173 Gene, 107 (1991) 173-174 0 1991 Elsevier Science Publishers B.V. All rights reserved. 037%1119/91/$03.50

GENE 06112

Nucleotide sequence of the gene encoding yeast C-8 sterol isomerase*

(Ergosterol biosynthesis; Succharomyces cerevisiae; recombinant DNA)

Beth A. Arthington”, JoAnn Hoskins’, Paul L. Skatrudb and Martin Bard”

a Department of Biology, Indiana University-Purdue University at Indianapolis, Indianapolis. IN 46202 (U.S.A.), and b Lilly Research Labora- tories, Lilly Corporate Center, Indianapolis, IN 46285 (U.S.A.) Tel. (311)276-7081

Received by J.A. Gorman: 19 June 1991 Revised/Accepted: 16 July/6 August 1991 Received at publishers: 20 August 1991

SUMMARY

The ERG? gene encoding the Sffcc~urorn~ce~ cerevisiae C-8 sterol isomerase, an enzyme involved in plant, animal, and fungal sterol biosynthesis was sequenced. A large open reading frame comprising 222 amino acids was observed.

The ERG2 gene codes for the C-8 sterol isomerase which results in unsaturation at C-7 in the B ring of sterols derived from plants (stigmasterol and @-sitosterol), animals (choles- terol), and fungi (ergosterol). We have previously reported the cloning of this gene from Saccharomyces cerevisiae and the location of the gene to an approximate l.l-kb DNA fragment of the original clone (Ashman et al., 1991). Dis- ruption of the genomic ER G2 gene by insertion of the LEU2 gene into the NdeI site within the coding region indicated that this gene was not essential, even though the C-8 sterol isomerase is a major target of morpholine ~tibiotics (Baloch et al., 1984). The erg2 disrupted strain was nystatin resistant and synthesized sterols consistent with the ab- sence of C-8 sterol isomerase activity (accumulation of ergosta-8-en3&ol, ergosta-5,8,22-trien-38-01, and ergosta- 8,22-3fl-01 sterols). The nt sequence of the ERG_? gene is presented in Fig. 1. A large ORF of 222 aa was observed which could encode a polypeptide of 24.9 kDa. Due to the additional methionines 17 and 30 residues downstream in

Co~es~~nde~ce to: Dr. M. Bard, Department ofBiology, Indiana Univer- sity-Purdue University at Indianapolis, 723 W. Michigan St., Indi- anapolis, IN 46202 (U.S.A.) Tel. (317)274-0593; Fax (317)274-2846. * On request, the authors will supply detailed experimental evidence for the conclusions reached in this Brief Note.

Abbreviations: aa, amino acid(s); ERGZ, gene encoding C-8 sterol isomerase; kb, kilobase( nt, nucleotide(s); oligo, oligodeoxyribonu- cleotide; ORF, open reading frame; S., Succharornyces.

the same reading frame, polypeptides of 206 aa and 193 aa are also possible. The largest ORF includes a hydrophobic region (aa 3 through 21) which may serve to anchor the enzyme to the endoplasmic reticulum (Kyte and Doolittle, 1982). Scanning the SwissProt data base did not reveal any signi~~~t aa homolo~es. The promoter sequence does not contain a TACTTT motif such as that present in a number of early ergosterol biosynthetic genes (Oulmouden and Karst, 1991).

ACKNOWLEDGEMENTS

This work was supported in part by a NIH grant to M.B. (lR15GM45959). We thank Dr. M. Goebl for scanning his data bank for aa sequence homologies.

REFERENCES

Ashman, W.H., Barbuch, R.J., Ulbright, C.E., Jarrett, H.W. and Bard, M.: Cloning and disruption of the yeast C-8 sterol isomerase gene. Lipids 26 (1991) 628-632.

Baloch, RI., Mercer, E.I., Wiggins, T.E. and Baldwin, B.C.: Inhibition of ergosterol biosynthesis of Saccharomyces cereviriae and V&ago maydis by tridemorph, fenpropimorph, and fenpropidin. Phyto- chemistry 23 (1984) 2219-2226.

Kyte, J. and Doolittle, R.F.: A simple method for displaying the hydropa- thic character of a protein. J. Mol. Biol. 157 (1982) 105-132.

Oulmouden, A. and Karst, F.: Nucleotide sequence of the ERG12 gene of &xccharamyces cerevisiae encoding mevalonate kinase. Curr. Genet. 19 (1991) 9-14.

174

-291 . CTAGAGTCTGCTATGTTGATTCTGCTTATTATCAACGAAACTTAT

. CCGCAAGGRAAACTACCGGTGCTATCGTTCTCGTTTGGAT

BalII/PstT.---..- TT~GGATAGATCTGCAGCGCCATGGTATATAAGAGAAAGATG

-238

-178

-118

-~58 5kz.u.L *

GGTTCAGATCTCTCTTGTCGCTCAATCAATCAAACTAAGACTAGCCCAGACCATTATAGC 47 TG 3 Met ' f

AAGTTTTTCCCACTCCTTTTGTTGATTGGTGTTGTTGTA~CTACATTATG~CGTATTGTTC LysPhePheProLeuLeuLeuLeuIleGlyValValGlyTyrIle~etAsnValLeuFhe

. . . ACTACCTGGTTGCCAACCAATTACATGTTCGATCCAAAAA ThrThrTrpLeuProThrAsnTyrMetPheAspProLysThrLeuAsnGlurIeCysAsn . . TCGGTGATTAGCAAACACAACGCAGCAGAAGGTTTATCCA SerValIleSerLysHisAsnAlaAlaGluGLyLeuSerThrGfuAspLeuLeuGlnAsp

GTCAGAGACGCACTTGCCTCTCATTACGGGGACGlLATACATC~CAGGTACGTC~G~ ValArgAspAlaLeuhlaSerHisTyrGlyAspGluTyrIleAsnArgTyrValLysGlu

. GARTGGGTCTTCAACAATGCTGGTGGTGCGATGGG~~~TGAT~ATC~TACA~GCTT~C GluTrpValPheAsnAsnAlaGlyGlyAlaMetGlyGl~e~Ile~le~uHisAlaSer . Scaf - 0 GTATCCGAGTACTTAATTCTATTCGGAACCGCTGTTGGTACTG~~CACACA~TGTT VpllSerGluTyrLeu~leLeuPheGlyThrAlaValG~yT~rGluGlyHisThrGlyV~I

. CACTTTGCTGACGACTATTTTACCATCTTACATGGTACGCAAATCGCAGCATTGCCATA~ HisPheAlaAspAspTyrPheThrIleLeuHisGlyThrGlnIleAlaAlaLeuProTyr

(I ~CACTGAAGCCGAAGTTTACACTCCTGGTATGACTCATChCTTG~G~~GATAC~C AlaThrGluAlaGluValTyrThrProGlyMetThrHisHisLeuLysLysGlyTyrAla . . . AAGCAATACAGCATGCCAGGTGGTTCCTTTGCCCTTGAATTGGCTCAAGGCTGGATTCCA LysGlnTyrSerMetProGlyGlySerPheAl.aLeuGluLeuAlaGlnGlyTrpIlePro . ScaI . . TGTATGTTGCCATTCGGGTTTTTGGACACTTTCTCCAGTACTCTTGATTTATACACTCTh CysMetLeuProPheGlyPheLeuAspThrPheSerSerThrLeuAspLeuTyrThrLeu . . TATAG~CTGTCTACCTGACT~~A~GA~AT~GT~G~~TTGTTG~C~G TyrArgThrValTyrLeuThrAlaArgAspMetGlyLyshsnLeuLeuGlnAsnLySLyS . TT Gik'

CTGCCCGACCTCACGAAATCAGTCATGCGGTAGTCCATTAT~TAT~CTATT Phe

, ATTATTATTACTATTACTATTATTATTATTATTATTATTATCTATTGTTTATATTCTCTT

TAGCTTTTTATCACCTACGACGACGGATATATTCGGCGTTT~~GTG~~CG

TCTTTTGGTGGACATGAACTCAATCTTCTTCTTTGAAA

cr

63 21

123 41

183 61

243 81

303 101

363 121

423 141

483 161

543 181

603 2QY.

663 221

123

783

843

903

905

Fig. 1. The nt sequence of the ERG2 gene and deduced aa sequence. The start codon of the 222-aa ORF and the stop codon are boxed; possible TATA motif and restriction sites are overlined. Those met~ionines which might correspond to the start codons are in capital letters (MET). To sequence both strands of the ERG2 gene, TacTrack Deaxa Sequencing System from Promega (Madison, WI) employing Sanger’s dideoxyribonucleotide triphosphate chain-termination method was used. A 1.5-kb DNA fragment containing the ERG2 gene was subcloned into the bacteriophages M13mp18 and M13mp19 and used to transfect Eschenkhiu coii strain DHSGL using standard protocols. Single-stranded phage DNA was isolated and used as a template for

sequencing. The universal primer complementary to the multiple cloning site of the phage M 13 was used to prime the first sequencing reaction. Oligo primers complementary to the most distal sequence were then used to prime all subsequent reactions. The procedure was carried out until both strands were read. The University of Wisconsin Genetics Computer Group sequence software analysis package was used to determine the 222-aa ORF and to generate Kyte-Doolittle hydrophobicity plot (not shown).