Evolutionary Origin in the Yeast Saccharomyces diastaticus

JOURNAL OF BACTERIOLOGY, Feb. 1985, p. 574-582 Vol. 161, No. 20021-9193/85/020574-09$02.00/0Copyright © 1985, American Society for Microbiology

Polymorphic Extracellular Glucoamylase Genes and TheirEvolutionary Origin in the Yeast Saccharomyces diastaticus

ICHIRO YAMASHITA,* TOHRU MAEMURA, TAKUSHI HATANO, AND SAKUZO FUKUIDepartment of Fermentation Technology, Faculty of Engineering, Hiroshima University, Shitami,

Higashi-Hiroshima 724, Japan

Received 21 June 1984/Accepted 1 October 1984

DNA coding for extracellular glucoamylase genes STAI and STA3 was isolated from DNA libraries of twoSaccharomyces diastaticus strains, each carrying STAI or STA3. Cells transformed with a plasmid carryingeither the STAI or STA3 gene secreted glucoamylases having the same enzymatic and immunological propertiesand the same electrophoretic mobilities in acrylamide gel electrophoresis as those of authentic glucoamylases.Southern blot analysis of genomic DNA from S. diastaticus and a glucoamylase-non-secreting yeast,Saccharomyces cerevisiae, revealed that the STAI and STA3 loci of S. diastaticus showed a high degree ofhomology, and that both yeast species (S. diastaticus and S. cerevisiae) contained DNA segments highlyhomologous to those of the extracellular glucoamylase genes. Restriction maps of the homologous DNAsegments suggested that the extracellular glucoamylase genes of S. diastaticus may have arisen fromrecombination among the resident DNA segments in S. cerevisiae.

Saccharomyces diastaticus carrying any one of the un-linked STA genes (STAI, STA2, or STA3) produces extra-cellular glucoamylase isozyme I, II, or III, respectively, andferments starch (19, 24). Glucoamylase I was purified andshown to be composed of two nonidentical glycosylatedsubunits, H and Y. The molecular weights of these proteinmoieties are 41,000 and 3,400, respectively (26). Since weare interested in the genetic and biochemical controls forextracellular enzyme production, we isolated glucoamylasenonproducing (amy) mutants from wild-type strains of S.diastaticus carrying STAI and analyzed their genetic char-acteristics (23). A mutation named amyl was located inSTAI. Another mutation named amy2 was apparently aregulatory mutation, because it was involved in expressionof both mating-type regulated genes, STAI, and a floccula-tion gene, FLO8 (22). S. diastaticus is closely related toSaccharomyces cerevisiae since haploid cells of these spe-cies are able to mate and they are genetically similar to eachother (19, 24). Thus, S. diastaticus might be considered to bederived from S. cerevisiae by the acquisition of the extra-cellular glucoamylase genes. The genetic separation be-tween the two species, however, is not simple since S.cerevisiae exclusively carries an inhibitory gene (INHI)against glucoamylase production (24). Therefore, genotypesfor glucoamylase production of S. diastaticus and S. cere-visiae are designated as STA inh° and sta° INHl, respec-tively.

Recently, we succeeded in cloning a glucoamylase-pro-ducing gene from a DNA library of a S. diastaticus straincarrying STAJ (21). It was suggested that the cloned DNAsegment contains a structural gene for the extracellularglucoamylase because Schizosaccharomyces pombe trans-formed with a plasmid carrying this DNA segment secretedglucoamylase having the same enzymatic characteristics(optimal pH and temperature) as those of the S. diastaticusglucoamylase (25). To extend our understanding of regula-tory mechanisms for glucoamylase production (synthesisand secretion), and to provide some information on poly-morphism of STA genes and on the mechanisms of gene

* Corresponding author.

evolution in the two yeast species (S. diastaticus and S.cerevisiae), we cloned the STA1 and STA3 genes from DNAlibraries of two S. diastaticus strains carrying STAI orSTA3, respectively.

In this paper, we show that the glucoamylase genes at theSTAJ and STA3 loci in S. diastaticus are highly homologousby DNA hybridization and have similar restriction maps,and that S. diastaticus and S. cerevisiae contain DNAsequences highly homologous to those of the extracellularglucoamylase genes. We discuss the evolutionary origin ofthe extracellular glucoamylase genes and suggest that theymay have arisen by recombination among resident genes inS. cerevisiae.

In the accompanying paper (27), we have determined thecomplete nucleotide sequence of the STAJ gene.

MATERIALS AND METHODS

Strains, media, and genetic methods. Genetic techniquesand media have been described previously (23). The yeastand bacterial strains used and their relevant genotypes arelisted in Table 1.

Preparation of DNA. Plasmid DNA was prepared by thealkaline-sodium dodecyl sulfate method of Birnboim andDoly (2). Total genomic DNA of yeast was isolated by amodification of the method of Cryer et al. (8).

Construction of S. diastaticus recombinant plasmid librar-ies. Recombinant plasmid libraries were constructed essen-tially as described by Carlson and Botstein (5).

Yeast and Escherichia coli transformations. The procedureused for the transformation of yeast has been describedpreviously by Beggs (1), except that 0.2 mg of Zymolyase60,000 (Kirin Brewery Co.) per ml was used instead ofhelicase and ca. 0.1 ,ug of DNA was used in each transfor-mation. Bacterial transformation of CaCl2-treated cells wasperformed as described by Cohen et al. (6).

Southern blot analysis. Transfer of DNA fragments fromagarose gels to nitrocellulose paper was as described bySouthern (16). Hybridization conditions and the labeling ofDNA for hybridization probes by nick translation were asdescribed by Rigby et al. (15).

574

POLYMORPHIC STA GENES IN YEAST 575

TABLE 1. Yeast and bacterial strainsOrganism' Strain Genotypeb Source

S. diastaticus 5106-9A a leu2 arg4 STA1 H. Tamakiinh°

YIY2-12D a leu2-3,112 his2 Our collectionlys2 STA1 inh°

YIY263 a his2 arg4 STAI Our collectioninh°

5301-17B a Iys7 STA3 inh° H. TamakiYIYSTA3-2 a leu2-3,112 his2 Our collection

his4 STA3 inh°YIYD a leu2-3,112 his4 Our collection

lys7 sta° inh°YIY342 a ura3 Iys7 sta° Our collection

inh°YIY345 a ura3 leu2-3,112 Our collection

his4 sta° inh°YIY320 a ura3 his2 amyl-l Our collection

inh°YIY321 a ura3 Iys2 amyl-I Our collection

inh°YIY324 a ura3 amyl-2 inh° Our collection

S. cerevisiae AH22 a leu2-3,112 his4 A. Toh-ecanI sta° INHI

E. coli JA221 F- recAl leuB6 A. Toh-eAtrpE5 lacYhsdR- hsdM+

a All S. diastaticus strains listed are meiotic progenies derived from thecross between prototype strains of S. diastaticus and S. cerevisiae (19). Forthe sake of convenience, we classified strains carrying STA inh° and sta° inh°as S. diastaticus.

b Polaina and Wiggs (14) reported a gene, STAIO, similar to the INHI gene,but they provided no evidence for its dominant character or its linkagerelationship to other genes.

Analysis of glucoamylase. Cells were cultured in YPSmedium, and the culture supernatants were obtained bycentrifugation. The secreted enzymes were partially purifiedby using a CM-Sephadex c-50 column (Pharmacia FineChemicals) as described previously (26). Glucoamylase ac-tivity was assayed as described previously (23).The enzyme was analyzed by 7% acrylamide gel elec-

trophoresis in the absence of sodium dodecyl sulfate (26).The gel was cut into slices of 2-mm width. To detect theenzyme activity, each slice was incubated in 0.1 ml of 10 mMphosphate buffer (pH 6.8) at 10°C overnight, and a sample ofeach incubation mixture was removed for enzyme assay.

Endo-,-N-acetylglucosaminidase H (Endo H; SeikagakuKogyo Co.) was used to remove glycosyl chains fromglucoamylase. The reaction mixture contained 2 U ofglucoamylase, 5 x 10-3 U of Endo H, and 10 mM sodiumacetate buffer (pH 5.5). The reaction was carried out at 37°Cfor 5 h.

Monoclonal antibodies. All procedures for screening mono-clonal antibodies were performed by the method of Koehlerand Milstein (11). Purified glucoamylase I (0.1 mg) (26) in 0.3ml of phosphate-buffered saline buffer was emulsified withan equal volume of complete Freund adjuvant and injectedintraperitoneally into BALB/c mice, followed in 2 weeks byanother immunization in Freund incomplete adjuvant. Threedays after final immunization, mice were killed by cervicaldislocation and the spleens were removed aseptically into asterile petri dish containing RPMI 1640 medium (NissuiSeiyaku Co.). The spleen cells were fused with mousemyeloma NS-1 cells in the presence of polyethylene glycol4000 (50% [wt/vol]). Standard HAT selection was carried

out, followed by screening for antibody production byprecipitation of glucoamylase activity with staphylococcalprotein A (20). Cells from the positive wells were cloned bylimit dilution. The cloning was repeated twice from wellscontaining single clusters of hybridoma cells, and againtested for the presence of antiglucoamylase antibodies. Weobtained two hybridomas, F3.2C7 and F3.2E12, from oneimmunized mouse. The two hydriboma cells produced onlyIgG2a specific to protein moieties of glucoamylases I and III(data not shown).

RESULTSMolecular cloning of STAI and STA3 DNA. To obtain the

DNA sequences encoding STA1 and STA3, we constructedtwo recombinant plasmid libraries that were representativeof the genomes of two S. diastaticus strains, each of whichcarries a single active STA gene at the STAI or STA3 locus.Total genomic DNA from S. diastaticus strains 5106-9A(STAI) and 5301-17B (STA3) was partially digested withrestriction endonuclease Sau3A, and fragments larger than 5kilobases (kb) were recovered from sucrose gradient centrif-ugation. The Sau3A-digested DNA fragments from STAJand STA3 strains were inserted by ligation into the uniqueBamHI sites of the cloning vectors pYI1 (21) and YEp13 (4),respectively, both of which carry ampicillin-resistant (Apr)and tetracycline-resistant (Tcr) genes for E. coli and also theyeast LEU2 gene. The resulting recombinant DNA mole-cules were used to transform E. coli to Apr. Each librarycontained more than 25,000 Apr clones, 95% of which weretetracycline suseptible. Selection for plasmids carrying theSTA genes was carried out in yeast. Recombinant plasmidDNA from each library was used to transform a yeast strainYIYD (a leu2-3,112 sta°) to leucine prototrophy. Transfor-mants carrying a plasmid capable of complementing sta°were then selected by their ability to form halos aroundcolonies on the Leu+ selection plates containing starch,since halo formation reflects glucoamylase-producing orstarch-fermenting abilities (21). Five and two halo-formingtransformants were obtained from the STAI and STA3libraries, respectively. They were subcultured to singlecolonies on YPSB (rich) agars, and the halo-forming colo-nies were again cultured to single colonies on the samemedium to allow other plasmids present in the transformantto segregate out. From the resulting halo-forming clones, theplasmids were recovered and then transferred again tobacteria by transformation to Apr. Recombinant plasmidswere purified from bacterial cultures and used to transformthe yeast recipient strain (YIYD) to Leu+. The plasmidstransforming the recipient cells to Leu+ Sta+ were selectedand identified as plasmids carrying putative STA genes. Weobtained seven plasmids that allow the sta° cells to produceglucoamylase activity; five of them (pSTA1, pSTA1-4-10,pSTA1-7-18, pSTA1-14-8, and pSTA1-16-4) were derivedfrom the STAI library, and the remaining two plasmids(pSTA3-6-4 and pSTA3-7-2) were derived from the STA3library. The plasmid DNA was subjected to restrictionanalysis. Several restriction sites were mapped to determinethe extent of overlap among the seven cloned yeast DNAsegments. Restriction maps of the plasmids are shown inFig. 1. All plasmids included a common 2.7-kb region.To localize the region responsible for STA activity, we

subcloned restriction endonuclease- or exonuclease (BAL31)-digested fragments of the original inserts (plYl to pIY9;Fig. 1). The smallest sequence essential for STA activity inthe STAI insert was clearly demonstrated to be a 2.5-kbfragment, denoted "STAl" in Fig. 1. Although only two

VOL. 161, 1985

576 YAMASHITA ET AL.

PLASM ID

B B KpSTA1

B/S B K

pSTAl-4-10AB K

pSTA1-7-18 t

B KpSTA1-14-8

RESTRICTION MAP

S Pt ES PvBt HB BiSIiI 11

S PtES PvBt HB Btl5 .S PtES PvBt HB Bt

II I1

S PtES PvBtHBB/S11 1 i 11 I

GLUCOAMYLASE -PRODUCING

ACTIV I TY

Hp B Pt -Pt B/S

Hp B Pt Pt

+

B

+

B/S S PtES PvBtHBB/SI I 11 l 11 11 I

S PtES PvBtHB BtII I II II

+Hp B

K S PtES PVBtHBB/SI I11 III1

S PtES PVBt HB Bt Hp B Pt Pt B/SI III I I It I I

B K S PtES PVBtHBIII II I I

PtES PVBt H B B/SIII I1 11

ES PvBt H B B/S11 .1LJ

S PvBt HB B/SI 1 l

STA 1

B/S B KpSTA3-6-4

B/S E B KpSTA3-7-2 i

B KpIY8

B KplY9

S PtES PvBt H B Bt Hp BPtIII I I I I I I

S PtES PVBt H B Bt Hp BI II 1I II

S PtES PvBt H B Bt Hp BI

S PtES PvBt H BIII I1

1 kb

FIG. 1. Restriction maps of the inserted yeast segments and localization of the essential STA region. Plasmids pSTA1, pSTA1-4-10,pSTA1-7-18, pSTA1-14-8, pSTA1-16-4, pSTA3-6-4, and pSTA3-7-2 are original plasmids. Plasmids plYl to pIY9 are subcloned plasmids.Analysis of PvuII-digested pSTA1 on acrylamide gel electrophoresis revealed two closely spaced PvuII sites (indicated by an asterisk);however, the experiment was not done in the other plasmids. The fragments A (1.9 kb), B (1.15 kb), C (1.15 kb), and D (1.3 kb) of pSTA1-4-10were subcloned in the vector pBR322. The restriction sites for EcoRI (E), BamHI (B), Hindlll (H), Sall (S), KpnI (K), PstI (Pt), PvuIl (Pv),BstEII (Bt), HpaI (Hp), and BamHI-Sau3A boundary (B/S) are indicated.

fragments were subcloned in the case of STA3 insert, thesequence of STA3 corresponding to the 0.2-kb BamHI-Sau3A segment of STAI was essential for STA activity.

It should be noted that STAI and STA3 DNA segmentsshowed identical restriction maps. Southern blot analysiswas performed to examine whether DNA sequences of STA1and STA3 show homology to each other. Four restrictionfragments derived from one (pSTA1-4-10) of the STAI-carrying recombinant plasmids were subcloned in the vectorpBR322 (3) to construct plasmid subclones A, B, C, and D(Fig. 1). Use of the subclones of STAI as hybridizationprobes confirmed that STA3 exhibited significant sequence

homology to STAI (Fig. 2).Analysis of gene products. Yeast cells carrying STAI or

STA3 in the genomes and the recipient sta° cells transformedwith pSTAl-4-10 or pSTA3-6-4 were cultured in YPS me-

dium at 28°C for 3 days, and the culture supematants wereobtained by centrifugation. The secreted glucoamylases werepartially purified by CM-Sephadex c-50 column chromatog-

raphy and used for further analysis. Glucoamylases secretedfrom the transformants, containing pSTA1-4-10 or pSTA3-6-4, showed the same enzymatic characteristics, such as

dependencies on pH (Fig. 3) and temperature (Fig. 4) andthermodenaturation kinetics (Table 2), to those ofglucoamylases I and III, except that half-times of thermo-denaturation at 55°C fluctuated among the four enzymes.Endo H-digested (deglycosylated) enzymes, however,showed identical thermodenaturation kinetics. The glycosylmoieties appear to contribute to the thermal stability of theenzyme.

Native and deglycosylated enzymes were analyzed by 7%acrylamide gel electrophoresis in the absence of sodiumdodecyl sulfate (Fig. 5). After electrophoresis, the gel was

sliced, and the enzymes extracted from each slice were

assayed for activity. When native enzymes were electropho-resed, only the enzyme from the strain 5106-9A (STAJ)moved in the gel, whereas the enzymes from another strain(YIY2-12D) of STAI, the strain 5301-17B (STA3), and the

J. BACTERIOL.

pSTA1-16-4B K

plY1

pIY2

p IY3

pIY4

pIY5

plY6

pIY7

+

Pt B

+


Pr(pS

FIG. 2.STA3. PlaBamHI plpapers, anisegments cof STA3.

transformmobilitymoved inImmun

ined byF3.2E12)III (Tablcants, conprecipitat

100

0

FIG. 3.glucoamyl5301-17Bwith pSTMcllvaine0.1 with E

obe A B C D nic determinants in the protein components of the enzymes,iTA , 3 , 3 1 3 13 secreted from the transformants, are related to those ofTA 1 1 3 1 3 1 3v glucoamylases I and III.

All these results and Southern blot data (Fig. 2) suggestedthat glucoamylases I and III and those secreted from thetransformants with pSTA1-4-10 or pSTA3-6-4 have similaramino acid sequences, and that addition of glycosyl chainsto protein moieties and modification of the glycosyl chainsare different among S. diastaticus strains.

Southern blot analysis of genomic DNA from S. diastaticuskb and S. cerevisiae. We carried out Southern blot analysis of19--nggenomic DNA from S. diastaticus strains carrying STAI or

STA3, S. cerevisiae (sta° INHI), and the recipient strain1.3- (sta° inh°). The same results were obtained from STAI and1.15-X X It 7 STA3 strains, and also from sta° INHI and sta° inh° strains.

In the experiment shown in Fig. 6, yeast DNA was digestedwith BamHI and probed with nick-translated, 32P-labeledpBR322 DNA containing the cloned subfragments A, B, C,

Southern blot analysis of the plasmids carrying STAI or and D (Fig. 1). The data show hybridization of a 4.2-kbLsmids pSTA1-4-10 and pSTA3-6-4 were digested with BamHI fragment of STA to probes A, B, and C, and[us Sall, electrophoresed, transferred to nitrocellulose hybridization of a 1.3-kb BamHI fragment of STA to probeLd hybridized with probes A, B, C, and D (Fig. 1). DNA D, confirming that the cloned DNA fragments were derivedif STAI also hybridized with the corresponding sequences from S. diastaticus. The unexpected results are that the

DNA from STA and sta° cells shared 1.3, 3.7, and 7.5-kbBamHI fragments.

iants with pSTA1-4-10 or pSTA3-6-4 showed little To further localize the deleted region (0.5 kb in all) of thein the gel. However, all deglycosylated enzymes 3.7-kb fragment (Asta DNA), STA and sta° genomic DNAsthe gel with the same electrophoretic mobilities. were digested with BamHI plus Sall and analyzed with theological cross-reactivity of the enzyme was exam- probes A, B, and C (Fig. 7). STA and sta° DNA sharedusing two monoclonal antibodies (F3.2C7 and 1.9-kb BamHI-SalI, 1.15-kb Sall-Sall, and 0.65-kb BamHI-specific to protein moieties of glucoamylases I and Sall fragments, but only STA DNA contained a 1.15-kbD 3). Glucoamylases secreted from the transform- BamHI-SalI fragment, indicating that the 0.5-kb deleteditaining pSTA1-4-10 or pSTA3-6-4, were immuno- sequence of Asta DNA was located in the 1.15-kb BamHI-Led with both antibodies, indicating that the antige- SalI fragment of STA DNA. It should be noted that the

7.5-kb fragment appeared by hybridization with the probe Ccontaining the 1.15-kb BamHI-SalI fragment of STAI. The

,T,,,, data also showed that the 7.5-kb BamHI fragment was notcut with Sall.

Integration of the cloned STA DNAs into homologous chro-_ .k o mosomal DNA sequences. To determine whether the cloned

A STAI and STA3 DNA integrate into chromosomal DNA ofamyl and sta° cells, we allowed plasmids containing STAI or

A\^ STA3 DNA to integrate into the genomes by recombinationwith homologous sequences, and determined their sites of

A,o\. insertion. For this analysis, we constructed URA3 plasmids,YIpS-STA1 and YIp5-STA3, by subcloning 5.5-kb BamHIpartial digests of pSTA1-4-10 or pSTA3-6-4 into the integrat-ing vector, YIpS (17). To stimulate insertion at homologous

A sequences of amyl (or STAJ) and Asta DNA, YIpS-STA1and YIpS-STA3 were cleaved at one site of the cloned yeast

A segment with KpnI. The linear full-length molecules wereused for transformation of amyl ura3 (YIY320 or YIY324)

A and sta° ura3 (YIY342) strains to Sta+ Ura+. We could notobtain any transformants if the uncut plasmids were used inthe transformation. Orr-Weaver et al. (13) reported that theintegration of complex plasmids can be targeted to a specific

3.0 4. 5.0 6.0 7.0 chromosomal site by digestion within the corresponding3.0 4.0 5.0 6.0 7.0 8.0 region of the plasmid. Most Sta+ Ura+ transformants,

containing YIp5-STA1 or YIpS-STA3, contained single Sta+pH Ura+ determinants since meiotic products derived from the

f crosses of the integrants with a strain YIY321 (amyl ura3)lases secreted from strains 5106-9A (STAI) (0) and showed 2(Sta+ Ura+):2(Sta Ura) segregation. The Sta+(STA3) (A), and transformants (recipient strain, YIYD) Ura+ integrants containing YIpS-STA1 or YIpS-STA3 at oneA1-4-10 (0) or pSTA3-6-4 (A) were examined in the site of the chromosomal DNA were crossed with strainsbuffer (pH 3.4 to 8.0; final ionic strength was adjusted to YIY263 (STAI URA3) or YIYSTA3-2 (STA3), and meiotic

(CI) at 40°C. products were analyzed (Table 4).

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LJlK

VOL. 161, 1985


TABLE 2. Thermodenaturation kinetics of glucoamylaseaHalf time (min)

Glucoamylasesecreted from strain (genotype): Temp nzyme Deglyco-(OC) Naie sylatedenzympe enzyme

5106-9A (STAI) 40 >120 >12045 >120 NDb50 >120 ND55 44 4060 12 865 4 3

5301-17B (STA3) 40 >120 >12045 >120 ND50 >120 ND55 65 4060 17 865 4 3

YIYD (sta°) with pSTA1-4-10 40 >120 >12045 > 120 ND50 >120 ND55 38 3660 14 765 4 3

YIYD (sta°) with pSTA3-6-4 40 >120 >12045 > 120 ND50 >120 ND55 80 3960 18 865 5 3

a Thermodenaturation kinetics of native and deglycosylated glucoamylasesfrom strains 5106-9A (STAI) and 5301-17B (STA3), and transformants (recipi-ent, YIYD) with pSTA14-10 or pSTA3-64 were examined. Enzyme solution(0.5 ml) in 10 mM phosphate buffer (pH 6.8) was preincubated at thetemperatures indicated. Samples were removed, and the remaining activitieswere immediately assayed at 40'C in 0.1 M sodium acetate buffer (pH 5.0).

b ND, Not determined.

YIp5-STA1 and YIp5-STA3 integrated into amyl-2 (orSTAI) and an unknown locus (probably Asta DNA), but notinto STA3 or URA3. The results confirmed the sequencehomology between KphI-flanking regions ofSTA1 and STA3,and the fact that the cloned STA1 DNA segments werederived from the STAI locus. The results also suggested thatthe Asta locus is not allelic to STA1 or STA3, nor linked to

100 Fox

I-I-

wI-

:

-J

wUJCK

50

030 40 50 60 70

TEMP., °CFIG. 4. Temperature dependency of glucoamylase. Temperature

dependencies of glucoamylases secreted from the strains describedin the legend to Fig. 3 were examined at 30 to 65°C in 0.1 M sodiumacetate buffer (pH 5.0). Symbols are identical to those used in Fig.3.

URA3. When the recipient was amyl-1, neither YIp5-STA1nor YIp5-STA3 integrated into amy -l, since the amyl-lmutant contained a large deletion including the KpnI regionof STAI (data not shown). It is noted that YIp5-STA1integrated at amyl-2 more frequently than at the Asta locus,whereas YIp5-STA3 did not do so, suggesting that thesequence around the KpnI region of STAI showed a lesserdegree of homology to the corresponding sequence of AstaDNA than that sequence of STA3 did.

Recovery of homologous DNA sequences to STA DNA fromsta° cells. YIp5-STA3ABst was constructed by self-ligation of

TABLE 3. Cross-reactivity of glucoamylases secreted from transformants with anti-S. diastaticus glucoamylase monoclonal antibodiesaRemaining activity (% of initial)

Monoclonal Dilution Glucoamylase secreted from the transformantantibody Glucoamylase I Glucoamylase IIIWith pSTA1-4-10 With pSTA3-6-4

F3.2C7 lox 71 46 67 59Sx 2 0 8 61x 2 0 0 0

F3.2E12 lox 27 43 38 45Sx 1 0 0 7lx 0 0 0 0

lG6D1O 1 x 100 100 100 100

aHybridoma cells were cultured in RPMI 1640 medium, and the culture supernatants were used for the experiments. The culture supernatants were diluted withthe same medium. Each (0.4 U) of the native glucoamylases secreted from S. diastaticus strains 5106-9A and 5301-17B, carrying STAI and STA3 respectively, inthe genomes, and frorn the transformants (recipient, YIYD) with pSTA1-4-10 or pSTA3-6-4 was incubated at 0°C for 60 min with a series of diluted antibodiessecreted from the hybridoma cells F3.2C7 and F3.2E12, which had been attached to staphylococcal protein A, and then centrifuged. Samples of the resultingsupernatants were assayed for the remaining glucoamylase activity. In a control experiment with a culture fluid of hybridoma cells (IG6D1O) which produced noanti-glucoarpylase antibody, nonspecific adhesion could not be detected.

A0

oA0

A0 0

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NATIVE DEGLYCOSYLATED Probe A B C100

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100

50

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100

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10 20

SLICE No.10 20

FIG. 5. Acrylamide gel electrophoresis of glucoamylase. Nativeand deglycosylated glucoamylases from strains 5106-9A (STAI),YIY2-12D (STA1), and 5301-17B (STA3), and transformants (recip-ient strain, YIYD) with pSTA1-4-10 (pSTA1) or pSTA3-6-4 (pSTA3)were analyzed by 7% acrylamide gels in the absence of sodiumdodecyl sulfate. Symbols: native (0) and deglycosylated (A) en-zymes, with the exception of the enzyme from YIY2-12D (native,0; deglycosylated, A).

a 10-kb fragment of BstEII-digested YIp5-STA3 (Fig. 8).Cells of a strain YIY345 (sta° ura3) were transformed withBstEII-digested YIp5-STA3ABst to Ura+. One of five Ura+integrants obtained was Sta+. Genomic DNA was preparedfrom a mixed culture of the Ura+ Sta+ and Ura+ Sta-transformant cells, digested partially with BamHI or com-pletely with KpnI, and ligated. The ligated DNA was used totransform E. coli to Ap'. Plasmid DNA was purified from thebacterial cultures and subjected to Southern blot and restric-tion analyses. We obtained six plasmids consisting of thevector YIp5 and yeast chromosomal DNAs that hybridizedto the STAI DNA (Fig. 9). One (pstaK) was derived fromKpnI-digested chromosomal DNA; and the other five (psta7,pstal2, pstal5, pstal6, and pstal8) were denrved fromBamHI-digested chromosomal DNA. Restriction maps of the plas-mids and their expected origin from chromosomal DNAsequences in the integrant cells are shown in Fig. 8. Fromthe restriction maps of the plasmids (psta7, pstal5, pstal6,

STA I

STAI stal STAl stoa STAI stal STAI sta°

kb _ w -

kboJu ._S _*

1.3-

*0 4

I.

STA3

FIG. 6. Southern blot analysis of genomic DNAs from S. dias-taticus and S. cerevisiae. Genomic DNAs were prepared from S.diastaticus strains 5106-9A (STAI) and 5301-17B (STA3), S. cere-visiae strain AH22 (sta° INHl), and the recipient strain YIYD (sta°inh/). The DNAs were digested with BamHI and processed forhybridization with the probes A, B, C, and D (Fig. 1). Results from

pSTA I strains 5106-9A (STA1) and AH22 (sta°) are shown since the sameresults were obtained from strains 5106-9A and 5301-17B and fromstrains AH22 and YIYD.

ASTAU sta*

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_0 O

.i tw.

FIG. 7. Southern blot analysis of the deleted region in thegenomic DNA of S. cerevisiae. Genomic DNAs were prepared fromS. diastaticus strains 5106-9A (STAI) and 5301-17B (STA3), S.cerevisiae strain AH22 (sta° INHI), and the recipient strain YIYD(sta' inh°). The DNAs were digested with BamHI plus Sall andprocessed for hybridization with the probes A, B, and C (Fig. 1).Results from strains 5106-9A (STAI) and AH22 (sta°) are shownsince the same results were obtained from strains 5106-9A and5301-17B and from strains AH22 and YIYD.

DT

kAAA"I AAAAAAA&AA&AA

I I I A*AA&AI

II ~ ~ ~ " AAAAA~AAAA pSTA 3 Probe

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TABLE 4. Integration of the cloned STA DNAs into homologous chromosomal DNA sequences'

YIp5-STA1 YIp5-STA3Recipient (genotype) STAI STA3 URA3 Other locus STAI STA3 URA3 Other locus

STAJSTA3 URA3 ~~~~~(Asta) STI ST3 URS(sta)

YIY320 (amyl-J ura3) 0 0 0 7 0 0 0 6YIY324 (amyl-2 ura3) 6 0 0 1 2 0 0 4YIY342 (sta° ura3) 0 0 0 6 0 0 0 5

a Recipient cells were transformed with KpnI-digested YIp5-STA1 or Kpnl-digested YIp5-STA3. The Sta' Ura+ integrants containing YIp5-STA1 or YIp5-STA3 at one site of the chromosomal DNA were crossed with strain YIY263 (STAI URA3) or YIYSTA3-2 (STA3), and meiotic products were analyzed. Linkagerelationships of the integration sites to the authentic STAI, STA3, and URA3 loci were determined by segregation patterns. When the integration site is linked toSTAI, all meiotic segregants derived from the cross between the integrant and the authentic strain carrying STAI show the glucoamylase-producing ability,whereas if they are not, three different segregation patterns (parental ditype, nonparental ditype, and tetratype) of the Sta+ phenotype are observed. Values arethe numbers of integrants in which the plasmids were integrated at the indicated loci.

pstak, and pstal8), we could speculate how YIp5-STA3ABst sequence (denotintegrated into chromosomal DNA. BstEII-digested YIp5- 1.15-kb BamHI-'STA3ABst should integrate at the 7.5-kb plus 1.3-kb BamHI It was shownfragment in the sta° chromosomal DNA by homologous "s2" in Fig. 8) N

recombination, a pathway which has the RAD52 gene prod- contained the sauct as a component (13). The restriction map of the 7.5-kb corresponding seBamHI fragment showed that the fragment contained a How the plasi

Pv

Pv PvPt Y PS-STA3t

&BstPt TA35- Pt Pt Yp5-T3Pt IstEll_ 0 t ktEII =<~Bst

BsEl LIGATION EHS

B K S PtESPVBtBtHPBB K S PtESPvBtBBtHPB B K S PtESPvBtHPB

UstEll~~~~~~~~~~~~-l

E~~~~~~~~~~~~~t

B KSPtE/P\BKSBPtES

BstEll \ 3.7B

Pt 4;P PtBtl

B K S P 7ESPVBt B1 HpB

H Pv,Pt PtPtH PvKB p

B - 7.5 -BB41-3-*-B- 5.4 -B - - 6.5 *-B-3'BH Pv,Pt PtPs BtH PVKIt S Pt Pv Pt EH| K S ESP Hr Ir 1II I ^kA _ _M 1 I

I 1-1. | l111 oi I

A

--_ psta15 _

ted "sl" in Fig. 8) carrying most of theSaII sequence of STA DNA.that the 1.3-kb BamHI fragment (denotedwhich was attached to the 7.5-kb fragmentame DNA sequence as that of the 1.3-kbegment of STA DNA.imids pstal2 and pstal8 carrying 3.7- and

BB*o 5.4 ---wB -a- 3.7 bB

|S Pt Pv Pt E H LPVSEPt S K

pstal2B 3.7 -B - 5.4 -[PVSEPt S K |SPt Pv Pt E H

Pv

Pt TYp5-StA3 Pt

ES

B K S Pt ESPvBtBt Ip BI i

H PV.Pt PtPvBtH pvK BtB 7.5 B<1 .3 B

B-- 5.4 BB-- 4.2 -B

A_________pstal8

psta 7

psta Kpstal6

FIG. 8. Integration of BstEII-digested YIp5-STA3/Bst into the chromosomal DNA of sta° cells and recovery of homologous DNAsequences to STA DNA from the integrant cells. YIp5-STA3ABst was constructed by self-ligation of the 10-kb fragment of BstEII-digestedYIp5-STA3. YIp5-STA3ABst was digested with BstEII and used for transformation of sta° cells. The integrated vectors and their flankingregions were recovered from genomic DNAs of the integrant cells as described in the text. Possible chromosomal DNA sequences of recipientand integrant cells were deduced by restriction maps of the recovered plasmids. Two possible orientations of the 3.7 and 5.4-kb (YIp5)fragments of pstal2 in the integrant chromosomal DNA are shown. DNA sequences of the fragments sl, s2, and Asta (described in the text)are boxed. The absence of BstEII site in the plasmid psta7 and the generation of a BamHI site in the plasmid pstal8 are marked with openand closed triangles, respectively. The DNA sequences of YIp5 are indicated as follows: solid line, pBR322; zigzag line, URA3 gene; closedbox, Apr gene; open box, Tcr gene. The sizes ofDNA fragments (in kb) are indicated. The restriction sites for EcoRI (E), BamHI (B), HindIlI(H), Sall, (S), KpnI (K), PstI (Pt), PvuII (Pv), BstEII (Bt), and Hpal (Hp) are also indicated.

J. BACTERIOL.

I


Probe C

1 2 3 4

Probe D

7 $ 11 12

*: V

75.*s- &4 -

- 4.2- 327

1.3..1*-------..... ..... ..

FIG. 9. Southern blot analysis of the plasmids recovered from the sta° cells transformed with BstEII-digested YIp5-STA3ABst. Plasmidswere digested with BamHI and processed for hybridization with probes C and D (Fig. 1). Lanes: 1 and 7, psta7; 2 and 8, pstal5; 3 and 9,pstaK; 4 and 10, pstal6; 5 and 11, pstal8; 6 and 12, pstal2.

4.2-kb BamHI fragments, respectively, were generated isuncertain. It should be noted, however, that the 0.5-kbBstEII-BamHI fragment carried by the plasmid pstal8 wasderived from the chromosomal DNA of the sta° recipientcells, because YIp5-STA3ABst carries no 0.5-kb BstEII-BamHI sequence. It may be speculated that YIp5-STA3ABstintegrated into the sl sequence of the 7.5-kb BamHI frag-ment as well (Fig. 8). If it is true, direct repeat sequenceswhich could not be detected by Southern blotting shouldexist at the two integration sites in the sl and s2 sequences.Involvement of the repetitive sequences in evolution of STAgenes will be discussed later. The 3.7-kb BamHI fragment ofthe plasmid, pstal2, may be derived from chromosomalDNA of the recipient cells, since the restriction map of thefragment agreed with the Southern blot data for genomicDNA of the sta° cells (Fig. 6 and 7).Recombination could occur between the BstEII-digested

STA DNA of the plasmid and the chromosomal DNA of thesta° recipient cells, after partial exonuclease-like digestion(10, 18) of the linearized donor plasmid DNA up to thesequence which can be recombined with the recipient coun-terpart. The absence of the BstEII site (marked with an opentriangle, Fig. 8) in the 6.5-kb BamHI fragment of psta7 andgeneration of the BamHI site (marked with a closed trianglein Fig. 8) in pstal8 may be explained by the mechanismdescribed above.

DISCUSSIONFrom the starch-fermenting yeast S. diastaticus, we have

cloned STA1 and STA3 genes encoding extracellularglucoamylases. Using as probes the subcloned fragments ofSTA1, Southern blot analysis clearly showed that STAI andSTA3 DNA of S. diastaticus contained almost identicalDNA sequences and that S. cerevisiae carrying no activeSTA gene also contained the highly homologous DNA seg-ments (sl, s2, and Asta) to the cloned STA DNAs. Recently,we have cloned these DNA sequences from a genomiclibrary of S. cerevisiae. Details will be published elsewhere.

xX5J

FIG. 10. A possible scheme for the generation of STA genes. TheDNA fragments (sl, s2, and Asta) homologous to STA DNA exist inS. cerevisiae cells. The sl and s2 fragments should be fused byrecombination at the direct repeat sequences (-) with subsequentdeletion of the spacer region. The fused s1-s2 fragment recombinedwith the Asta fragment, resulting in generation of an STA gene. Thepolymorphic STA genes were generated by translocation of thenewly evolved STA gene to unlinked regions of chromosomal DNA.

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One hypothesis for the generation of STA genes is shown inFig. 10. The sl and s2 fragments contain direct repeat. It wasdeduced from the data for integration of BstEII-digestedYIp5-STA3ABst into the sl and s2 sequences (Fig. 8). Thetwo fragments, sl and s2, should be fused by recombinationbetween the direct repeats with subsequent deletion of theinterstitial region. The fused sl-s2 segment could recombinewith the Asta fragment at the homologous sequences whichwere detected by Southern blot analysis (Fig. 6 and 7),resulting in generation of an STA gene. Generation ofpolymorphic STA genes may be explained by translocationof the newly evolved STA gene to other unlinked regions ofchromosomal DNA.Colonna and Magee (7) reported that S. cerevisiae synthe-

sizes an intracellular glucoamylase which is specificallyexpressed in sporulating ala diploid cells and plays a keyrole in spore maturation. Recently, however, we reportedthat extracellular production of glycoamylase from S. dias-taticus is repressed by the mating-type constitution a/a: a/adiploid cells produce greatly diminished amount of theenzyme compared with those of a, a, ala, and a/a cells (22).It is easy to imagine that the extracellular glucoamylase genein S. diastaticus may have evolved from an intracellularglucoamylase gene in S. cerevisiae, since differential expres-sion of closely related genes is well documented in highereucaryotes, fetal and adult hemoglobin genes in mammals(12), and 5S rDNA expressed in oocyte and somatic cells ofXenopus (9). In the accompanying paper (27), we havedetermined the complete nucleotide sequence of the STAIgene and revealed that the corresponding DNA sequence ofthe STAI gene to the Asta DNA codes for the functionaldomain of the extracellular glucoamylase. The Asta DNAmay code for sporulation-specific glycoamylase. An experi-mental support of this hypothesis is that the sporulation-spe-cific glucoamylase was immunoprecipitated by the anti-ex-tracellular glucoamylase antibodies (F3.2C7 and F3.2E12)(data not shown).

ACKNOWLEDGMENTSWe are very grateful to A. Toh-e for helpful discussions and

advice. We thank H. Tamaki for providing S. diastaticus strains. Wealso thank T. Miyakawa and E. Tsuchiya for help with generaltechniques for screening monoclonal antibodies.

This work was supported in part by grants from the Ministry ofEducation, Science and Culture of Japan.

LITERATURE CITED1. Beggs, J. D. 1978. Transformation of yeast by a replicating

hybrid plasmid. Nature (London) 275:104-109.2. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction

procedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

3. Bolivar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L.Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow. 1977.Construction and characterization of new cloning vehicles. II. Amultipurpose cloning system. Gene 2:95-113.

4. Broach, J. R., J. N. Strathern, and J. B. Hicks. 1979. Transfor-mation in yeast: development of a hybrid cloning vector andisolation of the CAN] gene. Gene 8:121-133.

5. Carlson, M., and D. Botstein. 1982. Two differentially regulatedmRNAs with different 5' ends encode secreted and intracellularforms of yeast invertase. Cell 28:145-154.

6. Cohen, S. N., A. C. Y. Chang, and L. Hsu. 1972. Non-

chromosomal antibiotic resistance in bacteria: genetic transfor-mation of Escherichia coli by R-factor DNA. Proc. Natl. Acad.Sci. U.S.A. 69:2110-2114.

7. Colonna, W. J., and P. T. Magee. 1978. Glycogenolytic enzymesin sporulating yeast. J. Bacteriol. 134:844-853.

8. Cryer, D. R., R. Eccieshall, and J. Marmur. 1975. Isolation ofyeast DNA. Methods Cell Biol. 12:39-44.

9. Ford, P. J., and R. D. Brown. 1976. Sequences of 5S ribosomalRNA from Xenopus mulleri and the evolution of SS gene-codingsequences. Cell 8:485-493.

10. Imai, Y., K. Suzuki, I. Yamashita, and S. Fukui. 1983. High-fre-quency transformation of Saccharomyces cerevisiae with lineardeoxyribonucleic acid. Agric. Biol. Chem. 47:915-918.

11. Koehler, G., and C. Milstein. 1975. Continuous cultures of fusedcells secreting antibody of predefined specificity. Nature (Lon-don) 256:495-497.

12. Nienhuis, A. W., and G. Stamatoyannopoulos. 1978. Hemoglobinswitching. Cell 15:307-315.

13. Orr-Weaver, T. L., J. W. Szostak, and R. J. Rothstein. 1981.Yeast transformation: a model system for the study of recom-bination. Proc. Natl. Acad. Sci. U.S.A. 78:6354-6358.

14. Polaina, J., and M. Y. Wiggs. 1983. STAIO: a gene involved inthe control of starch utilization by Saccharomyces. Curr. Genet.7:109-112.

15. Rigby, P. W. J., M. Dieckmann, C. Rhodes, and P. Berg. 1977.Labeling deoxyribonucleic acid to high specific activity in vitroby nick translation with DNA polymerase I. J. Mol. Biol.113:237-251.

16. Southern, E. M. 1975. Detection of specific sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98:503-517.

17. Struhl, K., D. T. Stinchcomb, S. Scherer, and R. W. Davis. 1979.High-frequency transformation of yeast: autonomous replica-tion of hybrid DNA molecules. Proc. Natl. Acad. Sci. U.S.A.76:1035-1039.

18. Suzuki, K., Y. Imai, I. Yamashita, and S. Fukui. 1983. In vivoligation of linear DNA molecules to circular forms in the yeastSaccharomyces cerevisiae. J. Bacteriol. 155:747-754.

19. Tamaki, H. 1978. Genetic studies of ability to ferment starch inSaccharomyces: gene polymorphism. Mol. Gen. Genet.164:205-209.

20. Tanaka, S., S.-Z. Hu, T. S.-F. Wang, and D. Korn. 1982.Preparation and preliminary characterization of monoclonalantibodies against human DNA polymerase a. J. Biol. Chem.257:8386-8390.

21. Yamashita, I., and S. Fukui. 1983. Molecular cloning of aglucoamylase-producing gene in the yeast Saccharomyces.Agric. Biol. Chem. 47:2689-2692.

22. Yamashita, I., and S. Fukui. 1983. Mating signals controlexpression of both starch fermentation genes and a novelflocculation gene FLO8 in the yeast Saccharomyces. Agric.Biol. Chem. 47:2889-2896.

23. Yamashita, I., and S. Fukui. 1984. Isolation of glucoamylase-non-producing mutants in the yeast Saccharomyces diastaticus.Agric. Biol. Chem. 48:131-135.

24. Yamashita, I., and S. Fukui. 1984. Genetic background ofglucoamylase production in the yeast Saccharomyces. Agric.Biol. Chem. 48:137-141.

25. Yamashita, I., and S. Fukui. 1984. Secretion of Saccharomycesdiastaticus glucoamylase from Schizosaccharomyces pombe.Agric. Biol. Chem. 48:1931-1932.

26. Yamashita, I., T. Hatano, and S. Fukui. 1984. Subunit structureof glucoamylase of Saccharomyces diastaticus. Agric. Biol.Chem. 48:1611-1616.

27. Yamashita, I., K. Suzuki, and S. Fukui. 1985. Nucleotidesequence of the extracellular glucoamylase gene STAI in theyeast Saccharomyces diastaticus. J. Bacteriol. 161:567-573.

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Evolutionary Origin in the Yeast Saccharomyces diastaticus