Vol. 166, No. 2JOURNAL OF BACTERIOLOGY, May 1986, p. 484-4900021-9193/86/050484-07$02.00/0Copyright © 1986, American Society for Microbiology

Cloning and Expression of a Saccharomyces diastaticusGlucoamylase Gene in Saccharomyces cerevisiae and

Schizosaccharomyces pombetJ. A. ERRATT* AND A. NASIM

Molecular Genetics Section, Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario,Canada KIA OR6

Received 1 October 1985/Accepted 3 February 1986

A recombinant plasmid pool of the Saccharomyces diastatcus genome was constructed in plasmid YEp13 andused to transform a strain of Saccharomyces cerevistae. Six transformants were obtained which expressedamylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of thesefragments were cloned in the same orientations and had ideittical restriction maps, which differed from the mapof the STAI gene (I. Yamashita and S. Fukui, Agric. Biol. Chemi. 47:2689-2692, 1983). The glucoamylaseactivity exhibited by aU S. cerevisiae transformants was approximately 100 times less than that of the donorstrain. An even lower level of activity was obtained when the recombinant plasmid was introduced intoSchizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragmenthybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX(STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetradanalysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kbhomologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present ineach of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S.cerevisiae and S. diasticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kbspecies was present only in the RNA from sporulating cultures of S. cerevisiae.

Saccharomyces diastaticus is closely related to Saccha-romyces cerevisiae, except that S. diastaticus has the abilityto produce and secrete glucoamylase (1). The genetics andbiochemistry of glucoamylase production have been wellstudied, most recently by Erratt and Stewart (11-13),Tamaki (26), and Yamashita and Fukui (27-30). The inde-pendent investigations of Erratt and Stewart (11) and Tamaki(26) have led to two nomenclatures for designating the genesfor starch fermentation. Tamaki (26) demonstrated the ex-istence of three polymeric genes for starch fermentation:STAI, STA2, and STA3. Erratt and Stewart (11-13) usedDEX to designate the gene for dextrin (starch) fermentation;they also found three polymeric genes for dextrin fermenta-tion, one of which was shown to be allelic to STA3. Re-cently, it was deternined that DEXI is allelic to STA2 andDEX2 is allelic to STAI (lOa).As a continuation of our earlier work on glucoamylase

from S. diastaticus, this yeast was used as a donor for thecloning of a glucoamylase gene into S. cerevisiae by func-tional complementation, using plasmid YEp13 (4) as thecloning vehicle. Functional complementation has been usedsuccessfully for cloning other genes in S. cerevisiae (3, 16)and, more recently, for cloning the STAJ, DEXI (STA2), andSTA3 genes from S. diastaticus (19, 28, 31). We establishedthat the gene cloned in this study was different from theSTAI, DEXI (STA2), and STA3 genes by comparing theirrestriction maps. Based on the data presented in this paper,we conclude that the cloned glucoamylase gene may be

* Corresponding author.t National Research Council of Canada publication no. 25512.

related to the sporulation-specific glucoamylase gene from S.cerevisiae (8) because both genes share a common restric-tion map; however, the cloned gene was not preferentiallytranscribed during sporulation in S. diastaticus.

MATERIALS AND METHODS

Organisms, DNAs, and enzymes. The strains used in thisstudy are listed in Table 1. Plasmid DNA was prepared by themethod described by Godson and Vapnek (14), with minormodifications. Yeast DNA was prepared by using standardprocedures (9). The restriction enzymes, alkaline phospha-tase, and T4 ligase were purchased from BoehringerMannheim Biochemicals or Bethesda Research Laborato-ries, Inc., and were used as directed by the manufacturers.

Construction of a pool of S. diastaticus DNA sequences inplasmid YEp13. Yeast DNA from S. diastaticus J3120-13Cwas partially cleaved with BamHI, and the resulting frag-ments were separated by size on a sucrose gradient (10 to30%) (18). Fragments ranging in size between 5 and 15kilobases (kb) (0.4 ,ug) were ligated to BamHI-digestedYEp13 (0.2 ,ug) treated with calf intestinal alkaline phospha-tase. After ligation, the DNA was used to transform Esche-richia coli RR1 to ampicillin resistance (Amp9. Approxi-mately 1.2 x 103 Ampr colonies were selected, of which 85%were tetracycline sensitive (Tets). The average insert sizedetermined from 12 plasmid isolates was approximately 7.6kb. The transformed colonies were pooled, and plasmidDNA was isolated to yield the S. diastaticus yeast bafik.

Transformation of S. cerevisiae and Schizosaccharomycespombe. The standard yeast transformation procedure (3) was

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TABLE 1. Species and strains used

Species and strain Known genotype

Saccharomyces diastaticusJ3120-13C .................... a DEXI DEX2 STA3J132b .................... aDEXIJ210c ..................... o DEXIJ1741c ..................... o DEX2J1744b.................... a DEX2 adel ade2 uralJ2064c .................... a STA3 his4 leu2J4000 .................... a/a DEXIIDEXI3a .....................aDEXI3b .................... a STA33c .................... aDEX2 STA33d ......................a DEXI DEX23a-la ..................... DEXI3a-lc ..................... DEXI3b-la ..................... STA33b-lb ..................... STA33c-la ..................... STA33c-lb ..................... DEX23c-lc ..................... DEX23c-ld ..................... STA33d-10a ..................... DEXI3d-lOb ..................... DEX23d-lOc ..................... DEX23d-lOd ..................... DEXI

Saccharomyces cerevisiae3a-lb ..................... dex3a-ld ..................... dex3b-lc ..................... dex3b-ld ..................... dexLL20 ...................... a leu2-3 leu2-1J his3-11

his3-15AH22.................... a leu2-3 leu2-112 his4-519AP1 .................... a/oxadell + ade2-21ade2-R8

urall+ his71+ lys2I+tyrll+ galll+ ura31+leull+ cyh2l+

NRC 5044 ...................... a/a adelladel arg61arg6trp21trp2

NRC 5045 .................... a/a thr41thr4 his4lhis4ura3lura3 metlmet

Schizosaccharomyces pombeNRC 2447 ..................... h- leul-32

Escherichia coli RR1 ..................F- hsdS20 (hsdR hsdM)recA + ara-14 proA2lac Yl gaIK2 rps-20 (Strr)xyl-5 mtl-l supE44 X-

used, with minor modifications, to transform S. cerevisiaeLL20 with the S. diastaticus plasmid bank. The followingmethod was developed to screen the transformants forglucoamylase activity. S. cerevisiae protoplasts were regen-erated on a minimal medium containing 1% (wt/vol) dex-trose, 2% (wt/vol) starch, 20 ,ug of histidine per ml, and 1.2M sorbitol in the overlay agar. After the protoplasts wereregenerated, the plates were incubated at 4°C for 2 to 3 daysto allow the starch to precipitate, which caused the plates toturn white. The colonies that expressed glucoamylase activ-ity hydrolyzed the starch around them; therefore, the areasaround these colonies remained clear. The plates were thenscreened for such clear zones around the colonies.Schizosaccharomyces pombe was transformed with the plas-mid containing the glucoamylase gene by the method de-scribed by Beach et al. (2).

Small-scale preparations of yeast DNA. Miniplasmid prep-

arations of yeast DNA were obtained by the method de-scribed by Nasmyth and Reed (22). This DNA was used forgel electrophoresis and E. coli transformations.

Glucoamylase assay. Glucoamylase activity was measuredby determining the hydrolysis of starch to glucose, asdescribed previously (12). For tetrad analysis of glucoam-ylase activity, the method described by Searle and Tubb (24)was used with minor modifications.

Genetic analysis. For genetic analysis, standard techniquesof mating, inducing sporulation, dissection, and tetrad anal-ysis were used (21).

Southern blot analysis. DNA fragments (5 ,ug each) wereseparated in 0.7% agarose gels or 7.2% polyacrylamide gelsin 60 mM Tris-5 mM sodium acetate-1 mM EDTA buffer(pH 8.1) and transferred to nitrocellulose as described bySouthern (25). The probe was the cloned glucoamylasefragment labeled by nick translation with [a-32P]dATP(Amersham Corp.) by the procedure of Rigby et al. (23).Hybridization was done under stringent conditions in 6xSSC (lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate)-0. 1% sodium dodecyl sulfate-Sx Denhardt solutionat 65°C. This was followed by washes at 65°C in 6xSSC-0.1% sodium dodecyl sulfate. The nitrocellulose filterswere then exposed to Kodak XAR-5 film at -700C withKodak intensifying screens.Northern blot analysis. For Northern blot analysis, the

homothallic and heterothallic diploid strains were grown invegetative and sporulation media as described by Colonnaand Magee (8). Total RNA and poly(A)+ RNA were isolatedas previously described (10). The sporulating and vegetative

....-I~ ~ ~ ~ a -.4...._4~

...~~ 7-W.

7X rz X XX

FIG. 1. Restriction map of the cloned fragment in plasmidYEp(DEX)4. The plasmid containing the cloned glucoamylase genewas digested with various restriction enzymes, and the resultingfragments were separated in a 0.7% agarose gel. (A) Ethidiumbromide stain of the gel; (B) hybridization of the 3.9-kb BamHIfragment from plasmid YEp(DEX)4 to the restriction fragments.The restriction map of the BamHI fragment in YEp(DEX)4 is shownat the bottom. Digestion was with BamHI, Sall, and EcoRI (lanes1); BamHI, Sall, and PstI (lanes 2); BamHI, Sall, and KpnI (lanes3); BamHI and HindIlI (lanes 4); BamHI and PvuII (lanes 5);BamHI and EcoRI (lanes 6); BamHI and PstI (lanes 7); BamHI andSall (lanes 8); BamHI and KpnI (lanes 9); and BamHI (lanes 10).

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486 ERRATT AND NASIM

poly(A)+ RNA and poly(A)- RNA samples were electropho-resed through agarose (1.5%) containing formaldehyde (2.2M) (15, 17). After transfer to nitrocellulose, the filters werehybridized to the nick-translated 3.9-kb glucoamylase frag-ment in 50% deionized formamide-5 x SSC-1 x Denhardtsolution (6). The filters were washed at room temperature in2x SSC-0.1% sodium dodecyl sulfate followed by washes in0.1x SSC-0.1% sodium dodecyl sulfate. The nitrocellulosefilters were then exposed to Kodak XAR-5 film at -70°Cwith Kodak intensifying screens.

RESULTSCloning of a glucoamylase gene by complementation in S.

cerevisiae. S. cerevisiae LL20 was transformed with the S.diastaticus plasmid bank. Of the 2 x 104 Leu+ transformantsobtained, 9 were selected which had small, clear zones afterincubation of the plates at 4°C for 2 to 3 days. Six of theoriginal nine transformants (0.03% of the total Leu+ trans-formants) showed glucoamylase activity upon subculturingon a selective medium.

Recovery of the plasmid from S. cerevisiae transformants.Rapid plasmid preparations from the six S. cerevisiae trans-formants were used to retransform E. coli RR1. Therecloned plasmids were isolated and used to retransform S.cerevisiae LL20. Each plasmid preparation produced ap-proximately the same number of transformants (300/,ug ofDNA). All of the transformants from the six plasmid sam-ples, YEp(DEX)3, YEp(DEX)4, YEp(DEX)5, YEp(DEX)6,YEp(DEX)7, and YEp(DEX)9, exhibited glucoamylase ac-

A

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24 48 72 96 120 144 168B

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Time (Hours)FIG. 2. Growth curves and glucoamylase activity in the media of

the transformed S. cerevisiae strain and the donor S. diastaticusstrain. (A) Glucoamylase activity; 1 U equals the amount of enzymerequired to release 1 mg of glucose from starch in a cell-free systemwith 1 ml of culture medium incubated at 30°C for 1 h. (B) Growthcurves. Symbols: 0, S. diastaticus grown in complete medium with2% starch; *, S. diastaticus grown in buffered minimal medium with2% starch; A, S. cerevisiae LL20[YEp(DEX)4] grown in completemedium with 2% starch; *, S. cerevisiae LL20[YEp(DEX)4] grownin buffered minimal medium with 2% starch.

FIG. 3. Hybridization of cloned glucoamylase gene to genomicDNAs from S. cerevisiae and S. diastaticus. DNAs from S. cerevi-siae (lane A) and S. diastaticus (lane B) were digested to completionwith BamHI. After electrophoresis the DNAs were transferred tonitrocellulose and probed with the nick-translated 3.9-kb glucoam-ylase gene fragment.

tivity. However, no expression of glucoamylase activity wasobtained in E. coli.A restriction map of the cloned glucoamylase fragment in

plasmid YEp(DEX)4 is shown at the bottom of Fig. 1. Theethidium-bromide-stained gel of plasmid YEpDEX(4) di-gested with several endonucleases is shown in Fig. 1A, andSouthern blot analysis with the cloned fragment from plas-mid YEp(DEX)4 as the probe is shown in Fig. 1B. Identicalresults were obtained for all six isolates.A PstI digest revealed that all inserts were cloned in the

same orientation. Although this strongly suggests that tran-scription was initiated outside the insert, when the insert wascloned in the opposite orientation, expression of glucoam-ylase activity was also obtained.

Determination of glucoamylase activity in transformed S.cerevisiae. The donor S. diastaticus strain and the trans-formed S. cerevisiae strains were inoculated into 100 ml ofbuffered minimal medium (pH 6.8) containing 2% dextroseand supplemented with histidine (20 ,ug/ml) and were incu-bated at 30°C on a rotary shaker for 4 days. These cultureswere used to inoculate minimal medium containing histidineor complete medium (1% yeast extract, 2% peptone), bothcontaining 2% starch as the sole carbon source. The gluco-amylase activity found in the media was monitored up to 1week. In the complete medium, glucoamylase activity wasdetected from the S. cerevisiae transformants (Fig. 2; onlythe results from strain LL20[YEp(DEX)4] are shown). Onthe average, the six strains produced glucoamylase activityat seven times the basal level of strain LL20. However, theaverage glucoamylase activity was still 100 times less thanthat found in the medium of the donor strain, S. diastaticus.There was a 10-fold difference between the growth of thedonor strain and that of the transformed strains; therefore,the differences in activity could not be totally accounted forby the differential growth rates. Intracellular glucoamylaseactivity was also measured, and it was determined that theclones did not contain appreciably higher levels than did thedonor strain. When the donor strain was grown in bufferedminimal medium, the glucoamylase activity was approxi-mate two times less than the activity found in the complete

A 8

4. 4 kb ,

3. 9 kb P

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 12 18 19 20 21 22 6

4. 4 kb3. 9 kb I

FIG. 4. Southern blot analysis of the parents and meiotic segregants in crosses involving the different DEX (STA) genes. The S. diastaticusdonor strain was crossed with S. cerevisiae LL20, and a tetrad (no. 3 ) in which all four spores expressed glucoamylase activity was chosenfor further crosses. Each spore from this tetrad was crossed with S. cerevisiae AH22 or LL20 to segregate the three different DEX (STA) genesinto different haploid cultures. DNA was isolated from each spore culture of the original tetrad and from the spore cultures from a tetrad fromthe second cross. These DNA samples were digested with BamHI, separated on a 0.7% agarose gel, blotted to nitrocellulose, and probed withthe nick-translated glucoamylase gene fragment. Hybridization of the glucoamylase probe to one tetrad and the parental samples from eachof the second crosses are shown in the figure. Lanes contained samples from the following S. diastaticus and S. cerevisiae strains: 1, 3a; 2,3a-la; 3, 3a-lb; 4, 3a-lc; 5, 3a-ld; 6, AH22; 7, 3b; 8, 3b-la; 9, 3b-lb; 10, 3b-lc; 11, 3b-ld; 12, LL20; 13, 3c; 14, 3c-la; 15, 3c-lb; 16, 3c-lc;17, 3c-ld; 18, 3d; 19, 3d-10a; 20, 3d-lOb; 21, 3d-10c; 22, 3d-10d. See Table 1 for the species and relevant genotypes of these strains.

medium; however, the growth in the minimal medium wasalso reduced by two times. The transformed strains, how-ever, did not grow in the buffered minimal medium withstarch as the sole carbon source, and there was no extracel-lular glucoamylase activity.

Hybridization of YEp(DEX)4 insert to S. diastaticus and S.cerevisiae genomic DNAs. The 3.9-kb YEp(DEX)4 insert wasused as a probe to analyze the BamHI genomic DNA blots ofS. diastaticus and S. cerevisiae. Both yeasts contained a3.9-kb fragment which was homologous to the cloned se-quence (Fig. 3). In addition, the S. diastaticus DNA con-tained a second homologous fragment at approximately 4.4kb (Fig. 3).

S. diastaticus J3120-13C contains three polymeric genescontrolling glucoamylase activity. To determine whether themultiple bands found with DNA from this strain corre-sponded to differences in the three genes, crosses wereperformed to segregate the genes into three different haploidcultures. Strain J3120-13C was first crossed with S. cerevi-siae LL20, and a tetrad was chosen in which all four sporesproduced glucoamylase. A second set of crosses was per-formed with the spore cultures from this tetrad and S.cerevisiae LL20 or AH22. S. diastaticus tester strains wereused to identify the DEX (STA) genes from the abovecrosses. The strains and their relevant genotypes are shownin Table 1. DNAs from the parents and meiotic segregantswere digested with BamHI and probed with the 3.9-kbcloned fragment. The segregation of glucoamylase activity inthe tetrads was also determined. Two observations could bemade (Fig. 4). (i) Regardless of which DEX (STA) gene theculture contained, two homologous fragments were identi-fied, at 4.4 and 3.9 kb. (ii) There was cosegregation ofglucoamylase activity with the 4.4-kb fragment. The strainsthat did not exhibit glucoamylase activity contained only onehomologous sequence, at 3.9 kb.The difference in the sizes of the two BamHI fragments

homologous to the probe could have resulted from a restric-tion site polymorphism or a major rearrangement of theDNA. To determine whether the difference was caused by a

polymorphism at the BamHI site, the DNAs from S. cere-visiae LL20 and S. diastaticus J3120-13C were digested tocompletion with a number of enzymes which cleave outsidethe BamHI restriction sites. If the additional hybridizationsequence was a result of restriction site polymorphism,cutting outside the BamHI restriction sites would haveeliminated the size difference. However, in each case S.cerevisiae contained one homologous sequence, whereas S.diastaticus contained two homologous fragments, one whichwas identical in size to the S. cerevisiae fragment and onewhich was larger. These results indicate that the difference

A B C 0 E

I 11 I II 1 II I II I II

23. I kb sR9. 4 kb6. 7 kb - _ ^

4. 4 kb

2. 3 kb2. 0 Nb

0. 5 kb

FIG. 5. Southern blot analysis of genomic DNAs from S. cere-visiae and S. diastaticus. DNAs from S. cerevisiae (lanes I) and S.diastaticus (lanes II) were digested to completion with BamHI andthen with KpnI (lanes A), Sall (lanes B), PstI (lanes C), EcoRI (lanesD), or PvuII (lanes E). The digested DNAs were separated on a0.7% agarose gel, blotted to nitrocellulose, and probed with thenick-translated 3.9-kb fragment.

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in the sizes of the two fragments lay within the BamHIrestriction sites and was probably caused by an insertion.To determine the site of this DNA rearrangement, the

DNAs from both species were digested to completion withBamHI and then with a number of restriction enzymes whichcleave within the 3.9-kb BamHI fragment. By using the3.9-kb fragment as a probe and comparing the homologousrestriction fragments from S. cerevisiae and S. diastaticusDNAs, it was possible to determine the approximate loca-tion of the DNA insertion. For example, the BamHI-KpnIdouble digest of DNA from S. cerevisiae resulted in twofragments of 1.0 and 2.9 kb (Fig. 5), representing the left- andright-hand sides of the 3.9-kb fragment as shown in Fig. 1.However, with a similar digest, the DNA from S. diastaticusresulted in three fragments, two of which were identical insize to the fragments seen with S. cerevisiae and one whichwas 3.4 kb. This additional fragment was approximately 0.5kb larger than the 2.9-kb fragment, and this indicates that the500-base-pair (bp) insertion in the 4.4-kb fragment occurredbetween the KpnI and BamHI restriction sites (the right-hand side). By comparing the rest of the double digests in asimilar manner, it could be concluded that the 0.5-kb insertoccurred between the PvuII and BamHI restriction sites, atthe right-hand side. There was a difficulty, however, ininterpreting the BamHI-SalI double digests. Based on therestriction map and the data presented in Fig. 5, the 0.65-kbfragment should have contained the 0.5-kb insert in the S.diastaticus genomic DNA, which would have resulted in anadditional band at 1.15 kb. This band would not have beenresolved from the 1.2-kb fragment under the experimentalconditions. However, an additional band was detected at2.55 kb. No 2.55-kb BamHI-SalI fragment was observed inthe DNA digests from three other strains of S. diastaticuscontaining either the DEXI, DEX2, or STA3 gene. There-fore, this additional band from the S. diastaticus J3120-13culture could have represented a variation within this strain.

I II

A B C D A B C D

255S .

18 S '

FIG. 6. Northern blot analysis ofRNAs isolated from vegetativeand sporulating samples of S. cerevisiae and S. diastaticus diploids.Total RNA was isolated from S. cerevisiae AP1 (lanes A and B) andS. diastaticus J4000 (lanes C and D) grown under either vegetative(lanes A and C) or sporulating (lanes B and D) conditions. TheRNAs were fractionated into poly(A)+ (lanes I) and poly(A)- (lanesII) samples, and 10 ,ug was electrophoresed on a 1.5% agarose gel.After transfer to nitrocellulose, the RNA was probed with thenick-translated glucoamylase gene fragment. The positions of the25S and 18S yeast rRNA are shown.

A B

a b c d e f

4 13.5 kb

_in 43.9 kb

LFIG 7. Expression of glucoamylase in Schizosaccharomyces

pombe. S. pombe was transformed with plasmid YEp(Dex)4, andthe transformants and the untransformed control were incubated onminimal medium containing 2% starch and 1% dextrose for 7 days(A). After 2 to 3 days at 4°C, clear halos were observed around thetransformed cultures (I), whereas no clearing was seen with theuntransformed cultures (II). (B) Southern blot analysis of rapidDNA preparations made from five transformed S. pombe colonies(lanes a to e) and one untransformed control (lane f). After BamHIdigestion, the DNA was probed with the glucoamylase gene frag-ment. The transformed cultures contained homologous fragmentscorresponding in size to the 3.9-kb insert and the linear form of therecombinant plasmid, whereas the untransformed culture containedno homologous DNA.

In addition to determining the site of the DNA insertion inthe 4.4-kb fragment, the data presented in Fig. 5 show thatthe 3.9-kb homologous fragment from S. cerevisiae had thesame restriction map as the 3.9-kb BamHI fragment from S.diastaticus.

Hybridization of YEp(DEX)4 insert to S. diastaticus and S.cerevisiae RNAs. Total RNA was isolated from vegetativeand sporulating heterothallic diploid cultures of S. cerevisiaeAP1 and S. diastaticus J4000. No detectable homology wasfound with RNA isolated from the vegetatively growing S.cerevisiae (Fig. 6); however, one homologous band at 1.5 kbwas obtained with RNA isolated from sporulating cultures.With S. diastaticus, two homologous bands, at 1.5 and 2.1kb, were obtained with RNAs from both the vegetative andsporulating cultures. No homology was detected betweenthe 3.9-kb fragment and any of the poly(A)- RNA samples(Fig. 6). No homology was detected between the 3.9-kbfragment and poly(A)+ RNA isolated from homothallic dip-loid strains of S. cerevisiae grown under vegetative orsporulating conditions.

Heterologous gene expression. The plasmid YEp(DEX)4was used to transform Schizosaccharomyces pombe, and thetransformants were screened for clear zones on regenerationagar containing starch. The transformed cultures were ableto hydrolyze the starch around the colony, whereas theuntransformed cultures could not (Fig. 7A). However, un-like the cultures of the S. cerevisiae transformants, noglucoamylase activity was detected in the growth medium.DNA was prepared from five transformed colonies and oneuntransformed control. After digestion with BamHI, theDNA was probed with the 3.9-kb BamHI fragment fromYEp(DEX)4. The five transformants contained two homol-ogous sequences, at 3.9 and 13.5 kb (Fig. 7B); the size of thelarger fragment was consistent with the linear form of the

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recombinant plasmid. No homology was detected in theuntransformed culture.

DISCUSSIONIn this paper we describe the cloning of a glucoamylase

gene from S. diastaticus. An S. diastaticus genomic library,constructed in plasmid YEp13, was used to transform astrain of S. cerevisiae containing a leu2 double mutation.Hybrid YEp13 plasmids carrying glucoamylase genes wereidentified by simultaneous complementation of the leu2mutation and acquisition of glucoamylase activity. Glucoam-ylase activity was also weakly expressed when these plas-mids were cloned into Schizosaccharomyces pombe, dem-onstrating that a structural gene for glucoamylase wascloned.The glucoamylase genes STAI, DEXI (STA2), and STA3

from S. diastaticus have been cloned into S. cerevisiae in asimilar manner to that described in this paper (19, 28, 31).The cloned BamHI fragment of the gene described here wasapproximately 3.9 kb, which was smaller than the smallestBamHI fragments of the STAI and STA3 inserts (28, 31).Differences were found in the restriction map between thegene cloned in this study and the STAI and STA3 genes (28,31). No HindIII site was found in this fragment, and only onePvuII site was detected, whereas the STAI and STA3 genescontain one HindIII site and two PvuII sites, approximately180 bp apart. The distance betWeen the second PvuII siteand the BamHI site in the STA1 gene has been reported to beapproximately 670 bp (32). However, only 300 bp separatedthe PvuII and BamHI sites in the fragment cloned in thisstudy. The double digests confirmed that the 500-bp inser-tion in the 4.4-kb fragment lay between the PvuII andBamHI sites. Although subcloned fragments of the 4.4-kbSTAI fragment were used as probes, Yamashita et al. (31)also did a SalI-BamHI double digest to localize the deletedregion in their 3.7-kb S. cerevisiae homologous fragment.Their results were consistent with the deletion being be-tween the Sall and BamHI sites. In this study, the DNArearrangement was localized to the area between the PvuIIand BamHI sites.When the 3.9-kb cloned fragment was used as a hybrid-

ization probe against BamHI-digested genomic DNAs fromS. cerevisiae and S. diastaticus, a hybridization signal wasobtained at 3.9 kb for both samples. However, S. diastaticusalso contained an additional band at 4.4 kb which segregatedwith the glucoamylase activity regardless of which DEX(STA) gene was being analyzed. Homology within a genefamily in S. cerevisiae has also been demonstrated with theMAL and SUC loci (5, 7, 20). Yamashita et al. (31) foundthree fragments common to S. diastaticus and S. cerevisiaeBamHI-digested DNAs. Their 3.7-kb fragment is probablythe same as the 3.9-kb fragment identified in this study; their1.3-kb fragment would not be picked up with the 3.9-kbfragment as the probe; and their 7.5-kb fragment was seen insome of the Southern blot analyses and is particularly clearin Fig. 4. The size of the hybridization fragment which theyfound to be specific to S. diastaticus was 4.2 kb, which isvery similar to the 4.4-kb size predicted here. With ClaI-digested S. diastaticus DNA, three fragments, at 10.0, 10.2,and 11.0 kb, are homologous to the DEXI (STA2) clonedfragment, whereas with CiaI-digested S. cerevisiae DNAonly one homologous sequence, at 10.0 kb, has been de-tected (19). Both Yamashita et al. (31) and Meaden et al. (19)speculated that the homologous sequence in S. cerevisiae isthe gene for the sporulation-specific glucoamylase reportedby Colonna and Magee (8).

The 3.9-kb BamHI fragment cloned in this study had arestriction map which was different from the restriction mapof the STAI, DEXI (STA2), and STA3 genes but identical tothat of the 3.9-kb homologous sequence in S. cerevisiae.Based on the Northern blot analysis, a homologous RNAtranscript was found in S. cerevisiae only during sporulation.Therefore, we speculate that the fragment cloned in thisstudy is related to the S. cerevisiae sporulation-specificglucoamylase gene; however, in S. diastaticus it is not clearfrom the Northern blot analysis whether the gene is ex-pressed only during sporulation.

ACKNOWLEDGMENTS

We thank G. G. Stewart and P. T. Magee for providing yeast strainsneeded for this investigation. We also thank V. L. Seligy for helpfulcomments on the manuscript.

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