Gene, 107 (1991) 155-160 0 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/91/SO3.50

15.5

GENE 06118

The gene encoding squalene epoxidase from ~~c~~ar~~yces cerevisiae: cloning and characterization (Recombinant DNA; terbinafine; sterol biosynthesis; antifungals; ERG1 gene)

Anita Jandrositz, Friederike Turnowsky and Cregor HGgenauer Institutftir ~i~obiQio~.e, Karl-FraPlzens-Univers~t~tGraz. A-8010 Graz (AustriaJ Received by J. Marmur: 11 February 1991 Revised/Accepted: 16 May/26 June 1991 Received at publishers: 26 August 1991

SUMMARY

The gene (ERGI) encoding squalene epoxidase (ERG) from Sacchuromyces cerevisiae was cloned. It was isolated from a gene library, prepared from an ahylamine-resistant (AIR) S. cerevisiae mutant, by screening transformants in a sensitive strain for AIR colonies. The ERG tested in a cell-free extract from one of these transformants proved to be resistant to the Al derivative, terbinafine. From this result, we concluded that the recombinant plasmid in the transformant carried an allelic form of the ERG1 gene. The nucleotide sequence showed the presence of one open reading frame coding for a 55 190-Da peptide of 496 amino acids. Southern hyb~dization experiments altowed us to localize the ERG1 gene on yeast chromosome 15.

INTRODUCTION

Sterols are characteristic lipid membrane components of eukaryotic cells. Fungi as lower eukaryotic organisms produce ergosterol, while cholesterol, its close chemical homologue, occurs in animals tissues. However, both substances are formed by very similar biosynthetic processes, the early steps being identical. The characteristic feature of sterol biosynthesis is the cyclization reaction of squalene to lanosterol by a specific enzyme, squalene cyclase. The substrate for this enzyme is an oxidized metabolite of squalene, 2,3-oxidosqualene (squalene epoxide). The formation of

Correspondence to: Dr. F. Turnowsky, Institut fur Mikrobiologie, Universit% Graz, Universitiltsplatz 2, A-8010 Graz (Austria) Tel.(+43)316-3805620; Fax(+43)316-382130. Abbreviations: aa, amino acid(s); Al, ~lyl~ine(s); bp, base pair(s); d, deletion; ERG, squalene epoxidase; ERGI, gene encoding ERG; ERGf-3, allelic form ofERG coding for a resistant ERG; kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; R, resistant; R,, relative migration distance on TLC plates; S., Saccharontyces; ss, single strand(ed); Tb, terbinafine; TLC, thin-layer chromatography; [ ] denotes plasmid-carrier state.

squalene epoxide is a key reaction in sterol formation because absence of oxygen prevents sterol biosynthesis. A simpli~ed scheme of ergosterol biosynthesis, starting from the important intermediate mevalonate, is shown in Fig. 1A. Squalene epoxidase (ERG) has been purified from rat liver as a constituent of the particulate microsome fraction, where also the enzymes for all subsequent reactions are localized. The rat liver 51-kDa enzyme consists of a single polypeptide chain (Ono et al., 1982). For the epoxidation reaction the presence of NADPH and FAD is required. Since the rat liver ERG contains no heme and is also not inhibited by classical inhibitors of cytochrome P450 it is believed to transfer oxygen by a mechanism different from that of cytochrome P450. The rat liver enzyme requires a protein cofactor for maximal activity. Knowledge of the reaction mechanism of ERG from yeast and other fungi is very incomplete. No extensive purification of the enzyme has been described. However, unlike in the rat liver system, partially pure enzyme preparations from yeast microsomes could not be stimulated by other cytoplasmic proteins (Jahnke and Klein, 1983). ERG is the target of a novel class of antimycotic drugs, the allylamines (Al) (Paltauf et al., 1982; Ryder and DuPont, 1985). They

156 are particularly cases (Petranyi

effective in the treatment of dermatomyet al., 1984). As a result of the inhibition of

ERG, Candida and Trichophyton species accumulate quantities of squalene, up to 600-fold over normal (Paltauf

et al., 1982). In addition

SW&W

Squakne

large levels

to the lack of ergosterol, AlLYlnMlNES

this squalene accumulation may be one of the factors leading to cell death. As a first step of a program to study the ERG enzyme of yeast in detail we cloned its gene. We took advantage of the availability of AIR mutants of S. cerevisiae and we utilized them for the selection of the clones.

EXPERIMENTAL

AND

ewxide

,111

..&

-

-

Yx&T

DISCUSSION

The strategy for cloning the ERG gene, which was designated ER GI by Karst and Lacroute (1977), included (I ) the generation of AIR yeast mutants, (2) selection of mutants which produced an AIR ERG, (3) preparation of a gene library from one of these mutants and (4) transformation of this library into a sensitive yeast strain followed by selection of AIR transformants. Fig. 1. Ergosterol

(a) Generation of AIR mutants For the generation of resistant mutants the haploid S. cerevisiae strain A2 and the most active Al derivative, Tb (Fig. 1B) were used. Mutations were introduced by UV irradiation of a cell suspension until only 1 y0 of the cells survived. The suspension of the survivors was spread on Tb-containing agar plates. Of 250 AIR colonies only 15 could be cultivated in liquid media in the presence of Tb. Three of these mutants produced an AIR ERG as was shown by the analysis described below. The minimal inhibitory concentration for the wt was 12.5 pg Tb per ml, that for the mutants M8, M27 and M3 1 was 100,100 and 50 pg Tb per ml, respectively. (b) Screening for mutants with AIR ERG The screening procedure involved the preparation of cellfree extracts from all 15 mutants which were able to grow in drug-containing liquid media. The cell-free extracts were incubated in the presence of [ “C]mevalonate as described by Ryder et al. (1984). The products of the reaction were extracted with petroleum ether and analyzed by TLC. The positions of the radioactive spots on the TLC plates were determined. Squalene and lanosterol as reference compounds could easily be discriminated by their R,values. We did not attempt to separate all the cyclic sterol metabolites. When extracts from the wt S. cerevisiae strain A2 were tested by this method, incubation in the presence of 100 pg Tb/ml caused a 76% decrease in radioactivity in the spot corresponding to sterols (Fig. 2A). This result is in accord with the previously described inhibition of ERG from Cundida albicans by this antifungal

of ergosterol

biosynthesis

biosynthesis.

and its inhibitors.

The steps leading

(A) Simplified to the formation

scheme of me-

valonate as well as the intermediate stages after the cyclization reaction are omitted. The Al-sensitive reaction is specifically designated. (B) Structure and Petranyi

of Tb (SF86-327).

The compound

is described

by StWz

(1984).

compound (Ryder and DuPont, 1985). In contrast, the three mutants, M8, M27 and M31, showed only a slight reduction of radioactivity in the sterol spot when the reaction was performed in the presence of Tb. The data for the mutant M8 are shown in Fig. 2B. We calculated an inhibition of 24% of the sterol formation. The AIR mutants which contained a sensitive enzyme were not investigated further. We assume that their resistance phenotype was due to an impaired uptake of the drug. (c) Generation of a gene library from the mutant Succharomyces cerevisiae A2-M8 Chromosomal DNA was prepared from S. cerevisiue A2-M8 and partially digested with Sau3A. The fragment mixture was ligated to the shuttle vector plasmid YEp351 which had been linearized with the restriction enzyme BumHI. Escherichia coli cells were transformed with the ligation mixture, the transformants selected with epicillin and plasmid DNA was prepared from a pool of approx. 30 000 colonies. (d) Transformation of a sensitive yeast strain S. cerevisiue A2 sphaeroplasts were transformed with this gene library and selected in a first step for growth on

157 0

EL

S

F

I

tt

t

t

a minimal medium lacking leucine. The transformants were pooled and grown in Tb-containing liquid minimal medium

I

‘.”

for several generations. The cells were subsequently spread on Tb-containing minimal plates lacking leucine. The plasmid DNA of individual AIR yeast colonies was obtained after propagation in E. coli and subjected to restriction analysis. Out of 20 transformants, seven contained a 4.8-kb insert that always showed the same restriction pattern indi-

!

0

0

15

10

5

25

20

(cpmI~lOoO 71

-0

I5

5

Fig. 2. Squalene formed

epoxidase

according

incubating

(ERG)

to Ryder

1 &i

assay

for 3 h. Cell extract

mixture

acid

a substrate-

period, the reaction cocktail

for 10 min either

was started

(Ryder

its AIR mutants Darmstadt,

after growth

by the addition

and suspended

or from one of (Merck,

harvestingand washing, the cells were

in 0.1 M KHZPO,

pH 7.4 The volume

buffer was equal to that of the pellet. The cells were broken the suspension Melsungen

three

times

homogenizer.

for 1 min with

The homogenate

10000 xg and the supernatant around

saved.

10 mg/ml. The reaction

15% KOH

in 90% ethanol

lipids were extracted

dissolved (No. 5719, lanosterol

and heating

Merck,

compounds.

concentration

F.R.G.).

On

each

was

of 1 ml of The non-

ether, the orgaThe residue

and applied to a silica-gel thin-layer

Darmstadt,

and squalene

in a Braunfor 10 min at

for 30 min at 90°C.

and dried in vacua.

of the

by shaking

by the addition

with 2 ml of petroleum

with water

in 30 ~1 cyclohexane

beads

The protein

was terminated

saponifiable

nic phase was washed

glass

was centrifuged

plate

was plate

ergosterol,

(Sigma, St. Louis, MO) were applied as reference

The plates were developed

in chloroform.

The radioactive

spots were localized by dividing the length of each lane into l-cm-wide strips, scraping off the silica gel from each strip and counting them after mixing with scintillation

fluid (Ready

CA) in a liquid scintillation vs. migration

distance.

protein +

counter.

The curves

The reference

compounds

, Beckman, represent

lar extract of one of the yeast transformants was tested for the presence of an AIR enzyme. As described in section b radioactive mevalonate was used as a substrate for the enzymatic reaction with and without drug. The products were separated by TLC and quantitated by measuring their radioactivities. The data are shown in Table I. Clearly, the enzymatic activity of the transformant A2[pAF22] is as resistant to Tb as is that of the parent mutant A2-M8. We interpret this result to mean that the insert of pAF22 indeed carries the ERG1 gene. Subclones of pAF22, namely pAF22 1 and pAF222, but not pAF223, conferred AIR phenotype when transformed into S. cerevisiae A2 (Fig. 3). This allowed a rough localization of the gene between the PstI and the rightward Sac1 sites of the insert. Therefore we sequenced the DNA segment extending from the multiple cloning site to the PstI site.

of 60 ~1 of

et al., 1984). The cell lysate was

in 500 ml Sabouraud-Bouillon

F.R.G.) at 30°C. After

with

or with solvent alone. After the

from either S. cerevisiae A2: (a, leu2, hti3, cad)

prepared

pelleted

of a cell lysate at 30°C

(930 ~1) was preincubated

cofactor

was perof 1 ml by

(specific activity 2 @i/mol)

in the presence

100 pg ofTb in 10 ~1 of dimethylsulfoxide preincubation

by TLC. The assay

et al. (1984) in a total volume

DL[2-‘4C]mevalonic

with a salt- and cofactor

25

20

[cm]

‘O

cating that they were of the same clonal origin. One recombinant plasmid was selected and designated pAF22. Sensitive S. cerevisiae A2 cells were again transformed with pAF22 DNA which resulted in a large number of AIR yeast colonies. This proved that the insert of pAF22 was responsible for the AIR phenotype. However, in order to demonstrate that we had indeed cloned the ERG1 gene, the cellu-

(e) Nucleotide sequence and analysis of ORFs The sequence was established by the dideoxy chain-termination method (Sanger et al., 1977) using both universal and specific oligo primers. Both strands were sequenced in the ss form. The sequence was analyzed using the GCG software package (Devereux et al., 1984). Only one complete ORF comprising 1488 bp was found. It codes for a 55 190-Da protein of 496 aa (Fig. 4). This M, is in good agreement with that of the rat liver ERG, which was determined by conventional methods (Ono et al., 1982). In addition to the ERG1 gene we found the beginning of another ORF in the opposite direction. Data banks were searched for homologies with the aa sequences derived from both gene ERGI and the incomplete ORF. No homologies were detected. The 296-bp DNA segment between the ERG1

Palo Alto, radioactivity

were visualized

under

a UV lamp and their positions are marked by arrows. Note that the scales ofthe ordinates differ in panels A and B. Abbreviations: E, ergosterol; F,

solvent front; L, lanosterol;

0, origin; S, squalene.

ERG assay with an

extract from S. cerevisiae A2 (wt) (panel A) or A2-M8 (AIR mutant) (panel B) in the presence (0) and absence (*) of Tb (100 pg/ml).

158 TABLE

I

Inhibition

of ERG in cell-free extracts

S. cerevisiae strain”

from wt, AIR mutant

Radioactivity

Saccharomyces cerevisiae strains

and transformed

in % of total TLC counts”

Uninhibited

reaction

y0 inhibition Inhibited

Sterols

Squalene

of sterol synthesis

reaction’

Sterols

Squalene

A2

33

63

8

88

76

A2-M8

46

42

35

55

24

A2[pAF22]

53

41

43

53

19

were those described

in Fig. 2 legend.

i) M8 is the AIR mutant. h The assay conditions c Tb concentration:

100 ug/ml.

[ERGlQr] ORF genotype resistance

+ s

SC

ESCS

SmP I==+

+

i

nd X

nd

F$

nd

P

-1ida

Fig. 3. Physical

map of the insert of pAF22

pAF22 was obtained

Chromosomal

AIR mutant by standard procedures digested with Sau3A. The fragment of the shuttle plasmid

YEp351,

DNA was extracted

linearized

with BumHI.

E. co/i MC1061

hsdR, hsdkf, rpsL} was

1982). The aa sequence of Fig. 4 is derived from an allelic form of ERG1 coding for an AIR enzyme. We have not yet sequenced the wt gene. The aa sequence was analyzed for hydrophobic domains by the method of Kyte and Doolittle (1982) (Fig. 5). At the C terminus a hydrophobic domain starting at aa 464 could be detected. The microsomal loca-

with the ligation mixture and spread on epicillin (a 1,4-cyclo-

hexadienyl

analogue

of ampicillin,

plates. Approximately DNA

from the

(Ausubel et al., 1987) and partially mixture was ligated with the DNA

{oraD 139, A(aru, leu)7697, A(lac)X74,galU,galK, transformed

Plasmid

a gene library from S. cerevisiae A2-M8

by screening

(a, leu2, his3, cunl, ERGI-3).

and its subclones.

gene and the beginning of the other ORF is A + T-rich. The most prominent feature within this DNA stretch is a run of 23 T’s, interrupted by two C’s, extending from nt 333-358 (Fig. 4). Such stretches are commonly found in intergenic regions in the nuclear genome of yeast. They can function bidirectionally as promoter elements, if combined with downstream TATA boxes (Struhl, 1985a,b). A TATA box for the ERG1 gene may be present within a run of nine consecutive TA repeats, ranging from nt positions 369-386. T-rich upstream regions have been associated with constitutively expressed genes because of their perturbation of local nucleosome structure formation. However, recent studies have shown that in yeast poly(dA-dT) stretches are recognized by specific binding proteins (Winter and Varshavsky, 1989; Lue et al., 1989). Hence, it remains to be tested whether expression of gene ERG1 is constitutive or regulated. It is of special interest if gene ERG1 is activated by oxygen or repressed by glucose. Downstream from the gene, at nt position 2070, the sequence ATAAA could function as polyadenylation signal (Zaret and Sherman,

was

sphaeroplasts

Biochemie

30000 transformants

prepared. (Ausubel

library.

The transformants

taining

SD medium

S. cerevisiae A2

Kundl, Austria)-containing were collected cells

were

et al., 1987) and transformed were allowed

(Sherman

to regenerate

and plasmid

converted

into

MC1061.

The individual

endonuclease

samples

insert is shown in the figure. Subclones

in soft agar con-

SmaI treatments

et al., 1982) with 19 aa, lacking

leucine.

represent

and pAF226

were spread on To-containing

agar

plates. Twenty independent colonies were selected and grown in liquid SD medium without leucine but in the presence of Tb. Plasmid DNA was and the DNA propagated

in E. coli

were analyzed

by restriction

and recombined

were prepared

with appropriately

map of its

by PstI, SocI, and cleaved YEp351, to

give pAF221, pAF222 and pAF223, respectively. Only the former two confer Tb resistance to sensitive host cells. In the figure, open boxes

three times in fresh medium. Aliquots

from each of the cultures

DNA

and pAF22 was selected. The physical

with the plasmid

Colonies were collected, pooled and grown in liquid SD medium without leucine in the presence of 100 ,ng of Tb per ml. The culture was passaged

prepared

digestion

inserts,

thin lines vector

are recombinants

used only for preparing

DNA.

Plasmids

with pBluescript

ss DNA.

The two ORFs

arrows. B, BarnHI; E, EcoRI; H, HindHI; Sm, SmuI; X, X&I; nd, not determined;

pAF224,

SK( + /-).

pAF225

They were

are represented

P, Psrl; S, Sau3A; + , AIR; -, AIS.

by

SC, SacI;

159 100

GATCG7GGCGMtlGGGM7CGttCtCCMtCtCltCTACCUUCCAyCGGCWUTltGCGTCGCtltMTGCWItACtGC:GTAGCGGGCCTfCGTATA

1

RPSNP,lRCARRGFYRRIPTAKIR~4RLPGEY 200

GCTCGGCCG*GCTCCTAC~GG~GCAGtGtATCGGACAGAGClGAlATMCAC~tACGClCCTAGYC~lGCATGCCGtGGClGCTCTCGGTCCE

101

L

E

A

s

s

t

E

F

A

L

L

T

D

5

L

A

Siv

C

Y

A

R

R

L

HI(ORF

201

GTAtMGtCTTAGACMTAGtCTTACCTtGCATGTAT~l~TCTltIGlAlllMTClAlIAlATGTT?ClAlGCtltlTTTTCClATTGTlGITTGC

300

301

tt7tCCTTTTCCTtATT7CTTtCTAGCllCTMllTTClTTCTTlTTllllttTTtlTCAll~tlAlATAt~AtA~tCA~CMttGtC

400

LO1

CAGTATtGAACAAtACAGGTTAltlCGAACMTTG

~TCACAGAAMACATAlCGAGAAAAGGGtCAlG7CTGCTGTTAACGtTGCACCtG ERGI

w

s

A

500 v

N

v

A

P

E

MlTtAttMtGCCGACMCA,WITtACCTACWTGCtJiTTGTCAfCGGTGCTGGTGTTATCGGTCCITGTGyTGCTACTGGTCTAGCMG~GGGTM

501

600

LIYADWtlTYDAlVlGAGVIGPCVATGLARKGK GAAAGtTCTlATCGTAW*CGTWCTGGGCTATCCCTt*TAGMttGTTGGTtUTTGATGCMCUGCTGGtGTTAWGEATTUGMGtCtGGGTATG

601

700

KVLI”ERDYAMPDRIVGELY4PGGVRALRSLGN AtTCIU?CTATCMW*UTCGMGUTI’ICETGTTACCGGTTATACffilCTTTTT~CGGC~~GTT~TATTCUTACCCTTAWGGCCt*TA

701

a00

I4SINNIEAyPVTGYfVFFNGE4VDlPyPYKADI tCCCtlUIGtt~TTGMGGACttGGTC~GATGGTM7GACMGGTCTTGGUG*WGC*CtATtCACATCAIIGGATTACGMGATGLTG~G

801

900

PKVEKLKDLVKDGNDKVLEDSTlHIKDYEDDER 1000

AGAMGGGGTGTTGCtTttGtTUlGGTA~TTCtt~CMCtTGAGWCATtACtGCt~CIGCC~TGTTACTAGAGtG~GGtMCtGTAtT

901

ERGVAFVNGRFLNNLRNITA9EPNVTRVOGNCt tAGAtATYGMGGATWAAAGMtWGGTtGTTGGTGCCMGGItGACATTWtGGCCGTGGCMGGtG~tTC*MGCCCACTTWCATTtATCTGTG

1001

1100

EiLKDEKNEVVGAKVDIDGRGKVEFKAHLtflCD 1200

ACGGTAlCTTTT~CGTTTUWMGWUTTGUCCUGACCAtGTTCCMCTGTCGGTTCTTCGtTtGTCGGIATGlCTTTGTTCMTGClMG~lCC

1101

GIFSRFRKECNPDHVPTVGSSFVGMSLFNAKNP tGCTCCtAtGCACGGTUCGTtAtTTtlGGTAGtGATCATATGC~TCtTGGtTlACC~TCAGtCCAGMG~CM~tCCtttGTGClTACMC

1201

1300

APMHGNVIFGSDNMPILVY4ISPEETRlLCAyN tCTCUMGGtCCCAGCTGATATCMGAGtTGGATWTTMGG*tGtCCMCCTtTCATTCC*UWGtCTACGTCCTTCATTtCATGMGCCGlCAGCC

1301

1400

SPKVPADIKSWWIKDVPPFlPKSlRPSFDEAVSO *AGGTAMTTtACIGCTATGC~CTCClACTt~CAGCTA~C~C~CGl~CTGGTAtGTGtGtlAtCffit~CGClCT~tAt~~CAtCC

l&O1

1500

GKFRAWPNSYLPARONDVlGXCYIOD*LYWIHP 1600

ATt~CtGGTGGTGGTATt~tGTC~TltG~T~TGlTGTCTTGTT~lTM~tAGGT~CCtA~CtT~GC~~CGT~GGTTtTG~T

1501

LtGGGWyVGLWDVVLLlKKIGDLDFSDREKVLD 1700

GM?TACtAWCTACUTTTCGWCIM~GTTAC~TTCCGTtAllMCGTTTTGtCAGtGGCTttGlAttCTtlGttCGCtGCTC*CAGCCAtMCT

1601

ELLDYNFERKSYDSVINVLSVALYSLFAADSDNL t~CGCITTACUMIGGTTGTTTChMtATTTCC*MGAGGltCCCATlGTGTWCUACCCGttCUyttCTGTCTGGTGTCTTGCCM*GCCtTT

1701

1800

KAL4KGCFKYF4RGGDCVNKPVEfLSGVLPKPL 1900

GCMtT~C~AGGGTTtTfTTCGCtGTCGCTTTTTAUCUtTtACTTCMCATGtUWICGTGGtTtClTGG~lTAC~TGGCTtTATTGGMGGT

lLlO1

4LTRVFFAVAFYl~YLNXEERGflGLPKALLEG 2000

AlTATGATTTTtATUlCAGCTATlA~GlATTUCCC~TTTTTGTllGCT~GTTUTTGGTTMCtA~GClTAtMGG~~GAGGATAG~~CG~

1901

IUIL17AIRVfTPFLFGELIG’ 2100

2001

WA*CAttMGCTGCACCTtTtTTtlllATtACA~GTCGGCTtG~GGCttGTATAGTACATtAC~~~~TCtTAtlttTAtTtATTACTtA

2101

TtTAtlTtACAtAtTtTCAAAAAAA

2201

GGAAAMTGGCTTGATATGCTTCMUtATGCttAGAWTTUTATtCGGtTATCMTTMCWTAlTACTTCCtTMGTGAtAtTAMTCMGCTTGCC

2301

GGATTCTGCAG

2200

TTCACAtATUTTTATTATtMCCGMGTGTTttATACTTTTtGftCtITCCTt~tGCCtCCAACAGAMA

2300

2311

Fig. 4. The nt sequence of a Suu3A-Psi1 fragment from S. cerevisiae A2-MS. The Sau3A site at the be~nning of the sequence is the ligation site with the vector DNA. The &I site at the end of the sequence is that which occurs in the middle of the original insert of pAF22. The derived aa sequences are represented by the one-letter code (aa are aligned with the first nt of each codon). ERGI begins at nt position 476 and ends at 1963. Promoter sequences and the putative polyadenylation site are underlined, the direction of translation is indicated by arrowheads above the ATG codons. An asterisk indicates a stop codon. The sequence reported in this article has been assigned the accession No. M64994 by GenBank.

tion of ERG (Jahnke and Klein, 1983) would require either a direct membrane anchor of the protein, or else be due to a strong association with another membrane protein. It remains to be seen whether this hydrophobic stretch indeed functions as a membrane anchor or whether a second protein, possibly as part of the electron transfer chain, attaches squalene epoxidase to the membrane.

probe was synthesized from the PstI fragment of pAF22 1. It was hybridized to a commercially available blot of S. cerevisiae chromosomes separated by pulsed-tield electrophoresis (Clontech Inc., Palo Alto, CA). One clear signal corresponding to chromosome 1.5was identified (data not shown). (g) Conclusions Plasmid pAF22 carries an allelic form of the gene for squalene epoxidase as was shown by transforming a sensitive yeast strain with this DNA, and subsequently analyzing

(f) Location of ERG1 in the yeast genome Gene ERG1 was localized on chromosome 15 of the S. cerevisiae nuclear genome by Southern blotting. A DNA 0

100

200

F

,‘,“““““,,,

.

.

.

.

Y

.

.

.

.

HPhobic

0 -3

HPhiiiC

;i

“

*

‘tL”’

‘2ba.

*

*

.&’

“

.&’

.

.

.

Fig. 5. Kyte-Doolittle hydropathy analysis (UWCGC Sequence Analysis Software Package, Madison, WI). A window of 9 aa was used.

160 the cellular extract for an AIR ERG. The size of the protein derived from the nt sequence corresponds well with that of the ERG isolated from rat liver. The enzymatic activity in rat liver seems to be associated with a single polypeptide chain, although in addition a cytoplasmic protein is required for full activity. In fungi the situation is less clear. The ERG1 gene product as the principal enzymatic compound may require interactions with other, possibly membrane proteins, for full oxygen transfer function. The gene which we have cloned will allow us to overexpress and purify the protein to study such important questions as its enzymatic activity, the requirement for other accessory proteins and its binding potential for allylamines.

ACKNOWLEDGEMENTS

This work was supported by the Fonds zur Forderung der wissenschaftlichen Forschung, grant No. P6715. We are grateful to A. StUtz, Sandoz Forschungsinstitut Vienna, for a sample of terbinafine (Tb). Strains and plasmids were a kind gift of A. Hartig. We thank G. Koraimann, A. Spok and P. Remler for their help with the computer work.

REFERENCES Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K.: Current Protocols in Molecular Biology. Wiley, New York, 1987. Devereux, J., Haeberli, P. and Smithies, 0.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Jahnke, L. and Klein, H.P.: Oxygen requirements for formation and activity of the squalene epoxidase in Saccharomyces cerevisiae. J. Bacterial. 155 (1983) 488-492. Karst, F. and Lacroute, F.: Ergosterol biosynthesis in Saccharomyces

cerevbiae. Mutants deficient in the early steps of the pathway. Mol. Gen. Genet. 154 (1977) 269-277. Kyte, J. and Doolittle, R.F.: A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157 (1982) 105-132. Lue, N.F., Buchmann, A.R. and Komberg, R.D.: Activation of yeast RNA polymerase II transcription by a thymidine-rich upstream element in vitro. Proc. Natl. Acad. Sci. USA 86 (1989) 486-490. Ono, T., Nakazono, K. and Kosaka, H.: Purification and partial characterization of squalene epoxidase from rat liver microsomes. Biochim. Biophys. Acta 709 (1982) 84-90. Paltauf, F., Daum, G., Zuder, G., Hogenauer, G., Schulz, G. and Seidl, G.: Squalene and ergosterol biosynthesis in fungi treated with naftifine, a new antimycotic agent. Biochim. Biophys. Acta 712 (1982) 268-273. Petranyi, G., Ryder, N.S. and Sttitz, A.: Allylamine derivatives: new class of synthetic antifungal agents inhibiting fungal squalene epoxidase. Science 224 (1984) 1239-1241. Ryder, N.S. and DuPont, M.-C.: Inhibition of squalene epoxidase by allylamine antimycotic compounds. Biochem. J. 230 (1985) 765-770. Ryder, N.S., Seidl, G. and Troke, P.F.: Effect of the antimycotic drug nafiifine on growth of and sterol biosynthesis in Candida albicans. Antimicrob. Agents Chemother. 25 (1984) 483-487. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Sherman, F., Fink, G.R. and Hicks, J.B.: Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, p. 62. Struhl, K.: Nucleotide sequence and transcriptional mapping of yeast pedib-his3-dedl gene region. Nucleic Acids Res. 13 (1985a) 8587-8601. Struhl, K.: Naturally occurring poly(dA-dT) sequences are upstream promoter elements for constitutive transcription in yeast. Proc. Natl. Acad. Sci. USA 82 (1985b) 8419-8423. Sttitz, A. and Petranyi, G.: Synthesis and antifungal activity of (E)-N(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl1-naphthalenemethanamine (SF86-327) and related allylamine derivatives with enhanced oral activity. J. Med. Chem. 27 (1984) 1539-1543. Winter, E. and Varshavsky, A.: A DNA binding protein that recognizes oligo(dA). oligo(dT) tracts. EMBO J. 8 (1989) 1867-1877. Zaret, K.S. and Sherman, F.: DNA sequence required for efhcient transcription termination in yeast. Cell 28 (1982) 563-573.