Grrre. 118 (1992) 131-136 0 1992 Elscvicr Science

GENE

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131

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06597

SDHl, the gene encoding the succinate from Saccharomyces cerevisiae (Nucleotide

sequence;

petite mutants;

Karen B. Chapman

Received

by F. Barany:

mitochondrial

*, Sharon D. Solomon*

17 February

1992: Accepted:

proteins;

dehydrogenase

citric acid cycle: electron

flavoprotein

subunit

transport)

and Jef D. Boeke

10 April 1992; Rcccivcd

at publishers:

12 May 1992

SUMMARY

We describe the isolation, sequence and construction of a disruption of the yeast SDHl gene, encoding the flavoprotein subunit of succinate dehydrogenase. This is the first eukaryotic flavoprotein subunit-encoding gene to be fully sequenced. The deduced amino acid (aa) sequence is 50”; identical to the Escherichiu coli enzyme sequence. The yeast gene encodes an N-terminal extension of 45 aa relative to the E. co/i sequence which may act as a mitochondrial targeting signal. Disruption of the gene results in the inability to respire, assayed as the inability to utilize the nonfermentable carbon source, glycerol. This is the expected phenotype for disruption of an essential component of the yeast citric acid cycle.

INTRODIICTION

Succinate is oxidized to fumarate by succinate dehydrogenase (EC 1.3.99.1) a multisubunit enzyme of the citric acid cycle, inside the mitochondria of eukaryotic cells, as well as in bacterial cells. The oxidation of succinate is carried out by the large 70-kDa flavoprotein subunit of the enzyme, which contain FAD covalently bound to a histidyl residue. The abstracted protons are then passed to an iron-

Correspondence to: Dr. J.D. Boeke, Department Genetics,

Johns

Hopkins

School

more, MD 21205, USA, Tel. (301)955-2481; * Present addresses: Pasteur,

F-75724

(K.B.C.) Paris

Department

CEDEX

of Molecular

of Medicine.

Biology and

725 N. Wolfe St., BaltiFax(301)550-6718.

of Molecular

15, France,

Biology, Institut

Tel. (33-1)45-68-8000;

(S.D.S.) Harvard University, College of Arts and Sciences, MA 02138. USA, Tel. (617)493-4648.

Cambridge,

Abbreviations: aa, amino acid(s); bp, base pair(s); DBRI, gene encoding dcbranching enzyme; FAD, flavin adenine dinuclcotide; kb, kilobase or 1000 bp; mt, mitochondrial;

nt, nucleotide(s);

SDHI, gent encoding subunit.

Sdhl.

Sdhl;

succinate

ORF, open reading frame: dehydrogenase

flavoprotein

sulfur protein subunit of about 27 kDa, which in turn passes them on to the electron transport chain. Together with two small peptides (14 and 16 kDa) whose precise roles are unknown, the two catalytic subunits of succinate dehydrogenase form complex II of the electron transport chain. With only four subunits, complex II is probably the sinplest complex in the electron transport chain, and yet its assembly and functions are only beginning to be invcstigated in eukaryotic systems. Complex II is found in the inner mitochodrial membrane and is the only component of the citric acid cycle that is so localized. A related enzyme, fumarate reductase, which carries out the same reaction as succinate dehydrogenase but in reverse, is known from bacterial systems. Although these proteins and their genes arc now well known in prokaryotic systems, until now only the ironsulfur protein gene has been cloned from eukaryotic systems. Both the yeast (Sacchnmqves c’erevisine; Lombard0 et al., 1990) and human (Kita et al., 1990) sequences of this subunit are known. We describe the fortuitous isolation of the S. cerevisiae gene encoding the flavoprotein subunit of succinate dehydrogenase, and its subsequent characterization.

133 EXPERlMENT.AL

(a)

Boekc. 1991) (Fig. 1). When this partial ORF was translated and compared to an aa sequence database. a high degree of similarity was noted to the E. cwli succinatc dehydrogenase ~avoprotein subunit (Wood et al., 1984). Since the original DBRI clone isolated (pKC28) contained approx. 10 kb of genomic DNA upstream from the DBRI locus, we characterized this adjacent pent. A 2.X-kb BnmHI-C%I fragment in&ding the DNA upstream from

AND DISCUSSION

Gene nomenclature We propose

that the gene encoding the flavoprotcin subunit of succinate dehydrogenaso be called SDHl, the ironsulfur protein, SDH2, and the genes for the twc smaller subunits, SDH3 and SBH4, in the order of descending molecular weight. This wouid conform to the standards already set in bacterial systems and to current nomcnclat~~ral rules in yeast genetics (Sherman, 19X1).

the DBRI gene was subcloned into plasmid pBS2,‘SK- for DNA sequencing (pKC60), and into plasmid pRS3 16 (generating plasmid pKC59) for construction of a disrLlpti~~n mutation and for definition of the SDHI gene by complcmentation of the disruption mutation.

tb) Gene isolation and subcloning In the process of characterizing the yeast DBRI locus, wc noted an ORF 5’ to the DBRl gene (Chapman and 799 Stu I

1

745 Bgl II 459 Nae I BamH I 390 Egl II 969 Kpn I 118 Spel 689 eta I ’

1328 Ava I/

2133 Cla I ’ 1984 PVUII f925 EC& I 1767 Bgr II

I

3568 BamH 3209 BsiX I 3807 Bsrn I 3529 Sph I

2833 Cia I 2666 Cla I ’

pKC28 pKC36

pKC-59160

1984 Pvu II 1925 EC& I

1 BamH I 118 Spel

psst

Asdh I :.%/IS3 of the SDHl:DBRI

Fig. I. Structure

of pKC28 \\-hich complcn~cnt

loci. The SDHI

the petite phcnotqpe

from pKC28

cloned into the Bm~Hi

&IwHI-C/OI

fragment

nloving the sequcnccs

cloned into pRS316

fr;ignxxt

Liagnxcnt cloned and replaced

h! li$t

containing

into pBSII-SK.

pcnc

rcprcsent

tract (undcrhned

pSS

fragnlcnt

Fi_r. 2. Tbc nt scyuencc of the SDHi dcrlincd aa near the left wrow

Plasmid

1 is derived

dcrivcd

-305

to -303

(Strntagcne.

from plasmid

pKC36

clcavagc

pKC36

and pKC5Y.

the 3.5.kh BrrwHI

pKC5Y and pKCh0 contain

&c%i::HfS3 construction

Piasrnid pKC61J

hct\%ccn the &ill

(kindlq provided by P. Hitter)

site for the nit processing

in the 200 block) upstre;um front the ORF.

The

contains

fmgmcnt

the wnc

2.X-kh

was gcncratcd

h> rc-

with a 2.kb BwIHI fragnrcnt containing the and Bockc, lYY1). Plasrnlds pKCS9 contolns ;I 2.5-hh

and dcduccd aa soqucnoc. The arrow indicates put&c

the consensus

Plasmids

into pRS316.

from pKCSY. with the scqucnccs

boxes. Two subclones. pKC36

these scqucnccs

(Chqxnan

cioncd

shaded Plaanid

La Jolla, CA), rcspcctivcl~.

390 and 1767. and replacing

cnrytnc

and putative TAT/\

sequcncc upstrcarn from the f>BKI gent.

contains the s;uw Brrw HI-C%rI

sitcs ai position containing

390 :tnd 1767 rctnoved

the H/S_? gene.

mt import prcscqucncc clca~agc sites; the thrw w-

(Hendrick boxa

et al., IWO). Sequence

(doubly underlined).

The scqucncc

originall)

fcaturcs

in&&

gent is indicated in boldface. Plasmlds pKC59

and pKCh0

from SDHl

rcportcd (Chapman

and Hockc. (ac-

is iIld~~~~t~d(undcrli~~~d in the 1200 block). The ,ATG of the d~~~~nstr~;~nl

were scqucnccd indcpcndcntl):

thus. i(X)“,, of the sequcncc inibrmntwn

from both strands. Nested dclctions wcrc gcncrated in both plasmids and wquenced a:, double-strsndcd and custom aynthctic olieodcou)rihonuclcotldes.

:I

During the course 01‘ this

should read .4C. not CA. The corrcctcd scqucncc is presented in this figure and has been subnwtted to GcnBank

cession No. ~~~6~~)Y). 4 directly rcpcatcd scqucnce dwnutrwm fjHK1

bq the darkly

1989). Methods.

and Hlctcr.

from pinamid pSZ62-Yhrr

\4ork wc noted an error in the previously determined IYYI) ;~t positions

arc indicated

pKC2X has been dcscrihcd

the .SL>Hl gene dcrivcd Plasmid

with a 2-kh BwvHI

long polypurinc

and pBSII,‘SK

shading.

ORFs

nrutnnt, arc drawn below pKC28.

pRS3 16 (Sikorski

between the two BglII sites at positions

yeast HIS.7 gent. indicated Brr~Hl-C&I

site ofplasnlid

and DBRl

of the &//I/

DNA,

\\a

obtained

a:, described. using bi 13 univct-sal primer

133

134

(c) The nt sequence The 2.8-kb Bur??H1-C%11 fragment was sequenced in its entirety on both strands (Fig. 2). The part of this scquencc that cncodcs DBRI has already been reported (Chapman and Boeke. 1991). A large ORF encoding a 64 1-aa protein (,M, 70 185) was found upstream from DBRI and in the same transcriptional orientation. This conceptual translation product can be readily aligned with E. co/i succinate dchydrogcnase (flavoprotein subunit), and can also be aligned with the related enzyme fumarate reductase from E. coli (Cole. 1982) and Proreus vu1g~wi.s(Cole. 1987) (Fig. 3). The yeast Sdhl protein is most similar to the reported partial sequence of the bovine Sdh protein (GenBank accession No. M60879; M.A. Birch-Machin. L. Farnsworth and D.M. Turnbull) (Table I). Yeast SDHl more closely rcscmbles the bacterial succinatc hydrogcnase aa sequence than the fumarate reductase sequcncc. The SDHI ORF contains a 4%aa N-terminal extension relative to the bacterial proteins. The aa composition of this pcptide, rich in basic aa such as Ala, Leu, Ser, and Thr. is typical of a mt targeting sequence (Pon and Schatz. 1991). Matrix-targeted and inner membrane-targeted nuclcarencoded mt proteins generally have two cleavage sites for the MasEMas2 processing cnzymc that removes the targcling peptide from incoming proteins (Pon and Schatz, 1991). There is a perfect match to a recently proposed internal (first) cleavage site proposed by Hendrick et al. ( 1989) within the putative leader sequence (Fig. 2). An Arg residue just upstream from the region where the yeast protein begins to rcscmble the bacterial proteins suggests the location of the second cleavage, which would generate the mature N terminus of Sdh 1 (Kalousek et al., 1988) (Fig. 2). Based on these sequence features, it is very likely that the tirst AUG codon in the ORF is in fact the start codon for this protein. (d) Phenotype of an SDHI::HIS3 gene disruption The sequence of SDHI strongly suggested its identity; howcvcr. we wished to confirm this genetically by creating a gene disruption. The expected phenotype of an s&l mutant is inability to respire, also known as the petite phenotype, because petite cells make small colonies that are unable to grow on nonfcrmentablc carbon sources such as glycerol and ethanol. The previously isolated s&2 mutants are petite (Lombard0 et al., 1990). The gene disruption was made by inserting a B~mHI fragment containing the yeast HIS3 gene into the SDHI coding region of plasmid pKC59.

Fig. 3. Sdh I aa scqucncc

alignment.

The >east Sdh

T,ZBL.E I

Protein ,’ Bovine Sdh

E. w/i Sdh 6. ol with a gap v.cight of 3.0 and

;I gap Icngth weight of 0. I. Residues 4X-62 I of Sdh I u crc compnrcdto thealigned residues (Fig. 3). cxccpt for the bo>inc partial xqucncc. which Ma\ aligned up to its C-tcmmmal end (equivalent to an positlon 15.1 of !cast Sdhl)

generating plasmid pSS 1. The insert of this plasmid \?‘as released by digesting with C/al and 1VotI. and the DNA was used to transform yeast strain YH8 (MA Ta wu_?-167 his3 D200 trpl D 1 lezr2 D 1) to histidine prototrophy. Of the five transformant colonies that were obtained, four bverc petite. The petite transformants formed small colonies on glucose-containing media such as YPD and SD (Sherman ct al., 1986), and were unable to grow on YPGE plates (Sherman et al., 1986) containing the nonfermentable carbon sources, glycerol and ethanol. The fifth transformant had no growth defect on any medium. The structure of the SDHl locus in the genomic DNA was examined by genomic blotting using an SDHl probe in three of the transformants, two of which were petite and one ivith no growth defect. In the latter case. the SDHI locus showed no sign of rearrangement, whereas the pattern of bands expected for the SDHl::HIS3 disruption was observed in the other two transformants, KC122 and KC123. Therefore, \ve conclude that the integration cvcnt in the nonpctite transformant occurred elsewhcrc in the genome. The inability of As&l strains KC122 and KC123 COgrow on YPGE medium (containing the nonfermentable carbon sources, glycerol and ethanol) is documented in Table III. Recently, results similar to this were obtained by another group that independently constructed an .s&l gene disruption (Robinson et al., 1991). Finally. plasmid pKC59 \vas introduced into the Asdhl::HIS3 strain KC122 in order to determine whether the intact SDHI gene indeed resided on the Barl?HI-&I fragment. Transformants were selected on uracil-less SC medium. and all transformants \vere found to have regained

I scqucncc wab manually aligned (using the GCG LINEUP program:

Madison.

Wisconsin)

with other

\uccinate dchydrogcnasc and fumaratc rcductase scqucnccs obtained from GcnBank. The aa found in at Icast two ofthc aligned sequnccs at a gibcn positron arc shaded. The number- 12 at the beginning of the bovine scqucncc represents B 12-w extension (part of the presumed mt targeting scqucncc) that is not shoun.

Scqucnccs

E co/i fumarnw

shown are (top tv bottom) >wst Sdh

reductnsc

(FRECO).

I (YEAST). partial bovine scqucncc (BOVINE). E. w/i sucanatc

and P. ~/~qrri.t furnaratc

reductaae

(I’RPVII).

dchydrogenasc

(E. COLI).

135

0

YEAST BOVINE E. COLI FRECO FRPVU

YEAST BOVINE E. COL FRECO FRPVU

49

M LS LKKSALS 12RARR LA LTC

50 HEYDCVV IG.A H:E~:D.AVVVG, R’cF:DAV:V-I:G# F Q A i, LA,! V&4 fNAD.1 A:-I’IG’A

YEAST BOVINE E. COLI FRECO FRPVU

y EAST BOVINE E. COLI FRECO FRPVU

YEAST E. COL FRECO FRPVU

K LT LLRNTRT TKWSAAWQTG

SADGKYHI ID AISAOYPVVD MK LPV MQT MU7

QGSVNGSASR NKRSSAKVSD .,

FTSSALVRQT TRSFHFTVDG

SHTV,

LAE. .AGYKf LSE. .AGFNT ISQ. .SGQTC AAQANPN’AKI A’P;‘fA&FQLKI S i:-#.y:M.T R E A? ALHY MTfQ;a;P ;4’fE’y,MCKTGR. VYDYFVHHCP VVDYFVEHCP

149 K$,l I ELEHYC; ASVVELENY.G EA.I:Lft~HMG TEMTQizELWG T.EMTQiE CWG

KEYGKGAQAY

RTCAV

199 WALLHTLYGQ

KNFG.“;EQt; MK.i E

RTAAA:ADRTG RT.WfA’RD-KTG R’i-WF’A:AI%KG

HALL.HTFYQQ FHMLHTLFQT FHM:LHT LPQT

HN.GEV-VGVI NQDGAVVGCT V.DDGH VbG LV VDEGH’ARGVV

AYNQEDCTIH ALCIETGEVV AMNHM:E.GTLV AI NMM’EGTKV

249 RFRAHKflIA Yf K&R&TV LA QIRAN’AVVMA QiRhNAVIMA

N’WKW_H:M.Y.DTY N-WRWH,F:YDTY NW~WHMYDT.V SFEY M,F’HDTV SYDFHF’NDTV 150 VFFSRTENGK MPFS LP,F$.FI.LDDGR CP!$‘S:RRPDGS CP$‘JSRKEIJGS. 200 ALR. HDT’HFF NI_K.NHTTIF $ LCIFPQI QR;F. SCKYPQI~QRF

AAm:;

$ #TV AAQGGI SHTVS AQGG I SHTVAAEGG. SHTVAAEGG.

ADRTG

299

uiPsC;t YGSGC

YEAST E. COLI FRECO FRPVU

CkSAHTCTGD TtN:kH-J NTi;D NTNGG.‘I, VTGD NiiNGr;‘iVTGD

GNAMVSRAGF GVGM A I RA@ G’M’GM’ALS,H,GV GMG I ALRH’GV

P, LQDLEFVQF RVQDMCMWQF PLRDM EF.VQY PLRDMEFVQY

YEAST E. COLI FRECO FRPVU

GF’LVNS EGER GYL-i.NkHGEl? G I ;I% NKN G Y R 6-i L.Vd KDGYR

FHERYA.. FM:ERYA. Y CQDYGMGP E Y LQDY c LGP E

PTAK. PNAK TPLGEPKNKY TPL~KPENKY

349 DLACRRVV.S D’LAGRDVVA ME LGRRDKVS ME LQPRDKVS

YEAST E. COLI FRECO FRPVU

R,G:V I;iK K K D.H R@ZDGFW%PW N T.;I:.S T”PRGDY R-T 1 KTH RGDV

MYLQLSHLPP AKkKLDHt’GK YYLDLRHCGE VtiiDiRHLGA

EVLKERLPGI EV.i;ESRtP%I KK LWERR’I-P 6’1 KK-cH.&‘RLP’F 1

399 SETAAI FAGV LELSRTFAHV CELAKAY VGY CE LAKAY VGV

YEAST E. COLI FRECO FRPVU

PTKWNGEXLT FTKVTGQALT ET:....... ET........

I DEETGEDKV V N cKG;EDV DQNCCTR. NQRTETR.

449 IPG.LMACGEA VRG:LFAVGE I X KG.LfAV’GEC 1 KGLFAVGEC

YEAST E. COLI FRECO FRPVU

VFGR,A,VA. HT V,FGR,AAGLH L VFGRL:GGEQA V FGRLAGEEA

VADTLCIPGLP QES I AEQGAL TERAATAGNG VRRAQEATPA

499 HKPLPSDLGK RDAS ESDV E NEAAI EAQAA NASALDAQTR

N.ANEs:RST’AE N’NRNQEDPVA &QDGG:E:NWAK NQKGsENWAQ

i.R.MNMKQTMQ t RKALQECMQ I RDEMG L6M.E I RDERGEAM E

KDVsVFRTOS HNF$V:FREGD EGCG i ,YRTP’E EGLG:YRTR E

549 SLDEEVRNIT AMAKBLEQLK LM-Q$T ! D$ LA LMQKT 1 DK I-7

TTDRS’MIWNS LD.DTSS E FNT 1:TBTssVFN-f IKDPSsV-FNT

DLVETLELQN QRVECLEXDN DLi_VT’I~E:LGH DLLYKIF’l%F

tLTCASQT,AY .LMETAYAT’AV GLNV_AECMA:H GLDVAFCM,AH

599 SAANRKESRC SANFRTESRG SAMARKESRG SAFWRKESRG

NRDDEHWMKH DRIJDENWLCH E R D FJVNF.F.K H E’#DD.VN.F;LKH

TLSWQKDVAA s LY LP T LAFRDADGT TLAFY NPEGA

PVTLKYRRV ESESMTRRSV TRLfY,SDVKI F’RLEYSDVK

YEAST E. COLI FRECO FRPVU

500 ES1 AN.LDKtR AS.LDRLNRWN GVEQR-CKDFV D I EDN LKKLM

HPTGIAGAGV H~TGLPGSG1 HPTGLT=.XGI

V

I_”

YEAST E. COLI FRECO FRPVU

AVEKTFDDVK VI REPL$tNAR E-LQERFK-RV’R F.-LKFRFKHVE

YEAST E. COLI FRECO FRPVU

600 AlIAR. EDYR AHSR. F’DFR A Ht:a-R:Lx.7EGCT A~HQR:LD-EGCT

YEAST E. COLI FRECO FRPVU

650 S V:E:p T V ?:A!.. AFPPK I RTY TLPRA$,RVYG KSAPAKRVYG

649

619

GE-ADAAQKAE %ATAODK.

AANKKLKANG QNKEKANG

t

I

DHTLDEKECR N. .MEPKLRP

136 TABLE Strains

II used in this atudl

Strains~’

Plasmid’

Parcnl

strain

YHX

’ YH8 (XU. 1990); all other strains. this work. Media used wcrc and SD (qcast mlropcn base/dcxtrosc). The mcdin \%ere prcparcd as dcxribcd

(yeast catrllct,pcptonc~dcxtrosc),

YPD

For sclcction of transformants. by Sherman ct al. (1986).

pRS3 I6

KC122

pKCS9

KC122

YPGE

SC medium lacking the approprlatc

(leaat

supplement

cxtr3ct:‘pcptonc,gl~ccrol,

cth;mplc mcntation

analysis

of the Atdhl::HIS_~ disruption

mutation

and complc-

b> pKCS9

Strain

Rclcvnnt

genotype

Plnsmid

Growth YPD.’

on

Growth YPGE”

on

K., Oka, H., Gcnnls,

Human

complex

cloning

of iron-sulfur

R.B.. .Ackrcll. B.A.C.

II (succinatc-ubiquinone (Ip) subunit

and Kaaahara.

oxidorcductasc):

of liver mitochondria.

Blochcm.

Biophqa. Rcs. Commun. 166 (1990) 101-10X. Lombardo. A.. Carinc. K. and SchctTter. 1.: Cloning and CJI;lr3CtCrILiltIo(1 of the iron-sulfur

subunit

gcnc

of succinatc

dchydrogcnasc

from

Sat,chtr,-on?~‘~~.scrw\iritrr. J. Biol. Chem. 265 ( 1990) 104 I9- 10113. Pon. L. and Schatz. G.: Biogcncsis of !cast mitochondria. In: Broach,

4+

KC‘102

SIIHI

none

+++

KC122

&Ill i ::HIS3

none

+++

J.R.. Pringle. J.R. and Jones,

KCIZJ

Aw/l~I ::HIS3

pRS316

+++

KC125

Ardhi::H/S3

pKC59

+++

lular Biology of the Yeast Srrc,c,hirn)n~~,c,es.Cold Spring Harbor oratory. Cold Spring Harbor. NY, 1991. pp. 333-406.

++

Robinson.

’ hlcdla \\crc prepared

aa described

(Sherman

ct al.. 19X6).

Sherman, w.wrr:

K.B. and Boekc, J.D.: jcaat

opcron encoding

Isolation

dcbranching sequence

the fumaratc

divorgcncc

reductasc

of the mcmbranc

scquencc

reductasc

and charactcriLntion

cnrymc.

analysis

I’.. Hendrick.

matrix proteasc\ mntrlx cn,ymcs.

J.N.. Jones, L1.W. and of the Yeast .Str~/~trn~-

Cold Spring Harblv

Labor-ator!.

F.. Fink, G.R.

and Hicks. J.B.: Method5

in Yeast Gcnctics.

strains dcaigned for efficient manipulation crw~~itiire. Genetics 122 (1989) 19-27. \Vood. D.. Darlison. qucnce

encoding

anchors

and abacncc

coding for the flavoprotcin

167

M.G., Wilde, R.J. and Guest, J.R.: Nuclcotldc the Ha\oprotcm

dchydrogenase

I clcmcnt transpowon

mini-TyI

clcments.

and hqdrophohic

of Ewherichitr m/i. Biochcm. ofscqucnccs

subunits J.

rcquircd in

war the long terminal

w

of the

222( 19X3) cJ(for?casr

repcats~ analqsia 01

Mol. Cell. Biol. IO (1990) 1695-2702.

of an

NOTE

ADDED

IN PROOF

(I 987)

subunit of the

of fivcheri~~hi~~w/i. Eur. J. Biochcm.

J.P. and Roacnbcrg.

Ty

of DN 4 in ,Srr(,r,htrn)~,nr,(~,\

of the frd

122(I 982)

L.E.: Surq of N-terminal precursor proteins: leader

pcptidcs cleaved b> two matrix proteases share a three-amin[, motif. Proc. NatI. ,Icad. SCI. I’S/\ X6 (1989) 3050-4060. Kalousck.

Bmlog)

dixuptcd

J. Blol. Chem.

of Pn~wuc w/~y~ri~. Extcnsivc

gent. Eur. J. Biochcm.

Hcndrlck. J.P.. Hodges. P.E!. nnd Roscnbcrg, protcol! tic cleawgc \itcs in mitochondiral

of the

Cell 65 (1991) 483-392.

and comparative

Irtl-linked UU/IC’ ccphalospormasc 3s i-488. Colt, S.T.: Nuclcvtidc

In: Strathern.

subunit.

NY. 1981, pp. 639-640.

Xu, H. and Bockc. J.D.: Localization

S.T.: Nuclcotidc

fumarntc 17%381.

Life Cycle and Inheritance.

Cold Spring Harbor,

succinatc 519-534.

REFERENCES

sequcncc

nomcnclaturc.

flavoprotcin

J.R. (Eds.). The Molecular

Lah-

Cold Spring Hal-bor Laboratory. Cold Spring Harbor. NY. 1980. Sikorskl, R.S. and Hxter. P.: A qstcm of shuttlc \cctors and ~casc host

We thank Immo Scheffler for helpful discussions, R. Jensen and A. Gabriel for comments on the manuscript and EmeriLa Caputo for technical assistance.

Colt,

F.: Gcnctic

Broach.

and Ccl-

A. and Lcm~rc, B.D.: Isolutlon

of a Su~~/rtrn~nt~.~e.rww.~‘l.iww mutant

for the succinate dehgdrogcnasc 266 (1991) 21347-21350.

Shaman, /\CKNO\VLEDGEMENTS

gcnc cncoding

EM’. (Eds.). The Molecular

K.hl.. \on Kicckebusch-Giick.

and characterization

the growth characteristics of the parental strain, i.e., fast growth on glucose-containing media and the ability to grow on YPGE (Table III). We conclude that a functional SDHI gent indeed resides on this restriction fragment.

C’hapman.

M.: cDNA

awl

L.C.: Two mltochondrial

act scqucntially in the processing of mammalian Proc. Natl. Acad. Sci. L’S/\ X5 (10X8)75?6-7540.

While this paper was in press, an independent report of the sequence of SDHl was published [Robinson, K.M. and Lemire. B.D.: Isolation and nucleotidc sequence of the Saccharom~res cerevisiue gene for the succinate dehydrogenase flavoprotein subunit. J. Biol. Chem. 267 (1992) IOlOl-10107]. The sequences are identical with the exccption of a silent third base difference at position 1433 and three sequence differences in the 3’ untranslated region (positions 2269. 2346, and 2411).

SDH1, the gene encoding the succinate dehydrogenase flavoprotein subunit from Saccharomyces cerevisiae.

We describe the isolation, sequence and construction of a disruption of the yeast SDH1 gene, encoding the flavoprotein subunit of succinate dehydrogen...
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