AU759732B2 - Method for screening antimycotic substances using essential genes from S. cerevisiae - Google Patents

Description

WO 99/55907 PCT/EP99/02722 METHOD FOR SCREENING ANTIMYCOTIC SUBSTANCES USING ESSENTIAL GENES FROM S. CEREVISIAE The present invention relates to a method for screening for antimycotic -substances in which essential genes from mycetes, particularly from Saccaromyces cerevisiae (S.cerevisiae) as well as functionally similar genes from -other mycetes, or the corresponding encoded proteins, are used as targets.

The spectrum of known fungal infections stretches from fungal attack of skin or nails to potentially hazardous mycotic infections of the inner organs; Such infections and resulting diseases are known as mycosis.

Antimycotic substances (fungistatic or fungicidal) are used for treatment of mycosis. However, up to now, relatively few substances with pharmacological effects are known, such as Amphotericin B, Nystatin, Pimaricin, Griseofulvin, Clotrimazole, 5-fluoro-cytosine and Batraphene. The drug treatment of fungal infections is extremely difficult, in particular because both the host cells and the mycetes, are eukaryotic cells. Administration of drugs based on known antimycotic substances results S. therefore often in undesired side-effects, for example Amphotericin B has a nephrotoxic effect. Therefore, there 2 is a strong need for pharmacologically efficient substances usable for the preparation of drugs, which are suitable for prophylactic treatments of immunodepressive states or for the treatment of an existing fungal infection. Furthermore, the substances should exhibit a specific spectrum of action 30 in order to selectively inhibit the growth and proliferation of mycetes without affecting the treated host organism.

The aim of the present invention is to provide a method for the identification of antimycotic substances. An 35 essential feature of this method is that essential genes from mycetes are used as targets for the screening.

The present invention thus concerns a method for the screening of antimycotic substances wherein an essential gene from mycetes or a similar mycete gene having a sequence identity, at the nucleotide level, of at least 50%, more preferably of at least and most preferably of at least 70% with the essential gene, or the corresponding encoded protein, is used as target and wherein the essential gene is YIL019w as herein described.

According to one embodiment of the method of the invention mycete cells which express the essential gene, or a functionally similar mycete gene, to a different level are incubated with the substance to be tested and the growth inhibiting effect of the substance is determined.

According to another embodiment, said target gene or the corresponding target gene encoded protein is contacted in vitro with the substance to be tested and the effect of.

the substance on the target is determined.

According to another embodiment, the screened substances inhibit partially or totally the functional expression of the essential genes or the functional activity of the encoded proteins.

According to another embodiment, the mycete species are selected from the group comprising Basidiomycetes, Ascomycetes and Hyphomycetes.

According to another embodiment of the method of the invention said functional similar genes are essential genes from Candida Spp., preferably Candida albicans, or *oo 06/02/03,mcl 1623.speci,2 WO 99/55907 PCT/EP99/02722 3 from Aspergillus Spp., preferably from Aspergillus fumigatus.

According to a further embodiment of -the above method said mycete cells are haploid S.cerevisiae cells.

According to a particular embodiment of the method of the invention the essential genes of S.cerevisiae are identified by integrating by homologous recombination a selection marker at the locus of the gene to be studied.

The present invention also concerns a method as described above wherein the functionally similar genes are identified by: a)providing a S.cerevisiae mutant strain in which the gene of S.cerevisiae to be investigated is either integrative or extrachromosomal under the control of a regulated promoter, b)culturing said mutant strain under growth conditions in which the regulated promoter is active, c)transforming the mutant strain with a cDNA or genomic DNA that has been prepared from the heterologous mycete-species and that has been integrated into an appropriate vector, d)altering the culture condition, so that the regulated promoter is switched off and only S.cerevisiae cells which contain a functionally similar gene can survive, e)isolating and analyzing the cDNA or genomic DNA.

The invention thus discloses that in a first step, essential genes from S.cerevisiae are identified. The invention also discloses that, essential genes from other mycetes are identified starting from the identified essential genes in S.cerevisiae. In order to identify essential genes of S.cerevisiae, individual genomic genes are eliminated through homologous recombination. If the DNA segment thus eliminated concerns an essential gene, then the deletion is lethal for the S.cerevisiae cells in haploid form.

A method, wherein the studied S.cerevisiae gene is replaced by a marker gene can be used to generate the WO 99/55907 Pr'T/fP99/n2722 4 corresponding genomic deletion of S.cerevisiae and to determine the S.cerevisiae cells containing the deletion.

As a selection marker a dominant selection marker kanamycin resistance gene) or an auxotrophic marker can for example be used. As an auxotrophic marker, it is possible to use genes coding for key enzymes of amino acid or nucleic base synthesis. For example, one can use as a selection marker the following genes from S.cerevisiae gene encoding for the metabolic pathway of leucine (e.g.LEU2-gene), histidine HIS3-gene) or tryptophan TRP1 gene) or for the nucleic base metabolism of uracil URA3-gene).

Auxotrophic S.cerevisiae strains can be used.

These auxotrophic strains can only grow on nutritive media containing the corresponding amino acids or nucleotide bases. All laboratory S.cerevisiae strains, containing auxotrophic markers can for instance be used. When diploid S.cerevisiae strains are used, then the corresponding marker gene must be homozygously mutated. Strain CEN.PK2 or isogenic derivates thereof can be used.

Strains containing no suitable auxotrophic marker can also be used such as prototrophic S.cerevisiae strains.

Then a dominant selection marker e.g. resistance gene, such as kanamycin resistance gene can be used. A loxP-KanMX-loxP cassette can advantageously be used for this purpose.

For the homologous recombination replacing the whole DNA sequence or part thereof of a S.cerevisiae gene, DNA fragments are used wherein the marker gene is flanked at the and 3'-ends by sequences which are homologous to the and 3'-ends of the studied S.cerevisiae gene.

Different processes can be used for the preparation of the corresponding DNA fragments which are also appropriate for the deletion of any specific S.cerevisiae gene. A linear DNA-fragment is used for the transformation of the suitable S.cerevisiae strain. This fragment is integrated into the S.cerevisiae genome by homologous recombination. These processes include:

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WO 99/55907 PrT/.P90/n2722 1. "Conventional method" for the preparation of deletion cassettes (Rothstein, R.J. (1983) Methods in Enzymology, Vol. 101, 202-211).

2. "Conventional Method" using the PCR technique ("modified conventional method").

3. SFH (short flanking homology)- PCR method (Wach, A. et al. (1994) Yeast 10: 1793-1808; Giltner, U. et al.

(1996) Nucleic Acids Research 24:2519-2524).

1. In the "conventional method" for the preparation of deletion cassettes in the S.cerevisiae genome, the gene to be studied is either already present in an appropriate vector or is integrated in such a vector. With this method, any pBR- pUC- and pBluescript®-derivates can be used for example. A major part of the target gene sequence is eliminated from the vector, for instance using appropiate restriction sites, conserving however the and regions of the studied gene inside the vector. The selected marker gene is integrated between the remaining regions.

2. In the modified form of this "conventional method", PCR is used. This method allows amplification of the and 5'-terminal regions of the coding sequence of the studied S.cerevisiae gene. This method amplifies selectively both terminal regions of the studied gene, therefore, two PCR-reactions must be carried out for each studied gene, amplifying once the 5'-end, and once the 3'end of the gene. The length of the amplified terminal DNAfragment depends on the existing restriction sites. The amplified terminal ends of the studied gene have generally a length of 50 to 5000 base pairs preferably a length between 500 and 1000 bp.

As template for the PCR-reactions, genomic DNA of S.cerevisiae or wild-type genes can be used. The primerpairs (a sense and an antisense primer, respectively) are constructed so that they correspond to the 3'-end and the 5'-end sequence of the studied S.cerevisiae gene.

Especially, the primer is selected such as to allow its integration by way of appropriate restriction sites.

As vectors, pBR- pUC- and pBluescript®-derivates can be used. In particular vectors already containing a WO 99/55907 PCT/IEPoo/n722 6 gene encoding the selection marker, are appropriate. In particular, vectors can be used, which contain genes of the selection marker HIS3, LEU2, TRP1 or URA3.

The DNA segments of the studied S.cerevisiae gene, obtained by PCR, are integrated in the vector at both sides of the selection marker, so that subsequently, as in the "conventional method", the selection marker is flanked on both ends by DNA sequences which are homologous to the studied gene.

3. Homologous recombination in S.cerevisiae takes place in a very efficient and precise manner and the length of the DNA sequence homologous to the studied S.cerevisiae gene flanking the selection marker gene can in fact be considerably shorter than with the "modified conventional method". The flanking ends homologous to the studied S.cerevisiae gene need to present a length of only about 20-60 bp, preferably 30-45 bp. The SFH-PCR method is particularly advantageous as the laborious cloning step can be obviated.

A PCR reaction is carried out on a DNA-template containing the gene for the selection marker to be used, wherein the primers are constructed so that the DNA sequence of the sense primer is homologous to the 5'-end of the selection marker sequence and so that the primer presents in addition at its 5'-end a region of preferably nucleotides, which corresponds to the sequence of the studied S.cerevisiae gene. The antisense primer is constructed in an analogous manner, i.e. it is complementary to the 3'-end of the gene sequence of the selection marker, wherein this primer contains at its end a region of also preferably 40 nucleotides, which corresponds to the complementary strand of the 3'-terminal coding sequence of the studied gene.

For the amplification of S.cerevisiae genes to be studied by the SFH-PCR method, vectors containing the gene for the auxotrophic marker or selection marker can be used.

Especially, plasmid pUG6 is used as the template. This plasmid contains a loxP-KanMX-loxP cassette (GXltner, U. et al. (1996) Nucleic Acids Research 24: 2519-2524). In other WO 99/55907 PCT/EP99/02722 7 terms, the Kanamycin resistance gene is flanked at both ends by a loxP sequence (loxP-KanMX-loxP cassette). This cassette is advantageous in that the Kanamycin resistance gene can be eventually eliminated from the S.cerevisiae genome after integration of the loxP-KanMX-loxP cassette into the S.cerevisiae gene to be studied. Cre-recombinase of bacteriophage P1 can be used for this purpose. Crerecombinase recognizes the loxP sequences and induces elimination of the DNA located between the two loxP sequences by a homologous recombination process. As a result only one loxP sequence remains and the so-called marker regeneration occurs, i.e. the S.cerevisiae strain may be transformed again using the loxP-KanMX-loxP cassette. This is particularly advantageous, when at least two functionally similar genes are to be deleted in order to obtain a lethal phenotype.

With the PCR-method, the PCR reaction primers are at the 3'-end a preferably 20 nucleotide long sequence, which is homologous to the sequence situated left and/or right of the loxP-KanMX-loxP cassette, and at the 5'-end a preferably 40 nucleotide long sequence, which is homologous to the terminal ends of the gene to be studied.

Using the three methods, one obtains linear deletion cassettes containing the gene encoding the selection marker, which is flanked on both sides by homologous sequences of the gene to be studied. The deletion cassettes are used for the transformation of diploid S.cerevisiae strains. The diploid strain S.cerevisiae CEN.PK2 (Scientific Research Development GmbH, Oberursel) can be used for example for this purpose.

[CEN.PK2 Mata/MAT a ura3-52/ura3-52 leu2-3, 112/leu2-3, 112his3Al/his3Al trpl-289/trpl-289 MAL2-8c/MAL2- 8 c SUC2/SUC2] The strain CEN.PK2 is prepared and cultivated using known methods (Gietz, R.D. et al. (1992) Nucleic Acids Research 8: 1425; Guldener, U. et al. (1996) Nucleic Acids Research 24:2519-2524).

WO 99/55907 prT/IPo9/n7 8 The cells of the S.cerevisiae strain used are transformed according to known processes with an appropriate DNA quantity of the linear deletion cassette Sambrook et al. 1989) Thereafter, the medium in which the cells are cultivated is replaced by a new medium, a so-called selective medium, which does not contain the corresponding amino acid g. histidine, leucine or tryptophan) or nucleic base g. uracil) or, when using a deletion cassette containing the kanamycin resistance gene, by a medium containing geneticin (G418®) a complete medium (YEPD) containing geneticin). Alternatively, the transformed cells may be plated on agar plates prepared using the corresponding media. Thereby, one selects the transformed cells, in which a homologous recombination occured, since only those cells can grow under these modified conditions.

However, in most cases, only one of the two copies of the gene in the double chromosome set of a diploid S.cerevisiae strain is replaced by the DNA of the deletion cassette during the transformation, resulting in a heterozygote-diploid S.cerevisiae mutant strain, wherein one copy of the gene studied is replaced by a selection marker, while the other copy of the gene is maintained in the genome. This presents the advantage that in case of a deletion of an essential gene, due to the existence of the second copy of the essential gene, the mutant S.cerevisiae strain is still viable.

The proper integration of the deletion cassette DNA at the predetermined chromosomal gene locus (gene locus of the gene to be studied) may be checked by Southern-Blot Analysis (Southern, E.M. (1975) J. Mol. Biol. 98:503-517) or by diagnostic PCR analysis using specific primers (Gildener, U. et al. (1996) Nucleic Acids Research 24:2519- 2524) The genetic separation of individual diploid cells may be monitored by tetrad analysis. To this end, reduction division (meiosis) is induced in the diploid cells, especially heterozygote mutant strains, using known methods such as nitrogen impoverishment on potassium acetate plates

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WO 99/55907 PCT/EP99/02722 .7PCT.P99/.2722 9 (Sherman, F. et al. (1986) Cold Spring Harbor Laboracory Press, Cold Spring Harbor, N. Guthrie, C. and Fink, G.R. (1991) Methods in Enzymology, Vol 194. Academic Press, San Diego, 3-21; Ausubel, F. M. et al. (1987) Current Protocol in Molecular Biology John Wiley and Sons, Inc., Chapter 13). Meiosis results only in asci with four ascospores (segregated), which can be indivualized after partial enzymatic digestion of the ascospore wall with zymolyase (Ausubel et al. (1987)) by way of micromanipulators SINGER). For example when a tetrad analysis is carried out on a heterozygote-diploid mutant strain in which an essential gene present in the double chromosome set is replaced on one chromosome by homologous recombination, then only two segregated ascospores are viable, namely those which carry the essential gene. The two remaining segregated ascospores are not viable because they lack the essential gene.

In order to check if the genes studied by this method are really essential or if the homologous recombination leads to an alteration of an essential gene adjacent to the gene locus of the gene studied, the heterozygote diploid S.cerevisiae mutant strain is transformed with a centromere plasmid containing said studied gene.

A tetrad analysis is carried out on the transformants. When four instead of two viable segregates are obtained, then the studied gene contained in the centromere plasmid can complement the defect of the two non-viable haploid S.cerevisiae cells/mutant strains, which demonstrates that the studied S.cerevisiae gene is essential.

Preferably, plasmids present in low copy number, e.g. one or two copies per cell are used as centromere plasmids. For example plasmids pRS313, pRS314, pRS315 and pRS316 (Sijkorski, R. S. and Hieter, P. (1989) Genetics 122: 19-27) or similar plasmids can be used for this purpose. Preferably, the studied genes are integrated in said plasmids including their and 5'-end non-coding regions.

WO 99/55907 PrT/IFPO/n'7T Individual S.cerevisiae genes may be studied using the above-described method, their sequences being totally or partially known. The complete genomic sequence of S.cerevisiae was made accessible to the public via the WWW (World Wide Web) on April 24, 1996.

Different possibilities exist to have access to the S.cerevisiae genomic DNA sequence via the WWW.

MIPS (Munich information Centre of Protein Sequence) Address http://speedy.mips.biochem.mpq.de/mips /yeast/ SGD (Saccharomyces Genome Database, Stanford) Address http://genome-www.stanford.edu/Saccharomyces YPD(Yeast Protein Database, Cold Spring Harbor) Address http://www.proteome.com/YPDhome.html The complete S.cerevisiae DNA sequence is also accessible via FTP (file transfer protocol) in Europe (e.g.

at the address: ftp.mips.embnet.org) in the U.S.A.

(address: genome-ftp.stanford.edu) or in Japan (address: ftp.nig.ac.jp).

The complete S.cerevisiae DNA sequence was published in Nature, special issue No 387, 1997.

essential genomic S.cerevisiae genes have been identified by this way. These essential genes are listed in table 1. Table 1 contains the systematic gene name of the essential genes (corresponding to the denomination under which the corresponding DNA sequences are accessible in databanks), the deleted nucleotides and the corresponding amino acids of the essential genes (position 1 is taken as reference, this latter corresponding to the A of the probable initiation codon ATG of the ORF). The deleted nucleotides correspond to nucleotides deleted in the gene and the deleted amino acids correspond to the amino acids missing in the encoded protein. Furthermore aa corresponds to the total number of amino acids present in the encoded protein. The numbers of deleted nucleotides do not necessarily correspond to 3 times the numbers of deleted amino acids; this is explained by the fact that a gene is bigger than the encoded reading frame for amino acids.

YMR134w for example encodes a protein of 237aa, the WO 99/55907 PCT/EP99/02722 .PCTEP99/.2722 11 deletion starts at nucleotide 5 (councing starts from HrG) and continues until nucleotide 740, this also includes part of the terminator region which does not encode aa, so the deletion of the aa starts from aa 2 until the end of the protein which is aa 237. Furthermore, the information available concerning the functions of respective genes or of the encoded proteins and/or homologies/similarities to other genes or proteins are indicated. The primers used for the PCR reaction to prepare the DNA fragments appropriate for the deletion of the genes are listed in table 2, where S1 and S2 are the forward and reverse primers, respectively, and the bold letters corresponding to the nucleotides of the respective gene.

The data of table 1 emphasize that despite the fact Sthat the S.cerevisiae gene DNA sequences are known, very little is known today about the function, the characteristic properties of these genes, the essential function of these genes, or the proteins encoded by the same.

According to one embodiment of the method, essential genes of S.cerevisiae are used to identify corresponding functionally similar genes in other mycetes.

By functionally similar genes in other mycete species, is meant genes which have a function similar or identical to that of the identified essential genes of S.cerevisiae. Functionally similar genes in other mycetes may, but need not be homologous in sequence to the corresponding essential S.cerevisiae genes. Functionally similar genes in other mycetes may exhibit only moderate sequence homology at the nucleotide level to the corresponding essential S.cerevisiae genes. By moderate sequence homology it is meant in the present invention genes having a sequence identity, at the nucleotide level, of at least 50%, more preferably of at least 60% and most preferably of at least In addition, functionally similar genes in other mycetes may, but need not encode proteins homologous in sequence to the proteins encoded by the essential S.cerevisiae genes. Functionally similar proteins in other WO 99/55907 PCT/EP99/02722 12 mycetes may exhibit moderate protein sequence homology to the proteins encoded by the essential S.cerevisiae genes.

By moderate protein sequence homology is meant in the present invention proteins having a sequence identity, at the amino-acid. level, of a least 40%, preferably of at least 50%, more preferably of at least 60% and most preferably of at least Genes homologous in sequence may be isolated according to known methods, for example via homologous screening (Sambrook, J. et al. (1989) Molecular Cloning Cold Spring Harbor Laboratory Press, or via the PCR technique using specific primers from genomic libraries and/or cDNA libraries of the corresponding mycetes.

According to one embodiment, genes homologous in sequences are isolated from cDNA libraries. In order to find out functionally similar genes in other mycetes, mRNA is isolated from mycete species to be studied according to known methods (Sambrock et al. 1989) and cDNA is synthesized according to known methods (Sambrock et al.

1989; or cDNA synthesis kits, e.g. from STRATAGENE).

The prepared cDNA is directionally integrated in a suitable expression vector.

For example, synthesis of the first cDNA strand may be carried out in the presence of primers having appropriate restriction sites in order to allow a subsequent cloning in the proper orientation with respect to the expression vector promoter. As restriction sites, any known restriction site may be used. As a primer, for instance the following primer, 50 nucleotides long may be used: 5'-GAGAGAGAGAGAGAGAGAGAACTAGTXXXXXXTTTTTTTTTTTTTTTTTT-3' The sequence (X) 6 represents an appropriate restriction site, for example for XhoI.

After two-strand synthesis, the cohesive ends of the double stranded cDNA are filled (blunt end) and the cDNA ends are then ligated using a suitable DNA adaptor sequence. The DNA adaptor sequence should contain a restriction site which should be different from the restriction site used in the primer for the synthesis of WO 99/55907 PCT/EP99/02722 13 the first cDNA strand. The DNA adaptoc may comprise for example complementary 9- or 13-mer oligonucleotides, whose ends represent the cohesive end of a restriction site.

These ends may be for example a EcoRI-site: XXXXXGGCACGAG 3' 3' XCCGTGCTC The single-stranded X in the adaptor sequence represent the cohesive end of a restriction site.

The cDNA provided with corresponding adaptor sequences is then cleaved using restriction endonuclease, whose recognition site was used in the primer for the synthesis of the first cDNA strand, for example XhoI. The cDNA thus obtained would have according to this example 3'- XhoI and 5'-EcoRI protruding ends and could be therefore directionally integrated into an expression vector cleaved with XhoI and EcoRI.

As expression vectors, among others, E.

coli/S.cerevisiae shuttle vectors, i.e. vectors usable in E. coli as well as in S.cerevisiae are suitable. Such vectors may then be amplified for instance in E. coli. As expression vectors, those which are present in a high copy number as well as those present in a low copy number in S.cerevisiae cells can be used. For this purpose, for example vectors selected in the group consisting of pRS423 pRS426 (pRS423, pRS424, pRS425, pRS426) and/or pRS313pRS316 (pRS313, pRS314, pRS315, pRS316) (Sikorki, R.S. and Hieter, P. (1989) Genetics 122: 19-27; Christianson T. W.

et al. (1992) Gene 110: 119-122) are suitable.

Expression vectors should contain appropriate S.cerevisiae promoters and terminators. In case they do not have these elements, the corresponding promoters and terminators are inserted in such a way that a subsequent incorporation of the generated cDNA remains possible.

Particularly suitable are the promoters of S.cerevisiae genes MET25, PGK1, TPI1, TDH3, ADHI, URA3. One may use promoters of the wild-type gene in non modified form as well as promoters which were modified in such a way that certain activator sequences and/or repressor sequences were eliminated. As terminators, for example the terminators of WO 99/55907 PCT/EP99/02722 14 the S.cerevisiae genes MET25, PGK1, TPI1, TDH3, ADHI, URA3 are suitable.

According to another embodiment, genes homologous in sequence are isolated from genomic libraries. Genomic DNA libraries from mycetes can be prepared according to procedures known (for example as described in Current Protocols in Molecular Biology, John Wiley and Sons, Inc).

For example, genomic DNA from mycetes can be prepared using known methods for yeast cell lysis and isolation of genomic DNA (for example commercially available kits from BiolOl, Inc) The genomic DNA can be partially digested using a restriction enzyme such as Sau3AI and the fragments are size-selected by agarose gel electrophoresis. DNA fragments having for example a size of 5-10kb are then purified by classical methods (as for example, using Gene Clean kit from BiolOl) and inserted in a E.coli/yeast shuttle vector such as YEP24 (described e.g. by Sanglard Kuchler K., Ischer Pagani Monod M. and Bille J., Antimicrobial Agents and Chemotherapy, (1995) Vol.39 Noll, P2378-2386) cut by a restriction enzyme giving compatible ends (for example BamHI for Sau3AI-cut genomic DNA) The resulting expression library can be amplified in E.coli.

However any known method, appropriate for the preparation of a genomic library, can be used in the present invention.

In order to find the genes in the studied mycete species, which are functionally similar to essential genes of S.cerevisiae, one S.cerevisiae essential gene is placed under control of a regulated promoter, either as an integrative or extrachromosomal gene.

1. For the integration of a regulated promoter in the S.cerevisiae genome, one replaces the native promoter of the selected essential gene by the regulated promoter, for example by homologous recombination via PCR (Gildener et al. (1996). The homologous recombination via PCR can be carried out for example in the diploid S.cerevisiae strain CEN.PK2. The successfull integration into one chromosome can be checked in haploid cells following tetrad analysis.

WO 99/55907 PCT/EP99/02722 Using the tetrad analysis, one obtains four viable ascospores, wherein in two haploid segregates, the selected essential gene is placed under the control of the native promoter, while the essential gene in the two remaining segregates is placed under the control of the regulated promoter.

The last mentioned haploid segregates are used for the transformation with the cDNA or the genomic DNA present in the recombinant vector.

2. Using the extrachromosomal variant, the selected essential S.cerevisiae gene, is first inserted in a suitable expression vector, for example a E.coli/ S.cerevisiae shuttle vector. For this purpose, the essential gene may be amplified via PCR from genomic S.cerevisiae DNA starting from the ATG initiation codon up to and including the termination codon. The primers used for this purpose may be constructed in such a way that they contain recognition sites for appropriate restriction enzymes, facilitating a subsequent insertion under control of a regulated promoter in an expression vector.

The recombinant expression vector with the plasmid copy of the essential S.cerevisiae gene under the control of a regulated promoter is subsequently used for the transcomplementation of the corresponding mutant allele.

The corresponding mutant allele may be selected from the heterozygote-diploid mutant strains prepared by eliminating, partially or totally, by homologous recombination an essential mycete gene listed in table 1 (first column of table as described above.

The expression vector with the selected essential S.cerevisiae gene is transformed in the corresponding heterozygote-diploid mutant strain carrying instead of the selected essential S.cerevisiae gene, a selection marker gene. The transformants are isolated by selection based on the auxotrophic marker contained in the expression vector used. The thus transformed heterozygote-diploid mutant strains are submitted to a tetrad analysis. One obtains four viable segregates. By retracing the corresponding markers of the mutant allele and the expression vector, the WO 99/55907 PCT/EP9/02722 16 transformed wild-type segregates may be distinguished from segregates which do not contain the genomic copy of the essential gene. Segregates, which do not contain the genomic copy of the selected essential gene, are designated as trans-complemented haploid mutant strains. They are subsequently used for transformation with cDNA or genomic DNA libraries from other mycete species present in appropriate vectors.

As regulated promoters, inducible or repressible promoters may be used. These promoters can consist of naturally and/or artificially disposed promoter sequences.

As regulated promoters, for example the promoters of GAL1 gene and the corresponding promoter derivatives, such as for example promoters, whose different UAS (upstream activation sequence) elements have been eliminated (GALS, GALL; Mumberg, J. et al. (1994) Nucleic Acids Research 22:5767-5768) may be used. As regulated promoters, promoters of gluconeogenic genes may also be used, such as e.g. FBP1, PCK1, ICL1 or parts therefrom, such as e.g. their activation sequence (UAS1 and/or UAS2) or repression sequence (URS, upstream repression sequence) (Niederacher et al. (1992), Curr. Genet. 22: 636-670; Proft et al. (1995) Mol. Gen. Gent. 246: 367-373; Schller et al.

(1992) EMBO J; 11: 107-114; Guarente et al. (1984) Cell 36: 503-511).

A S.cerevisiae mutant strain modified in this manner can be cultivated under growth conditions, in which the regulated promoter is active, so that the essential S.cerevisiae gene is expressed. The S.cerevisiae cells are then transformed with a representative quantity of the library containing the studied mycete species cDNA or genomic DNA. Transformants express additionally the protein whose coding sequence is present in the recombinant vector.

The method contemplates that the growth conditions may be modified in such a way as to inhibit the regulated promoter, under the control of which is the selected essential gene. Especially, growth conditions may be changed by replacing the growth medium. When for example the GAL1 promoter or a derivate thereof is used, one can WO 99/55907 PCT/EP0O/02722 17 replace the galactose-containing medium (induced state) by a glucose-containing medium (repressed state).

These modified conditions are lethal. for the S.cerevisiae cells in which the recombinant vector does not carry the functionally similar genomic DNA or cDNA of the studied mycete species. On the contrary, the S.cerevisiae cells in which the recombinant vector expresses a functionally similar coding sequence of the studied mycete species, are viable, since in these cells the lethal metabolic defect is complemented by the protein encoded by the functionally similar gene.

The method contemplates that the recombinant vector (the plasmid) is isolated from the surviving transformants using known method (Strathern, J.N. and Higgins, D.R.

(1991). Plasmids are recovered from yeast into Escherichia coli shuttle vectors in: Guthrie, C. and Fink, G.R. Methods in Enzymology, Volume 194. Guide to yeast genetic and molecular Biology. Academic Press, San Diego, 319-329) and the cDNA or genomic DNA is analyzed using DNA-analysis methods such as DNA sequencing. (Sanger et al. (1977), Proc. Natl. Acad. Sci. USA 74: 5463-5467) The method contemplates that essential S.cerevisiae genes may be used for the identification of functionally similar genes and/or genes homologous in sequence in other mycetes, especially essential genes functionally similar and/or homologous in sequence in mycetes pathogenic to human, animal and plants. For this purpose for example mycetes of the classes phycomycetes or eumycetes may be used, in particular the subclasses basidiomycetes, ascomycetes, especially mehiascomycetales (yeast) and plectascales (mould fungus) and gymnascales (skin and hair fungus) or of the class of hyphomycetes, in particular the subclasses conidiosporales (skin fungus) and thallosporales (budding or gemmiparous fungus), among which particularly the species mucor, rhizopus, coccidioides, paracoccidioides (blastomyces brasiliensis), endomyces (blastomyces), aspergillus, penicilium (scopulariopsis), trichophyton (ctenomyces), epidermophton, microsporon, piedraia, WO 99/55907 P"T/ P9Q/0m 7) 18WO 99/55907 PCT/ 7 hormodendron, phialophora, sporotrichon, cryptococcus, candida, geotrichum and trichosporon.

Of particular interest is the use of Candida Spp.

especially Candida albicans, Candida glabrata, Aspergillus Spp., especially Aspergillus fumigatus, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliens and Sporothrix schenckii.

The method contemplates that essential mycete genes are used to identify substances which may inhibit partially or totally the functional expression of these essential genes and/or the functional activity of the encoded proteins. Substances may be identified in this fashion, which inhibit mycetes growth and which can be used as antimycotics, for example in the preparation of drugs.

A particular feature of this method is that essential mycete genes or the corresponding encoded proteins are used as targets for the screening of the substances. The method contemplates that essential S.cerevisiae genes as well as functionally similar genes and/or genes homologous in sequence of other mycetes or the corresponding encoded proteins may be used as targets.

According to one embodiment of the screening method of the invention, mycetes cells are provided, which contain the essential gene used as target, and those cells are incubated with the substance to be tested. By this way, the growth inhibitory effect of this substance with respect to the essential target gene is determined.

The mycetes cells which express the essential target gene to a different degree are used, and these cells are then incubated with the substance to be tested and the growth inhibitory effect of this substance is determined.

The method includes the use of two or more mycetes cells, or strains derived therefrom, which differ in that they express the essential target gene to a different degree.

For example, two, three, four, five, ten or more mycetes cells or the corresponding mycetes strains may be comparatively analysed with respect to the growth WO 99/55907 PCT/EP99/02722 19 inhibitory effect of a substance used in a defined concentration. Through such concentration series, antimycotic substances may be distinguished from cytotoxic or inactive substances.

A particular embodiment of the method includes the use of haploid mycetes cells/ strains for the screening, especially haploid S.cerevisiae cells/ strains.

The method contemplates the integration of the essential gene selected as a target in a suitable expression vector.

As expression vectors E.coli/S.cerevisiae shuttle vectors are for example suitable. Especially vectors differing in their copy number per cell may be used.

Therefore, one may use vectors, which are present in the transformed S.cerevisiae cells in a high copy number, or one can also use those with a low copy number. One embodiment comprises the use of expression vectors which allow the integration of the target gene in the S.cerevisiae genome.

For example the vectors pRS423, pRS424,pRS425, pRS426, pRS313, pRS314, pRS315, pRS316, pRS303, pRS304, pRS305, pRS306 (Sikorki and Hieter, 1989; Christianson et al. 1992) are appropriate as expression vectors.

The vectors of the series pRS423 pRS426 are present in a high copy number, about 50 100 copies/ cell.

On the contrary, the vectors of the series pRS313 pRS316 are present in a low copy number (1 2 copies cell).

When expression vectors from these two series are used, then the target gene is present as an extrachromosomal copy. Using the vector of the series pRS303 pRS306 allows the integration of the target genes into the genome. Using these three different expression vector types allows a gradual expression of the studied functionally similar essential gene.

The method includes that the growth inhibitory effect of substances with respect to mycetes cells/strains is comparatively determined using expression vectors differing for instance in the copy number of the vector/ cell.

WO 99/55907 PCT/IPOO/n'279' Such cells may express the essential target gene to a different degree and may exhibit a graduated reaction with respect to the substance.

The method includes also, that a target gene expression of different strength is obtained in different mycetes cells (regulated overexpression) by insertion of the target gene in the expression vector between specific selected S.cerevisiae promoters and terminators.

S.cerevisiae promoters which are constitutively expressed, but with different strength, are suitable. Examples for such promoters are native promoters of S.cerevisiae genes PGK1, TPI1, TDH3, ADH1, URA3, TRP1, as well as corresponding derivatives therefrom, for example promoter derivatives without specific activator and/or repressor sequences.

Regulated promoters are also appropriate for the graduated over-expression of the target gene. The native promoters of the GAL1 genes and/or corresponding derivates thereof, for example promoters, in which different UAS elements have been eliminated. (GALS, GALL; Mumberg et al.

(1994) Nucleic Acids Research 22: 5767-5768) as well as promoters of gluconeogenic genes, for example the promoters FBP1, PCK1, ICL1, or parts thereof, for example their activator- (UAS1 or UAS2) or repressor- (URS) sequences are used in corresponding non activable and/or non repressible test promoters (Schiller et al. (1992) EMBO J. 11: 107-114) Guarente et al. (1984) Cell 36: 503-511; Niederacher et al.

(1992) Curr. Genet. 22: 363-370; Proft et al. (1995) Mol.

Gen. Genet. 246: 367-373).

In the expression vector terminator for example the terminator sequence of S.cerevisiae genes MET25, PGK1, TPI1, TDH3, ADHI, URA3 may be used.

The method includes that by the use of cleverly selected expression vector types and/or the preparation of suitable expression vectors, eventually using promoters of different strength and differently regulated promoters, a series of expression vectors may be constructed, all containing the same target gene, but differing in that they express the target gene to a different extent.

WO 99/55907 PCT/EP99/02722 21 The method includes the transformation of the expression vector in haploid wild-type cells of S.cerevisiae. The thus obtained S.cerevisiae cells/strains are cultivated in liquid medium and incubated in the presence of different concentrations of the tested substance and the effect of this substance on the growth behaviour of the cells/strains expressing the target gene to a different degree is comparatively analysed. The method also includes that haploid S.cerevisiae cells/strains, transformed using the respective expression vector type without target gene, are used as a reference.

The method includes that the screening of the substances can be carried out in different media using regulated promoters, especially GALl promoter and its derivates (GALS and GALL), since the expression degree may be largely influenced by the choice of the respective medium. Thus, the expression degree of the GALl promoter decreases in the following fashion: 2 galactose 1 galactose 1 glucose 2 glycerine 2 glucose.

The effect of the substances inhibiting the growth of wild-type cells of S.cerevisiae, may be partially or totally compensated by the overexpression of the functionally similar gene of another mycete species.

According to one embodiment, the method for screening antimycotic substances is carried out in vitro by contact of an essential or functionally similar gene or the corresponding encoded protein with the substance to be tested and determination of the effect of the substance on the target. Any in vitro test appropriate for determining the interaction of two molecules, such as a hybridization test or a functional test, can be used enzymatic tests which are described in details in Bergmeyer H.U., Methods of Enzymatic Analysis, VCH Publishers) If the screening is carried out using the encoded protein as the target, then the corresponding essential gene is inserted by any suitable method known in the art, such as PCR amplification using a set of primers containing appropriate restriction sites, (Current Protocol in Molecular Biology, John Wiley and Sons, Inc) into an expression system, such WO 99/55907 PCT/EP99/02722 22 as E. coli, Baculovirus, or yeast, and the expressed protein is then completely or partially purified by a method known in the art. Any purification method appropriate for the purification of expressed proteins, such as affinity chromatography can be used. If the target protein function is known, a functional test can then be carried out in which the effect of the antimycotic substance on the protein function is determined. If the protein function is unknown, substances which can interact with the target protein, e.g. which bind to the encoded protein, can be tested. In such a case a test such as protection of the target protein from enzymatic digestion by appropriate enzymes can be used.

The method also includes the identification of genes which are functionally similar and/or homologous in sequence to essential S.cerevisiae genes from humans, animals or plants. The corresponding human, animal or plant genes may optionally be used as target genes in the method in order to test if antimycotic substances exhibit an effect on these target genes.

A particular advantage of the method is that in this way substances may be identified which efficiently inhibit mycetes growth and also the influence of these substances on corresponding functionally similar genes and/or genes homologous in sequence to essential S.cerevisiae genes from human, animal or plants may be determined.

The method includes also the possibility to check the existence of functionally similar genes and/or human, animal or plant genes homologous in sequence to the corresponding essential mycete genes, for example by checking homology of the identified essential mycete genes or parts thereof with human, animal or plant sequence genes available in data banks. In this way, it is possible to select at an early stage from the identified essential mycete genes, depending on the aim, those for which no functionally similar gene and/or no human gene homologous in sequence exist, for example.

WO 99/55907 PCT/EP99/02722 23 Thereby, the method offers a plurality of possibilities to identify selectively substances with antimycotic effects, with no harmful effect on human beings.

For example, it is possible to identify substances usable for the preparation of drugs for the treatment of mycosis or prophylaxis in immunodepression states. These substances may be used for example for the manufacture of drugs usable for the treatment of mycotic infections, which occur during diseases like Aids or Diabetes. Substances which may be used for the fabrication of fungicides, especially of fungicides which are harmless for humans and animals, can also be identified.

Furthermore, the method offers the possibility to Sidentify antimycotic substances, which selectively inhibit growth of specific mycete species only.

The screening method is particularly advantageous inasmuch as it is sufficient to know whether the genes are essential, one does not need any additional information regarding the function of the essential genes or the function of the encoded proteins. In addition, it is particularly advantageous for the identification of functionally similar genes to essential S.cerevisiae gene, in other mycetes where the DNA sequence is not available for many of these genes.

Examples Example 1 Preparation of a deletion cassette for ORF YML114c, by the classical method using PCR (modified classical method) 1)Construction of the plasmids pBluescript®KS+ vector(Stratagene; the sequence of which is available on Genbank®X52327) is used as the starting vector for the preparation of the other plasmids.

The vector is cleaved with NotI and the singlestranded ends are subsequently eliminated by incubation with Mung Bean exonuclease. By religation of DNA fragments, the pKS+ANotI vector is thus obtained WO 99/55907 PCT/EP99/02722 24 (corresponding to the pBluescript®KS+ without the NotI restriction site).

pKS+ANotI is cleaved with PstI and BamHI and the DNA oligonucleotide, synthesized from the pK3/pK4 primer pair described below, is ligated in the opened plasmid. The pKS+neu plasmid thus prepared contains between PstI and BamHI restriction sites, the following novel restriction sites NotI, StuI, SfiI and NcoI PstI-NotI-StuI-SfiI- NcoI-BamHI) 5'-GCGGCCGCAAGGCCTCCATGGCCG-3' PK3 5'-GATCCGGCCATGGAGGCCTTGCGGCCGCTGCA-3' PK4 The URA3 gene of S.cerevisiae is amplified via PCR, by use of the primer-pair PK9 and PK10, described below, and an Ycplac33 vector DNA (Gietz, R. D. and Sugino, A. (1988) Gene 74: 527-534) as matrix. The amplified DNA is cleaved with BamHI and NotI and subsequently inserted in pKS+neu which has been cleaved by BamHI and NotI. The plasmid thus obtained is named pPK9/10.

NotI..

5'-ATCTGCAGCGGCCGCAAACATGAGAATTGGGTAATAACTG-3' PK9 PstI SfiI..

5'-ATGGATCCGGCCATGGAGGCCTTCAAGAATTAGCTTTTCAATTCATC-3' BamHI 2)Preparation of the deletion cassette The 5'-region of ORF YML114c was amplified by PCR using genomic DNA of S.cerevisiae as template and both primers YML114c-Asp718 and YLM114c-EcoRI, described below.

YML114c-Asp718: 5'-GCTGGTACCCGTCGGTCTCTTTACC-3' YLM114c-EcoRI: 5'-TTGGAATTCATTGCCCTTTATGAGTCC-3' The PCR fragment was subsequently cut with the restriction enzymes Asp718 and EcoRI. The resulting 613BP fragment was inserted in pPK9/10 linearized with Asp718 and EcoRI generating plasmid pYML114c-A.

WO 99/55907 PCT/EP99/02722 The 3'region of ORF YML114c was amplified by PCR using genomic DNA of S.cerevisiae as template and both primers YML114c-BamHI and YLM114c-SacI, described below.

YML114c-BamHI:5'-ATCGGATCCGCCAACAATGACAGCG-3' YLM114c-SacI: 5'-GTTGAGCTCTGAGCGTTTGTCCTTG-3' The PCR fragment was subsequently cut with BamHI and SacI.

The resulting 535bp fragment was inserted in plasmid pYML114c-A linearized with BamHI and SacI generating pYML114c-B.

This latter plasmid was used for transformation of S.cerevisiae after linearization with Aspl78 and SacI.

Examples 2-90: Construction of deletion cassettes for the remaining genes listed in table 1 Using the method disclosed in example 1, the deletion cassettes of each of the essential genes can be constructed using as primers those disclosed in table 2.

Example 91: S.cerevisiae cells from strain CEN.PK2 are transformed using each about 5 pg DNA of the linear deletion cassette of examples 1 to 90 according to known methods (Gietz et al. 1992; Guldener et al. 1996). The transformation reaction medium is plated on plates on the corresponding selective media. In this manner, the transformants are selected, in which homologous 2 i. recombination occured, since only these cells can grow under these modified conditions.

The recombinant cells were submitted to a tetrad analysis in the following conditions: Reduction division (meiosis) was induced in the heterozygote mutant strain using known methods (Guthrie C. and Fink,G.R. (1991) Methods in Enzymology, Vol 194, Academic Press, San Diego).

The resulting asci were submitted to partial enzymatic digestion with zygmolyase to digest the ascospore wall and separated using a micromanipulator (SINGER Instruments).

This analysis demonstrated that all the above-mentioned genes are essential for the growth of S.cerevisiae.

The present invention also applies more specifically to the following genes: YML114c, YLR186w, YLR215c, YLR222c, YLR243w, YLR272c, YLR275w, YLR276c, WO 99/55907 WO 9955907PCT/EP99/02722 YLR317w, YMLO23c, YMR093w, YMR281w, YDR196c, YDR449c, YDR398w, YDR413c, YDR201w, YPL063w, YPL146c, YFRO03c, YPRO82c, YLR359w, YML049c, YMR131c, YMR288w, YDR299w, YDR472w, YDR246w, YDR429c, YDR434w, YPL024w, YIL091c, YFR027w, YPR085c, YLR373c, YML077w, YMR 185w, YMR290c, YDR365c, YDR499w, YDR236c, YDR468c, YDR181c, YPL020c, YILO83c, YFR042w, YPR105c, YLR424W, YML093w, YMR212c, YMR211w, YDR396w, YDR141c, YDR361c, YDR489w, YDR53 1w, YPL012w, YILO19w, YIRO l0w, YPR112c, YL R437c, YML127w, YMR213w, YMRO49c, YDR4 07 c, YDR324c, YDR367w, YDR527w, YPL126w, YPLOO7c, YIL104c, YPRO48w, YPR137w, YLR440c, YMR 032w, YMR218c, YMR134w, YDR416w, YDR325w, YDR339c, YDR288w, YPL093w, YPL233w, YFLO24c, YPR072w, YPR143w, YPR144c and YPR169w.

TABLE 1: ESSENTIAL GENES Systematic aa deleted deleted comments ORF name nucleotides amino acids YMR049c 807 18-2277 6-759 weak similarity to A.thaliana PRL1 protein YMR134w 237 5-740 2-237 hypothetical protein YDR196c 241 174-543 59-181 similarity to C.elegans hypothetical protein T05G5.5 YDR299w 534 41-1560 14-520 hypothetical protein; nuclear localization (see http://paella.med.vale.edu/YGAC/qenes localization.html) YDR365c 628 45-1384 16-462 weak similarity to Streptococcus M protein YDR396w 166 141-460 48-154 hypothetical protein YDR407c 1289 48-3810 17-1270 weak similarity to Myolp YDR416w 859 151-2540 51-847 synthetic lethal with YDR449c 440 21-1270 8-424 hypothetical protein YDR472w 283 41-810 14-270 similarity to P.falciparum 41-2 protein antigen YDR499w 747 41-2100 14-700 weak similarity to hypothetical C.elegans protein, M.genitalium peptide chain release factor 1 and YJL149w YDR141c 1698 51-4850 18-1617 hypothetical protein YDR324c 717 79-2288 27-763 weak similarity to beta transducin from S. pombe and other WD-40 repeat containing proteins YDR325w 1051 110-3109 37-1037 hypothetical protein YDR398w 643 41-1880 14-627 similarity to human KIAA0007 gene YDR246w 219 41-580 14-194 hypothetical protein YDR236c 218 30-489 11-163 similarity to hypothetical A. thaliana protein YDR361c 283 43-812 15-271 hypothetical protein YDR367w 221 354-643 119-215 hypothetical protein YDR339c 189 40-529 14-177 weak similarity to hypothetical protein YOROO4w YDR413c 191 81-500 28-167 weak similarity to NADH dehydrogenase;or YDR412w YDR429c 274 86-645 29-215 TIF35; Vornlocher,H.-P., Hanachi,P. and Hershey,J.W.B.

Cloning and Characterization of the Two Large Subunits of Yeast Translation Initiation Factor eIF3.

Unpublished; translation initiation factor eIF3 (p33 subunit) Systematic ORF name deleted nucleotides deleted amino acids comments YDR468c 224 123-602 42-201 TLG1; member of the syntaxin family of t-SNAREs; tlg mutants seems to have a defect in the retrieval pathway to the TGN; viable YDR489w 294 131-630 44-210 hypothetical protein YDR527w 439 41-1260 14-420 weak similarity to Plasmodium yoelii rhoptry protein, or YDR526c YDR288w 303 41-800 14-267 hypothetical protein YDR201w 165 130-319 43-107 hypothetical protein YDR434w 534 41-1400 13-467 Similarity to S.pombe hypothetical protein YDR181c 481 194-1323 65-441 SAS4 (viable); involved in silencing at telomeres YDR531w 367 41-850 14-284 Similarity to hypothetical A. thaliana and C. elegans proteins YLR186w 252 4-750 2-250 strong similarity to S. pombe hypothetical protein C18G6.07C YLR215c 360 31-970 11-324 Similarity to rat cell cycle progression related D123 protein; there are few domains identical to the D123 protein YLR222c 817 8-2378 3-793 similarity to Dip2p YLR243w 272 41-700 14-234 strong similarity to YOR262w YLR272c 1176 15-3384 6-1128 similarity to hypothetical human ORF YLR275w 110 32-360 11-90 contains intron; strong similarity to human snRNPchain D2 involved in systemic lupus erythematosus; ident fied as part of the U1 complex by mass spectrometrie, PNAS 94: 385-390 (1997) Neubauer G. et al.

YLR276c 594 44-1733 15-578 similarity to RNA helicases; identified as part ot the Ul complex by mass spectrometrie, PNAS 94: 385-390 (1997) Neubauer G. et al.

YLR317w 144 4-403 2-135 YLR359w 482 120-1399 41-467 strong similarity to adenylosuccinate lyase YLR373c 901 14-2693 5-898 similarity to hypothetical protein YGR071c 0 00 Systematic aa deleted deleted comments ORF name nucleotides amino acids YLR424w 708 109-2098 37-700 weak similarity to Stulp YLR437c 133 7-376 3-126 YLR440c 709 18-1978 7-660 YML023c 556 81-1640 28-547 weak similarity to Nmd2p YML049c 1361 258-3967 87-1323 weak similarity to monkey UV-damaged DNA-binding protein YML077w 159 41-390 13-130 YMLO93w 899 29-2642 9-881 similarity to P falciparum liver stage antigen LSA-1 YML114c 510 11-1410 3-470 YML127w 581 65-1704 21-568 weak similarity to Loslp YMR032w 669 46-2002 15-668 weak similarity to S. pombe YMR093w 513 41-1300 13-434 weak similarity to Pwp2p YMR131c 511 11-1410 3-470 similarity to human retinoblastoma-binding protein YMR185w 981 65-2914 21-972 YMR212c 782 56-2287 18-763 weak similarity to myosin YMR213w 590 58-1533 19-511 similarity to S. pombe putative transcription factor YMR218c 1102 157-3253 52-1085- YMR281w 304 26-760 8-254 YMR288w 971 131-2670 43-890 strong similarity to S. pombe und C. elegans proteins YMR290c 505 11-1471 3-491 strong similarity to Myc-regulated DEAD box protein YMR211w 475 72-1341 25-447 weak similarity to beta tubulins YFL024c 832 47-2406 16-802 EPL1 (viable); weak similarity to YMR164c and Galllp YFROO3c 155 106-315 36-105 hypothetical protein YFR027w 281 40-649 14-217 hypothetical protein YFR042w 200 344-873 115-291 hypothetical protein YIL091c 721 44-1953 15-651 weak similarity to YIL083c 365 46-1005 16-335 hypothetical protein YIL019w 346 81-1000 27-334 hypothetical protein YIL109c 926 42-2721 14-907 SEC24 (lethal); component of COPII coat of ER-Golgi vesicles Systematic ORF name deleted nucleotides deleted amino acids comments i YIL104c 507 133-1082 45-361 similarity to hypothetical S. pombe protein YIR010w 576 41-1500 14-500 hypothetical protein YIR015w 144 85-274 29-92 hypothetical protein YPL126w 896 41-2700 14-900 weak similarity to fruit fly TFIID subunit YPL093w 647 151-1900 51-634 similarity to M.jannaschii GTP-binding protein, GTP1/OBG-family, weak similarity to other GTP-binding proteins YPL063w 476 126-1385 42-462 similarity to hypothetical protein YLR019w, YLL010c and S.pombe hypothetical protein SPAC2F7.02c YPL024w 241 41-550 14-184 NCE4 (viable); negative regulator of CTS1 expression YPL020c 621 44-1813 15-605 weak similarity to Smt4p YPL012w 1228 41-3630 14-1210 hypothetical protein YPL007c 588 55-1614 19-538 hypothetical protein YPL233w 216 41-610 14-204 hypothetical protein YPL146c 455 46-1325 16-442 weak similarity to myosin heavy chain proteins YPR048w 623 41-1650 14-550 similarity to M.domestica NADPH--ferrihemoprotein reductase and mammalian nitric-oxide synthases YPR072w 560 42-1541 14-514 NOT5 (viable); component of the NOT protein complex YPR082c 143 140-279 47-93 weak similarity to Ypklp YPR085c 448 277-1166 93-389 hypothetical protein YPR105c 861 74-2543 25-848 hypothetical protein YPR112c 887 52-2521 18-841 similarity to RNA-binding proteins YPR137w 573 41-1680 14-560 weak similarity to YPR143w 250 41-710 14-237 hypothetical protein YPR144c 552 107-1616 36-539 similarity to YDR060w and C.elegans hypothetical protein YPR169w 514 201-1490 201-1490 hypothetical protein WO 99/55907 31 TABLE 2: Primers used for gene deletions PCT/EP99/02722 Gene deletions on chromosome 13 Name Sequence 5'-3' YDR472w-Sl ATG TCT CAA AGA ATA ATT CAA CCA AGC GCA TCT GAC CAA CCA OCT GAA GCT TCG TAC GC YDR472w-S2 AGC CAA ATC TCA AAC CTT CCC TGT CAA GCA CTT GCC TGT CGC ATA GGC CAC TAG TGG ATG TG YDR499w-Sl ATG AGA CGA GAA ACG GTG GGT GAA TTT TCT TCA GAT GAC GCA OCT GAA GCT TCG TAC GC YDR499w-S2 CGT ACT TTA CTT GCA TTA TTC TCC CCG TTC TTT TAT TCA AGC ATA GOC CAC TAG TOG ATG TG YMRO49c-Sl CAG ACT ATT GAT TAC TTT ATG ACC GGT TAG TTT CTT TAG TCA OCT GAA OCT TCG TAC GC YMRO49c-S2 TCT GTT CTA ACA TAA CTA GGT CAA TGA TGG CTA AGA ACA AGC ATA GOC CAC TAG TOG ATC TG YMRl34w-Sl GCA AAG TGT GGT ATA GAA AAA GAA CCA AAG GCC GGT ATG TCA OCT GAA OCT TCG TAC Ge YMR134w-S2 TGT GTG TGT GCC TAC CTG CAT GTA TGC ATT TAG CAA TTG AOC ATA GOC CAC TAG TGG ATC TO YMLO23c-Sl CAC GCA ATG GTG CAC ATT ATT TTG TTG AAC TCA CTG AGA ACA OCT GAA OCT TCO TAC Ge YMLO23c-S2 ATT AGT TAC TTA TTC TAT AAT TAC ACT TTT ATC ATG AAC GOC ATA GOC CAC TAO TGG ATC TO YMLO49c-Sl AAT TCC TGC TCA TTC AAG GAA AGT CTC AGG AAA TTT TCA CCA OCT OAA OCT TCG TAC GC YML49c-S2 ACT CCT GCA TCG GAC ACT TCG TCG ATC TGG AAG CAG GGT COC ATA GOC CAC TAG TGG ATC TO YMLO77w-Sl ATG GGG ATA TAT TCA TTT TGG ATC TTT GAT AGG CAT TGT ACA OCT GAA OCT TCO TAC Ge YMLO77w-S2 TTC TAT TGG TGA TCT TTC TTG TCC CTT GAC CTC TCA TTT COC ATA GOC CAC TAG TGG ATC TO YMLO93w-Sl GCT AAC TTA AAT ATG GCA AAA AAG AAA TCT AAG AGC AGA TCA OCT GAA OCT TCO TAC Ge YMLO93w-S2 CAA AGG ATC AAT AAC TTG GCC TGG CTT AGT CAT GAT TCT COC ATA GOC CAC TAO TGG ATC TO YMLl14c-S1 AAC GTG TAA TTG AGG GAC TCA TAA AGG GCA ATG ACT TCC ACA OCT GAA OCT TCO TAC Ge YMLl14c-S2 GAC TTG TAG TAG CAT CGA TAT TGG TTG TGT TAT GTG CTA COC ATA GGC CAC TAO TGG ATC TO YML127w-Sl CCG CTA AAT GGT ACT CCA GTA AGC GAG GCA CCC GCC ACA ACA OCT GAA OCT TCG TAC Ge YMLl27w-S2 ATA ACC CCG ACG TGT TTT CCA TGT ATT CAG ACA ATG CTA AOC ATA GOC CAC TAG TGO ATC TO WO 99/55907 WO 9955907PCT/EP99/02722 Gene deletions on chromosome 13 Name Sequence 5'-31 YMRO32w-S. CTA CAG TTA TGA AGC TTG TTT TTG GGA CCC AAA CGA CAA TCA GCT OAA GCT TCG TAC GC YMRO32w-S2 CAG AAA ACT AGT AAA ATT GAT ATA CAT CGA GAT CAA AGA CGC ATA GGC CAC TAG TGG ATC TO YMRO93w-S. ATG TCG ACT GCT AGG, CCT AGA ATA ATC ACT TCG AAG GCT CCA OCT OAA GCT TCG TAC GC YMRO93w-S2 AAG CAC CAA TTC AGT AGC GGC TCT AAT GTA GAT TCA TCT CGC ATA GGC CAC TAG TGO ATC TO YMR13lc-Sl CTT TAA CTT CCT TTT GCC AGT GAA CAA ACA ATA ATT GTG GCA OCT GAA GCT TCG TAC GC YMR13lc-S2 GGT CTA TCG AGG TCA ACG AGG AAC AAG ATA GAG TGG TCT CGC ATA OGC CAC TAG TGO ATC TG YMR185w-52. ATC AAC ATA CAC GAT ATA TTG AAT ACA AGA CCG AAG CTC ACA GCT OAA GOT TCG TAO GC YMRl85w-S2 GTA ATG GGT TAT AAA CTA TCT AGT ACG GTT AAA AGC TTG TGC ATA OGC CAC TAG TGG ATC TO YMR212c-S1 CCT CTT GAA CTT AAA GAA TGT AAA TCT TCA TTT GCG TCT TCA OCT OAA OCT TOO TAC 00 YMR212c-S2 CGG ATG ATG TTC ACA CCA .AAA CAT CAG AAA CTG GTC AAT COC ATA GOC CAC TAG TOO ATC TG YMR2 13w-Si ATA CGT GAA AGG, CGG TGT ATG GAC CAA TGT GGA GGA TCA GCA OCT OAA OCT TCO TAC GC YMR2l3w-S2 GCT GTA ACT GTT CAA TAG ACT CCA CTT TTG ATT GGA TCG AGC ATA 000 CAC TAG TOG ATC TO YMR2l8c-Sl GAC TCA AAT GCA TTA GAG TGA TCA ACT CTA CAA CTT TTA CCA OCT OAA OCT TCO TAC GC YMR2l8c-S2 GAA GGC ATT TGA CGG AAC TGT ACG AAC GGT TAA CAG GCT TOC ATA OOC CAC TAG TOO ATC TO YMR281w-S1 CTG AAG AAA AGT TAA ATG AAG ATG TTG AGG CGT ACA AAG GCA OCT OAA OCT TCO TAC GC YMR281w-S2 AGT ACG TAT TGT GCA TGT GTA TTC ATA AGT GAA AGC TTG TOC ATA OOC CAC TAG TOO ATC TG YMR288w-Sl GAA AAC CTG CAG AAA GAA GCT GCA CGT ATT GGT GAG AAC GCA OCT OAA OCT TCO TAC GC YMR288w-S2 CCA AAC CTT CTA AAA TAC GCA TAA TAG CAT GTG GTG AAG TOC ATA GOC CAC TAG TOO ATC TO YMR29Oc-Sl TGA GTT TTA CGT CTT TTG GTA TTT GGC GTT TTT CCA CTG GCA OCT GALA OCT TCO TAC GC YMR29Oc-S2 GAT AAG, CTG AGC AAT ATT AAC AGG AGA AGT ATG GCT ACC COC ATA 000 CAC TAO TOO ATC TO YMR21lw-Sl AGA GAG CAA ACC ATT TGA CTA CTC AAT TCT TCA ATA TAC ACA OCT OAA OCT TCG TAC GC YMR211w-S2 ATT TCA ATC ATC TTA CTC CGT GAA TCA GGT TCG GAA TGA TOC ATA GOC CAC TAG TOO ATC TO WO 99/55907 PCT/EP99/02722 Gene deletions on chromosome 4 Name Sequence 5'-3' YDRl96c-Sl ATG CTT ATG ATC AAA TTG TGT TAT ACT TCA AGG ACA AAA TCA GCT GAA GOT TOG TAO GO YDR196c-S2 TTT CAA TCT GTT CGT ATA AGT CAA CCA ATG TGC TGT TAT TGO ATA GGO CAC TAG TGG ATO TG YDR299w-Sl ATG GAA AAA TCA CTA GCG GAT CAA ATT TCC GAT ATC GCC ACA GCT GAA GOT TOG TAC GO YDR299w-S2 CAA AGA TTT GGA TAT CAT CGT TTT TAA CAG CCT CTA ATT CGO ATA GGC CAC TAG TGG ATO TG YDR365c-Sl CTG GAG AGA ACC CAA AGA AGG AAG GTG TAG ATG CTA GGT TOA GOT GAA GOT TOG TAO GO YDR365c-S2 TTA GTA TGC TTT TTA TTA ACA GAT TTC AAC TTG CTT TTC TGO ATA GGC CAC TAG TGG ATO TG YDR396w-Sl CAG ATA CAC TAT TGT GGT GTA ATC TGG ACC TTG ACT GTC TOA GOT GAA GOT TCG TAO GO YDR396w-S2 TAG AGA AAA CAC TGA ATG ATC TTA GCG ACC GTA CAA AAG AGO ATA GGC CAC TAG TGG ATO TG YDR4O7c-Sl TTC TTA AGC ATT TCC CA-A GCT ATG TTG GCC CAT CTA AGA TOA GOT GAA GOT TOG TAO GO YDR4O7c-S2 AAT AAC AGA CAA GAT AAC GTT TTC AGA GTC GAA CTG GAT TGO ATA GGC CAC TAG TGG ATO TG YDR4l6w-Sl ACT TAC ATG GAA AAG ATA TAT CGA GTA TTG GAA AGA GGA GOA GOT GAA GOT TOG TAO GO YDR4l6w-S2 TCA AAT ATC TAG TTC TAT TTC ATC TGG ATT A-AT CGA ATA TGO ATA GGC CAC TAG TGG ATO TG YDR449c-S1 CAC ATC ACC GAT TTC TAA TAA TGT CGA AGA CAA GAT ACT A-A GOT GAA GOT TOG TAO GO YDR449c-S2 ATA ATT AAA TCT AGA ATT TTA TAC CTA GGA TCA TCT TCT GGO ATA GGC CAC TAG TGG ATO TG YDRl4lc-Sl TTC GTA ATC TTT GAA TTC TGC GAT TTC ATC TAC CAG CGC GOA GOT GAA GOT TOG TAO GO YDR141c-S2 CAC TAA AGC CCC TTA CAA TTG ACT CAA ATA ATA AAC AAC TGO ATA GGC CAC TAG TGG ATO TG YDR324c-Sl AAG AAG CCT GAA AAT ACG AAA CAA ACC GGT GAA GAT GAC COA GOT GAA GOT TOG TAO GO YDR324c-S2 AAA CACTAA CTT TGG TTG AAT AAA CGC CTT TTG TTT GGA GGO ATA GGC CAC TAG TGG ATO TG YDR325w-Sl GAC ATT A-AT ACG AAA ATC TTT AAC TCA GTT GCT GAA GTA TOA GOT GAA GOT TOG TAO GO YDR325w-S2 ACC TCG CTG AAA GAC TCT GAA TCC TTA TCT TCT TCA TCT AGC ATA GGC CAC TAG TGG ATO TG YDR398w-Sl ATG GAT TCT CCT GTT CTA CAG TCC GCT TAT GAC CCA TCA GOA GOT GAA GOT TOG TAO GO YDR398w-S2 A-AC GTC ACT ATA TCC GGC TTC CTC CTC GCC GTC GCT CTG CGO ATA GGC CAC TAG TGG ATO TG WO 99/55907 PCT/EP99/02722 Gene deletions on chromosome 4 Name Sequence 5'-3' YDR246w-S1 ATG GCC ATC GAA ACA ATA CTT GTA ATA AAC AAA TCA GGC GCA GCT GAA GOT TOG TAO GO YDR246w-S2 AAC AGG TTA GAT CTT ATA GGC ATT TCC ATT GAG TAA GAT GGO ATA GGO CAC TAG TGG ATC TG YDR236c-Si CTA AAA TAT TGA ACT TGA CCC TGG CCC CAT AAA AAT CAT TOA GOT GAA GCT TOG TAO GC YDR236c-S2 TTG AAG TGT TGA TGT TTACGT GGA CTA TTT ATG TTT CGT TGC ATA GGO CAC TAG TGG ATC TG YDR361c-S2 TTA CCA AGT GGA AAT TTC TGT TTC CAA TTC ATC GAT ACT TGC ATA GGC CAC TAG TGG ATO TG YDR36lc-Sl GGT TCA AGC TAT CAA ATT AAA TGA TTT AAA AAA TAG GAA GCA GCT GAA GOT TCG TAO GO YDR367w-Si ATC TGC GTA CTT TAT ACA ATC GAT ACC ATT TCC ACT TGT TOA GOT GAA GCT TCG TAC GC YDR367w-S2 GTT TTG TTC TAC GTC ATC CCT ATC AAC TAA ATA TTT GGG GGO ATA GGC CAC TAG TGG ATO TG YDR339c-Sl TAT GGG TAA AGC TAA GAA AAC AAG AAA GTT TGG CCT CGT ACA GOT GAA GOT TOG TAC GC YDR339c-S2 TAA AAG ACA TCT GGC AAT TTT TCA ATG ACG TAT GCG TGA CGO ATA GGO CAC TAG TGG ATO TG YDR413c-Sl TTC TTT GGT TTA TTC TTC GTT CAT TTT TGG TCA AAT ATC TCA GCT GAA GOT TOG TAC GC YDR413c-S2 ACA AAA GAA AGC ACA AGA GTT TAT TAA GGA GCA GGA AAG GGC ATA GGO CAC TAG TGG ATO TG YDR429c-Sl TCT AGA TCT ATC ATT ACA TAC AAG ATT GAA GAC GGT GTC ACA GOT GAA GOT TOG TAO GC YDR429c-S2 TTT CTT TGT TTC TAA CGA CAG AAA CTC TTG GAA TGG GTG CGO ATA GGO CAC TAG TGG ATC TG YDR468c-Sl GTO ACA ATA OTG CTG GTG ATG ACG ATC AAG AGG AGG AAA TCA GCT GAA GCT TCG TAC GC YDR468c-S2 CAA GAC GAC AAT AAG AAG TCC TAT ACA ACA ATC GTC GTA TGC ATA GGO CAC TAG TOG ATO TG YDR489w-Sl ACT ACC CAC AGA GAT GCA AAT ACA ATA GTG GGT TCG TCC TCA GOT GAA GOT TOG TAO GO YDR489w-S2 AGT CGG GOT CAT CTA TCA TGT TTA CGC TAO CTT CTG TAT OGC ATA GGC CAC TAG TGG ATC TG YDR527w-Sl ATG GAC TTA CTG GGC GAT ATA GTG GAG AAA GAT ACA TCT GOA GOT GAA GOT TOG TAO GO YDR527w-S2 CCC CAC CGC CTT GTT TCC ATA ACC AAA GTG CAT CAA TAG CGO ATA GGC CAC TAG TGG ATO TG YDR288w-Si ATG AGT TCT ATA GAT AAT GAC AGC GAT GTG GAT TTA ACA GOA GOT GAA GOT TOG TAO GO YDR288w-S2 GCC CAT GAT TTC TTG CAC CAA TTT TTC AAG AGA CTC TAG TGO ATA GGC CAC TAG TGG ATO TG WO 99/55907 WO 9955907PCT/EP99/02722 Gene deletions on chromosome 4 Name Sequence 5'-3' YDR2Olw-S1 CCC ATG TCT GGA CTA TTC AGA GCA TCA TCG TCA TCC ATA CCA GCT GAA GCT TCG TAC GC YDR2Olw-S2 AAA AGG GTT TTC CGT TTA GTT CCC GAA TAT GAT GTT GAA AGC ATA GGC CAC TAG TGG ATC TO YDR434w-Sl ATG TCC AAT GCA AAT CTA AGA AAA TGG GTT GGT TTT TGC TCA GCT OAA GCT TCG TAC GC YDR434w-S2 TAA AGG TAA ATA CAC AGC TAT CAT GTO CTC TTG TOO OAA GGC ATA GGC CAC TAG TGG ATC TG YDRl8lc-Sl AGG ATA AAC CCA AA.T GCT GGA CAT CTA AGG AAA TCT AAG, TCA OCT GAA GCT TCO TAC GC YDR18lc-S2 TAG TTG GGT TTG AAT CGT TAT CAC GGG AGA ACA TTG CTT TGC ATA OGC CAC TAG TOO ATC TO YDR53lw-Sl ATG CCG CGA ATT ACT CAA GAG ATA TCT TAC AAT TGC GAT TCA OCT GAA OCT TCG TAC GC YDR53lw-S2 AAA TAA GCT ATT TGC CCA ATA TTG TTG GAG ATG GCG AAT AOC ATA OOC CAC TAO TGO ATC TO WO 99/55907 PCT/EP99/02722 Gene deletions on chromosome 12 Name Sequence 5'-3' YLR186w-Sl CTA GTC ACC AAG AAG AAA ACC CGT AAA ATC GTA GGT CAT GOA OCT GAA GOT TCG TAO GC YLRl86w-S2 ATA CAA AGA GGA TGC CAA GTA GAC TTA AAC ACT ATA AAA TGC ATA GGC CAC TAG TGG ATO TO TTA CTT ATT GAT GTC CTC ACA AGA ATA TAC AAC TTT TAT ACA GOT GAA GOT TOG TAC GC YLR215c-S2 AGC TCT CGG ATT GCT TCA GGA TTT AAA CTA GCT TCT ACG AGO ATA GGC CAC TAG TGG ATO TG YLR222c-S1 CTC TCA ACG GTA GTA AGC CAT ACT ACG TAC AAT ATG GAT CCA GCT GAA GOT TOG TAC GO YLR222c-S2 AAT ATG TAA CTT TGT TCA ACT AAG TTA TCA ACC CTT GTG AGC ATA GGC CAC TAG TOG ATO TO YLR243w-Sl ATG TCT CGC GTT GGT GTC ATG GTA TTA GGA CCT GCA GGT GOA GOT GAA GOT TCG TAO GO YLR243w-S2 GAT AAT ATG GTT TCT ATA CTG TCA GGA TTA TTA GAT TCC AGO ATA GGC CAC TAO TOG ATO TO YLR272c-S1 TTT GGG TCT CGC ACT TTC TCA GTC TTC CAA CTA ATT TCT COA GOT GAA GOT TOG TAO GO YLR272c-S2 GGT AAC TGA CTT CGT TAC TTT ATG AGA TGT CCG GCT TTA GGO ATA GGC CAC TAG TOG ATO TO YLR275w-Si CCG TTT TAT CAT GTC GTA TGT TTG ATC TTA ACC ATT TTT ACA GOT GAA GOT TOG TAO GO YLR275w-S2 CAA CGA TAA CTG AAT CAC CTC TTA AGA ATA GTT TAC TTA TGC ATA GGC CAC TAG TGG ATO TO YLR276c-S1 CTT CAA CGG GTC TAC TTT ACC ATT CTT TGG CTT ACT GAC TOA GOT GAA GOT TOG TAO GO YLR276c-S2 AGC TAT GAG AAA AAG TCT GTG GAA GGC GCT TAT ATT GAC GGC ATA GGC CAC TAG TOG ATO TO YLR3l7w-S1 CTG CCA TCT TCT GCC ACC ACT TTG TCC TTC TTT CTT GAT GOA GOT GAA GOT TOG TAO GO YLR317w-S2 GAA GTA AAC TAA CTA GTA AAG TAG GCT AAT TCG AAA CGA TGC ATA GGC CAC TAG TGG ATO TO YLR359w-S1 GGC TAT TGC TGA GAA GGA ATT GGG CTT AAC TGT TGT TAC ACA GOT GAA GOT TOG TAO GO YLR359w-S2 AAC TTG ACT TGT TCA TCG TTT AGG TAC TTT TGG AAA GGT TGC ATA GGC CAC TAG TOG ATO TO YLR373c-S1 ACA CAC AGG TAC AGA GTG CTG AAA GAG GAT TGG TGT TGC COA GOT GAA GOT TOG TAC GC YLR373c-S2 CAA ACA GAC TTT GTT CCT TTG TAT GTC CTA TGG AAG ATA CGC ATA GGC CAC TAG TGO ATO TG YLR424w-S1 GAC ATG ACA TAC ACT AAT GAT GCC TTG AAA ACT AGT AGC GOA GCT GAA GOT TOG TAO GO YLR424w-S2 ATA GGT ACT TTC TAG AGG TCA AGG GCC CAT AAA TAA ATT GGC ATA GGC CAC TAO TGG ATO TO WO 99/55907 PCT/EP99/02722 Gene deletions on chromosome 12 Name Sequence 5'-31 YLR437c-Sl ATT GTG CAA GTC TGT TAA AGT CTT CTC TTG GAT CCA TTA ACA GCT GAA GCT TCG TAO GC YLR437c-S2 CAT CAC ACA CTA ATA CAG GAA CAA ACA AGA CTT AAT GGA CGC ATA GGC CAC TAG TGG ATO TG YLR44Oc-Sl TTG CCA AGA AAA TTG CAG TAA AAA TGT TGG AAG, AGC AAC TCA GCT GAA GOT TOG TAO GC YLR44Oc-S2 GCT CCA ATT CTA GTG TGC TCC ATT GCG ATG TAA CAA TTT CGC ATA GGO CAC TAG TGG ATC TG WO 99/55907 PCT/EP99/02722 Gene deletions on chromosome 6 Name Sequence 5'-3' YFLO24c-Sl TGA TGA ATT TTT CTG GGT TAT AGA AGA GTT CTG TTT CGC TCA GOT GAA GOT TOG TAO GO YFLO24c-S2 ACA CCT TCA AAC GCT ATA GAG ATC AAT GAC GGT TCG CAT AGO ATA GGC CAC TAG TGG ATO TG YFROO3c-S1 TGT GGA AGA GGT TCC CGC AGT TTT GCA GCT TCG AGC AAC TOA GOT GAA GOT TOG TAO GO YFROO3c-S2 ATC TTC TTT GTC TAC GTT CGT TAA AGT CAA GAT CCT TCT CGO ATA GGC CAC TAG TGG ATO TG YFRO27w-Sl AAT GAA AGC TAG GAA ATC GCA GAG AAA AGC GGG CAG TAA ACA GOT GAA GOT TOG TAO GO YFRQ27w-S2 AAT TTG GTT GCG ATA CCC AAC TTC CTT GCT GTC CTG CAC AGO ATA GGC CAC TAG TGG ATO TG YFRO42w-S1 AGT TTG CAC CAA TGG CAA TAT GCC TGT GAT AAA GAT AAG GOA GOT GAA GOT TOG TAO GO YFRO42w-S2 CAT GGA AGT TAT TTG GTT GCT TAG ATT CCA CGG GTT CAA AGO ATA GGC CAC TAG TGG ATO TG WO 99/55907 WO 9955907PCT/EP99/02722 Gene deletions on chromosome 9 Name Sequence 51-3' YILlO9c-Sl TGT CTC ATC ACA AGA AAC GTG TTT ACC CAC AAG CTC AGC TCA GCT GAA GCT TCG TAC GC YIL1O9c-S2 TCA TGA TTT GTA AGA ATT CTC TGT AAC TTT COT TAT TCA AGC ATA GGC CAC TAG TGG ATC TG YILO91c-Sl AGT GAC AGT TCT GTG AGG GAA AAG AAT GAT AAT TTC CGT GCA GCT GAA GCT TCG TAC GC YILO9lc-S2 CAT TGT AAA ATT CAG GAT TGT TTG GAG GCT TAT AAA AAA CGC ATA GGC CAC TAG TGG ATC TG YILO83c-S1 ACC TCT ACC CGT OCT CAA CAG ACC TCA AAT TCA TAC GTC TCA GCT GAA GCT TCG TAC GC YILO83c-S2 CGA TGA CTT CTG OGA TTA TCA TCT CTT CAA TGA TAT GOT 0CC ATA GGC CAC TAG TGG ATC TG YIL0l9w-Sl TTC AAA GA). AAG CTT TTG AAA OTC AGT TCG GAT CTT TAG ACA GCT GAA GCT TCG TAC GC YIL0l9w-S2 TAG CGA CGG OAT TTC TTT TTG CCA TTA AAT TTA CCA CTC CGC ATA GGC CAC TAG TGG ATC TG YILlO4c-Sl TCC CCT TAC TAT TTA AGA TTA AGA TTT CCT CAC GA.

TTA ACA GCT GAA GCT TCG TAC GC YILlO4c-S2 CCT OAT ACC TOT AAT GAT TGC AAT CTT GAT GAG AGA GCT GGC ATA GGC CAC TAG TGG ATC TG YIROlOw-Sl ATG AGT CTG GAA CCC ACA CAA ACG GTC TCC GOT ACG CCG CCA GCT GAA GCT TCG TAC C YIROlOw-S2 ACC TTA OCT CAT CGA CTT TTG TTT CTA ACT TTG TCT CA). CCC ATA CCC CAC TAG TGG ATC TG OCG AAC CAG GAC CAT TTC CAT AGA TTA AAC TAC CTC TAC CCA GCT GAA GCT TCG TAC GC YIR0l5w-S2 ATT TCC AGT TTT TTT 000 GTC CAT AAC AAC CGA TGG CAT TGC ATA GGC CAC TAG TGG ATC TG WO 99/55907 PCTIEP99/02722 Gene deletions on chromosome 16 Name Sequence 5'-3' YPL233w-Sl ATG TCA CAA GGT CAG TCC AAA AAA CTG GAC GTA ACT GTT GCA GCT GAA GCT TOG TAO GC YPL233w-S2 CAA TCC TCC TCC AGG AAG TCC ATT AAG CGC TTG ACC TTT TGC ATA GGC CAC TAG TGG ATO TG YPLl46c-Sl TCC AAC TAA TCT AACCAA GAA ACC ATC TCA ATA CAA ACA GOA GOT GAA GOT TCG TAC GO YPL146c-S2 TTT GAA GTC CTT ATG TGT CCA CTT TTC AGT GAT TTT CTG CGC ATA GGO CAC TAG TGG ATC TG YPL126w-S1 ATG ACG CAA TCC CTA GGT ATC GAA CAG TAT AAA CTG TCA GOA GOT GAA GCT TOG TAO GO YPL126w-S2 TAT GTT AAT ACT TTC ATC ACA CGA TCA AAA AAT GTA TCC AGO ATA GGC CAC TAG TGG ATC TG YPLO93w-S1 CAA GAT TAC AAG AAT CAG AGCGTT CTA TAT GCG TAA AGT TOA GOT GAA GCT TOG TAO GO YPLO93w-S2 CGG AAA TCT GTC TTA CCG ACA CCA CGC TTA CCA CTG AAT AGC ATA GGC CAC TAG TGG ATO TG YPLO63w-S1 TTC AAG CAT CCA GAA GAC TTT TAA CAA GTT ATT CAA GTT TOA GOT GAA GOT TOG TAO GO YPLQ63w-S2 GGA TTC AGC AAT CTT CTT CTT TTT CTT TTC CTC TTC AAA TGO ATA GGC CAC TAG TGG ATO TG YPLO24w-S1 ATG TCT TTT TCA TCT ATC TTA TCA CAG GAT ATC ACA GAT GOA GOT GAA GOT TOG TAO GO YPLO24w-S2 ACT TGT GAG TCC TTC AAT ATG AAA ACG CCC CTA TTG AAC AGO ATA GGC CAC TAG TGG ATO TG YPL020c-S1 TCA GTT GAA GTA GAT AAG CAC CGG AAC ACA CTA CAG TAT COA GOT GAA GOT TOG TAO GO YPL020c-S2 TCG GTT AAA ATC AAA TGG GCA ATA AAT CTT CTC ATC CTA AGO ATA GGC CAC TAG TGG ATO TG YPLO12w-S1 ATG GAT CAA GAC AAA GTT GCT TTT CTT TTA GAG CTG GAG GOA GOT GAA GOT TOG TAO GO YPLO12w-S2 ATT TGA ACT TTGGAC CTT TCT TAT TAT GTT TGC CAA TCT TGO ATA GGC CAC TAG TGG ATO TG YPLOO7c-S1 CCC GTT GTT TGC TCA CGG GAC ATA TCT TTA CAT GCG TTG TOA GOT GAA GOT TOG TAO GO YPLOO7c-S2 GGA CTT ATT GGT AGA TAG AAA GGA ATT TGA GGA TTG GAA GGO ATA GGC CAC TAG TGG ATO TG YPR048w-Si ATG TCA TCG AGC AAG AAA ATC GTC ATC CTC TAT GGA TCG GOA GOT GAA GOT TOG TAO GO YPR048w-S2 AAT TAG TTA TTT CCT CAC CTA ATC TCC ATA AGT AGT CTT GGO ATA GGC CAC TAG TGG ATO TG YPRO72w-S1 TGT CTC AAA GAA AGC TAC AAC AGG ATA TCG ATA AGC TTT TOA GOT GAA GOT TOG TAO GO YPRO72w-S2 AGA TTT GCC CAT TCC TGG TGG TA CTT TTC GAT TTC TTT AGO ATA GGC CAC TAG TGG ATO TG WO 99/55907 WO 9955907PCTIEP99/02722 0@ 0 000 0000 0 0000 00 0 0@ *0 00 0 0 0@ 0e 0@ @0 S 0 0000 0 0000

S

0S0 S 0000 0

S

S

@000 0 0050 0S S 0 S

S..

Gene deletions on chromosome 16 Name Sequence 51-3' YPRO82c-Sl CTT CGA TTG CTG AAA GAG TAA GGA ACT TTG CAG TTA TTT ACA GOT GAA GOT TOG TAO GO YPRO82c-S2 CAA TAA AGT TCA ACT TGT TGT TGT TCC CTG TAC CAA AAT CGC ATA GGC CAC TAG TGG ATC TG CTG TAC ATT CTT TOG AAA GAC TCC ATG CTG CGA ATT TTT GCA GOT GAA GOT TOG TAO GO YPRO85c-S2 TOO CAC TTT ATA GTT ATG GGA TTT CGA GOT GGA TTO GGT AGO ATA GO CAC TAG TGG ATO TG AGO TOG ATO ATO GAG GGO CAA TTG TCT AAA AAT OTA GOA ACA GOT GAA GOT TOG TAO GO YPR1O5c-S2 CTG TGT TOT ATO AAT OTT OAT ATT TOT AGO TTT AAT TOT TGO ATA GGO CAC TAG TGG ATO TG YPR1l2c-Sl CAT TGT CAA GGG TTT GOO OGT OTA TOT AAO AGA TGA TAA TOA GOT GAA GOT TOG TAO GO YPRll2c-S2 GAA ACC TTO GTT TTO TTO ATO ATO CAC ATO CAG TTT OTT TGO ATA GGO CAC TAG TGG ATO TG YPR137w-Sl ATG TOA GAT GTT ACC CAA CAG AAA AAG AGG AAA AGA TOO ACA GOT GAA GOT TOG TAO GO YPR137w-S2 AAA AGO OTG TTT GGT CAA TGA CAG OTG AAT ATA TAO OAT TGO ATA GGO CAC TAG TGG ATO TG YPR143w-Sl ATG GGO TOO AAG CAC AGA GTA GAO ACT AAG GAT AAG AAA ACA GOT GAA GOT TOG TAO GO YPR143w-S2 'TTO ATT GTO GOT TOO TGO GGO AGO TTT AAO TAA ATO CAA AGO ATA GGO CAC TAG TGG ATO TG YPRl44c-Sl TTC CAG AAA ATG TTA OTO AAT TGG AAG AAG ATG AGA CAG ACA GOT GAA GOT TOG TAO GO YPR144c-S2 ICCA TGO TAO COO AGG CAA GTA GAO GTT ACC TTG GGA TGA OGO ATA GGO CAC TAG TGG ATO TG YPR169w-S1 ITTT TAO ATO CTG AAO TGC OCA TTA TAA TAA OTG GOT GOA GOT GAA GOT TOG TAO GO YPR169w-S2 OTT

GTT

OTT

GGC

GAT COO ATG OTO ATA CAG GTO ATA GGO CAC TAG TGG ATC TG OTT TTT TTT Where the terms "comprise", "comprises","cmrsdorcmpingae 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.

EDITORIAL NOTE APPLICATION NUMBER 38207/99 The following Sequence Listing pages numbered 1 to 40 are part of the description.

WO 99/55907 SEQUENCE LISTING <110> HOECHST MARION ROUSSEL <120> METHOD FOR SCREENING ANTIMYCOTIC SUBSTANCES USING ESSENTIAL GENES FROM S.CEREVISIAE <130> 16363PC RUU 7 <140> <141> PCT/EP99/02722 <150> 98402254.1 <151> 1998-09-11 <150> 98401007.4 <151> 1998-04-24 <160> 180 <170> Patentln Ver. 2.1 <210> <211> <212> <213> 1 59

DNA

Artificial sequence Description of Artifici al Sequence: primer YLR2 43w-Si <220> <223> <400> 1 atgtctcgcg ttggtgtcat ggtattagga cctgcaggtg cagctgaagc ttcgtacgc 59 <210> 2 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR272c-S1 <400> 2 tttgggtctc gcactttctc agtcttccaa ctaatttctc cagctgaagc ttcgtacgc <210> 3 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR275w-S1 <400> 3 ccgttttatc atgtcgtatg tttgatctta accattttta cagctgaagc ttcgtacgc WO 99/55907 PCT/EP99/02722 2 <210> 4 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR275w-S2 <400> 4 caacgataac tgaatcacct cttaagaata gtttacttat gcataggcca ctagtggatc tg 62 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR276c-S1 <400> cttcaacggg tctactttac cattctttgg cttactgact cagctgaagc ttcgtacgc 59 <210> 6 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR276c-S2 <400> 6 agctatgaga aaaagtctgt ggaaggcgct tatattgacg gcataggcca ctagtggatc tg 62 <210> 7 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR317w-S1 <400> 7 ctgccatctt ctgccaccac tttgtccttc tttcttgatg cagctgaagc ttcgtacgc 59 <210> 8 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR359w-S1 WO 99/55907 PCT/EP99/02722 3 <400> 8 ggctattgct gagaaggaat tgggcttaac tgttgttaca cagctgaagc ttcgtacgc 59 <210> 9 <211> 62 <212> DNA <213> Artificial Sequence tU <220> <223> Description of Artificial Sequence: primer YLR359w-S2 <400> 9 gttcatcgtt taggtacttt tggaaaggtt gcataggcca ctagtggatc tg 62 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR373c-S1 <400> acacacaggt acagagtgct gaaagaggat tggtgttgcc cagctgaagc ttcgtacgc 59 <210> 11 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR3 73 c -S2 <400> 11 caaacagact ttgttccttt gtatgtccta tggaagatac gcataggcca ctagtggatc tg 62 <210> 12 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer s0 YLR424w-S1 <400> 12 gacatgacat acactaatga tgccttgaaa actagtagcg cagctgaagc ttcgtacgc 59 <210> 13 <211> 62 <212> DNA <213> Artificial Sequence WO 99/55907 PCT/EP99/02722 4 <220> <223> Description of Artificial Sequence: primer YLR424w-S2 <400> 13 ataggtactt tctagaggtc aagggcccat aaataaattg gcataggcca ctagtggatc tg 62 <210> 14 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR437c-S1 <400> 14 attgtgcaag tctgttaaag tcttctcttg gatccattaa cagctgaagc ttcgtacgc 59 <210> <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR437c-S2 <400> catcacacac taatacagga acaaacaaga cttaatggac gcataggcca ctagtggatc tg 62 <210> 16 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR440c-S1 <400> 16 ttgccaagaa aattgcagta aaaatgttgg aagagcaact cagctgaagc ttcgtacgc 59 <210> 17 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR440c-S2 <400> 17 gctccaattc tagtgtgctc cattgcgatg taacaatttc gcataggcca ctagtggatc tg 62 WO 99/55907 PCT/EP99/02722 <210> 18 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YFL024c-S1 <400> 18 tgatgaattt ttctgggtta tagaagagtt ctgtttcgct cagctgaagc ttcgtacgc 59 <210> 19 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML049c-S2 <400> 19 actcctgcat cggacacttc gtcgatctgg aagcagggtc gcataggcca ctagtggatc tg 62 <210> <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YFL024c-S2 <400> acaccttcaa acgctataga gatcaatgac ggttcgcata gcataggcca ctagtggatc tg 62 <210> 21 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YFR003c-S1 <400> 21 tgtggaagag gttcccgcag ttttgcagct tcgagcaact cagctgaagc ttcgtacgc 59 <210> 22 <211> 62 <212> DNA <213> Artificial Sequence <220> WO 99/55907 PCT/EP99/02722 6 <223> Description of Artificial Sequence: pcimei YFR003c-S2 <400> 22 atcttctttg tctacgttcg ttaaagtcaa gatccttctc gcataggcca ctagtggatc tg 62 <210> 23 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YFR027w-S1 <400> 23 aatgaaagct aggaaatcgc agagaaaagc gggcagtaaa cagctgaagc ttcgtacgc 59 <210> 24 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YFR027w-S2 <400> 24 aatttggttg cgatacccaa cttccttgct gtcctgcaca gcataggcca ctagtggatc tg 62 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YFR042w-S1 <400> agtttgcacc aatggcaata tgcctgtgat aaagataagg cagctgaagc ttcgtacgc 59 <210> 26 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YFR042w-S2 <400> 26 catggaagtt atttggttgc ttagattcca cgggttcaaa gcataggcca ctagtggatc tg 62 <210> 27 WO 99/55907 PCT/EP99/02722 7 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIL109c-S1 <400> 27 1C tgtctcatca caagaaacgt gtttacccac aagctcagct cagctgaagc ttcgtacgc 59 <210> 28 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML077w-Sl <400> 28 atggggatat attcattttg gatctttgat aggcattgta cagctgaagc ttcgtacgc 59 <210> 29 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIL109c-S2 <400> 29 tcatgatttg taagaattct ctgtaacttt cgttattcaa gcataggcca ctagtggatc tg 62 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIL091c-S1 <400> agtgacagtt ctgtgaggga aaagaatgat aatttccgtg cagctgaagc ttcgtacgc 59 <210> 31 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIL091c-S2 <400> 31 WO 99/55907 PCT/EP99/02722 8 cattgtaaaa ttcaqgattg tttggaggct tataaaaaac gcataggcca ccagtggatc tg 62 <210> 32 2Z <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YILO8 3 c -S <400> 32 acctctaccc gtgctcaaca gacctcaaat tcatacgtct cagctgaagc ttcgtacgc 59 <210> 33 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YILO83c-S2 S <400> 33 cgatgacttc tgggattatc atctcttcaa tgatatggtg gcataggcca ctagtggatc tg 62 <210> 34 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YILO19w-S1 <400> 34 ttcaaagaaa agcttttgaa agtcagttcg gatctttaga cagctgaagc ttcgtacgc 59 <210> <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIL0l9w-S2 <400> tagcgacggg atttcttttt gccattaaat ttaccactcc gcataggcca ctagtggatc tg 62 <210> 36 <211> 59 <212> DNA <213> Artificial Sequence WO 99/55907 PCT/EP99/02722 9 <220> <223> Description of Artificial Sequence: primer YIL104c-S1 <400> 36 tccccttact atttaagatt aagatttcct cacgaattaa cagctgaagc ttcgtacgc 59 <210> 37 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIL104c-S2 <400> 37 cctgatacct gtaatgattg caatcttgat gagagagctg gcataggcca ctagtggatc tg 62 <210> 38 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIR010w-S1 <400> 38 atgagtctgg aacccacaca aacggtctcc ggtacgccgc cagctgaagc ttcgtacgc 59 <210> 39 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML077w-S2 <400> 39 ttctattggt gatctttctt gtcccttgac ctctcatttc gcataggcca ctagtggatc tg 62 <210> <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIR010w-S2 <400> accttagctc atcgactttt gtttctaact ttgtctcaac gcataggcca ctagtggatc tg 62 WO 99/55907 PCT/EP99/02722 <210> 41 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIR015w-S1 <400> 41 gcgaaccagg accatttcca tagattaaac tacctctacc cagctgaagc ttcgtacgc 59 <210> 42 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YIR015w-S2 <400> 42 atttccagtt tttttggggt ccataacaac cgatggcatt gcataggcca ctagtggatc tg 62 <210> 43 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL233w-S1 <400> 43 atgtcacaag gtcagtccaa aaaactggac gtaactgttg cagctgaagc ttcgtacgc 59 <210> 44 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL233w-S2 <400> 44 caatcctcct ccaggaagtc cattaagcgc ttgacctttt gcataggcca ctagtggatc tg 62 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer WO 99/55907 PCT/EP99/02722 11 YPL146c-S1 <400> tccaactaat ctaaccaaga aaccatctca atacaaacag cagctgaagc ttcgtacgc 59 <210> 46 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL146c-S2 <400> 46 tttgaagtcc ttatgtgtcc acttttcagt gattttctgc gcataggcca ctagtggatc tg 62 <210> 47 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL126w-S1 <400> 47 atgacgcaat ccctaggtat cgaacagtat aaactgtcag cagctgaagc ttcgtacgc 59 <210> 48 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL126w-S2 <400> 48 tatgttaata ctttcatcac acgatcaaaa aatgtatcca gcataggcca ctagtggatc tg 62 <210> 49 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL093w-S1 <400> 49 caagattaca agaatcagag cgttctatat gcgtaaagtt cagctgaagc ttcgtacgc 59 <210> <211> 59 <212> DNA WO 99/55907 PCT/EP99/02722 12 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML093w-Sl <400> gctaacttaa atatggcaaa aaagaaatct aagagcagat cagctgaagc ttcgtacgc 59 <210> 51 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL093w-S2 <400> 51 cggaaatctg tcttaccgac accacgctta ccactgaata gcataggcca ctagtggatc tg 62 <210> 52 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL063w-S1 <400> 52 ttcaagcatc cagaagactt ttaacaagtt attcaagttt cagctgaagc ttcgtacgc 59 <210> 53 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL063w-S2 <400> 53 ggattcagca atcttcttct ttttcttttc ctcttcaaat gcataggcca ctagtggatc tg 62 <210> 54 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL024w-S1 <400> 54 atgtcttttt catctatctt atcacaggat atcacagatg cagctgaagc ttcgtacgc 59 WO 99/55907 PCT/EP99/02722 13 <210> <211> 62 <212> DNA <213> Artificial Sequence ;220> <223> Description of Artificial Sequence: primer YPL024w-S2 <400> acttgtgagt ccttcaatat gaaaacgccc ctattgaaca gcataggcca ctagtggatc tg 62 <210> 56 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL020c-S1 <400> 56 tcagttgaag tagataagca ccggaacaca ctacagtatc cagctgaagc ttcgtacgc 59 <210> 57 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL020c-S2 <400> 57 tcggttaaaa tcaaatgggc aataaatctt ctcatcctaa gcataggcca ctagtggatc tg 62 <210> 58 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPL012w-S1 <400> 58 atggatcaag acaaagttgc ttttctttta gagctggagg cagctgaagc ttcgtacgc 59 <210> 59 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer WO 99/55907 PCT/EP99/02722 14 YPLO12w-S2 <400> 59 atttgaactt tggacctttc ttattatgtt tgccaatctt gcataggcca ctagtggatc tg 62 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPLOO7c-S1 <400> cccgttgttt gctcacggga catatcttta catgcgttgt cagctgaagc ttcgtacgc 59 <210> 61 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMLO93w-S2 <400> 61 caaaggatca ataacttggc ctggcttagt catgattctc gcataggcca ctagtggatc tg 62 <210> 62 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPLOO7c-S2 <400> 62 ggacttattg gtagatagaa aggaatttga ggattggaag gcataggcca ctagtggatc tg 62 210> 63 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPRO4 8w- SI <400> 63 atgtcatcga gcaagaaaat cgtcatcctc tatggatcgg cagctgaagc ttcgtacgc 59 <210> 64 WO 99/55907 PCT/EP99/02722 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR048w-S2 <400> 64 LO aattagttat ttcctcacct aatctccata agtagtcttg gcataggcca ctagtggatc tg 62 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR072w-S1 <400> tgtctcaaag aaagctacaa caggatatcg ataagctttt cagctgaagc ttcgtacgc 59 <210> 66 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR072w-S2 <400> 66 agatttgccc attcctggtg gtaacttttc gatttcttta gcataggcca ctagtggatc tg 62 <210> 67 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR082c-S1 <400> 67 cttcgattgc tgaaagagta aggaactttg cagttattta cagctgaagc ttcgtacgc 59 <210> 68 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR082c-S2 WO 99/55907 PCT/EP99/02722 16 <400> 68 caataaagtt caacttgttg ttgttccctg taccaaaatc gcataggcca ctagtggatc tg 62 <210> 69 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR085c-S1 <400> 69 ctgtacattc tttcgaaaga ctccatgctg cgaatttttg cagctgaagc ttcgtacgc 59 <210> <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR085c-S2 "5 <400> tcccacttta tagttatggg atttcgagct ggattcggta gcataggcca ctagtggatc tg 62 <210> 71 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML114c-S1 <400> 71 aacgtgtaat tgagggactc ataaagggca atgacttcca cagctgaagc ttcgtacgc 59 <210> 72 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR105c-S1 <400> 72 agctcgatca tcgagggcca attgtctaaa aatctagcaa cagctgaagc ttcgtacgc 59 <210> 73 <211> 62 <212> DNA <213> Artificial Sequence WO 99/55907 PCT/EP99/02722 17 <:220> <223> Description of Artificial Sequence: primer YPR1O5c-S2 <400> 73 ctgtgttcta tcaatcttca tatttctagc tttaattctt gcataggcca ctagtggatc tg 62 <210> 74 <211> 59 <212> DNA <213> Artificial Sequence <220> l~<223> Description of Artificial Sequence: primer YPR112 c-Si <400> 74 cattgtcaag ggtttgcccg tctatctaac agatgataat cagctgaagc ttcgtacgc 59 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPRl37c-S1 <400> atgtcagatg ttacccaaca gaaaaagagg aaaagatcca cagctgaagc ttcgtacgc 59 <210> 76 <211> 62 <212> DNA <213> Artificial Sequence <220> .<223> Description of Artificial Sequence: primer YPR112c-S2 <400> 76 gaaaccttcg ttttcttcat catccacatc cagtttcttt gcataggcca ctagtggatc tg 62 <210> 77 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR143w-Sl <400> 77 atgggctcca agcacagagt agacactaag gataagaaaa cagctgaagc ttcgtacgc 59 <210> 78 WO 99/55907 PCT/EP99/02722 18 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR143w-S2 <400> 78 ttcattgtcg cttcctgcgg cagctttaac taaatccaaa gcataggcca ctagtggatc tg 62 <210> 79 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR144c-S1 <400> 79 ttccagaaaa tgttactcaa ttggaagaag atgagacaga cagctgaagc ttcgtacgc 59 <210> <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YPR144c-S2 <400> ccatgctacc ccaggcaagt agacgttacc ttgggatgac gcataggcca ctagtggatc tg 62 <210> 81 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML114c-S2 <400> 81 gacttgtagt agcatcgata ttggttgtgt tatgtgctac gcataggcca ctagtggatc tg 62 <210> 82 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer WO 99/55907 PCT/EP99/02722 19 YPR16 9w-Sl <400> 82 ttttacatcc tgaactgccc attataataa ctggctttgg cagctgaagc ttcgtacgc 59 <210> 83 <211> 62 <212> DNA <213> Artificial Sequence 220> <223> Description of Artificial Sequence: primer YPRl69w-S2 <400> 83 cttcttgatc ccatgctcat acaggtcctt ttttttgttg gcataggcca ctagtggatc tg 62 <210> 84 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML127w-Sl <400> 84 ccgctaaatg gtactccagt aagcgaggca cccgccacaa cagctgaagc ttcgtacgc 59 <210> <211> 62 <212> DNA <213> Artificial Sequence 220> <223> Description of Artificial Sequence: primer YML12 7w -S2 <400> ataaccccga cgtgttttcc atgtattcag acaatgctaa gcataggcca ctagtggatc tg 62 <210> 86 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMRO 32w-Si <400> 86 ctacagttat gaagcttgtt tttgggaccc aaacgacaat cagctgaagc ttcgtacgc 59 <210> 87 <211> 62 <212> DNA WO 99/55907 PCT/EP99/02722 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR032w-S2 <400> 87 <210> <211> <212> <213> <220> <223> 88 59

DNA

Artificial Sequence Description of Artificial Sequence: primer YMR093w-S1 <400> 88 <210> <211> <212> <213> <220> <223> 89 62

DNA

Artificial Sequence Description of Artificial Sequence: primer YMR093w-S2 <400> 89 <210> <211> <212> <213> <220> <223> 59

DNA

Artificial Sequence Description of Artificial Sequence: primer YMR131c-S1 <400> <210> <211> <212> <213> <220> <223> 91 62

DNA

Artificial Sequence Description of Artificial Sequence: primer YMR131c-S2 <400> 91 <210> <211> <212> <213> 92 59

DNA

Artificial Sequence <220> WO 99/55907 PCT/EP99/02722 21 <223> Description of Artificial Sequence: primer YMR185w-S1 <400> 92 atcaacatac acgatatatt gaatacaaga ccgaagctca cagctgaagc ttcgtacgc 59 <210> 93 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR185w-S2 <400> 93 gtaatgggtt ataaactatc tagtacggtt aaaagcttgt gcataggcca ctagtggatc tg 62 <210> 94 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR212c-S1 <400> 94 cctcttgaac ttaaagaatg taaatcttca tttgcgtctt cagctgaagc ttcgtacgc 59 <210> <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR499w-S1 <400> <210> 96 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR212c-S2 <400> 96 cggatgatgt tcacaccaaa acatcagaaa ctggtcaatc gcataggcca ctagtggatc tg 62 <210> 97 <211> 59 <212> DNA WO 99/55907 PCT/EP99/02722 22 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR213w-SI <400> 97 atacgtgaaa ggcggtgtat ggaccaatgt ggaggatcag cagctgaagc ttcgtacgc 59 <210> 98 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR2l3w-S2 <400> 98 gctgtaactg ttcaatagac tccacttttg attggatcga gcataggcca ctagtggatc tg 62 <210> 99 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR218c-Sl <400> 99 gactcaaatg cattagagtg atcaactcta caacttttac cagctgaagc ttcgtacgc 59 <210> 100 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR218c-S2 <400> 100 gaaggcattt gacggaactg tacgaacggt taacaggctt gcataggcca ctagtggatc tg 62 <210> 101 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR472w-Sl <400> 101' atgtc tcaaagaata attcaaccaa gcgcatctga ccaaccagct gaagcttcgt WO 99/55907 PCT/EP99/02722 23 acgc 64 <210> 102 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YML023c-S2 <400> 102 attagttact tattctataa ttacactttt atcatgaacg gcataggcca ctagtggatc tg 62 <210> 103 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR222c-S2 <400> 103 aatatgtaac tttgttcaac taagttatca acccttgtga gcataggcca ctagtggatc tg 62 <210> 104 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR243w-S2 <400> 104 gataatatgg tttctatact gtcaggatta ttagattcca gcataggcca ctagtggatc tg 62 <210> 105 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR272c-S2 <400> 105 ggtaactgac ttcgttactt tatgagatgt ccggctttag gcataggcca ctagtggatc tg 62 <210> 106 <211> 59 <212> DNA <213> Artificial Sequence WO 99/55907 PCT/EP99/02722 24 <220> <:223> Description of Artificial Sequence: primer YML 4 9c -Si ':400> 106 aattcctgct cattcaagga aagtctcagg aaattttcac cagctgaagc ttcgtacgc S9 <210> 107 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR3 17w- S2 <400> 107 gaagtaaact aactagtaaa gtaggctaat tcgaaacgat gcataggcca ctagtggatc tg 62 <210> 108 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYPR143w-Sl <400> 108 aaaagcctgt ttggtcaatg acagctgaat atataccatt gcataggcca ctagtggatc tg 62 <210> 109 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR472w- S2 <400> 109 agccaaatct caaaccttcc ctgtcaagca cttgcctgtc gcataggcca ctagtggatg tg 62 <210> 110 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR281w-S1 <400> 110 ctgaagaaaa gttaaatgaa gatgttgagg cgtacaaagg cagctgaagc ttcgtacgc 59 WO 99/55907 PCT/EP99/02722 <210> 111 <211> 62 <212> DNA J <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYMR281w-S2 <400> 111 agtacgtatt gtgcatgtgt attcataagt gaaagcttgt gcataggcca ctagtggatc tg 62 <210> 112 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR288w-S1 <400> 112 gaaaacctgc agaaagaagc tgcacgtatt ggtgagaacg cagctgaagc ttcgtacgc 59 <210> 113 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYMR288w-S2 <400> 113 ccaaaccttc taaaatacgc ataatagcat gtggtgaagt gcataggcca ctagtggatc tg 62 <210> 114 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYMR290c-S1 <400> 114 tgagttttac gtcttttggt atttggcgtt tttccactgg cagctgaagc ttcgtacgc 59 <210> 115 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer WO 99/55907 PCT/EP99/02722 26 YDR499W-S2 <400> 115 cgtactttac ttgcattatt ctccccgttc ttttattcaa gcataggcca ctagtggatg S tg 62 <210> 116 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR29Oc-S2 <400> 116 gataagctga gcaatattaa caggagaagt atggctaccc gcataggcca ctagtggatc tg 62 ~0 <210> 117 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMR2llw-Sl <400> 117 agagagcaaa ccatttgact actcaattct tcaatataca cagctgaagc ttcgtacgc 59 <210> 118 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYMR2llw-S2 <400> 118 atttcaatca tcttactccg tgaatcaggt tcggaatgat gcataggcca ctagtggatc tg 62 <210> 119 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR1 96 c-Sl <400> 119 atgcttatga tcaaattgtg ttatacttca aggacaaaat cagctgaagc ttcgtacgc 59 <210> 120 <211> 62 WO 99/55907 PCT/EP99/02722 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR196c-S2 <400> 120 tttcaatctg ttcgtataag tcaaccaatg tgctgttatt gcataggcca ctagtggatc tg 62 <210> 121 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR299w-S1 <400> 121 atggaaaaat cactagcgga tcaaatttcc gatatcgcca cagctgaagc ttcgtacgc 59 <210> 122 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR299w-S2 <400> 122 caaagatttg gatatcatcg tttttaacag cctctaattc gcataggcca ctagtggatc tg 62 <210> 123 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR365c-S1 <400> 123 ctggagagaa cccaaagaag gaaggtgtag atgctaggtt cagctgaagc ttcgtacgc 59 <210> 124 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR365c-S2 WO 99/55907 PCT/EP99/02722 28 <400> 124 ttagtatgct ttttattaac agatttcaac ttgcttttct gcataggcca ctagtggatc tg 62 <210> 125 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR396w-S1 <400> 125 cagatacact attgtggtgt aatctggacc ttgactgtct cagctgaagc ttcgtacgc 59 <210> 126 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMRO49c-S1 <400> 126 cagactattg attactttat gaccggttag tttctttagt cagctgaagc ttcgtacgc 59 <210> 127 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR396w-S2 <400> 127 tagagaaaac actgaatgat cttagcgacc gtacaaaaga gcataggcca ctagtggatc tg 62 <210> 128 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR47c-S1 <400> 128 ttcttaagca tttcccaagc tatgttggcc catctaagat cagctgaagc ttcgtacgc 59 <210> 129 <211> 62 <212> DNA <213> Artificial Sequence WO 99/55907 PCT/EP99/02722 29 <220> <223> Description of Artificial Sequence: primer YDR4O7c-S2 <400> 129 aataacagac aagataacgt tttcagagtc gaactggatt gcataggcca ctagtggatc tg 62 <210> 130 ~0 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR4 16w-Si <400> 130 acttacatgg aaaagatata tcgagtattg gaaagaggag cagctgaagc ttcgtacgc 59 2 0 <210> 131 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR416w-S2 <400> 131 tcaaatatct agttctattt catctggatt aatcgaatat gcataggcca ctagtggatc tg 62 <210> 132 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR449c-S1 <400> 132 cacatcaccg atttctaata atgtcgaaga caagatacta cagctgaagc ttcgtacgc 59 <210> 133 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR449c-S2 <400> 133 ataattaaat ctagaatttt atacctagga tcatcttctg gcataggcca ctagtggatc tg 62 WO 99/55907 PCT/EP99/02722 <210> 134 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR141c-Sl <400> 134 ttcgtaatct ttgaattctg cgatttcatc taccagcgcg cagctgaagc ttcgtacgc 59 <210> 135 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR141c-S2 <400> 135 cactaaagcc ccttacaatt gactcaaata ataaacaact gcataggcca ctagtggatc tg 62 <210> 136 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR324c-S1 <400> 136 aagaagcctg aaaatacgaa acaaaccggt gaagatgacc cagctgaagc ttcgtacgc 59 <210> 137 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYMR049c-S2 <400> 137 tctgttctaa cataactagg tcaatgatgg ctaagaacaa gcataggcca ctagtcgatc tg 62 <210> 138 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR324c-S2 WO 99/55907 PCT/EP99/02722 31 <400> 138 aaacactaac tttggttgaa taaacgcctt ttgtttggag gcataggcca ctagtggatc tg 62

DJ

<210> 139 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR325w-Sl <400> 139 gacattaata cgaaaatctt taactcagtt gctgaagtat cagctgaagc ttcgtacgc 59 <210> 140 <211> 62 <212> DNA <2131> Artificial Sequence <220> <223> Description of Artificial Sequence: prinerYDR325w-S2 <400> 140 acctcgctga aagactctga atccttatct tcttcatcta gcataggcca ctagtggatc tg 62 <210> 141 <211> 59 <212> DNA i~<213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR3 98w-Si <400> 141 atggattctc ctgttctaca gtccgcttat gacccatcag cagctgaagc ttcgtacgc 59 <210> 142 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR398w-S2 <400> 142 aacgtcacta tatccggctt cctcctcgcc gtcgctctgc gcataggcca ctagtggatc tg 62 <210> 143 <211> 59 WO 99/55907 PCT/EP99/02722 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR246w-S1 <400> 143 atggccatcg aaacaatact tgtaataaac aaatcaggcg cagctgaagc ttcgtacgc 59 <210> 144 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR246w-S2 <400> 144 aacaggttag atcttatagg catttccatt gagtaagatg gcataggcca ctagtggatc tg 62 <210> 145 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR236c-S1 <400> 145 ctaaaatatt gaacttgacc ctggccccat aaaaatcatt cagctgaagc ttcgtacgc 59 <210> 146 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR236c-S2 <400> 146 ttgaagtgtt gatgtttacg tggactattt atgtttcgtt gcataggcca ctagtggatc tg 62 <210> 147 <211> 62 <212> DNA <213> Artificial Sequence <226> <223> Description of Artificial Sequence: primerYDR361c-S2 <400> 147 WO 99/55907 PCT/EP99/02722 33 ttaccaagtg gaaatttctg tttccaattc atcgatacct gcataggcca ctagtggatc tg 62 <210> 148 <211> 59 <212> DNA <213> Artificial Sequence <220> i0 <223> Description of Artificial Sequence: primer YMR134w-S1 <400> 148 gcaaagtgtg gtatagaaaa agaaccaaag gccggtatgt cagctgaagc ttcgtacgc 59 <210> 149 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR361c-S1 <400> 149 ggttcaagct atcaaattaa atgatttaaa aaataggaag cagctgaagc ttcgtacgc 59 <210> 150 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR367w-S1 <400> 150 atctgcgtac tttatacaat cgataccatt tccacttgtt cagctgaagc ttcgtacgc 59 <210> 151 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR367w-S2 <400> 151 gttttgttct acgtcatccc tatcaactaa atatttgggg gcataggcca ctagtggatc tg 62 <210> 152 <211> 59 <212> DNA <213> Artificial Sequence <220> WO 99/55907 PCT/EP99/02722 34 <223> Description of Artificial Sequence: primerYDR339c-Sl <400> 152 gctaagaaaa caagaaagtt tggcctcgta cagctgaagc ttcgtacgc 59 <210> 153 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR339c-S2 1s 400> 153 taaaagacat ctggcaattt ttcaatgacg tatgcgtgac gcataggcca ctagtggatc tg 62 <210> 154 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR413c-Sl <400> 154 ttctttggtt tattcttcgt tcatttttgg tcaaatatct cagctgaagc ttcgtacgc 59 <210> 155 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR413c-S2 <400> 155 acaaaagaaa gcacaagagt ttattaagga gcaggaaagg gcataggcca ctagtggatc tg 62 <210> 156 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR429c-S1 <400> 156 tctagatcta tcattacata caagattgaa gacggtgtca cagctgaagc ttcgtacgc 59 <210> 157 <211> 62 WO 99/55907 PCT/EP99/02722 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR429c-S2 <400> 157 tttctttgtt tctaacgaca gaaactcttg gaatgggtgc gcataggcca ctagtggatc tg 62 <210> 158 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR468c-S1 <400> 158 gtcacaatac tgctggtgat gacgatcaag aggaggaaat cagctgaagc ttcgtacgc 59 <210> 159 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYMR134w-S2 <400> 159 tgtgtgtgtg cctacctgca tgtatgcatt tagcaattga gcataggcca ctagtggatc tg 62 <210> 160 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR468c-S2 <400> 160 caagacgaca ataagaagtc ctatacaaca atcgtcgtat gcataggcca ctagtggatc tg 62 <210> 161 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR489w-S1 WO 99/55907 PCT/EP99/02722 36 <400> 161 actacccaca gagatgcaaa tacaatagtg ggttcgtcct cagctgaagc ttcgtacgc 59 <210> 162 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR489w-S2 <400> 162 atctatcatg tttacgctac cttctgtatc gcataggcca ctagtggatc tg 62 <210> 163 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR527w-S1 <400> 163 atggacttac tgggcgatat agtggagaaa gatacatctg cagctgaagc ttcgtacgc 59 <210> 164 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR527w-S2 <400> 164 ccccaccgcc ttgtttccat aaccaaagtg catcaatagc gcataggcca ctagtggatc tg 62 <210> 165 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR288w-S1 <400> 165 atgagttcta tagataatga cagcgatgtg gatttaacag cagctgaagc ttcgtacgc 59 <210> 166 <211> 62 <212> DNA <213> Artificial Sequence WO 99/55907 PCT/EP99/02722 37 <220> <223> Description of Artificial Sequence: primer YDR288w-S2 <400> 166 gcccatgatt tcttgcacca atttttcaag agactctagt gcataggcca ctagtggatc tg 62 <210> 167 <211> 59 <212> DNA <213> Artificial Sequence 1: <220> <223> Description of Artificial Sequence: primer YDR2 01w-Si <400> 167 cccatgtctg gactattcag agcatcatcg tcatccatac cagctgaagc ttcgtacgc 59 <210> 168 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR2O1w-S2 <400> 168 aaaagggttt tccgtttagt tcccgaatat gatgttgaaa gcataggcca ctagtggatc tg 62 <210> 169 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR434w-S1 <400> 169 atgtccaatg caaatctaag aaaatgggtt ggtttttgct cagctgaagc ttcgtacgc 59 <210> 170 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YMLO2 3c-Si <400> 170 cacgcaatgg tgcacattat tttgttgaac tcactgagaa cagctgaagc ttcgtacgc 59 WO 99/55907 PCT/EP99/02722 38 <210> 171 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR434w-S2 <400> 171 taaaggtaaa tacacagcta tcatgtgctc ttgtgggaag gcataggcca ctagtggatc tg 62 <210> 172 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR181c-S1 <400> 172 aggataaacc caaatgctgg acatctaagg aaatctaagt cagctgaagc ttcgtacgc 59 <210> 173 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR181c-S2 <400> 173 tagttgggtt tgaatcgtta tcacgggaga acattgcttt gcataggcca ctagtggatc tg 62 <210> 174 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YDR531w-S1 <400> 174 atgccgcgaa ttactcaaga gatatcttac aattgcgatt cagctgaagc ttcgtacgc 59 <210> 175 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYDR531w-S2 WO 99/55907 PCT/EP99102722 39 <400> 175 aaataagcta tttgcccaat attgttggag atggcgaata gcataggcca ctagtggatc tg 62 <210> 176 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYLRl86w-S1 <400> 176 ctagtcacca agaagaaaac ccgtaaaatc gtaggtcatg cagctgaagc ttcgtacgc 59 <210> 177 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer YLR186w-S2 <400> 177 atacaaagag gatgccaagt agacttaaac actataaaat gcataggcca ctagtggatc tg 62 <210> 178 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYLR2l5c-S1 <400> 178 ttacttattg atgtcctcac aagaatatac aacttttata cagctgaagc ttcgtacgc 59 <210> 179 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primerYLR2l5c-S2 <400> 179 agctctcgga ttgcttcagg atttaaacta gcttctacga gcataggcca ctagtggatc tg 62 <210> 180 <211> 59 <212> DNA WO 99/55907 PCTIEP99/02722 '213> Artificial Sequence <220> <~223> Description of Artificial sequence: primerYLR222c-Sl '<400> 180 ctctcaacgg tagtaagcca tactacgtac aatatggatc cagctgaagc ttcgtacgc 59

Claims (10)

1. A method for the screening of antimycotic substances wherein an essential gene from mycetes or a similar mycete gene having a sequence identity, at the nucleotide level, of at least 50%, more preferably of at least 60% and most preferably of at least with the essential gene, or the corresponding encoded protein, is used as target and wherein the essential gene is YIL019w as herein described.

2. The method of Claim 1, wherein mycete cells which express the essential gene, or the said similar mycete gene, to a different level are incubated with the substance to be tested and the growth inhibiting effect of the substance is determined.

3. The method of Claim 1, wherein said target gene or the corresponding target encoded protein is contacted in vitro with the substance to be tested and the effect of the substance on the target is determined.

4. The method according to any one of Claims 1 to 3, wherein the screened substances partially or totally inhibit the functional expression of the essential gene or the functional activity of the encoded protein. The method according to any one of Claims 1 to 4, wherein the mycete species are selected from the group comprising Basidiomycetes, Ascomycetes and Hyphomycetes.

6. The method according to any one of Claims 1 to 5, wherein said similar 20 genes are essential genes from Candida Spp, or Aspergillus Spp.

7. The method according to Claim 6, wherein said similar genes are essential genes from Candida albicans, or Aspergillus fumigatus.

8. The method according to any one of Claims 1 to 7, wherein the said similar genes are identified by: a) providing a S.cerevisiae mutant strain in which the gene of S.cerevisiae to be investigated is either integrative or extrachromosomal under the control of a regulated promoter, b) culturing said mutant strain under growth conditions in which the regulated promoter is active, 30 c) transforming the mutant strain with cDNA or genomic DNA that has been prepared from the mycete-species to investigate and that has been integrated into an appropriate vector, d) altering the culture condition, so that the regulated promoter is %y-1,switched off and only S.cerevisiae cells which contain the said similar gene can survive, 06/02/03,mcl 1623.claims,42 e) isolating and analyzing the cDNA or genomic DNA.

9. The method according to Claim 8, wherein the said similar gene encodes a protein having a sequence identity, at the amino-acid level, with the corresponding S.cerevisiae essential gene encoded protein of at least 40%, preferably of at least more preferably of at least 60% and most preferably of at least The method according to any one of Claims 1 to 9, wherein said mycete cells are haploid S.cerevisiae cells.

11. The method according to any one of Claims 1 to 4 or 10, wherein the essential genes of S.cerevisiae are identified by integration through homologous recombination of a selection marker at the locus of the gene to be studied.

12. The method according to Claim 1 substantially as hereinbefore described with reference to any one of the Examples herein. DATED this 6 th day of February, 2003 HOECHST MARION ROUSSEL By Their Patent Attorneys: CALLINAN LAWRIE 06/02/03,mcl 1623.claims,43