YEAST

VOL.

6: 193-204 (1990)

The Alcohol Dehydrogenase System in the Yeast, Kluyveromyces lactis MICHELE SALIOLA, JEFFREY R. SHUSTERt AND CLAUD10 FALCONE* Department of Cell and Developmental Biology, University of Rome, ‘La Sapienza’. Citta Universitaria, Piazzale A . Moro., 00185 Roma, Italy tChiron Corporation, Emeryville, California 94608, U . S . A . Received 3 July 1989; revised 15 November 1989

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADHgenes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomycespombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named KIADHI. The other gene, homologous to KIADHI in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondria1 targeting presequence. We named this gene KlADH.3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresisresolved K . lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source. KEY WORDS - Kluyveromyces

lactis; alcohol dehydrogenase; gene regulation.

INTRODUCTION The yeast alcohol dehydrogenase (ADH) system has been extensively studied in Saccharomyces cerevisiae where structural and several regulatory genes have been identified. Three structural genes have been isolated and sequenced in this yeast: (1) A D H l encoding the fermentative enzyme that is preferentially expressed on glucose (Bennetzen and Hall, 1982; Denis et al., 1983). ( 2 ) ADH2 encoding the oxidative enzyme that is repressed by the presence of glucose and derepressed in the absence of glucose by the positive activators ADRl and ADR6 (Bemis and Denis, 1988; Biter et al., 1985; Blumberg et al., 1988; Cherry e f al., 1989; Ciriacy, 1979; Denis et al., 1981; Denis and Young, 1983; Denis, 1984, 1987; Eisen et al., 1988; Irani et al., 1987; Russell et al., 1983; Taguchi and Young, 1987; Yu et al., 1989) and (3) ADH3 encoding an activity localized in the mitochondrion (Young and Pilgrim, 1985). All three genes show a very high sequence identity. Recently a new gene, ADH4, encoding an ADH activity but not homologous to the previously mentioned ones, has been identified in S . cerevisiae *Addressee for correspondence. 0749-503X/90/030193--12 $06.00 0 I990 by John Wiley & Sons Ltd

(Paquin and Williamson, 1986; Walton et al., 1986; Williamson and Paquin, 1987). In this paper we report results of the study of the ADH system of the yeast Kluyveromyces lactis. We have identified and sequenced two K. lactis genes homologous to the ADH genes of S. cerevisiae and compared their coding and 5’-flanking regions in the two yeasts. The comparison of related gene sequences between organisms can be an important step in understanding gene regulation. This has recently been demonstrated in the galactosemelibiose regulons of S . cerevisiae and of the galactose-lactose regulons of K. lactis. The two sets of regulons share several common features and are both positively regulated by GAL4 in S. cerevisiae (for a general review see Johnston, 1987) and by LAC9 in K. lactis (Salmeron and Johnston, 1986; Wray et al., 1987). These genes share limited regions of homology and their gene products probably bind similar upstream activation sequences to activate transcription (Breunig and Kuger, 1987; Leonard0 et al., 1987; Ruzzi et al., 1987; Salmeron and Johnston, 1986; Wray et al., 1987). Recent work has demonstrated that LAC9 can complement gal4 mutant strains of S. cerevisiae and GAL4 can

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M. SALIOLA, J. R. SHUSTER A N D C. FALCONE

Figure I . K . lucfis has multiple ADH isozymes. (A) Southern hybridization of ADHZ to K . /ac/isgenomic DNA. The 980 bp EcoRV-BamHI fragment from plasmid pBR 322-ADR2-BSa (Williamson et al., 1981), encompassing almost the entire coding sequence of the ADHZ gene of S.cerevisiae, was nick-translated and hybridized to a BamHI digestion of K . lactis DNA. The fragments (2 pg of DNA in each lane) were electrophoresed on a 1% agarose gel and transferred to H yBond-N membranes. (B) Electrophoretic analysis of ADH activities in K . luctis cells grown on 7oi0 glucose and 2% ethanol to the early stationary phase. Samples of cell extracts, corresponding to 10 pg of protein, were electrophoresed on non-denaturing polyacrylamide gels and the ADH isozymes were revealed as bands by staining the gels for the enzyme activity (see Materials and Methods).

activate the galactose-lactose regulons in lac9 mutant K . lactis strains (Riley et al., 1987; Salmeron and Johnston, 1986;Wrayetal., 1987). Interest in K . lactis has been recently enhanced by development of an efficient transformation system based on stable vectors (Bianchi er al., 1987; Chen et al., 1986; Falcone et al., 1986). MATERIALS AND METHODS Strains and media

K . lactis strain 23591152, MATa met KI K2, was derived from strain CBS 2359 (Wesolowski et al., 1982) and strain SDI 1 has been previously described (Das and Hollenberg, 1982). Cells were grown at 28-30°C in YP medium (1 % yeast extract, I % peptone) containing 2% glucose for standard stock cultures. Glucose-repressed cultures were grown in 7-8% glucose and respiring cells in 2-3% ethanol.

K. lactis genomic banks One genomic bank was a kind gift of Dr. Micheline Wesolowski (Institut Curie, Orsay, France). The bank was constructed by cloning a Sau3A partial digestion of the DNA of K . lactis, strain 23591152, into the BamHI site of the replacement vector lambda-147 (Loenen and Brammar, 1980). The DNA bank was amplified in Escherichia coli strain BJ5 183. Inserts containing alcohol dehydrogenase sequences were identified by plaque hybridization to ADHZ DNA probes of S. cerevisiae and then subcloned in pBR322. The second genomic bank was constructed from Sau3A-digested SD11 DNA. The KlADHI gene was isolated by complementation of the Adh- strain of S. cerevisiae (J.R.S., in preparation). Plasmid cloning

Restriction enzyme digestions, purification and ligation of DNA were performed according to

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ALCOHOL DEHYDROGENASE SYSTEM IN THE YEAST

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Figure 2. Restriction maps of two K . lacris genomic fragments that hybridize to the S. cerevisiue ADH2 gene. Both fragments were cloned in the BamHl site of pBR322. The localization and the orientation of the ADH sequences in the inserts 12-V-14 and 23-V-55 are indicated by the solid bars. The asterisk indicates the uncertain position of the restriction site. The BamHI site mapped at the left of the insert 23-V-55 was generated during the cloning procedure of the Sau3A genomic fragments into the BamHI site of the vector lambda-147.

Maniatis et al. (1982). Plasmid amplification was obtained in E. colistrain HBlOl.

Preparation of DNA and R N A Total nucleic acids were prepared from 20-50 ml yeast cultures. Cells were converted to spheroplasts with zymolyase and total DNA was prepared following the rapid isolation method described by Davis et al. (1980). Total nucleic acids were prepared using the method of Schultz (1978). Poly-A' RNA was prepared with oligo-dT cellulose and Northern analysis was performed using glyoxylated nucleic acid (Maniatis et al., 1982). DNA labeling and sequencing DNA probes were labeled by nick-translation according to Rigby et al. (1977). Sequencing was performed following both the chemical method of Maxam and Gilbert (1980) and the terminator method of Sanger et al. (1977). ADH activity analysis 10 ml cultures of cells were grown to the early stationary phase in repressing (7% glucose) and derepressed (2% ethanol) conditions. Cells were broken with glass beads in Eppendorf tubes, centri-

fuged and the supernatants were analysed by electrophoresis on non-denaturing acrylamide gels. Gel preparation and the electrophoretic conditions were essentially as described by Williamson et al. (1980). ADH isozymes were revealed by staining the gels for enzyme activity (Lutstorf and Megnet, 1968). RESULTS

Isolation and identijication of the ADH genes The genomic DNA of K. lactis, strain 235911 52, was digested with several restriction enzymes and the fragments containing ADH genes were identified by Southern hybridization analysis. The probe used in these experiments was a DNA fragment containing almost the entire coding sequence of the ADH2 gene of S. cerevisiae. The results of the hybridization of this probe to the BamHI genomic fragments, reported in Figure lA, showed that three fragments, 4.5, 7.5 and 8.0 kbp in length, strongly cross-hybridized to ADH2 sequences of S. cerevisiae. A fourth fragment, about 16.0 kbp in length, hybridizing to a lesser extent to ADH2, was also evident in the autoradiographs. This fragment was not the product ofa partial digestion and, when cut with other restriction enzymes, produced one or more smaller

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M. SALIOLA, J. R. SHUSTER AND C . FALCONE

-275

-250

-225

AACACCTGTTGCAGGGTGG~ATGTATTTTTCTCAAAGTGTGCTA~TTTCACACCAGCTAGAAATCAGCTG -200

-175

TCTTACTTGTATACAATTAGACCA~CCATTTGGTCTTCTGGAATATGTA~ATAAACACCCGGTCGATTCT -150

-125

-100

GACA;~TCCATCCACTTTTGTAGTAGGTCT~TCTATATCCATTTGTACAATGTTG~TTCTGTTTTGCCCTA -75

Hind1II -25 CATCATCATCAAGCAAAAACAATAGTTTcA A T T G ~AT C AAAC AAGCTTTAAACAC A C ~ G C T C T A AC T +1 25 50 TAAAAAAAGAT A A A~ T G GCGCATCTATCCCAGAAACT C CTTCTA C G ~ C G G GG T 1

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75

100

125

BclI

AAGGACATCCCAGTTCCAAAGCCAAAGGCT~ACGAACTTTTGATC AACGTCAAGT~C

TGAATTGCAATAC

150

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TCCGGTGTCTGTCACACCGATTT~CACGCATGGAAGGGTGACTGGCCT~TGCCAACCAAATTGCCATTAG 200

225

250

TTG~TGGTCACGAAGGTGCTGGTGTCGT~GTTGCTATGGGTG~CGTC~G~GCTGGAAGATTGGTGA 300

275

325

CTTCGCTG~TATCAAATGGTTGAACGGTTCTTG~ATGTCCTGTGAATACTGTGAATTG~CCAACGAATCC 350

375

400

AACTGTCCAGAAG~TGACTTGTCCGGTTACACTCACGA~GGTTCTTTCCAACAATACGCTACT~CTGATG 425

450

475

CCGTTCAAGCTGCCAAGA~CCCAGTCGGTACTGACTTGGCTG~GTTGCTCCAGTGCTATGTGCTGGT~T 500

525

CACCGTTTACAAGGCCCTATCCGCCAACTTGAAGGCCGGTGACTG~GTCGCCATCTCTGGTGCTGCT 550

575

600

GGT~GTCTAGGTTCTCTAGCTGTCCAAT~CGCCAAGGCCATGGGTTACAGAGT~TTGGGTATCGATGCTG 625

650

675

GTGAAGAA;\AGGCTAAGTTGTTCAAGGACTTGG~TGGTGAATACTTCATTGATTTCACCAAGTCCAAGAA 700

725

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CATCCCAGAAGAA~TCATCGMGCTACCAAGGGTGGTG~TCACGGTGTCATC~CGTCTCTGTCTCC~ RI 775 800 825 TTCGCTATCGAACMTCT~CCAACTACGTCAGATCTAACGGTA~CGTCGTATTGGTCGGTCTACCAAG~G 850

a75

ACGCCAAGTGTAAGTCCGATGTC~TTMCCAAGTTGTGAAGTCCATCT~CATTGTCGGTTCTTACGTCGG 925

900

950

TAA~AGAGCTGACACCAGAGAAGCCATT~ACTTCTTCTCCAGAGGTCTAGTC~GGCTCCAATCCACGTC 975

1025

1000

GTTGGTTT~TCCGAACTACCATCCATCTACG~GATGG~GGGTGCTATCGTCG~TAGATACGTC~ SalI

1050

1075

.........

TCGACACTTCAAA~TAATGAAATCTCTTCCGCATTCAA~TCATGACTTTTTTC.. Figure 3A.

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ALCOHOL DEHYDROGENASE SYSTEM IN T H E YEAST

-150 -125 GATTCGCTCCGTTGAAACAATT~TTTAAGGGTTCAGAATACTATAGT~TTACTACAGTTGCTAATATTTA -100 -75 -50 CG~CCGAGCTTTGTGACAGAACT~AAACCAAAACCGTCTTT~G~TCA~GTGCTTTTCC -25 +1 25 TCcc CCA&XAATTAAAACAAGAAATATTAAG~TGTT - GAGATTGACTTCCGCCA G A ~ ATTGTTTC C ccc 50 75 100

ATTGCGTAAGGETGCTTTTGGTTCCATCAGAACCTT;~GCTACCTCTGTGCCAGAAACCC~GGGTGTT 125.

175

200

ATTTTCTATGAGAATG~TGGTAAATTGGAATACAAGGACAT~CCAGTTCC~GCCAAAGCCAAATEAAA 275 300 TCTTGATCAACGTCAAGTACT~CGGTGTGTGTCATACCGATTTGCA~GCATGGAAGGGTGACTGGCCATT 325 350 375 G ~ A C C A A G T T G C CATTGGTC GGTG~TCACGAAGGTGCTGGTGTCGTTGT!?GCTATGGGTGAAAACGTC 400 425 450 AAGGGC!?GGAACATTGGTGACTTTGCGGGTA~CAAATGGTTGAACGGTTCTTGTAT~TCCTGTGAATACT 475 500 525 GTGAATTGTCC~TGAATCCAACTGTCCAGATGCTG;~CTTGTCTGGTTACACCCACGATGG!?TCTTTCCA ACAA.

................................................................. Figure 3B.

Figure 3 . Nucleotide sequences of K . luctis A D H genes. (A) Nucleotide sequence of KIADHI. The sequence was determined from strain SD11 and also from bases -234 to +285 in strain 2359/152. The only difference observed was located at position - 23 where G is replaced by A in strain SD11. The underlined sequences beside the ATG and TAA start and stop codons are discussed in the text. The arrows at position - 74 and - 77 indicate two main transcription start points. Unique sites for some restriction enzymes are also indicated. (B) Nucleotide sequence of the KIADH3 gene. The reported sequence is from strain 2359, I52

fragments which strongly hybridized to the probe (data not shown). The presence of multiple ADH cross-hybridizing sequences suggests the presence of multiple isozymes. By analogy to the ADH system of S. cerevisiae, these isozymes may be differentially regulated by the carbon source of the growth medium. To verify this hypothesis, we analysed the electrophoretic patterns of the ADH activities in K . luctis cells grown on glucose and ethanol. The results, shown in Figure lB, reveal qualitative and quantitative differences in isozyme pattern between the two physiological conditions. In the extract from ethanol-grown cells, six main bands were revealed by staining of the gels for ADH activity. Two of them, indicated by the arrows in the figure, were very low in extracts from glucose-grown cells. In extracts from glucose-grown cells, the two slower migrating bands were much more abundant than the corresponding ones observed on ethanol, indi-

cating a preferential expression of some A D H gene(s) in the presence ofglucose. Post-translational modifications, the non-denaturing gel electrophoresis conditions, and/or heteromeric isozymes (Ciriacy, 1975) may account for the number of ADH bands (that exceed the number of A D H genes observed by Southern blot analysis). Since the A D H 2 gene of S. cerevisiae strongly hybridized to the genomic DNA of K. lactis, we used this gene to probe a K . l a d s genomic bank for the isolation of the genes encoding the alcohol dehydrogenase activities in this yeast. The genomic bank, constructed in the replacement vector lambda-147, was kindly supplied from Dr. M. Wesolowski (Institut Curie, Orsay, France). Recombinant DNA molecules containing ADH sequences were identified by plaque hybridization and further characterized by restriction enzyme digestions and Southern hybridizations. Two of these recombinant DNAs carried

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M. SALIOLA, J. R. SHUSTER A N D C. FALCONE

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Figure 4. Comparison between the ADH isozymes of K . luctis and S. cerevisiue. The S. cerevisiue sequences are from Young and Pilgrim (1985). The amino acid sequence of each protein was deduced from the corresponding nucleotide sequence. The aminoterminal extensions present in the mitochondrial enzymes of K. luctis and S. cerevisiue are reported in the first two rows. The KlADH 111 sequence is incomplete (dots) and refers to the first 155 amino acids of the protein. The comparison between the different enzymes is based on the sequence of KlADH I and, except for the two mitochondria1 presequences which are entirely reported, only the differences in the amino acid composition are indicated.

BamHI inserts showing different restriction patterns which strongly hybridized to ADH2 (not shown). These fragments were then subcloned into the BamHI site ofpBR322. Two plasmids, p12-V-14

and p23-V-55, each containing one of the two inserts were isolated. A more detailed restriction enzyme analysis of the cloned fragments was performed and the ADH-coding regions were localized

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ALCOHOL DEHYDROGENASE SYSTEM IN THE YEAST

Figure 5 . Localization of K . lacris ADH genes to genomic DNA fragments. Chromosomal DNA of K . lurris (about 2pg per lane) was digested with BamHI and the fragments were separated on I % agarose gel and transferred to HyBond-N membranes. The genomic fragments were hybridized to the nick-translated probes carrying 5’-flanking and coding regions of the cloned genes. In the case of KlADHf, the 1700 bp BamHIHind111 fragment (lane 1) and the 800 bp HindIII-EcoRI fragment (lane 2) from insert 23-V-55 were used as non-coding and coding probes. In the case of KlADH3, the two probes were the 750 bp DdeI fragment (lane 3) and the 1000 bp HincII-DdeI fragment (lane 4) from insert 12-V-14.

by hybridization to 5’ and 3’ probes from ADH2 DNA (see Figure 2). Sequence of the ADH genes

Figure 6 . Localization of K. lacris ADH genes on K . lactis chromosomes. Chromosome separation was performed by Dr F. Sor by the OFAGE technique (Sor, 1988). DNA probes encompassing coding and 5‘-flankingregions of KlADHf and KlADH3 were hybridized to the separated chromosomes transferred onto nitrocellulosefilters. The KlADHf probe(1ane 1) was the 2000 bp EcoRI-EcoRV fragment from insert 23-V-55 and the KIADH3 probe (lane 2) was the 1750 bp Hinc I1 fragment from insert 12-V- 14. I n lane 3 the ethidium bromide staining of the separated chromosomes is shown.

The DNA sequence of the KlADHl gene is shown in Figure 3A. An uninterrupted open reading frame (ORF) of 1050 nucleotides was found that is about 8 1 YOand 80% identical in sequence to the ADHI and ADH2 genes of S. cerevisiae respectively. It is interesting to note that the only difference observed in the sequences of KlADHl from two different strains of K . lactis was an A or G at position -23 in the promoter region. The KlADHl OR F could encode a protein of 350 amino acids, with an apparent molecular weight of 37 000 daltons. Only dispersed identity can be observed between the promoter regions of the ADHgenes from K . lactis and S. cerevisiae, indicating different control sequences in the expression of these genes. Nevertheless, a number of sequence motifs common to ADHI, ADH2 and to other yeast promoters have been observed the KlADHI gene. Analysis of the 5‘-flanking region shown (Figure 3A) revealed two eukaryotic promoter consensus sequences at position - 177

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M. SALIOLA, J. R. SHUSTER AND C . FALCONE

ADH

URA

1 2 3 4

5 6 7 8

DNA

-D

Figure 7. Northern analysis of the KlADHI transcript in K. lucris. Strain 2UV21 (a ura3 mutant of K. lucfis strain SDl1) was grown in YEP+8% glucose or YEP+ 3% ethanol. The K l A D H l hybridization probe (lanes 14)was a synthetic 18-mer complementary to a region 3’ to the KlADHl structural gene, 5’-TAAACGGAGAGAGGAGGG, kinased with gamma-[’2P]ATP.The URA3 hybridization probe (lanes 5-8) was a nick-translated portion of the K. luctis URA3 structural gene(Shuster et ul., 1987). The lanescorrespond to the following, listed as RNA type/carbon source for growth: lanes 1 and 5, total RNA/ glucose; lanes 2 and 6, total RNA/ethanol; lanes 3 and 7, p01y-A~ RNA/glucose; lanes 4 and 8. poly-A’ RNA/ethanol.

to

-

170 (TATATAAA) and position - 122 to

- 118 (TATAT). The sequence TCAAG, located between position - 76 and - 72 in this gene, has been postulated to represent the CAP sequence in S . cerevisiue (Russell er ul., 1983). Two main transcription start points, indicated in Figure 3A by the arrows, have been located both by S1 mapping and primer extension techniques (not shown) at the adenine residues at position -77 and -74 in the region of the TCAAG sequence. The CACACA sequence, which has been found in front of the ATG start codon in several genes of S . cerevisiue and presumed to be required for highly efficient translation (Stiles et ul., 1981), is also present in the KlADHI gene at positions -30 and -25. Although no conclusion can be drawn by comparison of the KlADHl gene to ADH genes of S. cerevisiue on the basis of the nucleotide sequence, we refer to this gene as KlADHl in that its transcriptional regulation is similar to the A D H l gene in S . cerevisiae (see transcription analysis section).

We report, in Figure 3B, the nucleotide sequence from insert 1 2-V- 14 encompassing the 5’-portion of an O R F homologous to ADH genes and about 170 nucleotides of its upstream region. The putative ATG initiation codon (at position + 1 in the sequence) is followed by a stretch of about 90 nucleotides before reaching a region of very high sequence identity to KlADHl and the ADHgenes of S. cerevisiue. The amino acid sequences of the K . lactis ADH genes predicted from the nucleotide sequences, have been compared to the sequences of ADH I, ADH 11, and ADH 111 isozymes of S. cerevisiue. From this analysis (Figure 4), the ADHgene from insert 12-V14 can be identified as one coding for a putative mitochondria1 activity. In analogy to S. cerevisiue, we will refer to this gene and its product as KIADH.3 and KIADH 111 respectively. Beside a potential TAATA element consensus sequence located between positions -111 and -107, no other characteristic motifs of the yeast promoters,

ALCOHOL DEHYDROGENASE SYSTEM IN THE YEAST

nor significant sequence identity to the ADH3 promoter, have been observed in the 5’-upstream region of this gene. Both predicted ADH 111 and KlADH 111 protein sequences share an amino-terminal extension of 27 amino acids. In S. cerevisiae this extension has been shown to be responsible for the mitochondria1 matrix targeting of this protein (Pilgrim and Young, 1987; Van Loon and Young, 1986). The identity at the amino acid level between the two presequences was 30% and only the very amino-terminal part of these extensions is conserved in the two yeasts. Outside the presequences, a strong identity can be observed between the reported ADH sequences. KlADH I was 85%,83% and 66% identical in the amino acid sequence to ADH I, ADH I1 and ADH I11 of S. cerevisiae respectively. With respect to the S. cerevisiae isozymes that are 348 amino acids long, KlADH I showed two additional alanine residues located immediately after the first methionine. Such alanine residues are also present in ADH I11 at the end of the presequence. As can be seen in Figure 4, the identity between the ADH isozymes is higher in the amino-terminal regions, the substitutions being localized mainly in the second third of the protein sequences. The identity in the amino acid sequence between KlADH I and the ADH enzyme of Schizosaccharornyces pombe is 50% (Russel and Hall, 1983); the same value has been observed between the S. pornbe and the S. cerevisiae enzymes.

20 1 this cloned gene and the BamHI genomic fragment of 4.5 kbp. We further investigated the localization of these ADH genes on the chromosomes of K. lactis. Probes from KlADHl and KlADH3 DNAs were hybridized to chromosomes that had been separated by the orthogonal-field-alternation gel electrophoresis (OFAGE) technique. The strips of the separated chromosomes were a kind gift from Dr F.Sor (Institut Curie, Orsay, France). The results, reported in Figure 6, show that KlADHl preferentially hybridized to the first (lane 1) and KlADH3 to the fourth (lane 2) of the six separated chromosomes of K. lactis. An additional hybridization signal could be observed with both probes on the faster migrating chromosome(s). From these results we conclude that thestructural genescodingfor the ADH isozymesare not clustered in K. lactis and are located on different chromosomes.

Localization of the KlADH genes in the genome of K. lactis

Transcription analysis The expression of the ADH genes in S. cerevisiae is regulated at the transcriptional level and is dependent on the carbon source. To determine if this was also the case in K. lactis, we studied the transcription of the KlADHl gene under different physiological conditions. The results of this analysis are shown in Figure 7. The level of KlADHl RNA was five to ten times higher in RNA isolated from glucose-grown cultures (lanes 1 and 3) than in RNA from ethanolgrown cultures (lanes 2 and 4)when compared with hybridization detected for K . lactis URA3 RNA. This result is similar to that observed for the ADHI gene of S. cerevisiae (Denis et al., 1983).

In order to find a correlation between the cloned genes and the genomic fragments containing ADH sequences (see Figure 1A), DNA probes containing coding and 5’-flanking regions were prepared from the inserts 12-V-14 and 23-V-55 and hybridized to the BamHI chromosomal fragments (Figure 5). In the case of the insert 23-V-55, the probe corresponding to the non-coding region specifically hybridized to the BamHI genomic fragment of 7.5 kb (lane 1). The probe encompassing the coding region of the gene hybridized preferentially to the same 7-5kb fragment (lane 2) and to a lesser extent, due to the cross-hybridization, to the other BamHI fragments carrying ADH sequences previously detected with the ADH2 probe of S. cerevisiae (see Figure 1A). In the case of the insert 12-V-14, the specific hybridization ofthe 5’-flankingprobe (lane 3) and the preferential hybridization of the coding probe (lane 4) permitted us to find a correspondence between

DISCUSSION The ADH system in K. lactis is encoded by multiple ADH genes. The exact number of these genes is unknown; however, four possible structural genes have been detected by Southern blot hybridization with ADH probes from both S. cerevisiae and K. lactis. In this respect, K. lactis is closer to S . cerevisiae than to S. pornbe, in which only one ADH gene is present (Russel and Hall, 1983). Another common feature shared by K. lactis and S. cerevisiae is the regulation of the expression of the ADH isozymes. The analysis of ADH activities in K. lactis extracts demonstrated that differently migrating bands are present depending on the carbon source. Some of these bands show higher intensities when cells are grown on glucose, while other bands are present only from ethanol-grown cultures. In the latter case, the activities may be repressed by glucose

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or induced by ethanol. These results suggest that K . luctis, similar to S. cerevisiue, may contain a fermentative enzyme(s) for the conversion of acetaldehyde to ethanol, and an oxidative enzyme(s) for the catabolism of alcohols. We have identified structural genes comprising part of the K . Iuctis multienzymatic ADH system. Two ADH genes have been isolated from a genomic bank and further characterized. Sequencing of KlADHl revealed an uninterrupted ORF of 1050 nucleotides. The O R F is about 81% and 80% identical to ADHl and ADH2 of S. cerevisiue respectively. KlADHl is only 60% identical in nucleotide sequence and 50% identical in amino acid sequence to the ADH gene of S. pombe. Analysis of the 5’-flanking region of KlADHl revealed consensus sequences for two TATA boxes and other elements which are present in many S. cerevisiue promoters. Two transcription start points have been located at positions - 74 and - 77, representing a relatively long distance compared to higher eukaryotic promoters. No long dyad sequences nor d(A)n tracts are present in the upstream region of KlADHI. In the promoter region of ADH2 a 22 base pair dyad sequence, located about 200 nucleotides upstream to the ATG, is involved in the derepression of this gene (Beter et ul., 1985; Shuster et ul., 1986; Yu et ul., 1989). A clearer picture on the nature of the KlADHl gene comes from transcription analysis in cells grown on glucose and ethanol. When a 3’flanking region, not containing KlADHl coding sequences was used as a probe in Northern experiments, a hybridization signal was observed that was five to ten times higher in glucose-grown cells than in ethanol-grown cells. This result indicates that KlADHl is preferentially expressed under fermentative conditions. One cannot exclude the possibility of a lower stability ofthe messenger RNA during respiratory metabolism. The regulation of KlADHl mRNA agrees with the regulation of ADH activity as determined by protein gel electrophoresis. The cloning of KlADHl into S. cerevisiae-K. luctis shuttle vectors will provide an interesting picture of the functioning and regulation of the KlADHl promoter in the two yeasts. Although we have isolated a KlADHl gene by complementation for ADH activity in S. cerevisiue and thus it can be active (J.R.S. in preparation), we have not investigated its regulation in detail. The second gene that we isolated has been named KIADH3 in analogy to ADH3 of S. cerevisiue. Analysis of the KIADH3 nucleotide sequence revealed an O R F homologous to KlADHl with an

M. SALIOLA, J. R. SHUSTER A N D C. FALCONE

additional extension at the S’(amino-terminal) end. In S. cerevisiue, a similar presequence is present in ADH 111which is responsible for the mitochondrial targeting of the protein. In both yeasts, they are 27 amino acids long but do not show extensive sequence identity. In fact, only the first six amino acids, with the exception of a leucine residue that is absent in the S. cerevisiae presequence, are tightly conserved. The KlADH 111 presequence presents the main characteristics that are common to ADH 111 and to other mitochondrial presequences of S. cerevisiue: (1) the presence of positively charged residues, (2) the absence of acidic residues, (3) the frequent occurrence of the hydroxylated amino acids serine and threonine, and (4) the marked amphipilicity (for recent reviews see Hart1 et al., 1989 and Roise and Shatz, 1988). The ADH genes are not clustered in K. lucris. Hybridization analysis of the two cloned genes with the chromosomes separated by the OFAGE technique revealed that at least KlADHl and KlADH3 are located on different chromosomes. We are now interested in the isolation of the other structural ADH genes of K , luctis and in the identification of the regulatory genes involved in the control of this complex multi-genic system. The possibility of exchanging structural and regulatory genes between K. luctis and S. cerevisiue could be of great interest for the study of the evolution of the ADH system in two different yeasts. A number of other Kluyveromyces Iuctis genes have been isolated (Shuster et ul., 1987; Stark and Milner, 1989; Wesolowski-Louvel et ul., 1988) and it will be of interest to investigate the regulation of these genes in S. cerevisiue as well as K . luctis. ACKNOWLEDGEMENTS This research was supported, in part, by the Commission of the European Communities (BAP 0061-I), by CNR (Progetto Finalizzato Biotecnologie e Biostrumentazione) and Pasteur-Fondazione Cenci Bolognetti. We would like to thank L. Frontali for helpful discussions, F. Castelli and Donna Moyer for technical assistance, and Micheline Wesolowski and Frederic Sor (Institut Curie, Orsay) for the generous gifts of the K . lactis gene bank and the OFAGE-separated chromosomes. REFERENCES Bemis, L. T. and Denis, C. (1988). Identificationof functional regions in the yeast transcriptional activator ADR 1. Mol. Cell. Biol. 8,2 125-2 13 1.

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