Proc. Natl. Acad. Sci. USA Vol. 93, pp. 186-190, January 1996 Genetics

Cloning and characterization of ERG25, the gene encoding C-4 sterol methyl oxidase (fungi/sterol biosynthesis) M. BARD*t, D. A. BRUNER*, C. A. PIERSON*, N. D. LEES*, B. BIERMANN*, L. FRYEt, C. KOEGEL§, AND R. BARBUCH§ *Department of Biology, Indiana University-Purdue University at Indianapolis, Indianapolis, IN 46202; tDepartment of Chemistry, Rensselaer Polytechnic Institute, Troy, NY 12180; and §Marion Merrell Dow Pharmaceutical Inc., Cincinnati, OH 45215 Communicated by David B. Sprinson, St. Luke's-Roosevelt Hospital Center, New York, NY, July 27, 1995 (received for review June 9, 1995)

ABSTRACT We have cloned the Saccharomyces cerevisiae LANOSTEROL C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to ERG1 1 remove the two C-4 methyl groups leading to cholesterol * (animal), (fungal), and stigmasterol (plant) bio- 4,4-DIMETHYLCHOLESTA-8,14,24-TRIENOL synthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy- ; ERG24 mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 4,4-DIMETHYLZYMOSTEROL 309-amino acid polypeptide (calculated molecular mass, 36.48 ; ERG25, ERG(?) kDa). The amino acid sequence shows a C-terminal endoplas- mic reticulum retrieval signal KKXX and three histidine-rich ZYMOSTEROL clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The ERG6, ERG2, ERG3 sterol profile of an ERG25 disruptant was consistent with the * ERG5, ERG4 erg25 allele obtained by mutagenesis. ERGOSTEROL In the synthesis of sterols, required components of eukaryotic membranes, an initial sterol (lanosterol in animals and fungi FIG. 1. Steps in ergosterol biosynthesis and encoding genes. and cycloartenol in plants) undergoes three demethylations prior to formation of the end product sterol. The first de- genes encoding the enzymes used in C-4 demethylation. In one methylation occurs directly with lanosterol or cycloartenol and report, a Chinese hamster ovary cell line auxotrophic for results in removal of the C-14 methyl group. The fungal cholesterol was observed to accumulate sterol with a C-4 demethylation is performed by the product of the ERG11 gene carboxylic acid group, indicating a deficiency in the decarbox- (Fig. 1). The remaining demethylations occur with the sequen- ylase (7). In addition, the fungal pathogen Cryptococcus neo- tial removal of the two C-4 methyl groups and result in the formans has been shown to accumulate 3-keto sterol after formation of zymosterol in animals and fungi (Fig. 1). exposure to the antifungal itraconazole, thus providing a The steps involved in removal of the C-4 methyl groups are potential screen for isolating strains deficient in 3-keto reduc- not well-defined in yeast but have been described in some tase activity (3, 8). This report describes the isolation of a yeast detail in animals by Gaylor's group (1-3). As depicted in Fig. mutant (erg25), which is auxotrophic for sterol and accumu- 2, the demethylation is specific for the C-4a methyl group and lates 4,4-dimethylzymosterol, indicating a defect in the C-4 is initiated by the C-4 methyl oxidase, which converts the sterol methyl oxidase. Complementation with a yeast genomic methyl group to the alcohol, the aldehyde, and finally to the library has permitted the isolation, sequencing, and disruption carboxylic acid (Fig. 2, structures a-d). This sequential oxida- of the yeast ERG25 gene.l tion was shown to require NADH and oxygen and cytochrome b5 as an electron carrier between the NADH and the oxidase. However, Maitra et al. (4) were unable to show, using 3H MATERIALS AND METHODS release from C-30 4,4-[3H]dimethylzymosterol, that cyto- Strains, Media, Transformation, and Mutagenesis. The chrome b5 was involved in C-4 methyl oxidation. In the next following yeast strains were used: SGY688 (MATa erglA.:URA3 reaction, the carboxyl group is removed by a second enzyme, ura3-52 hem3-11 trpl ade2-11 leu2 lys2), WAl (MATa ura3-52 the C-4 decarboxylase, resulting in formation of a keto group leu2-3,112 adeS his7-2), WA6 (same as WAl but MATa), and at the C-3 position (structure e). A third enzyme, 3-keto upc2 (9). CP2 (MA Ta ergl.:URA3 trp] his3 upc2 ade2-11) was reductase, then reduces the keto group to the required alcohol a segregant from a cross between SGY688 and WA6 followed (structure f). This process is repeated with the second methyl by a cross to upc2, a mutation that allows for aerobic uptake group after its transposition to the a position (5). of sterol. CP2 was plated onto lanosterol-supplemented me- The yeast Saccharomyces cerevisiae has long served as a dium to select for isolates (CP3) capable of specifically utilizing model system for studies in sterol biosynthesis. To this point, this sterol as a substrate for ergosterol synthesis. CP3 was most genes in yeast ergosterol synthesis have been at least grown at 30°C on yeast complete medium (1% yeast ex- partially characterized by direct cloning of the genes or by tract/2% peptone/2% glucose) supplemented with ergosterol analysis of mutations (6). The exceptions to this are the three Abbreviation: ORF, open reading frame. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in $The sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U31885). 186 Downloaded by guest on September 29, 2021 Genetics: Bard et al. Proc. Natl. Acad. Sci. USA 93 (1996) 187

ERG 25 ERG 25 (a) - (b)

4,4-dimethylzymosterol

4-methylzymosterol-4-carboxylic acid

3-keto-4-methylzymosterol 4a-methylzymosterol FIG. 2. Sterol structures and reactions involved in removal of the C-4a methyl group. or an ergosterol intermediate (0.002%) and Tween 80 (0.5%). cells were plated onto ergosterol medium and replica plated to Minimal medium consisted of 0.67% yeast nitrogen base, 2% lanosterol-containing medium. Putative ergosterol mutants glucose, and amino acid and nitrogenous base supplements as could not grow on lanosterol. a 0.8% complete synthetic mixture (CSM) addition (Bio 101). The genomic library of S. cerevisiae in vector YCp5O was Ergosterol and commercial grade lanosterol were purchased obtained from the laboratory of David Botstein (11). Approx- from Sigma. Pure lanosterol (>95%) was obtained from imately 5000 yeast transformants were selected for uracil Steraloids or as a gift from David Nes (Texas Tech). prototrophy on minimal medium lacking uracil (CSM-ura) CP3 yeast cells were grown overnight in complete medium but containing ergosterol and then screened for ergosterol supplemented with ergosterol and mutagenized with ethyl prototrophy on yeast minimal medium without ergosterol. according to standard procedures (10). The Plasmid DNA was extracted from yeast cells by standard methanesulfonate methods (10) and transformed into Escherichia coli DH5a for kill rate for an average experiment was 90-95%. Mutagenized plasmid amplification, restriction digests, and subcloning of DNA fragments. DH5a was grown in LB medium supple- mented with ampicillin (50 mg/liter) and DNA was isolated by A the alkaline lysis method (12) and purified using Qiagen columns. Plasmid Constructions and Gene The 2.4- and 0 Disruption. (I) 3.5-kb BamHI fragments from pERG25-1 were isolated from low melting point agarose gels and subcloned into the yeast 0 vector to generate plasmids pIU704 and pIU705, respectively. pIU707 contains the 1.35-kb Dra I fragment from pIU705 filled in and subcloned into the Sma I site of pRS316 (13). pIU709 contains a 0.4-kb Xba I deletion of pIU705. To disrupt the opening reading frame (ORF) of ERG25, a 1.2-kb Xba I fragment containing the URA3 gene was ligated into the XbaI site of pIU709 to generate pIU802. The 2.2-kb Dra I fragment was excised from a low melting point agarose gel and used to transform the wild-type diploid WA1/6 (14). Segregants were obtained by tetrad analysis. 18 20 22 DNA Sequencing. The 3.5-kb BamHI fragment containing the entire ERG25 gene was cloned into the Bluescript vector Retention Time pBS (KS+) in both orientations. The nucleotide sequences of FIG. 3. GC analyses of sterols accumulating in erg25 grown in the the insert of both strands were determined by the chain- presence of ergosterol. Peak A, ergosterol; peak B, lanosterol; peak C, termination method using Sequenase version 2.0 and deoxy- dimethylzymosterol . adenosine 5'-[a-[35S]thio]triphosphate. Initially, DNA was se- Downloaded by guest on September 29, 2021 188 Genetics: Bard et al. Proc. Natl. Acad. Sci. USA 93 (1996)

10(1, 1 69 Sterol Analyses. Sterols were isolated as nonsaponifiables as described (15). GC analyses of nonsaponifiables were per- formed on a HP5890 series II GC equipped with the HP chemstation software package. The capillary column (HP-5) was 15 m x 0.25 mm x 0.25 jam film thickness and was HO programmed from 195°C to 300°C (3 min at 195°C and then an il increase at 5.5°C/min until the final temperature of 300°C was reached and held for 4 min). The linear velocity was 30 cm/sec using nitrogen as the carrier gas, and all injections were run in () the splitless mode. GC/MS analyses were done with a Varian 3400 gas chromatograph interfaced to a Finnigan MAT TSQ II 700 mass spectrometer. The GC separations were done on a fused silica column, DB-5 15 m x 0.32 mm x 0.25 ,um film 412 thickness, programmed from 50°C to 250°C at 20°C/min after ,' 1 a 1-min hold at 50°C. The oven temperature was then held at 95 250°C for 10 min before programming the temperature to 09 300°C at 20°C/min. Helium was the carrier gas with a linear 4( - velocity of 50 cm/sec in the splitless mode. The mass spec- trometer was in the electron impact ionization mode at an 14 7 electron energy of 70 eV, an ion source temperature of 150°C, and scanning from 40 to 650 atomic mass units at 0.5-sec intervals.

_() 1S3~~~~~~~~9II RESULTS AND DISCUSSION 3 Isolation of the S. cerevisiae erg25 Mutant. Since no DNA source of the C-4 sterol methyl oxidase gene was available, ~I'i (( ., .(( (( mutagenesis was performed to isolate a yeast strain unable to li demethylate at C-4. Presumably, the mutant strain would be I (0 blocked so that 4,4-dimethylzymosterol or an oxidized deriv- I711 -_ ative of this sterol accumulated. The yeast strain CP2 contain- ing the auxotrophic ergl mutation (squalene epoxidase) was FIG. 4. Mass spectrum of GC/MS peak identified as 4,4- used. This strain could grow aerobically in medium supplied dimethylzymosterol. with ergosterol (the end product yeast sterol) or even choles- terol but not with the sterol intermediate lanosterol. Selection quenced with T3 and T7 primers followed by primers corre- for the utilization of lanosterol resulted in a CP3 strain able to sponding to newly sequenced DNA. Sequence data were import this sterol into the cell and convert it to ergosterol. aligned with DNAsIs (Hitachi) software. Ethyl methanesulfonate mutagenesis resulted in strain DC1,

Plasmid Complementation s ,_ E = m m. 'Gm c E -a N. &. .0 CL W._ of erg25 C C o oI &I y x =n I I 11 11 I I1 pIU705 ERG25-

plU707

CL m~~~~~~~~~~~~~~~~~~~~~~&

ER25 = - -oe = 11 11 I I~ pIU709

=_ E = = '; j 0c mCu M0 1 . 4...0 a# m o *=D L. ._ m in W) Cm oo c x I. LS s co 11.. .- I mmmmd plU802 nt URR3

500 bp

FIG. 5. Restriction map of the ERG25 gene and construction of plasmid subclones. The genomic clone is pERG25-1. All subclones are vector-based YCp5O except pIU707, which is in pRS316. The ability of each plasmid to complement erg25-25C is denoted by + or -; nt, not tested. Dashed line in pIU709 represents a deletion. Downloaded by guest on September 29, 2021 Genetics: Bard et al. Proc. Natl. Acad. Sci. USA 93 (1996) 189

which could no longer grow on lanosterol or other 4,4-dimethyl -186 TCTATATAATATATAATACAGGAGGATCTATTCTTTTTTTTCTTCTTCTCTTCTTCTCTC -126 CCTITTITCTCTCTCTTCGTTTCTTTCCTTCCAGTAAATCTTTATATTAGTTGTAACTTT sterols (4,4-dimethyl-8,14-cholestadien-3,B-ol). GC analysis of -66 TTCTCTTTAGATAGTAGCATAGAGGACTAAGGAAAAGTAGTACAGCCATAAAAAAAAGAG DC1 grown on ergosterol indicated an additional peak iden- -6 GAAAAGATGTCTGCCGTTTTCAACAACGCTACCCTTTCAGGTCTAGTCCAAGCAAGCACC tified as 4,4-dimethylzymosterol (Fig. 3). DC1 was crossed to 1 M S A V F N N A T L S G L V Q A S T wild-type strain WA6 so that the ergosterol mutation (erg25- 54 TACTCACAAACTTTGCAAAATGTCGCCCATTACCAACCTCAATTGAATTTCATGGAGAAA 25C) would segregate from erg]. The sterol profile of erg25- 18 YS Q T L Q N VA H Y Q P Q L N F M E K 25C grown on ergosterol is presented in Fig. 3 with peaks A and 114 TACTGGGCCGCATGGTACAGTTACATGAACAATGATGTTTTGGCCACCGGTCTAATGTTC C representing ergosterol and 4,4-dimethylzymosterol, respec- 38 Y W AA W Y S Y M N N D V L AT G L M F tively. Peak B represents low levels of accumulation of lanos- 174 [TTITATTGCATGAATTTATGTATTTCTTTAGATGTTTGCCATGGTTCATCATCGACCAA terol and the retention times of these two sterols are consistent 58 F L L H E F M Y F F R CL P W F II D Q with Kuchta et al. (16) and Maitra et al. (17). 234 ATTCCATACTTTAGAAGATGGAAGTTACAACCAACTAAGATTCCAAGTGCTAAGGAACAA GC/MS of erg25 Sterol Profile. Identification of the sterol 78 I P Y F RR W K L Q P T K I P S A K E Q eluting immediately after lanosterol in erg25-25C as 4,4-di- 294 CTATACTGTTTGAAATCCGTTCTTCTATCTCATTTCTTGGTCGAGGCCATCCCTATCTGG methylzymosterol was confirmed by its mass spectrum and its 98 L Y C L K S V L L S H F L V E A I P I W relative retention time. The molecular ion was observed at 412 354 ACCTTCCACCCAATGTGTGAAAAATTAGGTATTACTGTCGAAGTTCCATTCCCATCTTTG Da and the fragmentation pattern (Fig. 4) was identical to the 118 T F H P M C E K L G I T V E V P F P S L spectrum of 4,4-dimethylzymosterol observed by Byskov et al. 414 AAAACAATGGCTCTAGAAATTGGTCTATTCTTCGTCTTGGAAGATACATGGCATTACTGG (18), who based their identification of lanosterol and 4,4- 138 K T M A LE I G L F F V L E D T W H Y W dimethylzymosterol on mass spectra and 13C NMR. Further- 474 GCTCACCGTCTATTCCACTACGGTGTCTTCTACAAGTACATTCACAAGCAACATCACAGA more, Maitra etal. (17), using an OV- 17 column, observed that 158 A H R L F H Y G V F Y K Y I H K 0 H H R 534 TACGCTGCTCCATTCGGTCTTTCTGCTGAATATGCTCATCCTGCTGAAACTTTGTCTTTG 4,4-dimethylzymosterol eluted as a shoulder on the lanosterol Y A A P F G L S A E Y A P A E T L S L peak and since OV-17 and DB-5 are both methyl/phenol- 178 H 594 GGTTTTGGTACCGTTGGTATGCCAATTCTTTACGTCATGTACACTGGTAAATTACACTTG coated siloxanes, the relative retention time of the 4,4- 198 T V G P I Y T K L L dimethylzymosterol to lanosterol should be comparable for G F G M L Y V M G H 654 TTCACTCTATGTGTATGGATCACCCTAAGATTATTCCAAGCTGTTGACTCTCATTCTGGT both columns. This analysis is also completely consistent with 218 F T L C V W I T L R L F Q A V D S H S G the retention times and mass spectral data of Kuchta et al. (16) 714 TATGACTTCCCATGGTCTTTGAACAAGATCATGCCATTCTGGGCTGGCGCTGAACACCAC using an OV-1 capillary column. 238 Y F P W S L N K I M P F W A G A E H H Cloning and Disruption ofERG25. Mutant strain erg25-25C D 774 GATTTGCATCATCACTACTTTATTGGTAACTACGCTTCCTCTTTCAGATGGTGGGATTAC was transformed and grown on minimal medium plates con- 258 L H H H Y F I G N Y A S S F R W W D Y taining ergosterol with all nutrients except uracil. All trans- D 834 TGTCTAGACACTGAATCTGGTCCAGAAGCTAAGGCCTCCAGAGAAGAAAGAATGAAGAAG formants were then replica plated onto the same medium 278 C L D T E S G P E A K A S R EE R M K K except that ergosterol was omitted. Two plasmids, pERG25-1 894 AGAGCTGAAAACAATGCTCAAAAGAAGACTAACTAAGAGAAGAAACATACTTCAAAAAAA and pERG25-2, restored wild-type growth to erg25-25C. Ex- 298 R A E N N A Q K K T N - posure of these cells to 5-fluoroorotic acid confirmed that 954 AAAAAAAGAAAAACAACAAAAAAACGTATAAAATGAAATAAATTTCGAACCGCTTTTTTT complementation was plasmid mediated and not due to re- 1014 CCi11 ACTTTGTTTGACCTCCCCTAACTCTTTCTTTTTACCTTCACAAT version. pERG25-1 and pERG25-2 contained inserts of 9.3 1074 TGTTTAlIAATATATGATA AAATAATTCAGGTTAATTCTAAAAACTATATTGCA 1134 ATCTTTAAATATATGTATACACGTTCATTACCACGTATACATATCCATATATATATATA and 11.1 kb, respectively, and both contained identical BamHI 1194 TATATATACATACTATrTATGATAAAITTAACAAGCAAACCCATTATTAAATGCTATA fragments of 2.4 and 3.5 kb. Fig. 5 shows that the ERG25 gene is contained within the 3.5-kb BamHI fragment (pIU705) as FIG. 6. Nucleotide sequence of the ERG25 gene and deduced well as in the 1.35-kb Dra I fragment (pIU707). pIU705 was amino acid sequence. First nucleotide of the start codon is numbered deleted for an internal 0.4-kb Xba I fragment and this newly 1 and all upstream sequences are negatively numbered including two generated plasmid pIU709 failed to complement. possible TATA motifs beginning at -182 and -84 bp. Start codon of An ERG25 null allele was generated as follows. The Xba I the 308 amino acid ORF is overlined as are the stop codon and the fragment of pIU709 was replaced by an -1.2-kb Xba I TATA motifs. Histidine-rich motifs are underlined. fragment containing the URA3 gene. The entire 2.2-kb frag- three histidine motifs-H'59RLFH, H172KQHH, and ment of pIU802 was used to disrupt a wild-type diploid yeast H256HDLHHH. In addition, ERG3, another iron nonheme strain WA1/6. Upon sporulation, the recovered erg25A::URA3 enzyme, contains three histidine-rich clusters (D. Gachotte was auxotrophic for ergosterol and accumulated the C-4,4- and P. Benveniste, personal communication). Shanklin et al. dimethylsterols, and when mated with erg25-25C the diploid (22) surmise that these histidine residues may act as ligands for did not complement. the iron atom(s) contained in these enzymes. These observa- DNA Sequence of the ERG25 Gene and Predicted Amino tions support the conclusion that ERG25 is the structural gene Acid Sequence of the C-4 Methyl Sterol Oxidase. We deter- for the C-4 methyl oxidase. mined 2.2 kb of DNA sequence from the complementing between 3.5-kb BamHI fragment cloned into Bluescript pBS (SK+). Two other cases of amino acid sequence similarity This DNA fragment revealed a potential ORF of 309 amino ergosterol biosynthetic genes are the C-24(28) reductase acids. Two putative TATA box promoter sequences appear at (ERG4 encoded) and the sterol A.14-reductase (ERG24 en- -182 and -84 bp (Fig. 6). The gene encodes a predicted coded) in which there is 34% identity over the last 100 36.48-kDa protein similar to the 29-kDa protein reported for C-terminal amino acids. However, the conserved sequence the purified enzyme from rat liver microsomes (4). The may simply reflect that both enzymes are reductases. There is Kyte-Doolittle hydropathy plot (19) suggests at least three and also 16.7% identity between ERG3 and ERG4 in a 97-amino possibly four large hydrophobic regions and a C-terminal acid overlap. hydrophilic region. The former is suggestive of a membrane- The ORF showed 17.7% identity with ERG3 We thank Bristol-Myers Squibb for the yeast strain SGY688, Dr. Leo bound enzyme. Parks for upc2, and Dr. David Stocum for use of various laboratory (C-5 sterol desaturase; see ref. 20) over a 282-amino acid equipment. We also thank Drs. Irmi Becker and Matthias Rose of overlap and both ORFs have the C-terminal retrieval signal for Martinsried Institute for Protein Sequencing for confirming the amino endoplasmic reticulum resident enzymes, KKXX (21). Shan- acid sequence of ERG25. This work was supported by a Johnson & klin et al. (22) reported that eukaryotic membrane desaturases Johnson Focused Giving Award to M.B. and N.D.L. and National as well as the bacterial alkane hydroxylase and xylene mono- Institutes of Health Grants iR15 GM 45959-01 and iROl A138598-01 contain three histidine-rich motifs HX(3or4)H, to M.B. B.B. was supported by National Science Foundation Grant HX(2or3))HH, and HX(20r3)HH. The ERG25 ORF contains MCB-922099 to Drs. D. Crowell and S. Randall. Downloaded by guest on September 29, 2021 190 Genetics: Bard et al. Proc. Natl. Acad. Sci. USA 93 (1996)

1. Miller, W. L., Kalafer, M. E., Gaylor, J. L. & Delwicke, C. V. 11. Rose, M. D., Novick, P., Thomas, J. H., Botstein, D. & Fink, (1967) Biochemistry 6, 2673-2678. G. R. (1987) Gene 60, 237-243. 2. Williams, M. T., Gaylor, J. L. & Morris, H. R. (1977) Cancer Res. 12. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 37, 1377-1383. Cloning (Cold Spring Harbor Lab. Press, Plainview, NY). 3. Faust, J. L., Trzaskos, J. M. & Gaylor, J. M. (1988) in Biology of 13. Sikorski, R. S. & Hieter, P. (1989) Genetics 122, 19-27. Cholesterol, ed. Yeagle, P. L. (CRC, Boca Raton, FL), pp. 19-38. 14. Rothstein, F. J. (1983) Methods Enzymol. 101, 202-211. 4. Maitra, U. S., Mohan, V. P., Kochi, H., Shankar, V., Adlersberg, 15. Molzahn, S. W. & Woods, R. A. (1972) J. Gen. Microbiol. 72, M., Liu, K.-P., Ponticorvo, L. & Sprinson, D. B. (1982) Biochem. 339-348. Biophys. Res. Commun. 108, 517-525. 16. Kuchta, T., Bartkova, K. & Kubinec, R. (1992) Biochem. Biophys. 5. Fukushima, H., Grinstead, G. F. & Gaylor, J. L. (1981) J. Biol. Res. Commun. 189, 85-91. 17. Maitra, U., Mohan, V. P. & Sprinson, D. B. (1989) Steroids 53, Chem. 256, 4822-4826. 597-605. 6. Lees, N. D., Skaggs, B., Kirsch, D. R. & Bard, M. (1995) Lipids 18. Byskov, A. G., Andersen, C. Y., Nordholm, L., Thogersen, H., 30, 221-226. Guoliang, X., Wasserman, O., Andersen, J. V., Guddal, E. & 7. Plemenitas, A., Havel, C. M. & Watson, J. A. (1990) J. Biol. Roed, T. (1995) Nature (London) 374, 559-562. Chem. 265, 17012-17017. 19. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. 8. Vanden Bossche, H., Marichal, P., LeJune, L., Coene, M.-C., 20. Arthington, B. A., Bennett, L. G., Skatrud, P. L., Guynn, C. J., Gorrens, J. & Cools, W. (1993)Antimicrob. Agents Chemother. 37, Barbuch, R. J., Ulbright, C. E. & Bard, M. (1991) Gene 102, 2101-2105. 39-44. 9. Lewis, T. L., Keesler, G. A., Fenner, G. P. & Parks, L. W. (1988) 21. Shin, J., Dunbrack, R. L., Lee, S. & Strominger, J. L. (1991) Proc. Yeast 4, 93-106. Natl. Acad. Sci. USA 88, 1918-1922. 10. Sherman, F., Fink, G. R. & Hicks, J. (1986) Methods in Yeast 22. Shanklin, J., Whittle, E. & Fox, B. G. (1994) Biochemistry 33, Genetics (Cold Spring Harbor Lab. Press, Plainview, NY). 12787-12794. Downloaded by guest on September 29, 2021