Gene, 95 (1990) 91-98

Elsevier

91

GENE 03754

T h e A D E 2 gene f r o m Saccharom.w.es cerevisiae: s e q u e n c e a n d new vectors (Recombinant DNA; phosphoribosylamino-imidazole-carboxylase; exonuclease III; nuclease S 1; deletion analysis; autonomously replicating sequence; yeast plasmids; red pigment)

Agathe Stotz and Patrick Under

Department of Microbiology. Biozentrum. CH-4056 Basel (Switzerland) Received by T.A, Bickle: 18 April 1990 Revised: 8 June 1990 Accepted: I I June 1990

SUMMARY

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevbiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single Bglll fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schl~osacchammyces pombe and to the proteins encoded by the purEK operon of Escherichia coll.

INTRODUCTION

Mutants in the S. cerevbtae gene ADE2 (encoding phosphoribosylamino-imidazole-carboxylase, EC 4.1.1.21) have widely been used because they accumulate a red pigment (Ephrussi et al., 1949; Reaume and Tatum, 1949; Roman, 1956). In several studies this effect has been used in combination with other genes as a means to visualize processes occurring in vivo. Hieter et 8.1.(1985) have used this phenotype in an elegant way to follow the fate of cenCorrespondenceto: Dr. P. Linder, Department of Microbiology, Biozentrum, Klingelbergstrasse 70, 4056 Basel (Switzerland) Tel. (061) 2538 80; Fax (061)256760; E-mail: linder%urz.unibas.eh@cernvax.

Abbreviations: aa, amino acids; ade, mutations in the biosynthesis of adenine; ADE2, gene encoding phosphoribosylaminc~imidazole-carboxylase; AICAR, 5'.phosphoribosyl-5-amino-4-imidazole-carboximide; AR$, autonomously replicating sequence; bp, base pair(s); CEN, centromer; dideoxy, 2',Y-dideoxy-NTP; dNTP, deoxynucleotide triphosphate; ds, double strand(ed); exolll, exonuclease 1Ii; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani broth; nt, nueleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PEG, polyethyleneglycol; Pollk, Klenow (large) fragment of E. coil DNA polymerase i; pur£K, operon encoding phosphoribosylamino-imidazole-earboxylase; ss, single strand(ed); wt, wild type; [ ], denotes plasmid.carrier state. 0378-1119190/$03,50 0 1990 Elsevier ~'~ciencePublishers B.V. (Biomedical Division)

tromere plasmids in mitosis. They have used the suppressor gene SUPll, which suppresses the ade2-101 mutation and leads therefore in a homozygote diploid strain to a white colony, if the suppressor is present in more than one copy, to a pink color if the suppressor is present in one copy and to a red color ifno suppressor is present. By visual checking of the colonies the fate of the plasmid can be followed. A similar study using a different combination of markers has been described by Koshland et al. (1985). In this study the authors have taken advantage of the fact that ade3 mutations are epistatic to ade2 mutations, i.e., double ade2 ade3 mutations are white. In this context, the segregation of the ADE3 gene on a plasmid in an ade2 ade3 background can easily be analyzed. In yet another system, Brushi and Howe (1988) have used the ADE8 gene cloned on a plasmid in an adel ade2 ade8 background to follow in rive recombination events. Since the ade8 mutation is also epistatic over ade2 the cells shift from red to white in this system if the wt ADE8 gene on the plasmid is lost due to a recombinational event. The visualization of sectored colonies in a strain carrying an ochre suppressor and the ade2-101 allele has also been used for the isolation ofochre mutations in essential genes (Riles and OIson, 1988). in this system the ade2-101 mutation is overcome by a suppressor of ochre

92 mutations. Due to the instability of the plasmid bearing the suppressor gene, the colonies are sectored. If, however, an essential gene is also dependent on the presence of the ochre suppressor, no segregation of the plasmid and thus no sectoring can occur. Beside these various methods to study in vivo processes using the ade2 mutations, many laboratory yeast strains have an ode2 mutation and the availability of this gene for E. coil-yeast shuttle vectors and for gene replacement experiments would be extremely useful. Despite extensive searches in literature (MEDL) and nucleic acid databanks (EMBL, release 21) no source of a suitable ADE2 fragment or sequence could be found. Therefore, the aim of this study was to characterize a minimal ADE2 fragment for complementation of an ade2 mutation and to establish its primary sequence. Using these data we planned then to construct a fragment that should be of general use in molecular genetic experiments in $. cerevbiae. Recently the ADE6 gene from Schizosaccharomyces pombe has been sequenced (Szankasi et al., 1988). This gene is the equivalent to the ADE2 gene in S. cerevisiae,Two genes from the pur£K operon in E. coli have also been sequenced recently (Tiedeman et al., 1989; Watanabe et al., 1989).

RESULTS AND DISCUSSION

(a) Subeloning of the ADE2 gene from pFL39.AD£2 The plasmid pFL39.ADE2 carries an insert of approx. 5.2 kb complementing the ode2.1 mutation in the strain C W 0 4 (MATer ade2.1 his3-11,15 leu2.3,112 trpl-I ura3.1 eanl.lO0 (Benroques et ai., 1986)). To obtain a suitable restriction fragment for cloning, we carried out restriction

analysis of this plasmid. Subcloning of Hindlll and NsiI fragments revealed that none of the Hindlll fragments could complement the ade2-1 mutation, but the Nsil fragment (4.2 kb), cloned in the Pstl site of pFL39 could (data not shown). Therefore, we concluded that all the information necessary for complementation must be contained within this fragment. As it has been reported that one ofthe HindIll fragments confers the ability to replicate in yeast (Sasnauskas et al., 1987), we subcloned this NsiI fragment into the Pstl site ofplasmid pUC7 and transformed CWO4. The efficiency of transformation of this plasmid was compatible with the presence of an ARS element in this sequence (data not shown). The plasmids used in this study are listed in Table I. (b) Genetic linkage with AD£2 Although it is clear from the sequence comparison (see section d) with the ADE6 gene from Schizosaccharomyces pombe (Szankasi et aL, 1988) that this gene indeed codes for the phosphoribosyl-aminoimidazole-carboxylase we cheeked the linkage of the fragment with the genomic ADE2 locus. For this purpose, ADE2 was cloned into an integrative vector, pEMBLYi32, derived from pEMBLYi21 (Baidari and Cesareni, 1985) between the EcoRl and Sail sites, using a deletion subclone (pASZg, see section e). The resulting plasmid was cut at a unique Hpal site within the ADE2 gene and transformed into JY425 (MATa hb4-34 leu2.3,112 ura3-52; Cold Spring Harbor Laboratory course on Yeast Genetics, 1988). Integration of the plasmid pEMBLYi32.ADE2 by homologous recombination was verified by Southern analysis of genomic DNA (data not shown). The resulting strain JY425[pEMBLYi32-ADE2] was then crossed with strain CWO4 and tetrad analysis was

TABLE I Plasmids used in this study Name

Parents

Description

Source F, Lacroute

pTZIgR pTZ 19B pUCT-ADE2 pTZI9B

ARS.CEN in ClaI linker inserted at nt 747 in pUCI9, TRP! (EcoRI.Pstl) with BglII linker inserted at nt 629 in pUCI9 Multipurpose vector containing a fl phage origin of replication Chromosomal fragment complementing ade2 inserted in pFL39 Bglll linker at coordinate 416 of pTZIgR unidentified ARS element inserted in Bg/II site of pTZI9B Nsil fragment cloned in Pstl site of the polylinker ADE2 8ene inserted in the Sell site of the polylinker of pTZlgB used for

pFL39

pTZ ! 8R/pTZ19R pFL39-ADE2 pTZI9B pASZ5 pASZ6 pASZ7 pASZ8 pASZ9 pASZ 10

pASZI! pEMBLYi32 pEMBLYi32-ADE2

Pharmaeia F. Lacroute this study this study this study this study

exonueleasedeletions pTZ 19B pASZ$ pTZI9B pFL39

ADE2 (after first exolIi deletion) AD£2 (after second exolI! deletion) Bglll fragment (clone gt 0) inserted in BglII site of pTZ 19B BgiiI fragment ~ ! inserted in BgiI! site of pFL39 Derivative of pEMBLYi21 by deletion of Pstl site in the URA3 gene EcoRI-Sail from pASZ8 cloned in the polylinker of pEMBLYi32

this study this study this study this study G. Cesareni this study

93 performed. In 29 tetrads all Ade ÷ spores were also Urn "~, indicating that the two genes segregate 2:2. In a few tetrads, however, we found spores which were A d e - and Ura +, but all these cells remained white, indicating that an additional mutation was present. All these spores were found to carry indeed a wt ADE2 allele by crossing with other ade2- strains (data not shown). It is thus clear from the present data, the complementation and the sequence comparison with the ADE6 gene of Scl~.osacclmromyces pombe that we have indeed analyzed the ADE2 gene of S. cerevbiae.

subcloning: Nsil fragment into Pstl

put7 ~'~'"~' recloning: Sail fragment into polylinker of oTZIgB

exonuclease digesUon: cut BamHI and Kpn I digest exoIII and SI rellgete

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, ,ARS

(e) exolll deletion analysis of the ADF.2 gene The NsiI-ADE2 fragment cloned in pUC7 is flanked by two Soft sites present on the polylinker of pUC7 (Fig. 1). We used these Soil sites to clone the entire fragment into the po|ylinker of pTZI9B ( = pTZI9R carrying a Bg/li linker at nt 416) and made exolil deletions. To obtain unidirectional deletions in the pASZ7 plasmid ( -- pTZ 19BADE2), it was cut with BamHI + ICpnl prior to exolll digestion. The resulting clones were selected according to their size by gel electrophoresis. Since we have shown that the aVsil fragment can replicate in yeast we retransformed some of them directly using the plasmids from the deletion analysis into strain CWO4. Smaller clones which no longer confered growth on selective medium to CWO4, were recloned in the replicating yeast vector pFL39. This way we were able to distinguish between absence of the A R S element and the ADE2 sequence. The two smallest clones corresponding to these two phenotypes, clones ADE2 # 0 (Ars +, Ade2 +) and ADE2 # 1 (Ars-, Ade2 + ) were chosen for further studies (2,5 kb and 2,2 kb, respectively). To delimit the sequence requirements for the ude2 complementation on the other side of the gene, we recloned the smaller fragment ADE2 # ! in pASZ5 (pTZIgB-ARS) in

ADE2#0

ADE2#I

Fig. I. Representationofthe plasmidsand the differentsteps used in the deletionanalysis.The fragmentsand plasmidsare not drawn to scale.The Arsii fragmenthas been subcloned into the Pstl site of the polylinkerof pUC7 resultingin pASZ6(pUC?-ADE2).From this plasmid,whichhas

two Soil sites flankingthe Pstl site of the polylinker,the gene has been recloned in the Sail site of pTZI9B (pASZT,designated as pTZi9BAD£2 for clarityof the figure).This plasmidhas b~en used to produce exolll deletions.The shortest clone still compiementin~the ode2 mutation was then recloned in the opposite orientation in pASZ$ (pASZ5 = the pTZ19Bplasmidcarryingan ARS elementin the Bg/ll site at nt 416) for exoili deletionfromthe other side.The two clonesdiffering by the presenceand absenceof the abilityto conferautonomousreplication are indicatedby clones #0 and # I, respectively.Mediafor growth of yeast and the transformationprocedure was descibed by Sherman et aL(I 986).The exolI!deletionanalysiswas carriedout accordingto the protocol of Shiraishi and Shimura (1988) except that, after the exolll digestion,the ss DIqAwas digestedby SI nuclease(50 units) in 200pl reaction volume(for ! #8 DNA) and prior to ligationa filling-inby Pollk with all four dNTPs was carried out. Clones obtained upon transformation ofJMI09 (Yaniseh-Perronet al., 1985)were analyzedfor their size by gel electrophoresis.The ss DIqA of clones of interest was sequenced using the T7 polymerasekit from Pharmaciaand analyzedon 6% sequencinggels.

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95

opposite orientation and exolll digestion was carried out on DNA linearized with Ban, HI or KpnI. Analysis of the resulting plasmids in yeast did not reveal any complementing clone much smaller than the parental. Thus the minimal size .of a complementing fragment was 2.5 kb (including the ARS function).

(d) Sequence analysis The exolll deletion products from beth orientations were subjected to dideoxy sequencing analysis taking advantage of the phasmid system to sequence ss DNA rather than dsDNA. Uncertain regions in the sequence were either subcloned or oligos were used as internal primers. The resulting sequence ofthe clone ADE2 # 0 is shown in Fig. 2. The two clones, A D E 2 # 0 and ADE2# 1, differ by the absence of 278 bp from the 5' end ofclone # 1 (Fig. 2). This locates at least part of the sequence requirement for replication in this region. We cannot, however, find a sequence which has eleven or ten matches to the 1l-bp ARS core element (5'-TATFTAT~TFr~-Y; Newlon, 1988), but we can find eight sequences with two mismatches with this consensus sequence. Complemcntation analysis with clones ctlt at the natural BgilI site at the 3' end of the sequence has shown that the sequence beyond this site is not required for function of the ADE2 gene. We therefore set the limit in the given sequence to this site, which is common to ADE2 # 0 and ADE2 # I and is also the site used in the fragments used in further studies (see below). Analysis of the sequence reveals an ORF of 571 aa from nt 650-2366. The sequence ofthis putative protein has been compared to the protein of the ADE6 gene from ScAizosaccharomyces pombe (Szankasi et al., 1988) and the two proteins Pure and PurK encoded by the purEK operon from E. coil (Tiedeman etal., 1989; Watanabe etal., 1989) (Fig. 3). It is clear fro~ the similarity that the two proteins of $. cerevisiae and $chizosaccharomyces pombe are closely related, with 57% identical aa. The homologies to Pure and PurK sequences are of 46~o and 32~o, respectively. It has been suggested that the Pure protein is the catalytic subunit and the PurK protein the CO2-binding protein (see Tiedeman et al., 1989). We have set out to complement a bacterial pure mutation by the yeast ADE2 gene (Nsil fragment cloned in pTZ 1913, Fig. 1). No complementation,

however, was observed (data not shown). This could be due to the absence of regulatory sequences for the expression of the gene in E. coli or simply to nonfunctionality of the yeast gene product in bacteria. We have also checked for the presence of regulatory sequences. The sequence 5' to the ATG is rich in A's and T's and one potential TATA sequence (TATAAA at nt 555) could be found, but the in vivo function has not been tested. No sequence similarities to the conserved dements in the URAI and URA3 genes (Losson et al., 1985) are present. In the search for known sequence elements we have, however, detected two potential binding sites for the Gcn4 protein, the regulator ofgeneral aa control (TGACTC at nt 454 and 497)(for review see Hinnebusch, 1988). Compared to other genes, known to be regulated by GCN4, the localization of these sequences would suggest that the expression of the ADE2 gene is (also) under general aa control. In this respect it is interesting that a side product of the His synthesis pathway (AICAR) is an intermediate of the purine biosynthesis pathway (Jones and link, 1982) and that ade3 mutants are also His- (Roman, 1956). (e) Construction of a yeast vector using the ADE2 gem To be able to use the ADE2 fragment in gene disruption experiments and as a selectable marker on a plasmid we decided to create Bglll fragments carrying the gene with and without the ARS sequence. Using site directed mutagenesis ofthe ADE2 # 0 and ADE2 # I clones we destroyed the internal Bglll sites by replacement of the A at nt 1243 by a G, which does not change the protein sequence (Fig. 2). The resulting 'Bglll-mutant' was checked for the absence of the Bg/ll site and functional activity in yeast. Digestions with Sau3A and Rsal show no rearrangements in the resulting fragments (data not shown). The absence of visible rearrangements and the functionality in vivo argue that the Bg/ll mutant does not carry any undesired secondary mutations. To obtain Bglll sites at both ends of the gene we added a Bglll linker to the 5' end of the sequenced fragment and used the natural Bgill site at coordinate 2512. To do so the clones ADE2 # 0 (Ars +, Ade2 ÷ ) and ADE2 # l (Ars -, Ade2 +) in pTZ19B, having the internal Bglll site destroyed, were cut with EcoRl, the overhanging ends were filled by Pollk and religated in the presence of Bglll linkers

end of the fragmentand the deducedproteinsequenceare indicated.The 5' borderof cloneADE2# i at nt 278 is also indicated,the Y bordersbeing identical.Phasmidparticleswereinducedin LB(containing0.001%thiamine/14/Atkanamycinper ml/100/Atampiciilinper ml)bysuperinfectianofhelper pl',age MI3K07 (presentin the mediumusedfor inoculationat approx. 109 phages/ml).Ph•smids wereprecipitatedwith a solutionof 38% PEG/I.15 M NaCI (125 pl into 1400#1 supernatant).Afterphenolextractionand ethanolprecipitationthe ss DNA was resuspendedin 10-20#1 TE. Sequence analysisbycomputerhas beendoneon a VAXusingtbe SequenceAnalysisSo,warePackage(version6.1,April 1989)ofthe Univ.ofWisconsinGenetics ComputerGroup(UWGCG;Devereuxet al., 1986).Databankcomparisonwiththe EMBL(release2!) and MIPSX(release15)havealsobeencarried out usingthis programpackage.The genomicsequencehas receivedthe accessionnumberM32013(EMBL/GenBankdatabases).

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Fig. 3. Similarity to the Ade6 protein from $chizosaccharomycespombe and the Pure and PurK proteins from £. coil The alignment was made by the GAP program and the output files were aligned using the LINEUP program of the UWGCG programs. The proteins Purl{ (aa 1-400) and Pure (aa

402-570) havebeenalignedin one linewithAde2,althoughthey are encodedin E• co//bytwo ORFs•The last 4 aa ($ K F G) of PutK rarenot indieat~.'d in the figure.Identicalaa are boxed. (10-mers). In addition, the Sacl site of the polylinker had been previously destroyed by $1 digestion• Resulting clones were analyzed for the presence of a new Bgill site and the fragments were cloned into the BamHI site ofpTZ19B for sequencing of the ends. The sequence of the ends is indicated by capital letters above the ADE2 sequence in Fig. 2. The ADE2 gene on a BgllI fragment is convenient for use as a se!ectable marker in the pFL plasmids. In these plasraids, constructed by F. Lacroute, a Bglll linker is present in pUCI9 at nt 629 for cloning of auxotrophic markers. In addition to this Bglll linker these plasmids also carry a ClaI

linker at nt 747, where it is possible to insert an ARS-CEN cassette or a 2# origin of replication (F. Lacroute, unpublished). The availability of the ADE2 fragment therefore extends the use of these plasmids considerably. The resulting plasmid is called pASZII (Fig. 4). It contains HindIII, H~cII and Xbal sites in common with the polylinker. The polylinker and the white/blue selection for inserts is still available in this plasmid. Although we have not resequenced the entire plasmids, it is possible to reassemble the sequence and to obtain the complete sequence of the vectors constructed. We also constructed a pTZI9R plasmid containing a

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(f) Conclusions (1) We have been able to determine the minimal sequen~ requirements for complementation of ade2 mutations in S. cerevisiae. (2) The sequence of the fragment has been determined. (3)A BgilI fragment suitable for the use in yeast-E, coli shuttle vectors as well as in gene disruption experiments has been constructed. (4)The plasmids are also useful due to the red color of ade2 strains.

ACKNOWLEDGEMENTS III

1000

4000--

ADE2 gene. To show that these plasmids are not restricted to the ade2 allele in strain CWO4 used throughout this study we also transformed other ade2 strains with the pFL39ADE2 plasmid. In the strains Sl4a (MATa ade2-5) and YM701 (MATa ade2-101 his3-200 iys2-801 trpl-901 tyrl -501 ura3-52) we have observed complementation as with CWO4 showing that the plasmids are generally useful.

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We would like to thank F. Lacroute for pFL39 and for making the ADE2 gene available to us. We are also very grateful to R. D01z for maintenance of the computer facilities and to M. Hall and A. Prat for stimulating discussions and critical reading of the manuscript. This work was supported by grants 3.568-0.87 and 31-26343.89 from the Swiss National Science Foundation to P. Linder.

REFERENCES 000

Hindtll

Fig. 4. Yeast-E. coltshuttle vectors containingthe ADE2 gene. Plasmid pASZI 1 has been derived frompFL39 by replacingthe TRPl 8eneby the ADE2 # I ( Ars-, Ade2÷) fragment.The extensions of the ADE2 fragment and the AR$.C£N fragment (ClaI) are indicated by the ranges within the circles.The pASZ 10plasmid contains the AD£2 # 0 fragment and is based on pTZ 19B.It can be used for the isolationof ss DNA upon snperinfection with a helper phage. The polylinkers in pASZII and pASZI0 are indicated by PL.

BglII linker at position 416 ( = pTZ 19B) for the use with the ADE2 # 0 fragment (resulting in plasmid pASZ 10; Fig. 4). This plasmid is suitable for induction ofss D N A packaging with a helper phage, for sequencing, exolII deletion or site-directed mutagenesis. Depending on the strains, this construct gives small colonies, presumably due to instability of the plasmid. We also tested the ADE2 marker for efficiency of selection in pFL39-ADE2 by comparison with the TRPI and the ADE2 markers. We cannot detect any significant reduction of the efficiency of plating when selecting for the

Baldari, C. and Cesarani, G.: Piasmids pEMBLY:new single-stranded shuttle vectorsfor the recoveryand analysisofyeast DNA sequences. Gene 35 (1985) 27-32. Banroques,J., Delahodde,A. and Jacq, C: A mitochondrialRNA maturase gene transferredto the yeast nucleuscan control mitochondrial mRNA splicing.Cell 46 (1986) 837-844. Bruschi,C.V. and Howe,G.A.: HighfrequencyFLP.independenthomolngous DNA recombinationof 2 pm plasmid in the yeast Saccharo. myces cerevbiae.Curr. Oenet 14 (1988) 191-199. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensiveset of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Ephrussi, B. Hottinguer,H. and Tavlitzki,J.: Actionde racriflavinesur les levurcs.Ann. Inst. Past. 76 (1949)419-442. Hieter, P., Mann, C, Schnyder,M. and Davis, R.W.: Mitotic stabilityof yeast chromosomes: a colony color assay that measures nondisjunction and chromosomeloss. Cell 40 (1985) 381-392. Hinnebusch, A.G.: Mechanismsofgene regulationin the general control of amino acid biosynthesis in $accharomycescerevbiae. Microbiol. Rev. 52 (1988) 248-273. Jones, E.W. and Fink, G.R.: Regulation of amino acid and nucleotide biosynthesisin yeast.In Strathern,J.N., Jones, E.W.and Broach,J.R. (Eds.), The MolecularBiologyof the Yeast $accharomyoe$cenevisiae: Metabolism and Gene Expression.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 181-299. Losson, R., Fuchs, R.P.P. and Lacroute, F.: Yeast promoters URAI and URA3: examples &positive control.J. Mol. Biol. 185 (1985)65-81.

98 Koshland, D., Kent, J.C. ~nd Hartwell, LH.: Genetic analysis of the mitotic transwission of minichromosomes, Cell 40 (1985) 393-403. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor La~,o~to~, Cold Spring Harbor, NY, 1972. Newlon, C.S.: Yeast chromosome replicationand segregation. Microbioi. Rev. 52 (1988) 568-601. Reaume, S.E. mid Tatum, E.L.: Spontaneous and nitrogen mustardinduced nutritional deficiencies in Saccharemyces cere~s/ae. Arch. Biochem. 22 (1949) 331-338. Riles, L and Olson, M.V.: Nonsense mutations in essential genes of Sacc~remyces cere~isiae.Genetics i 18 (1988) 601-607. Roman, H.: Studies of gene mutation in Succhuromyces. Cold Spring Harbor Syrup. Quant. Biol. 21 (1956) 175-185. Sasnanskas, K.V., Giadvilaite, A.A. and Janulaitis, A.A., Cloning of the ADE2 gene of Sacchammyces ceveplslueand localization of the ARS sequence. Gcnetika 23 (1987) 1141-1148. Sherman, F., Fink, G.R. and Hicks, J.B.: Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1986.

Shiraishi, H. and Shimura, Y.: A rapid and efficient method for targeted random mutagenesis. Gene 64 (1988) 313-319. Szankasi, P., Heyer, W.D., Schuchert, P. and Kohli, J.: DNA sequence analysis of the ode6 gene of $chizosucchuvomycespombe. Wild-typo and mutant alleles including the recombination host spot allele ade6-M26. J. MUl. Biol. 204 (1988) 917-925. Tiedeman, A.A., Keyhani, J., Kambolz, J., Danm, H.A., Gots, J.S. and Smith, LM.: Nucleotide sequence analysis of the purEK operon encoding 5'-phosphoribosyl-5-aminoimidazole carboxylase of Eschegchia coil KoI2. J. Bacteriol. 171 (1989) 205-212. Watanabe, W., Sampei, G., Aiba, A. and Mizobuchi, K.: Identification and sequence analysis of Escherichia coli pure and puvK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis. J. Bacteriol. 171 (1989) 198-204. Yanisch-Perron, C., Vicira, J. and Messing, L: Improved MI3 phage cloning vectors and host strains: nucleotide sequences of the Ml3mpl8 and pUCI9 vectors, Gene 33 (1985) 103-119.

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors.

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complemen...
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