AU775543B2 - Novel candida albicans genes and proteins coded by said genes - Google Patents

Description

Novel Candida albicans genes and proteins coded by these genes.

The present invention relates to novel Candida albicans genes and the proteins coded by these genes as well as the polynucleotides (RNA, DNA) coding for these proteins or for the polypeptide analogues of these proteins.

The present invention also relates to the process for the preparation of these polypeptides and polynucleotides, their use for studying pathogenic mycetes and in particular Candida albicans and for the preparation of inhibitors of the proteins coded by the genes of the present invention, these inhibitors being able to be used as antifungal agents. The present invention also relates to the pharmaceutical compositions containing such inhibitors.

Therefore the present invention relates in particular to novel proteins of Candida albicans and the nucleotide sequences coding for these proteins, their preparation and their uses.

Also hereafter the following abbreviations will be used: AA for amino acids, NA for nucleic acids, RNA for ribonucleic acid, mRNA for messenager RNA, RNase for ribonuclease, DNA for desoxyribonucleic acid, cDNA for complementary DNA, bp for base pairs, PCR for polymerase chain reaction, C.a. or C.

albicans for Candida albicans, E. coli for Escherichia coli and S. cerevisiae for Saccharomyces cerevisiae.

The term screening used hereafter corresponds to the anglosaxon term screening.

The term polynucleotides designates hereafter the polynucleotides of the present invention or the DNA sequences and also RNA coding for the proteins of the present invention and their homologues coding for proteins with the same function.

The term polypeptides designates hereafter the polypeptides of the present invention or the proteins of the present invention and their functional analogues or homologues as defined hereafter, therefore having the same functions.

2 The term mycete designates hereafter a eucaryote organism, spore carrier, the nutrition of which occurs by absorption, which is devoid of chlorophyll and which reproduces in a sexual or asexual fashion.

Mycoses are infections of man or animals which can be superficial or deep, caused by pathogenic fungi. In the case of deep mycoses, they can be very severe and with a grave pronosis.

Antimycotic substances with fungistatic or fungicidal effects are used in the treatment of mycoses. This treatment is difficult because few available antifungal substances exist for therapeutics and they often have side effects which 0 limit their use. For example, Amphotericin B which represents the treatment of choice for deep mycoses, has nephrotoxic side effets.

Therefore a strong demand exists for novel substances which are effective against pathogenic fungi and capable of being used in therapeutics against fungal infections. These substances could be used either in prophylaxis, in the case of severe states of immunodepression or in curative treatment of fungal infections. In addition, these substances should have a specific mode of action, allowing them to inhibit the growth or to kill the cells of mycetes without altering the essential functions of the human cells.

25 A subject of the present invention is to propose genes which can constitute novel targets for the identification of antifungal substances and in particular of substances allowing the treatment of the infections due to fungi of the Candida genus.

These genes are in particular essential genes which are indispensable to the survival and multiplication of the cells.

Different methods can be used for determining whether the product of a gene is essential to the survial of a mycete or essential to the establishment or maintenance of an infection. The identification of the essential character of a gene provides additional information concerning its function and allows selection of the genes the product of which constitutes a useful target for an antifungal substance. Examples of these methods are briefly summarized hereafter. These methods are described in the following publications: Guthrie C. and Fink G.R. Eds. Methods in Enzymology, Vol 194, 1991, 'Guide to Yeast Genetics and Molecular Biology', Academic Press Inc.

Pink A.E. Wheals and J.S. Harrison Eds. The yeasts, Vol.6, 1995, 'Yeast Genetics' Academic Press Inc.

Ausubel F. et al. Eds. 'Short Protocols in Molecular Biology', 1995, Wiley.

Brown A.J.P. and Tuite M.F. (Eds)'Yeast Gene Analysis' 0 Methods in Microbiology, Vol 26, 1998, Academic Press Inc.

Depending on the case, one or the other of the methods described will be used as a function of the sought result.

In particular, the operation can be carried out by a direct inactivation method of the gene or by a transitory inactivation method of the gene.

In the yeast S. cerevisiae, the most commonly used method consists in inactivating the studied gene in the yeast chromosome. The wild allele is inactivated by insertion of a genetic marker (for example an auxotrophic gene or a resistance marker). This insertion is obtained in general by the genic conversion method using linear deletion cassettes S 25 prepared according to known methods as described in Guthrie C. and Fink G.R. Eds. Methods in Enzymology, Vol 194, 1991, 'Guide to Yeast Genetics and Molecular Biology', Academic Press Inc. or in Gultner et al. Nucleic Acid Research, 1996, 24: 2519-2524.

The inactivation occurs in a diplod strain then meiosis is induced by standard methods such as for example growth in a nitrogen-poor medium and the four spores originating from individual ascus are isolated by micromanipulation. The inactivation of an essential gene translates into a loss of viability of two spores (in four) which have acquired the selection marker. The viability of these spores can be restored by the introduction into the strain of a centromeric or replicative plasmid carrying a copy of the wild gene.

II

The operation can also be carried out by transitory inactivation of the gene: the use of controllable promoters also allows the determination of whether a gene is essential to the survival of a cell. In order to do this, the native promoter of the gene is replaced by a promoter which is directly controllable on the chromosome or on an extrachromosomic plasmid. For example the GAL promoter or its derivatives or the tetO promoter can be used (Mumberg et al.

1994, Nucleic Acid Research, 22: 5767-5768; Belli et al.

1998, Yeast, 14: 1127-1138). The essential character of the studied gene can thus be observed when the promoter used is repressed, either in the haploid strains in the yeast S.

0cerevisiae, or after inactivation of the second allele in diploid micro-organisms such as C. albicans.

Starting from an essential gene known in a species, identification can be carried out of homologous genes or genes having a similar function in another species of mycete: known methods can be used to identify the homologous genes of a studied gene in another species of mycete (so-called 'orthologous' genes) or genes with a similar function to the studied gene. Examples of methods which can be used are set out hereafter. These methods are described in the following books: Sambrook et al. 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press.

Ausubel F. et al. Eds. 'Short Protocols in Molecular Biology', 1995, Wiley.

Guthrie C. and Fink G.R. Eds. Methods in Enzymology, Vol 194, 1991, 'Guide to Yeast Genetics and Molecular Biology', Academic Press Inc.

The operation can be carried out for example by screening by homology, by genic complementation or also by amplification by PCR using specific probes from genomic DNA libraries or from complementary DNA (cDNA) libraries of the pathogenic mycetes.

The genomic DNA or cDNA libraries can be prepared according to known methods and the polynucleotide fragments obtained are integrated in an expression vector, for example a vector such as pRS423 or its derivatives which are also as useful in the E. coli bacteria as in S. cerevisiae.

Screening of the bank will be done by standard in situ hybridization methods on a replica of the bacterial colonies.

The hybridization conditions are adapted to the stringency desired for the reaction, so as to identify the fragments with more or less high homology with the gene studied.

The genes of other species of mycetes can also be identified by known methods called 'genic complementation'.

For example, a strain of S. cerevisiae in which an identified essential gene has been placed under the control of a controllable promoter can be transformed by a representative S sample of a DNA or cDNA bank corresponding to the studied mycete such as C. albicans. When the yeasts are cultured under conditions such that the promoter is repressed, only the yeasts carrying a recombinant vector containing a functionally equivalent sequence of the studied mycete with the initial essential gene can survive. The sequence of the gene in the studied mycete is then identified by isolating the recombinant vector and by sequencing according to known methods. Similarly, the so-called 'plasmid shuffle' method allows selection of the yeasts which have lost the expression of the initial essential gene and containing a functionally equivalent sequence originating from another mycete.

S 25 The study can be carried out on different species: the functionally equivalent genes or homologues in sequence with an essential gene can be isolated in other mycetes and in particular in the different pathogenic mycetes affecting humans. For this the mycetes belonging to the Zygomycetes, Basidiomycetes, Ascomycetes and Deuteromycetes classes can in particular be used. Quite particularly, the mycetes belonging to the following sub-classes: Candida spp., in particular Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei. The mycetes also belonging to the following sub-classes: Aspergillus fumigatus, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatidis, Paracoccidioides brasiliensis and Sprorothrix schenckii.

The present invention therefore relates to the identification of antimycotic substances such as in particular anti-Candida albicans substances.

The present invention therefore relates to inhibitors of fungal proteins which can be used as antifungal agents.

Thus organisms are known such as the pathogenic yeast Candida albicans which cause infectious diseases in the human organism. With the purpose of finding the means of treating diseases, targets can be chosen such as for example intracellular and one or more proteins of the present invention coded by the genes of the present invention can be one or some of these targets.

The present invention thus allows isolation of the DNA and RNA polynucleotides coding for the proteins of Candida albicans and revelation of their nucleotide sequences.

The genes coding for the proteins of Candida albicans will be called as follows: CaDR472, caDR489, CaDR527 in the form of two different alleles namely 1CaDR527 and 2CaDR527, CaFL024, CaNL260 and CaDR361.

The nucleotide sequences of these genes (and of the two alleles for CaDR527) are given in the sequence listing hereafter and are called respectively as follows: SEQ ID No. for CaDR472, S- SEQ ID No. 1 for CaDR472, 25 SEQ ID No. 3 for the CaDR489,llele ofCaDR527 namely CaDR527, 25 SEQ ID No. 5 for the 1 st allele of CaDR527 namely 1 CaDR527, SEQ ID No. 7 for the 2 n d allele of CaDR527 namely 2CaDR527, SEQ ID No. 9 for CaFL024, SEQ ID No. 11 for CaNL260, and SEQ ID No. 13 for CaDR361.

The polypeptide sequences of the proteins coded by the genes are called respectively as follows: SEQ ID No. 2 or PCaDR472 for the protein coded by CaDR472, SEQ ID No. 4 or PCaDR489 for the protein coded by CaDR489, 08/06/04.a I 2396.specipgs.6 SEQ ID No. 6 or IPCaDR527 for the protein coded by 1CaDR527, SEQ ID No. 8 or 2PCaDR527 for the protein coded by 2CaDR527, SEQ ID No. 10 or PCaFL024 for the protein coded by CaFL024, SEQ ID No. 12 or PCaNL260 for the protein coded by CaNL260, and SEQ ID No. 14 or PCaDR361 for the protein coded by CaDR361.

Therefore a subject of the present invention is isolated polynucleotides each containing a nucleotide sequence chosen from the following group: a) a polynucleotide having at least 50'% or at least 60% and preferably at least identity with a polynucleotide coding for a polypeptide having the same function and having an amino acid sequence homologous with SEQ ID No. 12 as defined above and hereafter, b) a complementary polynucleotide of polynucleotide a).

Therefore a subject of the present invention is the polynucleotides defined above such that these polynucleotides are DNA.

Therefore a subject of the present invention is the polynucleotides defined above such that these polynucleotides are RNA.

A more precise subject of the present invention is the polynucleotides as defined 25 above each comprising a nucleotide sequence of SEQ ID No. 11 as defined above and hereafter.

The present invention thus allows the isolation of the DNA sequences coding respectively for the proteins of Candida albicans PCaNL260 as described above.

The present invention also allows revelation of the nucleic acid sequences of the genes of the present invention and also the amino acid sequences of the proteins of PCaNL260 coded by these genes.

04/06/04.at 2396.specipgs,7 Therefore a subject of the present invention is the DNA sequences as defined by the polynucleotides above, characterised in that these DNA sequences are those of the genes coding respectively for the proteins of Candida albicans (having the same functions as the proteins PCaNL260), and each containing a nucleotide sequence of SEQ ID No. 11 as described above.

A subject of the present invention is also the DNA sequences of genes as defined above each coding for an amino acid sequence of SEQ ID No. 12.

The sequence SEQ ID No. 12 of the protein PCaNL260 therefore comprises 148 amino acids.

A particular subject of the present invention is the DNA sequences coding for the proteins of PCaNL260 as defined above as well as the DNA sequences which hybridize with these and/or present significant homologies with these sequences or with fragments of these and code for the proteins having the same functions.

A subject of the present invention is also the DNA sequences as defined above comprising modifications introduced by suppression, insertion and/or substitution of at least one nucleotide coding for proteins having the same activities as the proteins of PCaNL260 as defined above.

In particular a subject of the present invention is the DNA sequences as defined o above as well as the DNA sequences which have a nucleotide sequence homology of at o# least 50% or at least 60% and preferably at least 70% with said DNA sequences.

Therefore a subject of the present invention is also the DNA sequences as defined above as well as the DNA sequences which code for the proteins of similar functions of which the respective AA sequences have a homology of at least 40% and in particular of or of at least 50%, rather at least 60% and preferably at least 70% with the AA sequences coded by said DNA sequences.

04/06/04.a 12396 specipgs.8 By sequences which hybridize are included DNA sequences which hybridize with one of the DNA sequences above under standard conditions of high, medium or low stringency and which code for polypeptides having the same function. The stringency conditions are those carried out under conditions S* S *o* 08/06/04,atl 2396.specipgs,9 known to a person skilled in the art, such as those described by Sambrook et al Molecular cloning, Cold Spring Harbor Laboratory Press, 1989. Such stringency conditions are for example hybridization at 65 0 C, for 18 hours in a 5 x SSPE; x Denhardt's; 100pg/ml ssDNA; 1 SDS solution followed by washing 3 times for 5 minutes with 2 x SSC; 0.05 SDS, then washing 3 times for 15 minutes at 65 0 C in 1 x SSC; 0.1 SDS.

The high stringency conditions for example include hybridization for 18 hours at 65 0 C in a 5 x SSPE; 10 x Denhardt's; 100pg/ml ssDNA; 1 SDS solution, followed by washing twice for 20 minutes with a 2 x SSC; 0.05 SDS solution at 65 0 C followed by a final wash for 45 minutes in a 0.1 x SSC; 0.1 SDS solution at 65 0 C. Medium stringency conditions for example include a final washing for 20 minutes in a 0.2 x SSC, 0.1 SDS solution at 65 0

C.

By sequences which have significant homologies are included the sequences having a moderate or considerable identity of nucleotide sequence with one of the DNA sequences above and which code for a protein having the same function.

By sequence of similar DNA, is thus meant the DNA sequences which can belong to mycetes other than Candida albicans and in particular to S.c. and which are similar or identical to the DNA sequences of the genes of Candida albicans as defined above. These similar DNA sequences are not necessarily identical to the DNA sequences of the genes as defined above. The homology of sequence at the nucleotide level can be moderate or considerable. The present invention thus relates in particular to the DNA sequences which have an homology of nucleotide sequence of at least 50 of preferably at least 60 and even more preferably of at least with the sequences of the genes of the present invention.

Moreover, these similar DNA sequences do not necessarily code for identical proteins, at the level of the amino acid sequences to the proteins coded by the genes as defined above. Thus the present invention relates in particular to the DNA sequences which code for the so-called homologous proteins having an homology of amino acid sequence of at 11 least 40%, in particular 45%, preferably at least of 50%, more preferably at least of and yet more preferably at least of 70% with the proteins coded by the genes of the present invention.

Each gene of the present invention is represented as a single strand DNA sequence of SEQ ID No. 11, as indicated in the sequence listing hereafter, but it is understood that the present invention includes the complementary DNA sequence of this single strand DNA sequence and also includes the so-called double strand DNA sequence constituted by these two DNA sequences complementary to one another.

The DNA sequences as defined above are examples of the combination of codons coding for the amino acids corresponding respectively to the amino acid sequences of SEQ ID No. 12, as defined above, but it is also understood that the present invention includes any other arbitrary combination of codons coding for these same amino acid sequences.

For the preparation of the polynucleotides and in particular of the DNA sequences as defined above, the DNA sequences modified as indicated above or also the homologous DNA sequences as defined above, the techniques known to a person skilled in the art can 20 be used and in particular those described in the work by Sambrook, J. Fritsch, E.F. Maniatis, T. (1989) entitled: Molecular cloning, a laboratory manual, Laboratory, Cold Spring Harbor NY.

9 The homologous DNA sequences as defined above can in particular be isolated 25 according to the methods known to a person skilled in the art for example by the PCR technique using degenerated nucleotide primers to amplify this DNA from genome or cDNA libraries of the corresponding mycetes. The cDNA can also be prepared from mRNA isolated from mycetes of different species studied within the scope of the present invention such as Candida albicans but for example and quite as well: Candida stellatoidea, Candida tropicalis, Candida stellatoidea, Candida tropicalis, Candida 16/06/04.at 12396specipgs, 11

II

parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida glabrata, Candida lusianiae or Candida rugosa or also mycetes such as Saccharomyces cerevisiae or also the mycetes of Aspergillus or Cryptococcus type and in particular, for example, Aspergillus fumigatus, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliens and Sporothrix schenckii or also the mycetes of the phycomycetes or eumycetes classes in particular the subclasses of basidiomycetes, ascomycetes, mehiascomycetales (yeast) and plectascales, gymnascales (fungi of the skin and hair) or of the hyphomycetes class, in particular the subclasses conidiosporales and thallosporales amongst which the following species: mucor, rhizopus, coccidioides, paracoccidioides (blastomyces, brasiliensis), endomyces (blastomyces), aspergillus, menicilium (scopulariopsis), trichophyton (ctenomyces), epidermophton, microsporon, piedraia, hormodendron, phialophora, sporotrichon, cryptococcus, candida, geotrichum, trichosporon or also toropsulosis.

The polynucleotides of the present invention can thus be obtained by using the usual cloning and screening methods such as those of cloning and sequencing from fragments of chromosomal DNA extracted from cells or also originating from S 25 gene banks. For example, in order to obtain the polynucleotides of the present invention, a bank of chromosomal DNA fragments can be used. A probe corresponding to an oligonucleotide labelled with a radioactive element, preferably constituted by 17 or also 20 or more nucleotides and derived from a partial sequence can be prepared. The clones containing DNA identical to that of the probe can be identified in this way under stringent conditions. By the sequencing of the individual clones identified in this way, using the sequencing primers originating from the original sequence, it is then possible to extend the sequence in both directions in order to determine the complete gene sequence.

In a usual and efficient fashion, such sequencing can be carried out by using denatured double strand DNA prepared 13 from a plasmid. Such techniques are described by Maniatis, T. Fritsh, E.F. and Sambrook as indicated above. (Laboratory Manual, Cold Spring Harbor, New York (1989), in particular in 1.90 and 13.70 in the chapters on screening by hybridization and sequencing from denatured double strand DNA).

Within the scope of the present invention, a bank of chromosomal DNA fragments of Candida albicans can in particular be used as indicated hereafter in the examples described in the experimental part.

A detailed description of the operating conditions in which the present invention has been carried out is given below.

A quite particular subject of the invention is the polypeptides each having an amino acid sequence of SEQ ID No. 12 coded by the DNA sequences as defined above and the analogues of these polypeptides.

By polypeptide analogues, are understood polypeptides, the amino acid sequence 0* 20 of which has been modified by substitution, suppression or addition of one or more amino acids but which retain the same biological function. Such polypeptide analogues can be produced spontaneously or can be produced by post-transcriptional modification or also by modification of the DNA sequence of the present invention as indicated above, using techniques known to a person skilled in the art: amongst these techniques, the technique of 25 directed mutagenesis known to a person skilled in the art (Kramer et al., Nucl. Acids Res., 12, 9441 (1984); Kramer, W. and Fritz, Methods in Enzymology, 154, 350 (1987); Zoller, M.J. and Smith, M. Methods in Enzymology, 100, 468 (1983)) can in *6 particular be mentioned.

C Modified DNA synthesis can be carried out as indicated above and in particular by using well known chemical synthesis techniques such as for example the phosphotriester method [Letsinger, R.L. and Ogilvie, K. Am. CHEM. Soc., 91, 3350 (1969); Merrifield, Sciences, 150, 178 (1968)] or the phosphoamidite method [Beaucage, S.L. and Caruthers, M Tetrahedron Lett., 22, 1859 (1981); McBride, L.J. and 04/06/04.at 2396.specipgs. 13 14 Caruthers, M.H. Tetrahedron Lett., 24 245 (1983)] or also the combination of these methods.

The polypeptides of the present invention can therefore be prepared using techniques known to a person skilled in the art, in particular partially by chemical synthesis or also by the recombinant DNA technique by expression in a procaryotic or eucaryotic host cell as indicated hereafter.

A particular subject of the present invention is the process for the preparation of recombinant proteins PCaNL260 having the amino acid sequence of SEQ ID No. 12, as defined above, comprising, for the preparation of each of these proteins, the expression in an appropriate host of the DNA sequence as defined above coding for this protein then the isolation and the purification of said recombinant protein.

To produce the polypeptides of the present invention, recombinant DNA techniques using genetic engineering and cell culture methods known to a person skilled in the art can in particular be used. The following stages can then be carried out: firstly preparation of the appropriate gene, then incorporation of this gene into a vector, transfer of the carrier vector of the gene into an appropriate host cell, production of the polypeptide by expression of the gene, isolation of the polypeptide, the polypeptide thus produced can then be purified.

25 The polypeptides of the present invention obtained by expression of the polynucleotides of the present invention can be purified from cell cultures transformed by methods well known to a person skilled in the art such as precipitation with ammonium sulphate or ethanol, extraction under acid conditions, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, 30 hydroxylapatite chromatography and high performance liquid chromatography (HPLC).

Techniques well known to a person skilled in the art can be used to regenerate the protein when it is denatured during its isolation or purification.

The DNA sequences according to the present invention and in particular SEQ ID No. 11 can be prepared according to techniques known to a person skilled in the art in particular by chemical synthesis or by screening of a gene bank or a cDNA bank using synthetic oligonucleotide probes by known hybridization techniques, as well as amplification of DNA from isolated fragments or also by reverse transcriptase from messenger RNA (mRNA) 04/06/04,a1 2396 specipgs,1 4 The advantage of the technique comprising firstly the isolation of mRNA by extraction of the total RNA then the synthesis of cDNA from these mRNA by reverse transcriptase in particular rests on the fact that the mRNA does not contain introns even though these non-coding sequences can be present in the genomic DNA.

The usual cloning techniques known to a person skilled in the art and in particular described in the book by Sambrook, J. Fritsh, E. F. Maniatis, T. (1989) entitled: Molecular cloning: a laboratory manual, Laboratory, Cold Spring Harbor NY, can then be carried out.

In these techniques, cloning can be carried out by insertion of a fragment into a plasmid which can be provided with a suitable commercial kit then transformation of a bacterial strain by the plasmid thus obtained. In particular the XL1 Blue or DH5 alpha E.

coli strain can be used. The clones can then be cultured in order to extract the plasmid DNA according to standard techniques known to a person skilled in the art referred to above (Sambrook, Fritsh and Maniatis). The DNA sequencing of the amplified fragment contained in the plasmid DNA can then be carried out.

2 The polypeptides of the present invention can be obtained by expression in a host cell containing a polynucleotide according to the present invention and in particular a DNA sequence coding for a polypeptide of the present invention preceded by a suitable promoter sequence. The host cell can be a procaryotic cell, for example E. coli or a eucaryotic cell 25 such as yeasts such as for example Ascomycetes amongst which is Saccharomyces or also mammalian cells such as Cos cells for example.

A particular subject of the present invention is the expression vectors each containing one of the DNA sequences of the present invention as defined above.

oo In each of these expression vectors, such a DNA sequence is therefore in particular the DNA sequence of a gene of the present invention coding for a protein of Candida albicans and containing a nucleotide from SEQ ID No. 11.

In each of these expression vectors, such a DNA sequence is thus even more particularly that of the genes as defined above coding for one of the amino acid sequence of SEQ ID No. 12 as defined above and hereafter.

04/06/04.aI 2396.spcipgs. In each of the expression vectors of the present invention, such a DNA sequence is thus a DNA sequence as defined above coding for the proteins of PCaNL260, as well as the DNA sequences which hybridize with this and/or have significant homologies with this sequence or with the fragments of this or also the DNA sequences comprising modifications introduced by suppression, insertion and/or substitution of at least one nucleotide coding for a protein having the same activity.

In each of the expression vectors, such a DNA sequence is in particular a DNA sequence as defined above as well as similar DNA sequences which have a nucleotide sequence homology of at least 50 or at least 60 and preferably at least 70 with said DNA sequence or also similar DNA sequences which code for a protein, the AA sequence of which has an homology of at least 40 and in particular of 45 or of at least 50 rather at least 60 and preferably at least 70 with the AA sequence coded by said DNA sequence.

The expression vectors are vectors allowing the expression of the protein under the control of a suitable promoter. Such a vector can be a plasmid, a cosmid or viral DNA.

For the procaryotic cells, the promoter can for example be the lac promoter, the trp promoter, the tac promoter, the p-lactamase promoter or the PL promoter. For the yeast cells, the promoter can be for example the PGK promoter or the GAL promoter. For mammalian cells, the promoter can for example be the SV40 promoter or adenovirus promoters.

Baculovirus type vectors can also be used for the expression in insect cells.

The host cells are for example procaryotic cells or eucaryotic cells. The procaryotic cells are for example E. coli, Bacillus or Streptomyces. The eucaryotic host cells include 30 yeasts as well as cells of higher organisms, for example mammalian cells or insect cells.

The mammalian cells are for example hamster CHO or BHK cells and monkey Cos cells.

The insect cells are for example SF9 cells.

The present invention therefore relates to a process which comprises the expression of a polynucleotide according to the present invention coding for the proteins of PCaNL260, in a host cell transformed by a polynucleotide according to the present invention and in particular a DNA sequence coding for the amino acid sequence of SEQ ID No. 12. In the implementation of such a process, the host cell is in particular a eucaryote cell.

08/06/04,a 12396 specipgs.16 17 For the implementation of the present invention, the vectors used can for example be pGEX or pBAD and the host cell can be E. coli or for example the vector pYX222 and the host cell can be in particular Saccharomyces cerevisiae.

A particular subject of the present invention is the host cell transformed with a vector as defined above and containing a DNA sequence according to the present invention.

A subject of the present invention is therefore the process for the preparation of a recombinant protein according to the present invention, as defined above, in which the host cell is DH5 alpha E. coli or XL1-Blue E. coli or in particular Saccharomyces cerevisiae.

A detailed account of the conditions under which the operations indicated above can be carried out is given hereafter in the experimental part. A plasmid is thus obtained in which the gene of the present invention is inserted and this plasmid introduced into a host cell is then obtained by operating according to the usual techniques known to a person *skilled in the art.

A very precise subject of the present invention is the plasmids deposited on the 25 h May 1999 at the Collection Nationale de Cultures de Microorganismes (CNCM) INSTITUT PASTEUR 25, rue du Docteur Roux 75724 PARIS Cedex 15 under the number: 1-2212. Also deposited on the same date were 1-2214, 1-2215, 1-2216, 1-2217, 25 1-2211 and 1-2213.

1-2214 is the registration number of the strain CaDR472.10 constituted by the oooo bacteria XL 1-blue E. coli containing a plasmid carrying the gene of Candida albicans CaDR472 prepared as indicated in Example 1 of the present specification.

This gene therefore corresponds to the sequence CaDR472 of SEQ ID No. 1.

1-2215 is the registration number of the strain CaDR489.37 constituted by the bacteria XL I-blue E. coli containing a plasmid carrying the gene of Candida aibicans CaDR489 prepared as indicated in Example 2 of the present specification.

16/06/04,at 2396.specipgs, 17 18 This gene therefore corresponds to the sequence CaDR489 of SEQ ID No. 3.

1-2216 is the registration number of the strain CaDR527.2 constituted by the bacteria XL1blue E. coli containing a plasmid carrying the gene of Candida aibicans CaDR527 (allele 1) prepared as indicated in Example 3 of the present specification.

This gene therefore corresponds to the sequence 1CaDR527 of SEQ ID No. 1-2217 is the registration number of the strain CaDR527.3 constituted by the bacteria XL 1-blue E. coli containing a plasmid carrying the gene of Candida aibicans CaDR527 (allele 2) prepared as indicated in Example 3 of the present specification.

This gene therefore corresponds to the sequence 2CaDR527 of SEQ ID No. 7.

1-2211 is the registration number of the strain CaFL024.4 constituted by the bacteria XL -blue E. coli containing a plasmid carrying the gene of Candida albicans CaFL024 prepared as indicated in Example 4 of the present specification.

20 This gene therefore corresponds to the sequence CaFL024 of SEQ ID No. 9.

1-2212 is the registration number of the strain CaNL260.4 constituted by the bacteria XL1-blue E. coli containing a plasmid carrying the gene of Candida albicans CaNL260 of the present invention prepared as indicated in Example 5 of the present 25 specification.

This gene therefore corresponds to the sequence CaNL260 of SEQ ID No. 11.

1-2213 is the registration number of the strain CaDR361.3 constituted by the bacteria XL 1-blue E. coli containing a plasmid carrying the gene of Candida albicans CaDR361 prepared as indicated in Example 6 of the present specification.

This gene therefore corresponds to the sequence CaDR361 of SEQ ID No. 13.

16/06/04.at 12396.spcipgs, 18 19 The operating conditions under which the present invention was carried out are described hereafter in the experimental part.

Therefore a subject of the present invention is a screening process for antifungal products characterized in that it comprises a stage where the activity of the proteins of PCaNL260 as defined above is measured in the presence of each of the products the antifungal properties of which one wishes to determine and the products having an inhibitory effect on this activity are selected.

In particular, the genes coding for the proteins of PCaNL260 of the present invention being essential to the survival of the cells of Candida albicans, of the inhibitory substances of such proteins PCaNL260, could be of use as antifungal agents, either as medicaments or on the industrial level.

For example, to screen antifungal substances such as the substances active on Candida albicans, the activity of a protein coded by a gene of the present invention or one of its functional homologues is measured and the protein is put in the presence of each of 20 the products the antifungal properties of which one wishes to determine and the products having an inhibitory effect on this activity are selected.

Such screening can be carried out by measuring the activity of the proteins of PCaNL260 of the present invention in the presence of potential activators or inhibitors to be tested, for example by measuring in vitro in an appropriate reaction medium.

S• The activity of the proteins of the present invention can also be measured in vivo by an appropriate cell test. For example, the activity of PCaNL260 can be advantageously measured in cells of a mutant of Saccharornyces cerevisiae transformed by one of the 30 genes of the present invention and not expressing the homologous protein PYDR 472w, PYDR 489w, PYDR 577w, PYFL 024c, PYNL 260c and PYDR 361c of Saccharomyces cerevisiae.

The invention also encompasses the use of a product selected as indicated above for its inhibitory properties on one of the proteins of the present invention for obtaining of an antifungal agent.

08/06/04.all 2396 specipgs,19 The present invention is better understood using the experimental part which follows and which describes the cloning of genes CaDR472, CaDR489, 1CaDR527, 2CaDR527, CaFL024, CaNL260 and CaDR361.

Therefore a subject of present invention is the use of a product selected by the process of screening antifungal products as defined above for obtaining an antifungal agent.

A subject of the present invention is also the use of the genes of Candida albicans of the present invention or of the proteins coded by these genes as defined above for the selection of products having antifungal properties as defined above and used as inhibitors of the proteins of Candida albicans coded by these genes.

A subject of the present invention is also the pharmaceutical compositions containing as active ingredient at least one inhibitor of the proteins of Candida albicans of the present invention as defined above.

Such compositions can in particular be useful for treating topical and systemic fungal infections.

S: The pharmaceutical compositions indicated above can be administered by buccal, rectal, parenteral route or by local route as a topical application on the skin and mucous 25 membranes or by injection by intravenous or intramuscular route. These compositions can be solid or liquid and be presented in all the pharmaceutical forms commonly used in human medicine such as, for example, plain or sugar coated tablets, gelatin capsules, granules, suppositories, injectable preparations, ointments, creams, gels and aerosol **preparations; they are prepared according to the usual methods. The active ingredient can 30 be incorporated in excipients normally used in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting, dispersing or emulsifying agents, and preservatives.

The dose will be variable according to the product used, the subject treated and the disease in question.

08/06/04.at 2396.spccipgs.20 A particular subject of the present invention is thus the use of compositions as defined above as antifungal a a a a a a a a a.

a a a a..

04/06/04,ati 2396.specipgs,21 agents.

A subject of the present invention is also a method of inducing an immunological response in a mammal comprising the inoculation of this mammal with a polypeptide according to the present invention as defined above or a fragment of this polypeptide having the same function in order to produce an antibody protecting the mammal against the disease.

Therefore a subject of the present invention is also the use of a polypeptide as defined above or a fragment of this polypeptide having the same function for the preparation of a medicament intended to induce an immunological response in a mammal by inoculation with this medicament producing an antibody protecting the animal against the disease.

A subject of the present invention is also the antibodies directed against the polypeptides of the present invention as defined above or against a fragment of these polypeptides having the same function and coded by the polynucleotides of the present invention and in particular by a DNA sequence as defined above.

The polypeptides of the present invention can thus be used as immunogens to produce immunospecific antibodies of these polypeptides. The term antibody used designates antibodies which can equally be monoclonal, polyclonal, chimeric, single chain, non-human antibodies and human 25 antibodies, as well as Fab fragments, including the products of a Fab immunoglobulin bank. The antibodies produced against the polypeptides of the present invention can be obtained by administration of the polypeptides of the present invention or fragments carrying epitopes, their analogues or also animal cells, preferably non-human, by using routine protocols for the preparation of monoclonal antibodies. Such antibodies can be prepared by methods well known in this field such as those described in the book Antibodies, Laboratory manual Ed. Harbow and David Larre, Cold Spring Harbor laboratory Eds, 1988.

Therefore a quite particular subject of the present invention is an antibody directed against any one of the proteins PCaDR472, PCaDR489, 1PCaDR527, 2PCaDR527, PCaFL024, PCaNL260, PCaDR361 of the present invention or a fragment of these proteins. Such a fragment has in particular the same function as the protein from which it originated.

A subject of the present invention is also the use of genes CaDR472, CaDR489, lCaDR527, 2CaDR527, CaFL024, CaNL260 and CaDR361 of the present invention or of the proteins coded by these genes as defined above for the preparation of compositions which can be used for the diagnosis or treatment of diseases caused by the pathogenic yeast Candida albicans.

The present invention also relates to the use of the polynucleotides of the present invention as diagnostic reagents. The detection of a polynucleotide according to the present invention coding for one of the proteins of Candida albicans of the present invention or of its analogues in a eucaryote in particular a mammal and more particularly a human being, can constitute a means of diagnosing a disease: thus, such a polynucleotide according to the present invention and in particular a DNA sequence can be detected by a wide variety of techniques in a eucaryote in particular a mammal and more particularly a human being, infected by an organism containing at least one of the polynucleotides of the present invention. The nucleic acids for such a use as a diagnostic tool can be detected in infected cells or tissues, such as bone, blood, muscle, cartilage or skin. For this 25 detection, the genomic DNA can be used directly or also be amplified by PCR or another amplification technique. The RNA or DNA and cDNA can also be used with the same purpose. By amplification techniques, the line of the mycete present in a eucaryote in particular a mammal and more particularly a human being, can be characterized by analysis of the genotype. Deletions or insertions can be detected by a change in the size of the amplified product in comparison with the genotype of the reference sequence. The points of mutation can be identified by hybridization of the DNA amplified with the sequences, labelled by a radioactive element, of polynucleotides of the present invention. Perfectly complementary sequences can therefore be distinguished from duplexes which poorly resist digestion by nucleases. The DNA ii sequence differences can also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agent, or by direct DNA sequencing (reference: Myers et al. Science, 230: 1242 (1985)).

Sequence changes at specific locations can also be revealed by protection experiments against nucleases such as RNase I and Sl or by chemical cleavage methods (reference: Cotton et al., Proc Natl Acad Sci, USA, 85: 4397-4401 (1985).

Cells containing one of the polynucleotides of the present invention carrying mutations or polymorphisms can also be detected by a large number of techniques making it possible in particular to determine the serotype. For example, the RT-PCR technique can be used to detect the mutations. It is particularly preferable to use RT-PCR techniques in conjunction with automatic detection systems, such as for example the GeneScan technique. RNA and cDNA can be used in the PCR or RT-PCR techniques. For example, complementary primers of polynucleotides coding for the polypeptides of the present invention can be used to identify and analyse the mutations.

Primers can therefore be used to amplify an isolated DNA from the infected individual. In this way mutations in the DNA sequence can be detected and used to diagnose the infection and determine the serotype or the classification of the infectious agent. Such techniques are standard for a person skilled in the art and are described in particular in the manual 'Current Protocols in Molecular Biology', Ausubel et al, ed. John Wiley sons, Inc., 1995).

The present invention therefore relates to a process of diagnosing a disease and preferably a fungal infection caused by Candida albicans such as mycoses as indicated above, this process comprising the determination from a sample taken from an infected individual, an increase in the quantity of one of the polynucleotides of the present invention. Such a polynucleotide can in particular have a DNA sequence of the present invention as defined above.

Increases or reductions in the quantity of polynucleotides can be measured by techniques well known to a person skilled in the art such as in particular amplification, PCR, RT PCR, Northern blotting or other hybridization techniques.

In addition, a diagnosis method in accordance with the present invention consists of the detection of too large an expression of polypeptides of the present invention, in comparison with control samples constituted by normal, noninfected tissues used to detect the presence of an infection.

The techniques which can therefore be used to detect the quantities of proteins expressed in a host cell sample are well known to a person skilled in the art. For example radioimmunoassay or competitive-binding techniques, Western Blot analysis and ELISA test (ref Ausubel indicated above) can thus be mentioned.

A subject of the present invention is also a kit for the diagnosis of fungal infections comprising a DNA sequence according to the present invention as defined above or a similar sequence or a functional fragment of this sequence, the polypeptide coded by this sequence or a polypeptide fragment having the same function or an antibody directed against such a polypeptide coded by this DNA sequence or against a fragment of this polypeptide.

This kit can thus contain a DNA sequence according to the present invention as defined above either for example the DNA sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID 25 No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13 or a fragment of this sequence.

Such a kit can similarly contain a polypeptide according to the present invention or a fragment of this polypeptide and in particular one of the proteins according to the present invention having the AA sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No.

12 and SEQ ID No. 14 or also an antibody as defined above.

Such a kit can be prepared according to methods well known to a person skilled in the art.

The sequence listing SEQ ID No. 1 to SEQ ID No. 32 and Figures 1 to 6 hereafter show the following illustrations which allow a better description of the present invention.

Sequences SEQ ID No. 1 to SEQ ID No. 32 represent the nucleotide or peptide sequences indicated in the present invention.

Sequences SEQ ID No. 1 to SEQ ID No. 14 describe the nucleotide sequences of the genes of Candida albicans of the present invention and the peptide sequences of the proteins derived from these genes.

Sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13 thus respectively describe the nucleotide sequences of the genes of Candida albicans of the present invention: CaDR472, CaDR489, lCaDR527, 2CaDR527, CaFL024, CaNL260 and CaDR361.

Sequences SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ 0 ID No. 8, SEQ ID No. 10, SEQ ID No. 12 and SEQ ID No. 14 respectively describe the peptide sequences of the proteins derived from the genes of the present invention.

Thus, for example, sequences SEQ ID No. 1 and SEQ ID No.

2 respectively represent the nucleotide sequence of the gene CaDR472 and the peptide sequence of the protein derived from this gene namely PCaDR472.

Sequences SEQ ID No. 15 to SEQ ID No. 20 respectively represent the sequences of the 6 probes used for the preparation of the genes of Candida albicans of the present invention as indicated hereafter in the experimental part.

Sequences SEQ ID No. 21 to SEQ ID No. 32 respectively represent the sequences of the 2 x 6 oligonucleotides used to amplify the probes for the preparation of the genes of Candida albicans of the present invention as indicated hereafter in the experimental part.

Figures 1 to 6 hereafter each refer respectively to one of the 6 preparations of the genes of Candida albicans of the present invention namely: CaDR472, CaDR489, 1CaDR527/2CaDR527, CaFL024, CaNL260 and CaDR361, these preparations being described hereafter in the experimental part in Examples 1 to 6.

Each of Figures 1 to 6 describe the comparison of the protein derived from the probe used for the preparation of one of the genes of Candida albicans of the present invention (the 6 probes used having sequences SEQ ID No. 15 to SEQ ID No. 20) with the sequence of the gene of S.c. taken as a starting point of the preparation of this gene of Candida albicans.

Thus, with reference to Example 1 of the preparation of the gene CaDR472 of the present invention, Figure 33 represents the comparison of the protein derived from the probe of CaDR472 (SEQ ID No. 15) with the protein derived from the gene YDR472w of S. cerevisiae.

The experimental part hereafter allows the description the present invention without however limiting it.

Experimental part EXAMPLE 1: Cloning and sequencing of the gene CaDR472 0 (method A) The Stanford Internet site (http://candida.standford.edu/) allows direct access to the preliminary sequences of the genome of Candida albicans. One of these sequences has an homology with the gene YDR472w of S. cerevisiae. Two oligonucleotides have been chosen in this sequence namely: 5'CAATTTATTC ATGTTCGNAT CTGGAAATTG ATTTT3' called SEQ ID No.

21 and 5'CCAAATCTCA AACTCTCTCT AATTAAAAC3' called SEQ ID No.

22.

These two oligonucleotides are used to amplify the fragment of C. albicans. After cloning, a so-called probe S 25 sequence of CaDR472 of 320 base pairs close to the expected sequence was obtained: the probe of CaDR472 is called SEQ ID NO 15. The protein derived from the probe of CaDR472 (SEQ ID NO 15) was compared to that of YDR472w which demonstrates an identity of 48% between these two AA sequences: this comparison is represented in Figure 1.

The fragment of 320 base pairs of C. albicans was used as a probe for screening the gene bank of C. albicans: this bank of C.a. was prepared by partial digestion of the genomic DNA of C. albicans by Sau3AI and cloning in the vector YEP24 at the BamHI restriction site. The clones of the gene bank were then plated at the density of 2000 clones per dish: each dish is then covered with a nitrocellulose filter which is successively treated with: NaOH, 0.5M, for 5 minutes; Tris, 1M, pH 7.7, for 5 minutes; Tris 0.5M, pH 7.7, NaCl 1.25M, for 5 minutes. After drying, the filters are kept for two hours at 800C. Prehybridization and hybridization are carried out in a buffer of 40 formamide, 5xSSC, 20 mM Tris pH 7.7 IxDenhardt 0.1 SDS. The probe is then labelled with P32 using the Rediprime and dCTP 32p kit from Amersham UK.

Hybridization is carried out for 17 hours at 420C. The filters are then washed with IxSSC, 0.1 SDS, three times for 5 minutes at ambient temperature and then twice for minutes at 600C then subjected to an autoradiography overnight. The colonies corresponding to the spots obtained are isolated by a new plating at low density followed by hybridization: 8 positive clones are thus obtained (from 000) which are then sequenced using an ABI 377 device.

Sequences are compiled using ABI software then analyzed using a GCG software package. One of the 8 clones is shown to contain the complete coding sequence corresponding to the probe used: this gene is called CaDR472 and this sequence is called SEQ ID NO 1.

CaDR472 has 47.5 of nucleotides identical to YDR472w of S. cerevisiae.

For the translation to amino acids, account was taken of the fact that in C. albicans the CTG codon is translated to serine (there is one CTG codon in CaDR472). The protein S 25 derived from the gene CaDR472 (SEQ ID No. 1) namely SEQ ID No. 2 (PCaDR472) has 52.4 similarity in amino acids and 44 identity in amino acids with the protein derived from YDR472w.

The complete sequence of the gene CaDR472 contains a CTG codon.

EXAMPLE 2: Cloning and sequencing of the gene CaDR489 The operation is carried out as in Example 1 starting from preliminary sequences of the genome of Candida albicans from the Stanford Internet site (http://candida.standford.edu/). One of these sequences has an homology with the gene YDR489w of S. cerevisiae. Two oligonucleotides were chosen in this sequence namely: GGTGACTCAG AGCGTCTCAA CTATATTG3' called SEQ ID No. 23 and 5'TTTGATAAAC ACAGGCTGGT CTAAATCTGG CTC3' called SEQ ID No. 24.

These two oligonucleotides are used to amplify the fragment of C. albicans. After cloning, a so-called probe sequence of CaDR489 of 295 base pairs close to the expected sequence was obtained: the probe of CaDR489 is called SEQ ID No. 16. The protein derived from the probe of CaDR489 (SEQ ID No. 16) was compared to that of YDR489w which demonstrates an identity of 41% between these two AA sequences: this comparison is represented in Figure 2.

The fragment of 295 base pairs of C. albicans was used as probe for screening the gene bank of C. albicans prepared by partial digestion of the genomic DNA of C. albicans proceeding as in Example 1.

The cloning is carried out as indicated in Example 1 and after prehybridization and hybridization carried out as indicated in Example 1, 4 positive clones are obtained (from 000). The sequencing and analyzing of the sequences obtained as indicated in Example 1, and thus a clone is obtained shown to contain the complete coding sequence corresponding to the probe used: this gene is called CaDR489 and this sequence is called SEQ ID No. 4.

CaDR489 has 48.1 of nucleotides identical to YDR489w of S.

25 cerevisiae.

The protein derived from the gene CaDR489 (SEQ ID No. 3) namely SEQ ID No. 4 or PcaDR489 has 50 similarity in amino acids and 37 of identity in amino acids with the protein derived from YDR489.

The complete sequence of the gene CaDR489 contains a CTG codon.

EXAMPLE 3: Cloning and sequencing of the gene CaDR527 The operation is carried out as in Example 1 starting from preliminary sequences of the genome of Candida albicans from the Stanford Internet site (http://candida.standford.edu/). One of these sequences has an homology with the gene YDR527w of S. cerevisiae. Two oligonucleotides have been chosen in this sequence namely: TGAGATTTGG CTTTAAAGGC GA3' called SEQ ID No. and 5'GGTCTTTTTT CCATCAGCTG CCTCTGTTAT TG3' called SEQ ID No.

26.

These two oligonucleotides are used to amplify the fragment of C. albicans. After cloning, a so-called probe sequence of CaDR527 of 392 base pairs close to the expected sequence was obtained: the probe of CaDR527 is called SEQ ID No. 17. The protein derived from the probe of CaDR527 (SEQ ID No. 17) was compared to that of YDR527w which demonstrates an identity of 41% between these two AA sequences: this comparison is represented in Figure 3.

The fragment of 392 base pairs of C. albicans was used as probe for the screening of the gene bank of C. albicans prepared by partial digestion of the genomic DNA of C.

albicans proceeding as in Example 1.

The cloning is carried out as indicated in Example 1 and after prehybridization and hybridization carried out as indicated in Example 1, 7 positive clones are obtained (from 000). The sequencing and analysis of the sequences obtained is carried out as indicated in Example 1.

Thus two clones obtained are each shown to contain a complete coding sequence each corresponding to an allele of the probe used: this gene is called CaDR527 and the two alleles are thus called lCaDR527 and 2CaDR527 and their S 25 respective sequences are respectively called SEQ ID No. 5 and SEQ ID No. 7.

It is noted that the genes of the alleles 1CaDR527 and 2CaDR527 (SEQ ID No. 5 and SEQ ID No. 7) differ by 13 nucleotides.

The gene CaDR527 (1st allele) has 53.8 of nucleotides identical to YDR527w of S. cerevisiae.

The proteins derived from these alleles namely SEQ ID No. 6 (PCaDR527) for the 1st allele 1CaDR527 and SEQ ID No. 8 for the 2nd allele 2CaDR527 differ between themselves by amino acids.

The protein derived from the gene CaDR527 (SEQ ID No. 6) has 58.9 similarity in amino acids and 47.9 identity in amino acids with the protein derived from YDR527.

The complete sequence of the gene CaDR527 does not contain a CTG codon.

EXAMPLE 4: Cloning and sequencing of the gene CaFL024 (method B) The Stanford Internet site (http://candida.standford.edu/) allows direct access to the preliminary sequences of the genome of Candida albicans. One of these sequences has an homology with the gene YFL024c of S. cerevisiae. Two oligonucleotides were chosen in this sequence namely: 5' ATTCCCACAC CGGACGCTTC 3' called SEQ ID No. 27 and 5'GACAACTCCT CGTACGATAG 3' called SEQ ID No. 28.

These two oligonucleotides are used to amplify the fragment of C. albicans. After cloning, a so-called probe sequence of CaFL024 of 335 base pairs close to the expected sequence was obtained: the probe of CaFL024 is called SEQ ID No. 18. The protein derived from the probe of CaFL024 (SEQ ID No. 18) was compared to that of YFL024c which demonstrates a similarity of 62 and an identity of 58 between these two AA sequences: this comparison is represented in Figure 4.

This fragment of 335 base pairs of C. albicans was used as probe for screening a gene bank of C. albicans: this bank of genes of C.a. was prepared by partial digestion of the genomic DNA of C. albicans by SauIIIA and cloning in the vector YEP-24 at the BamHI restriction site. The clones of S 25 the gene bank were then plated at a density of 2000 clones per dish: each dish is then covered with a nitrocellulose filter which is successively treated with: 1.5 M NaCl/ 0.5 M NaOH for 5 minutes; 1.5 M NaCl/0.5 M Tris-HCl pH 7.2/1 mM EDTA for 3 minutes, twice.

The DNA is then 'crosslinked' to the filter (Amersham Life Science, ultraviolet crosslinker).

The probe (100 ng) is then labelled with P32 using the Rediprime and dCTP kit (Amersham Life Science).

Prehybridization and hybridization of the filters are carried out in a buffer of 30 of formamide, 5 x SSC, 5 of Denhardt's solution, 1 SDS, 100 pg/ml of salmon sperm DNA and a concentration of the probe of 10(6) cpm/ml: the hybridization is carried out at 42 0 C for 16 hours.

The filters are then washed three times, for 5 minutes each time, at ambient temperature with 2 x SSC/ 0.1 SDS then three times with 1 x SSC/ 0.1 SDS for 20 minutes each time at 60 0 C. The filters are subjected to an autoradiography overnight. The colonies corresponding to the positive clones (spots obtained) are isolated and subjected to a second screening by a new plating at low density followed by hybridization: 6 clones are thus obtained (from 144 000) which are then sequenced using an ABI 377 device. Sequences are compiled using ABI software then analyzed using a GCG software package. One of the 6 clones is shown to contain the complete coding sequence corresponding to the probe used: this gene is called CaFL024 and this sequence called SEQ ID NO 9.

CaFL024 has 49.1 of nucleotides identical to YFL024c of S. cerevisiae.

There are 2 CTG codons in CaFL024. The protein derived from the gene CaFL024 (SEQ ID No. 9) namely SEQ ID No. (PCaFL024) has 51.8 similarity in amino acids and 44.0 identity in amino acids with the protein derived from YFL024c.

EXAMPLE 5: Cloning and sequencing of the gene CaNL260 The operation is carried out as in Example 4 starting from preliminary sequences of the genome of Candida albicans 25 on the Stanford Internet site (http://candida.standford.edu/). One of these sequences has an homology with the gene YNl260c of S. cerevisiae. Two oligonucleotides were chosen in this sequence namely: AGATAATGTATTAAATTTAG 3' called SEQ ID No. 29 and 5'CTCTTAATTTATTTCTTGCC 3' called SEQ ID No. These two oligonucleotides are used to amplify the fragment of C. albicans. After cloning, a so-called probe sequence of CaNL260 of 326 base pairs close to the expected sequence was obtained: the probe of CaNL260 is called SEQ ID No. 19. The protein derived from the probe of CaNL260 (SEQ ID No. 19) was compared to that of YNL260c which demonstrates a similarity of 56.7 and an identity of 40.3 between these two AA sequences: this comparison is represented in Figure The fragment of 326 base pairs of C. albicans was used as probe for screening the gene bank of C. albicans prepared by partial digestion of the genomic DNA of C. albicans proceeding as in Example 4.

The prehybridization and hybridization are carried out as indicated in Example 4, 2 positive clones are obtained (from 40 000). The sequencing and analysis of the sequences obtained are carried out as indicated in Example 4, and a clone is thus obtained shown to contain the complete coding sequence corresponding to the probe used: this gene is called CaNL260 and this sequence is called SEQ ID No. 11.

CaNL260 has 47.6 of nucleotides identical to YNL260c of S. cerevisiae.

The protein derived from the gene CaNL260 (SEQ ID No.

11) namely SEQ ID No. 12 (PCaNL260) has 50.7 similarity in amino acids and 32.6 identity in amino acids with the protein derived from YNL260c.

There is no CTG codon in CaNL260.

EXAMPLE 6: Cloning and sequencing of the gene CaDR361 The operation is carried out as in Example 4 starting from preliminary sequences of the genome of Candida albicans: The Stanford Internet site (http://candida.standford.edu/) allows direct access to the preliminary sequences of the S 25 genome of Candida albicans.

One of these sequences has an homology with the gene YDR361c of S. cerevisiae. Two oligonucleotides were chosen in this sequence namely: CCTCAAATTGATTTCCATGC 3' called SEQ ID No. 31 and 5'GTGGAATCACTTCAACTGGC 3' called SEQ ID No. 32.

These two oligonucleotides are used to amplify the fragment of C. albicans. After cloning, a so-called probe sequence of CaDR361 of 374 base pairs close to the expected sequence was obtained: the probe of CaDR361 is called SEQ ID No. 20. The protein derived from the probe of CaDR361 (SEQ ID No. 20) was compared to that of YDR361c which demonstrates a similarity of 52.4 and an identity of 40.0 between these two AA sequences: this comparison is represented in Figure 6.

The fragment of 374 base pairs of C. albicans was used as probe for screening the gene bank of C. albicans prepared by partial digestion of the genomic DNA of C. albicans by Saull/A and cloning in the vector YEP 24 (selection marker Trp) at the Bam HI restriction site.

The prehybridization and hybridization are carried out as indicated in Example 4, 4 positive clones are obtained (from 40 000). The sequencing and analysis of the sequences obtained are carried out as indicated in Example 4, and thus a clone is obtained which is shown to contain the complete coding sequence corresponding to the probe used: this gene is called CaDR361 and this sequence called SEQ ID No. 13.

CaDR361 has 53.9 of nucleotides identical to YDR361c 15 of S. cerevisiae.

SCaDR361 there is no CTG codon in CaDR361.

Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

e 0 O 0 0 0 EDITORIAL NOTE APPLICATION NUMBER 55390/00 The following Sequence Listing pages numbered 39 to 80 are part of the description. The claims pages follow on pages "35" to "37".

39 SEQUENCE LISTING <110> Hoechst Marion Roussel <120> Novel genes of Candida albicans and the proteins coded by these genes.

<130> 2517 PCT SEQUENCES IN FRENCH <140> <141> <150> <151> FR 9907250 1999-06-09 <160> 32 <170> PatentIn Ver. 2.1 <210> <211> <212> <213> <220> <221> <222> <220> <221> <222> 1 747

DNA

Candida albicans

CDS

(747) modified base (136) (138) <400> 1 atg tca aat gac gat ata ata ctc cca tca gtt tca tcc tta tcg aaa Met Ser Asn Asp Asp Ile Ile Leu Pro Ser Val Ser Ser Leu Ser Lys 1 5 10 ota act ata Len Thr Ile ata gga oca Ile Gly Pro gat gta toa aaa Asp Val Ser Lys gga ttt gga Gly Phe Gly tac aat Tyr Asn cog tcc Pro Ser ata tca aat act att Ile Ser Asn Thr Ile 40 acc cta gaa tct toa ctg gta tta Thr Leu Glu Ser Ser Ser Val Len tta aat Leu Asn aaa ogt aca ata tca Lys Arg Thr Ile Ser 55 tta aca cca aca tca tct gao tcc att Leu Thr Pro Thr Ser Ser Asp Ser Ile tat gat aga aat att Tyr Asp Arg Asn Ile tcg tta tca ttt ttg Ser Leu Ser Phe Len ato aog aaa aag oca Ile Thr Lys Lys Pro ttt tgt gag att att Phe Cys Glu Ile Ile 90 gaa ato aao tta Glu Ile Asn Leu agt tgg goa cac tot aat Ser Trp Aia His Ser Asn too aaa ggo att Ser Lys Gly Ile 100 oaa gat tta gaa Gin Asp Leu Glu ogt tta aao gga Arg Leu Asn Gly tta ggt tat Len Gly Tyr 110 oaa ata ggt Gin Ile Gly 115 oaa oga tat otc gaa Gin Arg Tyr Leu Glu 120 ttg tgt aaa ata aga gaa ggt ttt Leu Cys Lys Ile Arg Giu Giy Phe 125 aaa aao agt aaa oga gag Lys Asn Ser Lys Arg Glu 130 oat ggt cog tto tgg aaa His Giy Pro Phe Trp Lys 145 150 att aga ott ttg gaa Ile Arg Leu Len Glu 135 ttg att ttt ggt aaa Leu Ile Phe Giy Lys 155 atg tta oaa ttt att Met Len Gin Phe Ile 140 act got aat gaa tta Thr Aia Asn Glu Len 160 gaa aaa tcg caa Glu Lys Ser Gin gat ttg ccc aat gaa Asp Leu Pro Asn Glu 165 aga ttt att agt ata Arq Phe Ile Ser Ile 185 atg att gtg gag Met Ile Val Glu aat gtg 528 Asn Val 175 gac tta 576 Asp Leu cca tta tta Pro Leu Leu aat tgt tca Asn Cys Ser 195 aat Asn 180 cct aag gag tat Pro Lys Giu Tyr ggC Gly 190 gca ttt gtt gcg ggt Ala Phe Vai Ala Gly 200 ata att gag gga Ile Ile Giu Gly ctt gat aat 624 Leu Asp Asn agt gga Ser Gly 210 ttc aat gcc gat gtt Phe Asn Ala Asp Val 215 aca gca cac acg gtc Thr Aia His Thr Vai 220 gct aca gat gca 672 Ala Thr Asp Ala aat Asn 225 cca tta aga aca gta Pro Leu Arg Thr Val 230 ttt ttg atc aag ttt gac gat tct gtt Phe Leu Ile Lys Phe Asp Asp Ser Val 235 att aga gag agt Ile Arg Glu Ser ttg aga ttt gga taa Leu Arg Phe Gly 747 245 <210> 2 <211> 249 <212> <213> Candida aibicans <400> 2 Met Ser Asn Asp Asp Ile Ile Leu Pro Ser Val Ser Ser Leu Ser Lys 1 5 10 Leu Thr Ile Asn Asp Val Ser Lys Ser Giy Phe Giy Tyr Asn Pro Ser 25 Ile Giy Pro Ile Ser Asn Thr Ile ThrLe Gi Se SrSrVa Lu Leu Glu Ser Ser Ser Val Leu Lys Ser Leu Asn Tyr Asp Arg Thr Ile Ser Thr Thr Pro Thr Asp Ser Ile Arg Asn Ile Ser Ile 70 Phe Lys Lys Pro His 75 Ser Ile Asn Leu Ser Leu Ser Phe Leu Gin Cys Giu Ile Trp Ala Ser Lys Giy Asp Leu Glu Asn 105 Leu Leu Asn Giy His Ser Asn Leu Gly Tyr 110 Giu Gly Phe Gin Ile Giy 115 Lys Asn Ser Arg Tyr Leu Cys Lys Ile Arg 125 Le u Lys Arg Giu 130 Gly Ile 135 Le u Leu Leu Glu Gin Phe Ilie Pro Phe Trp Lys 150 Leu Ile Phe Gly Lys 155 Met Ala Asn Giu Leu 160 Lys Ser Gin Pro Asn Giu Ile Val Giu Asn Vai 175 Pro Leu Leu Asn Arg Phe 180 Asn Cys Ser Ala Phe Val 195 Ser Giy Phe Asn Ala Asp 210 Asn Pro Leu Arg Thr Val 225 230 Ile Ser Ile 185 Ile Lys Giu Tyr Gly Asp Leu 190 Leu Asp Asn Ala Gly 200 Thr Ile Giu Gly Al a 205 Al a Val1 215 Ph e Ala His Thr Val1 220 Asp Thr Asp Ala Leu Ile Lys Phe 235 Asp Ser Val Ile Arg Glu Ser Leu Arg Phe Gly 245 <210> <211> <212> <213> <220> <221> <222> <220> <221> <222> 3 711

DNA

Candida albicans

CDS

modified base (577)..(579) <400> 3 atg gat Met Asp att gac gat Ile Asp Asp att tta aaa gaa ttt Ile Leu Lys Glu Phe 10 gaa gag tct tca aaa gat Glu Glu Ser Ser Lys Asp aac tta tat caa gac ttg Asn Leu Tyr Gin Asp Leu gaa aag att agc Glu Lys Ile Ser agt aaa aca tcg tct Ser Lys Thr Ser Ser 25 cta aga Leu Arg atg atc aac gaa cgt Met Ile Asn Glu Arg atg get ccg gaa Met Ala Pro Glu ttg cca tac Leu Pro Tyr aaa caa Lys Gin gat tta atg tcc act Asp Leu Met Ser Thr 55 gtt tta aca atg atg tct aac caa caa Val Leu Thr Met Met Ser Asn Gin Gin caa Gin tat tta tta gaa tct Tyr Leu Leu Glu Ser 70 cac gaa tat ggt His Glu Tyr Gly gat Asp 75 atg aat ggc gac agt Met Asn Gly Asp Ser ggt gta tta tcc gga gac Gly Val Leu Ser Gly Asp ttt aaa tta caa Phe Lys Leu Gin 90 cta atg att atc gaa Leu Met Ile Ile Glu act 288 Thr gat tta gag cgt Asp Leu Giu Arg 100 ctc aac tat att Leu Asn Tyr Ile gtt Va1 105 cga tta tac ata cga Arg Leu Tyr Ile Arg 110 act cga 336 Thr Arg ttg agt aag Leu Ser Lys 115 ttg aat aaa ttt act Leu Asn Lys Phe Thr 120 att ttt tac atc aat Ile Phe Tyr Ile Asn 125 gaa agc agt 384 Glu Ser Ser caa aat Gin Asn 130 gat aat tta ttg tcc Asp Asn Leu Leu Ser 135 aaa gag gaa aga gat Lys Giu Giu Arg Asp 140 tat ata cac aaa 432 Tyr Ile His Lys tat ttc cag att ttg act caa Tyr Phe Gin Ile Leu Thr Gin 145 150 tta tat aac aac Leu Tyr Asn Asn 155 tgt ttc ctc aaa aaa 480 Cys Phe Leu Lys Lys 160 cta cca caa atg ttg Leu Pro Gin Met Leu 165 acc tat ttg gat gac Thr Tyr Leu Asp Asp 170 acc agt ggt gga Thr Ser Gly Gly caa Gin 175 tca 528 Ser atg atc gtt gag Met Ile Val Glu 180 cca gat tta gac cag Pro Asp Leu Asp Gin 185 cct gtg ttt atc aaa tgt acc Pro Vai Phe Ile Lys Cys Thr 190 ctg gaa gtc Ser Giu Vai 195 cca ata tta cta Pro Ile Leu Leu gat Asp 200 tac gac ggt gct aca Tyr Asp Gly Ala Thr 205 gag ata gat Glu Ile Asp tta gaa Leu Glu 210 tta ata aaa aag Leu Ile Lys Lys gga Gly 215 gtc tac gtg gtg aaa Val Tyr Val Val Lys 220 tac agc cta gtc Tyr Ser Leu Vai aaa aga tat att gat att qga gat gtq qta ttg ata tqa Lys Arq Tyr Ile Asp Ile Giy Asp Vai Val Leu Ile 225 230 235 <210> 4 <211> 237 <212> <213> Candida albicans <400> 4 Met Asp Ile Asp Asp 1 5 Ile Leu Lys Giu Giu Giu Ser Ser Lys Asp Giu Lys Ilie Leu Arq Aia Se r Ser Lys Thr Ser Ser 25 Ile Asn Leu Tyr Gin Asp Leu Leu Pro Tyr Met Ile Asn Giu Met Ala Pro Giu Lys Gin Asp Leu Met Ser Thr 55 Val Leu Thr Met Met Ser Asn Gin Gin Tyr Leu Leu Giu Ser His Giu Tyr Gly Met Asn Gly Asp Ser Gly Vai Leu Ser Asp Phe Lys Leu Gin Leu Met Ile Ile Giu Thr Asp Leu Giu Leu Ser Lys 115 Arg 100 Leu Asn Tyr Ilie Vai 105 Arg Leu Tyr Ilie Arg Thr Arg 110 Giu Ser Ser Leu Asn Lys Phe Ile Phe Tyr Ile Gin Asn 130 Asp Asn Leu Leu Ser 135 Lys Giu Giu Arg Asp 140 Tyr Ilie His Lys Phe Gin Ile Leu Phe Gn li Leu Gin Leu Tyr Asn Cy Ph Le Ls Cys Phe Leu Lys Leu Pro Gin Met Leu 165 Thr Tyr Leu Asp Asp 170 Thr Ser Giy Giy Gin Ser 175 Met Ile Vai Ser Giu Vai 195 Pro Asp Leu Asp Pro Val Phe Ile Lys Cys Thr 190 Giu Ile Asp Pro Ile Leu Leu Asp 200 Tyr Asp Giy Aia Th r 205 Leu Giu 210 Leu Ilie Lys Lys Giy 215 Vai Tyr Vai Val Lys 220 Tyr Ser Leu Vai Arg Tyr Ile Asp Giy Asp Vai Val Leu Ile 235 <210> <211> 1383 <212> DNA <213> Candida aibicans <220> <221> <222>

CDS

(1)..(1383) <400> atg gat ttc ata gga gag att ata gag Met Asp Phe Ilie Giy Gu Ile Ilie Giu cat gag aca gag gca cct aaa 48 His Giu Thr Giu Aia Pro Lys gga ttc ccc gaa ctt aaa aaa 96 Giy Phe Pro Giu Leu Lys Lys gaa cca acc cca aaa ccc aca att ggt Giu Pro Thr Pro Lys Pro Thr Ile Giy 25 tta aaa qaa Leu Lys Glu aag aaa gtc tca Lys Lys Val Ser tgg agg caa aag Trp Arg Gin Lys caa cag gaa Gin Gin Glu cag agc Gin Ser aca act tcc cca aaa Thr Thr Ser Pro Lys 55 act act gaa atc cgt tca gag gct tcc Thr Thr Giu Ile Arg Ser Giu Aia Ser aaa Lys att cac caa gaa Ile His Gin Glu aat Asn 70 atc gag aag atg gct Ile Giu Lys Met Ala 75 caa atg tca gag gaa Gin Met Ser Giu Glu ggt tta gat cct aaa Gly Leu Asp Pro Lys gag att ttg caa Glu Ile Leu Gin tta att gaa agt Leu Ile Giu Ser 100 gag cgt gag gag tta Glu Arg Giu Giu Leu ttg att ggt aga tcc Leu Ile Giy Arg Ser 105 aag aaa agg gaa gca aca gac Lys Lys Arg Giu Ala Thr Asp 110 cat gaa cac His Giu His 115 aat gga cat gct cat Asn Giy His Ala His 120 gaa cat gca gag Glu His Ala Glu gga tac cat gga Gly Tyr His Gly 125 tgg att Trp Ile 130 gga tca atg aaa act Gly Ser Met Lys Thr 135 tct gaa gga tta aca gat tta tct caa Ser Giu Gly Leu Thr Asp Leu Ser Gin 140 tta Leu 145 gat aag gaa gat gtg Asp Lys Glu Asp Vai 150 gac cgt gca ttg ggt Asp Arg Ala Leu Gly 155 ggc agt aat acg aaa Gly Ser Asn Thr Lys 170 ata agt tca tta tcc Ile Ser Ser Leu Ser 160 aaa gtc gct ttc gac Lys Val Ala Phe Asp 175 480 528 tta tct gaa cct gag Leu Ser Giu Pro Glu 165 gat aat atc Asp Asn Ile aag Lys 180 acg gtt aaa ttt Thr Val Lys Phe gaa Glu 185 gat ttg gat Asp Leu Asp gat gga Asp Gly 190 att gaa 576 Ile Glu ttg gat cca Leu Asp Pro 195 aat gga tgg gag gac Asn Gly Trp Glu Asp 200 gtt act gat gtc aat gaa tta gtt Val Thr Asp Val Asn Glu Leu Val 205 cct aat Pro Asn 210 aat gat cac att Asn Asp His Ile gca Ala 215 cct gac gat tac cag att aat cct gat Pro Asp Asp Tyr Gin Ile Asn Pro Asp 220 agc Ser 225 gat gaa gaa gga ttg Asp Glu Glu Gly Leu 230 aat aat act gtt cat Asn Asn Thr Val His 235 ttt aca aaa ccc aaa Phe Thr Lys Pro Lys 240 cag cca gat ttg Gin Pro Asp Leu gat Asp 245 ata aat gat ccc gat ttc ttt gat aag Ile Asn Asp Pro Asp Phe Phe Asp Lys 250 cta cat Leu His 255 gag aaa tac tat Glu Lys Tyr Tyr 260 cct gat ttg cct aaa Pro Asp Leu Pro Lys 265 gaa aca gaa aag ttg tca tgg Glu Thr Glu Lys Leu Ser Trp 270 atg aca cag Met Thr Gin 275 cca atg cca aaa caa Pro Met Pro Lys Gin 280 ttg tct acc gtt Leu Ser Thr Val tat Tyr 285 gaa tca ata Glu Ser Ile tct gat Ser Asp 290 atg aga ttt gac ttt Met Arg Phe Asp Phe 295 aaa gga gat tta att gaa ttg ggt cca Lys Gly Asp Leu Ile Glu Leu Giy Pro 300 gag Glu 305 gga gaa gaa cca Gly Glu Glu Pro aaa Lys 310 gat agt tca tcc gaa Asp Ser Ser Ser Glu 315 ata cct act tat atg Ile Pro Thr Tyr Met 320 gga ctt cat cat Gly Leu His His cat His 325 tcg gag aac cca Ser Glu Asn Pro cat His 330 atg gca Met Ala ggt tat aca ttg Gly Tyr Thr Leu 335 1008 ggt gag ttg Gly Glu Leu cat tta gcc aga tcg His Leu Ala Arg Ser 345 act tta get gga caa aga tgc Thr Leu Ala Gly Gin Arg Cys 350 1056 ttg agc att Leu Ser Ile 355 caa aca tta ggg Gin Thr Leu Gly aga Arg 360 atc tta cat aaa ttg gga tta cat Ile Leu His Lys Leu Gly Leu His 365 1104 aaa tac Lys Tyr 370 agt ata cta cca aaa Ser Ile Leu Pro Lys 375 aca gac tca gat gat cag agt ttt aca Thr Asp Ser Asp Asp Gin Ser Phe Thr 380 1152 gaa atc aaa caa Glu Ile Lys Gin cta Leu 390 tca ctt gac ttt gaa Ser Leu Asp Phe Glu 395 gat atg atg tgg gac Asp Met Met Trp Asp 400 1200 ttg ata gac caa tta Leu Ile Asp Gin Leu 405 cga atc att gaa aca Arg Ile Ile Glu Thr 410 ata aca gag gca gct gat Ile Thr Glu Ala Ala Asp 415 1248 gaa aaa aag acc Glu Lys Lys Thr 420 aga aac tta tct gtc Arg Asn Leu Ser Val 425 aga aat tat gca ata Arg Asn Tyr Ala Ile 430 gag gca 1296 Glu Ala ttg tgg tta Leu Trp Leu 435 tat aga act gga ggt Tyr Arg Thr Gly Gly 440 gga aga cca gag ata Gly Arg Pro Glu Ile 445 gca gtt cag aaa taa Ala Val Gin Lys 460 act aaa caa 1344 Thr Lys Gin acc gaa gag gat ttg ata gca caa Thr Glu Glu Asp Leu Ile Ala Gin 1383 <210> 6 <211> 461 <212> <213> Candida aibicans <400> 6 Met Asp Phe 1 Giu Pro Thr Ile Gly 5 Pro Lys Giu Ile Ile Giu Giu Thr Giu Ala Pro Lys Pro Thr Ile Gly 25 Trp Phe Pro Giu Leu Lys Giu Gin Ser Thr Lys Ile His Lys Lys Vai Ser Arg Arg Gin Lys Leu Lys Lys Gin Gin Glu Giu Ala Ser Giu Thr Ser Pro Lys Thr 55 Gin Giu Asn Ile Giu 70 Gin Giu Arg Giu Giu Ser Leu Ile Giy Arq 100 Thr Giu Ile Ile Leu Lys Met Ala Gin 75 Leu Leu Lys Giy 90 Ser Lys Lys Arg 105 Giu His Ala Giu Met Ser Giu Arg Giu Leu Ile Giu His Giu His 115 Trp Ilie Gly Leu Giu Gly 125 Asp Asp Pro Lys Ala Thr Asp 110 Tyr His Gly Leu Ser Gin Giy His Ala His 120 Ser Ser Met Lys Giu Giy Leu 130 Leu Asp 145 Lys Giu Asp Arg Ala Leu Gly 155 Ser Ser Leu Leu Ser Giu Pro Giu Giy Giy Ser Asn Thr Lys Lys Val Ala Phe Asp 165 170 175 Asp Asn Ile Leu Asp Pro 195 Thr Val Lys Phe Asp Leu Asp Asp Gly Ile Glu 190 Glu Leu Val Asn Gly Trp Glu Asp 200 Val Thr Asp Val Pro Asn 210 Asn Asp His Ile Al a 215 Pro Asp Asp Tyr Gin Ile Asn Pro Asp 220 Phe Thr Lys Pro Lys Asp Glu Glu Gly Asn Asn Thr Val Gin Pro Asp Leu Asp 245 Ile Asn Asp Pro Asp 250 Phe Phe Asp Lys Leu His 255 Giu Lys Tyr Met Thr Gin 275 Pro Asp Leu Pro Lys 265 Glu Thr Glu Lys Leu Ser Trp 270 Glu Ser Ile Pro Met Pro Lys Leu Ser Thr Val Tyr 285 Ser Asp 290 Met Arg Phe Asp Phe 295 Lys Gly Asp Leu Ile Glu Leu Gly 300 Ile Pro Thr Tyr Pro Glu 305 Gly Gly Glu Giu Pro Leu His His His 325 Asp Ser Ser Ser Giu 315 Met 320 Ser Glu Asn Pro Met Ala Gly Tyr Thr Leu 335 Gly Giu Leu Leu Ser Ile 355 Al a 340 His Leu Ala Arg Ser 345 Thr Leu Ala Gly Gin Arg Cys 350 Gly Leu His Gin Thr Leu Gly Ile Leu His Lys Leu 365 Lys Tyr 370 Ser TIe Leu Pro Lys 375 Thr Asp Ser Asp Gin Ser Phe Thr Asp 385 Glu Ile Lys Gin Ser Leu Asp Phe Glu 395 Asp Met Met Trp Asp 400 Leu Ile Asp Gin Leu 405 Arg Ile Ile Glu Ile Thr Glu Ala Ala Asp 415 Glu Lys Lys Leu Trp Leu 435 Thr 420 Arg Asn Leu Ser Val 425 Arg Asn Tyr Ala Ile Glu Ala 430 Thr Lys Gin Tyr Arg Thr Gly Gly Arg Pro Glu Thr Glu 450 Glu Asp Leu Ile Ala 455 Gin Ala Val Gin <210> 7 <211> 1383 <212> DNA <213> Candida albicans <220> <221> CDS <222> (1)..(1380) <400> 7 atg gat Met Asp 1 gaa cca Glu Pro tta aaa Leu Lys ttc ata gga Phe Ile Gly 5 gag att ata gag Glu Ile Ile Glu cat gag aca gag gca cct aaa His Glu Thr Glu Ala Pro Lys 10 acc cca Thr Pro aaa ccc aca att Lys Pro Thr Ile ggt gga ttc ccc gaa ctt aaa aaa Gly Gly Phe Pro Glu Leu Lys Lys 25 tgg agg caa aag caa caa cag gag Trp Arg Gin Lys Gin Gin Gin Glu gaa Glu aag aaa gtc tca aga Lys Lys Val Ser Arg 40 cag agc Gin Ser aca act tcc cca Thr Thr Ser Pro aaa Lys 55 act act Thr Thr gaa atc Glu Ile cgt tca gag gct tcc Arg Ser Giu Ala Ser att cac caa gaa Ile His Gin Glu aat atc gag Asn Ile Glu aag atg gct caa atg tca gag gaa Lys Met Ala 75 Gin Met Ser Glu Glu gag att ttg caa gag Glu Ile Leu Gin Glu tta att gaa agt ttg Leu Ile Giu Ser Leu 100 cgt gag gag tta Arg Giu Giu Leu att ggt aga tcc Ile Giy Arg Ser 105 aag ggt tta gao cct aaa Lys Gly Leu Asp Pro Lys aag aaa agg gaa gca Lys Lys Arg Glu Ala 110 aca gac Thr Asp cat gaa cac ast gga cat got His Giu His Asn Gly His Ala 115 gaa cat gca gag gga tac cat gga Glu His Ala Giu Gly Tyr His Gly 125 tgg att Trp Ile 130 gga toa atg aaa Gly Ser Met Lys act tot gsa ggs tta Thr Ser Giu Gly Leu 135 gac cgt got ttg ggt Asp Arg Ala Leu Gly 155 aca gat tta tot caa Thr Asp Leu Ser Gin 140 tta Leu 145 gat aag gaa gat gtg Asp Lys Giu Asp Val 150 ata agt tca tta too 480 Ile Ser Ser Leu Ser 160 tta tot gaa cct gag Leu Ser Giu Pro Glu 165 ggt ggo ago aat Gly Gly Ser Asn acg Thr 170 aaa aaa gto got tto Lys Lys Val Ala Phe 175 gao 528 Asp gat aat ato aag Asp Asn Ile Lys 180 acg gtt aaa ttt gaa Thr Vai Lys Phe Glu 185 got ttg gat gat gsa att gaa Ala Leu Asp Asp Giu Ile Glu 190 576 ttg gat cca Leu Asp Pro 195 aat gga tgg gag gac Asn Gly Trp Glu Asp 200 gtt act Val Thr gat gtc aat Asp Val Asn 205 gaa tta gtt Glu Leu Val cct aat Pro Asn 210 aat gat cac att Asn Asp His Ile gca Ala 215 cct gac gat tac cag att aat cct gat Pro Asp Asp Tyr Gin Ile Asn Pro Asp 220 gat gaa gaa gga Asp Glu Glu Gly ttg aat aat act gtt cat Leu Asn Asn Thr Val His 230 235 ata aat gat ccc gat ttc Ile Asn Asp Pro Asp Phe 250 ttt aca aaa ccc Phe Thr Lys Pro aaa Lys 240 cag cca gat ttg gat Gin Pro Asp Leu Asp 245 ttt gat aag Phe Asp Lys cta cat 768 Leu His 255 tca tgg 816 Ser Trp gag aaa tac Glu Lys Tyr tat Tyr 260 cct gat ttg cct aaa Pro Asp Leu Pro Lys 265 gaa aca gaa aag Glu Thr Glu Lys ttg Leu 270 atg aca cag Met Thr Gin 275 cca atg cca aaa caa ttg tct aca gtt Pro Met Pro Lys Gin Leu Ser Thr Val 280 gaa tca ata 864 Glu Ser Ile tct gat Ser Asp 290 atg aga ttt gac ttc Met Arg Phe Asp Phe 295 aaa gga gat tta att Lys Gly Asp Leu Ile 300 agt tca ttc gaa ata Ser Ser Phe Glu Ile 315 gaa ttg agc gca 912 Glu Leu Ser Ala cct act tat atg 960 Pro Thr Tyr Met 320 gag Glu 305 gga gaa gaa cca aaa Gly Glu Glu Pro Lys 310 gga ctt cat cat cat Gly Leu His His His 325 tcg gag aac cca Ser Glu Asn Pro cat His 330 atg gca ggt tat Met Ala Gly Tyr aca ttg Thr Leu 335 1008 ggt gag ttg gca Gly Glu Leu Ala 340 cat tta gcc aga His Leu Ala Arg tcg Ser 345 act tta gct Thr Leu Ala gga caa aga tgc Gly Gin Arg Cys 350 1056 ttg agc att Leu Ser Ile 355 caa aca tta ggg aga Gin Thr Leu Gly Arg 360 ata tta cat aaa ttg gga tta cat Ile Leu His Lys Leu Gly Leu His 365 1104 aaa tac Lys Tyr 370 agt ata cta cca aaa Ser Ile Leu Pro Lys 375 aca gac tca gat Thr Asp Ser Asp gat gaa atc aaa caa cta Asp Glu Ile Lys Gin Leu 385 390 ttg ata gac caa tta cga Leu Ile Asp Gin Leu Arg 405 tca ctt gac ttt gaa Ser Leu Asp Phe Glu 395 atc att gaa aca ata Ile Ile Glu Thr Ile 410 gat cag agt ttt aca Asp Gin Ser Phe Thr 380 gat atg atg tgg gac Asp Met Met Trp Asp 400 aca gag gca get gat Thr Glu Ala Ala Asp 415 1152 1200 1248 gaa aaa aag acc aga aac tta tct gtc aga aat tat gca Glu Lys Lys Thr Arg Asn Leu Ser Val Arg Asn Tyr Ala ata gag gca Ile Glu Ala 430 1296 420 425 ttg tgg tta Leu Trp Leu 435 tat aga act gga ggt Tyr Arg Thr Gly Gly 440 gga aga cca gag ata act aaa caa Gly Arg Pro Glu Ile Thr Lys Gin 445 1344 acc gaa g Thr Glu G 450 <210> 8 <211> 460 <212> <213> Can ag gat ttg ata gca lu Asp Leu Ile Ala 455 caa gca gtt cag Gin Ala Val Gin aaa taa 1383 Lys 460 dida albicans <400> 8 Met Asp Phe Ile Gly Glu Ile Ile Giu 1 5 Giu Pro Thr Pro Lys Pro Thr Ile Giy 25 Leu Lys Giu Lys Lys Val Ser Arg Trp 40 Gin Ser Thr Thr Ser Pro Lys Thr Thr 55 Lys Ile His Gin Giu Asn Ile Giu Lys 70 Giu Ile Leii Gin Giu Arq Giu Giu Leu Leu Ile Giu Ser Leu Ile Gly Arg Ser 56 His Giu Thr Giu Aia Pro Lys 10 Gly Phe Pro Giu Leu Lys Lys Arg Gin Lys Gin Gin Gin Giu Giu Ile Arg Ser Giu Aia Ser Gin Met Leu 90 Lys Aia Lys Met Ser Giu Giy Leu Asp Pro Lys Lys Arg Giu 100 As n 105 Giu His Giu His 115 Trp Ile Gly Giy His Aia His Ala Giu Gly 125 Asp Ala Thr Asp 110 Tyr His Giy Leu Ser Gin Ser Met Lys 130 Leu Asp Th r 135 Asp Giu Gly Leu Th r 140 Ile Lys Giu Asp Arg Ala Leu Gly 155 Lys Ser Ser Leu Ser Giu Pro Giu 165 Thr Gly Ser Asn Lys Val Ala Phe Asp 175 Ile Giu Asp Asn Ile Lys 180 Val Lys Phe Giu 185 Leu Asp Asp Giu 190 Leu Asp Pro 195 Asn Gly Trp Glu Asp 200 Val Thr Asp Val Asn 205 Giu Leu Val Pro Asn 210 Asn Asp His Ile Ala 215 Pro Asp Asp Tyr Gin 220 Ile Asn Pro Asp Asp Glu Glu Gly Leu 230 Asn Asn Thr Val Phe Thr Lys Pro Lys 240 Gin Pro Asp Leu Ile Asn Asp Pro Asp 250 Phe Phe Asp Lys Leu His 255 Giu Lys Tyr Met Thr Gin 275 Tyr 260 Pro Asp Leu Pro Giu Thr Giu Lys Leu Ser Trp 270 Giu Ser le Pro Met Pro Lys Gin 280 Leu Ser Thr Val Tyr 285 Ser Asp 290 Met Arg Phe Asp Phe 295 Lys Gly Asp Leu Giu Leu Ser Ala Gi u 305 Giy Giu Giu Pro Lys 310 Asp Ser Ser Phe Giu 315 le Pro Thr Tyr Met 320 Gly Leu His His His 325 Ser Giu Asn Pro Met Ala Gly Tyr Thr Leu 335 Giy Giu Leu Leu Ser Ile 355 His Leu Ala Arg Ser 345 Thr Leu Ala Gly Gin Arg Cys 350 Gly Leu His Gin Thr Leu Giy Ile Leu His Lys Leu 365 Lys Tyr 370 Ser Ile Leu Pro Lys 375 Thr Asp Ser Asp Asp 380 Gin Ser Phe Thr Asp 385 Giu Ilie Lys Gin Leu 390 Ser Leu Asp Phe Asp Met Met Trp Leu Ile Asp Gin 58 Arg Ile Ile Glu Thr 410 Ile Thr Glu Ala Ala Asp 415 Glu Lys Lys Leu Trp Leu 435 Thr 420 Arg Asn Leu Ser Val 425 Arg Asn Tyr Ala Ile Glu Ala 430 Ile Thr Lys Gin 445 Tyr Arg Thr Gly Gly Arg Pro Glu Thr G1 <210> <211> <212> <213> <220> <221> <222> <220> <221> <222> <220> <221> <222> -u 00 Glu Asp Leu Ile Ala 455 Gin Ala Val Gin 9 2262

DNA

Candida albicans

CDS

(1)..(2262) modified base (1093)..(1095) modified base (1828)..(1830) <400> 9 atg gca gca gca cca cca cca cca gcg aaa aac cag ggt aag gca aaa Met Ala Ala Ala Pro Pro Pro Pro Ala Lys Asn Gin Gly Lys Ala Lys cag cat gtt aca ggt gcc agg ttc cgt cag cga aaa atc tcg gta aag Gin His Val Thr Gly Ala Arg Phe Arg Gin Arg Lys Ile Ser Val Lys 25 cag ccc ttg Gin Pro Leu act att tat aaa cag Thr Ile Tyr Lys Gin 40 aga gac cta cct act cta gat agc Arg Asp Leu Pro Thr Leu Asp Ser aat gag Asn Glu tta gag cct agt caa Leu Glu Pro Ser Gin 55 gtc cat cat tta aat tct aat gcg tca Val His His Leu Asn Ser Asn Ala Ser tca tca aca caa caa Ser Ser Thr Gin Gin ccg aga gac ctt cat Pro Arg Asp Leu His 75 gca gtt gaa act ggg Ala Val Glu Thr Gly gtt gac aag aat Val Asp Lys Asn gct gca caa aaa Ala Ala Gin Lys 100 gag gaa gag gaa gtg Glu Glu Glu Glu Val gca ctt ttg ggt tcg Ala Leu Leu Gly Ser 105 ctt cag caa gtt Leu Gin Gin Val atc aat Ile Asn aaa aaa gaa gaa aaa age agt Lys Lys Glu Glu Lys Ser Ser 110 gat atg tat Asp Met Tyr 115 att ccc aca ccg Ile Pro Thr Pro get tcg agg ata tgg Ala Ser Arg Ile Trp 125 ccc gag gca Pro Glu Ala cac aag His Lys 130 tat tac aag gat caa Tyr Tyr Lys Asp Gin 135 aag ttc aag cag cca Lys Phe Lys Gin Pro 140 gag aca tat atc 432 Glu Thr Tyr Ile aag Lys 145 ttt agt gcg aca gta Phe Ser Ala Thr Val 150 gag gac aca gtg Glu Asp Thr Val ttt tat aga gag Phe Tyr Arg Glu 170 ggt gtg gag tac aat Gly Val Glu Tyr Asn 155 aca cta tgc aag tac Thr Leu Cys Lys Tyr 175 atg 480 Met 160 tat 528 Tyr gac gag gta gat Asp Glu Val Asp ccc aaa aag Pro Lys Lys aac aag tca gat Asn Lys Ser Asp aac aat Asn Asn cga aag tgt Arg Lys Cys 190 act gaa Thr Glu 576 ttg gag ttt Leu Giu Phe 195 gaa aca atc tgt gac Giu Thr Ile Cys Asp 200 aag ttg gaa aag Lys Leu Giu Lys acc Thr 205 att gaa gca 624 Ile Giu Ala cga caa Arg Gin 210 ccg ttt ttg tct atg Pro Phe Leu Ser Met 215 gac ccc agc aac att Asp Pro Ser Asn Ile 220 cta tcg tac gag 672 Leu Ser Tyr Giu gag Giu 225 ttg tcg tcg tac att Leu Ser Ser Tyr Ile 230 gtg gat cag ttt Vai Asp Gin Phe acc aat ggt ggg Thr Asn Giy Gly 250 aaa agt gca gtg aaa aca Lys Ser Ala Vai Lys Thr 235 240 aat cta gag tat ata tcq Asn Leu Giu Tyr Ile Ser 255 agc aac ccg tat att Ser Asn Pro Tyr Ile 245 acg aca gct Thr Thr Aia tta aaa Leu Lys 260 gag aga ttg Giu Arg Leu aag gaa ata aag Lys Giu Ile Lys tat gaa ccg Tyr Giu Pro 270 ttt gtt act Phe Val Thr 275 att ttt gat aag aac Ile Phe Asp Lys Asn 280 caa atg tcc aca agt gcg gtg aga Gin Met Ser Thr Ser Ala Vai Arg 285 cct att Pro Ile 290 ccc aaa ttg ttt gag Pro Lys Leu Phe Giu 295 ttg ttc ggc aga cct gtt tat gat cat Leu Phe Gly Arg Pro Val Tyr Asp His 300 aag gag aga aaa ata Lys Giu Arg Lys Ile 310 gaa aga aag ggc aaa Giu Arg Lys Gly Lys 315 acc atc cag ccc aca Thr Ile Gin Pro Thr 320 otc aaa ttt gaq Leu Lys Phe Glu gat Asp 325 cct aac tcg aac Pro Asn Ser Asn gaa Glu 330 aag gaa aac gac Lys Giu Asn Asp aat gac Asn Asp 335 1008 cca tat ata Pro Tyr Ile ttc aga cga ogt gaq Phe Arg Arg Arg Glu 345 ttt agg caa gca aga aag acg Phe Arg Gin Ala Arg Lys Thr 350 1056 aga aga gcc Arg Arg Ala 355 gat aca att ggt gca Asp Thr Ile Gly Ala 360 gag aga ata aga ctg Glu Arg Ile Arg Ser 365 atg oaa aag Met Gin Lys 1104 tcg ttg Ser Leu 370 cac cgo goa cgt gat His Arg Ala Arg Asp 375 ttg ata atg agt gtt agt gaa aga gag Leu Ile Met Ser Val Ser Giu Arg Glu 380 1152 otc aaa otc gao aat Leu Lys Leu Asp Asn 390 ttt caa gca gag cat Phe Gin Ala Giu His 395 gaa ttg ttt aaa gcc Glu Leu Phe Lys Ala 400 1200 agg tgc got aoo aag Arg Cys Ala Thr Lys 405 got tgt aag agg gag Ala Cys Lys Arg Glu 410 otc aat ato aag ggt gao Leu Asn Ile Lys Gly Asp 415 1248 gaa tao ttg tto Glu Tyr Leu Phe 420 ttt ccg oat aaa aag Phe Pro His Lys Lys 425 aag aaa att gtt ogt Lys Lys Ile Val Arg 430 act gaa 1296 Thr Glu gat gaa gaa Asp Giu Glu 435 agg gag aag aag Arg Glu Lys Lys gaa aag aag aag Glu Lys Lys Lys oaa gac caa gaa Gin Asp Gin Glu 445 1344 ott gca Leu Ala 450 otc aag oaa oaa oaa Leu Lys Gin Gin Gin 455 gca ota cag oaa oag oag oaa oaa oca Ala Leu Gin Gin Gin Gin Gin Gin Pro 460 1392 caa cca cca caa Gin Pro Pro Gin caa Gin 470 gca cca tca aaa Ala Pro Ser Lys caa Gin 475 gat ggt aca Asp Gly Thr tca acg Ser Thr 480 age cag cct tat gtc Ser Gin Pro Tyr Val 485 ctt gtt aca gtt tcg Leu Val Thr Val Ser 500 cgt get gtg ttg gag Arg Ala Val Leu Glu 515 aaa ctc cca ccc gca Lys Leu Pro Pro Ala 490 aaa gtt cca gat atg gat Lys Val Pro Asp Met Asp 495 1440 1488 1536 1584 ttg gta tta aag Leu Val Leu Lys 505 aaa ttg cgc aag Lys Leu Arg Lys 520 gaa aag aac gaa acc atc aaa Glu Lys Asn Glu Thr Ile Lys 510 aga aag gaa cac gac aag gga Arg Lys Glu His Asp Lys Gly 525 ttt atc Phe Ile 530 aat ttg aca gac gat Asn Leu Thr Asp Asp 535 ccg tat cag cca Pro Tyr Gin Pro ttt ttc gat att tca 1632 Phe Phe Asp Ile Ser 540 tat tcg tcg att gcg 1680 Tyr Ser Ser Ile Ala 560 aat agg gcc gaa gag Asn Arg Ala Glu Glu 550 ttg agc cat att Leu Ser His Ile ccg Pro 555 gcc aca cac tat cac Ala Thr His Tyr His 565 ctt aaa aag cta ctt Leu Lys Lys Leu Leu 580 caa ttc aac aca tcg Gin Phe Asn Thr Ser 570 aac tac atg aac gac caa 1728 Asn Tyr Met Asn Asp Gin 575 tta cct ggt gta aaa acg 1776 Leu Pro Gly Val Lys Thr 590 gaa gag aaa Glu Glu Lys aaa Lys 585 ttt ttg ggt Phe Leu Gly 595 tct aac ggg gag ttg Ser Asn Gly Glu Leu 600 gta cca tcg aag gca ttt cca cat Val Pro Ser Lys Ala Phe Pro His 605 1824 ttg ctg Leu Ser 610 tcg ttg ctt gag Ser Leu Leu Glu aag tat aag gcg Lys Tyr Lys Ala gag acg caa gat Glu Thr Gin Asp 635 aca Thr 620 agt ggg tat att Ser Gly Tyr Ile cga tta ttg caa agc gtg Arg Leu Leu Gin Ser Val 630 ttt agt tca tac aco Phe Ser Ser Tyr Thr 640 1872 1920 1968 2016 aat ggc ttt aaa Asn Giy Phe Lys gcg ttt ccc cag Ala Phe Pro Gin 660 gat gtt gag cca aaa Asp Val Giu Pro Lys 645 aga ata cgt cga aga Arg Ile Arg Arg Arg 665 gaa Glu 650 aca aat gaa cct Thr Asn Giu Pro gtt atg Val Met 655 gtg ggc agg gct ggc agg gtt Val Giy Arg Ala Gly Arg Val 670 ttt ttg qac Phe Leu Asp 675 cac cag caa gag His Gin Gin Glu tac Tyr 680 ccg caa ccg aat Pro Gin Pro Asn cag caa gac Gin Gin Asp 2064 aca gat Thr Asp 690 att aaa Ile Lys 705 cgt gtg qga ggt atc Arg Vai Gly Giy Ile 695 cca gat gtg tat tgt aaa gag gat gcc Pro Asp Val Tyr Cys Lys Glu Asp Ala 700 2112 cga tta cag Arg Leu Gin tca aag tgg aag ttc Ser Lys Trp Lys Phe 710 ttg gat cct tca aag Leu Asp Pro Ser Lys 730 gat aca gaa tat aaa aca Asp Thr Giu Tyr Lys Thr 715 720 ttg aat ggt att agt cca Leu Asn Giy Ile Ser Pro 735 act gaa cca ttt Thr Giu Pro Phe tct acg caa tcg Ser Thr Gin Ser 740 2160 2208 2256 2262 att aga ttt ggg tct Ile Arg Phe Gly Ser 745 atg ttg ttg aat aga Met Leu Leu Asn Arg 750 aca cgt Thr Arg aaa tag Lys <210> <211> 754 <212> <213> Candida albicans <400> Met Ala Ala Ala Pro Pro Pro Pro Ala 1 5 Lys Asn Gln Gly Lys Ala Lys 10 Gin His Val Thr Gly Ala Arg Phe Arg 25 Gln Pro Leu Thr Ile Tyr Lys Gin Arg 40 Gin Arg Lys Ile Asp Leu Pro Thr Ser Val Lys Leu Asp Ser Ser Asn Ala Ser Asn Glu Leu Glu Pro Ser Gin 55 Val His His Leu Arg Asp Leu His 75 Asn Ser Ser Ser Thr Gin Gin 70 Pro Ala Val Glu Thr Gly Val Asp Lys Asn Glu Glu Glu Val Leu Gin Gin Val Ile Asn Ser Ser Ala Ala Gin Ala Leu Leu Gly Ser 105 Lys Lys Glu Glu Asp Met Tyr 115 Ile Pro Thr Pro Ala Ser Arg Ile Trp 125 Pro Glu Ala His Lys 130 Tyr Tyr Lys Asp Gin 135 Lys Phe Lys Gin Pro Glu Thr Tyr Ile 140 Val Glu Tyr Asn Met Lys 145 Phe Ser Ala Thr Val 150 Glu Asp Thr Val Gly 155 Asp Giu Val Asp Lys Phe Tyr Arg Giu 170 Thr Leu Cys Lys Tyr Tyr 175 Pro Lys Lys Leu Giu Phe 195 Lys 180 Asn Lys Ser Asp Giu 185 Asn Asn Arg Lys Cys Thr Giu 190 Ile Giu Ala Giu Thr Ile Cys Asp 200 Lys Leu Glu Lys Thr 205 Arq Gin 210 Pro Phe Leu Ser Asp Pro Ser Asn Leu Ser Tyr Glu Giu 225 Ser Leu Ser Ser Tyr Asn Pro Tyr Ile 245 Ilie 230 Val Asp Gin Phe Lys 235 Ser Ala Val Lys Thr 240 Vai Thr Asn Giy Asn Leu Giu Tyr Ile Ser 255 Thr Thr Aia Phe Val Thr 275 Leu 260 Lys Giu Arg Leu Se r 265 Lys Giu Ile Lys Tyr Giu Pro 270 Aia Val Arg Ilie Phe Asp Lys As n 280 Gin Met Ser Thr Pro Ilie 290 Pro Lys Leu Phe Leu Phe Giy Arg Pro 300 Val Tyr Asp His Trp 305 Lys Giu Arg Lys Ile 310 Giu Arg Lys Gly Lys 315 Thr Ile Gin Pro Th r 320 Leu Lys Phe Glu Pro Asn Ser Asn Lys Giu Asn Asp Asn Asp 335 Pro Tyr Ilie Cys 340 Phe Arg Arg Arg Giu 345 Phe Arg Gin Ala Arg Lys Thr 350 Arg Arg Ala Asp Thr Ile Giy Ala Giu Arg Ile Arg Ser Met Gin Lys Ser Leu 370 Ile Leu His Arg Ala Arg Lys Leu Asp Asn 390 Ala Thr Lys Ala 405 Leu Phe Phe Pro Asp 375 Phe 66 Leu Ile Met Ser Val Ser Glu Arg Glu 380 Gin Ala Glu His Glu Leu Phe Lys Ala 395 400 Lys Arg Glu Leu Asn Ile Lys Gly Asp 410 415 Lys Lys Lys Lys Ile Val Arg Thr Glu Cys Cys Glu Tvr His Asp Glu Glu 435 Leu Ala Leu 450 Pro Gin Pro 465 420 Arg 425 Lvs Ara Glu Glu Lys Lys Lys Lys 440 Ala 430 Asp Gin Glu Gin Gin Pro Lys Gin Gin Gin 455 Ala Leu Gin Gin Gin 460 Asp Pro Gin Gin 470 Lys Pro Ser Lys Gin 475 Lys Gly Thr Ser Thr 480 Asp Ser Leu Gin Pro Tyr Val 485 Val Thr Val Ser 500 Leu Pro Pro Val Pro Asp Leu Val Leu Lys 505 Lys Asn Glu Arg Ala Val 515 Phe Ile Asn 530 Thr Asn Arg 545 Leu Glu Lys Leu Arg Lys Arg Lys 520 Leu Thr Asp Asp Pro Tyr Gin Pro 535 Ala Glu Glu Leu Ser His Ile Pro 550 555 Glu His 525 Phe Phe 540 Tyr Ser Thr Ile Lys 510 Asp Lys Gly Asp Ile Ser Ser Ile Ala 560 Ala Thr His Tyr His Gin Phe Asn Thr Ser Asn Tyr Met Asn Asp Gin 565 Leu Leu Lys Lys Phe Leu Gly 595 Leu Ser Ser Giu Giu Lys Leu Pro Gly 575 Val Lys Thr 590 Phe Pro His Gly Tyr Ile Asn Gly Giu Leu 600 Lys Pro Ser Lys Al a 605 Ser Leu Leu Giu 610 Giu Arg Giu 615 Val1 Tyr Lys Ala Leu Leu Gin Giu Thr Gin 625 Asn Asp 635 Th r Ser Ser Tyr Gly Phe Lys Asp 645 Arg Giu Pro Lys Glu 650 Val1 Asn Giu Pro Val Met 655 Ala Phe Pro Phe Leu Asp 675 Thr Asp Arq Gin 660 His Ile Arg Arg Gly Arg Ala Gin Gin Glu Gin Pro Asn Gly Arg Vai 670 Gin Gin Asp Giu Asp Ala Val Gly Gly 690 Ile Lys Ile 695 Lys Asp Val Tyr Cys 700 Thr Arg Leu Gin Ser 710 Leu Trp Lys Phe Giu Tyr Lys Giu Pro Phe Ser 725 Asp Pro Ser Lys 730 Asn Gly Ile Ser 735 Ser Thr Gin Ser Ile Arq Phe Gly Ser Met Leu Leu Asn Arg Thr Arg 745 750 Lys <210> 11 <211> 447 <212> DNA <213> Candida aibicans <220> <221> CDS <222> <400> 11 atg tca gat ata gat Met Ser Asp Ile Asp 1 5 ata gat aat gta Ile Asp Asn Val tat gaa tta gga ttt aaa gaa ggt caa Tyr Giu Leu Gly Phe Lys Giu Gly Gin 25 tat tta gaa gga aaa gaa tat ggt tat Tyr Leu Glu Gly Lys Glu Tyr Gly Tyr 40 tta aat tta gaa gaa gaa caa Leu Asn Leu Giu Glu Giu Gin 10 ata caa gga aca aaa gat caa Ile Gin Gly Thr Lys Asp Gin caa act gga ttt caa cga ttt Gin Thr Gly Phe Gin Arg Phe tta atc Leu Ile att ggt tat att caa Ile Giy Tyr Ile Gin 55 gaa tta atg aaa ttt tgg tta tcc cat Glu Leu Met Lys Phe Trp Leu Ser His ata Ile gat caa tat aat Asp Gin Tyr Asn aac Asn 70 tct tct tca ctt cgg Ser Ser Ser Leu Arg 75 aat cat ttg aat aat Asn His Leu Asn Asn aat gga gat aaa gaa Asn Giy Asp Lys Glu ttg gaa gat att atg Leu Giu Asp Ile Met qca caa att tct ata Ala Gin Ile Ser Ile 90 gtt gaa gat tat gaa aaa aat att aaa aag gca aga aat aaa tta aga Val Giu Asp Tyr Giu Lys Asn Ile Lys Lys Aia Arg Asn Lys Leu Arg gtg ata qct Val Ile Ala 115 agt ata act aaa Ser Ile Thr Lys act tqg aaa att gat Thr Trp Lys Ile Asp 125 tca ttg gat 384 Ser Leu Asp aat ttg Asn Leu 130 gtg aaa gaa qta Val Lys Glu Val gg t Gly 135 gga act tta caa gtt Gly Thr Leu Gin Vai 140 agt qaa aac ccc 432 Ser Giu Asn Pro 0 gat gat atg tgg tga 447 Asp Asp Met Trp 145 <210> 12 <211> 149 <212> <213> Candida albicans <400> 12 Met Ser Asp Ile Asp 1 5 Ile Asp Asn Val Leu 10 Asn Leu Giu Giu Giu Gin Tyr Glu Leu Tyr Leu Glu Gly Phe Lys Giu Gly Gin 25 Ile Gin Gly Thr Lys Asp Gin Gin Arg Phe Gly Lys Giu Tyr Tyr Gin Thr Gly Leu Ile Ile Giy Tyr Ile Gin 55 Giu Leu Met Lys Phe Trp Leu Ser Asn His Leu Asn His Asp Gin Tyr Asn Asn Ser Ser Ser Leu Asn Leu Giu Asp Ile Ala Gin Ile Ser Ile Thr Asn Giy Asp Lys Giu Lys Val Glu Asp Val Ile Ala 115 Glu Lys Asn Ile Ala Arg Asn Lys Leu Arg 110 Ser Leu Asp Ser Ile Thr Lys Glu 120 Thr Trp Lys Ile Asp 125 Asn Leu 130 Val Lys Glu Val Gly Thr Leu Gin Val 140 Ser Glu Asn Pro Asp Asp Met Trp 145 <210> <211> <212> <213> <220> <221> <222> 13 966

DNA

Candida albicans

CDS

<400> 13 atg ggt Met Gly 1 aaa aga aga Lys Arg Arg 5 gta gat gaa gaa tct Val Asp Glu Glu Ser 10 gat tca gat att Asp Ser Asp Ile gat gtt 48 Asp Val agt tca acc gat Ser Ser Thr Asp tca gaa act gaa tta Ser Glu Thr Glu Leu 25 gaa agc aca caa Glu Ser Thr Gin caa caa caa Gin Gin Gin caa caa Gin Gin gaa ggt gct act aca Glu Gly Ala Thr Thr att caa gaa act gtt gat gtt gat Ile Gin Glu Thr Val Asp Val Asp ttt gat Phe Asp ttt ttt gat tta aat Phe Phe Asp Leu Asn 55 cct Pro caa att gat Gin Ile Asp ttc cat gct act aag Phe His Ala Thr Lys aat Asn ttt tta aga caa tta Phe Leu Arg Gin Leu 70 ttt ggt gat gat aat Phe Gly Asp Asp Asn 75 gga gaa ttt aat tta 240 Gly Glu Phe Asn Leu agt qaa ata gcc Ser Glu Ile Ala att aaa act gaa Ile Lys Thr Glu 100 gat Asp tta att tta cqa gaa Leu Ile Leu Arg Glu 90 aat tcc gtg ggg aca tca 288 Asn Ser Val Gly Thr Ser ttt gca att tta aqt gta 336 Phe Ala Ile Leu Ser Val 110 gga atg gaa agt gat Gly Met Giu Ser Asp 105 att aat tta Ile Asn Leu 115 act aat aat tta aat Thr Asn Asn Leu Asn 120 gtg goc gtg att aaa caa ttg att Val Ala Val Ile Lys Gin Leu Ile 125 gaa tat Glu Tyr 130 att tca aat aaa acc Ile Ser Asn Lys Thr 135 aaa tot aaa act gaa ttc aat att att Lys Ser Lys Thr Glu Phe Asn Ile Ile 140 ttg Leu 145 aaa aaa ttg tta acc Lys Lys Leu Leu Thr 150 aat cag aac gat act Asn Gin Asn Asp Thr 155 act aga gat agg aaa Thr Arg Asp Arg Lys 160 ttt aaa act gga tta ata Phe Lys Thr Gly Leu Ile 165 att agt gaa Ile Ser Glu aga ttt ata aat atg cca gtt Arg Phe Ile Asn Met Pro Val 170 175 ott tta caa gaa atg gaa aaa Leu Leu Gin Giu Met Giu Lys 190 gaa gtg att cca Glu Val Ile Pro 180 cca atg tat aaa atg Pro Met Tyr Lys Met 185 got gaa gat Ala Giu Asp 195 got cat gaa aat Ala His Giu Asn tat Tyr 200 gaa ttt gat tat Glu Phe Asp Tyr ttt tta att ata Phe Leu ile Ile 205 tca aga Ser Arg 210 gtt tat caa tta Val Tyr Gin Leu gat cca gtg gaa Asp Pro Val Glu aga qaa gat gaa gat Arg Giu Asp Giu Asp 220 aaq aat aag aaq aaq Lys Asn Lys Lys Lys 240 ca c His 225 gaa aaa gaa tcc aat Giu Lys Giu Ser Asn 230 cgt aaa aag Arg Lys Lys aaq aac Lys Asn 235 aaa ttg gct aat aat Lys Leu Ala Asn Asn 245 gaa cca aaa cca ata Giu Pro Lys Pro Ile 250 gaa atg gat tat ttc cat Glu Met Asp Tyr Phe His 255 ctt gaa qat Leu Giu Asp att ttg gaa tca Ile Leu Glii Ser act caa ttt aaa Thr Gin Phe Lys gga ata ttt Gly Ile Phe 270 gaa tat aat Glu Tyr Asn 275 aat gaa aat aaa caa Asn Glu Asn Lys Gin 280 gaa aca gat tca aga aga gta ttt Giu Thr Asp Ser Arg Arg Val Phe 285 act gaa Thr Glu 290 tat ggt att gat cct Tyr Gly Ile Asp Pro 295 aaa tta agt tta atc tta att gat aaa Lys Leu Ser Leu Ile Leu Ile Asp Lys 300 gat Asp 305 aat tta gct aaa tca Asn Leu Aia Lys Ser 310 gtc att gaa atg gaa Val Ile Giu Met Giu 315 caa caa ttc cca cct Gin Gin Phe Pro Pro 320 cca taa Pro <210> 14 <211> 322 <212> <213> Candida aibicans <400> 14 Met Gly Lys Arq 1 Ser Ser Thr Asp Gin Gin Gin Glu Phe Asp Phe Phe Asn Phe Leu Arg Ser Giu Ile Ala Arq Val Asp Giu Giu Ser Asp Ser Asp Ile Asp Val 5 10 Ser Glu Thr Glu Leu Giu Ser Thr Gin Gin Gin Gin 25 Giy Ala Thr Thr Ile Gin Giu Thr Vai Asp Val Asp 40 Asp Leu Asn Pro Gin Ile Asp Phe His Ala Thr Lys 55 Phe Gly Gin Leu 70 Aso Leu Gly Asp Asp Asn 75 As n Giu Phe Asn Leu Ile Leu Arg Gly Glu 90 Pro Ser Val Gly Thr Ser Ile Lys Thr Ile Asn Leu 115 Giu Tyr Ile Met Giu Ser Phe Ala Ile Asn Asn Leu Asn 120 Lys Ala Val Ile Lys 125 Ph e Leu Ser Vai 110 Gin Leu Ile Asn Ile Ile Ser Asn Lys 130 Leu Lys Thr 135 As n Ser Lys Thr Giu 140 Thr Lys Leu Leu Thr 150 Ile Gin Asn Asp Arg Asp Arg Lys Thr Giy Leu 165 Ile Ser Giu Arg 170 Ile Asn Met Pro 175 Giu Val Ile Pro Pro Met Tyr Lys Met Leu Leu Gin Giu Met Giu Lys 180 185 1 -74 Glu Phe Asp Ala Glu Asp Ala His Glu Asn Tyr 195 200 Ser Arq Val Tyr Gin Leu Vai Asp 210 215 His Glu Lys Glu Ser Asn Arq Lys 225 230 Lys Leu Ala Asn Asn Giu Pro Lys 245 Leu Glu Asp Gin Ile Leu Glu Ser 260 Giu Tyr Asn Asn Glu Asn Lys Gin 275 280 Thr Glu Tyr Gly Ile Asp Pro Lys 290 295 Asp Asn Leu Ala Lys Ser Val Ile 305 310 Pro Tyr Phe 205 Arq Giu Leu Ile Ile Pro Val Giu Asp Glu Asp Lys Lys Asn Lys 235 Pro Ile Giu Met 250 Asn Thr Gin Phe 265 Glu Thr Asp Ser Asn Lys Lys Asp Tyr Phe His 255 Lys Gly Ile Phe 270 Arg Arg Val Phe 285 Leu Leu Ser Leu Ile 300 Giu Met Giu Gin 315 Ile Asp Lys Gin Phe Pro <210> <211> 320 <212> DNA <213> Candida aibicans <400> caatttattc atggtccgtt ctggaaattq atttttgqta aaactqctaa tgaattaqaa aaatcgcaag atttqcccaa tgaatatatg attgtggaga atqtqccatt attaaataga 120 tttattaqta tacctaaqqa gtatggcgac ttaaattgtt cagcatttgt tgcggqtata 180 attgagqgag cacttgataa tagtqqattc aatgccgatg ttacagcaca cacggtcgct 240 acaqatgcaa atccattaag aacagtattt ttgatcaagt ttgacgattc tgttttaatt 300 agagagagtt tgagatttgg 320 <210> 16 <211> 295 <212> DNA <213> Candida albicans <400> 16 qttcatgttt qqtgactcag aqcgtctcaa attgaqtaag ttgaataaat ttactatttt tttattgtcc aaaqagqaaa gagattatat atataacaac tgtttcctca aaaaactacc tggtggacaa tcaatgatcg ttgagccaga ctatattgtt ttacatcaat acacaaatat acaaatgttg tttagaccag ogattataca gaaagcagtc ttccagattt acctatttgg cctgtgttta tacgaactcg aaaatgataa tgactcaatt atgacaccag tcaaa <210> 17 <211> 392 <212> DNA <213> Cand-ida albicans <400> 17 atctctgata tgagatttgg ctttaaaggc gatttaattg aattggctcc agtgggaqat gcaccaaaaq atagttcatc cqacatacgt actcatatgg gactccatca tcattcqqag 120 accccacata tggcaggtta tacattgggt gaqttgqccc atttagccag atcqacttta 180 76 gctqgacaaa gatqcttgag cattcaaaca ttaggqaqaa tcttccataa attgggatta 240 cataaataca qtatactacc aaaccagctc aatqatcaga gttttacaqa tgaatcaaaa 300 ctatcacttq actttgaaga tagatqtgqg acttqataqa ccaattacqa atcattgaaa 360 caataacaga ggcagctgat gqaaaaaaga cc 392 <210> 18 <211> 335 <212> DNA <213> Candida albicans <400> 18 attcccacac cggacgcttc gagqatatgg cccgaggcac acaagtatta caagqatcaa aagttcaagc aqccagaqac atatatcaaq tttagtgcga cagtagagga cacagtgqgt gtggagtaca atatqqacga ggtagatqaa aaqttttata gagagacact atgcaagtac tatcccaaaa agaaaaacaa gtcagatgag aacaatcgaa agtgtactga attggagttt gaaacaatct gtgacaagtt ggaaaagacc attgaagcac gacaaccgtt tttgtctatg gaccccagca acattctatc gtacgaggag ttgtc <210> 19 <211> 326 <212> DNA <213> Candida albicans <400> 19 agatataqat aatgtattaa atttagaaga agatcaatat gaattaggat ttaaagaagg tcaaatacaa ggaacaaaag atcaatattt agaaggaaaa gaatatqgtt atcaaactgq 120 77 atttcaacqa tttttaatca ttggttatat tcaagaatta atgaaatttt qqttatccca 180 tatagatcaa tataataact cttcttcact tcgqaatcat ttgaataatt tggaagatat 240 tatqgcacaa atttctataa cgaatgqaga taaagaaqtt qaagattatq aaaaaaatat 300 taaaaagqca agaaataaat taagag 326 is <210> <211> 374 <212> DNA <213> Candida albicans <400> cctcaaattq atttccatqc tactaaqaat ttttaagaca ttatttgqtg atgataatqq aqaatttaat ttaagtqaaa taqccgattt aattttacga gaaaattccg tgqqqacatc 120 aattaaaact gaaggaatgq aaagtgatcc atttqcaatt ttaagtgtaa ttaatttaac 180 taataattta aatqtggccg tqattaaaca attgattqaa tatattttaa ataaaaccaa 240 atctaaaact qaattcaata ttattttgaa aaaattqtta accaatcaga acqatactac 300 tagagataqq aaatttaaaa c tqqattaat aattaqtgaa aqatttataa atatgccagt 360 tgaagtgatt ccac 374 <210> 21 <211> <212> DNA <213> Candida albicans <400> 21 caatttattc atqttcgnat ctggaaattg atttt <210> 22 <211> 29 <212> DNA <213> Canciida albicans <400> 22 ccaaatctca aactctctct aattaaaac 29 <210> 23 <211> 38 <212> DNA <213> Candida albicans <400> 23 gttcatgttt qgtqactcag agcgtctcaa ctatattq 38 <210> 24 <211> 33 <212> DNA <213> Candida aibicans <400> 24 tttgataaac acaggctggt ctaaatctqg ctc 33 <210> <211> 32 <212> DNA <213> Candida albicans <400> atctctqata tqagatttgg ctttaaaggc ga 32 <210> 26 <211> 32 <212> DNA <213> Candida albicans <400> 26 ggtctttttt ccatcagctg cctctqttat tg 32 <210> 27 <211> <212> DNA <213> Canclida albicans <400> 27 attcccacac cgqacgcttc <210> 28 <211> <212> DNA <213> Candida albicans <400> 28 gacaactcct cgtacgatag <210> 29 <211> <212> DNA <213> Candida albicans <400> 29 agataatgta ttaaatttag <210> <211> <212> DNA <213> Candida albicans <400> ctcttaattt atttcttqcc <210> 31 <211> <212> DNA <213> Candida albicans <400> 31 cctcaaattg atttccatgc <210> 32 <211> <212> DNA <213> Candida albicans <400> 32 qtggaatcac ttcaactggc

Claims (26)

1. An isolated polynucleotide containing a nucleotide sequence selected from the group consisting of: a) a polynucleotide having at least 50% identity with a polynucleotide coding for a polypeptide having the same function and having an amino acid sequence homologous with the sequence of SEQ ID No. 12, and b) a complementary polynucleotide of polynucleotide a).

2. The polynucleotide according to claim 1 having at least 60% identity with said polynucleotide coding for a polypeptide.

3. The polynucleotide according to claim 1 having at least 70% identity with said polynucleotide coding for a polypeptide.

4. The polynucleotide according to any one of claims 1 to 3 in the form of DNA. The polynucleotide according to any one of claims 1 to 3 in the form of RNA.

6. The polynucleotide according to claim 4 comprising the nucleotide sequence of SEQ ID No. 11.

7. The polynucleotide according to claim 4 coding for the proteins of Candida albicans having the same functions as the proteins of PCaNL260, and containing a nucleotide sequence from SEQ ID No. 11.

8. The polynucleotide according to claim 7 coding for the amino acid sequence of S 20 SEQ ID No. 12.

9. The polynucleotide according to claim 7 or claim 8 coding for the protein of PCaNL260, and DNA sequences which hybridize with these and/or have homologies of at least 50% with said polynucleotides.

10. The polynucleotide according to any one of claims 7 to 9 comprising 25 modifications introduced by suppression, insertion and/or substitution of at least one nucleotide coding for the protein having the same activities as the protein of PCaNL260.

11. The polynucleotide according to any one of claims 7 to 10 comprising DNA sequences which code for the proteins with similar functions the respective amino acid sequences of which have an homology of at least 40% with the amino acid sequences coded by said polynucleotides.

12. The polynucleotide of claim 11 having a homology of at least 45% with the amino acid sequences. 15/06/04,atI 2396.specipgs.35

13. The polynucleotide of claim 11 having a homology of at least 50% with the amino acid sequences.

14. The polynucleotide of claim 11 having a homology of at least 60% with the amino acid sequences.

15. The polynucleotide of claim 11 having a homology of at least 70% with the amino acid sequences.

16. An isolated polypeptide having an amino acid sequence from SEQ ID No. 12 coded by the DNA sequences according to any one of claims 7 to 11 and analogues of these polypeptides.

17. Process for the preparation of recombinant proteins having the amino acid sequences of SEQ ID No. 12 comprising, for the preparation of each of these proteins, the expression in an appropriate host of a DNA sequence coding for this protein according to any one of claims 7 to 11 then the isolation and purification of said recombinant protein.

18. Expression vectors each containing one of the polynucleotide sequences according to any one of claims 7 to 11.

19. Host cell transformed with a vector according to claim 18. Process as defined in claim 17 in which the host cell is DH5 alpha E. coli or XL1- Blue E. coli.

21. Process as defined in claim 18 in which the host cell is Saccharomyces cerevisae. 20 22. A plasmid deposited at the CNCM under the number 1-2212.

23. Screening process for antifungal compounds characterised in that the activity of the polypeptide of PCaNL260 as defined in claim 16 is measured, in the presence of the compounds, to determine the antifungal properties of said compounds wherein the compounds having an inhibitory effect on this activity are selected. 25 24. Use of a compound selected by the process according to claim 23 to inhibit o Candida or fungi.

25. Use of the polynucleotides according to any one of claims 7 to 15 or use of the polypeptide according to claim 16 for the selection of compounds having antifungal properties as inhibitors of the proteins of Candida albicans.

26. Use of a polypeptide as defined in claim 16 or a fragment of this polypeptide having the same function for the preparation of a medicament intended to induce an immunological response in a mammal by inoculation of this medicament producing an antibody which allows said mammal to be protected against the disease. 15/06/04.at 12396 spccipgs.36

27. An isolated antibody specific for the polypeptide according to claim 16 or a fragment of the polypeptide having the same function.

28. The antibody according to claim 27 specific for the proteins of PCaNL260 or a fragment thereof.

29. Use of the polynucleotides of CaNL260 according to any one of claims 7 to 15 or of any one of the polypeptides coded by these genes according to claim 16 for the preparation of compositions which can be used for the diagnosis or treatment of diseases caused by the pathogenic yeast Candida albicans. Kit for the diagnosis of fungal infections comprising a polynucleotide according to any one of claims 7 to 11 or a sequence having a similar function or a functional fragment of this sequence, the polypeptide coded by this sequence or a polypeptide fragment having the same function or an antibody directed against such polypeptide coded by this DNA sequence or against a fragment of this polypeptide.

31. The isolated polynucleotide of claim 1 substantially as described in any one of the Examples herein. DATED this 16 h day of June, 2004 20 AVENTIS PHARMA S.A. By their Patent Attorneys: CALLINAN LAWRIE S. *ee e* *e *•e ***ee *ee* 15/06/04,atl 2396 specipgs37