Vol. 149, No. 3JOURNAL OF BACTERIOLOGY. Mar. 1982. p. 1064-107()0021-9193/82/031 064-07$02 00/0

Isolation of a Maltase Structural Gene from Saccharomycescarlsbergensis

H. J. FEDEROFF.' J. 1). COHEN.' T. R. ECCLESHALL.' R. B. NEEDLEMAN,2 B. A. BUCHFERER.'J. GIACALONE.' AND J. MARMUR'*

Department of Biochemistry, Albert Einstein Co/llege of Mcdicine, Bronx, New Yortk 10461,1 and Departmentof Biochemistry, Wayne State University. Schiool of Medicine, Detr-oit, Michigan 482012

Received 23 June 1981/Accepted 13 November- 1981

The maltase structural gene MAL6 of the yeast Saccharomyces carlshergensishas been cloned by transformation of a maltose nonfermenting recipient strainwith autonomously replicating chimeric recombinant plasmids. One recombinantplasmid, pMAL26, was shown by positive hybridization translation, as well as bySouthern and Northern blot experiments, to carry the MAL6 structural gene.

A monomeric cx-glucosidase, maltase, is in-duced in the yeast Saccharomyces carlsbergen-sis upon the addition of maltose to the growthmedium (21). The induction of maltase synthesisin yeast is dependent on any one of at least fiveunlinked loci (MALI. MAL2, MAL3, MAL4,and MAL6). Naumov, in analyzing the progenyof crosses between various naturally occurringmaltose-negative and maltose-positive isolatesof Saccharomyces, concluded that maltose fer-mentation requires the presence of two genes,termed MALp and MALg, and that each MALlocus was composed of these two linked genes(19, 20). A maltose-negative strain could arise ifit lacked either a functional MALp or a function-al MALg gene. A strain carrying MALg andMALp, either linked or at unlinked loci, allowsthe cell to utilize maltose as a carbon source.Strains having the genotype mnallp MALgJMAL2p mal2g, for example, grow in maltose-containing medium, suggesting that the MALgor MALp gene product(s) or both act in trans(20). In fact, Naumov determined the genotypeof the MAL6 strain CB1I (the strain used in thisstudy to prepare the clone bank) to be mnallpMALJg mal2p MAL2g MAL6p MAL6g (20).That is, this maltose-positive strain containedthree functional MALg genes and one functionalMALp gene. Previously, ten Berge and co-work-ers had carried out extensive mutagenesis stud-ies, using strain CBI1 and selected mutantswhich were temperature sensitive for growth onmaltose medium (26). None of these mutants,which mapped in the MAL6 locus, possessed athermolabile maltase. Moreover, nonconditionalmal6 mutants had basal (uninduced) levels ofmaltase activity. Naumov 's analysis of tenBerge's mal6 mutants indicated that they lackeda functional MALp gene (20). Thus, the simplestrationalization of the above observations was toascribe a regulatory function to the MALp gene

and to consider the MALg gene as encoding themaltase structural gene.The transformation of appropriate yeast mu-

tants with recombinant plasmid vectors whichcomplement the defect in the mutants has facili-tated the detection, cloning, and isolation ofyeast genes (6, 18). This report describes thecomplementation of a maltase-negative strain bydirect selection for maltose utilization aftertransformation by a pooled collection of autono-mously replicating plasmid vectors, each con-taining a yeast nuclear DNA fragment.

MATERIALS AND METHODS

Bacterial and yeast strains. The following strainswere used in this investigation: Escherichia coli K-12strain RR1I1 (F- pro leuB tili ac Y StrW hsdR hsdM); S.carlsbergensis CB11 (a adel i/ll/p MALIg mal2pMAL2g MAL6p MAL6g). a maltose-fermenting strain:and S. (erevisile JC27 (c. leu2-3 leiu2-112 his4), amaltose-nonfermenting strain derived from a crossbetween the Mal strain HF1066A (cx leu2-3 leu2-112his4) and strain CBtl. The mal genotype of JC27 wasdetermined by allele testing in crosses with CB11, aCB11 derivative carrying a mnalp gene and a number ofmnalp and mialg strains characterized and provided tous by G. Naumov. The results of this genetic analysisdemonstrated that strain JC27 lacks a functionalMALp gene but carries one or more functional MALggenes (data not shown).

Media. L medium (17) supplemented when requiredwith 20 Fg of chloramphenicol, ampicillin, or tetracy-cline per ml was used for culturing bacterial cells.Media for the growth of yeast cells were: YEPD (1%yeast extract, 2% peptone, 2% glucose) and SMal, theselective medium used to discriminate between Mal'and Mal cells (0.7% yeast nitrogen base [Difco Labo-ratories] without amino acids, supplemented with 50p.g of leucine per ml, 50 pg of histidine per ml, and 2%maltose).

Plasmids. Two E. (olilS. cerevisiae hybrid plasmids,pYT11 and pYT14. were previously constructed in ourlaboratory. They consist of the E. co/i plasmid pBR325(4) joined to the yeast 2-km DNA plasmid (14) at their

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unique PstI restriction enzyme cleavage sites. The twoplasmids represent the two possible orientations ofpBR325 relative to the 2-,um DNA plasmid. Bothplasmids retain two drug resistance markers, Tetr andCamr, coding respectively for resistance to tetracy-cline and chloramphenicol. Unique cleavage sites forthe restriction endonucleases BamHI and Sall arepresent within the Tetr gene and can be used forcloning purposes. By inserting a yeast gene (LEU2)into each plasmid, they were both tested and shown tobe capable of transforming leu2 yeast cells with highefficiency (data not shown). As is the case with similarhybrid plasmids containing the yeast 2-p.m DNA repli-con (2, 11), both plasmids behave in yeast as autono-mously replicating extrachromosomal genetic ele-ments exhibiting mitotic instability. The two plasmidvectors were used to construct a yeast genomic DNAlibrary as described below.

Construction of a yeast genomic DNA library. TotalDNA was isolated from the maltose-fermenting strainCB as previously described (8) and mechanicallysheared into fragments of an average size of 15 kilo-base pairs. Plasmid pYTl and pYT14 DNAs. isolat-ed as described below, were linearized by treatmentwith endonuclease BamHI. Polydeoxythymidylate[poly(dT)], and polydeoxyadenylate [poly(dA)] homo-polymers were added to the 3' termini of, respectively,the yeast DNA and plasmid DNAs, as describedelsewhere (22). Poly(dT)-tailed yeast DNA was an-nealed to poly(dA)-extended pYTl 1 and pYT14 DNA,and the mixture was used to transform E. coli RRIOl.Approximately 4,500 transformants of phenotypeCamr Tets Amps were selected and combined intoeight pools, each consisting of about 500 independentclones.

Isolation of plasmid DNA. Plasmid DNA was ampli-fied in growing bacterial cultures by addition of spec-tinomycin (150 ,ug/ml). Extraction and purification ofplasmid DNA were achieved by using the cleared lysateand CsCl-ethidium bromide density gradient centrifu-gation method of Guerry et al. (12).

Transformation of E. coli and yeast with plasmidDNA. Bacterial transformation of CaCl2-treated cellswas performed as described previously (7). For thetransformation of Mal- yeast cells to a Mal' pheno-type, the procedure described by Hinnen et al. (13)was used. The medium for the selection of Mal' yeasttransformants was SMal supplemented with 1 M sorbi-tol, 0.02% glucose, and 3% agar. When yeast transfor-mation was performed with DNA isolated from clonepools, 100 ,ug of DNA was used per transformation.Transformation with individual recombinant plasmidswas achieved with 1 to 5 ,ug of DNA.

Isolation of RNA. Yeast RNA was isolated by a

modification of the method developed by Sripati andWarner (25). Cells were grown in YEP, supplementedwith an appropriate carbon source, to early logarith-mic phase (1 x 107 to 5 x 107 cells per ml). The cellswere collected by centrifugation and washed with ice-cold sterile deionized water. Cells were resuspendedat a density of 2 g (wet weight)/ml in LETS buffer,which contained 10 mM Tris-hydrochloride (pH 7.4)-10 mM EDTA-100 mM lithium chloride-0.2% sodiumdodecyl sulfate (SDS). The cellular suspension was

homogenized with glass beads (0.45 to 0.50 mm indiameter; 1:2 [vol/vol] beads-cell suspension) for 20 s

with cooling in a Braun homogenizer. The cell lysate

was centrifuged at 12,000 x g for 10 min, and thesupernatant was removed, adjusted to 0.1% (vol/vol)with respect to diethylpyrocarbonate, and placed onice for 10 min. RNA was deproteinized by shakingwith an equal volume of chloroform-isoamyl alcohol(24:1, vol/vol) for 3 to 5 min. The mixture was centri-fuged at 12,000 x g for 20 min, and the aqueous phasewas removed. The organic phase was reextracted withan equal volume of LETS buffer, and after centrifuga-tion (12,000 x g for 20 min), this aqueous phase was

pooled with the first one. The combined aqueousphase was extracted by adding 1.5 volumes of redis-tilled, neutralized, water-saturated phenol and 0.5volume of chloroform-isoamyl alcohol (24:1, vol/vol),mixing in a Vortex mixer (3 to 5 min), and centrifugingat 12,000 x g for 20 min. The RNA was precipitated byadding 0.08 volume of 5 M lithium chloride and 2volumes of absolute ethanol and storing for a minimumof 5 h at -20°C.

Polyuridylate [poly(U)]-Sepharose was used for thepreparation of poly(A)+ RNA. Between 0.3 and 0.4 goffreeze-dried poly(U)-Sepharose was swollen in 30 to40 ml of 1 M lithium chloride for 60 min. The Sephar-ose was washed on a sterile scintered-glass funnel with40 ml of 0.1 M lithium chloride and suspended in 20 mlof the same salt solution. The slurry was poured into a

small sterile column and washed with 20 ml each ofL4ETSF (40 mM lithium chloride, 10 mM EDTA, 10mM Tris-hydrochloride [pH 7.5], 0.2% SDS, 5% de-ionized formamide), 90% formamide buffer (10 mMTris [pH 7.5], 10 mM EDTA, 0.2% SDS, 90% deion-ized formamide), and again with L4ETSF buffer. TotalRNA was dissolved in L4ETSF buffer at a concentra-tion of 500 ,ug/ml, heated to 60°C for 5 min, and rapidlycooled in an ice-water bath. The RNA sample was

applied to the column and washed with the L4ETSFbuffer until no further optically absorbing (at 260 nm)material was eluted (usually 20 to 30 column volumes).Poly(A)+ RNA was eluted from the column with 2 to 3column volumes of 90% formamide buffer. Thepoly(A)+ RNA was ethanol precipitated and stored at- 200C.

TABLE 1. Mal' transformation of yeast strain JC27(MalVY

No. of Mal'Source of DNA used in transformation colonies

obtained

Pool 1 .............................. 580Pool 2 .............................. 25Pool3 .............................. 40Pool 4.............................. 315Pool 5 .............................. 0Pool 6 .............................. 0Pool 7 .............................. 0Pool 8 .............................. 0No DNA ........................... 0

a Maltose-nonfermenting (Mal-) strain JC27 wasused as a recipient in transformation. JC27 was trans-formed with recombinant plasmid DNA prepared fromeach of the eight clone pools (see text). Mal' coloniesappeared after 5 to 6 days of incubation at 30°C in SMmedium.

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FIG. 1. Hybridization of radiolabeled pMAL26DNA to fractionated RNA. A 25-,ug portion of totalRNA was denatured in a buffer containing 50 mMboric acid-5 mM sodium borate-10 mM sodium sul-fate-1 mM EDTA-20 mM methyl mercuric hydroxide-20% glycerol, pH 8.2. Denatured RNA samples (10 p.geach) were loaded on a 2.0% agarose gel in this buffer,except that glycerol was omitted and the methylmercuric hydroxide concentration was decreased to 5mM. The gel was run for 700 V-h, using borate buffer(50 mM boric acid, 5 mM sodium borate) with buffercirculation. The RNA was transferred to diazotizedpaper and hybridized with 105 cpm of nick-translatedpMAL26 DNA per cm2 (5 x 107 cpm/,ug). (Lane 1)RNA prepared from uninduced cells of strain CB11;(lanes 2 and 3) RNA prepared from strain CB 1I. I and 2h after the addition of maltose. The migration positionof 18S and 25S rRNA's was determined by hybridizingthe blot with nick-translated cloned yeast rDNA (3,23).

RESULTS AND DISCUSSIONTo construct a clone bank from a donor yeast

strain that carries the MAL6p MAL6g locus,randomly sheared fragments of DNA from strainCB11 were inserted by the poly(dA-dT) connec-tor method (22) into the cloning vectors pYT11and pYT14, which in turn were chimeras of

FIG. 2. Identification of maltase structural genesequences on pMAL26 by positive hybridizationtranslation. A 30-,ug portion of polyadenylated RNAfrom maltose-grown cells of strain CB11 were hybrid-ized to pMAL26 DNA which had been heated to 700Cto relax the supercoiled molecules. Hybridization wasperformed in a solution containing 0.1 M PIPES [pi-perazine-N,N'-bis(2-ethanesulfonic acid)] (pH 7.8),0.01 M EDTA, 0.4 M NaCl, and 70% formamide for 1h at each of the following temperatures: 55, 52, 49, and46°C. After cooling to room temperature, unhybrid-ized RNA was partitioned from R-loops and duplexDNA by chromatography on Bio-Gel A 150 m, equili-brated with a buffer containing 0.01 M Tris-hydrochlo-ride (pH 7.4)-0.8 M NaCl-0.001 M EDTA. The frac-tions containing R-loops were pooled and ethanolprecipitated. RNA was melted from the hybrids byheating to 100°C for 3 min before translation in a yeastcell-free translation system (10). Approximately three-quarters of the translation reaction was immunopre-cipitated with maltase antibody raised in rabbits to thepurified enzyme. Total and immunoprecipitated trans-lation products and controls were run on a 10% SDS-polyacrylamide gel (15) and autoradiographed (5).(Lane 1) Total R-looped RNA cell-free translationproducts; (lane 2) immunoprecipitated cell-free trans-lation products of R-looped RNA; (lane 3) cell-freetranslation products from total CB11 polyadenylatedRNA; (lane 4) immunoprecipitated total CB11 RNAcell-free translation products; (lane 5) endogenouscell-free translation products (no added RNA).

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YEAST MALTASE STRUCTURAL GENE 1067

pBR325 (4) and the yeast 2-km plasmid (14)joined at their unique PstI restriction sites. Thecloned yeast DNA was then amplified in E. coliRR101. The recombinant plasmid DNA isolatedfrom pools of 500 E. coli (pYT) transformantswas used to transform the Mal- strain JC27.Several pools gave higher numbers of Mal'yeast transformants than did other pools orcontrols lacking added DNA. These data arepresented in Table 1.Mal+ colonies were identified as transform-

ants and distinguished from possible revertantsin the following manner: several Mal' colonieswere picked from the original transformationplates and purified by streaking on SMal plates.Purified Mal+ colonies were again picked, andthe stability of their Mal+ phenotype was testedafter growth on nonselective medium (YEPD).Most of the Mal+ colonies obtained with theDNA from pools 1, 2, 3, and 4 (Table 1) werefound to be highly unstable during mitoticgrowth as they segregated mostly Mal- clones.This mitotic instability is an expected character-istic of transformants obtained with nonintegrat-ing vectors such as pYT11 and pYT14 (11).DNA prepared from several Mal+ yeast trans-

formants grown under selective conditions

Eco RI FRAGMENTS A

(SMal medium) was used to transform E. coli tochloramphenicol resistance (Camr) since eachrecombinant pYT DNA molecule still retainedthis drug resistance marker. Plasmid DNAs iso-lated from several individual E. coli Camr trans-formants were then tested for their ability totransform JC27 to the Mal' phenotype. Theplasmid pMAL26 was subsequently identifiedby this approach. The CB11 genomic DNAfragment carried by pMAL26 thus complement-ed the defect in strain JC27 so as to allow thestrain to grow with maltose as a carbon source.

It is unlikely that transformation to the Mal'phenotype was due to a suppressor carried bythe plasmid since extracistronic suppression wasnot detected in genetic crosses between donor(CB11) and recipient (JC27) strains.To determine whether pMAL26 contained any

sequences complementary to the maltose-induc-ible RNA, pMAL26 DNA was hybridized tototal cellular RNA that had been immobilized bythe procedure of Alwine et al. (1). It had beenshown previously that the maltase mRNA mi-grated with an apparent sedimentation coeffi-cient of 17S (H. J. Federoff, Ph.D. thesis, AlbertEinstein College of Medicine of Yeshiva Univer-sity, New York, N.Y., 1979). Total RNA pre-

MALTASE STRUCTURAL GENE

B IFI C I H III E I D A

2)JM DNA pBR325 YEAST DNA INSERT

lpBR325

IlKb

I =EcRI2 Av= I

4 z Pi5 -bQQI6 Sal I7=PstI(a)8 =Hnd (a)

FIG. 3. Restriction map of plasmid pMAL26. Plasmid DNA was digested with the appropriate restrictionendonucleases, using the reaction conditions suggested by the supplier (Bethesda Research Laboratories,Bethesda, Md.). When necessary, multiple digests were performed. In addition, several individual restrictionfragments were purified from agarose gels (27) and subjected to restriction analysis. Restriction digests wereanalyzed on 0.8% agarose gels run in a buffer containing 40 mM Tris acetate (pH 7.8)-0 mM sodium acetate-1mM EDTA. The approximate location of the structural gene for maltase was derived from the experiments andresults described in the legent to Fig. 4. 1, EcoRI; 2, AvaI; 3, BglII; 4, PvuII; 5, HpaI; 6, SalI; 7, PstI (a); 8,HindlIl (a). (a) indicates that not all PstI and HindIII sites are shown.

II I I 111111 II1ItIIII I IIlIII1752 1 2 4 17 41 2 31 81 1642 1 3 53 12 5486 24

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pared from induced and uninduced cells of strainCB11 was fractionated by electrophoresis on anagarose gel containing the denaturant methylmercuric hydroxide and transferred to DBMpaper. The immobilized RNA was then hybrid-ized to 3P-labeled pMAL26 DNA prepared bynick translation (23). The result shown in Fig. 1indicates that the plasmid hybridized to a malt-ose-inducible RNA which migrated to the posi-tion expected for 17S RNA. This result indicatedthat the plasmid contained either all or part of aDNA sequence complementary to a maltose-inducible RNA species. To show that pMAL26hybridized to a transcript which was indeedmaltase mRNA, a positive hybridization-transla-tion assay was performed. Supercoiled pMAL26DNA was relaxed by heat treatment, denatured,and hybridized (under conditions which favor R-loop formation) to excess polyadenylated RNAprepared from maltose-grown cells of strainCB11. Specific R-loop and duplex DNA werepartitioned from nonhybridized RNA by themethod of Woolford and Rosbach (28). After theRNA was dissociated from the R-loops, it wastranslated in the presence of [35S]methionine in ayeast cell-free protein-synthesizing system (10).The cell-free translation products were immuno-precipitated with maltase antibody, and the totalimmunoprecipitated cell-free translation prod-ucts were displayed on a 10% SDS-polyacryl-amide gel. The autoradiogram of this Eel (Fig. 2)showed that immunoprecipitable maltase wassynthesized in vitro from RNA complementaryto pMAL26 DNA. Identical results were ob-tained with a wheat germ cell-free protein-syn-thesizing system (data not shown).As a first step in determining the location of

the maltase structural gene within the 11.4-kilobase genomic insert of pMAL26, a restric-

FIG. 4. Mapping the maltase structural gene inpMAL26 DNA. The DNAs of plasmids pMAL26 andpYT11 (parent vector) were digested with restrictionenzymes, using conditions specified by the commer-cial supplier. DNA digests were fractionated on a 1%agarose gel and transferred to a nitrocellulose sheet(24). Polyadenylated RNA prepared from maltose-induced cells of strain CB11 was fractionated on asucrose gradient, and the 17S region was collected.The RNA was fragmented in 40 mM Tris-hydrochlo-ride, pH 9.0, at 80°C for 15 min, ice chilled, and thenend labeled with [fy-32P]ATP and T4 polynucleotidekinase (16). Radioactivity labeled RNA was hybrid-

ized to the nitrocellulose sheet in modified Denhardtsolution (9), which contained 5x SSC ( x SSC is0.015 M sodium citrate, pH 7.0, plus 0.15 M sodiumchloride), 50% formamide, 0.5% SDS, 10 ,ug ofsheared denatured calf thymus DNA per ml, 10 mMsodium phosphate buffer (pH 6.8), 0.02% Ficoll (Phar-macia Fine Chemicals, Inc.), 0.02% polyvinylpyrroli-done, and 0.02% bovine serum albumin (fraction VSigma Chemical Co.) The hybridization was carriedout at 45°C for 16 h. After extensive washing inhybridization solution, the nitrocellulose sheet wasexposed to Kodak SB5 X-ray film in the presence of aLightning-Plus (DuPont Corp.) intensifying screen. (A)Ethidium bromide stain pattern of the restriction frag-ments of pMAL26 and pYT11. (B) Autoradiogram ofthe nitrocellulose sheet bearing DNA transferred fromthe gel depicted in (A) after hybridization with -32P-labeled 17S polyadenylated RNA. No hybridizationwas expected, or seen, in the control lanes containingonly pYT1I DNA.

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YEAST MALTASE STRUCTURAL GENE 1069

tion map was constructed (Fig. 3). Polyadenylat-ed RNA prepared from maltose-induced cells ofstrain CB11 was fractionated by sucrose gradi-ent centrifugation, and the maltase mRNA-en-riched 17S region was collected. This 17S RNAfraction was then fragmented, radiolabeled using[y-32P]ATP and T4 polynucleotide kinase, andused to hybridize to a Southern blot of an

agarose gel containing various restriction en-

zyme digests of pMAL26 DNA. The ethidiumbromide-stained gel and the autoradiogram ofthe hybridized blot are shown in Fig. 4A and B,respectively. Analysis of this experiment indi-cates that the maltase structural gene is con-

tained within EcoRI fragments D and E. Thesefragments define a maximum coding region of4.7 kilobase pairs. Hybridization-translation ex-

periments by preparative R-looping with EcoRIfragment D have confirmed that it containssequences complementary to maltase mRNA(data not shown).

Since the sequences complementary to themature maltase mRNA are contained whollywithin the DNA insert, the adjacent regions maycontain other genes of the MAL locus. Theobserved complementation of malp strains bypMAL26 does not necessarily imply that theplasmid contains a functional MALp gene. Theregulation of the plasmid-borne maltase struc-tural gene may be different from that of the genelocated at a chromosomal site. In addition, or

alternatively, the cumulative effect of basal levelRNA synthesis from a multicopy plasmid mightproduce enough maltase mRNA, and hencemaltase, in the absence of a regulatory gene toallow the cells to utilize maltose. If these twopossibilities do not hold, then it is likely that thegenomic insert contains a MALp gene, specifi-cally MAL6p, since the DNA was derived fromstrain CB11. Furthermore, if it is assumed thatcomplementation of JC27 is due to a functionalMALp gene on pMAL26, one cannot rigorouslyexclude the possibility that the cloned maltase-coding sequences are nonfunctional. These pos-sibilities are currently being resolved by sub-cloning smaller fragments of the original 11.4-kilobase insert, determining the minimum sizeand DNA base sequences of the fragmentswhich can complement malp and malg strainsand finding their relationship to fragments whichhybridize to maltase mRNA.The availability of the cloned maltase struc-

tural gene will facilitate the study of the regula-tion of transcription and turnover of maltasemRNA in wild-type and mutant strains. Theexpression of chromosomal and plasmid-associ-ated maltase genes will be compared underrepressed and induced conditions. The clonedDNA should also be helpful in elucidating theorganization of the MAL6 locus.

ACKNOWLEDGMENTS

Support for H.J.F. was from the American Cancer Society,SPF-18. J.D.C. was supported by Public Health Servicefellowship 1F32 GM06209 from the National Institutes ofHealth. Support for T.R.E. was from Public Health Servicegrant 1T32 AG00052 from the National Institutes of Health.R.B.N. was the recipient of a Research Development Awardfrom Wayne State University. Support for J.M. was fromPublic Health Service grant 5R01 CA12410 from the NationalCancer Institute.

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