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Sequence of a gene coding for a cytoplasmic alcohol dehydrogenase from Kluyveromyces marxianus ATCC 12424
Biochimica et BiophysicaActa, 1173 (1993) 99-101 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00
BBAEXP 90495
99
Short Sequence-Paper
Sequence of a gene coding for a cytoplasmic alcohol dehydrogenase from Kluyveromyces marxianus ATCC 12424 1 Jean-Marc Ladri~re, Jean Delcour and Jean Vandenhaute Facultds UniversitairesN-D de la Paix, Laboratoire de GdndtiqueMoldculaire, Namur (Belgium) (Received 21 December 1992)
Key words: Alcohol dehydrogenase; ADH; Glucose repression; (Kluyveromyces) Using a Saccharomyces cerevisiae ADH1 probe, a gene coding for a cytoplasmic alcohol dehydrogenase from Kluyveromyces marxianus ATCC 12424 (formerly K. fragilis) has been cloned. This gene is able to restore alcoholic fermentation in an ADH-null strain of S. cerevisiae and its encoded protein shows strong similarity with other yeast alcohol dehydrogenases (from S. cerevisiae, Schizosaccharomyces pombe and its close relative K. lactis). The product of the gene expressed in S. cerevisiae co-migrates on native gel with a K. marxianus ADH isozyme which is more active in cells growing on non-fermentable carbon sources than on glucose. This behaviour is in contrast with that of the two cytoplasmic ADH isozymes of K. lactis which are both more active in glucose-growing thanin ethanol-growing cells.
Acetaldehyde reduction to ethanol is the last step of alcoholic fermentation in yeast. Conversely, ethanol oxidation is the first reaction of ethanol respiration pathway. The enzymes catalyzing these reactions and their structural genes have been well studied in Saccharomyces cerevisiae. ADH1 encodes the fermentative enzyme and is expressed in a semi-constitutive way, its transcription being induced by glucose [1]. A D H 2 is a glucose repressed gene and codes for the respiratory enzyme [2]. A D H 2 being highly repressed by glucose, S. cerevisiae metabolizes the ethanol produced by fermentation only when glucose is exhausted from the culture medium [3]. S. cerevisiae has two further A D H genes: ADH3, coding for a mitochondrion-targeted enzyme presumably involved in ethanol oxidation [4], and A D H 4 which displays no significant similarity with any other characterized yeast A D H gene [5]. K. lactis has four A D H genes, two of them coding for cytoplasmic isozymes (ADH1 and A D H 2 ) [6,7] and the two others coding for mitochondrial isozymes ( A D H 3 and A D H 4 ) [8]. Their regulation and physiological role are currently under investigation. In rag2 strains of K~ lactis (rag2 is a mutation in the gene coding for phosphoglucose isomerase), ADH1 and A D H 2 are semi-
Correspondence to: J. Vandenhaute, Laboratoire de G~n6tique Mol6culaire, Facult6s Universitaires N-D de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium. i The sequence data presented in this paper have been submitted to the EMBL Data Library under the accession number X60224.
constitutively expressed and glucose induced, whereas A D H 4 is specifically induced by ethanol and disruption of either of the four genes has no effect on growth on glucose or ethanol [9]. In a wild-type strain, A D H 4 is constitutively expressed and disruption of ADH1 leads to a decrease in ethanol production and the inability to grow on glucose in the presence of a respiratory inhibitor [9]. Nothing is known about the A D H system of its close relative K. marxianus. It has been reported that' this yeast has, in contrast to S. cerevisiae, the ability to metabolize simultaneously glucose and ethanol [3]. The K. marxianus gene for the respiratory A D H could thus be insensitive to glucose repression or less sensitive than S. cerevisae ADH2. This raises interest on the cloning of this gene and the study of its regulation. A probe generated by random primed synthesis on a 758-bp fragment of S. cerevisiae ADH1 open reading frame was used to screen a K. marxianus A T C C 12424 genomic library established in the shuttle vector YEp351 [10,11]. Several different plasmids were isolated and used to transform a leu2 derivative of the S. cerevisiae ADH-null strain 302.21 (MATa, his4, trpl, leu2::TRP1, adhl, adh2, adh3). This strain is unable to perform alcoholic fermentation and metabolizes glucose only via respiration [12]. Plasmid p G I K 10.9 gave transformants able to grow on plates containing glucose and the respiratory inhibitor antimycin A, demonstrating that alcoholic fermentation was restored. Moreover, these transformants lost the ability to grow on a medium containing the suicide substrate allyl
100 i0 30 50 ~GTATCTTCATCTATGGTATACCTTTTTTTGCCACTGG~CATGTATTATTATTACTAT 70 90 ii0 TGT~TTGT~TTGTTATTGTTTTTAAATCCCC~GCAC~CTTAAATT~GT~GTGGT 130 150 170 T~TCTACATTTAI~CAAAA.TCATACAC~CAC~TGGCTATTCCAGAAACTCAAAAG M A I P E T Q K 190 210 230 GGTGTTATCTTCTACG~CACGGTGGTGAGTTGC~TAC~GGACATTCCAGTTCCAAAG G V I F Y E H G G E L Q Y K D I P V P K 250 270 290 CCAAAGCCAAATG~CTTTTGATC~CGTT~GTACTCTGGTGTGTGTCACACCGATTTG P K P N E L L I N V K Y S G V C H T D L 310 330 350 CACGCATGGC~GGTGACTGGCCATTGGACACC~GTTGCCATTGGTCGGTGGTCACG~ H A W Q G D W P L D T K L P L V G G H E 370 390 410 GGTGCTGGTATTGTTGTTGCCATGGGTGAG~CGTTACTGGGTGGGAAATCGGTGACTAT G A G I V V A M G E N V T G W E I G D Y 430 450 470 GCTGGTATC~GTGGTTG~CGGTTCCTGTATGTCTTGTGAGGAGTGTGAGTTGTCG~C A G I K W L N G S C M S C E E C E L S N 490 510 530 G~CCAAACTGTCCA~GGCCGACTTGTCTGGTTACACTCACGACGGTTCTTTCC~C~ E P N C P K A D L S G Y T H D G S F Q Q 550 57O 590 TACGCTACCGCTGACGCTGTGCAGGCTGCCAG~TTCCAAAG~CGTCGACTTGGCCGAG Y A T A D A V Q A A R I P K N V D L A E 610 630 650 GTTGCCCC~TCTTGTGTGCCGGTGTTACCGTGTAC~GGCTTTG~GTCTGCCCACATC V A P I L C A G V T V Y K A L K S A H I 670 690 710 ~GGCTGGTGACTGGGTCGCCATCTCCGGTGCATGTGGTGGTCTAGGTTCCTTGGCCATC K A G D W V A I S G A C G G L G S L A I 730 750 770 C~TACGCC~GGCTATGGGTTACAGAGTGCTAGGTATCGATGCTGGTGACGAAAAGGCC Q Y A K A M G Y R V L G I D A G D E K A 790 810 830 AAATTGTTC~GG~TTGGGCGGTG~TACTTCATCGACTTCACC~GACC~GGACATG K L F K E L G G E Y F I D F T K T K D M 850 87O 890 GTAGCAG~GTCATTGAGGCCACC~TGGTGTGGCCCACGCTGTCATT~CGTGTCTGTG V A E V I E A T N G V A H A V I N V S V 910 930 950 TCCG~GCCGCCATCTCTACCTCTGTCTTGTACACCAGATCAAACGGTACCGTCGTCTTG S E A A I S T S V L Y T R S N G T V V L 970 990 I010 GTCGGTTTGCCAAGAGACGCCC~TGT~GTCTGATGTCTTC~CC~GTCGTC~GTCC V G L P R D A Q C K S D V F N Q V V K S 1030 1050 1070 ATCTCCATTGTTGGTTCTTACGTTGGT~CAGAGCAGACACCAGAG~GCCCTAGACTTC I S I V G S Y V G N R A D T R E A L D F 1090 Iii0 1130 TTCTCCAGAGGTTTGGTC~GGCTCC~TT~GATTCTCGGCTTGTCTG~TTGGCATCC F S R G L V K A P I K I L G L S E L A S 1150 1170 1190 GTTTACGAC~GATGGTCAAGGGCCA~TCGTTGGTAG~TTGTCGTTGACACTTCCAAA V Y D K M V K G Q I V G R I V V D T S K 1210 1230 1250 TAAACAA~GGACCCTTTTGATATGGT~TA~TTAACT~CTAAACTTTTTATGACTTT
TABLE I
Amino acid sequence identity and similarity of K. marxianus ADH with S. cerevisiae [4,14,15], K. lactis [6,7,8] and Schizosaceharomyces pombe [16] ADHs The algorithm used was the GAP program [17]. Mitochondrial ADHs were considered without their presequence. Identity
Similarity
(%)
(%)
S. cerevisiae
ADH I ADH II ADH III
79.3 79.3 77.9
87.6 87.1 85.9
K. lactis
ADH ADH ADH ADH
85.9 86.8 78.7 76.4
91.7 91.4 87.6 84.4
Schizosaccharomyces pombe
ADH I
49.7
67.2
I II III IV
electrophoresis and compared with the A D H produced in S. cerevisiae strain 302.21 [pGIK 10.9] (Fig. 2). The K marxianus A D H co-migrates with the major isozyme present in /¢ marxianus growing on non-fermentable carbon sources. Its activity, though it remains the major one, decreases when cells are grown on glucose. Glucose seems to be responsible for this decrease, since cells grown in 2% ethanol/7% glucose display the same isozyme profile as in 7% glucose. This behaviour is similar to that of the glucose-repressible S. cerevisiae A D H II isozyme [2] but is different from that
o
c5
o~,
K.m. 10gg
l~g
I
]
10gg 50~g 10~g 50~g
10gg 10~g 10gg 10~g
I I
I 2%E 2%E 2%P 2%Gly +7%G
Fig. 1. Nucleotide and deduced amino acid sequence of K ma~ianus ADHgene.
alcohol, oxidized by A D H to the toxic compound acrolein [13]. A sequencing primer was designed by alignment of S. cerevisiae and K. lactis ADH1 open reading frame 5' region [6,14]. The entire sequence of the gene was established on both strand (Fig. 1). The predicted protein is 348-amino acid long and should be localised in the cytoplasm as it does not possess the amino terminal extension of mitochondrion-targeted ADHs [8]. Percentages of similarity and identity with other yeast ADHs are given in Table I. The K. marxianus A D H displays the highest similarity with the K. lactis A D H I and A D H II but is not more closely related to either of these isozymes. The A D H isozymes produced by K. marxianus on different carbon sources were revealed by native gel
I 2%G
7%G
Fig. 2. Native PAGE analysis of ADH isozymes from K. marxianus strain ATCC 12424 (noted K.m.) and S. cerevisiae strain 302.21 (noted S.c.) transformed or not by pGIK 10.9 grown on different carbon sources (2%G: 2% glucose; 7%G: 7% glucose; 2%E/7%G: 2% ethanol+7% glucose; 2%E: 2% ethanol; 2%P: 2% pyruvate; 2%Gly: 2% glycerol). Soluble extracts were run on 7% acrylamide gel and stained for ADH activity according to Lutstorf and Megnet [13]. Amounts of proteins are given on the top of the lanes.
101 of the two K. lactis cytoplasmic ADHs which are both semi-constitutively expressed and glucose-induced [9]. The molecular identity between the bands comigrating must first be established before drawing conclusions about the physiological role of the K. rnarxianus ADH described in this report. It can be speculated that its presence at high glucose concentrations would explain the ability of K. marxianus to simultaneously metabolize glucose and ethanol. Further studies will determine whether glucose repression takes place at the transcriptionnal level as in S. cerevisiae A D H 2 [2]. S. cerevisiae A D H 1 gene and strain 302.21 were a kind gift from Dr. Elton T. Young. J-M Ladri~re holds an IRSIA bursary. References 1 Denis, C.L., Ferguson, J. and Young, E.T. (1983) J. Biol. Chem. 258, 1165-1171. 2 Denis, C.L., Ciriacy, M. and Young, E.T. (1981) J. Mol. Biol. 148, 355 -368. 3 W6hrer, W., Forstenlehner, L. and R6hr, M. (1981) in Current developments in yeast research (Stewart, G.G. and Russel, I., eds.), pp. 405-410, Pergamon press, Toronto.
4 Young, E.T. and Pilgrim, D. (1985) Mol. Cell. Biol. 5, 3024-3034. 5 Williamson, V.M. and Paquin, C.E. (•987) Mol. Gen. Genet. 209, 374-381. 6 Saliola, M., Shuster, J.R. and Falcone, C. (1990) Yeast 6, 193-204. 7 Shain, D.H., Salvadore, C. and Denis, C.L. (1992) Mol. Gen. Genet. 232, 479-488. 8 Saliola, M., Gonnella, R., Mazzoni, C. and Falcone, C. (1991) Yeast 7, 391-400. 9 Mazzoni, C., Saliola, M. and Falcone, C. (1992) Mol. Microbiol. 6, 2279-2286. 10 Myers, A.M., Tzagoloff, A., Kinney, D.M. and Lusty, C.J. (1986) Gene 45, 299-310. 11 Laloux, O., Cassart, J.P., Delcour, J., Van Beeumen, J. and Vandenhaute, J. (1991) FEBS Lett. 289, 64-68. 12 Williamson, V.M., Bennetzen, J.L., Young, E.T., Nasmyth, K. and Hall, B.D. (1980) Nature 283, 214-216. 13 Lutstorf, U. and Megnet, R. (1968) Archives Biochem. Biophys. 126, 933-944. 14 Bennetzen, J.L. and Hall, B.D. (•982) J. Biol. Chem. 257, 30183025. 15 Russell, D.W., Smith, M., Williamson, V.M. and Young, E.T. (1983) J. Biol. Chem. 258, 2674-2682. 16 Russel, P.R. and Hall, B.D. (1983) J. Biol. Chem. 258, 143-149. 17 Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395.
BBAEXP 90495
99
Short Sequence-Paper
Sequence of a gene coding for a cytoplasmic alcohol dehydrogenase from Kluyveromyces marxianus ATCC 12424 1 Jean-Marc Ladri~re, Jean Delcour and Jean Vandenhaute Facultds UniversitairesN-D de la Paix, Laboratoire de GdndtiqueMoldculaire, Namur (Belgium) (Received 21 December 1992)
Key words: Alcohol dehydrogenase; ADH; Glucose repression; (Kluyveromyces) Using a Saccharomyces cerevisiae ADH1 probe, a gene coding for a cytoplasmic alcohol dehydrogenase from Kluyveromyces marxianus ATCC 12424 (formerly K. fragilis) has been cloned. This gene is able to restore alcoholic fermentation in an ADH-null strain of S. cerevisiae and its encoded protein shows strong similarity with other yeast alcohol dehydrogenases (from S. cerevisiae, Schizosaccharomyces pombe and its close relative K. lactis). The product of the gene expressed in S. cerevisiae co-migrates on native gel with a K. marxianus ADH isozyme which is more active in cells growing on non-fermentable carbon sources than on glucose. This behaviour is in contrast with that of the two cytoplasmic ADH isozymes of K. lactis which are both more active in glucose-growing thanin ethanol-growing cells.
Acetaldehyde reduction to ethanol is the last step of alcoholic fermentation in yeast. Conversely, ethanol oxidation is the first reaction of ethanol respiration pathway. The enzymes catalyzing these reactions and their structural genes have been well studied in Saccharomyces cerevisiae. ADH1 encodes the fermentative enzyme and is expressed in a semi-constitutive way, its transcription being induced by glucose [1]. A D H 2 is a glucose repressed gene and codes for the respiratory enzyme [2]. A D H 2 being highly repressed by glucose, S. cerevisiae metabolizes the ethanol produced by fermentation only when glucose is exhausted from the culture medium [3]. S. cerevisiae has two further A D H genes: ADH3, coding for a mitochondrion-targeted enzyme presumably involved in ethanol oxidation [4], and A D H 4 which displays no significant similarity with any other characterized yeast A D H gene [5]. K. lactis has four A D H genes, two of them coding for cytoplasmic isozymes (ADH1 and A D H 2 ) [6,7] and the two others coding for mitochondrial isozymes ( A D H 3 and A D H 4 ) [8]. Their regulation and physiological role are currently under investigation. In rag2 strains of K~ lactis (rag2 is a mutation in the gene coding for phosphoglucose isomerase), ADH1 and A D H 2 are semi-
Correspondence to: J. Vandenhaute, Laboratoire de G~n6tique Mol6culaire, Facult6s Universitaires N-D de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium. i The sequence data presented in this paper have been submitted to the EMBL Data Library under the accession number X60224.
constitutively expressed and glucose induced, whereas A D H 4 is specifically induced by ethanol and disruption of either of the four genes has no effect on growth on glucose or ethanol [9]. In a wild-type strain, A D H 4 is constitutively expressed and disruption of ADH1 leads to a decrease in ethanol production and the inability to grow on glucose in the presence of a respiratory inhibitor [9]. Nothing is known about the A D H system of its close relative K. marxianus. It has been reported that' this yeast has, in contrast to S. cerevisiae, the ability to metabolize simultaneously glucose and ethanol [3]. The K. marxianus gene for the respiratory A D H could thus be insensitive to glucose repression or less sensitive than S. cerevisae ADH2. This raises interest on the cloning of this gene and the study of its regulation. A probe generated by random primed synthesis on a 758-bp fragment of S. cerevisiae ADH1 open reading frame was used to screen a K. marxianus A T C C 12424 genomic library established in the shuttle vector YEp351 [10,11]. Several different plasmids were isolated and used to transform a leu2 derivative of the S. cerevisiae ADH-null strain 302.21 (MATa, his4, trpl, leu2::TRP1, adhl, adh2, adh3). This strain is unable to perform alcoholic fermentation and metabolizes glucose only via respiration [12]. Plasmid p G I K 10.9 gave transformants able to grow on plates containing glucose and the respiratory inhibitor antimycin A, demonstrating that alcoholic fermentation was restored. Moreover, these transformants lost the ability to grow on a medium containing the suicide substrate allyl
100 i0 30 50 ~GTATCTTCATCTATGGTATACCTTTTTTTGCCACTGG~CATGTATTATTATTACTAT 70 90 ii0 TGT~TTGT~TTGTTATTGTTTTTAAATCCCC~GCAC~CTTAAATT~GT~GTGGT 130 150 170 T~TCTACATTTAI~CAAAA.TCATACAC~CAC~TGGCTATTCCAGAAACTCAAAAG M A I P E T Q K 190 210 230 GGTGTTATCTTCTACG~CACGGTGGTGAGTTGC~TAC~GGACATTCCAGTTCCAAAG G V I F Y E H G G E L Q Y K D I P V P K 250 270 290 CCAAAGCCAAATG~CTTTTGATC~CGTT~GTACTCTGGTGTGTGTCACACCGATTTG P K P N E L L I N V K Y S G V C H T D L 310 330 350 CACGCATGGC~GGTGACTGGCCATTGGACACC~GTTGCCATTGGTCGGTGGTCACG~ H A W Q G D W P L D T K L P L V G G H E 370 390 410 GGTGCTGGTATTGTTGTTGCCATGGGTGAG~CGTTACTGGGTGGGAAATCGGTGACTAT G A G I V V A M G E N V T G W E I G D Y 430 450 470 GCTGGTATC~GTGGTTG~CGGTTCCTGTATGTCTTGTGAGGAGTGTGAGTTGTCG~C A G I K W L N G S C M S C E E C E L S N 490 510 530 G~CCAAACTGTCCA~GGCCGACTTGTCTGGTTACACTCACGACGGTTCTTTCC~C~ E P N C P K A D L S G Y T H D G S F Q Q 550 57O 590 TACGCTACCGCTGACGCTGTGCAGGCTGCCAG~TTCCAAAG~CGTCGACTTGGCCGAG Y A T A D A V Q A A R I P K N V D L A E 610 630 650 GTTGCCCC~TCTTGTGTGCCGGTGTTACCGTGTAC~GGCTTTG~GTCTGCCCACATC V A P I L C A G V T V Y K A L K S A H I 670 690 710 ~GGCTGGTGACTGGGTCGCCATCTCCGGTGCATGTGGTGGTCTAGGTTCCTTGGCCATC K A G D W V A I S G A C G G L G S L A I 730 750 770 C~TACGCC~GGCTATGGGTTACAGAGTGCTAGGTATCGATGCTGGTGACGAAAAGGCC Q Y A K A M G Y R V L G I D A G D E K A 790 810 830 AAATTGTTC~GG~TTGGGCGGTG~TACTTCATCGACTTCACC~GACC~GGACATG K L F K E L G G E Y F I D F T K T K D M 850 87O 890 GTAGCAG~GTCATTGAGGCCACC~TGGTGTGGCCCACGCTGTCATT~CGTGTCTGTG V A E V I E A T N G V A H A V I N V S V 910 930 950 TCCG~GCCGCCATCTCTACCTCTGTCTTGTACACCAGATCAAACGGTACCGTCGTCTTG S E A A I S T S V L Y T R S N G T V V L 970 990 I010 GTCGGTTTGCCAAGAGACGCCC~TGT~GTCTGATGTCTTC~CC~GTCGTC~GTCC V G L P R D A Q C K S D V F N Q V V K S 1030 1050 1070 ATCTCCATTGTTGGTTCTTACGTTGGT~CAGAGCAGACACCAGAG~GCCCTAGACTTC I S I V G S Y V G N R A D T R E A L D F 1090 Iii0 1130 TTCTCCAGAGGTTTGGTC~GGCTCC~TT~GATTCTCGGCTTGTCTG~TTGGCATCC F S R G L V K A P I K I L G L S E L A S 1150 1170 1190 GTTTACGAC~GATGGTCAAGGGCCA~TCGTTGGTAG~TTGTCGTTGACACTTCCAAA V Y D K M V K G Q I V G R I V V D T S K 1210 1230 1250 TAAACAA~GGACCCTTTTGATATGGT~TA~TTAACT~CTAAACTTTTTATGACTTT
TABLE I
Amino acid sequence identity and similarity of K. marxianus ADH with S. cerevisiae [4,14,15], K. lactis [6,7,8] and Schizosaceharomyces pombe [16] ADHs The algorithm used was the GAP program [17]. Mitochondrial ADHs were considered without their presequence. Identity
Similarity
(%)
(%)
S. cerevisiae
ADH I ADH II ADH III
79.3 79.3 77.9
87.6 87.1 85.9
K. lactis
ADH ADH ADH ADH
85.9 86.8 78.7 76.4
91.7 91.4 87.6 84.4
Schizosaccharomyces pombe
ADH I
49.7
67.2
I II III IV
electrophoresis and compared with the A D H produced in S. cerevisiae strain 302.21 [pGIK 10.9] (Fig. 2). The K marxianus A D H co-migrates with the major isozyme present in /¢ marxianus growing on non-fermentable carbon sources. Its activity, though it remains the major one, decreases when cells are grown on glucose. Glucose seems to be responsible for this decrease, since cells grown in 2% ethanol/7% glucose display the same isozyme profile as in 7% glucose. This behaviour is similar to that of the glucose-repressible S. cerevisiae A D H II isozyme [2] but is different from that
o
c5
o~,
K.m. 10gg
l~g
I
]
10gg 50~g 10~g 50~g
10gg 10~g 10gg 10~g
I I
I 2%E 2%E 2%P 2%Gly +7%G
Fig. 1. Nucleotide and deduced amino acid sequence of K ma~ianus ADHgene.
alcohol, oxidized by A D H to the toxic compound acrolein [13]. A sequencing primer was designed by alignment of S. cerevisiae and K. lactis ADH1 open reading frame 5' region [6,14]. The entire sequence of the gene was established on both strand (Fig. 1). The predicted protein is 348-amino acid long and should be localised in the cytoplasm as it does not possess the amino terminal extension of mitochondrion-targeted ADHs [8]. Percentages of similarity and identity with other yeast ADHs are given in Table I. The K. marxianus A D H displays the highest similarity with the K. lactis A D H I and A D H II but is not more closely related to either of these isozymes. The A D H isozymes produced by K. marxianus on different carbon sources were revealed by native gel
I 2%G
7%G
Fig. 2. Native PAGE analysis of ADH isozymes from K. marxianus strain ATCC 12424 (noted K.m.) and S. cerevisiae strain 302.21 (noted S.c.) transformed or not by pGIK 10.9 grown on different carbon sources (2%G: 2% glucose; 7%G: 7% glucose; 2%E/7%G: 2% ethanol+7% glucose; 2%E: 2% ethanol; 2%P: 2% pyruvate; 2%Gly: 2% glycerol). Soluble extracts were run on 7% acrylamide gel and stained for ADH activity according to Lutstorf and Megnet [13]. Amounts of proteins are given on the top of the lanes.
101 of the two K. lactis cytoplasmic ADHs which are both semi-constitutively expressed and glucose-induced [9]. The molecular identity between the bands comigrating must first be established before drawing conclusions about the physiological role of the K. rnarxianus ADH described in this report. It can be speculated that its presence at high glucose concentrations would explain the ability of K. marxianus to simultaneously metabolize glucose and ethanol. Further studies will determine whether glucose repression takes place at the transcriptionnal level as in S. cerevisiae A D H 2 [2]. S. cerevisiae A D H 1 gene and strain 302.21 were a kind gift from Dr. Elton T. Young. J-M Ladri~re holds an IRSIA bursary. References 1 Denis, C.L., Ferguson, J. and Young, E.T. (1983) J. Biol. Chem. 258, 1165-1171. 2 Denis, C.L., Ciriacy, M. and Young, E.T. (1981) J. Mol. Biol. 148, 355 -368. 3 W6hrer, W., Forstenlehner, L. and R6hr, M. (1981) in Current developments in yeast research (Stewart, G.G. and Russel, I., eds.), pp. 405-410, Pergamon press, Toronto.
4 Young, E.T. and Pilgrim, D. (1985) Mol. Cell. Biol. 5, 3024-3034. 5 Williamson, V.M. and Paquin, C.E. (•987) Mol. Gen. Genet. 209, 374-381. 6 Saliola, M., Shuster, J.R. and Falcone, C. (1990) Yeast 6, 193-204. 7 Shain, D.H., Salvadore, C. and Denis, C.L. (1992) Mol. Gen. Genet. 232, 479-488. 8 Saliola, M., Gonnella, R., Mazzoni, C. and Falcone, C. (1991) Yeast 7, 391-400. 9 Mazzoni, C., Saliola, M. and Falcone, C. (1992) Mol. Microbiol. 6, 2279-2286. 10 Myers, A.M., Tzagoloff, A., Kinney, D.M. and Lusty, C.J. (1986) Gene 45, 299-310. 11 Laloux, O., Cassart, J.P., Delcour, J., Van Beeumen, J. and Vandenhaute, J. (1991) FEBS Lett. 289, 64-68. 12 Williamson, V.M., Bennetzen, J.L., Young, E.T., Nasmyth, K. and Hall, B.D. (1980) Nature 283, 214-216. 13 Lutstorf, U. and Megnet, R. (1968) Archives Biochem. Biophys. 126, 933-944. 14 Bennetzen, J.L. and Hall, B.D. (•982) J. Biol. Chem. 257, 30183025. 15 Russell, D.W., Smith, M., Williamson, V.M. and Young, E.T. (1983) J. Biol. Chem. 258, 2674-2682. 16 Russel, P.R. and Hall, B.D. (1983) J. Biol. Chem. 258, 143-149. 17 Devereux, J., Haeberli, P. and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395.