CA2191067A1 - Dna encoding gls1 - Google Patents

Abstract

The DNA encoding glucan synthesis gene 1 (GLS1) is cloned and used in an in vitro assay to screen for compounds that modulate 1,3.beta.-D glucan synthase activity.

Description

2~91~67 ~W095/32982 r_-,.x,,.r-~7 TITLE OF T.T~E .TNVENTION
DNA ENCOD.TNG GLS l CROSS RELATED TO OTHER APPLICATIONS
This is a contin~ ion of U.S. Serial Number 08/249,420 filed May 26, 1994, now pending.
BACKGROUND OF T.T-T.E INVENTION
A DNA molecule containing a gene which reverses a mutant phenotype of a strain of Saccharomyces cerevisiae is isolated and purified. The gene is GLSI (glucan _ynthesis gene 1). GLSI
encodes a subunit of 1,3-13-D glucan synthase. The protein enco~ed by GLSI lc~lc~c~ a target for drug therapy for fungal disease. Tlle invention includes homologues of GLS I is isolated from other fu.ngi, 5 such as Aspergillus fumigatus, Candida albicans, Schizosaccharn~omyces pombe and Phytophthora infestans.
Understanding the mode of action of ll,e-~t;-llic compounds requires a variety of experimental approaches. One approach involves the isolation of organisms resistant or sensitive to test 20 compounds. Such Ol~;~li~lllS may be used to isolate genes encoding the drug targets.
The fungal cell wall is a complex structure composed of a number of polymers: chitin, a- and ,~-glucans, and mannoproteins. The fungal cell wall is involved in a variety of vital cellular processes:
25 vegetative growth, morphogenesis, uptake and secretion of macromolecules and protection against osmotic changes are affected by changes in the composition and integrity of the cell wall. Antifungal compounds which act via the inhibition of cell wall synthesis (a Rrocess essential to fungi and absent from m~mm~ n cells) may have high 30 fungicidal activity and low toxicity to m~mm~ n cells.
One class of ~-glucan inhibitors is comprised of lipopeptide antibiotics such as aculeacin A, echinocandin B and the pneumocandins.
These compounds are cyclic hexapeptides that contain a non-polar fatty acid side chain. Echinocandins are fungicidal because they inhibit 2191l~6,7 WO 95132982 , , r~ r~ '''7 ~

synthesis of 1,3-,~-D glucan, which disrupts the integrity of the cell wall and causes lysis of yeast cells. In vitro erhinnr:~n(lin~ inhibit polymerization of glucose into 1,3-,B-D glucan.
Another class of ,B-glucan synthesis inhibitors comprises the 5 pap~ r~n~iins and rh~ti~lr~n~in These compounds contain a glycoside component connected to an aromatic ring system and two long chain fatty acids. These compounds have the same mode of actron as the erhinnr~n-1ins It has been shown that Pneumocystis carinii has ~-glucan in the wall of its cyst form (Matsumoto, Y., et al., 1989, J. Protozool. 36:21S-22S). Inhibitors of ,~-glucan synthesis, such as p~rlll~n~nflins and echinocandins, may be useful in the treatment of P.
carinii infections. In a rat model of P. carinii pneumonia, L-671,329 (an echinocandin) arld L-687,781 (a papulacandim) were both effective 15 in reducing the number of cysts in the lungs of infected rats (D.M.
Schmatz et al., l990, PNAS 87:5950-5954). These results suggest that ~-glucan synthesis is a target for the i-l~ntifir~tion of lI-ela~,eulics useful in the treatment of P. carinii infections.
There have been a number of efforts to isolate drug-20 resistant yeast strains affected in ,~-glucan synthesis. The mutants that have been isolated include acul (Mason, M.M., et al., 1989, Cold Spring Harbor Laboratory, Abstract # 154), and papl (Duran, A., et al., 1992, Profiles in Biotechnology (T.G. Villa and J. Abalde, Eds.) Serivicio de Publicaciones, Universidad de Santiago, Spain. pp.
25 221-232).
In the present work a more potent erhinoc~ndin (L-733,560) was used as a selective agent to isolate mutant strains specifically affected in glucan synthesis. One mutant (strain MS14) is echinocandin-resistant and is also supersensitive to the chitin synthase 30 inhibitor nikkomycin Z. The mutation in MS14 maps to the F~KSI gene and is de~ig~n~t~d fks l -4. Another mutant (strain MS I ) is resistant to echinocandins and ~u~ lsi~ive to both p~rlll~nt1in and rapamycin.
Strain MS I was used to clone the GLS I gene.

219106~
~ WO 95132982 PCT/IJS951065S7

- 3 -SU~MARY OF THE INVENTION
A DNA molecule encoding a protein involved in biosynthesis of 1,3-~-D glucan (GLSI) is identified, cloned, expressed and used in assays to screen for antifungal compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Segregation pattern (2:2) of meiotic product~
infiir~ting single gene mutations conferring resistant to echinocandins.
Cells representing the 4 meiotic products of tetrads resulting from a crosses between MSl and the wild-type strain GG100-14D were spotted on media containing 7.5 ~LM of L,733-560. Following growth at 28C
for two days, two of four segregants were able to grow in~ic~fin~ the 2:2 segregation of the drug-resistance phenotype.
Figure 2. Straim MSI (glsl-l mutant) was tested for 15 ~ L~ e to the echinocandin L-733,560 by the broth microdilution assay.
Figure 3. Morphological defects in strain MS1 (glsl-l mutant) showing aggregates of cells (A) tbat start to Iyse towards tbe center of the aggregate (B) before all the cells in the 20 aggregate Iyse (C&D).
Figure 4. Effect of L-733,560 and Nikkomycin Z on 1,3-~-D glucan synthesis and chitin synthesis. Membrane extracts prepared from X2180-lA (wild-type) and from MSI (glsl-l mutant) were used to catalyze 1,3-~B-glucan synthase reactions (A) and chitin 2s synthase reactions (B). UDP-glucose (A) and N-acetyl~l--r~ minl~ (B) were used as substrates.
Figure 5. Effect of GTP~S on 1,3-~-glucan synthase activity. Membrane extracts from wild-type and mutant strains were used to prime 1,3-,~-glucan synthase reactions in the presence of increasing concentrations of GTP~S.
Figure 6. Localization of the glsl-l minimllm compl~m~ntin~ fragment. A partial restriction map of the 17-kb clone cr,nt~inin~ glsl-l compl~m~ntin~ activity is depicted (A). The direction of transcription of GLSI is indicated by the arrow. The yeast genomic W0 95/32g82 F ~ .'C -''7 ~

DNA fragments ~ s~llL~d by the lines below the restriction map (B) were inserted into the centromeric plasmid YCP50. The recombinant plasmids were transformed into MSI (glsl-l mutant). Transformed cells were tested for complementation of the echinoc~n-lin-resistance 5 phenotype. The plasmids as represented from the top to the bottom are pJAC2, pHF, pEF. Abbreviations for restriction enzymes: D, DpnI; E, EcoRI; H, HindIII; K, KpnI.
Figure 7. GLS1 mediates sensitivity to el hint-ç~n(lin~
Figure R. Hydropathy plot of the 348 amino acid GLSI
gene product.
Figure 9. Nucleotide and predicted amino acid sequences of the S. cerevisiae GLSI.
Figure 10. GLSl complements the papulacandin- and the y~ l-sllrc.~ ivi~y phenotypes of the glsl-1 mutation.
5 (A) Effect of rapamycin on cells carrying the glsl-l mutation.
(B) Effect of papulacandin B on the same mutant cells. The mutant cells transformed with GLSI on a plasmid reverse sensitivity to both drugs.
Figure 11. Southern hybridization of genomic DNA. To 20 test whether homologues of the cloned GLSI exist in other fungi, DNA
probes from the GLS1 gene of S. cerevisiae were hybridized to genomic DNA from several heterologous species. The existence of GLSI
homologues in several species, including Candida albicans, Aspergillus fumigatus, Pneumocysts carinii and Schizosaccharomyces pombe is 25 shown.
DETAILED DESCRIPTION OF THE INVENTION
A DNA molecule encoding a protein involved in biosynthesis of 1,3-,~-D glucan (GLSl) is i(l~n~ifi~-l cloned, expressed 3 and used in assays to screen for antifungal compounds.
Antifungal compounds are used for treatment of fungal infections in animals, including humans. There is increasing demand for safer and more effective antifungal compounds. Because the structure of the fungal cell wall differs from the structure of ~.. _ :.. _ . : _. : . .... . , . . _.

2Iqla~7 ,~ Wo 95/32~82 r~ 7 S
m~mm~ n cell membranes, compounds that specifically interfele with the m~inf~n~nce or biosynthesis of the fungal cell wall are targets of drug screens.
Cell wall biosynthesis is filn~mrnt~l to the ~ l,r~
5 and growth of fungi and plants. Cell walls provide skeletal support and merh~nir~l protection of the protoplasts from the environment.
Functions such as the selective uptake of macromolecules, osmoreglll~finn~ cell growth and cell division occur in the cell wall.
Enzymatic activities related to hydrolysis of extracellular nutrieots and o turnover of the cell wall macromolecules during morphogenesis are associated with the extracellular matrix.
Polysaccharides account for as much as 80-90% of the cell wall of S. cerevisiae. The major cell wall polymers are glucan and marman; in addition, small amounts of chitin are present(Cabib, E.
15 1991, Antimicrob. Agents Chemother. 35:170-173). It is believed that glucan supports and ~"-;"l~i"s rigidity of the cell wall while mannoproteins regulate its permeability (Zlotnik et al., 1984).
Three types of glucan account for 30-60% of the cell wall of S. cerevisiae (Fleet, G. H., 1985, p. 24-56. In M. R. McGinnis (ed 20 Current Topics in Medical Mycology Vol. 1. Springer, Verlag, New York). The major form of glucan (60% of the total) is insoluble in alkali or acetic acid, is a branched 1-3-~ polymer, has fibrillar structures containing 3% of 1-6-,B interchain linkages, and lacks 1-6-~interresidue linkages. A second form of glucan (32% of the total) is 25 soluble in dilute alkali, has an amorphous structure and contains mainly 1-3-,B linkages with some 1-6-,B linkages. A minor form of glucan (8%
of the total) is acid-soluble, highly-branched and contains mainly 1-6-~linkages.
FrhinrJc~n~1in~ interfere vrith cell wall biosynthesis, most 30 likely by inhibiting the synthesis of 1,3-,~-glucan. A key enzyme in the cell wall formation is the 1,3-~ glucan synthase. This enzyme is absent from animal cells, making it a target for development of antifuogal compounds. 1,3-,~ glucan synthase is a membrane-associated enzyme that uses UDP-glucose as a substrate and is sfim~ f~d by a detergent-2 1 q ~ Q6~
WO 95/32982 r~ c c- 7 soluble GTP-binding protein (Kang, M.S. and E. Cabib. Proc. Nafl.
Acad. Sci. USA 83:5808-5812).
The ~rhni~ P,c used to isolate drug-resistant mutants are similar to those used to isololate auxotrophic, ~ ,u~ ul~-sensitive, and 5 UV-sensitive mutants, such as described (Sherman ef al., 1986).
The GLSI gene may be isolated from a chromosomal DNA
library by complementation of a mutation (glsl-l) which renders cells resistant to echinocandins (Sherman et al., 1986). The GLSI gene may be isolated from chromosomal DNA by preparing a library of DNA
o fr~mPntc in a DNA cloning vector and screening individual clones for the presence of GLS 1. For example, a library of S. cerevisiae genomic DNA from strain GRF88 in the plasmid YCp50 can be obtained from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, as ATCC 37415.
A plasmid library may be prepared by isolating chromosomal DNA from pure cultures of the microu~ isllls. The chromosomal DNA is fragmented for example, by partial digestion with one or more restriction endonuclease enzymes. The resulting DNA
fragments are separated by size and may then fragments are inserted into a cloning vector.
The cloning vector is cut with at least one restriction endonuclease, treated with phl-Cph~t~cP and the DNA fragments are ligated with a DNA ligase. The cloning vectors are used to transform host cells c--mretPnt for the uptake of DNA. Host cells for cloning, 5 DNA processing, and expression include but are not limited to bacteria, yeast, fungi, insect cells and m~lmm~ n cells. Escherichia coli K-12 strainsRRl,HBlOl,JMlQ9,DHllS,orDH5aareusefulhostcells.
When about 5 x 104 independent genomic DNA fragments are ligated into a cloning vector, a library is formed. A complete library is likely 3 to contain a representation of the entire genome. Competent host cells which take up and stably maintain a recombinant DNA molecule in the transformation procedure can be identified by their ability to grow on medium supplemented with a plasmid-selective drug. For plasmid vectors containing the ampicillin resistance gene, ampicillin is the 095132982 21 9 l 06~ r~,l/lJ~,5.'C'''7 selective drug. To obtain full representation of a library, transformation mixtures are spread on agar plates and incubated under appropriate con(li~if)n.~. Transformed cells are resuspended from the surface of agar plates in a small volume of liquid medium. The cell 5 suspension is used to inoculate a larger volume of liquid medium supplemented with the selective drug, and incubated overnight at 37C.
Plasmid DNA is then extracted from the cells by methods known in the art.
Screens to identify the GLSI gene in the plasmid library can be devised. One strategy requires the use of a glsl-l mutant of S.
cerevisiae, such as strain D2-8B or strain D2-8D. Cells are made competent to take up DNA and then transformed with library DNA.
Transformants bearing the GLSI gene will exhibit a plasmid-dependent increase in sensitivity to a selective echimocandim.
Aliquots of the transformation mixture are plated or selective media. Colonies of tran~ ldllL~ may be collected, resllcp~-n(l~d in liquid medium, pooled, and stored frozen at -80C in medium supplemented with 25% glycerol. The titer, defined as the number of colony forming units per milliliter, is determined by 20 methods known in the art-T~entificq~ion of lldll:~l;)lllldll~; that contain the GLSI gene is accomplished by plating the library onto agar plates containing plasmid-selective medium such that a countable number of colonies grow on each plate. A portion of each colony is transferred to two agar 25 plates by replica plating: one plate contains plasmid-selective medium supplemented with a ~ c~lllldlion of the selective echinocandin which kills the cells with intermediate sensitivity, and a second plate contains plasmid-selective medium only. Positive clones grow normally on the plate without echinocandin but grow poorly or not at all on the 3 echinocandin-containing plate.
The e~hin~ nllin-sensitive phenotype of potential clones may be detected by a variety of tests. In one test, cells from a colony are patched directly onto the surface of plates containing different concentrations of the selective echinocandin. The test is scored after ~9~
wo ssl32ss2 r~ o~-~7 two days of incubation. Cells that grow poorly in the presence of the drug are potential positives and are likely to contain plasmids carryin~
the complementary gene.
In a second test, a portion of each colony is transferred by replica plating to an agar plate containing the selective echimoGandin at a concentration ~ dl~ly twice that used in the first test. Positive clones (clones that are sensitive to echinocandin) do not grow on these plates.
In a third test, cells from a colony are inoculated into o plasmid-selective liquid medium and grown to saturation. An aliquot of the saturated culture is used to inoculate fresh liquid medium supplemented with or without the selective echinocandin. Growth is measured by optical density at a wavelength of 600 nm. Colonies that do not grow in the presence of echinocandin are scored echinocandim-sensitive.
In another test, clones are tested in a broth microdilution assay, wherein a range of ~ lct;~ lions of the selective erhinoc~n(lin are tested. Positive clones are more sensitive to the selective echinocandin than the original resistant mutant.
Tests such as those described above may be used to screen a library of genomic DNA so as to identify a rec;o~,~bi~ l plasmid that contains a functional copy of the GLS1 gene. To ~ t~rrnin~ whether an increase in sensitivity to echinocandin is due to a plasmid-encoded copy of GLS 1, positive clones are cured of plasmid DNA and tested for a decrease in sensitivity to echinocandin. If imcreased echinocandin sensitivity is due to the presence of the plasmid, then plasmid loss results im the loss of this phenotype.
More direct proof that an increase in sensitivity to e~hint-ç~n-lin is due to the presence of a plasmid containing the GLS1 gene maybe obtained by isolating plasmid DNA from a positive clone.
Cells of E. coli competent to take up DNA are transformed with the pla~mid, and transformants are identified and isolated. Plasmid DNA is isolated from the transformed E. coli and then digested with restriction t-n(loml(~ s~.s to yield fragments of discrete sizes. The size of each -WO 9513298~ 2 1 9 1 0 6 7 P~ ~u~ , 5.r7 _ 9 _ fragment is estimated by conventional methods, such as gel electrophoresis. By digesting the plasmid with a variety of enzymes, a cleavage map is generated. The cleavage map is distinct and specific for the cloned fragment. A detailed cleavage map is sufficient to identify a particular gene within the genome. Fragments of the cloned gene, generated by digestion with ~n(lnnllrl~ , may be purified from agarose gels and ligated into vectors suitable for sequencing by methods known in the art. Vectors include, but are not limited to pUC18, pUCI9, YEp24, pGEM3Zf(+), pGEMSZf(+), and pGEM7Zf(-).
o The GLS I gene of S. cerevisiae may be used to isolate and characterize homologous genes in pathogenic fungi. Because other fungi, which include but are not limited to strains of C. neoformans, C.
albicans, A. fumigatlls, and Phytophthora infestans have I,3-13-D glucan in their cell walls, it is likely that a functional homologue of GLS I
exists in each of these fungi. Functional homologues of GLSI may exist in other organisms that have 1,3-,13-D glucans in their cell walls.
GLS1 homologues may be detected by isolating chromosomal DNA from a test organism. A portion of the isolated chromosomal DNA is cut with a number of restriction enzymes. The digested fragments of DNA are separated by gel electrophoresis. The fragments are then transferred to a solid membrane support. The membrane is then hybridized overnight with a labeled probe. Tlle blot is washed and then exposed to XAR-5 film and developed by conventional methods (Laskey and Mills (1977) FEBS Letlers, 82:
314-316). The conditions for washing the blot are such that only DNA
fragments with a high degree of homology (estimated at > 80%) will hybridize to the probe. The size and pattern of the digested fragments which hybridize with the probe generate a genomic map. For each organism, the map is sufficient to specifically identify the GLSI
homologue in the chromosome.
Mutations of the GLSI gene, including but not limited to glsl-l (st}ain MSl), gls1-2 (strain MS41) and gls1-3 (strain MS43), or disruptions or deletions of GLS 1, are useful for screening for glucan synthase inhibitors. Such a screen relies on the increase in W0 95132982 P~ 7 echinocandin-resistance and in papulacandin sensitivity of such mutants compared to an GLSI wild-type strain. Any technique capable of detecting this difference in sensitivity can be used. A zone of inhibition assay on agar plates is particularly useful.
C~oned GLSI cDNA may be recombinantly expressed by mo~ecular cloning into an expression vector ~ p a suitable promoter and other appropriate transcription regulatory e~ements, and transferred into prokaryotic or eukaryotic host cel~s to produce recombinant GLSI.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an alu,u-ululiaL~ host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, yeast, bluegreen algae, plant cells, insect cells and animal cells.
Specifically designed vectors al~ow the shuttling of DNA
between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector may contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA
sequence that directs RNA polymerase to bind to DNA and initiate RNA
synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
A variety of m~mm~ n expression vectors may be used to express recombinant GLSI in m~mm~ n cells. Comrnercially-available m~mm~ n expression vectors which may be suitable for recombinant GLS1 expression, include but are not limited to, pMClneo 3 (Stratagene), pXTI (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo~ATCC 371g8), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC
37565).

2~ 9 1 067 ~ W0 95132982 P~ ''7 DNA encoding GLS1 may also be cloned into an expression vector for expression in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to 5 bacteria, yeast, m~lmm~lizm cells and insect cells. Cell lines derived from m:lmm~ n species which may be suitable and which are commercially available, include but are not limited to, CV-I (ATCC
CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-Kl (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL
1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to Lldll~r(~lllldtion~ transfection, infection, protoplast fusion, and electroporation. The expression vectorc~",~ cells are clonally propagated and individually analyzed to deLtl-llille whether they produce GLSI protein. T~lr"lir;, Al;l-n of GLSI expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-GLSI antibodies, and the presence of 20 host cell-associated GLS I activity.
Expression of GLS I cDNA may also be perrormed using in vif~-o produced synthetic mRNA. Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes.
To determine the GLS I cDNA sequence(s) that yields optimal levels of enzymatic activity and/or GLSI protein, modifed GLS I cDNA molecules are constructed. Host cells are transformed with the cDNA molecules and the levels of GLSI RNA and protein are measured.
Levels of GLS1 protein in host cells are q~l~ntit~t~d by a variety of methods such as immllnc ~ffinity and/or ligand affinity t~orhniqll~s GLS I -specific affinity beads or GLS 1 -specific antibodies are used to isolate 35S-methionine labelled or unlabelled GLSI protein.

21~1067 W0 95132982 P~ .r ~7 Labelled GLS l protein is analyzed by SDS-PAGE. Unlabelled GLS l protein is detected by Western blotting, ELISA or RIA assays employing GLS l specific antibodies.
Following expression of GLS l in a recombinant host cell, GLSl protein may be recovered to provide GLSl in active form.
Several GLSI purification procedures are available and suitable for use.
Recombinant GLS l may be purified from cell Iysates or from conditioned culture media, by various combinations of, or individual application of fractionation, or chromatography steps that are known in the art.
In addition, recombinant GLS l can be separated from other cellular proteins by use of an immuno-affinity column made with monoclonal or polyclonal antibodies specific for full-length nascent GLS I or polypeptide fragments of GLS l .
The recombinant protein may be used to generate antibodies. The term "antibody" as used herein includes both polyclonal and monoclonal antibodies, as wel~ as fragments thereof, such as, Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten.
Monospecific antibodies to GLSI are purified from m~mm~ n antisera containing antibodies }eactive against GLS I or are prepared as monoclonal antibodies reactive with GLS l using standard techniques. Monospecific antibody as used herein is defined as a smgle antibody species or multiple antibody species with homogenous binding characteristics for GLS l . Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the GLSl, as described above.
Enzyme-specific antibodies are raised by illlllllllli~.;ll~ animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits 30 bemg preferred, with an appropriate concentration of GLS1 either with or without an immune adjuvant.
Monoclonal antibodies (mAb) reactive with GLS 1 may be prepared by conventional methods, such as by il"""",i,i"~ inbred mice with GLS 1.

~ WO 95132982 2 1 9 1 ~ 6 7 P~I~u~ ~ ~e7 In vitro production of anti-GLSI is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum tc obtain sufficient quantities of the specific mAb. The mAb are purified by tP~ hniq~ c known in the art.
Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays whicll include, but are not limited to, precipitation, passive :~gllltin~ion, enzyme-linked immunosorbent antibody (ELISA) technique and radioimm-~n~l~cs~y (RIA) techniques. Similar assays are used to detect the presence of GLS I in body fluids or tissue and cell extracts.
Methods such as those described above may be used to produce monospecific antibodies may be utilized to produce antibodies specific for GLSI polypeptide fragments or full-length nascent GLSI
PIYpeptide.
Kits containing GLS I cDNA, antibodies to GLS I or GLS 1 protein may be prepared. Such kits are used to detect DNA which hybridizes to GLSI DNA or to detect the presence of GLSI protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses, taxonomic ~t,~ lions and epidemiological studies.
The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of GLS I DNA, GLS I RNA or GLS I protein. The recombinant proteins, DNA molecules, RNA molecules and antibodies Iend themselves to the formulation of kits suitable for the detection and typing of GLSI. Such a kit would comprise a compartmPnt:~li7ed carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant GLS1 protein or anti-GLS I antibodies suitable for detecting GLS 1. The carrier may also contain means for detection such as labeled antigen or enzyme substrates or the like.
Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set 21q7067 WO 95/32982 r~ ., r -~-7 ~

of similar DNA oligonucleotides. Only one member of the set will be identical to the GLSI sequence but will be capable of hybridizing to GLS 1 DNA even in the presence of DNA
oligonucleotides with mism~trh~.c. The ~ d DNA
5 oligonucleotides may still hybridize to the GLSl DNA to permit identiflcation and isolation of GLS 1 encoding DNA.
DNA encoding GLSI from a particular organism may be used to isolate and purify homologues of GLSI from other organisms. To accomplish this, the first GLSl DNA may be mixed with a sample containing DNA encoding homologues of GLS l under d~p~ idlC hybridization conditions. The hybridized DNA complex may be isolated and the DNA encoding the homologous DNA may be purified ~I-e-crlwl-.
It is known that there is a .c~lhst~nti~l amount of 5 redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA
sçql-.o.ncç~ which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced 20 codons will be defined as a degenerate variation. Also included within the scope of this invention are mllt~tionc either in the DNA
sequence or the translated protein which do not sllhst~nti~lly alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for Iysine, or 25 asparagine for ~lllt~min~ may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the 30 DNA s~q~l~onr~.,c include, but are not limited to site directed mutagenesis.
Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate.
As used herein, a "functional derivative" of GLSI is a compound that possesses a biological activity (either functional or 2191~67 ~ WO 95132982 r~~ rr7 structural) that is sllh~t~nti~lly similar to the biological activity of GLSI. The term "functional derivatives" is intended to include the "fragments," "variants," "degenerate variants," "analogs" and "homologs" or to "chemical derivatives" of GLSI. The term "fragment" is meant to refer to any polypeptide subset of GLSl. The term "variant" is meant to refer to a molecule ~ub~ ally similar in structure and function to either the entire GLSI molecule or to a fragment thereof. A molecule is "substantially similar" to GLSl if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical. The term "analog" refers to a molecule sllhct~nti~lly similar in function to either the entire GLS I molecule or to a fragment thereof.
The present invention is also directed to methods for screening for compounds which modulate that expression of DNA or RNA encoding GLS1 as well as the function of GLSI protein in vivo.
Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
Compounds may modulate by increasing or ~tt~ml:~tin~ the expression of DNA or RNA encoding GLS I or the function of GLS I protein.
Compounds that modulate the expression of DNA or RNA encoding 25 GLSI or the function of GLS1 protein may be detected by a variety of assays. The assay may be a ~irnple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
The following examples are provided to further define the invention without, however, limiting the invention to the particulars of these examples.

~1~tQ` 7 WO 95132982 ~ r~ '''7 EXAMPLE I
Strains, Plasmids and Media The yeast strains used in this study are listed in Table 1.
5 The mutant strains are derived from strain X2180-lA.

Strain Relevant Properties X21 80-lA MATa GLS I (wt, EchS) MSI MATagl~l-l (EchR) MS41 MATa gls1-2 (EchR) MS743 MATa glsl -3 (EchR) GG100-14D MATaGLSl (wt,EchS) D2 MATa/MATa (MS41 x GG100-14D) D12 MATa/MATa (MSl x GG100-14D) D28 MATa/MATa (MS7-43 x GG100-14D) D132 MATa/MATa (D28-18C x D28-3B) D136 MATaJ~IATa (D28-18C x D2-1D) D137 MATa/MATa (D28-18C x D12-llD) D140 MATa/MATa (D28-9D x D2-lB) D141 MATa/MATa (D12-7D x D2-lB) D142 MATa/MATa (D2-2D x D2-lB) D2-lB MATa ura3-52 glsl-2 D2-lD MATa ura3-52 glsl-2 D2-2D MATa his3 glsl-2 D12-7D MATa his3 glsl-l D12-1 ID MATa ura3-52 glsl-3 D28-3B MATa his3 glsl-3 D28-9D MATa his3 gls1-3 D28-18C MATa ura3-52 his3 gls1-3 D2-5A MAToc his3 ura3-52 GLSl MS100 MATa his3 trpl glsl ::URA3-52 MS101 MATahis3 glsl::URA3-52 2191~7 WO 95/32982 r~ r---7 Strains GG100-14D and X2180-lA were obtained from K. Bostian and C. Ballou respectively. The mutants were generated in X2180-lA and outcrossed to GG100-14D. Abbreviations: wt, wild-type; Ech, echinocandin; S, sensitive; R, resistance.

Plasmid Description Source of cloned DNA
pJAC2 16-Kb GLSl clone in YCp50 GRF88 pJACl 4-kbGLSl clone inYCpS0 GRF88 pJAC4 4-Kb GLSl clone in YEP24 GRF88 The media used are as follows. YPAD medium contains 1% Bacto Yeast Extract, 2% Bacto-Peptone, 2%; Dextrose, 0.003%;
and adenine sulfate. Synthetic Dextrose (SD) Medium contains 0.67%
Bacto Yeast Nitrogen Base without amino acids (Difco), 2% De~trose and 2% Bacto Agar (Difco). Synthetic Complete (SC) medium is SD
medium supplemented with 20 mg each of adenine, histidine, and uracil, 60 mg of leucine, 30 mg of Iysine, and 20 mg of tryptophan per liter of medium. Sporulation medium is 2% Bacto Agar (Difco) and 0.3%
potassium acetate. Ura drop-out medium is SC medium without uracil.
Solid media are prepared with approximately 20 g/L agar.

DNA M~n~ulation and T-a.-~ro----alion Standard t~hni~lu~s of DNA manipulation were utilized (Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). E. coli strain DHSa (Hanahan, D.
1983. Studies on transformation of Escherichia coli with plasmids. J.
Mol. Biol. 166:557-580) was used as the host in bacterial transformation. Yeast transformation with the DNA libraries was performed by electroporation (Becker, D. and L. Guarente. 1991.
High-efficiency transformation of yeast by electroporation. In C.
Guthrie and G. Fink (eds.), Guide to yeast genetics and molecular WO 95132982 2 1 q 1 a 0 7 r~ c r~7 ~

biology, Methods Enzymol. 194:182-187.). All other yeast L~dll~rol,~lations with different plasmid subclones were by the alkali cation method (Ito, H., M. Fukuda, M. Murata, and A. Kimura. 1983.
Transformation of intact yeast cells with alkali cations. J. Bacteriol.
153:63-68). Plasmid DNA wa~ prepared from E. coli by the alkaline Iysis method (Sambrook, J., et al., supra). Plasmids were isolated from yeast for lldll~rol,l,dlion into E. coli as previously described (Hoffman, C. S. and F. Winston. 1987. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for lldl~ru~ dlion of Escherichia coli. Gene 57: 267-272). The wild-type gene, GLSI, which complements the echinocandin-resistance phenotype of the MS I
strain was isolated from a yeast genomic DNA library constructed in the ~e"LIu,ll~,lic shuttle vector YCp50 (Rose, M. D., P. Novick, J. H.
Thomas, D. Botstein, and G. R. Fink. 1987. Gene. oO:237-243) as described below.

The nucleotide sequences of the GLS 1 5' and the 3' ends were determined by the dideoxy chain termin~ti-)n method (Sanger, F.
S. Nicklen, and A. Coulson. 1977. "DNA sequencing with chain-lilld~illg inhibitors". Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977), using synthetic oligonucleotide primers complementary to specific regions of GLSl and a c~qll~n~ reagent kit (U.S. Biochemical Corp.).

PCR Amplification The polymerase chain reactions (PCR) were performed according to published procedures (Mullis, K. B. and F. A. Faloona, 1987. Specific synthesis of DNA in vifro via a polymerase-cataly~ed chain reaction. Meth. Enzymol. 155:335-350). Approximately 5 ng of genomic DNA from strain X2180-lA was used as a template. Synthetic oligonucleotide primers were designed to amplify a 1.5-kb fragment of 2 ' 9 1 ~67 ~ WO 95132982 1 I r~ .'C -''7 a region 5-kb to the left of the MAT locus on chromosome Ill. The sequence of the two primers were:
5'-TGACAGTAGTTTCACAAGTACTTAATATTGGAAATG-3' (SEQ
5 ID NO.:l) and 5'-TCAGATAATTTTATCGGTACCTTTTATATGTTAAAT-3' (SEQ
ID NO.:2).
Amplified DNA fragments were gel-purified, radiolabelled o by the random priming method (Feinberg, A. P and B. Vogelsteiln.
1984. Anal. Biochem. 137:266) a~nd used as probes to screen bacterial colonies cr",l~illill~ a yeast genomic DNA library in the centromeric vector YCp50 (Rose, M. D., et al., supra) following published protocols (Sambrook, J., et al., supra).

Gene Di.~ruption Chromosomal disruption of the GLSI gene was performed 20 by one-step gene disruption protocol (Rothstein, R. J. 1983. One-step gene disruption in yeast. Methods Enzymol. 101:202-211). Plasmid pJAC4 was constructed by cloning of a 1.6-kbp DpnI fragment fi-om pJAC2-1 into the HincII site of pUC18. Double digestion of pJAC4 with Nrul-EcoRV resulted in the excision of a 1.2-kb fragment containing all but the 40 C-terminal amino acids encoded by GLS 1. A
1.5-kb DNA fragment carrying the S. cerevisiae URA3 gene was isolated from the YCp50 plasmid as a Nrul-SmaI fragment. The purified fragment was treated with T4 DNA polymerase and blunt-end ligated into the Nrul-EcoRV sites of GLS 1 on plasmid pJAC4 resulting 30 in plasmid pJAC9 which contains a disruption deletion of GLSl.
Plasmid pJAC9 was propagated in E. coli, and the structure of the plasmid was verified by restriction analysis.
The glsl ::URA3 disruption fragment was purified from pJAC9 as 2.7 kb Xbal-HindlII fragment that was used to transform ura WO 9S132982 ;~ Q 6 7 r~ c ~ ~rr7 ~ 20 ~
3-S~ yeast strains containing a wild-type GLSI gene (strains GGI00-14D and D2-5A). Uracil ~ululul~u~Jllic yeast transformants were selected on SC medium lacking uracil. From each of the two transformed strains, one Ura+ t}ansformant was purified by single colony formation. Three Ura+ single colonies from each of the two transformants were tested for L-733,560-resistance and shown to exhibit drug resistance. These colonies were further ulld~ d and the integration event at the GLSI locus was confirmed by Southern hybridization (Sambrook, J., et al., s~pra). The two mutants resulting o from disruption the GLSI gene in GG100-14D and D2-5A were n lt~d MSI00 and MS101, respectively.
Total genomic DNA was isolated from stationary cultures of strains GG100-14D, D2-SA, MS100 and MS101. AL~I)lu~ ,ly 5 llg amoumts of each DNA were restriction digested with DraI. The digestion products were resolved on 1% agarose gels, followed by transfer to Zeta probe GT nylon membranes and hybridization according to the manufacturers protocols (Biorad Laboratories). A 32p radiolabeled probe of a DraI fragment, intemal to GLSI, was prepared by the random primer method (Feinberg, A. P and B. Vogelstein. 1984.
Anal. Biochem. 137:266).
~.~AMPLE 6 Liquid Broth Microdilution Assav To qll~ntit:~P the echinocandin sensitivity/resistance of the mutant strains, yeast cells grown to log phase were inoculated into 2 ml SC broth and incubated overnight at 28C. A flat bottom 96-well microtiter plate was seeded with 75 ~1 of SC medium in columns 2 through 12. To column 1, 150 1l1 medium was added. Echinocandin (L-733,560) was prepared at a concentration of 30 llg/ml solution in sterile distilled water. An aliquot (75 !11) Of el hinoç:ln~lin solution was added to column 3, and 75 ~I from c~lumn 3 was transferred to column

4, followed by mixing of the contents of each well. 75 !11 from column 4 was transferred into column 5, and the serial dilution was carried on to column 12 from which a 75 ~I was discarded. A dilution of 6x105 .

21ql~,7 ~ W0 95t32982 ~ ''-7 cells/ml in URA drop out medium was prepared from the yeast strains.
75 !11 cell suspension was then added to columns 2 through 12 giving 4.5 xlo4 cells/well. The plates were incubated at 28C for 24-48 hr.
Growth in the presence and absence of echinocandin was measured by absorbance at 600 nm.

Glucan synthase assay o Membrane extracts were prepared from mutant and wild-type cells grown to logarithmic phase (Kang and Cabib, PNAS, 83, 5808-5812, 1986). After homogenization with glass beads, unbroken cells and debris were removed by low speed centrifugation (1,000 x g for 5 mim). The sl~rPrnqtqnt fluids were centrifuged at 100,000 x g for 60 min, and the resulting pellets were washed with 2.5 ml (per gram of wet cells) of buffer containing 0.05 M potassium rhosFhqtP (pH 7.5), 0.5 mM DTT, and 1.0 mM PMSF. The washed pellet was r~ rPn~lPd in the same buffer containing 5% glycerol. This protein extract served as the source for both I ,3-~-glucan synthase and the chitin s~vnthases utilized in the enzymatic assays.
Protein concentrations were ~1Pt~rminPd using BCA protein assay reagent kit (Pierce Corp.), utilizing bovine serum album (BSA;
Pierce Corp.) as a standard. The 1,3-~-glucan synthase reactions were performed as previously described (Cabib, E., and M. S. Kang. 1987.
Fungal 1,3-b-glucan synthase, Methods Enzymol. 138: 637-642).
Briefly, an 80 ml reaction contained 125 mM Tris HCL (pH7.5), 0.25 mM dithiothretol, 30 mM KF, 0.3 M glycerol, 0.23% BSA, 0.125 mM
PMSF, 2 mM UDP-glucose, 10 mM guanosine 5'-(g-thio) triphosphate (GTP~S), 0.1 nmol UDP-[3H] glucose (Amersham; 4.5 Ci/mmol; ) plus 25 mg of membrane protein extract. The reactions were performed in the presence of 0.0, 0.1, 0.5, 5, 25 and 50 mM L,733-560 for dose titration of the drug. Following incubation at 25C for 150 min~tes, the [3H]-glucose incorporated into trichloroacetic acid-insoluble material was collected onto glass fiber filter~ (102 x 258 mm) and measured using a betaplate liquid scintillation counter (Cambridge Technologies . .

WO 95132982 2 1 9 1 0 6 7 P~l/IJ~ C 7 ~

Inc.; series 2800 harvester) at 25% efficiency. The product of such reaction~ wa.s verified by solubilization by lal~ a~ ase (Sigma, #L9259) but not by oc-amylase (Sigma, #A2643).
EXAMPLE
Chitin synthase assay A previously described chitin synthase assay was used (Kang, M. S., N. Elango, E. Mattia, J. Au-Young, P. W. Robbins, and o E. Cabib. 19~4. Isolation of chitin synthetase from Saccharomyces cerel~isiae. Purification of an enzyme by ~ lalJlll~ll~ in the reaction product. J. Biol. Chem. 2~9.14966-14972). Approximately 125 mg of membrane protein extract was trypsin-activated and used to catdlyze chitin synthase reactions. A reaction of 100 ml contained 50 mM Tris 15 HCI, pH 7.5, 40 mM MgCI, 32 mM N-acetylglucosamime (GlcNAc), 1 mM UDP-N-acetyl-[14C]~ n~min(~ (4x105 cpm/umol), 0.8 mg/ml digitonin. The reactions were performed in the presence of 0.0, 0.125, 0.5, 2 and 8 IlM of nikkomycin Z. After 30 minutes of incubation at 30C, the reaction products were ~ alcd with 10% trichloroacetic 20 acid and collected onto Whatman glass microfiber GF/A discs followed by counting of the incorporated ~14C]-GlcNAc.
EXAM~LE 9 25 Isolation of S~n.~lh~ Echinocandin-Resistant Mutants To isolate mutants in genes involved in biosynthesis of 1,3-,~-glucan, pneumocandin B compound, L-733,560 was used to identify resistant mutants in strain X2180-lA. Approximately 40 spontaneous mutants capable of colony formation in the presence of 7.5 mM of L-30 733,560 were isolated as follows: wild-type strain X2180-lA was grown to stationary phase in SD minimal medium. A~ v~ dl~ly 1-3 x 106 cells were spread on SD plates cnnt~inin~ 7.5, 15 or 45 mM L-733,560. Following incubation at 28C for four days, echinocandin-resistant colonies appeared at a frequency of 1-3 x 10-6. Mutant strains MSI, MS41 and MS14 were isolated by this procedure. MS14 contains 2 ~ 7 ~ W0 9~132982 ~ u,. ~ ''7 a mutation (fks1-4) in the FKS 1 gene. MS1 and MS41 contain gls 1-1 and gls1-2 mutations, respectively.

EMS Mutagenesis and Isolation of Strain MS43 Carrying the gls1-3 mutation YPAD broth (5 ml) was inoculated with an overnight culture of strain X2180-lA to give initial cell density of I x 106 cell per ml and inrllh~tr.~d at 30''C overnight. A 2.5 ml aliquot of the overnight culture was was-led twice in 50 mM KPO4 buffer, pH 7.0 by centrifugation, and resuspended in 10 ml of the same buffer. To a 5 ml aliquot of the washed cells, 150 ~11 of ethyl l~ llla,.csulfonate (EMS) was added. The suspension of treated cells was vortexed and incubated at

5 30''C for 1 hr. The other 5 ml of the washed culture was kept on ice untreated. To the 5 ml treated cells, an equal volume of a freshl~
prepared 10% (w/v) filter-sterilized sodium thiosulfate solution was added and mixed. Cells were collected, washed twice with sterile water, resuspended in 5 ml YPAD, and incubated at 24C for 4-6 hr.
20 Appropriate cell dilutions were plated on minimal medium (SD). The untreated culture was diluted and plated by the same way. After 3 days of growth at 24C, colonies were replica-plated onto YPAD medium with and without 0.001 ~lg/ml L-733,560 and incubated at 30C. Of 1000 colonies tested by this procedure, 10 colonies were resistant to L-25 733~560. One of these resistant strains, designated MS43, was shown tocontaim the gls1-3 mutation as described below.

30 Genetic An~ is Outcrosses were performed between each of three mutant straims (MSl, MS41, MS43) and the wild-type strain GG100-14D.
Tetrad analysis revealed that L-733,560-l~ ce segregated as a simgle trait in all three mutant strains (Fig. 1). Single gene mutations are = = =, = = , _ , . , _, W095132982 21 q 1 0 6 7 r~ 7 expected to segregate in a mendelian fashion 12:2) upon crossing of a mutant to a wild-type strain.
The mutations in the three mutant strains were tested for dominance or recessi~leness by mating the MATa echinocandin-resist~nt 5 mutant~ to the MATa echinocandin-sensitive strain C~100-14D ~wild-type). All three resulting MATa/MATa heterozygous diploid strains, D2 (MSI x GG100~T4D), D12 (MS41 x GG100-14D and D28 (MS43 x GG100-14D), exhibited sensitivity to L-733,560, in~ ring that strains MSI, MS41 and MS43 contain recessive mutations.
The recessive nature of these mutations was verified by the finding that the heterozygous diploids D2? D12 and D28 exhibited the wild-type rather than the mutant phenotype. Complementation tests were performed using drug-resistant segregants from the D2, D12 and D28 diploids. The diploids formed between resistant isolates carrying 5 mtlt:~ti~nC from MSI, MS41 or MS43 (D132, D136, D137, Dl407 D141 and D142) exhibited resistance to L-733,560, in~ir~tin~ lack of complementation among the three mutations.
These results indicate that the three independently isolated mllt~tif-ns glsl-l (strain MSl), glsl -2 (strain MS41) and glsl-3 (MS43) 20 comprise one complementation group. Mutations in different or unlinked genes can Cul~ a specific phenotype. Mutations in the same gene or in tightly-linked genes usually fail to complement each other and are, therefore, classified as one complementation group.

Genetic Mappin~ of the ~Isl Mutations Genetic analysis of the meiotic segregants of 37 tetrads resulting from ~U~I,/O~ lg MS41 to GG100-14D lead to mapping of the 30 glsl-l mutation to within 1.35 centi Morgan from the MAT locus on chromosome III (Table 2). Similar analysis re~lealed that both glsl-2 (strain MS41 ) and and glsl -3 (strain MS43) are linked to the MAT
locus. The parental ditype class of tetrads (PD) was the only class obtained from crosses between ~Gl~Q-14D and MSI (12 tetrads) or between GG100-14-D and MES43 (19 tetrads). If the mutations were in .. . . . . .. .. . .. . . . ... . . . . _ ... . . . . . . .

219~067 W0 95132982 r~ 7 unlinked loci or in genes located on different chromosomes, then a predominant tetratype class (T) of progeny would have been expected.
The phenotypes of the mutant and the wild-type strain used in the genetic crosses are given below:

Strain Phenotype MSl Mat a, EchinocandinR
MS41 Mat a, EchinocandinR
MS43 Mat a, EchinocandinR
o GG100-14D Mat a EchinocandinS
The tetrads resulting from uulu.~ g a MATa resistant mutant (strains MS1, MS41 or MS43) to a MATa sensitive wild-type (GG100-14D) should exhibit one of the three tetrad types as follows: 1. Parentalditypephenotypes(PD): Mata Frhinor:~n~ R
Mat a Echinocandinl S
The siblings exhibit the phenotypes of either parent.
2. Non-parental ditype phenotype~ (NPD): Mat a F.rhinrc~n(lin S
Mat a Frhinoc~n~iin R
The siblings exhibit phenotypes of neither parent.
3. Tetr~ e phenotypes (T): The siblings exhibit both parental and non-parental ditype phenotypes (Mat a Frhinor:~nrlinS, Mat a 25 EchinocandinR, Mat a EchinocandinR, Mat a EchinocandinS
The data obtained from outcrossing the three glsl mutants to the wild-type strain GG100-14D is ~u~u~ ;d in Table 2.

WO95/32982 2 ~ 9 ~ Q~7 r~ ,. .'0~''7 GENETIC MAPPING OF THE GLSI MUTATIONS
5 Interval Ascus Type Map Distance (cM) PD NPD T
glsl-l - MAT 36 0 1 1.35 glsl-2 - MAT 12 0 0 gls1-3 - MAT 19 0 0 A NPD ratio of <I indicates linkage. This data indicates that the threeglsl mutations are linked to MAT. The map distance between two loci is calculated as follows: 100 (1/2T) + NPD / Total number of tetrads The distance between glsl-l and MAT= 50/37 = 1.3~ centi Morgan.

Characterization of Strain MSI Mutant Strain MS I exhibits a bilateral mating defect, (i.e., mating 20 for the production of homozygous diploids containing two copies of glsl-l is very ineffecient). The resultant homozygous diploids do not form spores upon subculturing on sporulation media. The homozygous diploids are osmotically unstable and burst when suspended in water.
In contrast, mating between wild-type cells and cells 25 contslinin~ any of the three glsl mutations for production of heterozygous diploids is normal.
Morphologically, MSl cells show some aggregated cells (Fig. 3), multiple buds, and occasional flocculated growth in YPAD
medium at 30C. Strain MSI also grows slower in YPAD than its wild-30 type parental strain. This slow growth is characterized by a long lagperiod before the cells enter the division cycle.

2 1 ~ 7 WO9S/32982 P~,l/u~ 5. -'~'7 EXA~PLE 14 Effect of Antifun~al Dru~s on Strain MS I
Strain MS I did not exhibit multiple drug resistance when 5 tested against a panel of more than 30 inhibitors affectimg cell wa~ll, membrane, sterol, and protein synthesis.
In addition to its resistance to L-733,560 (Fig. 2), MS I
cells are more sensitive to the 1,3-~-glucan synthase inhibitor, pap~ nllin, and to the immunu~u~ rapamycin, than is the wild-type parental strain (Figure 10).

Levels of 1~3-~-Glucan Synthase and Chitin Synth~ Activities Crude enzyme preparations from cell ~ ...I,l~les were tested for 1,3-,~ glucan synthase and chitin synthase activities. The sensitivity of in vitro synthesis of those polymers to L-733,560 and Nikkomycin Z was ,~ d (Fig 4).
T_ese ~ showed that glucan synthesis activity of MSI is reduced (80% less activity) relative to wild-type cells.

Stimulation of 1,3-~-D-Glucan Synthesis by GTP~yS
2s To study activation of 1,3-~-glucan synthesis by GTPyS
incorporation of UDP-glucose into 1,3-~-D glucans, in absence and in presence of 3.3 uM GTP~S was measured. The glucan synthesis of the MSI mutant enzymatic activities was stimulated (~lU~ ld~ly 19-fold) by GTP~S. This contrasts the 6-fold stimulation by the MS I mutant 3 o enzyme (Fig. 4).
In another experiment the stimulation of the mutant and the wild-type membranes by different cnnc~ntr~ions of GTP~S was studied.
T_e MSI mutant enzymatic activity responded rather poorly to increasing concentrations of GTPyS (Fig. 5).
_, _ _ , . .. .. . . . . .

W095/32982 ~ 1 9 1 ~7 PCTIUS9S/06~57 Isolation of the GLS 1 Gene by Functional Complementation and by Hybridization The GLS I gene was cloned by complementation of the echinocandin-resistance phenotype. Yeast strain D2.8B (MAT a, ura3-52, glsl -l ) was transformed with a yeast genomic DNA library in the centromeric vector YcP50 (Rose, M. D., et al., 1987. Gene. 60:237-243), followed by selection of transformants on Ura-drop-out medium.
Transformants (2400) were picked onto master plates of Ura-drop out medium and replica-plated onto plates containing the same medium supplemented with 0.0 or 7.5 ~lM of L-733-560. Following incubation at 30C for 2-3 days, 4 sensitive colonies colonies were isolated. One of these four colonies, designated D2.~B (pJAC2-1) was shown to contain a comp!t~mentin~ plasmid, pJAC2-1. In a separate e~ , a 1.5 kb DNA fragment representing a sequence located at about 7 kb to the left of MAT was amplified by PCR, radiolabeled amd used as a probe to screen a YCpS0-based yeast genomic library by colony hybridization (Sambrook, J., et al., supra). Screening of approximately 4800 bacterial colonies by this procedure resulted in a hybridizing clone ill;llY a plasmid ~l~civn~t~d pJAC2-2.
pJAC2-1 and pJAC 2-2 contain DNA fragments that have identical restriction maps. pJAC2-2 was introduced into strain D2.8B.
Three yeast transformants were tested and shown to have acquired a wild-type level of sensitivity to L-733-560. Furthermore, ~ lllblalle extracts prepared from the llall~rollllalll~ reversed the low level of 1,3-,~-glucan synthase specific activity associated with the membranes of the untransformed mutant.
A yeast transformant ~ n:~tl~d D2.8B (pJAC2-2) was cured of its transforming plasmid, pJAC2-2, by three successive rounds of overnight growth in YPAD broth followed by plating on YPAD
plate~ for single colony formation. Cured clones lost the plasmid and exhibited resistance to L-733,560.

WO 9S132982 2 1 9 1 0 6 ~ PCT/US9S/065~7 D~i~.",ill~lion of the Glsl-l Minimum ComplPmPntin~ Fra~ment The glsl-l complPm~ntin~ region of pJAC2 was defined by 5 digesting with restriction enzymes that cut within the cloned insert DNA
(Fig. 6). Plasmids containing restriction fragments subcloned in YcP50 were propagated in E. coli DH5a, characterized with regard to restriction patterns and then introduced into mutdnt yeast strain MSD2.8B. The resulting yeast tran~ru"l~d~ were tested for growth rate and drug-resistance phenotype. The glsl-l comp!~m~ntin~ activity was present in approximately 4kb Kpnl fragment (Fig. 1). Furtl1er ~o~ lion analysis defined a 1.6 kbp DpnI fragment containing the GLS I gene.
Using restriction analysis and subcloning of smaller DNA
5 fr~nPnt~ from the origimal 17 kb library clone, a 4 kb KpnI fragment of pJAC2 was cloned in the single copy vector YCpLac33 to yield pJACI. pJACI complementedtheMSI mutantphenotypes.
By similar analysis a 1.6 kb DpnI fragment from pJAC2 was cloned into the HincII site of the bacterial vector pUC18 to produce 20 a plasmid ~ n~t~d pJAC4. A BamHVSphl 1.6 kb frdgment c~t~inin~
GLS I was purified from pJAC4 and subcloned into both YEp24 and YCp50 plasmids (digested with BamHI and SPHI) to yield pJAC5 and pJAC3 respectively. Both plasmids (pJAC3 and pJAC5) complemented the glsl-l drug-resistance/sensitivity phenotypes (Figures 7,8).

Nucleotide and Deduced ~mino Acid Sequence Analysis of GLS I
The dideoxy chain termination method was used to 30 ~i~tPrrninP the nucleotide sequence of the 5' end of GLS1. The sequence was determined for the first 200 bp and compared with the open reading frame (ORF) YCR34 on Chromosome m (Olwer, S. et al., 1992, Natul-e 357:38-46). The sequences were identical over the 200 bp compared. These results suggested the identity of GLSl and the YCR34 ....... . _ .. . .. _ _ . _ .... ...... _ . _ .. ... _ ... _ .. ... . ..... ... _ _ .

wog5~32s82 2 1 9 1 367 P~ S~ ' ~

ORF. The nucleotide and the predicted amino acid sequences of GLS l/YCR34 are shown in Figure 9.
The 600 bp of sequence in the 5' untranslated region of GLSI/YCR34 contains four candidate promoter 'TATA' boxes. In 5 addition, there are two candidate UAS elements with a strong homology to the yeast HAPl binding site. Thus, the promoter region of GLSI
shows a striking similarity to that of the yeast gene CYCI. The sequence matches of the 4 'TATA' elements are almost identical in the two genes. The UAS sequences also show a strong homology to the UASl of CYCI. This suggests that GLSI expression may be controlled in a fashion ~imilar to that of CYCI.
The 348 amino acid putative protein product of GLSIIYCR534 was compared to protein (l~t?l~a~es No ~i~nifir:~nt homology with known proteins was found. A hydropathy analysis was 5 performed on the 348-residue amino acid sequence, using the Kyte and Doolittle algorithm (Fig. 10). The putative protein product is basic (pl= 10.3) and hydrophobic. There are several leucine zipper motifs in the sequence, inrlic:ltin~ that the protein product may fold as a dimer.
EXA~PLE 20 GLS I Gene Disruption A chromosomal deletion of the GLS1 gene was generated by one-step gene disruption (Rothstein, R. J., 1983, Methods ~;nzymol.
25 1012:202-211) to test whether GLSI is essential. A 1.2 kb region of plasmid pJAC4, containing most of the GLS 1 coding sequence, was deleted as a Nrul-EcoRV fragment. The deleted region of pJAC4 was replaced by blunt-end ligation with a 1.5 kb DNA fragment containing the URA3 gene. The disrupted copy of GLS 1 was excised as a 2.7-kbp 30 HindII/Xbal fragment, and used to transform the two GLSl-contaiming wild-type strains GG100-14D and D2.5A.
The resulting Ura+ yeast transformants were tested for echinocandin-resistance. Two transformants (GGDglsl and D2.5ADglsl) acquired resistance to L-733,560 and were analy~ed further (Figure 9). The alteration of the GLS1 locus was confirmed by ~ W0 9513~98~ 2 1 q 1 Q 6 7 T~ '7 Southem hybridization analysis. The viability of the haploid strains with GLS I deletions indicate that the gene is not essential for growth.

GLSl Homologues Exist in Patho~enic Species Yeast 1,3-~-D glucan synthase can be fr~t~ti-)n~t~d into a soluble and insoluble fractions by treating yeast membrane preparations with salt and detergent. A glucan synthase activity can be reconstituted by mixing the two fractions in presence of GTP. Cabib and coworkers have demonstrated that the ~olubilized fraction is exch~n~hle between yeast and other fungi. This suggests a possible homology betwee glucan synthesis enzymes amongst fungi.
To test whether homologues of t_e cloned GLSI exist in 5 other fungi, genomic DNA from several heterologous species was prepared and a series of PCR and Southem hybridization analysis were perfommed. The results showed that GLSI homologues exists in other fungi, including Candida albicans, Aspergillus fumigatus, Schizosaccharomyces pombe. and Phytophthora infestans (Figure 11).

Isolation of GLSI Homologue~ from Pneumocystis Carinii Whole rat lungs from P. carinii -infected male 25 Sprague-Dawley rats are homogenized with a Brinkmann homogenizer, and DNA is isolated as described (P. A. Liberator, et al., 1992. J. Clin.
Mic~o. 30(11): 2968-2974). Two to five micrograms of purified DNA
are digested with a restriction endonuclease such as EcoRI, and the fragments are separated on an agarose gel. DNA is transferred to a 30 solid support such as nitrocellulose and probed by the method of Southem (Southem, E. M. 1975. J. Mol. Biol. 98:503-517) for fragments with homology to GLS1. By washing the blot at a red~ced stringency, weakly homologous genes can be i~iPntifit~l The P. carinii GLSI homologues are cloned by preparing a mini-library from the region of the agarose gel where the hybridizing WO 95/3~982 2 ~ 6 7 P~ '''7 fragment was visualized on the Southern blot. Following phenol:CHC13 extraction to remove t.~ A~ , DNA fragments from this area of the gel are ligated into an appropriate plasmid vector and transformed imto E. coli. The E. ~oli clones bearing the mini-library are spread onto 5 agar plates and probed for inserts homologous to GLS1 by in situ colony Iysis. DNA from individual transformants is transferred to nitrocellulose, hybridized to a radiolabeled GLS1 DNA fragment, washed, and exposed to film. Colonies containing an insert with homology to GLSl are visualized on the filln; plasmid DNA is then isolated from positive clones, propagated, and analyzed. DNA sequence analysis by standard methods is used to establish the extent of homology to GLS I, and functional homology may be demonstrated by expression in S. cerevisiae disrupted for GLS1.

tion of GLS1 Homologs from Phytopathogenic Fun~i To clone GLS1 homologs from phytopathogenic fungi such as Phytophthorn infestans, high molecular weight genomic DNA is 20 isolated by the method described by Atkins and Lambowitz (Mol. Cell.
Biol., 5; 2272-2278), partially digested by a restriction enzyme, and cloned into the Stratagene Vector Lambda-Dash using a cloning kit obtained from the m:~mlf~chlrer and methods of the art (Maniatis). The libraries are screened using probes from GLS1.

The GLS~ mutants (strains MS1, MS41 and MES7-43) are echinocandin-resistant and papulacandin-supersensitive, while the fl~sl-4 30 mutant (strain MS14) is echinoc~n~lin-resistant (50 fold more resistant than wild-type) and nikkomycin Z-~ul~e~ lsi~ive (1000 fold more sensitive). The GLSl and the fksl -4 mutants can be incorporated into an assay to screen and classify antifungal compounds with chitin and glucan synthase inhibitory effects, based on their dirr~lcllLial resistance/sensitivity to the echinocandins, papulacandin and 2 ~ 9 1 ~67 W095132982 P~~ -7 Nikkomycin Z. The data from this assay and the sizes of the zones of growth inhibition in millimeter is given in the following table:
5 Inhibitor lag/Disc Strain X2180 ~SI MS14 (WT) (gls I - I ) (fks 1-4) L,733-560 20 15 12 8vh (erhins)ç~n-1in) L-688-786 10 0 7vh 0.0 (e~hino~n~lin) Aculeacin 50 15 9 0.0 Papulacandin 50 10 20 lOh Nik~comycin Z 10 0.0 0.0 30.0 Rapamycin 12.5 0.0 1 3 0.0 The MS1 and MS14 yeast strains may be used in an assay to 20 screen for glucan and chitin synthesis inhibitors. This assay can also discriminate between different classes of glucan synthesis inhibitors like pap~ c~n~1in~ and echinocandins.
A compound that is active against MS14 but inactive a~ainst MSI and the wild-type strain is a "chitin synthase-type" of inhibitor. A
compound that is active against MS I but less active against the wild-type strain and MS14 is a "papulacandin type" of inhibitor. "Echinocandin-type" inhibitors would exhibit less activity on MSI cells and lesser activity on MS14 cells relative to the wild-type strain.

WO95132982 21 9 ~ 0 67 r~ c~ 7 Cloning of GLS 1 for Expression of the GLS 1 Polypeptide In Other Host Cell Systems (a) Cloning of GLSI cDNA into a bacterial expression vector. Recombinant GLSI is produced in a bacterium such as E. coli following the insertion of the optimal GLS I cDNA sequence into expression vectors designed to direct the expression of heterologous proteins. These vectors are constructed such that recombinant GLS 1 is synthesi~ed alone or as a fusion protein for subsequent manipulation.
Expression may be controlled such that recombinant GLSI is recovered as a so]uble protein or within insoluble inclusion bodies. Vectors such as pBR322, pSKF, pUR, pATH, pGEX, pT7-5, pT7-6, pT7-7, pET, pIBI (IBI), pSP6/T7-19 (GibcotBRL), pBluescript II (Stratagene), pTZ18R, pTZ19R (USB), pSE420 (Invitrogen) or the like are suitable for these purposes.
(b) Clonin~ of GLSI cDNA into a viral expression vector Reco.l.billdlll GLSI is produced in m~mm~ n host cells, such as HeLa S3 cells, after infection with vaccinia virus containing the GLSI cDNA
sequence. To produce GLSl:vaccinia virus, the GLSI cDNA is first ligated into a transfer vector, such as pSC11, pTKgptFIs, pMT601 or other suitable vector, then transferred to vaccinia virus by homologous recombination. After plaque purification and virus amplification, GLSl:vaccinia virus is used to infect m~mm~ n host cells and produce recombinant GLS I protein.

Process for the Production of a Glucan Synthase Subunit Peptide Recombinant GLSI is produced by (a) ~lall~rOIIIIillg a host cell with DNA encoding GLS 1 protein to produce a reGu--~ -à.ll host cell; (b) culturing the recombinant host cell under conditions which allow the production of glucan synthase subunit peptide; and (c) recovering the recombinant glucan synthase subunit peptide. The .. . . . ... . , ... ~ . . .. . . ... . . .

~ wogsl32g82 2 1 9 ~ 0 6 7 r~".,~ ~r 7 recombinant glucan synthase subunit is purified and characterized by standard methods.

Compounds that modulate glucan synthase subunit activity may be detected by a variety of methods. A method of identifying compounds that affect glucan synthase subunit comprises:
(a) mixing a test compound with a solution o containing glucan synthase subunit to forrn a mixture;
(b) measuring glucan synthase subunit activity in the mixture; and (c) comparing the glucan synthase subunit activity of the mixture to a standard.
Compounds that modulate glucan synthase subunit activity may be forrn~ t~d into pharm~re--tir~l compositions. Such ph~rm~rellti~:~l compositions may be useful for treating diseases or conditions that are characterized by fungal infection.

DNA which is structurally related to DNA encoding glucan synthase subunit is detected with a probe. A suitable probe may be 25 derived from DNA having all or a portion of the nucleotide sequence of the figures, RNA encoded by DNA having all or a portion of the nucleotide sequence of figures, degenerate oligonucleotides derived from a portion of the amino acid sequence of figures or an antibody directed against the peptide encoded by GLSl.

A kit useful for the detection and characterization of DNA
or RNA encoding glucan synthase subunit or glucan synthase subunit peptide is prepared by conventional methods. The kit may contain DNA
.. . . . . .. .. . . ........ . .. . . . . . . . . .

W0 9513298~ 2 ~ 6 7 P~ ,.'C--'7 ~

encoding glucan synthase subunit, recombinant glucan synthase subunit peptide, RNA corresponding to the DNA encoding glucan synthase subunit or antibodies to glucan synthase subunit. The kit may be used to 5 characterize test samples, such as forensic samples or epidemiological samples.

~0

Claims (16)

WHAT IS CLAIMED IS:

1. Isolated DNA encoding glucan synthase subunit or a functional derivative thereof.

2. The isolated DNA of Claim 1 having the nucleotide sequence of Figure 9 or a functional derivative thereof.

3. Isolated RNA encoded by the isolated DNA of Claim 1 or its complementary sequence.

4. An expression vector containing the isolated DNA of Claim 1.

5. A recombinant host cell containing the expression vector of Claim 4.

6. A process for the production of a recombinant glucan synthase subunit peptide, comprising:
(a) transforming a host cell with the isolated DNA
of Claim 1 to produce a recombinant host cell;
(b) culturing the recombinant host cell under conditions which allow the production of recombinant glucan synthase subunit peptide;
and (c) recovering and purifying the glucan synthase subunit peptide.

7. The purified recombinant glucan synthase subunit peptide produced by the process of Claim 6.

8. An isolated and purified glucan synthase subunit peptide or a functional derivative thereof.

9. The isolated and purified glucan synthase subunit peptide of Claim 8 which is encoded by the DNA of Figure 9.

10. The isolated and purified glucan synthase subunit peptide of Claim 9 having the amino acid sequence of Figure 9.

11. A method of identifying compounds that modulate glucan synthase subunit peptide activity, comprising:
(a) mixing a test compound with a solution containing glucan synthase subunit peptide to form a mixture;
(b) measuring glucan synthase subunit peptide activity in the mixture; and (c) comparing the glucan synthase subunit peptide activity of the mixture to a standard.

12. Compounds identified by the method of Claim 11.

13. Pharmaceutical compositions comprising the compound of Claim 12.

14. A kit comprising a reagent selected from the group consisting of the isolated DNA of Claim 1, recombinant glucan synthase subunit peptide or functional derivatives thereof and antibodies to recombinant glucan synthase subunit peptide or functional derivatives thereof.

15. An antibody immunologically reactive with the glucan synthase subunit peptide of Claim 8.

16. A method for treating an animal in need of such treatment of a condition comprising administering the compound of Claim 12 to the animal.