Gene, 88 (1990) 149-157

Elsevier

149

GENE 03445

T h e F U R 1 gene o f Sacckaromyces ceret, isiae: cloning, structure and expression o f wild-type and mutant alleles (Recombinant DNA; uracil phosphoribosyltransferase; nucleotide sequence enzymatic activities)

L. Kern', J. de Montlgny ", R. Jund" and F. Lacroute b ° £aboratoire de G~n~dque Physiologique, Institut de Biologie Mol~culaire et Cellulaire du C.N.R.$., F-67084 Strasbou~ Cedex (France), and b Centre de Gdnddque Mol~culaire du C.N.R.S., F-91191 Gif-sur-Ywue Cedex (France) Tel. 169 82 3180 Received by J.-P. Lecocq: 4 October 1989 Revised: 13 November 1989 Accepted: 15 November 1989

SUMMARY

The FURl gene of Saccharomyces cerevbiae encodes uracil phosphoribosyltransferase (UPRTase) which catalyses the conversion of uracil into uridine 5'-monophosphate (UMP) in the pyrimidine salvage pathway. The FUR ! gene is included in a 2.1 kb genomic segment of DNA and is transcribed into a I kb poly(A) ÷ mRNA. Sequencing has determined a 753 bp open reading frame capable ofencoding a protein of 251 amino acids. The FUR! genes for three recessive furl alleles, having different sensibilities to 5-fluorouridine (5-FUR) but identical levels of resistance to 5-fluorouracil (5-FU), were cloned and sequenced. Single bp changes located in different regions of the gene were found in each mutant. Two in vitro-constructed deletions of the FUR! gene have been integrated at the chromosomal locus, giving strains with 5-FUR R and 5-FUR n mutant phenotype. Assays of UPRTase, uridine kinase, uridine ribohydrolase and uridine 5'-monophosphate nucleotidase enzymatic activities, in extracts of strains where the FUR! gene is overexpressed or deleted, indicate that the FURl encoded protein possesses only UPRTase activity.

INTRODUCTION

UPRTase catalyses the conversion of uracil and 5-PRPP into UMP and PPi. This enzyme has a fundamental importance in the utilization of endogenous uracil formed by degradation of pyrimidine nt and in the utilization of exogenous uracil, cytosine and uridine for pyrimidine Correspondenceto: Dr. L. Kern, Laboratoire de G6n6tique Physiologique, Institut de Biologie Mol6culaire et Cellulaire du C.N.R.S., 15 rue Ren6 Descartes, F-67084 Strasbourg Cedex (France) Tel. 88417033; Fax 88610680. Abbreviations: aa, amino acid(s); bp, base pair(s); cpm, counts per minute; ,£ deletion; ds, double strand(ed); kb, 1000bp; FURl, eerie encoding UPRTase; 5-FU, 5.fluorouracil; 5-FUR, 5-fluorouridine; 5-FUMP, 5-fluoroUMP; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; OMP, orotidine 5'-monophosphate; OPRTase, orotate phosphoribosyl transferase (EC: 2.4.2.10); ORF, open reading frame; PPi, 0378-1119J90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

synthesis in Saccharomyces cerevisiae (Fig. I; Grenson, 1969; Jund and Lacroute, 19"/0). Mutants of S. cerevisiae lacking UPRTase activity were selected on the basis of resistance to 5-FU, an analog of uracil which is toxic when converted into 5-FUMP. Mutants in the FURl locus are resistant to I0- 2 M 5-FU whereas a wt strain is sensitive to 3 x I0 - 5 M 5-FU (Jund inorganic pyrophosphate; 5-PRPP, 5.phosphoribosyl-l-pyrophosphate; PRTase, phosphoribosyltransferase; R resistance/resistant; s, sensitivity/sensitive; S., Sacckaromyces; tsp, transcription start point(s); ss, single-strand(ed); UMP, uridine 5'-monophosphate; UMPase, UMP nucleotidase (ecto-5' nucleotidase, EC: 3.1.3.5); UPRTase, uracil phosphoribosyl transferase (EC: 2.4.2.9); Ukinase, uridine kinase (ATP: uridine 5'-phosphotransferase, EC: 2.7.1.48); Urhase, uridine ribohydrolase (uridine nucleosidase, EC: 3.2.2,3); UTP, uridine 5'-triphosphate; wt, wild type; YEPD and YNB, see MATERIALS AND METHODS, section a; [ ], denotes plasmid-carrier state.

150

(3Ur

Glutamlne + C ~ + A'rP .~

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CPSaN -Carl0amoylphosphate UflA2

ATCBSe

~

L r Ureidosl :cinic acid

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DHOaSO

Repression

URA~ . . . . . .

Reiroinhibitton --

Rcpressio,

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up "

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URAS,

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~,~FUR! ~

~'~ Uridine 5'.phasphate

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.

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Uridina S'-ffiphosphate . . . . . . . . . . . . CTPnn

i

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Uridine

r

Omtl acid

l=y,

•

R1

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URA7

Cytidina 5'-Iriphosphate

Fig. i. Pyrimidine biosynthesis in .Saccharomyces cerevisiae. The enzymes are abbreviated as follows: CPSase, carbamyiphosphate synthetase; ATCase, aspartate transcarbamylase; DHOase, dihydroorotase;

DHOdehase, dihydroorotatedehydrogenase;OPRTase, orom~e phosphoribosyl transferase; OMPdecase, orotidine Y-phosphate decarboxylase; UMPkase, uridine $'-phosphate kinase; UDPkase, uridine $'.diphosphate kinase; CTPsase, cytidine 5'-triphosphate synthetase; UPRTase, uracil phosphoribosyltransferase; Ukinase, uridine kinase; Urhase, uridine ribohydrolase;UMPase, UMP nucleotidase; cytdase, cytosinedeaminase;cytp,cytosinepermease;up, uridinepermease;urip, uridine permease.

and Lacroute, 1970). The block in UPRTase activity associated with a Ura- mutation prevents these recombinant strains from salvage by exogenous uracil and therefore makes furl mutants auxotrophic for uracil extremely useful in industry as autoselective strains on complete medium (Loison et al., 1986), A more detailed study of the furl mutants has revealed that, whereas they form a homogeneous class for 5-FU R, three groups have different levels of resistance to 5-FUR, an analog ofuridine. In previous reports (Grenson, 1969; Jund and Lacroute, 1970), it was demonstrated that in S. cerevisiae uridine is converted into UMP by a Ukinase encoded by the URKI gene. An important flow of uddine is hydrolysed into uracil by an Urhase (Carter, 1951) encoded by the URH gene, Assuming that 5-FUR is metabolised by these same enzymes, one can predict a 5-FURR in urkl and furl mutants. In this latter case, the resistance to exogenous 5-FUR can be considered as resistance to intracellular 5-FU. However, this scheme does not explain the different levels of 5-FURR in furl alleles. It

has been postulated that the FUR1 protein is a multifunctional protein bearing a Ukinase activity in addition to the UPRTase activity (Grenson, 1969) or some other uridine metabolism enzymatic activities. Nevertheless, Jund and Lacroute (1970) reported that in these three groups of furl mutants, the mechanism of5-FURR does not seem to be correlated to Ukinase and Urhase activities since these activities are similar in a wt strain and in furl mutants. Measuring the enzymatic activities in cells transformed with a multicopy plasmid carrying the FURl gene might allow detection of a cryptic o~ minor uridinemetabolising activity of the UPRTase protein. In S. cerevisiae, it has been suggested that the UPRTase encoded by the FUR1 gene might be able to catalyse the transformation of orotate to a M P with a lower efficiency than OPRTase encoded by the URA5 gene, thus explaining the leaky phenotype of ura5 mutants on YNB (Jund and Lacroute, 1972). PRTases using uracil as substrate are rare among the many PRTases described and no primary structure of UPRTase has been reported. Thus it was of interest to clone the FURl gene and to determine the primary structure of the UPRTase protein. Cloning of the FURl gene made it possible to obtain in viva deletions of the gene, giving null mutants. Furthermore, we have cloned and sequenced the furl-7, furl-8 and furl-9 alleles in order to cone!ate the primary structures of the UPRTase mutated proteins with the corresponding phenotypes.

MATERIALS AND METHODS

(a) Strains and media Yeast strains are isogenic derivatives of the wt strain FLI00 (ATCC 28383) except for GRF18 (a gift from G.R. Fink, Cambridge, MA, U.S.A.). The strain uraSfurl-71eu2 was obtained after crossing a strain a uraSfurl-7 derived from FL100 with GRF18 (edeu2his3). The phenotypes of the different furl alleles studied are shown in Table IIL The growth media used were YEPD (1% yeast extract, 2Yo peptone, 2% glucose) and YNB (0.67% yeast nitrogen base, 2% glucose) appropriately supplemented. Yeast genetic techniques described by Mortimer and Hawthorne (1966) were followed. Yeast cells were transformed according to Hinnen et ai. (1978) with minor modifications (Chevalier et eL, 1980). Escherichia coli strains used for selection and amplification of recombinant DNA was BJ5183 (F- recBC sbcB endA-l gai met thi bio hsdR), a gift from B. Jarry (Lab. de G6n6tique Mol~culaire des Eucaryotes, Strasbourg) and JMl03 (A(lac-pro) thi rpsL endA sbcBl5 hsdR4 supE [F' traD36 proAB + laclq lacZAMl5]), selected by J. Messing (University of Minnesota). Growth media for E. coil were Luria broth or minimal medium M9 described by

151 Davis and Mingioli (1950). E. coli cells were transformed as described by Mandel and Higa (1970). (b) Plasmids Plasmids are derivatives of pJDB207 (Beggs, 1978) and pUC19 (Norrander et al., 1983). We used the pFL plasmids, constructed by F. Lacroute, derivatives ofpUCl9 in which a Bg/ll linker was cloned in the Alul site at nt 629 and a ClaI linker was inserted in the AluI site at nt 747. The 0.83 kb EcoRI-Pstl segment of the TRPI gene, sufficient to restore the Trp* phenotype, was cloned under Bg/II linkers and introduced into the modified pUCI9. This integrative vector is named pFL35. All the polylinker sites are unique except for the Xbal site, which is also present in the TRPI segment. The Taql segment of CEN Vl (Phillipsen) and an uncharacterized A R S from the yeast 8enome were inserted between ClaI linkers, yielding a 0.81 kb ClaI ARS-CEN segment. This segment was introduced into pFL35 to obtain the low copy replicating vector pFL39. A 0.51 kb MstI segment of the 2 ~tD region was inserted between ClaI linkers and introduced into pFL352, to obtain the high-copy replicating vector pFL45. All plasmids used are shown in Table I.

(c) Enzyme assays Crude extracts were prepared and UPRTase activity was measured using the method described by Natalini et al. (1979). Ukinase, Urhase and UMPase activities were measured using the methods of Valentin-Hansen (1978). Magni et al. (1975) and Paglia and Valentine (1975), respectively. OPRTase activity was assayed using the spectrometric method of Lieberman et al. (1954). The amount of proteins in crude extracts was measured by the method of Bradford (1976)with bovine serum albumin as standard.

(d) Preparation and analysis of DNA Plasmid DNA from E. colicultures was prepared according to Clewell and Helinski (1969). Total yeast DNA from

S. cerevisiae was extracted according to Winston et al. (1983). Restriction endonuclease and ligation assays were carried out as indicated by the suppliers (Amersham, Biolabs, Boehringer Maunheim, BRL). Standard published procedures were used for transfer of restriction fragments to nitrocellulose paper, nick translation and hybridization (Maniatis et al., 1982).

(e) Preparation and amdysis of RNA The phenol-cresol method of Waldron and Lacroute (1975) was used to extract total yeast RNA. Poly(A) +RNA was obtained according to Aviv and Leder (1972). Hybridization of DNA to RNA fixed onto nitrocellulose fdters was carried out as described by Gillespie ~ d Spiegelman (1965). The procedure of Thom~ (1980) was used for Northern blotting.

RESULTSAND DISCUSSION (a) Cloning and sequeneiag of the FUR1 gene The FUR1 gene of S. cerevisiae encoding UPRTase was cloned by complementation of a uro3furl-71eu2 strain. A urn, mutant presents a leaky phenotype on YNB. As reported by Jund and Lacroute (1972), this phenotype could result from a weak OPRTase activity of the UPRTase since a recombinant uraSfurl-7 is lethal on YNB and can only grow on uridine and not on uracil. An Sau3A bank of wt DNA inserted into the Ban, HI site ofthe shuttle E. coilyeast plasmid pJDB207 was used to transform a ura$furl-71eu2 strain for ability to grow on YNB supplemented with uracil. Transformants were checked for their sensitivity to 5-FU and growth on YNB. One clone, 5-FU s, which grew slowly on YIqB was further analysed, it contained a plasmid (pB2) carrying a 3.8 kb insert which was not able to complement a ura,~ mutation. A 2.1 kb Clala-Clalc segment recloned into the Accl site of pFL39 (pPE) fully retains ability to restore 5-FU s in furl-7, furl.8

TABLE i Plasmids employedin the presentstudy Name

Vectora

Selectivemarker

Part of FUR1geneu

Naturec

pB2 pPE pHX pAC pAXA pLK.dI pLKAT

pJDB207 pFL39 pFL39 pFIA5 pFL39 pFL35 pFL35

LEU2 TRPI

Sau3A-Sau3A ClaIa-Clalc (=

Replicative Replicative Replicative Replicative Replicative Integrative Integrative

TRPI TRPI TRPI TRPi TRPI

AC) Hindlll.Clalc ( = HC) Clala.Clalc HC d(Accl-Xbal) HC d(Clalb-Xbal) AC d(H~dlll-Xbal)

a Plasmidsare describedin MATERIALSAND METHODS,sectionb. b AC = Ciala-Clale fragmentof FUR1 ~ne, HC = HindIll-Clalc fragmentof FUR1 gone (Fig. I). c Nature of yeast plasmids.

152 TABLE II

and furl-9 strains. The restriction map of the 2.1 kb Clala-Clalc segment and the sequencing strategy are shown in Fig. 2. Both strands of the segment were entirely sequenced. The sequence contains a single ORF of 753 nt (Fig. 3). The deduced protein contains 251 aa with a calculated Mr of 28.7 kDa. This data is in agreement with 27 kDa determined by Natalini et al. (1979) for the Furified baker's yeast enzyme. We note that UPRTase of E. coil K 12 is 23.5 kDa in size (Rasmussen et al., 1986), while that of Tetrahymena pyriformis is 36 kDa (Plunkett and Moner, 1978). The codon bias index of the UPRTase sequence, calculated according to Bennetzen and Hall (1982), is 0.46 which corresponds to an intermediate protein abundancy. We ha,~e compared the deduced aa sequence with the yeast OPRTase encoded by the URA$ gene (de Montigny et al., 1989) and with other PRTases. The only significant similarity found was the peptide ThrGlyGly in position 177-179 proposed to be a part of the putative 5-PRPP binding site (Hershey and Taylor, 1986). Uracil is a substrate for both UPRTase and the uracil transport protein encoded by the FUR4 gene (Jund et al., 1988). However, comparison of the deduced aa sequences of F U R l and FUR4 shared no detectable similarity between the primary structures of these two proteins.

Transcription from the two strands of FUR1 Strain

Amount of RNA hybridized to FURl a

FLI00

Coding strand

Noncoding strand

2.32 x 10 -Sb 96% c

0.11 X 4~ ~

10 - S b

a Exponentially growing cultures were labelled for 5 min with [3H] adenine at a final activity of 20/~Ci[ml (specific activity 19 Ci/mmol). Coding strand is the 1.4 kb HindIII-ClaIc segment cloned into M 13mpl 8. The noncoding strand is the same segment cloned into Ml3mpl9. b The rate of transcription is expressed as the fraction ofcpm retained by the DNA probe versus the input, and adjuste0 for the I kb probe. Amount of RNA hybridized to the noncoding strand is not significantly different from those hybridized to Ml3mpl9 used as standard. ¢ Percentage of RNA hybridized to the probe.

revealed at nt - 9 and -7 relative to ATG at nt + I (Fig. 5). The FURl mRNA half-life was determined by the Greenberg method (1972). We found a half-life of 7 min which is within the range (3-20 min) ofdecay rates reported for different yeast mRNA of structural genes (Hynes and Philipps, 1976).

(e) Construction of deletions in the FUR1 gene We have replaced the wt gene by two different deleted alleles by transplacement.excision (Scherer and Davies, 1979) in order to obtain non reverting strains of furl mutants and to determine resistance levels to 5-FUR in strains lacking the UPRTase protein. In the first case, the 1.4kb Hindlll-Clalc segment was cloned in plasmid pFL35. The internal 0.32 kb Clalb.Xbal segment was deleted. Clalb andXbaI sites were filled in and the resulting linear plasmid (pLKA I)was ligated. In the second case, the 2.1 kb Clala-Clalc segment was cloned in plasmid pFL35 and the internal 0.87 kb Hindlll-Xbal segment deleted. After filling in of the extremities and ligation, we obtained pLK,4T plasmid. To introduce the two deleted alleles, a

(b) Transcription studies The 1.4 kb HindIII-Clalc segment which contains the complete FURl ORF was cloned in both orientations into phages Ml3mpl8 and mpl9. The DNA cloned into M 13rap 18 hybridizes significantly with a radiolabeiled total RNA fraction of a wt strain, confirming that this ORF is transcribed (Table II). Northern blotting using the 1.4 kb HlndIII.Clalc segment as a probe reveals a single band of about I kb in both poly(A) ÷ and total RNA lanes from a wt strain (Fig. 4). In order to identify the tsp of FURl RNA, a ss HindllI-Accl DNA segment was 5' end-labelled at the AccI site and hybridized to the poly(A) ÷ RNA fraction of a wt strain. After S1 nuclease digestion, two different tsp are

v

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0,I kb Fig. 2. Restrictionmap and sequencing strategyfor the 2.1 kb Clala-ClaIc segment. The arrows indicatethe extent of the sequence obtained from each cloned fragment, The deduced O R F is shown by the large open arrow,

153 -894

ATCGATAAAAGAACTAATG'ITrCCCAAAGA A A T A ~ G G G A A T A A A G A A T A A T A G G

-794

GCACAGAGGAAC~TTGG

-694

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-594

AAACCAAGAAAACTTGAAAAATGTT CTGG AAAACATTTCTC AGGTGCAGATAG CTCAAA~A~AGA~C~

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-394

C T G A T G A A T C G T C C C A C T T G T C G T A T T A G A G ~ r G T C A A C G A C A C T C A C A A G G T A T T T A A T C A G C A A A A T C C C C G C C A C A A A C T A i-t-l-, T I - ~ T

-294

G tTx-rC T C A T G A C T ~ C C T A A T A A C A A T A C C T C A ~ C T A C T A G T A A T C G A C C T A T G T A A T T A T T T C A T A A A C T A T A A A G C

- 194

G G CCGG'I-FFFx~"TATAAGC T T A T CT CATCGCATAAAAAATCGACAGTTGTAATTATCTCCGGCGGAt.-FFI-t'CCt-I-I- t ~ T t

--94

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L

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VV CTG CCTCAAAAGAG~FATATAT~TGGTAAGAATCCTCTTCCAATACTAG CIT~'FITCTTt'PIT~ACCA~c M N

"tTFFFFt'rC A C T T C T T ~ C A A A G

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107

T G 'C r .G . ~G. A A C C A ~ . . . . . x • A A .G A A C G.T C T A C.T T G C T A . CCTCA . A A C A .A A C C A A T T G ~ 1 1 1 ~ T A C A C C A T CATCAffzAAATAAGAATACAACTAGACCTGA S S E P F K N V Y L L P Q T N Q L L G L Y T I I R N K N T T R P D

207

TTTCA'FFF~'CTACTCCG ATAG AATCATCAGA ~ G T T G G T ' r G A A G AAGGTITGAACCATCTACCTGTGCAAAAGC A A A T T G T G G A ~ h ~ C ~ F I F Y S D R I I R L L V E E G L N H L P V Q K Q I v E T D T

307

A A C T T C G A A G GTGTCTCATTCATGGGTAAAATCTGTGGTG'~-FI'CCATI~TCAGAGC T G G T G A A T C G A T G G A G C N F E G V S F H G K I C G V S I V R A G E S M E Q

A A ~ ~ G L R

~ T A ~ D C C

N T R

E ~ S

V

407

TGCGTATCGGTAAAA'Ft-F~'AATTCAAAGGGACGAGGAGACTG~ . - ~ - t - t - A C C A A A G T T ~ r T C r A C G A A A A A T T A C C A E u ~ G G A T A T A T C T G A A A ~ T A ~ T ~ R I G K I L I Q R D E E T A L P K L F Y E K L P E D I S r_ R Y V F

507

CCTATTAGACCCAATG ~CCAC C G G T G G T A G T G C T A T C A T G G C T A C A G A A G T C T T G A T T A A G A G A G G T G T T ~ G C ~ G AG A G ~ A ~ ~ C L L D P M L A T G G S A I M A T E V L I K R G V K P E R I Y F L N

607

CTAATCTGTAGTAAGGAA~TrGAAA/.ATACCATGCCGCCTTCCCAG~GGTCAGAATTGTTACTGGTGCCCT~~~~GT L I C S K E G I E K ¥ H A A F P E V R I V T G A L D R AACACCATCTTG~

G ~

L ~

D G

C

E

N

G

~

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707

ATCTAGTTCCAGGGTTGGGTGAt.-t-t- ~ 3 G T G A C A G A T A C T A C T G T G ~ T A A A T C A C A C C C G L V P G L G D F G D R Y Y C V *

T

807

ATCAA~-`'-~.~GG~FF~CTACTGT~FI~AAA'I~TCt`~t~rCTC~1-t~t.1~AAAtT1-t.tGTTG~CGTCTCTTCTACTAT~r~t~G~~t~t~'~T

907

TACc,~t`t-t"~`~GTAAAAATAATAT~`~CGTACCAATC~GTCA.t-t~t`ATAACAAATATGCI~rGAAAAATCTAACGACTCTGTI-7CITA~%~A~\T~

007

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107

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207

TTCCG CCACTTAGG ATTATCG AT

AGA T ~ C C C C G ~ T ~ C ~ C ~ T C ~ T A G T A T A T

~ 229

Fig. 3. Nt sequenceof the yeast FUR1gene and deducedaa sequencefor UPRTase. DNA fragmentswere sequencedaftercloninginto the repHcative forms of Ml3mpl8 and mpl9 (Norrander et al., 1983), by the dideoxynucleotide chain termination method described by Sanger et al. (1977), using the DNA polymerase I Klenow segment and the MI3 annealing primer from Amersharn ( 5 ' - d G T A A A A C G A ~ A G T - Y ) . Arrowheads indicate the two up determined by SI nuclease mapping. The putative TATA element is boxed. The yeast consensus sequence for termination is underlined and brackets show the AATAAA signal of higher eukaryotes. The nt and aa sequences were analysed using the UWGCG programs (Devereux et al., 1984) on a VAX 11/750 microcomputer.

tvpl-4 strain was transformed to prototrophy by pLKA 1 or pLKAT in the FUR1 insert by Nsil or by NcoI digestion, respectively, in order to direct integration into the FURl locus (Orr-Weaver et al., 1981). In each case, one Trp + clone was grown on YEPD in order to allow a recombinational event leading to the excision ofthe plasmid DNA. Trp- clones were obtained after nystatin enrichment. Half

of the Trp- clones were 5-FU R. Southern hybridization shows the replacement of the wt allele by the deleted ~1 or AT one (Fig. 6). These two mutants, furl-,~ l and furl-AT, display the same level of both 5-FU R 0 0 -2 M) and 5-FUR R (2 x 10-3 M). However, the 5-FUR R level is different than those reported for the furl-7, furl-8 and furl-9 mutants.

154

A

B

(;

A

A

C

I

2

3

kh Ik)

fi.75 1.2fi 2.26

1.98 b

0.56

•

__T •

T

Fig. 4. Northern blot of the transcripts encoded by the FURl gene.

100#8 oftotal RNA (lane B) and 25 #g of poly(A)÷RNA (lane A) from a wt strain wereanalysedin a 1% agarosegel,Hybridizationwas carried out with0.2 pg ofthe 32P-labelled1.4 kb HIndlll.ClalcDNA segmentof FURl. The Hlndlll restriction fragments of ADNA were used as size markers, Ukinase, Urhase and UMPase activities were measured using crude extracts from cultures grown on YNB. Table Ill shows that all furl mutants display wt Ukinase, Urhase and UMPase activities. (d) Cloning and sequencing of fur/mutant alleles In order to understand the different phenotypes of furl mutants, we have cloned the three alleles furl-7, furl-8 and furl-9. For that purpose, the Hindlll-ClaIc segment was cloned into the replicative vector pFL39. The 0.56kb Accl-Xbal segment was deleted and the resulting plasmid (pAXA) was used to transform S. cerevisiae strains furl-7trpl-4, furl.8trpl-4 and furl.9trpl-4 to tryptophan independence. In each experiment, 3 to 10 Trp ÷ transformants were selected. Plasmid DNA extracted from these prototrophic strains was amplified in E. coli BJ5183 and analyzed by restriction endonuclease digestion. We found two classes of plasmids: the parental plasmid and the gaprepaired plasmid (Orr-Weaver and Szostak, 1983; Stiles, 1983). Each mutant was transformed by repficative vectors

Fig. 5. Sl nuclease mapping of the 5' termini ofthe FURl mRNA in wt strain. Sequencing of the DNA strand used in SI nuclease mapping was performed according to Bencini et el. (1984). Analysis of ss DNA protected qainst nuclease S I by hybridizationwith RNA was carried out according to the procedure ofllerk and Sharp (1977). DNA/RNA hybrids were digested with 50, 100 and 250 units of SI nuclease (lanes 1, 2 and '3) for 45 rain at 37°C. then denatured and loaded onto a 5% DNA sequencing gel. Lanes G + A and A + C show nt sequence.

containing the corresponding mutant allele. The 5-FU R levels remain the same ( > 10-2 M) as compared to a nontransformant mutant. The 5-FUR~ levels are similar to those of each non transformant strain. These observations strongly suggest that the 5-FU R and 5-FURR are exclusively the result of the mutations in the AccI-XbaI part of the FURl gene. The AccI-Xbal segment of the gap repaired plasmids were sequenced. For this purpose, ds D N A tern-

155 A

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t).46

C

i)

: :-

E

F

(;

I!

I

J

K

modified T7 DNA polymerase (Tabor and Richardson, 1987) supplied by U.S. Biochemical Corp., Cleveland, OH. In these cases, we used three specific synthetic oligo primers consisting of 17met complementary to the coding strand of FURl DNA (nt 13-29: 5 ' - d A T r C T I T I W ~ TC-Y; 264-280: 5'-dATCTACCTGTGCAAAAG-Y; 514-530: 5°-dAGACCCAATGCrGC~CA-Y). In each mutant sequence, one single bp change was observed, leading to a single aa substitution: furl-7 corresponds to the replacement of a T by an A + 422, changing an lie codon into an Ash codon at aa 141; furl-8 corresponds to A + is3 .... > 1", changing Argsl into Ser; and furl-9 corresponds to A + e23.... > G, changing GIu 2°8 into Gly.

L

"

i b _,_6~ l .tin . . . . . . . . . . .

(t5 Conclusions (1) The yeast FURl gene encoding UPRTase has been cloned, sequenced and the aa sequence of the gene product deduced. The introduction of the cloned gene into a uraSfurl-7 leu2 mutant restores the UPRTase activity but transformed clones exhibit a leaky phenotype on YNB. Furthermore, OPRTase activity remains constant when uraSfurl-7trpl-4 strain is transformed by a low (pHX) or a high (pPE) copy repficating plasmid carrying FURl 8ene. These results confirm that in yeast a protein other than OPRTase encoded a URA5 gene must possess OPRTase activity (de Montigny e~ al., 1989) but that UPRTase is not this protein. (2) S:veral features of interest relating to the expression of the FURl gene are found in the untranslated regions of the gene. A putative Goldberg-Hogness box ('rATA box)

0.56

Fig. 6. Southern blot of total DNA from the deleted furl strains, lanes A, B, C, D: wt DNA digested by AccI, Clal, Hiodlll, Xbal; Lanes E, F, G, H: furl-J! DNA digested by Accl, Clal, llindlll, XbaI; Lanes I, J, K, L: furl.AT DNA digested byAccI, CInl,Hindlll, XbaI. The probe was the 2.1 kb Clala-Clalc 32P-labelled segment of the FURl gene. Two additional bands at 6.5 kb and 8 kb are systematically present in all the lanes and probably correspond to a~pe¢ific hybridization.

plates were used directly after alkaline denaturation (Chen and Seeburg, 1985) for sequencing with 'Sequenase', a

TABLE Ill Resistance levels to $-FU and S-FUR and assays of UPRTase, Ukinase, Urhase and UMPase activities in different strains Strains a

FLI00 furl-7 trpl.4 [pAC]

furl-7 furl-8 furl-9 furl-all

furl-dT udcl urh

Activities© 5.FUe/S.FUR e levels (M) b

UPRTase

Ukinase

Urhase

UMPase

3 x 10=5/2 × 10 -5 3 × 10-5/2 × 10 -5 > 10-2/1 × 10 -4 > 1 0 - 2 / • 10 -2 > 10-2/8 x 10 -4 • 1 0 - 2 / 2 × 10 -3 • 10-2/2 x 10 -3 3 x 10-5/> I0 -2

4.5 76 0 0 0 0 0 4.1

3.1 2.9 2.6 2.7 2.5 2.4 2.7 0.5

6.5 6.4 5.9 6.3 6.2 6.1 6.6 6.8

6.4 6.1 6.4 6.8 6.5 6.9 6.2 6.3

3 x 10-5/8 x 10 - 4

4.8

2.9

0

6.5

• All strains are described in RESULTS AND DISCUSSION. b Resistances were determined as previously described (Jund and Lacroute, 1970) and are expressed as concentrations of the analogue. ¢ Specific activities are expressed as nanomoles of product formed (UMP, uracil or uridine) per rain and per milligram of proteins. Each result is the average value of three independent assays. 0 indicates a value similar to an assay without enzymatic extract. Assays are based on different values of UMP, uracil and uridine when they are chromatographed on PEI-cellulose thin layer (Merck) using bidistilled water as a solvent. . . After . . the . development of the chromatogram on 15 cm, spots corresponding to reaction compounds, visualized by autoradiography are cut out, placed m scmtillauonwals, eleted for I h in 2 ml of 0.05N HCI and counted in I0 ml of scintillation mixture. In these conditions of chromatography, the migration distances for uracil, uridine and UMP are I0, 12 and 0 cm respectively.

156 5'-TATATATA-3 ° is located 54 bp upstream from the first initiation start site. Our predicted start codon is preceded at nt -3 by an A residue which is favourable to efficient translation in yeast ( C i ~ and Donahue, 1987) and in eukaryotes (Kozak, 1981). S1 nuclease mapping of the 5' ends of the FURl mRNA revealed two tsp. The distance between the 5' ends of the FURl mRNA and the putative AUG codon (9 and 7 nt) are shorter than those described in other yeast leader sequences which vary from 11 nt (Mat al; Astell et al., 1981) to 591 nt (GCN4, Hinnebush, 1984). The translated sequence Of FUR1 ends with a TAA stop codon. The signal TAA...CATGT...(AT) rich...TTT for transcriptional termination and polyadenylation fits quite well the yeast consensus sequence proposed by Zaret and Sherman (1982; 1984). In addition, the AATAAA polyadenylation signal typical of higher eukaryotes (Proudfoot and Brownlee, 1976)is found down. stream of the yeast consensus termination sequence. Furthermore, no splicing signals (Langford and Gallwitz, 1983) are found in this sequence. The full length of the messenger deduced from the positions of the start and stop signals is in good agreement with the size of I kb determined by Northern blotting. (3) The cloning of the FURl gene on a high copy replicating vector allowed us to assert that UPRTase protein does not carry a second activity which would be involved in uridine metabolism like Urhase, Ukinase or UMPase. Furthermore, these activities remain constant in the wt strain and in all our mutants, furl.7, furl-8, furl-9, furl-

dl and furl.AT. (4) Three furl mutant alleles were analyzed, cloned and sequenced. The nt sequences reveal one single point mutation in each mutant, furl-7, furl-8 and furl-9. These three mutations, which seem randomly distributed on the nt sequence, lead to the loss of UPRTase activity correlated to an equally high level of 5-FU R. The fact that each mutation is sufficient for the creation of the phenotype suggests. that the three mutated aa belong to regions which may contribute to a spatial functional domain. This hypothesis will be strengthened if other mutations leading to similar phenotypes will be shown close to the three identified mutations. Concerning the versatility of the 5-FUR R of the furl alleles, it would be attractive to consider the Ukinase and the UPRTase as a complex in which the aa residues defined by the three furl alleles play a key role for the maintenance of the Ukinase activity. But such an hypo. thesis is in strong contradiction with the fact that the deleted alleles furl.A 1 and furl-/tT have invariant Ukinase activity. Indeed, S-FUR metabolism seems to be more complex in yeast. Beside the three identified loci of resistance to S-FUR (which are URKI encoding an Ukinase, URH encoding an Urhase and FUll encoding an uridine permease), five other non allelic loci giving only specific resistance to 5-FUR

(FUI2-FUI6) were isolated but have not yet been correlated with a known enzymatic activity. (5) UPRTase has a key position in the utilization of exogenous pyrimidines and in the reutilization of pyrimidine bases in yeast and the molecular cloning of the FURl gene allows us to investigate the regulation of FUR1. Observations that theintracellular concentration of UTP is the same when UTP is formed from the biosynthetic pathway or from exogenous uracil suggest that UPRTase may be controlled to limit the amount of U M P in the cell. This point is currently under study. ACKNOWLEDGEMENTS

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