- like small G-protein Cdc42p (Cvrckova, F. et al . (1995) Genes Dev. 9, 1817-1830) which is involved in controlling cell polarity during budding and in response to pheromone. Like Ste20p and the mammalian homolog p21- activated kinase p65PAK) , Cla4p is able to phosphory- late and activate myosin-I, a mechanism that may contribute to the organization of the actin cytoskeleton.
Our finding that CaCLA4 expression in S. cerevisiae completely complements the Cla4p functions sug- gests that CaCla4p may have similar properties in C. albicans . Thus, CaCla4p may be required for myosin-I driven polarized growth during hyphal formation in a mechanism that may involve the C. albicans homolog of Cdc42p. Our complementation assays in S. cerevisiae suggest that CaCla4p may share an essential function with Cst20p, the C. albicans homolog of Ste20p (Figs. 6A and 6B) . This notion suggests, together with our findings that null mutants of CaCLA4 are completely non-pathogenic (Fig. 10) and null mutants of CST20 are reduced in virulence (Figs. 6A and 6B), that CaCla4p and Cst20p, and proteins such as CaCdc42p and CaBemlp interacting with these protein kinases, may be valid targets for the development of antifungal agents.
The present invention will be more readily un- derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I Screening test for inhibitors of CaCla4p and Cst20p An in vi tro assay containing the proteins
CaCla4p and/or Cst20p will be used to test compounds inhibiting their activity to render avirulent any fungi, which may be pathogenic.
The activity of the protein will be monitored to determine if the compounds tested do inhibit their biological activity, using myelin basic protein as a substrate.
In cases were a selective inhibition of CaCla4p and Cst20p and not to pδδ-^ would be desired, com- pounds testing positive for the inhibition of both CaCla4p and Cst20p will be tested to determine if they also inhibit the protein pδδ-^- . This would be useful in cases of pathogenic fungi infection such as for C. albicans were the fungi is to be rendered avirulent without affecting the normal protein of the patient p65^K.
In some cases of inflammation, it would be desirable to be provided with compounds inhibiting all three proteins, namely, CaCla4p, Cst20p and pδS-P^.
EXAMPLE II
Screening test for inhibitors of CaCla4p and CaCdc42p interactions An in vi tro assay containing the proteins
CaCla4p and CaCdc42p will be used to test compounds inhibiting their interactions.
CaCla4p may be solid phase bound and CaCdc42p will be in suspension free to interact with CaCla4p. A labeled antibody specific to CaCdc42p will be added to the assay to determine the presence of CaCdc42p bound to CaCla4p. The compounds tested to inhibit the CaCdc42p-CaCla4p interactions, should when tested positive, cause only a minute quantity of CaCdc42p to bind to CaCla4p interactions.
The analogous in vi tro assay will be used to test compounds that inhibit the interaction between Cst20p and CaCdc42p.
EXAMPLE III
Screening test for inhibitors of CaCla4p and CaBemlp interactions
An in vi tro assay containing the proteins CaCla4p and CaBemlp will be used to test compounds inhibiting their interactions.
CaCla4p may be solid phase bound and CaBemlp will be in suspension free to interact with CaCla4p. A labeled antibody specific to CaBemlp will be added to the assay to determine the presence of CaBemlp bound to CaCla4p. The compounds tested to inhibit the CaBemlp- CaCla4p interactions, should when tested positive, cause only a minute quantity of CaBemlp to bind to CaCla4p interactions.
The analogous in vi tro assay will be used to test compounds that inhibit the interaction between Cst20p and CaBemlp.
EXAMPLE IV
A two-hybrid CaCdc42p and CaCla4p interaction system in a humanized S. cerevisiae strain This screening assay is based on the assumption that the interaction of the small G-protein CaCdc42p with its cellular targets Cst20p and CaCla4p is essential for viability of C albicans cells. This essential function is reasonable to assume based on work that has been performed in S. cerevisiae (Leberer E. et al. (1997) Embo J. 16, 83-97). The two hybrid interaction system will use green fluorescent protein fused to the GAL1 promoter as a functional read out. This reporter gene will be integrated into a S. cerevisiae strain in which the STE20 and CLA4 genes have been replaced by the human homolog p65PAK. The CaCDC42 gene will be fused to the DNA binding domain of GAL4 , and the CaCLA4 gene will be fused to the activation domain of GAL4. Interaction of the two proteins will cause green fluo- rescence. Whereas inhibitors of the interaction will suppress fluorescence.
Non-specific inhibitors of the two-hybrid interaction system will be excluded by performing a parallel screen with unrelated fusion proteins known to inter- act. Compounds of general toxicity or inhibitors of the human homologs will also be excluded in this system because those compounds will not allow growth of the cells and therefore reduce the fluorescent readout in both parallel screens. A two-hybrid yeast strain carrying the GAL4-GFP fusion gene is constructed. This strain will be deleted for the CLA4 gene using the TRP1 marker as described (Leberer E. et al . (1997) Embo J. 16, 83-97). The STE20 gene will be replaced by the human PAK gene as described above. To replace the CDC42 gene by its human homolog, an integrating plasmid will be constructed carrying the HsCDC42 gene fused to a URA3 blaster gene and CDC42 flanking sequences. After linearization, the construct will be transformed into the PAK containing two-hybrid strain, and integrants will be selected on -ura medium. The URA3 gene will then be looped out on FOA medium. The various gene disruptions and gene replacements will be verified by Southern blot analyses . The two-hybrid vectors carrying the CaCDC42 gene fused to the GAL4-DNA binding domain and the CaCLA4 gene fused to the transcriptional activation domain of GAL4 will be constructed by standard procedures. To facilitate the interaction of the two proteins, we will use site-directed mutagenesis to create a mutation in the CAAX-box domain of CaCDC42p to prevent isopren- ylation and targeting of the fusion protein to the plasma membrane. We will evaluate and optimize the assay system and adapt the assay conditions to the scale used in microtiter plates for automated screening of compounds . EXAMPLE V
Detection of the presence of C. albicans using probes
The sequences of either one of the genes CaCLA4, CST20, CaCDC42 and CaBEMl may be used to derive probes for the detection of C. albicans using PCR techniques or hybridization assays.
EXAMPLE VI
Use of nucleotide sequences of CaCLA4, CST20, CaCDC42 and CaBEMl to identify homologue from other fungi
The nucleotide sequences of CaCLA4 , CST20,
CaCDC42 and CaBEMl may be used to identify and clone homologues from other fungi.
EXAMPLE VII
A S. cerevisiae-based screening system using CaSte20p and the pheromone signaling pathway as drug target
In this system, we will use green fluorescent protein (GFP) under transcriptional control of a pheromone inducible promoter { FUSl ) as a read out. The pheromone signaling pathway and thereby the reporter gene will be induced with pheromone in two different strains. First, in a strain in which STE20 is functionally replaced by the CaSTE20 gene. And second, in a strain in which STE20 is functionally replaced by the mammalian homolog PAK. Compounds that block the induc- tion of the reporter gene in the CaSTE20 strain but not in the PAK strain are expected to be specific inhibitors of the C. albicans kinase. This assay is very specific and is a positive selection of compounds that excludes the finding of compounds with inhibitory action against the mammalian homolog PAK or compounds of general toxicity.
The FUSl gene, including its promoter, will be isolated by the polymerase chain reaction (PCR) from genomic DNA of S. cerevisiae and fused to the GFP gene from Aequoria victoria on a yeast expression plasmid. The function of the reporter gene will be analyzed after transformation of a MATa yeast strain and induc- tion with pheromone.
The STE20 gene will be replaced in a supersensi- tive sstl yeast strain by the human PAK gene using homologous recombination. For this purpose, an integrating plasmid will be constructed carrying the PAK gene fused to a URA3 blaster gene and STE20 flanking sequences. The construct will be linearized and transformed into yeast, and integrants will be selected on - ura medium. The URA3 gene will then be looped out on FOA medium to gain back the ura3 marker. Correct inte- gration of the PAK gene will be confirmed by Southern blot analysis.
The humanized strain will then be transformed with the FUS1-GFP reporter gene and analyzed for a functional signaling pathway by measuring green fluo- rescence after induction with pheromone. The assay system will be evaluated, optimized and adapted to the scale used in microtier plates.
EXAMPLE VIII
Fluorescence resonance energy transfer (FRET) as probe for protein-protein interactions
The engineering of different GFP mutants with altered fluorescence characteristics allows the use of fluorescence resonance energy transfer (FRET) to probe protein-protein interactions (Heim and Tsien (1996) Curr. Biol. 6, 178-182). The FRET phenomenon consists in a fluorescence transfer between a donor and a receptor fluorochrome. If excitation and emission wave- lengths are compatible, the FRET is easily measurable. The main parameter of the reaction is the distance between donor and receptor, which must be in the range of nanometers. This is precisely the kind of values in protein-protein interactions.
We propose to develop a novel yeast assay system which uses FRET to measure the in vivo interaction between CaCdc42p and Cacla4p. The CaCDC42 gene will be fused to a GFP mutant that acts as donor, and the CaCLA4 gene will be fused to a mutant that acts as receptor. The yeast strain used as an expression sys- tem will be humanized as described in Example VII. Inhibitors of the interaction are expected to reduce energy transfer, and this reduction can be easily measured spectroscopically . The interaction of unrelated proteins known to interact will be used as a reference to exclude non-specific inhibitors of the assay system. Compounds inhibiting the interaction of the human homologs or of general toxicity will be excluded by inhibition of growth and therefore reduced fluorescence in both screens . The CaCDC42 gene will be fused to the gene encoding the GFPY66H mutant as donor, and the CaCLA4 gene will be fused to the gene encoding the GFP3657 mutant as receptor (Heim and Tsien (1996), Curr. Biol. 6, 178-182). The constructs will then be transformed into the humanized yeast strain described in Example VII, and the FRET phenomenon will be analyzed in yeast cultures using fluorescence spectroscopy. The conditions for the assay will be worked out and optimized. We will adapt the assay conditions to the scale used in microtiter plates for automated screening.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia- tions, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: National Research Council of Canada
(ii) TITLE OF THE INVENTION: CANDIDA ALBICANS PROTEINS
ASSOCIATED WITH VIRULENCE AND HYPHAL FORMATION AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 12
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SWABEY OGILVY RENAULT
(B) STREET: 1981 McGill College Ave . - Suite 1600
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(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/029,458
(B) FILING DATE: 30-OCT-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Cδte, France
(B) REGISTRATION NUMBER: 4166
(C) REFERENCE/DOCKET NUMBER: 2139-10PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 514 845-7126
(B) TELEFAX: 514-288-8389
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CGGGATCCAG ACCAACCACT CGAACTACT 29
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CGGGATCCGA AGGTGAACCA CCATATTTG 29
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GAAGATCTTG TAATCAATGT TCCCGTGGA 29
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GAAGATCTCA TCGTGATATT AAATCCGAT 29
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4492 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic RNA (ix) FEATURE :
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 355...4044 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GTACCCACTT TACAATCACT TACAAGTCAA ATAATTACAA CTTGACAATC CTCACTTTAA 60 GTCTAACGTA TATACGCGTA CACCATCTTA TACTCCACAT ACATATTGGA TTCAATTTTT 120 ATTTTATTGT TTAGTTTATA TCCAACCACT GACAATTACC AATAGTTTTC AATTAATATT 180 CACAATTTAA CTATTTGTTT GACAGCTGAA AAGAGATAAA AAAAGAATCA AGTGCTATAA 240 CTCACAAGGG CTAGAAATAA GTTTGCAAAA AACAAGTTTT AAAAATAGTA ACTGCACTTT 300 TGTTGACTCT TTCACCTCCC CATTGAATTT AACTGAACAC AAATAAAGCC TATC ATG 357
Met 1 AGC ATA CTT TCA GAG AAC AAT CCT ACA CCA ACA TCA ATA ACA GAT CCA 405 Ser lie Leu Ser Glu Asn Asn Pro Thr Pro Thr Ser lie Thr Asp Pro
5 10 15
AAT GAG TCT TCT CAT CTA CAC AAC CCA GAG TTA AAC TCT GGA ACG AGG 453 Asn Glu Ser Ser His Leu His Asn Pro Glu Leu Asn Ser Gly Thr Arg 20 25 30
GTT GCT TCT GGA CCT GGA CCT GGA CCT GAA GTT GAA TCA ACA CCA CTA 501 Val Ala Ser Gly Pro Gly Pro Gly Pro Glu Val Glu Ser Thr Pro Leu 35 40 45
GCA CCC CCA ACT GAG GTC ATG AAT ACT ACA TCA GCT AAT ACT TCT TCA 549 Ala Pro Pro Thr Glu Val Met Asn Thr Thr Ser Ala Asn Thr Ser Ser 50 55 60 65
TTA AGT TTA GGG TCT CCA ATG CAC GAG AAA ATA AAA CAA TTT GAT CAA 597 Leu Ser Leu Gly Ser Pro Met His Glu Lys lie Lys Gin Phe Asp Gin 70 75 80
GAC GAG GTT GAC ACT GGG GAA ACT AAT GAT AGG ACT ATA GAA TCT GGA 645 Asp Glu Val Asp Thr Gly Glu Thr Asn Asp Arg Thr lie Glu Ser Gly 85 90 95
TCT AGT GAT ATT GAT GAT TCA CAA CAA TCA CAT AAC AAC AAC AAC AAC 693 Ser Ser Asp lie Asp Asp Ser Gin Gin Ser His Asn Asn Asn Asn Asn 100 105 110
AAC AAC AAC AAC AAC AAC GAG AGC AAT CCA GAA TCA AGT GAA GGC GAT 741 Asn Asn Asn Asn Asn Asn Glu Ser Asn Pro Glu Ser Ser Glu Gly Asp 115 120 125
GAT GAA AAA ACC CAA GGA ATG CCT CCT CGA ATG CCA GGG ACA TTC AAT 789 Asp Glu Lys Thr Gin Gly Met Pro Pro Arg Met Pro Gly Thr Phe Asn 130 135 140 145
GTG AAA GGT TTG CAC CAA GGG GAT GAT AGT GAC AAT GAA AAA CAG TAC 837 Val Lys Gly Leu His Gin Gly Asp Asp Ser Asp Asn Glu Lys Gin Tyr 150 155 160
ACC GAG CTA ACT AAA TCA ATC AAT AAA CGT ACC AGT AAA GAT TCG TAT 885 Thr Glu Leu Thr Lys Ser lie Asn Lys Arg Thr Ser Lys Asp Ser Tyr 165 170 175 TCT CCT GGC ACA CTT GAA AGT CCC GGT ACT CTT AAT GCA TTG GAA ACA 933 Ser Pro Gly Thr Leu Glu Ser Pro Gly Thr Leu Asn Ala Leu Glu Thr 180 185 190
AAT AAT GTC TCA CCA GCA GTT ATA GAG GAA GAA CAA CAT ACA CTG TCT 981 Asn Asn Val Ser Pro Ala Val lie Glu Glu Glu Gin His Thr Leu Ser 195 200 205
TTG GAA GAT TTG TCA TTG TCC TTA CAA CAC CAA AAT GAA AAT GCA AGA 1029 Leu Glu Asp Leu Ser Leu Ser Leu Gin His Gin Asn Glu Asn Ala Arg 210 215 220 225
TTA TCT GCA CCC CGC AGT GCA CCG CCA CAG GTT CCG ACT TCA AAG ACA 1077 Leu Ser Ala Pro Arg Ser Ala Pro Pro Gin Val Pro Thr Ser Lys Thr 230 235 240
TCG TCA TTT CAC GAT ATG AGT CTG GTT ATA TCT TCA TCA ACT TCT GTG 1125 Ser Ser Phe His Asp Met Ser Leu Val lie Ser Ser Ser Thr Ser Val 245 250 255
CAT AAG ATA CCA TCA AAT CCA ACT TCA ACT CGA GGT TCT CAT TTA TCA 1173 His Lys lie Pro Ser Asn Pro Thr Ser Thr Arg Gly Ser His Leu Ser 260 265 270
AGT TAC AAA TCT ACA TTG GAC CCT GGG AAA CCT GCA CAA GCA GCA GCA 1221 Ser Tyr Lys Ser Thr Leu Asp Pro Gly Lys Pro Ala Gin Ala Ala Ala 275 280 285
CCA CCA CCA CCA GAA ATA GAC ATT GAC AAT TTA TTA ACC AAA AGT GAA 1269 Pro Pro Pro Pro Glu lie Asp lie Asp Asn Leu Leu Thr Lys Ser Glu 290 295 300 305
TTG GAT CTG GAA ACA GAC ACA TTG AGT AGT GCC ACA AAT TCT CCA AAC 1317 Leu Asp Leu Glu Thr Asp Thr Leu Ser Ser Ala Thr Asn Ser Pro Asn 310 315 320
CTT TTA AGA AAT GAT ACT TTA CAA GGA ATT CCA ACA AGA GAT GAC GAA 1365 Leu Leu Arg Asn Asp Thr Leu Gin Gly lie Pro Thr Arg Asp Asp Glu 325 330 335
AAT ATT GAT GAC CTG CCC CGT CAA CTA TCA CAA AAT ACT AGT GCG ACG 1413 Asn lie Asp Asp Leu Pro Arg Gin Leu Ser Gin Asn Thr Ser Ala Thr 340 345 350
TCA AGA AAT ACT TCG GGA ACA TCG ACT TCT ACA GTG GTG AAA AAT TCA 1461 Ser Arg Asn Thr Ser Gly Thr Ser Thr Ser Thr Val Val Lys Asn Ser 355 360 365
AGA TCT GGT ACG TCA AAA TCA ACC TCA ACC TCA ACT GCT CAT AAC CAA 1509 Arg Ser Gly Thr Ser Lys Ser Thr Ser Thr Ser Thr Ala His Asn Gin 370 375 380 385
ACA GCA GCA ATT ACT CCT ATA ATC CCG AGT CAC AAC AAG TTT CAT CAA 1557 Thr Ala Ala lie Thr Pro lie lie Pro Ser His Asn Lys Phe His Gin 390 395 400
CAA GTG ATA AAT ACC AAT GCA ACA AAT AGT TCA TCT TCA CTA GAA CCA 1605 Gin Val lie Asn Thr Asn Ala Thr Asn Ser Ser Ser Ser Leu Glu Pro 405 410 415 TTG GGG GTT GGC ATA AAT TCA AAT CTG TCT CCT AAA AGT GGG AAA AAG 1653 Leu Gly Val Gly lie Asn Ser Asn Leu Ser Pro Lys Ser Gly Lys Lys 420 425 430
CGG AAA AGT GGA AGT AAA GTC CGA GGT GTG TTT TCG TCA ATG TTT GGG 1701 Arg Lys Ser Gly Ser Lys Val Arg Gly Val Phe Ser Ser Met Phe Gly 435 440 445
AAA AAC AAG TCA ACG TCA TCA TCG TCG TCT TCA AAC TCA GGT CTG AAT 1749 Lys Asn Lys Ser Thr Ser Ser Ser Ser Ser Ser Asn Ser Gly Leu Asn 450 455 460 465
AGC CAC TCA CAG GAA GTC AAT ATT AAG ATC AGT ACT CCA TTC AAT GCC 1797 Ser His Ser Gin Glu Val Asn lie Lys lie Ser Thr Pro Phe Asn Ala 470 475 480
AAG CAC CTT GCC CAT GTG GGC ATT GAT GAT AAT GGT TCA TAC ACC GGT 1845 Lys His Leu Ala His Val Gly lie Asp Asp Asn Gly Ser Tyr Thr Gly 485 490 495
TTG CCA ATA GAG TGG GAA AGA TTA TTA TCT GCT AGT GGT ATT ACC AAG 1893 Leu Pro lie Glu Trp Glu Arg Leu Leu Ser Ala Ser Gly lie Thr Lys 500 505 510
AAG GAA CAA CAA CAG CAC CCA CAA GCA GTG ATG GAT ATA GTG GCG TTT 1941 Lys Glu Gin Gin Gin His Pro Gin Ala Val Met Asp He Val Ala Phe 515 520 525
TAT CAA GAT ACA AGT GAA AAC CCT GAT GAC GCT GCA TTT AAA AAG TTT 1989 Tyr Gin Asp Thr Ser Glu Asn Pro Asp Asp Ala Ala Phe Lys Lys Phe 530 535 540 545
CAT TTT GAT AAT AAT AAA AGT AGT TCG AGT GGT TGG TCT AAT GAA AAT 2037 His Phe Asp Asn Asn Lys Ser Ser Ser Ser Gly Trp Ser Asn Glu Asn 550 555 560
ACT CCA CCA GCA ACA CCG GGT GGG AGT AAC AGT GGC AGT GGC AGT GGT 2085 Thr Pro Pro Ala Thr Pro Gly Gly Ser Asn Ser Gly Ser Gly Ser Gly 565 570 575
GGC GGT GGC GCT CCT TCA AGT CCC CAT CGT ACA CCT CCT TCA TCG ATC 2133 Gly Gly Gly Ala Pro Ser Ser Pro His Arg Thr Pro Pro Ser Ser He 580 585 590
ATT GAA AAA AAC AAC GTT GAA CAA AAA GTG ATT ACC CCA TCT CAG TCA 2181 He Glu Lys Asn Asn Val Glu Gin Lys Val He Thr Pro Ser Gin Ser 595 600 605
ATG CCA ACA AAG ACA GAG AGT AAA CAG CTG GAA AAC CAG CAC CCA CAT 2229 Met Pro Thr Lys Thr Glu Ser Lys Gin Leu Glu Asn Gin His Pro His 610 615 620 625
GAA GAT AAT GCT ACT CAG TAT ACA CCA AGA ACA CCA ACA TCC CAT GTA 2277 Glu Asp Asn Ala Thr Gin Tyr Thr Pro Arg Thr Pro Thr Ser His Val 630 635 640 CAA GAG GGT CAA TTT ATT CCA AGT AGA CCA GCT CCG AAA CCA CCA TCA 2325 Gin Glu Gly Gin Phe He Pro Ser Arg Pro Ala Pro Lys Pro Pro Ser 645 650 655
ACA CCG CTT TCT TCC ATG AGT GTG TCA CAT AAA ACA CCT TCT TCG CAA 2373 Thr Pro Leu Ser Ser Met Ser Val Ser His Lys Thr Pro Ser Ser Gin 660 665 670
TCA TTA CCA AGG AGT GAT TCA CAA TCC GAT ATT CGT TCT TCA ACC CCT 2421 Ser Leu Pro Arg Ser Asp Ser Gin Ser Asp He Arg Ser Ser Thr Pro 675 680 685
AAA TCA CAT CAA GAT GTT TCG CCA AGC AAG ATC AAA ATT CGT TCA ATT 2469 Lys Ser His Gin Asp Val Ser Pro Ser Lys He Lys He Arg Ser He 690 695 700 705
TCG TCA AAA TCA TTA AAG TCA ATG CGG TCT AGA AAA AGT GGG GAT AAG 2517 Ser Ser Lys Ser Leu Lys Ser Met Arg Ser Arg Lys Ser Gly Asp Lys 710 715 720
TTT ACT CAT ATT GCA CCT GCT CCT CCA CCA CCA TCA TTA CCT TCA ATT 2565 Phe Thr His He Ala Pro Ala Pro Pro Pro Pro Ser Leu Pro Ser He 725 730 735
CCT AAA TCA AAG TCG CAT TCG GCA TCT TTG TCA AGT CAA TTG AGA CCA 2613 Pro Lys Ser Lys Ser His Ser Ala Ser Leu Ser Ser Gin Leu Arg Pro 740 745 750
GCA ACA AAT GGA TCA ACA ACT GCC CCT ATT CCA GCA AGT GCC GCG TTT 2661 Ala Thr Asn Gly Ser Thr Thr Ala Pro He Pro Ala Ser Ala Ala Phe 755 760 765
GGT GGT GAG AAT AAT GCT TTA CCA AAA CAA AGA ATA AAT GAG TTC AAG 2709 Gly Gly Glu Asn Asn Ala Leu Pro Lys Gin Arg He Asn Glu Phe Lys 770 775 780 785
GCT CAT AGA GCA CCT CCA CCA CCT CCA CTG GCA CCA CCT GCA CCA CCT 2757 Ala His Arg Ala Pro Pro Pro Pro Pro Leu Ala Pro Pro Ala Pro Pro 790 795 800
GTG CCT CCT GCT CCA CCA GCC AAT TTA TTA TCG GAA CAG ACT TCT GAG 2805 Val Pro Pro Ala Pro Pro Ala Asn Leu Leu Ser Glu Gin Thr Ser Glu 805 810 815
ATA CCT CAA CAA CGT ACT GCT CCT CTG CAA GCA TTA GCT GAT GTT ACT 2853 He Pro Gin Gin Arg Thr Ala Pro Leu Gin Ala Leu Ala Asp Val Thr 820 825 830
GCC CCA ACT AAT ATT TAT GAA ATT CAA CAA ACT AAA TAT CAG GAA GCA 2901 Ala Pro Thr Asn He Tyr Glu He Gin Gin Thr Lys Tyr Gin Glu Ala 835 840 845
CAA CAG AAA TTA CGT GAG AAG AAG GCT AGA GAA CTT GAA GAA ATA CAA 2949 Gin Gin Lys Leu Arg Glu Lys Lys Ala Arg Glu Leu Glu Glu He Gin 850 855 860 865
AGA CTA CGA GAG AAG AAT GAA AGA CAA AAT AGA CAA CAG GAG ACT GGG 2997 Arg Leu Arg Glu Lys Asn Glu Arg Gin Asn Arg Gin Gin Glu Thr Gly 870 875 880 CAA AAT AAT GCT GAC ACG GCT AGC GGT GGT AGT AAT ATT GCT CCA CCA 3045 Gin Asn Asn Ala Asp Thr Ala Ser Gly Gly Ser Asn He Ala Pro Pro 885 890 895
GTA CCT GTA CCA AAT AAA AAA CCG CCT TCT GGA TCT GGT GGT GGC CGT 3093 Val Pro Val Pro Asn Lys Lys Pro Pro Ser Gly Ser Gly Gly Gly Arg 900 905 910
GAT GCC AAA CAA GCA GCT TTG ATA GCC CAA AAG AAA CGA GAA GAA AAG 3141 Asp Ala Lys Gin Ala Ala Leu He Ala Gin Lys Lys Arg Glu Glu Lys 915 920 925
AAA CGT AAA AAC TTA CAA ATT ATT GCC AAA TTA AAG ACA ATT TGT AAT 3189 Lys Arg Lys Asn Leu Gin He He Ala Lys Leu Lys Thr He Cys Asn 930 935 940 945
CCT GGA GAT CCA AAT GAA TTA TAT GTT GAT TTA GTT AAA ATT GGT CAA 3237 Pro Gly Asp Pro Asn Glu Leu Tyr Val Asp Leu Val Lys He Gly Gin 950 955 960
GGT GCC TCC GGT GGA GTT TTC CTT GCT CAT GAT GTT CGT GAT AAA TCC 3285 Gly Ala Ser Gly Gly Val Phe Leu Ala His Asp Val Arg Asp Lys Ser 965 970 975
AAT ATT GTT GCC ATA AAA CAA ATG AAT TTA GAA CAA CAA CCT AAA AAA 3333 Asn He Val Ala He Lys Gin Met Asn Leu Glu Gin Gin Pro Lys Lys 980 985 990
GAA TTA ATT ATT AAT GAA ATT TTG GTT ATG AAA GGT AGT CTG CAT CCC 3381 Glu Leu He He Asn Glu He Leu Val Met Lys Gly Ser Leu His Pro 995 1000 1005
AAT ATT GTC AAT TTT ATT GAT TCA TAT CTT TTA AAA GGT GAT TTA TGG 3429 Asn He Val Asn Phe He Asp Ser Tyr Leu Leu Lys Gly Asp Leu Trp 1010 1015 1020 1025
GTG ATT ATG GAA TAT ATG GAA GGT GGA TCC CTT ACC GAT ATA GTG ACT 3477 Val He Met Glu Tyr Met Glu Gly Gly Ser Leu Thr Asp He Val Thr 1030 1035 1040
CAT AGT GTT ATG ACC GAA GGT CAA ATT GGA GTT GTA TGT CGT GAA ACT 3525 His Ser Val Met Thr Glu Gly Gin He Gly Val Val Cys Arg Glu Thr 1045 1050 1055
TTG AAA GGT CTT AAA TTT TTA CAT TCC AAA GGG GTT ATC CAT CGT GAT 3573 Leu Lys Gly Leu Lys Phe Leu His Ser Lys Gly Val He His Arg Asp 1060 1065 1070
ATT AAA TCC GAT AAT ATT TTA TTA AAT ATG GAT GGT AAC ATC AAG ATC 3621 He Lys Ser Asp Asn He Leu Leu Asn Met Asp Gly Asn He Lys He 1075 1080 1085
ACT GAT TTT GGG TTT TGT GCT CAA ATC AAT GAA ATC AAT CTG AAA CGT 3669 Thr Asp Phe Gly Phe Cys Ala Gin He Asn Glu He Asn Leu Lys Arg 1090 1095 1100 1105 ATC ACT ATG GTG GGT ACA CCA TAT TGG ATG GCA CCA GAA ATT GTT TCA 3717 He Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu He Val Ser 1110 1115 1120
CGT AAA GAG TAT GGT CCA AAA GTT GAT GTT TGG TCA TTA GGT ATC ATG 3765 Arg Lys Glu Tyr Gly Pro Lys Val Asp Val Trp Ser Leu Gly He Met 1125 1130 1135
ATT ATA GAA ATG TTA GAA GGT GAA CCA CCA TAT TTG AAT GAA ACT CCA 3813 He He Glu Met Leu Glu Gly Glu Pro Pro Tyr Leu Asn Glu Thr Pro 1140 1145 1150
TTG AGG GCA TTA TAT CTT ATT GCA ACT AAT GGT ACA CCA AAA TTA AAA 3861 Leu Arg Ala Leu Tyr Leu He Ala Thr Asn Gly Thr Pro Lys Leu Lys 1155 1160 1165
GAT CCT GAA TCT TTA AGT TAT GAT ATT AGA AAA TTT TTG GCA TGG TGT 3909 Asp Pro Glu Ser Leu Ser Tyr Asp He Arg Lys Phe Leu Ala Trp Cys 1170 1175 1180 1185
TTA CAA GTT GAC TTT AAT AAA AGA GCT GAT GCT GAT GAA TTA TTA CAT 3957 Leu Gin Val Asp Phe Asn Lys Arg Ala Asp Ala Asp Glu Leu Leu His 1190 1195 1200
GAT AAT TTT ATT ACT GAA TGT GAT GAT GTA TCG TCG TTA AGT CCA TTA 4005 Asp Asn Phe He Thr Glu Cys Asp Asp Val Ser Ser Leu Ser Pro Leu 1205 1210 1215
GTG AAA ATT GCT CGA TTG AAA AAA ATG AGT GAA TCT GAT TAATGAATGG TG 4056 Val Lys He Ala Arg Leu Lys Lys Met Ser Glu Ser Asp 1220 1225 1230
GAGTTATCCT AGAAATAAAA ACATTTAAAA AAAAAGAAGA AGAACAACAA GAACCCTAAA 4116
TTCTACTGCT GTCAATATAT TGGCTAATTT CCATTCTCGT TTCTATTTCT ATTTCGTTTT 4176
TATTCTTTGA ATTATTATTG TTAGTGGTAG AGATTTTTAC TAGTATATTT TTTTTATTCA 4236
TTTTTATATT TGTATTTATA TATATATTTT TCATTTAGTA TTTACTTACA CTGCAGTATC 4296
TTTCTTTTCT GTGTAGATGA TATGTAGTAA TAAGTTAACT TGTTCAAGAC AGTGAATGGA 4356
AATATATTAT AGCTTGACTA TATAAGGTGG AGAGCTGTAA TTGGCTTTCC GTATAGAAAA 4416
GTCTTGAACA AACGTTACCA GATTTCTGCT ATTCTTATTT GGTACGATTC GGGCGTATGA 4476
TAGGTTTATT GAGCTC 4492
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1230 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Ser He Leu Ser Glu Asn Asn Pro Thr Pro Thr Ser He Thr Asp
1 5 10 15
Pro Asn Glu Ser Ser His Leu His Asn Pro Glu Leu Asn Ser Gly Thr 20 25 30 Arg Val Ala Ser Gly Pro Gly Pro Gly Pro Glu Val Glu Ser Thr Pro
35 40 45
Leu Ala Pro Pro Thr Glu Val Met Asn Thr Thr Ser Ala Asn Thr Ser
50 55 60
Ser Leu Ser Leu Gly Ser Pro Met His Glu Lys He Lys Gin Phe Asp 65 70 75 80
Gin Asp Glu Val Asp Thr Gly Glu Thr Asn Asp Arg Thr He Glu Ser
85 90 95
Gly Ser Ser Asp He Asp Asp Ser Gin Gin Ser His Asn Asn Asn Asn
100 105 110
Asn Asn Asn Asn Asn Asn Asn Glu Ser Asn Pro Glu Ser Ser Glu Gly
115 120 125
Asp Asp Glu Lys Thr Gin Gly Met Pro Pro Arg Met Pro Gly Thr Phe
130 135 140
Asn Val Lys Gly Leu His Gin Gly Asp Asp Ser Asp Asn Glu Lys Gin 145 150 155 160
Tyr Thr Glu Leu Thr Lys Ser He Asn Lys Arg Thr Ser Lys Asp Ser
165 170 175
Tyr Ser Pro Gly Thr Leu Glu Ser Pro Gly Thr Leu Asn Ala Leu Glu
180 185 190
Thr Asn Asn Val Ser Pro Ala Val He Glu Glu Glu Gin His Thr Leu
195 200 205
Ser Leu Glu Asp Leu Ser Leu Ser Leu Gin His Gin Asn Glu Asn Ala
210 215 220
Arg Leu Ser Ala Pro Arg Ser Ala Pro Pro Gin Val Pro Thr Ser Lys 225 230 235 240
Thr Ser Ser Phe His Asp Met Ser Leu Val He Ser Ser Ser Thr Ser
245 250 255
Val His Lys He Pro Ser Asn Pro Thr Ser Thr Arg Gly Ser His Leu
260 265 270
Ser Ser Tyr Lys Ser Thr Leu Asp Pro Gly Lys Pro Ala Gin Ala Ala
275 280 285
Ala Pro Pro Pro Pro Glu He Asp He Asp Asn Leu Leu Thr Lys Ser
290 295 300
Glu Leu Asp Leu Glu Thr Asp Thr Leu Ser Ser Ala Thr Asn Ser Pro 305 310 315 320
Asn Leu Leu Arg Asn Asp Thr Leu Gin Gly He Pro Thr Arg Asp Asp
325 330 335
Glu Asn He Asp Asp Leu Pro Arg Gin Leu Ser Gin Asn Thr Ser Ala
340 345 350
Thr Ser Arg Asn Thr Ser Gly Thr Ser Thr Ser Thr Val Val Lys Asn
355 360 365
Ser Arg Ser Gly Thr Ser Lys Ser Thr Ser Thr Ser Thr Ala His Asn
370 375 380
Gin Thr Ala Ala He Thr Pro He He Pro Ser His Asn Lys Phe His 385 390 395 400
Gin Gin Val He Asn Thr Asn Ala Thr Asn Ser Ser Ser Ser Leu Glu
405 410 415
Pro Leu Gly Val Gly He Asn Ser Asn Leu Ser Pro Lys Ser Gly Lys
420 425 430
Lys Arg Lys Ser Gly Ser Lys Val Arg Gly Val Phe Ser Ser Met Phe
435 440 445
Gly Lys Asn Lys Ser Thr Ser Ser Ser Ser Ser Ser Asn Ser Gly Leu
450 455 460
Asn Ser His Ser Gin Glu Val Asn He Lys He Ser Thr Pro Phe Asn 465 470 475 480
Ala Lys His Leu Ala His Val Gly He Asp Asp Asn Gly Ser Tyr Thr 485 490 495 Gly Leu Pro He Glu Trp Glu Arg Leu Leu Ser Ala Ser Gly He Thr
500 505 510
Lys Lys Glu Gin Gin Gin His Pro Gin Ala Val Met Asp He Val Ala
515 520 525
Phe Tyr Gin Asp Thr Ser Glu Asn Pro Asp Asp Ala Ala Phe Lys Lys
530 535 540
Phe His Phe Asp Asn Asn Lys Ser Ser Ser Ser Gly Trp Ser Asn Glu 545 550 555 560
Asn Thr Pro Pro Ala Thr Pro Gly Gly Ser Asn Ser Gly Ser Gly Ser
565 570 575
Gly Gly Gly Gly Ala Pro Ser Ser Pro His Arg Thr Pro Pro Ser Ser
580 585 590
He He Glu Lys Asn Asn Val Glu Gin Lys Val He Thr Pro Ser Gin
595 600 605
Ser Met Pro Thr Lys Thr Glu Ser Lys Gin Leu Glu Asn Gin His Pro
610 615 620
His Glu Asp Asn Ala Thr Gin Tyr Thr Pro Arg Thr Pro Thr Ser His 625 630 635 640
Val Gin Glu Gly Gin Phe He Pro Ser Arg Pro Ala Pro Lys Pro Pro
645 650 655
Ser Thr Pro Leu Ser Ser Met Ser Val Ser His Lys Thr Pro Ser Ser
660 665 670
Gin Ser Leu Pro Arg Ser Asp Ser Gin Ser Asp He Arg Ser Ser Thr
675 680 685
Pro Lys Ser His Gin Asp Val Ser Pro Ser Lys He Lys He Arg Ser
690 695 700
He Ser Ser Lys Ser Leu Lys Ser Met Arg Ser Arg Lys Ser Gly Asp 705 710 715 720
Lys Phe Thr His He Ala Pro Ala Pro Pro Pro Pro Ser Leu Pro Ser
725 730 735
He Pro Lys Ser Lys Ser His Ser Ala Ser Leu Ser Ser Gin Leu Arg
740 745 750
Pro Ala Thr Asn Gly Ser Thr Thr Ala Pro He Pro Ala Ser Ala Ala
755 760 765
Phe Gly Gly Glu Asn Asn Ala Leu Pro Lys Gin Arg He Asn Glu Phe
770 775 780
Lys Ala His Arg Ala Pro Pro Pro Pro Pro Leu Ala Pro Pro Ala Pro 785 790 795 800
Pro Val Pro Pro Ala Pro Pro Ala Asn Leu Leu Ser Glu Gin Thr Ser
805 810 815
Glu He Pro Gin Gin Arg Thr Ala Pro Leu Gin Ala Leu Ala Asp Val
820 825 830
Thr Ala Pro Thr Asn He Tyr Glu He Gin Gin Thr Lys Tyr Gin Glu
835 840 845
Ala Gin Gin Lys Leu Arg Glu Lys Lys Ala Arg Glu Leu Glu Glu He
850 855 860
Gin Arg Leu Arg Glu Lys Asn Glu Arg Gin Asn Arg Gin Gin Glu Thr 865 870 875 880
Gly Gin Asn Asn Ala Asp Thr Ala Ser Gly Gly Ser Asn He Ala Pro
885 890 895
Pro Val Pro Val Pro Asn Lys Lys Pro Pro Ser Gly Ser Gly Gly Gly
900 905 910
Arg Asp Ala Lys Gin Ala Ala Leu He Ala Gin Lys Lys Arg Glu Glu
915 920 925
Lys Lys Arg Lys Asn Leu Gin He He Ala Lys Leu Lys Thr He Cys
930 935 940
Asn Pro Gly Asp Pro Asn Glu Leu Tyr Val Asp Leu Val Lys He Gly 945 950 955 960 Gin Gly Ala Ser Gly Gly Val Phe Leu Ala His Asp Val Arg Asp Lys
965 970 975
Ser Asn He Val Ala He Lys Gin Met Asn Leu Glu Gin Gin Pro Lys
980 985 990
Lys Glu Leu He He Asn Glu He Leu Val Met Lys Gly Ser Leu His
995 1000 1005
Pro Asn He Val Asn Phe He Asp Ser Tyr Leu Leu Lys Gly Asp Leu
1010 1015 1020
Trp Val He Met Glu Tyr Met Glu Gly Gly Ser Leu Thr Asp He Val 1025 1030 1035 1040
Thr His Ser Val Met Thr Glu Gly Gin He Gly Val Val Cys Arg Glu
1045 1050 1055
Thr Leu Lys Gly Leu Lys Phe Leu His Ser Lys Gly Val He His Arg
1060 1065 1070
Asp He Lys Ser Asp Asn He Leu Leu Asn Met Asp Gly Asn He Lys
1075 1080 1085
He Thr Asp Phe Gly Phe Cys Ala Gin He Asn Glu He Asn Leu Lys
1090 1095 1100
Arg He Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu He Val 1105 1110 1115 1120
Ser Arg Lys Glu Tyr Gly Pro Lys Val Asp Val Trp Ser Leu Gly He
1125 1130 1135
Met He He Glu Met Leu Glu Gly Glu Pro Pro Tyr Leu Asn Glu Thr
1140 1145 1150
Pro Leu Arg Ala Leu Tyr Leu He Ala Thr Asn Gly Thr Pro Lys Leu
1155 1160 1165
Lys Asp Pro Glu Ser Leu Ser Tyr Asp He Arg Lys Phe Leu Ala Trp
1170 1175 1180
Cys Leu Gin Val Asp Phe Asn Lys Arg Ala Asp Ala Asp Glu Leu Leu
185 1190 1195 1200
His Asp Asn Phe He Thr Glu Cys Asp Asp Val Ser Ser Leu Ser Pro
1205 1210 1215
Leu Val Lys He Ala Arg Leu Lys Lys Met Ser Glu Ser Asp 1220 1225 1230
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3496 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 432...3344 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GAATTCTTTT TAGAAGAGAA AGAAAAAATT CCCAAAAAAA AAAGATTTCA TTTAATTCCA 60
CGGGAACATT GATTACAACC ACGTCAACAG TTTCCCTTTT ATATTGAAAT CAACATTCAA 120
TTTTGTCTTT TTTTTTTTTT CATTGATTTT TCCCCAATCT TTTTATCTTC ATATTAATAT 180
TGGATATCAA TTACTAATAC TGTCAGGGAT AGTTTAGTAA ATATTTACAT TCTCCATTCA 240
ATCCTAAATT TTTTTTTATA TAGCTAGTTT TTGGTTGAAA AAAAAAAAAT AGGGGGAAGG 300
AAGTTTTTTT TTCTATTTAT TTAATTGTTT TGATTCCAAC CATATTGTAT ATTTGTCTTG 360 TCAGTTATAT TACTTTCTTG TTACTTAATT ATTAATTATT TGCTATATTA TTGAATTGAA 420 TCCTCAAAAG A ATG ACA AGT ATT TAT ACA TCA GAT TTG AAA AAC CAT AGA 470 Met Thr Ser He Tyr Thr Ser Asp Leu Lys Asn His Arg 1 5 10
CGT GCG CCA CCT CCA CCA AAT GGG GCA GCT GGC TCT GGC TCA GGT TCT 518 Arg Ala Pro Pro Pro Pro Asn Gly Ala Ala Gly Ser Gly Ser Gly Ser 15 20 25
GGC TCA GGT TCT GGT TCT GGT TCT GGC AGT TTG GCT AAT ATT GTT ACC 566 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Leu Ala Asn He Val Thr 30 35 40 45
AGT TCT AAT AGT CTT GGC GTA ACA GCA AAT CAA ACC AAA CCT ATT CAA 614 Ser Ser Asn Ser Leu Gly Val Thr Ala Asn Gin Thr Lys Pro He Gin 50 55 60
TTA AAT ATA AAT TCT AGC AAA CGT CAA TCA GGT TGG GTT CAT GTT AAA 662 Leu Asn He Asn Ser Ser Lys Arg Gin Ser Gly Trp Val His Val Lys 65 70 75
GAT GAT GGT ATT TTC ACA TCA TTT AGA TGG AAC AAA CGG TTT ATG GTT 710 Asp Asp Gly He Phe Thr Ser Phe Arg Trp Asn Lys Arg Phe Met Val 80 85 90
ATT AAT GAT AAA ACT TTA AAC TTT TAT AAA CAA GAA CCA TAT TCT AGT 758 He Asn Asp Lys Thr Leu Asn Phe Tyr Lys Gin Glu Pro Tyr Ser Ser 95 100 105
GAT GGT AAT TCC AAT TCT AAT ACC CCT GAT TTA TCA TTC CCA CTA TAT 806 Asp Gly Asn Ser Asn Ser Asn Thr Pro Asp Leu Ser Phe Pro Leu Tyr 110 115 120 125
TTA ATT AAT AAT ATT AAT TTG AAA CCA AAC TCC GGG TAT AGC AAA ACT 854 Leu He Asn Asn He Asn Leu Lys Pro Asn Ser Gly Tyr Ser Lys Thr 130 135 140
TCA CAA TCA TTT GAA ATT GTT CCC AAA AAC AAT AAT AAA TCA ATT TTG 902 Ser Gin Ser Phe Glu He Val Pro Lys Asn Asn Asn Lys Ser He Leu 145 150 155
ATT TCT GTT AAA ACC AAT AAT GAT TAT TTG GAT TGG CTA GAT GCA TTC 950 He Ser Val Lys Thr Asn Asn Asp Tyr Leu Asp Trp Leu Asp Ala Phe 160 165 170
ACC ACA AAA TGT CCT TTA GTA CAA ATT GGT GAA AAT AAT AGT GGT GTA 998 Thr Thr Lys Cys Pro Leu Val Gin He Gly Glu Asn Asn Ser Gly Val 175 180 185
TCA AGT AGT CAC CCT CAT TTA CAA ATT CAA CAT TTA ACC AAT GGT TCC 1046 Ser Ser Ser His Pro His Leu Gin He Gin His Leu Thr Asn Gly Ser 190 195 200 205
TTG AAC GGC AAC TCA TCT TCA TCA CCA ACA TCT GGA TTA TTA TCT TCT 1094 Leu Asn Gly Asn Ser Ser Ser Ser Pro Thr Ser Gly Leu Leu Ser Ser 210 215 220 TCA GTG CTA ACT GGA GGT AAT TCT GGC GTT TCT GGT CCT ATT AAT TTC 1142 Ser Val Leu Thr Gly Gly Asn Ser Gly Val Ser Gly Pro He Asn Phe 225 230 235
ACT CAT AAA GTA CAC GTG GGA TTT GAT CCT GCC AGT GGT AAT TTT ACT 1190 Thr His Lys Val His Val Gly Phe Asp Pro Ala Ser Gly Asn Phe Thr 240 245 250
GGA TTA CCA GAC ACT TGG AAA AGT TTA TTA CAA CAT TCG AAA ATC ACT 1238 Gly Leu Pro Asp Thr Trp Lys Ser Leu Leu Gin His Ser Lys He Thr 255 260 265
AAT GAG GAT TGG AAA AAA GAT CCT GTT GCT GTT ATT GAA GTT TTA GAA 1286 Asn Glu Asp Trp Lys Lys Asp Pro Val Ala Val He Glu Val Leu Glu 270 275 280 285
TTT TAT TCC GAT ATA AAT GGA GGT AAT TCA GCT GCT GGA ACT CCA ATT 1334 Phe Tyr Ser Asp He Asn Gly Gly Asn Ser Ala Ala Gly Thr Pro He 290 295 300
GGA TCA CCC ATG ATC AAT TCC AAA ACC AAC AAT AAT AAT AAT GAC CCT 1382 Gly Ser Pro Met He Asn Ser Lys Thr Asn Asn Asn Asn Asn Asp Pro 305 310 315
AAC AAT TAC TCA TCA ACC AAA AAC AAT GTC CAA GAG GCA AAT TTA CAA 1430 Asn Asn Tyr Ser Ser Thr Lys Asn Asn Val Gin Glu Ala Asn Leu Gin 320 325 330
GAA TGG GTA AAA CCT CCA GCA AAA TCT ACT GTC TCA CAA TTC AAA CCT 1478 Glu Trp Val Lys Pro Pro Ala Lys Ser Thr Val Ser Gin Phe Lys Pro 335 340 345
AGT CGA GCT GCA CCA AAA CCA CCA ACT CCA TAT CAT TTG ACA CAA CTA 1526 Ser Arg Ala Ala Pro Lys Pro Pro Thr Pro Tyr His Leu Thr Gin Leu 350 355 360 365
AAT GGC TCT TCC CAC CAA CAT ACA TCA TCA TCA GGC TCA TTA CCT AGT 1574 Asn Gly Ser Ser His Gin His Thr Ser Ser Ser Gly Ser Leu Pro Ser 370 375 380
TCT GGT AAT AAT AAT AAT AAT AAC AGC ACT AAC AAT AAT AAT ACT AAA 1622 Ser Gly Asn Asn Asn Asn Asn Asn Ser Thr Asn Asn Asn Asn Thr Lys 385 390 395
AAC GTT TCA CCA TTG AAT AAT TTG ATG AAT AAA TCT GAA CTT ATT CCT 1670 Asn Val Ser Pro Leu Asn Asn Leu Met Asn Lys Ser Glu Leu He Pro 400 405 410
GCT AGA AGA GCT CCA CCA CCT CCA ACA AGT GGC ACA TCT TCA GAT ACA 1718 Ala Arg Arg Ala Pro Pro Pro Pro Thr Ser Gly Thr Ser Ser Asp Thr 415 420 425
TAT TCT AAT AAG AAT CAT CAA GAT AGA TCT GGA TAT GAA CAA CAA CGT 1766 Tyr Ser Asn Lys Asn His Gin Asp Arg Ser Gly Tyr Glu Gin Gin Arg 430 435 440 445
CAA CAA CGT ACT GAC TCA TCA CAA CAA CAA CAA CAA CAA AAG CAA CAT 1814 Gin Gin Arg Thr Asp Ser Ser Gin Gin Gin Gin Gin Gin Lys Gin His 450 455 460 CAA TAT CAA CAG AAA TCC CAA CAA CAA CAA CAA CAA CCA CAA CAA CCA 1862 Gin Tyr Gin Gin Lys Ser Gin Gin Gin Gin Gin Gin Pro Gin Gin Pro 465 470 475
TTA TCT CTG CAT CAA GGT GGG ACT TCG CAT ATT CCG AAA CAA GTA CCT 1910 Leu Ser Leu His Gin Gly Gly Thr Ser His He Pro Lys Gin Val Pro 480 485 490
CCT ACA TTA CCA TCA TCT GGA CCA CCC ACT CAG GCT GCT TCA GGA AAA 1958 Pro Thr Leu Pro Ser Ser Gly Pro Pro Thr Gin Ala Ala Ser Gly Lys 495 500 505
TCA ATG CCA TCT AAA ATT CAT CCT GAT CTT AAG ATT CAA CAA GGC ACA 2006 Ser Met Pro Ser Lys He His Pro Asp Leu Lys He Gin Gin Gly Thr 510 515 520 525
AAT AAT TAT ATT AAG AGT AGC GGT ACT GAT GCT AAT CAA GTC GAT GGT 2054 Asn Asn Tyr He Lys Ser Ser Gly Thr Asp Ala Asn Gin Val Asp Gly 530 535 540
GAT GCT AAA CAA TTT ATT AAA CCA TTT AAT TTA CAA CTG AAA AAG AGT 2102 Asp Ala Lys Gin Phe He Lys Pro Phe Asn Leu Gin Leu Lys Lys Ser 545 550 555
CAG CAA CAA TTG GCA TCA AAA CAA CCG TCA CCA CCT TCA TCT CAA CAA 2150 Gin Gin Gin Leu Ala Ser Lys Gin Pro Ser Pro Pro Ser Ser Gin Gin 560 565 570
CAG CAA CAA AAA CCT ATG ACA TCA CAT GGA TTA ATG GGT ACA TCA CAT 2198 Gin Gin Gin Lys Pro Met Thr Ser His Gly Leu Met Gly Thr Ser His 575 580 585
TCA GTT ACT AAA CCA TTG AAT CCA GTC AAT GAT CCA ATC AAA CCA TTA 2246 Ser Val Thr Lys Pro Leu Asn Pro Val Asn Asp Pro He Lys Pro Leu 590 595 600 605
AAT TTG AAA TCA TCT AAA TCC AAA GAA GCA TTA AAT GAA ACT CTG GGG 2294 Asn Leu Lys Ser Ser Lys Ser Lys Glu Ala Leu Asn Glu Thr Leu Gly 610 615 620
GTG CTG AAA ACA CCA TCA CCT ACA GAT AAA TCA AAT AAA CCA ACT GCA 2342 Val Leu Lys Thr Pro Ser Pro Thr Asp Lys Ser Asn Lys Pro Thr Ala 625 630 635
CCT GCT AGT GGT CCT GCA GTG ACC AAA ACA GCT AAA CAA CTC AAG AAG 2390 Pro Ala Ser Gly Pro Ala Val Thr Lys Thr Ala Lys Gin Leu Lys Lys 640 645 650
GAA CGA GAA AGA TTG AAT GAT TTA CAA ATC ATT GCT AAA TTG AAA ACA 2438 Glu Arg Glu Arg Leu Asn Asp Leu Gin He He Ala Lys Leu Lys Thr 655 660 665
GTG GTT AAT AAT CAA GAT CCT AAA CCA TTA TTT AGA ATT GTT GAA AAA 2486 Val Val Asn Asn Gin Asp Pro Lys Pro Leu Phe Arg He Val Glu Lys 670 675 680 685 GCT GGT CAA GGT GCT AGT GGG AAT GTT TAT TTG GCG GAA ATG ATC AAA 2534 Ala Gly Gin Gly Ala Ser Gly Asn Val Tyr Leu Ala Glu Met He Lys 690 695 700
GAT AAT AAT CGA AAG ATT GCG ATT AAA CAA ATG GAT CTT GAT GCT CAA 2582 Asp Asn Asn Arg Lys He Ala He Lys Gin Met Asp Leu Asp Ala Gin 705 710 715
CCC CGT AAA GAG TTA ATA ATA AAT GAA ATC TTG GTT ATG AAA GAT AGT 2630 Pro Arg Lys Glu Leu He He Asn Glu He Leu Val Met Lys Asp Ser 720 725 730
CAA CAT AAA AAT ATT GTT AAT TTT TTG GAT TCT TAT TTA ATT GGT GAT 2678 Gin His Lys Asn He Val Asn Phe Leu Asp Ser Tyr Leu He Gly Asp 735 740 745
AAT GAA TTA TGG GTA ATT ATG GAA TAT ATG CAA GGT GGT TCA TTA ACG 2726 Asn Glu Leu Trp Val He Met Glu Tyr Met Gin Gly Gly Ser Leu Thr 750 755 760 765
GAA ATC ATT GAA AAT AAT GAT TTT AAA TTG AAT GAA AAA CAA ATT GCC 2774 Glu He He Glu Asn Asn Asp Phe Lys Leu Asn Glu Lys Gin He Ala 770 775 780
ACT ATA TGT TTT GAA ACC TTA AAG GGG TTA CAA CAT TTA CAT AAA AAA 2822 Thr He Cys Phe Glu Thr Leu Lys Gly Leu Gin His Leu His Lys Lys 785 790 795
CAT ATT ATT CAT CGT GAT ATT AAA TCC GAT AAT GTT TTA TTA GAT GCA 2870 His He He His Arg Asp He Lys Ser Asp Asn Val Leu Leu Asp Ala 800 805 810
TAT GGT AAT GTT AAA ATC ACT GAT TTT GGA TTT TGT GCT AAA TTA ACT 2918 Tyr Gly Asn Val Lys He Thr Asp Phe Gly Phe Cys Ala Lys Leu Thr 815 820 825
GAT CAA AGA AAT AAA CGT GCC ACA ATG GTG GGG ACA CCA TAT TGG ATG 2966 Asp Gin Arg Asn Lys Arg Ala Thr Met Val Gly Thr Pro Tyr Trp Met 830 835 840 845
GCA CCT GAA GTG GTT AAA CAA AAG GAA TAT GAT GAA AAA GTT GAT GTT 3014 Ala Pro Glu Val Val Lys Gin Lys Glu Tyr Asp Glu Lys Val Asp Val 850 855 860
TGG TCA TTG GGG ATT ATG ACT ATT GAA ATG ATT GAA GGA GAA CCA CCT 3062 Trp Ser Leu Gly He Met Thr He Glu Met He Glu Gly Glu Pro Pro 865 870 875
TAT TTG AAT GAA GAA CCA TTA AAA GCT TTA TAT CTT ATA GCT ACT AAT 3110 Tyr Leu Asn Glu Glu Pro Leu Lys Ala Leu Tyr Leu He Ala Thr Asn 880 885 890
GGT ACA CCA AAA TTG AAA AAA CCC GAA TTA TTA TCG AAT TCA ATT AAA 3158 Gly Thr Pro Lys Leu Lys Lys Pro Glu Leu Leu Ser Asn Ser He Lys 895 900 905
AAA TTC TTA TCA ATT TGT CTT TGT GTT GAT GTT AGA TAT CGT GCT AGT 3206 Lys Phe Leu Ser He Cys Leu Cys Val Asp Val Arg Tyr Arg Ala Ser 910 915 920 925 ACT GAT GAA TTA TTA GAA CAT TCA TTT ATT CAA CAT AAA TCA GGG AAA 3254 Thr Asp Glu Leu Leu Glu His Ser Phe He Gin His Lys Ser Gly Lys 930 935 940
ATT GAA GAA TTG GCA CCA TTA TTA GAA TGG AAA AAA CAA CAA CAA AAG 3302 He Glu Glu Leu Ala Pro Leu Leu Glu Trp Lys Lys Gin Gin Gin Lys 945 950 955
CAT CAA CAG CAT AAA CAA GAA ACA CTG GAT ACA GGA TTT GCA TAGAGATTG 3353 His Gin Gin His Lys Gin Glu Thr Leu Asp Thr Gly Phe Ala 960 965 970
AATATAGCCG TAGAAAACTG GTACTTTGGT TTTGGTATAA TATATTTATG TGATGTGTTG 3413 TGTGTATGGT TAGTTTAGAT TTGGATTTTT AGTTTTTTAG AGTTTAGTTT TTCAATTTTT 3473 AGTTTTAGAG ACAATATTCT AGA 3496
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 971 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Thr Ser He Tyr Thr Ser Asp Leu Lys Asn His Arg Arg Ala Pro
1 5 10 15
Pro Pro Pro Asn Gly Ala Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly
20 25 30
Ser Gly Ser Gly Ser Gly Ser Leu Ala Asn He Val Thr Ser Ser Asn
35 40 45
Ser Leu Gly Val Thr Ala Asn Gin Thr Lys Pro He Gin Leu Asn He
50 55 60
Asn Ser Ser Lys Arg Gin Ser Gly Trp Val His Val Lys Asp Asp Gly 65 70 75 80
He Phe Thr Ser Phe Arg Trp Asn Lys Arg Phe Met Val He Asn Asp
85 90 95
Lys Thr Leu Asn Phe Tyr Lys Gin Glu Pro Tyr Ser Ser Asp Gly Asn
100 105 110
Ser Asn Ser Asn Thr Pro Asp Leu Ser Phe Pro Leu Tyr Leu He Asn
115 120 125
Asn He Asn Leu Lys Pro Asn Ser Gly Tyr Ser Lys Thr Ser Gin Ser
130 135 140
Phe Glu He Val Pro Lys Asn Asn Asn Lys Ser He Leu He Ser Val 145 150 155 160
Lys Thr Asn Asn Asp Tyr Leu Asp Trp Leu Asp Ala Phe Thr Thr Lys
165 170 175
Cys Pro Leu Val Gin He Gly Glu Asn Asn Ser Gly Val Ser Ser Ser
180 185 190
His Pro His Leu Gin He Gin His Leu Thr Asn Gly Ser Leu Asn Gly
195 200 205
Asn Ser Ser Ser Ser Pro Thr Ser Gly Leu Leu Ser Ser Ser Val Leu 210 215 220 Thr Gly Gly Asn Ser Gly Val Ser Gly Pro He Asn Phe Thr His Lys 225 230 235 240
Val His Val Gly Phe Asp Pro Ala Ser Gly Asn Phe Thr Gly Leu Pro
245 250 255
Asp Thr Trp Lys Ser Leu Leu Gin His Ser Lys He Thr Asn Glu Asp
260 265 270
Trp Lys Lys Asp Pro Val Ala Val He Glu Val Leu Glu Phe Tyr Ser
275 280 285
Asp He Asn Gly Gly Asn Ser Ala Ala Gly Thr Pro He Gly Ser Pro
290 295 300
Met He Asn Ser Lys Thr Asn Asn Asn Asn Asn Asp Pro Asn Asn Tyr 305 310 315 320
Ser Ser Thr Lys Asn Asn Val Gin Glu Ala Asn Leu Gin Glu Trp Val
325 330 335
Lys Pro Pro Ala Lys Ser Thr Val Ser Gin Phe Lys Pro Ser Arg Ala
340 345 350
Ala Pro Lys Pro Pro Thr Pro Tyr His Leu Thr Gin Leu Asn Gly Ser
355 360 365
Ser His Gin His Thr Ser Ser Ser Gly Ser Leu Pro Ser Ser Gly Asn
370 375 380
Asn Asn Asn Asn Asn Ser Thr Asn Asn Asn Asn Thr Lys Asn Val Ser 385 390 395 400
Pro Leu Asn Asn Leu Met Asn Lys Ser Glu Leu He Pro Ala Arg Arg
405 410 415
Ala Pro Pro Pro Pro Thr Ser Gly Thr Ser Ser Asp Thr Tyr Ser Asn
420 425 430
Lys Asn His Gin Asp Arg Ser Gly Tyr Glu Gin Gin Arg Gin Gin Arg
435 440 445
Thr Asp Ser Ser Gin Gin Gin Gin Gin Gin Lys Gin His Gin Tyr Gin
450 455 460
Gin Lys Ser Gin Gin Gin Gin Gin Gin Pro Gin Gin Pro Leu Ser Leu 465 470 475 480
His Gin Gly Gly Thr Ser His He Pro Lys Gin Val Pro Pro Thr Leu
485 490 495
Pro Ser Ser Gly Pro Pro Thr Gin Ala Ala Ser Gly Lys Ser Met Pro
500 505 510
Ser Lys He His Pro Asp Leu Lys He Gin Gin Gly Thr Asn Asn Tyr
515 520 525
He Lys Ser Ser Gly Thr Asp Ala Asn Gin Val Asp Gly Asp Ala Lys
530 535 540
Gin Phe He Lys Pro Phe Asn Leu Gin Leu Lys Lys Ser Gin Gin Gin 545 550 555 560
Leu Ala Ser Lys Gin Pro Ser Pro Pro Ser Ser Gin Gin Gin Gin Gin
565 570 575
Lys Pro Met Thr Ser His Gly Leu Met Gly Thr Ser His Ser Val Thr
580 585 590
Lys Pro Leu Asn Pro Val Asn Asp Pro He Lys Pro Leu Asn Leu Lys
595 600 605
Ser Ser Lys Ser Lys Glu Ala Leu Asn Glu Thr Leu Gly Val Leu Lys
610 615 620
Thr Pro Ser Pro Thr Asp Lys Ser Asn Lys Pro Thr Ala Pro Ala Ser 625 630 635 640
Gly Pro Ala Val Thr Lys Thr Ala Lys Gin Leu Lys Lys Glu Arg Glu
645 650 655
Arg Leu Asn Asp Leu Gin He He Ala Lys Leu Lys Thr Val Val Asn
660 665 670
Asn Gin Asp Pro Lys Pro Leu Phe Arg He Val Glu Lys Ala Gly Gin 675 680 685 Gly Ala Ser Gly Asn Val Tyr Leu Ala Glu Met He Lys Asp Asn Asn
690 695 700
Arg Lys He Ala He Lys Gin Met Asp Leu Asp Ala Gin Pro Arg Lys 705 710 715 720
Glu Leu He He Asn Glu He Leu Val Met Lys Asp Ser Gin His Lys
725 730 735
Asn He Val Asn Phe Leu Asp Ser Tyr Leu He Gly Asp Asn Glu Leu
740 745 750
Trp Val He Met Glu Tyr Met Gin Gly Gly Ser Leu Thr Glu He He
755 760 765
Glu Asn Asn Asp Phe Lys Leu Asn Glu Lys Gin He Ala Thr He Cys
770 775 780
Phe Glu Thr Leu Lys Gly Leu Gin His Leu His Lys Lys His He He 785 790 795 800
His Arg Asp He Lys Ser Asp Asn Val Leu Leu Asp Ala Tyr Gly Asn
805 810 815
Val Lys He Thr Asp Phe Gly Phe Cys Ala Lys Leu Thr Asp Gin Arg
820 825 830
Asn Lys Arg Ala Thr Met Val Gly Thr Pro Tyr Trp Met Ala Pro Glu
835 840 845
Val Val Lys Gin Lys Glu Tyr Asp Glu Lys Val Asp Val Trp Ser Leu
850 855 860
Gly He Met Thr He Glu Met He Glu Gly Glu Pro Pro Tyr Leu Asn 865 870 875 880
Glu Glu Pro Leu Lys Ala Leu Tyr Leu He Ala Thr Asn Gly Thr Pro
885 890 895
Lys Leu Lys Lys Pro Glu Leu Leu Ser Asn Ser He Lys Lys Phe Leu
900 905 910
Ser He Cys Leu Cys Val Asp Val Arg Tyr Arg Ala Ser Thr Asp Glu
915 920 925
Leu Leu Glu His Ser Phe He Gin His Lys Ser Gly Lys He Glu Glu
930 935 940
Leu Ala Pro Leu Leu Glu Trp Lys Lys Gin Gin Gin Lys His Gin Gin 945 950 955 960
His Lys Gin Glu Thr Leu Asp Thr Gly Phe Ala 965 970
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1031 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 271...843 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CAACCAAACC AACTTTCATC CTTCTACCAA TATCTTCAAC AAAAGTTTTA TTCAATACTA 60
TTTTAAAAAT AACAGTGTTA CTCGTTCATT TGATTTGTTA ATAAGACTGA TTTACCCACT 120
TTTTAGTTCC TATAATCATA CAGATTTCTC GTCCTAAATC TATTTTTATT GTTATTTTTA 180
CTTTAGTTTT CACTTTTGCT TTCAGTTTTT TCTTTTTTTA GCACAAGAGA AAAGTATTCA 240 GCTCATAAAT AATTAATATA TCCATATATC ATG CAA ACT ATA AAA TGT GTT GTT 294
Met Gin Thr He Lys Cys Val Val 1 5
GTC GGT GAT GGT GCC GTT GGT AAA ACT TGC TTA TTA ATC TCG TAT ACC 342 Val Gly Asp Gly Ala Val Gly Lys Thr Cys Leu Leu He Ser Tyr Thr 10 15 20
ACT AGT AAA TTT CCA GCT GAT TAT GTT CCT ACT GTT TTT GAT AAT TAT 390 Thr Ser Lys Phe Pro Ala Asp Tyr Val Pro Thr Val Phe Asp Asn Tyr 25 30 35 40
GCT GTA ACC GTG ATG ATA GGA GAC GAA CCA TTT ACC TTG GGA TTA TTT 438 Ala Val Thr Val Met He Gly Asp Glu Pro Phe Thr Leu Gly Leu Phe 45 50 55
GAT ACT GCT GGT CAA GAA GAT TAC GAC AGA TTA AGG CCT TTG TCA TAT 486 Asp Thr Ala Gly Gin Glu Asp Tyr Asp Arg Leu Arg Pro Leu Ser Tyr 60 65 70
CCA TCG ACT GAT GTA TTC CTT GTT TGT TTT TCC GTC ATT TCT CCC GCT 534 Pro Ser Thr Asp Val Phe Leu Val Cys Phe Ser Val He Ser Pro Ala 75 80 85
TCG TTT GAA AAT GTT AAA GAA AAA TGG TTC CCA GAA GTT CAT CAC CAT 582 Ser Phe Glu Asn Val Lys Glu Lys Trp Phe Pro Glu Val His His His 90 95 100
TGT CCC GGT GTG CCA ATA ATT ATT GTC GGT ACC CAA ACT GAT TTA CGA 630 Cys Pro Gly Val Pro He He He Val Gly Thr Gin Thr Asp Leu Arg 105 110 115 120
AAC GAT GAT GTT ATT TTA CAG AGA TTG CAC AGA CAA AAA TTG TCC CCA 678 Asn Asp Asp Val He Leu Gin Arg Leu His Arg Gin Lys Leu Ser Pro 125 130 135
ATC ACC CAG GAA CAG GGT GAA AAA TTG GCT AAG GAA TTG AGA GCT GTC 726 He Thr Gin Glu Gin Gly Glu Lys Leu Ala Lys Glu Leu Arg Ala Val 140 145 150
AAG TAT GTT GAG TGT TCT GCA TTG ACT CAA AGA GGA TTG AAA ACA GTG 774 Lys Tyr Val Glu Cys Ser Ala Leu Thr Gin Arg Gly Leu Lys Thr Val 155 160 165
TTT GAC GAG GCT ATA GTA GCT GCA TTA GAA CCT CCT GTA ATT AAA AAA 822 Phe Asp Glu Ala He Val Ala Ala Leu Glu Pro Pro Val He Lys Lys 170 175 180
TCG AAA AAG TGT ACT ATT TTA TAGGTCGGCG ATACTAGAAG ATAGAGGATA TTGG 877 Ser Lys Lys Cys Thr He Leu 185 190
AAATAGGGCA TACATGAGAT ATTGAATATC TATCATTAAA TATATAATTA GTTTTTTTCT 937 AAAACCTATC TTTAGGTTTG ATCTCGTTTG ATGTGTTGGG CTGTTTCGCA AAACAGTGTT 997 CCAATCAATA AAAAGATGTG TGTAAGACTC TAGA 1031 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 191 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Gin Thr He Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys
1 5 10 15
Thr Cys Leu Leu He Ser Tyr Thr Thr Ser Lys Phe Pro Ala Asp Tyr
20 25 30
Val Pro Thr Val Phe Asp Asn Tyr Ala Val Thr Val Met He Gly Asp
35 40 45
Glu Pro Phe Thr Leu Gly Leu Phe Asp Thr Ala Gly Gin Glu Asp Tyr
50 55 60
Asp Arg Leu Arg Pro Leu Ser Tyr Pro Ser Thr Asp Val Phe Leu Val 65 70 75 80
Cys Phe Ser Val He Ser Pro Ala Ser Phe Glu Asn Val Lys Glu Lys
85 90 95
Trp Phe Pro Glu Val His His His Cys Pro Gly Val Pro He He He
100 105 110
Val Gly Thr Gin Thr Asp Leu Arg Asn Asp Asp Val He Leu Gin Arg
115 120 125
Leu His Arg Gin Lys Leu Ser Pro He Thr Gin Glu Gin Gly Glu Lys
130 135 140
Leu Ala Lys Glu Leu Arg Ala Val Lys Tyr Val Glu Cys Ser Ala Leu 145 150 155 160
Thr Gin Arg Gly Leu Lys Thr Val Phe Asp Glu Ala He Val Ala Ala
165 170 175
Leu Glu Pro Pro Val He Lys Lys Ser Lys Lys Cys Thr He Leu 180 185 190
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2231 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 291...2195 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
AAGCTTGTTT CTTATCTCCT TAGTATATTG TTTTACAACA CCACATACAC ATACACATAT 60 AGCCTTCATT AGCCTTCATT TTGACATATT TCAATAACAA TCAAGAACTA CAAGTCATAA 120 CTGACACACA TATAATATCT TAATTGTTAT TATAAATTTA TTCTTGATTA GATTTTAGAC 180 GGGCAGAAAC AAAAACGGAA AATCCAACTC ATCCCCGATA ACTACACACA TCTATATTAA 240 ATCATCTATT AGTCTATCAG TTATATCTCC CTCCCCTTTT CTTCTAACAA ATG ATT 296
Met He 1
AAG ACG TTT CGG AAA AGT AAA AGA CTG TCG AGT AAT TCA AGT TCA CCC 344 Lys Thr Phe Arg Lys Ser Lys Arg Leu Ser Ser Asn Ser Ser Ser Pro 5 10 15
AAG AAA ACA ATA TCT CGA GTA TCA TCA ACT TCA AGT AAT CAA ACA TCT 392 Lys Lys Thr He Ser Arg Val Ser Ser Thr Ser Ser Asn Gin Thr Ser 20 25 30
CAT GAT GGA ATA TTA CAA TCA CCT AAA AAA GTC ATT AGA GCT CTA TAT 440 His Asp Gly He Leu Gin Ser Pro Lys Lys Val He Arg Ala Leu Tyr 35 40 45 50
GAT TAT GAA CCT CAA GGT CCT GGA GAA TTG AAA TTT TTC AAA GGA GAT 488 Asp Tyr Glu Pro Gin Gly Pro Gly Glu Leu Lys Phe Phe Lys Gly Asp 55 60 65
TTT TTC CAT GTA TTA AAT GAT GTT GAT GAT GAA TTA CAT AAA GAA GCG 536 Phe Phe His Val Leu Asn Asp Val Asp Asp Glu Leu His Lys Glu Ala 70 75 80
GAA CGT AAT GGA TGG ATA GAA GCA ACA AAT CCA ATG ACT CAA CTT AAA 584 Glu Arg Asn Gly Trp He Glu Ala Thr Asn Pro Met Thr Gin Leu Lys 85 90 95
GGG ATG GTC CCC ATT AGT TAT TTT GAA ATA TTT GAT CGA TCT CGT CCT 632 Gly Met Val Pro He Ser Tyr Phe Glu He Phe Asp Arg Ser Arg Pro 100 105 110
ACA GTT ACA GCA TCA TCA AAC AGT TTT ACA AAT TCC ATT GAT ATT CAA 680 Thr Val Thr Ala Ser Ser Asn Ser Phe Thr Asn Ser He Asp He Gin 115 120 125 130
CAT CAA CAT CAA CAA GGA ATT CAC AAT GGA ACA GGA AAT CGA AAT TTA 728 His Gin His Gin Gin Gly He His Asn Gly Thr Gly Asn Arg Asn Leu 135 140 145
AAT CAA ACA TTA TAT GCT GTT ACA CTA TAT GAA TTT AAA GCT GAA CGA 776 Asn Gin Thr Leu Tyr Ala Val Thr Leu Tyr Glu Phe Lys Ala Glu Arg 150 155 160
GAT GAT GAA TTG GAT ATA ATG CCT AAT GAA AAT TTA ATT ATT TGT GCA 824 Asp Asp Glu Leu Asp He Met Pro Asn Glu Asn Leu He He Cys Ala 165 170 175
CAT CAT GAT TAT GAA TGG TTT ATT GCC AAA CCA ATA AAT CGA TTA GGT 872 His His Asp Tyr Glu Trp Phe He Ala Lys Pro He Asn Arg Leu Gly 180 185 190
GGA CCA GGT TTA GTA CCT GTT TCT TAT GTT AAA ATA ATT GAT CTT TTA 920 Gly Pro Gly Leu Val Pro Val Ser Tyr Val Lys He He Asp Leu Leu 195 200 205 210 AAC CCT AAT TCT CAT TAT ACA TCA ATT GAT ACA TCA AGG CGA TCA CAA 968 Asn Pro Asn Ser His Tyr Thr Ser He Asp Thr Ser Arg Arg Ser Gin 215 220 225
GTC ATA CAA GTA ATC AAT GGA TTT AAT ATA CCG ACA GTA GAA CAA TGG 1016 Val He Gin Val He Asn Gly Phe Asn He Pro Thr Val Glu Gin Trp 230 235 240
AAA AAT CAA ACT GCC AAA TAT CAA GCT TCA ACA ATC CCC CTT GGT TCA 1064 Lys Asn Gin Thr Ala Lys Tyr Gin Ala Ser Thr He Pro Leu Gly Ser 245 250 255
ATA TCA GGA AGT GGT ACT CCA CCA ACA TCA GCT AAT TCA CAA TAT TTT 1112 He Ser Gly Ser Gly Thr Pro Pro Thr Ser Ala Asn Ser Gin Tyr Phe 260 265 270
GAT AAT CAT ACT ATG ACT TCA AAT CGA TCA TCA CTG GGT TCA TCA ATT 1160 Asp Asn His Thr Met Thr Ser Asn Arg Ser Ser Leu Gly Ser Ser He 275 280 285 290
TCT ATT ATT GAA GCT AGT GTT GAT TCA TAT CAA TTA GAT CAT GGT CGA 1208 Ser He He Glu Ala Ser Val Asp Ser Tyr Gin Leu Asp His Gly Arg 295 300 305
TAT CAA TAT TCA ATA ACT GCT CGA TTA AAT AAT GGC AGA ATA AGA TAT 1256 Tyr Gin Tyr Ser He Thr Ala Arg Leu Asn Asn Gly Arg He Arg Tyr 310 315 320
TTA TAT CGA TAT TAT CAA GAT TTT TAT GAT TTA CAA GTG AAA TTA TTA 1304 Leu Tyr Arg Tyr Tyr Gin Asp Phe Tyr Asp Leu Gin Val Lys Leu Leu 325 330 335
GAA TTA TTT CCT TAT GAA GCT GGG AGA ATT GAA AAT TCT AAA AGA ATA 1352 Glu Leu Phe Pro Tyr Glu Ala Gly Arg He Glu Asn Ser Lys Arg He 340 345 350
ATT CCA TCT ATA CCA GGA CCT TTA ATT AAT GTC AAT GAT TCA ATA TCA 1400 He Pro Ser He Pro Gly Pro Leu He Asn Val Asn Asp Ser He Ser 355 360 365 370
AAA TTA CGA AGA GAA AAA TTG GAT TAT TAT TTA TCA AAT TTA ATT GCA 1448 Lys Leu Arg Arg Glu Lys Leu Asp Tyr Tyr Leu Ser Asn Leu He Ala 375 380 385
TTA CCT AGT CAT ATA TCT CGA TCA GAA GAA GTA TTA AAA TTA TTT GAT 1496 Leu Pro Ser His He Ser Arg Ser Glu Glu Val Leu Lys Leu Phe Asp 390 395 400
GTT TTA GAT AAT GGA TTT GAT CGA GAA ACT GAT GCT ATT AAT AAA CGA 1544 Val Leu Asp Asn Gly Phe Asp Arg Glu Thr Asp Ala He Asn Lys Arg 405 410 415
TTT TCT AAA CCA ATA AGT CAA AAA TCA AAT TCT CAT CAA GAT AGA TTA 1592 Phe Ser Lys Pro He Ser Gin Lys Ser Asn Ser His Gin Asp Arg Leu 420 425 430
TCT CAA TAT TCC AAT TTT AAC GTT TTA CAA CAA CAA CAA CAA CAA CAG 1640 Ser Gin Tyr Ser Asn Phe Asn Val Leu Gin Gin Gin Gin Gin Gin Gin 435 440 445 450 CAA CAA CAG CAA TAT GCT CAT CAT TCA AGA GGT TCT GAT AAT TCA CCT 1688 Gin Gin Gin Gin Tyr Ala His His Ser Arg Gly Ser Asp Asn Ser Pro 455 460 465
ACT AAT GAA TCA TCA GGT TCA AAT TTA ATT AAT TCT TCT TCT CAT AAT 1736 Thr Asn Glu Ser Ser Gly Ser Asn Leu He Asn Ser Ser Ser His Asn 470 475 480
GAT TCA TCA TTA TCT TCA TCA CCA CCA CCA CCA CCA CCA CAA ACT GTC 1784 Asp Ser Ser Leu Ser Ser Ser Pro Pro Pro Pro Pro Pro Gin Thr Val 485 490 495
ACC ACC ACG AAC ACC ACG AAC ACC ACC ATA ACC ACA GAC TCC TCA TCA 1832 Thr Thr Thr Asn Thr Thr Asn Thr Thr He Thr Thr Asp Ser Ser Ser 500 505 510
AAA CAA CCA AAA GCC AAA GTG AAA TTT TAT TTT GAT GAT GAT ATA TTT 1880 Lys Gin Pro Lys Ala Lys Val Lys Phe Tyr Phe Asp Asp Asp He Phe 515 520 525 530
GTA TTA TTA ATC CCA ACC AAT TTA CGA TTA CAA GAT TTA AAA TCA AAA 1928 Val Leu Leu He Pro Thr Asn Leu Arg Leu Gin Asp Leu Lys Ser Lys 535 540 545
TTA TTT AAA CGA TTA GAA TTG GAT ATT ACT TAT AAA TAT GAA AAA CCT 1976 Leu Phe Lys Arg Leu Glu Leu Asp He Thr Tyr Lys Tyr Glu Lys Pro 550 555 560
GAT CAA CAA CAA AAA CCT ACA TCA GAA TCA ATT CAT TTA TTT TTG AAA 2024 Asp Gin Gin Gin Lys Pro Thr Ser Glu Ser He His Leu Phe Leu Lys 565 570 575
AAT GAT TTT GAA GAT TTT TTA ATT GAA AAT GAA ACT AGC AAC AAC AAC 2072 Asn Asp Phe Glu Asp Phe Leu He Glu Asn Glu Thr Ser Asn Asn Asn 580 585 590
AAT CTG GAA ATT GAT TTC GAA AAT GAA ATT ATT AAA GAA AAA TTA GGA 2120 Asn Leu Glu He Asp Phe Glu Asn Glu He He Lys Glu Lys Leu Gly 595 600 605 610
GAA TTT GAA GTT AAT GAT GAT GAA AAA TTT CAA AGT ATT TTA TTT GAT 2168 Glu Phe Glu Val Asn Asp Asp Glu Lys Phe Gin Ser He Leu Phe Asp 615 620 625
AAA TGT AAA TTA ATG GTT TTA GTA TAT TAAACAGAGA TCAATAAGAG AGAGAGA 2222 Lys Cys Lys Leu Met Val Leu Val Tyr 630 635
GAGAGACAT 2231
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 635 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Met He Lys Thr Phe Arg Lys Ser Lys Arg Leu Ser Ser Asn Ser Ser
1 5 10 15
Ser Pro Lys Lys Thr He Ser Arg Val Ser Ser Thr Ser Ser Asn Gin
20 25 30
Thr Ser His Asp Gly He Leu Gin Ser Pro Lys Lys Val He Arg Ala
35 40 45
Leu Tyr Asp Tyr Glu Pro Gin Gly Pro Gly Glu Leu Lys Phe Phe Lys
50 55 60
Gly Asp Phe Phe His Val Leu Asn Asp Val Asp Asp Glu Leu His Lys 65 70 75 80
Glu Ala Glu Arg Asn Gly Trp He Glu Ala Thr Asn Pro Met Thr Gin
85 90 95
Leu Lys Gly Met Val Pro He Ser Tyr Phe Glu He Phe Asp Arg Ser
100 105 110
Arg Pro Thr Val Thr Ala Ser Ser Asn Ser Phe Thr Asn Ser He Asp
115 120 125
He Gin His Gin His Gin Gin Gly He His Asn Gly Thr Gly Asn Arg
130 135 140
Asn Leu Asn Gin Thr Leu Tyr Ala Val Thr Leu Tyr Glu Phe Lys Ala 145 150 155 160
Glu Arg Asp Asp Glu Leu Asp He Met Pro Asn Glu Asn Leu He He
165 170 175
Cys Ala His His Asp Tyr Glu Trp Phe He Ala Lys Pro He Asn Arg
180 185 190
Leu Gly Gly Pro Gly Leu Val Pro Val Ser Tyr Val Lys He He Asp
195 200 205
Leu Leu Asn Pro Asn Ser His Tyr Thr Ser He Asp Thr Ser Arg Arg
210 215 220
Ser Gin Val He Gin Val He Asn Gly Phe Asn He Pro Thr Val Glu 225 230 235 240
Gin Trp Lys Asn Gin Thr Ala Lys Tyr Gin Ala Ser Thr He Pro Leu
245 250 255
Gly Ser He Ser Gly Ser Gly Thr Pro Pro Thr Ser Ala Asn Ser Gin
260 265 270
Tyr Phe Asp Asn His Thr Met Thr Ser Asn Arg Ser Ser Leu Gly Ser
275 280 285
Ser He Ser He He Glu Ala Ser Val Asp Ser Tyr Gin Leu Asp His
290 295 300
Gly Arg Tyr Gin Tyr Ser He Thr Ala Arg Leu Asn Asn Gly Arg He 305 310 315 320
Arg Tyr Leu Tyr Arg Tyr Tyr Gin Asp Phe Tyr Asp Leu Gin Val Lys
325 330 335
Leu Leu Glu Leu Phe Pro Tyr Glu Ala Gly Arg He Glu Asn Ser Lys
340 345 350
Arg He He Pro Ser He Pro Gly Pro Leu He Asn Val Asn Asp Ser
355 360 365
He Ser Lys Leu Arg Arg Glu Lys Leu Asp Tyr Tyr Leu Ser Asn Leu
370 375 380
He Ala Leu Pro Ser His He Ser Arg Ser Glu Glu Val Leu Lys Leu 385 390 395 400
Phe Asp Val Leu Asp Asn Gly Phe Asp Arg Glu Thr Asp Ala He Asn 405 410 415 Lys Arg Phe Ser Lys Pro He Ser Gin Lys Ser Asn Ser His Gin Asp
420 425 430
Arg Leu Ser Gin Tyr Ser Asn Phe Asn Val Leu Gin Gin Gin Gin Gin
435 440 445
Gin Gin Gin Gin Gin Gin Tyr Ala His His Ser Arg Gly Ser Asp Asn
450 455 460
Ser Pro Thr Asn Glu Ser Ser Gly Ser Asn Leu He Asn Ser Ser Ser 465 470 475 480
His Asn Asp Ser Ser Leu Ser Ser Ser Pro Pro Pro Pro Pro Pro Gin
485 490 495
Thr Val Thr Thr Thr Asn Thr Thr Asn Thr Thr He Thr Thr Asp Ser
500 505 510
Ser Ser Lys Gin Pro Lys Ala Lys Val Lys Phe Tyr Phe Asp Asp Asp
515 520 525
He Phe Val Leu Leu He Pro Thr Asn Leu Arg Leu Gin Asp Leu Lys
530 535 540
Ser Lys Leu Phe Lys Arg Leu Glu Leu Asp He Thr Tyr Lys Tyr Glu 545 550 555 560
Lys Pro Asp Gin Gin Gin Lys Pro Thr Ser Glu Ser He His Leu Phe
565 570 575
Leu Lys Asn Asp Phe Glu Asp Phe Leu He Glu Asn Glu Thr Ser Asn
580 585 590
Asn Asn Asn Leu Glu He Asp Phe Glu Asn Glu He He Lys Glu Lys
595 600 605
Leu Gly Glu Phe Glu Val Asn Asp Asp Glu Lys Phe Gin Ser He Leu
610 615 620
Phe Asp Lys Cys Lys Leu Met Val Leu Val Tyr 625 630 635

Claims

WE CLAIM :
1. An in vi tro screening test for compounds to inhibit the biological activity of at least one protein selected from the group consisting of CaCla4p, Cst20p, CaCdc42p and CaBemlp, which comprises: a) at least one of said proteins; and b) means to monitor the biological activity of said at least one protein; thereby compounds are tested for their inhibiting potential .
2. The screening test of claim 1, wherein the inhibition of the interactions between CaCla4p and Ca Cdc42p is determined.
3. The screening test of claim 1, wherein the inhibition of the interactions between Cst20p and CaCdc42p is determined.
4. The screening test of claim 1, wherein the inhibition of the interactions between CaCla4p and CaBemlp is determined.
5. The screening test of claim 1, wherein the inhibition of the interactions between Cst20p and CaBemlp is determined.
6. A method for determining at least one gene involved in filamentous growth associated with virulence, which comprises using one protein selected from the group consisting of CaCla4p, Cst20, CaCdc42p and CaBemlp to determine said gene.
PCT/CA1997/000809 1996-10-30 1997-10-29 Candida albicans proteins associated with virulence and hyphal formation and uses thereof WO1998018927A1 (en)

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US10/093,524 US20030166886A1 (en) 1996-10-30 2002-03-11 Candida albicans proteins associated with virulence and hyphal formation and uses thereof

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Cited By (9)

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US7033621B1 (en) 1997-04-28 2006-04-25 Novogen, Inc. Isoflavone compositions produced from legumes
US6639064B1 (en) * 1999-09-17 2003-10-28 New York University NRIF3, novel co-activator for nuclear hormone receptors
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US7763660B2 (en) * 2003-02-05 2010-07-27 The University Of Vermont And State Agricultural College Inhibitors of Candida albicans
CN113502345A (en) * 2021-04-23 2021-10-15 江苏师范大学 Application of aspergillus flavus pathogenic gene cdc48 in screening of medicines for preventing and treating aspergillus flavus pollution

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