ADH2 REGULATION IN THE YEAST 
Saccharomyces cerevisiae 
 
BY 
 
MOJABATHO PORTIA KHOBOKO 
 
 
B.SC. HONS. (UFS) 
 
 
SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS  
FOR THE DEGREE 
 
 
MAGISTER SCIENTIAE  
 
 
IN THE FACULTY OF NATURAL AND AGRICULTURAL SCIENCES 
DEPARTMENT OF MICROBIAL, BIOCHEMICAL AND FOOD BIOTECHNOLOGY 
UNIVERSITY OF THE FREE STATE 
BLOEMFONTEIN 
SOUTH AFRICA 
 
 
MAY 2004 
 
STUDY LEADER: DR. J. ALBERTYN 
CO-STUDY LEADER: PROF. J.C. DU PREEZ 
 
ACKNOWLEDGEMENTS 
 
I wish to acknowledge the following people and institutions: 
 
• Dr Jacobus Albertyn. For his support, guidance, supervision through out 
my postgraduate studies and for giving me the opportunity to develop as a 
young scientist. 
 
• Prof James du Preez. For his advices and time. 
 
• The Molecular lab members. For their assistance and advice in molecular 
biology and more especially to Puleng and Evodia for the courage and 
support that they gave me and Olga for all the help and contribution in this 
study. 
 
• To all the lecturers and post graduate students in the Department of 
Microbial, Biochemical and Food Biotechnology for their assistance, 
support and friendship.  
 
• The NRF. For their financial Assistance. 
 
• The financial assistance of the Department of Labour (DoL) towards this 
research is hereby acknowledged. Opinions expressed and conclusions 
arrived at, are those of the author and are not necessarily to be attributed 
to the DoL. 
 
• My family. For their love, support and for allowing me to reach my goals, 
especially my mother (Jeanette) and brother (Justice) for not loosing their 
hope in me. To my better half (Lebogang) for being the pillar of my 
strength when times were tough. 
 
• To The Lord My Saviour. For blessing my life with the opportunities of 
becoming a better person. 
 ii 
 
 
 
 
 
 
 
 
 
All that a man achieves and all 
that he fails to achieve is the 
direct result of his thought… 
 James Allen  
 iii 
 
 
TABLE OF CONTENTS 
 
 
CHAPTER 1-INTRODUCTION 1 
 
CHAPTER 2-LITERATURE REVIEW 7 
1. INTRODUCTION 7 
2. ETHANOL METABOLISM 10 
3. ALCOHOL DEHYDROGENASES (ADH) OF S. CEREVISIAE 11 
3.1 ALCOHOL DEHYDROGENASE I  12 
3.2 ALCOHOL DEHYDROGENASE III 13 
3.3 ALCOHOL DEHYDROGENASE IV 15 
3.4 ALCOHOL DEHYDROGENASE V 15 
3.5 GLUTATHIONE-DEPENDENT FORMALDEHYDE DEHYDROGENASE (SFA1) 16 
3.6 ALCOHOL DEHYDROGENASE VI 17 
3.7 ALCOHOL DEHYDROGENASE VII 18 
4. ALCOHOL DEHYDROGENASE II AND ITS REGULATION 18 
4.1.CCR4-NOT 19 
4.2. ADR6 22 
4.3. ADR7, 8 AND 9 23 
4.4. ADR1 24 
4.5 CAT8 26 
4.6 CYCLIC AMP-DEPENDENT PROTEIN KINASE (CAPK)  28 
4.7 SNF1/CCR1/CAT1 30 
4.8 CAMP-INDEPENDENT PROTEIN KINASE (SCH9) 32 
5. ALCOHOL DEHYDROGENASE II REGULATION: AN OVERVIEW 33 
6. THE USE OF THE ADH2 PROMOTER 35 
7. REFERENCES 37 
 
CHAPTER3-THE CONSTRUCTION OF THE EXPRESSION CASSETTE 52 
1. ABSTRACT 51 
 iv 
2. INTRODUCTION 52 
3. MATERIALS AND METHODS  54 
3.1. STRAINS USED 54 
3.2. ISOLATION OF ESCHERICHIA COLI GENOMIC DNA 54 
3.3. ISOLATION OF S. CEREVISIAE GENOMIC DNA 54 
3.4. AMPLIFICATION OF THE DOWNSTREAM AND UPSTREAM REGION (FLANKING 
REGIONS) OF ADH2, LACZ, UPSTREAM+LACZ, DOWNSTREAM+LACZ AND 
THE EXPRESSION CASSETTE 55 
3.5. CLONING OF PCR PRODUCTS, CONSTRUCTION OF THE EXPRESSION 
CONSTRUCT AND TRANSFORMATION 57 
3.6. DETECTION OF Β-GALACTOSIDASE ACTIVITY FROM POSITIVE COLONIES 60 
3.7. β-GALACTOSIDASE ASSAY 60 
4. RESULTS AND DISCUSSIONS 62 
4.1. CONSTRUCTION OF THE EXPRESSION CASSETTE 62 
4.2. AMPLIFICATION OF THE ADH2 FLANKING REGIONS AND LACZ GENE 62 
4.3. CONSTRUCTION OF THE EXPRESSION CASSETTE USING YIP356R 66 
4.4. CONFIRMATION OF THE EXPRESSION CASSETTE 73 
4.5. PRELIMINARY Β-GALACTOSIDASE ACTIVITY ASSAY FROM THE 
TRANSFORMED YEAST 76 
5. REFERENCES 79 
 
CHAPTER 4-INVESTIGATION OF β-GALACTOSIDASE PRODUCTION UNDER THE 
CONTROL OF ADH2  81 
 
1. ABSTRACT 81 
2. INTRODUCTION 81 
3. MATERIALS AND METHODS 84 
3.1 YEAST STRAIN 84 
3.2 INOCULUM AND MEDIUM COMPOSITION 84 
3.3. ANALYTICAL METHODS 85 
4. RESULTS  87 
4.1 GROWTH PROFILE DURING GROWTH ON  GLUCOSE AND ETHANOL AS 
RESPECTIVE CARBON SOURCES  87 
4.1.1 GROWTH UNDER REPRESSING CONDITIONS ON GLUCOSE 88 
4.1.2 GROWTH UNDER DEREPRESSING CONDITIONS ON ETHANOL  90 
 v 
5. DISCUSSION 98 
6. REFERENCES 101 
 
CHAPTER 5-GENERAL CONCLUSIONS 105 
REFERENCES 107 
 
CHAPTER 6-SUMMARY 108 
CHAPTER 7-OPSOMMING 110 
 
 
 
 
 vi 
CHAPTER 1 
 
CHAPTER 1 
 
INTRODUCTION 
 
 
ADH2 belong to a large family of enzymes that catalyze the reversible transfer of 
hydrogen to the carbonyl group of aldehydes or related compounds. ADH2 
encode for an enzyme, which is essential during ethanol utilization by 
Saccharomyces cerevisiae, this enzyme catalyses the initial step during ethanol 
assimilation by reducing ethanol into acetaldehyde. The alcohol dehydrogenase 
(ADH) systems of various organisms e.g. yeast; fungi and human have been the 
subject of thorough analyses on the molecular structure and the mode of 
catalyses. The S. cerevisiae system is of exceptional attractiveness because of 
its genetic accessibility, available molecular techniques and ease in cultivation. 
The regulatory system governing the expression of ADH2 has been exploited as 
a model system for the analysis of regulation of glucose sensitive genes.  
 
The synthesis of many enzymes in S. cerevisiae is repressed when the cells are 
grown in a glucose based medium and derepressed when the cells are grown in 
medium containing a nonfermentable carbon source such as ethanol (Polakis 
and Bartley, 1965). The ADH2 gene of S. cerevisiae is not expressed when the 
cells are grown in medium containing glucose as a carbon source and 
derepressed over a 100-fold when the cells are shifted to a nonfermentable 
carbon source (Ciriacy, 1975). The absence of a fermentable carbon source 
signals yeast to activate the expression of genes controlling aerobic metabolism 
of alternative carbon sources.  
 
The repression ADH2 expression is not mediated by a repressor but primarily 
due to the absence of positive activation (Shuster et al., 1986). Numerous 
transcription factors allow the cell to generate energy and metabolites from non-
glucose carbon sources such as ethanol. Adr1p and Cat8p are the two 
 1 
CHAPTER 1 
transcription activators required for the activation of the expression of ADH2 
(Walther and Schüller, 2001), these transcription factors are not active during 
growth on glucose. Two cis–acting upstream activating sequences (UASs) have 
been described for the ADH2 gene. Both sequences confer glucose-regulated 
transcriptional regulation on this gene (Beier and Young, 1982; Shuster et al., 
1986; Beier et al., 1985; Yu et al., 1989). Adr1p binds to the UAS1 (Cook et al., 
1994) in the ADH2 promoter and Cat8p (Hedges et al., 1995) binds to the UAS2.  
Both Adr1p and Cat8p are active in the presence of Snf1p, a yeast homolog of 
AMP-activated protein kinase (Dombek et al., 1999). Glucose exerts its gene 
repression on ADH2 through the regulating the Snf1p activity. Snf1p is activated 
when the glucose level is low or absent in the medium (Jiang and Carlson, 1996). 
Snf1p regulates the expression of CAT8 and the transcription activation function 
of its encoded enzyme (Cat8p) and Snf1p regulates chromatin binding by Adr1p 
(Young et al., 2002). 
 
Saccharomyces cerevisiae has been used in the expression of heterologous 
genes. Regulated promoters are more frequently used because their activity can 
be controlled during the fermentation process. Due to being under glucose 
repression, the ADH2 promoter has been employed in regulated overexpression 
of heterologous genes in S. cerevisiae (Price et al., 1990). For example, 
TBV25H, a malaria transmission blocking vaccine candidate was expressed from 
S. cerevisiae under the control of this promoter (Noronha et al., 1998).  
 
The expression of ADH2 was recently found to be negatively affected by ethanol 
(du Preez et al., 2001), these results were unexpected as ethanol is the substrate 
of Adh2p and this enzyme has been classified as the major alcohol 
dehydrogenase isozymes involved in the conversion of ethanol to acetaldehyde 
during ethanol assimilation. It would therefore be expected that the expression of 
ADH2 would be induced by ethanol or if not induced, at least not be repressed by 
ethanol. Ethanol repression might thus have negative implications during the 
expression of heterologous genes using the ADH2 promoter.  
 2 
CHAPTER 1 
 
Because these results were obtained using an episomal multicopy plasmid with a 
promoter terminator expression cassette derived from the S. cerevisiae ADH2 
gene, these findings may not apply to the natural ADH2 promoter. Due to the 
extensive sequence identity between ADH1 and ADH2 (95% identity) it Is difficult 
to directly measure the ADH2 mRNA through northern blot analysis without cross 
binding of an ADH2 probe on ADH1 mRNA (Sierkstra et al., 1992). 
 
It therefore became the aim of this study to determine the effect of ethanol on the 
genomic copy of ADH2 in S. cerevisiae. E. coli LacZ, encoding β-galactosidase, 
was used as a reporter gene to construct an expression cassette for the study of 
the expression profile of ADH2 under different growth conditions.  The effect of 
ethanol was determined by cultivating S. cerevisiae containing an integrated 
copy of the expression cassette into three different ethanol concentrations: 5, 20 
and 30 g ethanol l-1. Glucose repression was studied through cultivation on 2 g 
glucose l-1. 
 3 
CHAPTER 1 
REFERENCES 
 
Beier D.R. and E.T. Young 1982. Characterisation of a regulatory region 
upstream of the ADR2 locus of Saccharomyces cerevisiae. Nature 
300:724-728 
 
Beier D.R., Sledziewski A. and Young E.T. 1985. Deletion analysis identifies a 
region, upstream of the ADH2 gene of Saccharomyces cerevisiae, which 
is required for ADR1-mediated derepression. Mol. Cell. Biol. 5:1743-1749 
 
Ciriacy M. 1975. Genetics of alcohol dehydrogenases in Saccharomyces 
cerevisiae  II. Two loci controlling synthesis of the glucose-repressible 
ADHII. Mol. Gen. Genet. 138:157-154 
 
Cook W.J., Chase D., Audino D.C. and Denis C.L. 1994.  Dissection of the 
ADR1 protein reveals multiple, functionally redundant activation domains 
interspersed with inhibitory regions: evidence for a repressor binding to the 
ADR1c region. Mol. Cell. Biol. 14:629-640. 
 
Dombek K.M., Voronkova V., Roney A. and Young E.T. 1999. Functional 
analysis of the yeast GLC-7 binding protein Reg1 identifies a protein 
phosphatase type 1-binding motif as essential for repression of ADH2 
expression. Mol. Cell. Biol. 19:6029-6040 
 
du Preez J.C., Maré J.E., Albertyn J. and Kilian S.G.  2001. Transcriptional 
repression of ADH2-regulated β-xylanase production by ethanol in 
recombinant strains of Saccharomyces cerevisiae. FEMS Yeast Res. 
1:233-40. 
 
 4 
CHAPTER 1 
Hedges D., Proft M. and Entian K.D. 1995. CAT8, a new zinc cluster-encoding 
gene necessary for derepression of gluconeogenic enzymes in the yeast 
Saccharomyces cerevisiae. Mol Cell Biol. 15: 1915-22. 
 
Jiang R. and Carlson M. 1996. Glucose regulates protein interaction within the 
yeast SNF1 protein kinase complex. Genes Dev. 10: 3105-3115 
 
Noronha S.B., Kaslow D.C. and Shiloach J. 1998. Transition phase in the 
production of recombinant proteins in yeast under the ADH2 promoter: an 
important step for reproducible manufacturing of malaria transmission 
blocking vaccine candidate.  J. Industrial Micro. Biotech. 20: 192-199 
 
Polakis E.S. and Bartley W. 1965. Changes in enzyme activities of 
Saccharomyces cerevisiae during aerobic growth on different carbon 
sources. Biochem. J. 97: 248-297 
 
Price V.L., Taylor W.E., Clevenger W., Worthingtons M. and Young E.T. 
1990. Expression of heterologous proteins in Saccharomyces cerevisiae 
using ADH2 promoter. Meth. Enzymol. 185: 308-318 
 
Sierkstra L.N., Verbakel J.M.A. and Verrips C.T. 1992. Analysis of transcription 
and translation of glycolytic enzymes in glucose limited cultures of 
Saccharomyces cerevisiae. J. Gen. Microbiol. 138: 2559-2566 
 
Walther K. and Schüller H.J. 2001.Adr1 and Cat8 synergistically activate the 
glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast 
Saccharomyces cerevisiae. Microbiol. 147: 2037-2044. 
 
Shuster J.J., Yu D., Cox R.V.L., Smith M. and Young E.T. 1986. ADR1-
mediated regulation of ADH2 requires an inverted repeat sequence. Mol. 
Cell. Biol. 6: 1894-1902 
 5 
CHAPTER 1 
 
Young E.T., Kacherovsky N. and Van Riper K. 2002. Snf1 protein kinase 
regulates Adr1 binding to chromatin but not transcription activation. J. Biol. 
Chem. 277: 38095-38103. 
 
Yu J., Donoviel M.S. and Young E.T. 1989. Adjacent activating sequence 
elements synergistically regulate transcription of ADH2 in Saccharomyces 
cerevisiae. Mol. Cell. Biol 9: 34-42 
 
 
 6 
CHAPTER 2 
 
 
CHAPTER 2 
 
LITERATURE REVIEW 
 
 
1. INTRODUCTION 
 
Saccharomyces cerevisiae (bakers’ yeast) is widely used in industry for biomass 
or heterologous protein production. Saccharomyces cerevisiae belongs to a 
group of facultative anaerobic yeasts. These microorganisms will ferment hexose 
sugars, like glucose and fructose, under both aerobic and anaerobic growth 
conditions. S. cerevisiae displays the Crabtree effect where, even when oxygen 
may be present, NADH generated during glycolysis is mainly oxidized by 
fermentation rather than by respiration (Walker, 1998). The Crabtree effect 
probably results from a multiplicity of factors, including the mode of sugar 
transport and the regulation of enzyme activities involved in respiration and 
alcoholic fermentation. In aerobic batch cultures of this yeast typically about 70% 
of the available glucose is fermented to ethanol and CO2, 20% is incorporated 
into biomass, 8% is used in glycerol production and only 2% will yield CO2 and 
H2O via the oxidative phosphorylation inside the mitochondria (Hohmann and 
Mager, 1997). Accordingly glycolytic flux is high and O2 consumption is low (Ruis 
and Schüller, 1995; Mager and de Kruijff 1995).  
 
Saccharomyces sp. will utilize a wide variety of compounds as carbon and 
energy sources. Glucose and fructose enter immediately into the glycolytic 
pathway where ATP is obtained from substrate phosphorylation and sugars are 
converted into pyruvate and then ethanol and CO2. As stated above, although 
only a small fraction of the fermentable carbon source is initially completely 
metabolized and much less ATP is produced than during respiratory growth, 
yeasts prefer fermentative growth. This may seem wasteful; however, since the 
ethanol as well as the acetate produced during fermentation will be metabolized 
 7 
CHAPTER 2 
 
in the post-diauxic growth phase, as soon as the fermentable sugar is exhausted, 
nearly all of the available carbon source will be used eventually. In addition, and 
perhaps more importantly, the production of high ethanol concentrations inhibits 
the growth of most other microorganisms allowing S. cerevisiae to eventually 
dominate in spontaneous fermentation. This success in fermentative growth and 
utilization of the produced ethanol is due to the alcohol dehydrogenase (ADH) 
isozymes available in the yeast. 
 
Alcohol dehydrogenase isozymes of S. cerevisiae are involved in the 
fermentative growth of the yeast and ethanol assimilation. Adh1p and Adh2p 
isozymes (major isozymes) function in ethanol production during fermentative 
growth and ethanol assimilation respectively. There are however, other isozymes 
which have been identified to function as Adh1p, namely Adh3p or as Adh2p, 
namely Adh5p. The function of Adh1p during fermentative glucose assimilation is 
to reduce acetaldehyde into ethanol and this reaction is the final step in the 
production of ethanol during fermentative growth. The initial reaction during 
ethanol assimilation is catalyzed by Adh2p, encoded by a glucose repressible 
ADH2. Nonfermentable carbon sources such as ethanol and acetate are 
metabolized via respiration in the TCA cycle with ATP being produced by 
oxidative phosphorylation (Finley and Chau, 1991). Adh2p oxidises ethanol into 
acetaldehyde which then is converted into acetate. This acetate can either be 
used in the TCA cycle or enter the gluconeogenic cycle. 
 
Yeast batch cultures grown on glucose show several well-defined growth phases 
due to multiple levels of metabolic regulation by the available carbon source. In 
the first phase characterized by rapid growth, glucose is fermented with 
concomitant repression of genes required for respiratory growth. When glucose is 
exhausted the culture enters a short adaptive lag-phase known as diauxic shift. 
During this phase the glucose-repressed genes become derepressed and the 
culture adopts its metabolism for the subsequent utilization of ethanol and other 
by-products of fermentation (Maeda et al., 1994). The promoters of these glucose 
 8 
CHAPTER 2 
 
repressible genes e.g. ADH2 are widely employed in the expression of 
heterologous proteins. 
 
 
 
FIGURE 1. Genetics of oxidative and gluconeogenic metabolism in the yeast S. 
cerevisiae. Structural genes known to be transcriptionally regulated 
when glucose is used as sole carbon source are depicted in bold 
(Schüller, 2003). 
 9 
CHAPTER 2 
 
2. ETHANOL METABOLISM 
 
Saccharomyces cerevisiae utilizes ethanol that is produced during glucose 
assimilation as a carbon source when glucose is depleted. Availability of high 
glucose concentrations causes this production of ethanol under aerobic growth 
conditions. Fermentation occurs even when oxygen is available due to crab tree 
effect. The production of ethanol by S. cerevisiae during fermentative growth 
involves a two-step reaction after the ultimate product of glycolysis (pyruvate) has 
been formed. During fermentative sugar metabolism, pyruvate is decarboxylated 
to acetaldehyde by pyruvate decarboxylase and subsequently reduced to ethanol 
by an alcohol dehydrogenase enzyme. The initial step in fermentation is 
catalyzed by pyruvate decarboxylase encoded by PDC1; the expression of this 
gene is induced by glucose (Butler and MacConnell, 1988). Pdc1p 
decarboxylates pyruvate into acetaldehyde, the penultimate product of 
fermentation. Adh1p catalyzes the second and final step where ethanol is 
produced; this enzyme reduces acetaldehyde into ethanol (van den Berg et al., 
1998) with ADH1 expression being induced by glucose. 
 
The ethanol that is produced during fermentation by the yeast is utilized after the 
depletion of glucose. During ethanol assimilation, ethanol is converted to acetyl-
CoA. Three enzymes are involved in this pathway; alcohol dehydrogenase 
(ADH), aldehyde dehydrogenase (ALD) and acetyl-CoA synthatase (ACS) are 
required for the production of acetyl-CoA, which is subsequently used for 
mitochondrial oxidation and cytoplasmic gluconeogenesis. Adh2p, which oxidises 
ethanol into acetaldehyde, catalyzes the initial step, Ald6 catalyzes the second 
step by converting acetaldehyde into acetate and the final step is catalyzed by 
Acs1p, which then converts acetate into acetyl-CoA (Fig. 1). The expression of 
these three genes is under glucose regulation. In addition, during ethanol 
assimilation the expression of these three genes is dependent on both Cat8p and 
Adr1p transcription activators (Walther and Schüller, 2001) (Fig. 2).  
 10 
CHAPTER 2 
 
 
 
FIGURE 2.  Regulatory pathway leading to transcriptional co-activation of ADH2, 
ALD6 and ACS1 by Cat8 and Adr1(Walther and Schüller, 2001). 
 
ADH2 has been known as a major isozyme involved in ethanol assimilation, 
however van den Berg et al. (1998) indicated that ADH2 expression is not 
derepressed on ethanol growth instead ethanol appears to repress ADH2 
expression and this expression is derepressed by acetate. Du Preez et al. (2001) 
have also observed that a high ethanol concentration represses the expression of 
this gene. 
 
3. ALCOHOL DEHYDROGENASES (ADH) OF S. CEREVISIAE 
 
When S. cerevisiae is grown on glucose almost all of the sugar is fermentatively 
used, regardless of the availability of oxygen, and a specific ADH isozyme serves 
to regenerate the glycolytic NAD+ by the reduction of acetaldehyde to ethanol. 
 11 
CHAPTER 2 
 
After depletion of the fermentable sugar, rapid oxidation of the accumulated 
ethanol occurs, again by the action of a specific ADH isozyme. The alcohol 
dehydrogenase isozymes of S. cerevisiae are responsible for the interconversion 
of acetaldehyde and ethanol. This reaction is the final step during alcohol 
fermentation in yeast and the initial step in the metabolism of ethanol in a wide 
variety of organisms (Walker, 1998). 
 
When grown on fermentable carbon sources such as glucose, yeast expresses 
an isozyme (Adh1p) which preferentially catalyses the conversion of the 
penultimate intermediate of the fermentative pathway, acetaldehyde to ethanol. 
ADH1 was first isolated and characterized by Racker (1955) and studied by 
Lutstorf and Megnet (1968). Adh1p (cytoplasmic isozyme), Adh3p (mitochondrial 
isozyme) and Adh4p (cytoplasmic isozyme) convert acetaldehyde into ethanol. 
ADH5 encodes for Adh5p, which is involved in the oxidation of ethanol to 
acetaldehyde like Adh2p. Sfa1p is a long chain alcohol dehydrogenase and it 
oxidizes ethanol and long chain alcohols. Adh6p and Adh7p are the only alcohol 
dehydrogenases not involved in interconversion between acetaldehyde and 
ethanol. 
 
3.1. ALCOHOL DEHYDROGENASE I  
 
The Adh1p isozyme is the classical fermentative enzyme (Racker, 1953), which 
serves to generate the glycol NAD+ by the reduction of acetaldehyde to ethanol. 
Adh1p is very similar to Adh2p (approximately 95% identity on nucleotide level).  
The expression of ADH1 was initially thought to be constitutive, but later it was 
found to be induced five to 10-fold by glucose or other fermentative carbon 
source and has also been shown to be repressed by growth on non-fermentative 
carbon sources (Denis et al., 1983). This change in expression is correlated with 
an increase in ADH1 mRNA level. The change in expression may be due to the 
negative role of Snf1p in the expression of this gene when glucose is depleted 
 12 
CHAPTER 2 
 
(Young et al., 2003). This was confirmed by the presence of higher levels of 
ADH1 mRNA in the snf1 mutant than in the wild type. 
 
The molecular basis of ADH1 expression and its regulation is not precisely 
known. High-level expression depends on a conserved sequence, designated 
UASRPG, found at various glycolytic genes and ribosomal protein genes. Deletion 
of this element reduces ADH1 expression 20-fold without changing its regulatory 
pattern (Tornow and Santangelo, 1990). The UASRPG box at position -664 binds 
the general transcription activator Rap1; this ternary complex is crucial for ADH1 
transcription activation (Tornow and Santangelo, 1990; Tornow et al., 1993).  
 
Adh1p is the second enzyme able to detoxify cellular formaldehyde, apparently 
via reduction to methanol and its action is weaker than that of Sfa1p. Due to its 
ability in mediating high expression levels, the ADH1 promoter has been used 
widely in artificial overproduction of proteins in S. cerevisiae (Ammerer, 1983). 
 
3.2. ALCOHOL DEHYDROGENASE III 
 
ADH3 is also known as the mitochondrial alcohol dehydrogenase, due to its 
activity being associated with this organelle. The Adh3p protein contains an N-
terminal extension of twenty-eight residues that is required for its localization to 
the mitochondrial matrix, and this extension is cleaved away during the 
translocation process. Adh3p is among the isozymes that belong to a larger 
group of alcohol dehydrogenase that all contain coordinately bound zinc near its 
catalytic centers (Ciriacy, 1997) and it is involved in the reduction of 
acetaldehyde to ethanol (Fig. 3).  
 
ADH3 is induced by ethanol and repressed by glucose. ADH3 is involved in the 
ethanol-acetaldehyde shuttle, consisting of mitochondrial and cytoplasmic 
isozymes of alcohol dehydrogenase. Since ethanol and acetaldehyde can diffuse 
 13 
CHAPTER 2 
 
freely across biological membranes, the net results of the ethanol-acetaldehyde 
shuttle would be the exchange of NADH and H+ for NAD+ (Fig.3).  
 
 
 
 
FIGURE 3. A scheme representing the respiratory chain of S. cerevisiae. Adh, 
alcohol dehydrogenase; bc1, bc1 complex; cytochrome c oxidase; 
Gpd, soluble glycerol-3-phosphate dehydrogenase; Gut2, 
membrane-bound glycerol-3-phosphate dehydrogenase; Nde, 
external NADH dehydrogenase; Ndi1, internal NADH 
dehydrogenase; Q:ubiquinone. Note that the ethanol¯acetaldehyde 
shuttle is reversible in principle (Bakker et al., 2000).  
 
This shuttle restores the mitochondrial redox balance under anaerobic conditions, 
where the NADH + H+ must be transported to the cytosol, where it can be 
oxidized by the formation of glycerol or be oxidized by Nde1p and Nde2p (Bakker 
et al., 2000). It can also compensate for Ndi1p by reoxidising the mitochondrial 
NADH +H+. 
 
 14 
CHAPTER 2 
 
3.3. ALCOHOL DEHYDROGENASE IV 
 
The ADH4 gene, encoding ADH activity, is not homologous to the previously 
mentioned alcohol dehydrogenase genes. It is unrelated to ADH1, ADH2, or 
ADH3 and its predicted amino acid sequence is most similar to the ADH2 gene of 
the fermentative bacterium Zymomonas mobilis. It is activated by zinc and not by 
ferrous ions, like structurally similar alcohol dehydrogenase from Zymomonas 
mobilis (Drweke and Ciriacy, 1988). Adh4p reduces acetaldehyde to ethanol and 
is transcriptionally repressed under glucose and ethanol growth conditions (van 
den Berg et al., 1998). ADH4 expression is activated by Zap1p (zinc-responsive 
transcriptional activator) that regulates the genes involved in zinc uptake (Yuan, 
2000). Transcription of ADH4 is induced in low-zinc media and this transcriptional 
induction requires Zap1p. This induction suggests that Adh4p functions under 
these conditions (Yuan, 2000). The main purpose for the ADH4 induction in low-
zinc level is to compensate for a decrease zinc-dependent alcohol 
dehydrogenase activity; because the genes encoding zinc-dependent alcohol 
dehydrogenases (ADH1, ADH2, ADH3 and ADH5) are repressed under zinc 
limiting conditions (Lyons et al., 2000). 
 
3.4. ALCOHOL DEHYDROGENASE V 
 
Adh5p is a zinc dependent alcohol dehydrogenase similar to the other alcohol 
dehydrogenases that have been mentioned. It has a high homology on 
nucleotide level with ADH1, ADH2 and ADH3 as well as the Zymomonas mobilis 
ADH2 gene (Ciriacy, 1997). Adh5p catalyzes the reaction where alcohol is 
converted to aldehyde or ketone e.g. where ethanol is oxidized to acetaldehyde.  
The transcription of this gene is constant in both glucose and ethanol growth 
conditions (van den Berg et al., 1998). 
 
 15 
CHAPTER 2 
 
3.5. GLUTATHIONE-DEPENDENT FORMALDEHYDE DEHYDROGENASE (SFA1) 
 
A 3.7 kb DNA fragment of yeast chromosome IV has been sequenced and 
contains the SFA1 gene which, when present on a multi-copy plasmid in S. 
cerevisiae, confers hyper-resistance to formaldehyde (FA). It belongs to the 
alcohol dehydrogenase class III (glutathione-dependent formaldehyde 
dehydrogenase) (Fernadez et al., 1995). This regulated gene codes for a long 
chain alcohol dehydrogenase (glutathione-dependent formaldehyde 
dehydrogenase) of S. cerevisiae. Sfa1p oxidizes long chain alcohols and can 
oxidize formaldehyde in the presence of glutathione. Wehner et al. (1993) have 
shown that Sfa1p is non-essential, suggesting that other enzymes of general 
metabolism are sufficient to survive exposure to FA <10-3 M in this organism.  
 
The predicted protein shows strong homologies to several mammalian alcohol 
dehydrogenases and contains a sequence characteristic of binding site for NAD+. 
Interestingly the protein has higher structural homology to several ADHs of man 
and rodents than to other known ADHs of S. cerevisiae (for example homology to 
ADHI is lower than 35% (Jörnvall, 1977). SFA1 requires (GSH) glutathione and 
NAD+ for the oxidation of FA (Wehner et al., 1993), while the oxidation of long-
chain alcohols is independent of GSH and the enzyme activity improves with 
chain length. FA is not a substrate as such, but after spontaneous reaction with 
GSH to produce S-hydroxymethylglutathione (Mason and Sanders, 1986) yields 
the best substrate for the SFA1-encoded enzyme. Since S-
hydroxymethylglutathione is the substrate of the FA-degrading enzyme, the 
enzyme-catalyzed reaction is the oxidation of a terminal alcoholic hydroxyl group 
residing on a tripeptide. This might be considered an analogous configuration of 
a long-chain alcohol. SFA1 is not only inducible by FA but also by ethanol and 
methylmethanesulphonate (MMS), two structurally chemicals and unlikely 
substrates (Wehner et al., 1993). Sfa1p is active towards ethanol only at high 
substrate concentrations (Fernandez et al., 1995). SFA1 was successfully 
employed in constructing a formaldehyde resistance expression cassette. When 
 16 
CHAPTER 2 
 
this cassette is introduced into yeast strains, they can resist up to 6 mM-
formaldehyde (van den Berg and Steensma, 1997) 
 
3.6. ALCOHOL DEHYDROGENASE VI 
 
The YMR318C gene was recently characterized by Larroy et al. (2002) and due 
to its substrate specificity and sequence characteristics the gene was called 
ADH6. ADH6 is a member of the medium-chain dehydrogenases/reductases 
(MDRs). MDRs constitute a wide protein family with many different activities 
including the cinnamyl alcohol dehydrogenase to which ADH6 belongs. ADH6 
encodes for an NADP(H)-dependent alcohol dehydrogenase (Larroy et al., 2002) 
and it has been classified as a member of the cinnamyl alcohol dehydrogenase 
family. Adh6p is active towards both aromatic and aliphatic aldehyde and both 
linear and branched-chain primary alcohols as substrates.  
 
Adh6p is strongly induced in yeast when galactose is used as a sole carbon 
source as compared to when glucose is the carbon source (Larroy et al., 2003). 
The expression of this gene seems to be repressed by glycerol (Larroy et al., 
2003). The major function of Adh6p is the reduction of aldehyde rather than the 
oxidation of alcohols (Larroy et al., 2002). This enzyme is capable of significantly 
eliminating veratraldehyde in S. cerevisiae, which is toxic to the cells. Reduction 
of this aldehyde to the corresponding alcohol is part of the lignin biodegradation 
process (Reiser et al., 1994). Since veratraldehyde is a compound in the 
ligninolysis pathway, Adh6p may give the yeast the opportunity to live in 
ligninolytic environments where products derived from lignin biodegradation may 
be available. The other potential function of Adh6p may be in the biosynthesis of 
fusel alcohols during amino acid assimilation (Larroy et al., 2002). Fusel alcohols 
confer major organoleptic properties to alcohol beverages during fermentation. 
The best substrates for Adh6p are pentanal and veratraldehyde, ethanol is a very 
poor substrate of this enzyme (Larroy et al., 2003). Unlike alcohol 
 17 
CHAPTER 2 
 
dehydrogenases previously mentioned in this literature, ADH6 is not involved in 
the interconversion of ethanol and acetaldehyde. 
 
3.7. ALCOHOL DEHYDROGENASE VII 
 
YCR105W gene was also recently characterized by Larroy et al. (2003) and it 
also encodes for a NADP (H) dependent alcohol dehydrogenase. This gene, 
designated ADH7, is also a member of the MDRs superfamily. Adh7p is 64% 
identical to Adh6p and its substrate specificity is similar to that of Adh6p. Adh7p 
has the highest catalytic efficiencies for the reduction reaction rather than for the 
oxidative reactions, suggesting that the enzyme would act as an aldehyde 
reductase rather than an alcohol dehydrogenase (Larroy et al., 2003). The close 
structural and functional similarities between Adh6p and Adh7p suggest common 
physiological roles for both enzymes. Adh6p and Adh7p are the only two 
members of the cinnamyl alcohol dehydrogenase family in yeast. 
 
4. ALCOHOL DEHYDROGENASE II AND ITS REGULATION 
 
The glucose repressible alcohol dehydrogenase (ADH2) from S. cerevisiae 
functions under non-fermentable growth conditions in the utilization of ethanol as 
carbon source. Yeast grown in media containing a fermentable carbon source 
such as glucose, repress the expression of ADH2 and yeast grown on non-
fermentable carbon source such as ethanol, derepress the level of Adh2p activity 
several hundred fold (Lutstorf and Megnet, 1968). The function of Adh2p in the 
cell is to oxidize ethanol formed during fermentation to acetaldehyde, which can 
then be metabolized via the tricarboxylic acid cycle in the mitochondria and also 
serves as an intermediate in gluconeogenesis (Young and Pilgrim, 1985). ADH2 
is not induced by ethanol as sometimes reported (Ciriacy, 1997). Adh2p has 
gained a specific attraction as its formation is under the control of carbon 
catabolite repression, which is considered as a central regulatory system in the 
control of many enzymes involved in carbon metabolism. Genetic analysis of 
 18 
CHAPTER 2 
 
ADH2 regulation has identified several genes that encode trans-acting factors, 
which are required for full derepression of ADH2 (Ciriacy, 1975; Ciriacy, 1977; 
Denis, 1984; Taguchi and Young, 1987), some of these such as CCR1 (SNF1) 
(Ciriacy, 1977), CCR2, CCR3 and CCR4 also affect other glucose repressible 
genes. 
 
Both positive regulatory genes, ADR1, CCR1-3 (Ciriacy, 1975), CCR4 (Denis, 
1984), CAT8 (Walther and Schüller, 2001) and ADR6 (Taguchi and Young, 1987) 
and the negative regulatory genes ADR4 (Ciriacy, 1975), CRE1 and CRE2 
(Denis, 1984) are involved in ADH2 regulation. Each of these will be discussed 
extensively in the following sections.  
 
4.1. CCR4-NOT 
 
The CCR4-NOT complex is one of several large groups of proteins involved in 
transcription (Liu et al., 1998). This complex affects the expression of many 
genes including ADH2, which are required for the assimilation of non-fermentable 
carbon sources (Denis, 1984; Denis and Malvar, 1990). It consists of at least 2 
complexes, 1.9 and 1.0 MDa in sizes that are distinct from other large, 
transcriptionally important groups of proteins such as SNF/SWI complex. CCR4-
NOT complexes contains Ccr4p, CafIp (Pop2p), the five NOT proteins and two 
other proteins. Not1p appears to be the core component of the complex. Caf1p 
binds to the central region of Not1p and links Ccr4p to the rest of the Not 
proteins. The C terminus of Not1p in turn contacts Not2p, Not4p and Not5p (Liu 
et al., 1998), whereas Caf1p is required for Ccr4p to associate with the 1.0 MDa 
complex (Liu et al., 1998; Bai et al., 1999), since Ccr4p cannot associate with the 
1.9 MDa complex in the absence of Caf1p (Fig. 4).  
 
 
 
 
 19 
CHAPTER 2 
 
 
 
FIGURE 4. Schematic representation of the Ccr-Not complex and the interaction 
with the Srb proteins. The Ccr-Not complex interacts with may different 
proteins such as proteins, as Dhh1p, TFIID, and the Ubc4 and Ubc5 
E2 enzymes, contributing to mRNA degradation, transcription initiation 
and ubiquitination respectively (Collart, 2003). In the case of ADH2  
expression, Ccr-Not complex may be involved in transcription initiation.  
 
Two additional factors (Caf4p and Caf16p), which physically interact with the 
CCR4-NOT complex, were identified by Liu et al. (2001). Caf4p interacted in the 
two-hybrid assay with Ccr4p and Not1p whereas Caf16p interacted only with 
Ccr4p.  
 
These two factors (Caf4p and Caf16p) were found to interact with Srb9p, Srb10p 
and Ssb11p, components of the RNA polymerase II holoenzyme (Song et al., 
1996) during ADH2 derepression (Liu et al., 2001). Defects in the Srb9, 10 and 
11 proteins affect ADH2 expression in a manner similar to that observed for 
defects in Ccr4p complex suggesting that the physical interactions between these 
protein groups represents shared regulatory interactions.  
 20 
CHAPTER 2 
 
 
Three possible models may explain the interactions between the CCR4-NOT 
complex and Srb9-11 proteins: In the first model the SRB proteins may stabilize, 
recruit or influence CCR4-NOT function. In the second model the CCR4-NOT 
proteins may regulate the function of Srb9-11 proteins and in the third model the 
physical connections between the CCR4-NOT complex and Srb9-11 derive from 
their proximity at the promoter in shared regulatory events.   
 
Ccr4p acts at a post chromatin remodeling step in affecting ADH2 derepression 
(Verdone et al., 1997). Evidence has been found that implicate the CCR4-NOT 
proteins in playing an important role in the control of transcriptional initiation 
(Denis and Malvar, 1990). In addition, the CCR4-NOT proteins exhibit multiple 
contacts to proteins playing important roles in controlling initiation (Liu et al., 
2001). Ccr4p was found not to affect the degradation rate of ADH2 mRNA or 
elongation through the ADH2 gene, indicating that the effects of Ccr4 on ADH2 
expression must be at the level of initiation of transcription, in addition, the 
CCR4-NOT proteins exhibit multiple contacts to proteins such as TFIID, Ada2p 
and Srb9-11p playing important roles in controlling initiation (Benson et al., 1998; 
Liu et al., 2001). Denis et al. (2001) provided genetic evidence that the CCR4-
NOT complex is involved in regulating transcriptional elongation. Defects in 
transcriptional elongation factors such as TFIIS in yeast elicit 6-azauracil (6AU) 
and mycophenolic sensitive phenotypes (Archambault et al., 1992). They 
observed that a deletion of CCR4, or any other component of the CCR4-NOT 
complex, gives rise to a 6AU sensitive phenotype and that ccr4 deletion or NOT1 
overexpression suppresses a spt5-242 defect that has been shown to be 
suppressed by the slowing of elongation. CCR4-NOT1 complex could aid in 
setting up processive polymerases at the promoter and thereby generate more 
active or increased numbers of elongating polymerases.  
 
Ccr4p activity is blocked by CRE1 and CRE2 genes, which are allelic to Spt10p 
and Spt6p respectively. SPT10 and SPT6 are two genes, which are believed to 
 21 
CHAPTER 2 
 
be involved in chromatin maintenance. Mutations in SPT10 and SPT6 allow for 
ADH2 transcription following growth on glucose (Denis, 1984). SPT10 and SPT6 
genes have been identified as playing important transcriptional roles in several 
systems. The mechanism in which Spt10p and Spt6p affects Ccr4p activity was 
examined by Denis (1984) and it was found that Spt10p and Spt6p did not control 
CCR4 mRNA or protein expression nor did Spt10p and Spt6p co-
immunoprecipitate with Ccr4p.  This suggested that Spt10p and Spt6p negatively 
regulates the transcription of ADH2 by acting through a factor that requires Ccr4p 
function, but do not regulate CCR4 expression, control its activity, physically 
interact with it or affect its binding to other factors. CCR4 and its negative 
effectors Spt10p and Spt6p do not require the upstream activation sequences for 
their function and the derepression activity is independent of ADR1.  
 
4.2. ADR6 
 
ADR6 was initially identified in a screen of mutants able to decrease Ty-activated 
ADH2 expression and it was shown to be required for ADH2 expression when 
glycerol was used as a derepressing carbon source (Taguchi and Young, 1987). 
ADR6, encoding a polypeptide of 1314 amino acids (O’Hara et al., 1988), is a 
transcription factor whose site of action is unknown. This factor may mediate 
ADH2 expression through binding to a sequence downstream of the ADH2 
upsteram activating sequence (UAS). ADR6 was found to be allelic to SWI1 by 
Peterson and Herskowitz (1992) a component of the SWI/SNF chromatin 
remodeling complex required for the transcription of a subset of genes in S. 
cerevisiae (Cairns et al., 1994). This gene was also found to be allelic to GAM3, 
a gene required for STAI and ADH2 transcription as well and for initiation of 
meiosis (Yoshimoto et al. 1992). 
 
The ADH2 sequence expression requirements for ADR6 and ADR1 action 
appear to differ, suggesting independent modes of action for these two elements. 
ADR1 gene product function in the regulation of ADH2 transcription initiation and 
 22 
CHAPTER 2 
 
the ADR6 gene product may act at some subsequent step but prior to the 
translation of ADH2 mRNA. It was demonstrated that Swi/Snf complexes could 
cause ATP-dependent disruption of nucleosome structure and could increase the 
binding of transcription factors to their sites on nucleosomal templates (Kingston 
and Narlikar, 1999), suggesting the role of ADR6/SWI1 in ADH2 derepression at 
this level.  However, observations obtained by Di Mauro et al. (2000) proved that 
ADR6/SWI1 is not required for nucleosome modification and transcription during 
ADH2 derepression, leaving the role of ADR6/SWI1 in ADH2 derepression to the 
possible roles suggested Taguchi and Young (1987) that ADR6/SWI1 may be 
involved in facilitation of ADH2 RNA transcription processing or maturation of 
ADH2 mRNA or stabilization of ADH2 mRNA.   
 
4.3. ADR7, 8 AND 9 
 
ADR7, ADR8 and ADR9 were identified by Karnitz et al. (1992) and they affect 
ADH2 expression under both repressing and derepressing growth conditions. 
Strains containing mutations in ADR7, ADR8 and ADR9, unlike wild type strains, 
allowed ADH2 expression in the absence of ADR1. These regulatory genes do 
not function through any known cis-acting element and do not affect ADH2 
mRNA or protein stability.  Possibly ADR7, ADR8 and ADR9 may play roles in 
analogous glucose mediated repressor pathway for ADH2.  
 
 23 
CHAPTER 2 
 
4.4. ADR1 
 
The Adr1p regulatory protein is a transcriptional activator of ADH2 and is 
required for a dramatic 500-fold increase in ADH2 activity. Genetic analysis of 
ADH2 regulation revealed that derepression of ADH2 activity and mRNA 
accumulation requires the product of an unlinked regulatory locus encoded by 
ADR1. 
 
ADR1 was identified by Ciriacy (1975) to be a possible ADH2 positive regulatory 
gene. Some recessive mutations of ADR1 prevent ADH2 derepression (Ciriacy, 
1975) and other semi-dominant mutations at ADR1 (adr1-5C) allow partially 
constitutive synthesis of ADH2 (Ciriacy, 1979). Nucleotide sequence analysis has 
shown that ADR1 encode for a protein of 1323 amino acids, of which the amino 
terminal 302 amino acids are sufficient to stimulate ADH2 transcription 
(Hartshorne et al., 1986). Adr1p is a large transcriptional factor containing a 
complex DNA binding domain consisting of C2H2 zinc fingers which are essential 
for DNA binding (Blumberg et al., 1987) and a proximal accessory region (PAR). 
A nuclear localization signal is located near the amino terminus and four 
transcription activating domains (TADs) have been identified by deletions and 
gene fusions to LexA (Cook et al., 1994) and GAL4 (Young et al., 1998). In vitro 
binding studies as well as in vivo co-immunoprecipitation suggests that these 
TADs interact with TFIIB, TFIID, Ada2p (transcription co-activator) and Gcn5p 
acetyltransferase (Chiang et al., 1996; Komarnitsky et al., 1998) showing the 
recruitment of transcription initiation complex (pol II complex) and chromatin 
remodeling by Adr1p to the ADH2 promoter.  Denis et al. (1981) proved that 
Adr1p does not act on the Adh2p protein but is involved in the expression of 
ADH2. Initial increase in functional ADH2 mRNA levels after derepression was 
shown to follow the same kinetics in the strain containing adr1 and adrI-5c alleles.   
 
ADR1 mediates ADH2 expression through a 22 base pair inverted repeat dyad 
sequence (Shuster et al., 1986; Yu et al., 1989); an upstream activator sequence 
 24 
CHAPTER 2 
 
(UAS1) located 230 base pairs from the transcription initiation site (Beier et al., 
1985). Activation of ADH2 expression from UAS1 requires the binding of two 
Adr1p monomers (Thukral et al., 1991). Each half of the inverted repeat is an 
independent functional binding site for one monomer of Adr1p. The central region 
of UAS1 provides non-base specific contacts for Adr1p and may prevent steric 
interference between the two Adr1p monomers bound in close proximity to one 
another (Cheng et al., 1994).  
 
The activity of the Adr1p protein is regulated in a carbon source dependent 
manner in strains, which do not regulate ADR1 mRNA levels (Blumberg et al., 
1988). Glucose regulation of ADR1 function appears to occur principally at the 
posttranscriptional level since ADR1 mRNA do not differ between glucose and 
ethanol grown cells. Adr1p is inactive as a transcriptional factor in the presence 
of glucose, although the intact protein is present and DNA binding appears 
normal (Taylor and Young, 1990). The activity of Adr1p is regulated positively by 
the Snf1 complex and negatively by cAMP-dependent protein kinase and the 
Reg1-Glc7 complex. 
 
Under derepressing conditions Adr1p primes the assembly of the pre-initiation 
complex and at the same time the disruption of the two nucleosomes (-1 & +1) 
protecting the TATA box and the RNA initiation sites (Verdone et al., 1997). Two 
distinct structural alterations occur during chromatin remodeling by Adr1p under 
derepressing conditions (Di Mauro et al., 2000). The first consists of a localized 
modification induced by the Adr1p DNA binding domain at the level of the 
nucleosome -1, this cleavage site contains the TATA box. It is located halfway 
between UAS1 where Adr1p binds to the TATA box. The second type of 
structural alteration, which is induced when the activation domain is present, is 
more stable and involves both nucleosome -1 and nucleosome +1 containing the 
RNA initiation site. This second structural alteration leads to a fully derepressed 
ADH2 promoter, which is functionally active. Two functions can thus be attributed 
to Adr1p: 
 25 
CHAPTER 2 
 
1) Adr1p reconfigures nucleosomes in the immediate vicinity of its binding 
site allowing the basal promoter elements to assume the most appropriate 
structure for the subsequent activation.  
2) Adr1p recruits the transcription machinery through its activation domain 
allowing mRNA accumulation. 
Adr1p contributes to the transcriptional derepression of ADH2 synergistically with 
Cat8p (Walther and Schüller, 2001) 
 
4.5 CAT8 
 
Cat8 is required for the activation of gluconeogenic genes through a carbon 
source-responsive element (CSRE) present in their promoters (Hedges et al., 
1995). The carbon source-responsive element functions as an activating 
promoter motif of gluconeogenic genes in S. cerevisiae.  The CAT8 gene 
encodes a transcription factor with a binuclear zinc cluster domain at its N 
terminus and a C terminal transcription activation domain. Expression of CAT8 as 
well as transcription activation by Cat8p is affected by carbon source (Rahner et 
al., 1996).  
 
Before CAT8 could be associated with the derepression of ADH2 the UAS2 was 
identified in ADH2 and it was found to have the CSRE required for Cat8p 
dependent derepression. The deletion of either UAS1 or ADR1 allowed a 
substantial (but not complete) derepression of ADH2, arguing for an additional 
UAS element and trans-activator. This led to the discovery of the UAS2 by Yu et 
al. (1989). The deletion of ADH2 sequences 5' to UAS1 identified a GC rich 20 
bp sequence referred to as UAS2. Its activity as a single copy was glucose 
regulated, Adr1p independent and comparable to that of a single copy of UAS1. 
The two UAS elements acted synergistically to activate expression 20-fold as 
compared with the activation measured with either element alone (Yu et al., 
1989). These results suggested that neither ADR1 nor the putative UAS2 binding 
factor alone can effectively activate ADH2 transcription. 
 26 
CHAPTER 2 
 
 
UAS2 was suggested to be a binding site for a protein that interacts with Adr1p 
bound to UAS1 (Donoviel et al., 1995) and DNA binding data suggested that 
Adr1p might bind to this site. However a functional CSRE, which specifically 
interacts with Cat8p, was identified within UAS2 (Walther and Schüller, 2001). 
Gel retardation experiments showed that Cat8p and the related zinc cluster 
protein Sip4p differ with respect to binding affinity to CSRE sequence elements, 
providing the evidence that Cat8p is the dominating regulator acting via UAS2. 
The Cat8p activator contributes to the transcriptional derepression of ADH2 
synergistically with Adr1p and together they mediate more than 98% of total 
ADH2 derepression. 
 
Cat8p activity is in turn regulated at the expression, as well as the transcription 
activating levels. Mig1p, a DNA binding zinc finger protein, represses the 
expression of CAT8. Mig1p recruits the Ssn6/Cyc8-Tup1 complex, which repress 
transcription (Treitel and Carlson, 1995). The activator function of Cat8p is 
inhibited by glucose and is dependent on Snf1p. A functional Snf1p complex is 
required for Cat8p function (Randez-Gil et al., 1997). The Snf1p complex 
regulates the activating function of Cat8p and is required for the phosphorylation 
of Cat8p in derepressed state (Fig. 5). 
 27 
CHAPTER 2 
 
 
 
FIGURE 5. Derepression of gluconeogenic structural genes by carbon source-
responsive element (CSRE)-binding factors, Cat8p and Sip4p. A 
dual function of the Snf1 protein kinase complex is proposed: 
deactivation of the Mig1p repressor allows biosynthesis of Cat8p 
and post-translational modification converts Cat8p into a 
transcriptional activator which subsequently stimulates expression 
of SIP4. Cat8p and Sip4p both contribute to derepression of 
gluconeogenic structural genes, with Cat8p being the more 
important activator. The activator of CAT8 transcription in the 
absence of Mig1p is unknown. It is also unclear whether Cat8p and 
Sip4p are directly phosphorylated by Snf1p. URE Upstream 
regulatory element (Schüller, 2003). 
 
4.6 CYCLIC AMP-DEPENDENT PROTEIN KINASE (CAPK)  
 
The cyclic AMP (cAMP) signal transduction pathway plays an important role in 
triggering changes in gene expression that is responsible for long-term 
adaptation to fluctuations in environmental conditions. The penultimate effector of 
 28 
CHAPTER 2 
 
this pathway, cAMP-dependent protein kinase (cAPK), can influence the level of 
gene expression either positively or negatively thereby controlling transcription. In 
S. cerevisiae, growth on glucose induces a cAMP signal that initiates a cascade 
of protein phosphorylation by cAPK (Matsumoto et al., 1982), resulting in 
increased glycolysis and inactivation of several enzymes involved in 
gluconeogenesis. cAPK also regulate the expression of ADH2 by 
phosphorylating its transcription factor Adr1p. Adr1p is a serine phosphoprotein 
whose activity appears to be negatively regulated by cAPK-dependent 
phosphorylation. Genetic evidence suggests that Adr1p is partially inactivated by 
cAMP-dependent protein kinase (Cherry et al., 1989). 
 
Three different genes TPK1, TPK2 and TPK3 encode the cAPK catalytic subunits 
in yeast. The activation of the cAPK phosphorylation system under glucose 
growth conditions appear to be mediated by a glucose-dependent signal 
transmitted through the adenyl cyclase effectors CDC25, RAS1 and RAS2 
(Mbonyi et al., 1988; Munder and Kűntzel, 1989). Yeast contains only one 
regulatory cAPK regulatory subunit encoded by the BCY1 gene (Toda et al., 
1987). ADH2 expression is inhibited in cells containing a deletion of the BCY1 
gene, and this inhibition can be restored by mutations in the TPK genes (Cherry 
et al., 1989; Denis et al., 1992). This suggests that unregulated cAPK activity 
inhibits an important step required for the activation of ADH2 expression. 
 
It was originally postulated that the dominant adr1C mutations causing enhanced 
ADH2 transcription under repressed conditions (Ciriacy, 1979), affected the cyclic 
AMP-dependent protein kinase phosphorylation site at ser-230 of ADR1, thereby 
inhibiting the inactivation of Adr1p (Cherry et al., 1989).  However Denis et al. 
(1992) provided evidence which indicates that this protein kinase inhibits ADR1 
function by a mechanism that is independent of ser-230. Dominant mutations in 
ADR1 (adr1C) which allow glucose-insensitive ADH2 transcription (Denis et al., 
1992) has been identified as amino acid substitutions between amino acid 227 
and 239 of ADR1.  It was suggested that cAPK inhibits ADH2 expression by 
 29 
CHAPTER 2 
 
phosphorylating either Adr1p or another protein required for Adr1p activity. The 
phosphorylation of Adr1p does not affect the binding of this transcription factor to 
the UAS1 (Taylor and Young, 1990) suggesting that during glucose repression, 
cAMP-dependent phosphorylation of Adr1p may inhibit its ability to interact with 
general transcription factors or RNA polymerase II. Dombek and Young (1997) 
suggested that cAPK may also inhibit ADH2 expression by decreasing the 
expression of ADR1 and indicated that cAPK acts through UAS1 in decreasing 
the abundance of Adr1. These observations suggests that the negative control of 
ADH2 expression by cAPK is exerted through several mechanisms, one being 
the inhibition of ADR1 transcription and the other being the direct phosphorylation 
of Adr1p. 
 
4.7 SNF1/CCR1/CAT1 
 
CCR1 is a cAMP-independent protein kinase involved in ADH2 regulation. It was 
found to be allelic to SNF1 (Denis, 1984) and CAT1 (Ciriacy, 1977) which 
encodes for a yeast protein kinase required for the transcription of several 
glucose-repressed genes including ADH2 (Denis, 1987). Snf1p protein kinase 
phosphorylates and controls factors required for ADH2 regulation and was 
thought to act independently of ADR1 (Denis and Audino, 1991). Genetic 
analysis indicates that SNF1/CCR1/CAT1 is required for Adr1p activation of 
ADH2 expression.  
 
A combination of genetic, two-hybrid and co-immunoprecipitation experiments 
indicated that Snf1p is complexed with Snf4p and one member of the Sip/Gal83 
class proteins (Celenza et al., 1989; Yang et al., 1994). The interactions between 
Snf1 and Snf4 appear to be carbon source regulated (Jiang and Carlson, 1996). 
In repressed cells the N-terminal kinase domain of Snf1p appears to interact with 
its C-terminal regulatory domain, which is thought to inhibit kinase activity. Upon 
depletion of glucose from growth medium, Snf4p binds to the kinase, displacing 
 30 
CHAPTER 2 
 
the regulatory domain and thereby freeing the Snf1p kinase domain from auto 
inhibition. This scenario is indicated in the Fig. 6 (Jiang and Carlson, 1996). 
 
 
FIGURE 6. Model for the regulation of the Snf1 complex by glucose. The bridging 
protein (Brp) between Snf1p and Snf4p can be Gal83p, Sip1p, Sip2p 
or some other, as yet, unidentified protein. Glucose affects the 
interaction between the catalytic domain (KD) and the regulatory 
domain (RD) of Snf1p, presumably by inhibiting the 
(auto)phosphorylation of Snf1p and/or activating its dephosphorylation. 
Glucose may act at the level of the corresponding kinase and 
phosphatase but may also alter the conformation of Snf1p or even 
Brp, making Snf1p a worse or better substrate for the corresponding 
enzyme. Hxk2p and Grr1p are required for transmitting the glucose 
signal (Jiang and Carlson, 1996).  
 
The Snf1 kinase complex is inactivated by Reg1-Glc7 phosphatase complex. 
Protein phosphatase type 1 (PP1) plays a key role in regulating a variety of 
processes in eukaryotic cells (Bollens and Stalmans, 1992) and the gene coding 
for the S. cerevisiae homologue of PP1 is GLC7. Glc7p is required for the 
appropriate regulation of a number of cellular processes, including glycogen 
biosynthesis, translation, cell cycle progression, chromosome segregation, 
meiosis and sporulation as well as repression of many glucose-regulated genes 
 31 
CHAPTER 2 
 
(Stark, 1996). Reg1p, a regulatory subunit that affects glucose repression, growth 
and glycogen accumulation (Entian and Zimmermann, 1980), confers substrate 
specificity for Glc7p. Reg1p interacts with the kinase domain of Snf1p, altering 
protein-protein interactions within the kinase complex (Ludin et al., 1998). Two-
hybrid experiments have suggested that Reg1p interacts weakly with the kinase 
domain of Snf1p in repressed cells and strongly in derepressed cells. Once 
bound, Glc7p dephosphorylates Snf1p thereby releasing Snf4p from the kinase 
regulatory domain and returning the complex to an autoinhibited state. The Reg1-
Glc7 phosphatase complex is part of the cytoplasmic machinery for resetting 
Snf1p kinase activity to a basal level implying that the activated form of Snf1p 
complex rapidly cycles between the nucleus and the cytoplasm (Dombek et al., 
1999).  
 
The Snf1 kinase complex relieves ADH2 derepression by activating Adr1p 
transcription factor.  The regulated Adr1p binding to the ADH2 promoter requires 
the Snf1p kinase complex and under repressing conditions Adr1p is non-
functional due to the autoinhibition of the Snf1p kinase complex exerted by the 
Reg1-Glc7 phosphatase complex (Young et al., 2002).  Snf1p kinase complex 
regulates chromatin binding by Adr1p and it was recently found to activate Adr1p-
dependent ADH2 derepression by allowing Adr1p to bind to UAS1 and does not 
enhance the activation potential of Adr1p (Young et al., 2002). Therefore the role 
of Snf1p in ADH2 derepression is to modify chromatin to allow Adr1p to bind to 
the ADH2 UAS1. Snf1p also regulates Cat8p expression and its transcription 
activation function. 
 
4.8 CAMP-INDEPENDENT PROTEIN KINASE (SCH9) 
 
SCH9 encodes for a cAMP-independent protein kinase and it was initially 
proposed to be involved in ADH2 repression. It was postulated that Sch9 protein 
kinase could functionally substitute for cAPK (Cameron et al., 1988). Denis and 
Audino (1991) provided evidence that Sch9p is required for ADH2 derepression 
 32 
CHAPTER 2 
 
and not ADH2 repression. Increased expression of SCH9 had no effect in the 
expression of ADH2 and strains carrying a sch9 disruption displayed a three to 
ten-fold decrease in Adh2p activity when compared to their isogenic parental 
strains. Sch9p does not act through ADR1 to derepress ADH2 expression (Denis 
and Audino, 1991) but appears to act in a pathway separate from that and not 
parallel to that involving cAPK. Since Sch9 does not act through UAS1 it may 
control factors that act through other activation sequences or through factors that 
control the general transcriptional machinery. 
 
5. ALCOHOL DEHYDROGENASE II REGULATION: AN OVERVIEW 
 
In the yeast S. cerevisiae, growth, metabolism and gene expression are highly 
regulated in response to carbon source availability. Glucose is the most abundant 
monosaccharide in nature and it is also the primary fuel for microorganisms. 
Yeast cells also prefer to use glucose as a carbon source. A major route by 
which glucose encourages its own use and stimulates fermentation is by 
regulating gene expression. When glucose is abundant the expression of a set of 
genes encoding glycolytic enzymes, glucose transporters, ribosomal proteins and 
other proteins is induced. At the same time the expression of a large set of genes 
is repressed (Gancedo, 1998; Vallari et al., 1992). This set includes genes 
involved in utilization of alternative carbon sources (i.e ADH2 in ethanol 
utilization), gluconeogenesis, the ticarboxylic acid cycle, respiration and other 
processes (Hohmann and Mager, 1997). When glucose is limiting, an important 
aspect of the cell's regulatory response is the release of glucose repression and 
the expression of a large set of genes including ADH2. When the concentration 
of glucose drops below 0.2%, expression of these genes is activated or 
derepressed. For repressible genes like ADH2, this change in the rate of 
expression can be 200-fold or greater (Celenza et al., 1989; Dombek et al. 1993; 
Griggs and Johnston, 1991). The repression of ADH2 expression by glucose is 
not due to the availability/binding of a repressor to prevent transcription but it is 
due to the unavailability or lack of function of the transcription activators (Cat8p 
 33 
CHAPTER 2 
 
and Adr1p). Although mutations in a variety of genes such as CCR4, ADR6 or 
CRE1 have an effect in the expression of ADH2, the corresponding proteins are 
not likely to be involved directly in the glucose signal (Gancedo, 1998). 
 
The expression of ADH2 under derepressing conditions is dependent on the two 
UASs (UAS1 and UAS2) located at the promoter. Two transcription activators, 
whose activity/expression is under glucose regulation binds to these UASs, 
therefore two major pathways appear to be involved in the glucose regulated 
ADH2 expression. The first one is exerted through Adr1p, which binds to the 
AUS1 (Shuster et al., 1986; Yu et al., 1989); the second one is exerted through 
Cat8p, which binds to the UAS2 (Walther and Schüller, 2001). A number of 
genes have been identified as playing integral roles in glucose repression 
(Gancedo, 1998). Among these are REG1, GLC7 and SNF1, the yeast 
homologue of the catabolic subunit of AMP-activated protein kinase (Hardie et 
al., 1998). The Snf1p function is activated when glucose is limiting (Wilson et al., 
1996). Snf1 is essential for the regulatory response to glucose starvation in yeast 
(Celenza and Carlson, 1986) and is required for the transcription of glucose 
repressed genes when glucose is limiting. Both of these pathways are affected 
by the Snf1 protein kinase complex. Snf1p and Reg1-Glc7 a protein 
phosphatase1 and its regulatory subunits are the central components of the two 
pathways for glucose repression of ADH2 expression. The pathway exerted 
through Cat8p is also affected by Mig1p, a transcriptional repressor; this means 
that CAT8 is regulated at a level of transcription and protein function.  In the first 
pathway, involving UAS1, Adr1p function is activated in the absence of glucose 
by Snf1p and the second pathway involves UAS2. Activation through UAS2 
involves the activation of Cat8p through phosphorylation. Snf1p activates the 
expression of CAT8 by inhibiting Mig1p through phosphorylation during glucose 
limitation (Treitel et al., 1998). The derepression of ADH2 is summarized in Fig. 
7.  
 34 
CHAPTER 2 
 
 
 
FIGURE 7. Model of the regulation of ADH2 by glucose. When glucose is limiting, 
the Snf1 kinase activates Cat8 and Adr1, which in turn activate the 
expression of ADH2, and inhibits Mig1 repressor function leading to 
the expression of Cat8.  
 
6. THE USE OF THE ADH2 PROMOTER 
 
S. cerevisiae is the most extensively studied non-mammalian eukaryote; its 
genetics, biochemistry, and life cycle are well known. Due to this it is an attractive 
host for the production of heterologous proteins. Expressed proteins can be 
specifically engineered for cytoplasmic localization or for extracellular export. In 
addition, yeasts are exceedingly well suited for large-scale fermentation for the 
production of large quantities of heterologous protein. Either constitutive or 
inducible promoters can be used for heterologous protein production depending 
on the type of the protein being produced. In principle, any promoter isolated 
from a yeast gene can be employed to achieve expression of a foreign coding 
 35 
CHAPTER 2 
 
sequence in yeast. However, since promoter control is a primary way in which 
the expression of genes is controlled in yeast, yeast promoters are usually 
subjected to some form of regulation that will also affect any derivative 
heterologous constructions (Hadfield et al., 1993).  
 
Inducible expression cassettes are employed for the regulated production of 
heterologous proteins and the ADH2 promoter has been used for this purpose 
due to its regulation through glucose repression. The ADH2 promoter is both 
powerful and tightly regulated and is therefore very useful in regulated 
overexpression of heterologous genes in S. cerevisiae (Price et al., 1990). In 
order to maintain repression of ADH2, cells must be grown in excess glucose 
until induction, which is effected by changing to the fresh medium containing a 
non-fermentable carbon source e.g. ethanol. Alternatively, ADH2 can be induced 
by culturing initially in a lower concentration of glucose which is gradually 
depleted (Romanos et al., 1992).  
 
Since the ADH2 promoter is repressed over 100-fold by glucose, it can be used 
for efficient expression of toxic proteins e.g. insulin-like growth factor (IGF-1). 
ADH2 promoter has been successfully used for the production of TBV25H, a 
malaria transmission blocking vaccine candidate (Noronha et al., 1998). This 
production was feasible only due to the properties of ADH2 promoter, as 
previously available fermentation procedures for the production of this protein 
have been unsatisfactory mainly because of irreproducibility. A 260 bp region 5' 
to the initiation site, which contains two UASs that are sufficient for full promoter 
activity and regulation, has been used in efficient expression vectors. 
 
Hybrid glycolytic ADH2 promoters have been constructed by using the ADH2 
UAS containing 22bp dyad 320bp upstream of the GAP TATA element, and were 
able to achieve tightly regulated production of peroxide dismutase (50D)-
proinsulin fusion (Romanos et al., 1992).  
 
 36 
CHAPTER 2 
 
7. REFERENCES 
 
Ammerer G. 1983. Expression of genes in yeast using the ADR1 promoter. 
Meth. Enymol. 101:192-201 
    
Archambault J., Lacroute F., Ruet A. and Freisen J.D. 1992. Genetic 
interaction between transcription elongation factor TFIIS and RNA 
polymarase II. Mol. Cell. Biol. 12:4142-4152 
      
Bai Y., Salvadore C., Chiang Y.C., Collart M.A., Liu H.Y. and Denis C.L. 1999. 
The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically 
and functionally separated from NOT2, NOT4, and NOT5. Mol. Cell. Biol. 
19:6642-6651. 
 
Bakker B.M., Bro C., Kotter P., Luttik M.A., van Dijken J.P., Pronk J.T. 2000. 
The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox 
shuttle in Saccharomyces cerevisiae. J. Bacteriol. 182:4730-4737. 
 
Beier D.R., Sledziewski A. and Young E.T. 1985. Deletion analysis identifies a 
region, upstream of the ADH2 gene of Saccharomyces cerevisiae, which is 
required for ADR1-mediated derepression. Mol. Cell. Biol. 5:1743-1749 
 
Benson J.D., Benson M., Howley P.M. and Struhl K. 1998. Association of 
distinct yeast Not2 functional domains with components of Gcn5 histone 
acetylase and Ccr4 transcriptional regulatory complexes. EMBO J. 17:6714-
6722. 
 
Blumberg H., Eisen A., Sledziewski A., Bader D. and Young E.T. 1987. Two 
zinc fingers of a yeast regulatory protein shown by genetic evidence to be 
essential for its function. Nature 328:443-445. 
 
 37 
CHAPTER 2 
 
Blumberg H., Hartshorne T.A. and Young E.T. 1988. Regulation of expression 
and activity of the yeast transcription factor ADR1. Mol. Cell. Biol. 8:1868-
1876. 
 
Butler G. and McConnell D.J. 1988. Identification of an upstream activation site 
in the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces 
cerevisiae. Curr. Genet. 14:405-412. 
 
Bollen M. and Stalmans W. 1992. The structure, role, and regulation of type 1 
protein phosphatases. Crit. Rev. Biochem. Mol. Biol. 27:227-281.  
 
Cameron S., Levin L., Zoller M. and Wigler M. 1988. cAMP-independent 
control of sporulation, glycogen metabolism, and heat shock resistance in 
S. cerevisiae. Cell. 53:555-566.  
 
Cairns B.R., Kim Y.J., Sayre M.H., Laurent B.C. and Kornberg R.D. 1994. A 
multi subunit complex containing the SWI1/ADR6, SWI2/SNF2, SWI3, 
SNF5, and SNF6 gene products isolated from yeast. Proc. Natl. Acad. Sci. 
91:1950-1954. 
 
Celenza J.L. and Carlson M. 1986. A yeast gene that is essential for release 
glucose repression encodes a protein kinase. Science 233:1175-1180 
 
Celenza J.L., Eng F.J. and Carlson M. 1989. Molecular analysis of the SNF4 
gene of Saccharomyces cerevisiae: evidence for physical association of the 
SNF4 protein with the SNF1 protein kinase. Mol. Cell. Biol. 9:5045-5054. 
 
Cheng C., Kacherovsky N., Dombek K.M., Camier S., Thukral S.K., Rhim E. 
and Young E.T. 1994. Identification of potential target genes for Adr1p 
through characterization of essential nucleotides in UAS1. Mol. Cell. Biol. 
14: 3842-3852 
 38 
CHAPTER 2 
 
 
Cherry J.R., Johnson T.R., Dollard C., Shuster J.R. and Denis C.L. 1989. 
Cyclic AMP-dependent protein kinase phosphorylates and inactivates the 
yeast transcriptional activator ADR1. Cell 56:409-419 
 
Chiang, Y. C., Komarnitsky, P., Chase, D. & Denis, C. L. 1996. ADR1 
activation domains contact the histone acetyltransferase GCN5 and the 
core transcriptional factor TFIIB. J. Biol. Chem. 271:32359-32365.  
 
Ciriacy M. 1975. Genetics of alcohol dehydrogenases in Saccharomyces 
cerevisiae  II. Two loci controlling synthesis of the glucose-repressible 
ADHII. Mol. Gen. Genet. 138:157-154 
 
Ciriacy M. 1977. Isolation and characterization of mutants defective in 
intermediary carbon metabolism repression. Mol. Gen. Genet. 154:213-220 
 
Ciriacy M. 1979. Isolation and characterization of further cis- and trans-acting 
regulatory elements involved in the synthesis of glucose-repressible alcohol 
dehydrogenase (ADHII) in Saccharomyces cerevisiae. Mol. Gen. Genet. 
176:427-431 
 
Ciriacy M. 1997. Alcohol dehydrogenases. p. 213-223. In F.K. Zimmermann and 
K.D. Entian (ed.), Yeast sugar metabolism. Technomic Publishing 
Company, Lancaster, Pa. 
 
Collart M.A. 2003. Global control of gene expression in yeast by the Ccr4-Not 
complex. Gene 313:1-16 
 
Cook W.J., Chase D., Audino D.C. and Denis C.L. 1994. Dissection of the 
ADR1 protein reveals multiple, functionally redundant activation domains 
 39 
CHAPTER 2 
 
interspersed with inhibitory regions: evidence for a repressor binding to the 
ADR1c region. Mol. Cell. Biol. 14:629-640. 
 
Denis CL. 1984. Identification of new genes involved in the regulation of yeast 
alcohol dehydrogenase II. Genetics. 108:833-844. 
 
Denis C.L. 1987. The effects of ADR1 and CCR1 gene dosage on the regulation 
of the glucose-repressible alcohol dehydrogenase from Saccharomyces 
cerevisiae. Mol. Gen. Genet. 208:101-106 
 
Denis C.L, Ferguson J. and Young E.T. 1983. mRNA levels for the 
fermentative alcohol dehydrogenase of Saccharomyces cerevisiae 
decrease upon growth on a nonfermentable carbon source. J. Biol. Chem. 
258:1165-1171. 
 
Denis C.L. and Audino D.C. 1991. The CCR1 (SNF1) and SCH9 protein 
kinases act independently of cAMP-dependent protein kinase and the 
transcriptional activator ADR1 in controlling yeast ADH2 expression. Mol. 
Gen. Genet. 229:395-399 
 
Denis C.L., Ciriacy M and Young E.T. 1981. A positive activator regulatory 
gene is required for accumulation of the functional mRNA for the glucose 
repressible alcohol dehydrogenase of Saccharomyces cerevisiae decrease 
upon growth on a nonfermentable carbon-source. J. Biol. Chem. 258:1165-
1171 
 
Denis C.L., Fontane S.C., Chase D., Kemp B.E. and Bemis L.T. 1992. ADRC 
mutations enhance the ability of ADR1to activate trancription by a 
mechanism that is independent by the effects on cyclic AMP-dependent 
protein kinase phosphorylation of ser-230. Mol. Cell. Biol. 12:1507-1514 
 
 40 
CHAPTER 2 
 
Denis C.L. and Young E.T. 1983. Isolation and characterization of the positive 
regulatory gene ADR1 from Saccharomyces cerevisiae. Mol. Cell. Biol. 
3:360-370 
Denis C.L. and Malvar T. 1990. The CCR4 gene from Saccharomyces 
cerevisiae is required for both nonfermentative and spt-mediated gene 
expression. Genetics. 124:283-291. 
 
Denis C.L., Chiang Y.C., Cui Y. and Chen J. 2001. Genetic evidence supports 
a role for the yeast CCR4-NOT complex in transcriptional elongation. 
Genetics 158:627-634. 
 
Di Mauro E., Kendrew S.G. and Caserta M. 2000. Two distinct nucleosome 
alterations characterize chromatin remodeling at the Saccharomyces 
cerevisiae ADH2 promoter.  J. Biol. Chem. 275 :7612-7618 
 
Dombek K.M., Cameir S. and Young E.T. 1993. ADH2 expression is repressed 
by REG1 independently of mutations that alter the phosphorylation of the 
yeast transcription factor ADR1. Mol. Cell. Biol. 17:1450-1458 
 
Dombek K.M. and Young E.T. 1997. Cyclic AMP-dependent protein kinase 
inhibits adh2 expression in part by decreasing expression of the 
transcription factor gene ADR1. Mol. Cell. Biol. 17:1450-1458 
 
Dombek K.M., Voronkova V., Roney A. and Young E.T. 1999. Functional 
analysis of the yeast GLC-7 binding protein Reg1 identifies a protein 
phosphatase type 1-binding motif as essential for repression of ADH2 
expression. Mol. Cell. Biol. 19:6029-6040 
 
Donoviel M.S., Kacherovsky N. and Young E.T. 1995. Synergistic activation of 
ADH2 expression is sensitive to upstream activating sequence 2 (UAS2) 
 41 
CHAPTER 2 
 
orientation, copy number UAS1-UAS2 helical phasing. Mol. Cell. Biol. 
15:3442-3449 
 
 
du Preez J.C., Maré J.E., Albertyn J. and Kilian S.G. 2001. Transcriptional 
repression of ADH2-regulated β-xylanase production by ethanol in 
recombinant strains of Saccharomyces cerevisiae. FEMS Yeast Res. 1:233-
240. 
 
Entian K.D. and Zimmermann F.K. 1980. Glycolytic enzymes and intermediates 
in carbon catabolite repression mutants of Saccharomyces cerevisiae. Mol. 
Gen. Genet. 177:345-350 
 
Fernandez M.R., Biosca J.A., Norin A., Jornvall H. and Pares X. 1995. Class 
III alcohol dehydrogenase from Saccharomyces cerevisiae: structural and 
enzymatic features differ toward the human/mammalian forms in a manner 
consistent with functional needs in formaldehyde detoxication. FEBS Lett. 
370:23-26 
 
Finley D. and Chau V. 1991. Ubiquitination. Annu. Rev. Cell. Biol. 7:25-69 
 
Gancedo J.M. 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. 
Rev. 62:334-361.  
 
Griggs D.W. and Johnston M. 1991. Regulation expression of the GAL4 
activator gene in yeast provides a sensitive genetic switch for glucose 
repression. Proc. Natl. Acad. Sci. 88:8597-8601 
 
Hadfield C. Raina K. K., Shashi-Menon K. and Mount R. C. 1993. The 
expression and performance of cloned genes in yeasts. Mycol. Res. 8:897-
944 
 42 
CHAPTER 2 
 
 
Hardie D.G., Carling G.D. and Carlson M. 1998. The AMP-activated/SNF1 
protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu. 
Rev. Biochem. 67:821-855 
Hartshorne,T.A., Blumberg,H. and Young,E.T. 1986. Sequence homology of 
the yeast regulatory protein ADR1 with Xenopus transcription factor TFIIIA. 
Nature. 320:283-287 
 
Hedges D., Proft M. and Entian K.D. 1995. CAT8, a new zinc cluster-encoding 
gene necessary for derepression of gluconeogenic enzymes in the yeast 
Saccharomyces cerevisiae. Mol. Cell. Biol. 15:1915-1922 
 
Hohmann S. and Mager W.H. Molecular biology intelligence unit (Yeast stress 
responses p. 9-21) R.G. Landes company Austi, Texas, USA. 1997 
Springer-verlag,Heidelberg, Germany 
 
Jiang R. and Carlson M. 1996. Glucose regulates protein interaction within the 
yeast SNF1 protein kinase complex. Genes Dev. 10:3105-3115 
 
Jörnvall H. 1977. Differences between alcohol dehydrogenases. Structural 
properties and evolutionary aspects. Eur. J. Biochem. 72:443-452. 
 
Karnitz L., Michael M. and Young E.T. 1992. The identification and 
characterization of three genes that affect expression of ADH2 in 
Saccharomyces cerevisiae. Genetics 132:351-359 
 
Kingston R.E. and Narlikar G.J. 1999. ATP-dependent remodeling and 
acetylation as regulators of chromatin fluidity. Genes Dev. 13:2339-2352.  
 
 43 
CHAPTER 2 
 
Komarnitsky P.B., Klebanow E.R., Weil P.A. and Denis C.L. 1998. ADR1-
mediated transcriptional activation requires the presence of an intact TFIID 
complex. Mol. Cell. Biol. 18:5861-5867. 
 
 
 
Larroy C., Fernandez M.R., Gonzalez E., Pares X. and Biosca J.A. 2002.  
Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene 
product as a broad specificity NADPH-dependent alcohol dehydrogenase: 
relevance in aldehyde reduction. Biochem. J. 361:163-172 
 
Larroy C., Pares X. and Biosca J.A. 2002. Characterization of a 
Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase 
(ADHVII), a member of the cinnamyl alcohol dehydrogenase family. Eur. J. 
Biochem. 269:5738-5745. 
 
Larroy C., Fernandez M.R., Gonzalez E., Pares X. and Biosca J.A. 2003. 
Properties and functional significance of Saccharomyces cerevisiae ADHVI. 
Chem. Biol. Interact. 143-144:229-238. 
 
Liu H.Y., Badarinarayana V., Audino D.C., Rappsilber J., Mann M. and Denis 
C.L. 1998. The NOT proteins are part of the CCR4 transcriptional complex 
and affect gene expression both positively and negatively. EMBO J. 
17:11096-11106. 
 
Liu H.Y., Chiang Y.C., Pan J., Chen J., Salvadore C. and Audino D.C., 
Badarinarayana V., Palaniswamy V., Anderson B. and Denis C.L. 2001. 
Characterization of CAF4 and CAF16 reveals a functional connection 
between the CCR4-NOT complex and a subset of SRB proteins of the RNA 
polymerase II holoenzyme. J. Biol. Chem. 276:7541-7548.  
 
 44 
CHAPTER 2 
 
Ludin K., Jiang R. and Carlson M. 1998. Glucose-regulated interaction of a 
regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in 
Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 95:6245-6250 
 
Lutstorf U. and Megnet R. 1968. Multiple forms of alcohol dehydrogenase in 
Saccharomyces cerevisiae. I. Physiological control of ADH-2 and properties 
of ADH-2 and ADH-4. Arch. Biochem. Biophys. 126:933-944. 
 
Lyons T.J., Gasch A.P., Gaither L.A., Botstein D., Brown P.O. and Eide D.J. 
2000. Genome-wide characterization of the Zap1p zinc-responsive regulon 
in yeast. Proc. Natl. Acad. Sci. 97:7957-7962.  
Maeda T., Wurgler-Murphy S.M. and Santio H. 1994. A two component system 
that regulates an osmosensing cascade in yeast. Nature 369:242-245 
 
Mager W.H. and de Kruijff A.J.J.  1995. Stress-induced transcription activation. 
BioEssys 159:506-531 
 
Mason R.P. and Sanders K.M. 1986. Formaldehyde metabolism in Escherichia 
coli. Detection by in vivo 13C NMR spectroscopy of S-(hydroxymethyl) 
glutathione as a transient intracellular intermediate. Biochem. 25:4504-4507 
 
Matsumoto K., Uno I., Oshima Y. and Ishikawa T. 1982. Isolation and 
characterization of yeast mutants deficient in adenylate cyclase and cAMP-
dependent protein kinase. Proc. Natl. Acad. Sci. 79:2355-2359. 
 
Matsumoto K. Yoshimatsu T. and Oshima Y. 1983. Recessive mutations 
conferring resistance to carbon catabolite repression of galactokinase 
synthesis in Saccharomyces cerevisiae. J. Bacteriol. 153:1405-1414 
 
Mbonyi K., Beullens M., Detremerie K., Geerts L. and Thevelein JM. 1988. 
Requirement of one functional RAS gene and inability of an oncogenic ras 
 45 
CHAPTER 2 
 
variant to mediate the glucose-induced cyclic AMP signal in the yeast 
Saccharomyces cerevisiae. Mol. Cell. Biol. 8:3051-3057. 
 
Munder T. and Kuntzel H. 1989. Glucose-induced cAMP signaling in 
Saccharomyces cerevisiae is mediated by the CDC25 protein. FEBS Lett. 
242:341-345 
 
Noronha S.B., Kaslow D.C. and Shiloach J. 1998. Transition phase in the 
production of recombinant proteins in yeast under the ADH2 promoter: an 
important step for reproducible manufacturing of malaria transmission 
blocking vaccine candidate. J. Industrial Micro. Biotech. 20:192-199 
 
O'Hara P.J., Horowitz H., Eichinger G. and Young E.T. 1988. The yeast ADR6 
gene encodes homopolymeric amino acid sequences and a potential metal-
binding domain. Nucleic Acids Res. 16:10153-10169. 
 
Peterson C.L and Herskowitz I. 1992. Characterization of the yeast SWI1, 
SWI2, and SWI3 genes, which encode a global activator of transcription. 
Cell. 68:573-583. 
 
Price V.L., Taylor W.E., Clevenger W., Worthingtons M. and Young E.T. 
1990. Expression of heterologous proteins in Saccharomyces cerevisiae 
using ADH2 promoter. Meth. Enzymol. 185:308-318 
 
Racker E. 1953. Methods in enzymology (Colowick S.P. and Kaplan N.O., eds), 
pp. 500-503, Academic press, New York. 
 
Rahner A., Scholer A., Martens E., Gollwitzer B. and Schüller H.J. 1996. Dual 
influence of the yeast Cat1p (Snf1p) protein kinase on carbon source-
dependent transcriptional activation of gluconeogenic genes by the 
regulatory gene CAT8. Nucleic Acids Res. 24:2331-2337. 
 46 
CHAPTER 2 
 
 
Randez-Gil F., Bojunga N., Proft M. and Entian K.D. 1997. Glucose 
derepression of gluconeogenic enzymes in Saccharomyces cerevisiae 
correlates with phosphorylation of the gene activator Cat8p. Mol. Cell. Biol. 
17:2502-2510. 
 
Reiser J., Muheim A., Hardegger M., Frank G. and Fietcher A. 1994. Aryl-
alcohol dehydrogenase from white-rot fungus Phanerochaete 
chrysosporium. Gene cloning, sequence analysis, expression, and 
purification of the recombinant enzyme. J. Biol. Chem. 269:28152-28159 
 
Romanos M.A., Scorer C.A. and Clare J.J. 1992. Foreign gene expression in 
yeast: a review. Yeast. 8:423-488. 
  
Ruis H. and Schüller C. 1995. Stress signaling in yeast. BioEssays 17:959-965 
 
Schüller H.J. 2003. Transcriptional control of nonfermentative metabolism in the 
yeast Saccharomyces cerevisiae. Curr. Genet. 43:139-160 
 
Shuster J.J., Yu D., Cox R.V.L., Smith M. and Young E.T. 1986. ADR1-
mediated regulation of ADH2 requires an inverted repeat sequence. Mol. 
Cell. Biol. 6:1894-1902 
 
Stark M.J. 1996. Yeast protein serine/threonine phosphatases: multiple roles and 
diverse regulation. Yeast. 12:1647-1675.  
 
Song W, Treich I., Qian N., Kuchin S.and Carlson M. 1996. SSN genes that 
affect transcriptional repression in Saccharomyces cerevisiae encode SIN4, 
ROX3, and SRB proteins associated with RNA polymerase II. Mol. Cell. 
Biol. 16:115-120 
 
 47 
CHAPTER 2 
 
Taguchi A.K.W. and Young E.T. 1987. The identification and characterization of 
ADR6, a gene required for sporulation and for expression of the alcohol 
dehydrogenase II isozyme from Saccharomyces cerevisiae. Genetics 
116:523-530 
 
Taylor W.E. and Young E.T. 1990. cAMP-dependent phosphorylation and 
inactivation of yeast transcription factor ADR1 does not affect DNA binding. 
Proc .Natl. Acad. Sci. 87:4098-4102 
 
Thukral S.K., Eisen A. and Young E.T. 1991. Two monomers of yeast 
transcription factor ADR1 bind a palindromic sequence symmetrically to 
activate ADH2 expression. Mol. Cell. Biol. 11:1566-1577. 
 
Toda T., Cameron S., Sass P., Zoller M. and Wigler M. 1987. Three different 
genes in S. cerevisiae encode the catalytic subunits of the cAMP-
dependent protein kinase. Cell. 50:277-287. 
 
Tornow J. and Santangelo G.M. 1990. Efficient expression of the 
Saccharomyces cerevisiae glycolytic gene ADH1 is dependent upon a cis-
acting regulatory element (UASRPG) found initially in genes encoding 
ribosomal proteins. Gene 90:79-85 
 
Tornow J., Zeng X., Gao W. and Santangelo G.M. 1993. GCR1, a 
transcriptional activator in Saccharomyces cerevisiae, complexes with 
RAP1 and can function without its DNA binding domain. EMBO J. 12:2431-
2437 
 
Treitel M.A. and Carlson M. 1995. Repression by SSN6-TUP1 is directed by 
MIG1, a repressor/activator protein. Proc. Natl. Acad. Sci. 92:3132-3136. 
 
 48 
CHAPTER 2 
 
Treitel M.A., Kuchin S. and Carlson M. 1998. Snf1 protein kinase regulates 
phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae. Mol 
Cell Biol. 18:6273-6280 
 
Vallari R.C., Cook W.J., Audino D.C. Morgan M.J., Jensen D.E., Laudano 
A.P. and Denis C.L. 1992. Glucose repression of the yeast ADH2 gene 
occurs through multiple mechanisms, including control of the protein 
synthesis of its transcriptional activator, ADR1. Mol. Cell. Biol. 12:1663-
1673 
 
van den Berg M.A., de Jong-Gubbels P. and Steensma H.Y. 1998. Transient 
mRNA responses in chemostat cultures as a method of defining putative 
regulatory elements: application to genes involved in Saccharomyces 
cerevisiae acetyl-coenzyme A metabolism. Yeast. 14:1089-1104. 
 
van den Berg M.A. and Steensma H.Y. 1997. Expression cassettes for 
formaldehyde and fluoroacetate resistance, two dominant markers in 
Saccharomyces cerevisiae. Yeast. 13:551-559. 
 
Verdone L., Cesari F., Denis C.L., Di Mauro E. and Caserta M. 1997. Factors 
affecting Saccharomyces cerevisiae ADH2 chromatin remodeling and 
transcription. J. Biol. Chem. 272:30828-30834. 
 
Walker, G. M. 1998. Yeast metabolism. In: Yeast, Physiology and Biotechnology. 
pp. 203-264. John Wiley and Sons, UK. 
 
Walther K. and Schüller H.J. 2001. Adr1 and Cat8 synergistically activate the 
glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast 
Saccharomyces cerevisiae. Microbiol. 147:2037-2044. 
 
 49 
CHAPTER 2 
 
Wehner E.P., Rao E. and Brendel M. 1993. Molecular structure and genetic 
regulation of SFA1, a gene responsible for resistance to formaldehyde in 
Saccharomyces cerevisiae, and characterization of its protein product. Mol. 
Gen. Genet 237:351-388 
 
Wilson W.A., Hawley S.A. and Hardie D.G. 1996. Glucose 
repression/derepression in budding yeast: SNF1 protein kinase is activated 
by phosphorylation under derepressing conditions, and this correlates with 
a high AMP:ATP ratio. Curr. Biol. 6:1426-1434. 
 
Yang X., Jiang R. and Carlson M. 1994. A family of proteins containing a 
conserved domain that mediates interactions with the yeast SNF1 protein 
kinase complex. EMBO J. 13:5878-5886 
 
Yoshimoto H., Ohmae M. and Yamashita I. 1992. The Saccharomyces 
cerevisiae GAM2/SIN3 protein plays a role in both activation and repression 
of transcription.  Mol. Gen. Genet. 233:327-330. 
 
Young E.T. and Pilgrim D. 1985.  Isolation and DNA sequence of ADH3, a 
nuclear gene encoding the mitochondrial isozyme of alcohol 
dehydrogenase in Saccharomyces cerevisiae. Mol. Cell. Biol.  5:3024-3034.  
 
Young E.T., Saario J., Kacherovsky N., Chao A., Sloan J.S. and Dombek 
K.M. 1998. Characterization of a p53-related activation domain in Adr1p 
that is sufficient for ADR1-dependent gene expression. J. Biol. Chem. 
273:32080-32087. 
 
Young E.T., Kacherovsky N. and Van Riper K. 2002. Snf1 protein kinase 
regulates Adr1 binding to chromatin but not transcription activation. J. Biol. 
Chem. 277:38095-38103.  
 
 50 
CHAPTER 2 
 
Young E.T., Dombek K.M., Tachibana C. and Ideker T. 2003. Multiple 
pathways are co-regulated by the protein kinase Snf1 and the transcription 
factors Adr1 and Cat8. J. Biol. Chem. 278:26146-26158.  
 
Yu J., Donoviel M.S. and Young E.T. 1989. Adjacent activating sequence 
elements synergistically regulate transcription of ADH2 in Saccharomyces 
cerevisiae. Mol. Cell. Biol. 9:34-42 
 
Yuan D.S. 2000. Zinc regulated genes in Saccharomyces cerevisiae revealed by 
transposon tagging. Genetics 156:45-58 
 
Zimmermann F.K., Kaufmann I., Rasenberger H. and Haubetamann P. 1977. 
Genetics of carbon catabolite repression in Saccharomyces cerevisiae: 
genes involved in derepression process. Mol. Gen. Genet. 151:95-103 
 
 
 51 
CHAPTER3 
 
CHAPTER3 
 
THE CONSTRUCTION OF THE EXPRESSION CASSETTE 
 
 
1. ABSTRACT 
 
ADH2 encodes for the main alcohol dehydrogenase isozyme that catalyses the 
conversion of ethanol to acetaldehyde during ethanol assimilation. Expression of 
this gene is known to be under glucose repression and is derepressed by growth 
on ethanol and other non-fermentable carbon sources. Results obtained by du 
Preez et al. (2001) showed, however, that this gene was repressed by growth on 
ethanol. These results were obtained using a multicopy expression vector with a 
promoter-terminator expression cassette derived from the S. cerevisiae ADH2 
gene. The aim of this study is the investigation of ethanol repression of ADH2 in 
the genome of S. cerevisiae. To achieve this, an expression cassette using LacZ 
as a reporter gene was constructed using the YIp356R shuttle vector. A 1000 bp 
up and downstream region, flanking the open reading frame of ADH2, was fused to 
the 5’-end and 3’-end of the LacZ gene. Attempts to replace the ADH2 ORF with 
the expression cassette were unsuccessful therefore the expression cassette was 
cloned into YIplac211 and integrated into the S. cerevisiae genome.  
 
2. INTRODUCTION 
 
The ADH2 gene encodes an enzyme which has been identified as the main 
alcohol dehydrogenase isozyme involved in ethanol assimilation (Ciriacy, 1975; 
Ciriacy, 1979; Lutstorf and Megnet, 1968). A study conducted by du Preez et al. 
(2001) revealed that the expression of ADH2 is, however, repressed under high 
ethanol growth conditions. In this study an episomal multicopy plasmid with a 
promoter-terminator expression cassette derived from the S. cerevisiae ADH2 
gene was used and ADH2 expression was studied using XYN2 as a reporter gene. 
 52 
CHAPTER3 
Thus the results observed could be due to the presence of negative regulatory 
elements, such as downstream repression sequences in the expression cassette 
and the possibility of multicopy effects causing transcriptional misregulation.  
 
Therefore it became a necessity that the effect of ethanol on the expression of this 
gene be studied in the native complete ADH2 gene, however, because of high 
sequence similarity between the ADH2 and ADH1 genes it is difficult to 
discriminate between the mRNA sequences of these genes, rendering it virtually 
impossible to use northern blots to investigate glucose repression of ADH2 without 
the interference of ADH1 mRNA.   
 
The aim of this study was the investigation of the effect of ethanol concentration on 
the expression of ADH2 by constructing an expression cassette with LacZ as a 
reporter gene. To accomplish this, an expression cassette was constructed where 
the LacZ gene is flanked by the upstream and downstream regions of ADH2.  This 
was followed by an attempt to replace the ADH2 gene in vivo through homologous 
recombination.  Due to the non-selective properties of LacZ, the donor cells used 
contained an ADH2 deletion, where the coding part of this gene was replaced with 
URA3   (URA3::ADH2)  (Fig. 1).  
 
Expression  Cassette Upstream Region   LacZ Downstream Region 
 
 
adh2∆::URA3 Upstream Region URA3 Downstream Region 
 
FIGURE 1. Scheme representing homologous recombination between the 
expression cassette and adh2∆::URA3. Attempts to replace ADH2 
ORF with the cassette were unsuccessful. 
 
 However, an attempt to replace the ORF of ADH2 with the expression cassette 
was unsuccessful and a second approach was used where S. cerevisiae was 
transformed with an integrative plasmid (YIplac211) containing the expression 
cassette.   
 53 
CHAPTER3 
3. MATERIALS AND METHODS  
 
3.1. STRAINS USED 
 
Saccharomyces cerevisiae W303 (MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 
ade2-1 can1-100 GAL SUC2); Escherichia coli Top 10 (Invitrogen, USA). 
 
3.2. ISOLATION OF ESCHERICHIA COLI GENOMIC DNA 
 
Bacterial cells were grown overnight in 5 ml Luria-Bertani (LB) broth (Sambrook et 
al., 1989) for 12 h. The culture was transferred into a 1.5 ml microcentrifuge tube, 
centrifuged for 5 min at 13,000 rpm and 500 ml Trizol (life technologies) added to 
the culture, mixed by pipetting up and down and centrifuged for 10 min at 13,000 
rpm. The supernatant was transferred into a new microcentrifuge tube and DNA 
was precipitated by adding 0.5 ml 100% ethanol and incubated for 5 min at room 
temperature. DNA was spooled with a pipette tip and transferred to another tube, 
centrifuged briefly, washed twice with 70% ethanol, centrifuged for 5 min at full 
speed, dried and resuspended in TE containing 50 µg RNAse ml-1. 
 
3.3. ISOLATION OF S. CEREVISIAE GENOMIC DNA 
 
Yeast cells were grown overnight in a 5 ml YEPD medium at 30 ºC, centrifuged for 
1 min at full speed and washed once with 1ml sterile water. The cells were 
resuspended in 500 µl lysis buffer (100 mM Tris pH 8, 50 mM EDTA, 1% SDS), 
acid washed glass beads were added and then mixed for 2 min by vortex. The 
liquid phase was transferred to another tube, 275 µl of 7 M ammonium acetate (pH 
7.0) was added and the tube was incubated at 65 ºC for 5 min then placed on ice 
for 5 min. Chloroform (500 µl) was added, mixed by vortex and centrifuged for 2 
min. The top phase of the supernatant was transferred to a new tube and DNA was 
precipitated with 1 ml isopropanol, incubated for 5 min at room temperature and 
 54 
CHAPTER3 
centrifuged for 5 min. The pellet was washed with 700 µl 70% ethanol, centrifuged 
for 10 min at 14000 rpm at 4 ºC, dried and resuspend in 50 µl water. 
 
3.4. AMPLIFICATION OF THE DOWNSTREAM AND UPSTREAM REGION 
(FLANKING REGIONS) OF ADH2, LACZ, UPSTREAM+LACZ, 
DOWNSTREAM+LACZ AND THE EXPRESSION CASSETTE 
 
TABLE 1.  Primers used in this study 
 
PCR Product Forward Orientation Reverse Orientation 
 ADH2-1F  ADH2-1R  
ADH2 
5'-CGG ATC AAC TGG CAC CAT 5'-TTT TCT CCC TCA CTG CAT 
CT-3' CCC-3 
Upstream  ADH2-4F  ADH2-2R  
Region 5’-ACT GCA GTT CCG GAT GAG 5’-TTG AGT TTC TGG TCT AGA 
GTT CCA G-3’ CAT TGT G-3’ 
Downstream  ADH2-2F  ADH2-4R  
Region 
5’-AGT AGG TAC CAT CTT TAG 5’-AGT CGA CTT TTC TCC CTC 
ATG ACA GTG-3’ ACT GCA TCC-3’ 
LacZ LACZ-4F  LACZ-4R  
5’-ATC TAG AAC CAT GAT TAC 5’-GGT ACC TTA TTT TTG ACA 
GGA TTC ACT G-3’ CCA GAC CAA C-3’ 
LacZ+Upstream ADH2-4F  LACZ-4R  
Region 5’-ACT GCA GTT CCG GAT GAG 5’-GGT ACC TTA TTT TTG ACA 
GTT CCA G-3’ CCA GAC CAA C-3’ 
Expression ADH2-4F  ADH2-1R  
construct 5’-ACT GCA GTT CCG GAT GAG 5'-TTT TCT CCC TCA CTG CAT 
GTT CCA G-3’ CCC-3 
 
 55 
CHAPTER3 
The general method followed for amplification is as follows. The amplification was 
performed in a total volume of 50 µl. The following reagents were added: dNTPs 
(final concentration of 0.2 mM), reverse primer (final concentration of 2 pmol), 
forward primer (final concentration of 2pmol), genomic/plasmid DNA, depending on 
the PCR product  (final concentration of 500 ng µl-1), 10X PCR buffer and sterile 
water up to a final volume of 50 µl. All the reagents were added in a 0.2 ml PCR 
tube and incubated for 5 min at 94 ºC before adding the enzyme mix (Taq 
polymerase) or Expand Long Template DNA polymerase mix (for PCR products 
with ≥ 3000 bp band sizes) to a final concentration of 0.02 U µl-1 (Roche).  
 
The polymarase chain reaction was performed under the following cycling 
programs for 30 cycles: 
Amplification of ADH2: 
Denaturation at 94 ºC for 30 sec, annealing at 54 ºC for 30 sec and 
extension at 68 ºC for 2.5 min repeated 30 times followed by a final 
step of 10 min at 68 ºC to complete elongation.  
Amplification of the flanking regions of ADH2: 
Denaturation at 94 ºC for 30 sec, annealing at 58 ºC for 30 sec and 
extension at 72 ºC for 1 min.  
LacZ amplification: 
Denaturation at 94ºC for 30 sec, annealing at 57 ºC for 30 sec and 
extension at 68 ºC for 2.5 min.  
Upstream region+LacZ: 
Denaturation at 94 ºC for 30 sec, annealing at 59 ºC for 30 sec and 
extension at 68 ºC for 2.5 min.  
Amplification of the expression cassette: 
Denaturation at 94 ºC for 30 sec, annealing at 59 ºC for 30 sec and 
extension at 68º C for 4 min.  
Confirmation of transformation by PCR:  
Denaturation at 94 ºC for 30sec, annealing at 59 ºC for 30 sec and 
extension at 68 ºC for 2.5 min.  
 
 56 
CHAPTER3 
Confirmation of the correct PCR product was achieved through restriction digest  
(to confirm the restriction sites included in the PCR product) and by subjecting the 
DNA to gel electrophoresis and visualisation under UV radiation. A 0.1% ethidium 
bromide stained 1% agarose gel was prepared by dissolving 0.5 g agarose gel 
through boiling in 50 ml TAE buffer (50X stock solution: 24.2 g Tris base, 57.1 ml 
glacial acetic acid, 37.2 g Na2EDTA.2H2O, up to 1 liter. pH 8.0).  With gel 
electrophoresis band sizes were compared with λDNA digested with EcoRI and 
HindIII (λIII). The profile of this molecular weight marker is given in Fig. 2.  
 
 
FIGURE 2. Profile of molecular weight marker λIII with corresponding sizes.  
 
3.5. CLONING OF PCR PRODUCTS, CONSTRUCTION OF THE EXPRESSION 
CONSTRUCT AND TRANSFORMATION 
 
Three plasmid vectors were used for cloning of PCR products: pGem-T-Easy was 
used to clone all PCR products, YIp356R shuttle vector (Myers et al., 1986) to 
clone the upstream region and downstream region of ADH2 for expression 
cassette construction and YIplac211 to clone the expression cassette followed by 
transformation into yeast. The expression cassette was constructed by digesting 
the flanking regions of ADH2 with XbaI/SalI and NcoI/NsiI restriction enzyme 
combination for cloning the upstream region and downstream region, respectively, 
into YIp356R. 
 57 
CHAPTER3 
 
DNA ligation was performed as follows. The plasmids and desired DNA fragments 
were added together in a 1:3 molar concentration together with ligation buffer to a 
final working solution of 1 X (from a 10 X concentrated stock solution supplied by 
the manufacturers). 1 µl T4 DNA ligase (final concentration of 0.2 U ml-1) was 
added and sterile distilled water up to a final volume of 20 µl. This was incubated 
at 14-16 °C overnight. 
 
The vectors were transformed into Top 10 E.coli competent cells. Competent cells 
were prepared according to the method of Inoue et al. (1990). Bacterial 
transformation was performed as follows: 
Plasmid DNA was added to 80 µl competent E.coli cells, placed on ice for 30 min 
followed by 40 sec at 42 °C and then for 2 min on ice. 800 µl LB media (containing 
100 µl of 1 M glucose per 5 ml media) was added. This was incubated at 37 °C for 
1 hour, and centrifuged for 2 min at 4000 rpm. The supernatant was removed and 
100 µl LB was added to the cell pellet. The cells were plated on LB plates 
containing Ampicillin, X-gal and IPTG (15 g agar to 1 liter of LB, allowed the 
medium to cool to 50 oC before adding Ampicillin to a final concentration of 100 µg 
ml -1, 2 ml of 100 mM IPTG l-1 and 400 µl X-gal l-1) and incubated overnight at 37 
°C. White colonies were inoculated into 5 ml LB medium containing Ampicillin (100 
µg ml-1 medium), and incubated overnight at 37 oC shaker. 
 
Small scale plasmid isolations were performed by taking 1 ml of the transformed E. 
coli cells grown overnight and centrifuged at 13000 rpm for 2 min. The pellet was 
resuspended in 200 µl STET buffer (16 g sucrose, 10 ml Triton X-100, 10 ml 1M 
Tris-HCl pH8 and 20 ml 0.5M EDTA pH 8 dissolved in 200 ml distilled water, 
autoclaved and stored at 4 oC), after the addition of 4 µl lysozyme the tubes were 
placed in boiling water bath for 60 sec followed by incubation on ice for 10 min and 
centrifuged for 10-15 min (13000-15000 rpm). The pellet was removed with a 
toothpick and 200 µl Isopropanol was added to the supernatant. This was left for 
10 min at -20 °C, centrifuged for 10 min at ~15000 rpm. The pellets were dried for 
10-15 min and resuspended in 50 µl TE + RNAse (50 µg ml-1). Screening of the 
 58 
CHAPTER3 
correct clones were performed through restriction digest with relevant restriction 
enzymes or PCR and assessed by gel electrophoresis. YIplac211, containing the 
complete expression cassette, was linearised by digesting with StuI followed by 
transformation into S. cerevisiae. 
 
Yeast transformation was performed as follows: The cells were inoculated in 5 ml 
YEPD medium (20 g peptone l-1, 10 g yeast extract l-1 and 20 g glucose l-1) and 
incubated overnight at 30 ºC. The cells suspension was transferred into 
microcentrifuge tubes and centrifuged at 4000 rpm for 2 min at room temperature 
and the supernatant was discarded. The cells were washed in 10 ml LiOAc/TE 
solution (0.1 M LiOAc, 10 mM Tris-HCl pH 8.0 and 1 mM EDTA), centrifuged at 
4000 rpm for 2 min at room temperature and the supernatant was discarded. The 
pellet was resuspended in 1 ml LiOAc/TE solution and mixed carefully by hand. 
Plasmid DNA (5-10 μl) together with 5 μl sheared salmon sperm DNA (to increase 
the efficiency of transformation) was added to 100 μl of cell suspension followed by 
incubation for 5 min at room temperature. 280 μl of PEG-4000 solution (50% PEG 
in LiOAc/TE solution) was added to the cell suspension and incubated for 45 min in 
a 30 °C waterbath. DMSO (40 μl ) was added and mixed by inverting the tube 4-6 
times. Heat shock was performed by placing the cell suspension in a 42 °C 
waterbath for 5 min followed by centrifugation at 4000 rpm for 2 min. The 
supernatant was discarded and the cells were washed by resuspending in 1 ml 
sterile dH2O and centrifuged at 4000 rpm for 2 min. The supernatant was 
discarded, the cell were resuspended in 0.5 ml sterile dH2O and 100 μl was plated 
on -URA selective medium (6.7g yeast nitrogen base l-1 without amino acids, 15 g 
agar l-1, 1 pellet NaOH, 20 g glucose l-1 and 100 ml of 10X amino acid dropout 
solution without L-Uracil (300 mg L-Isoleucine, 1500 mg L-Valine, 200 mg L-
Adenine hemisulfate salt, 200 mg L-Arginine HCL, 300 mg L-Lysine, 200 mg L-
Methionine, 500 mg L-Phenylalanine, 2000 mg L-Threonine, 300 mg L-Tyrosine, 
200 mg L-Histidine HCL monohydrate, 1000 mg L-Leucine and 200 mg L-
Tryptophan per L) and incubated for 3-5 days at 30 ºC. For 5-Fluorotic acid (FOA) 
selection, plates were prepared by media supplemented with 0.1-0.2% 5-FOA and 
10 mg Uracil l-1.  
 59 
CHAPTER3 
 
3.6. DETECTION OF Β-GALACTOSIDASE ACTIVITY FROM POSITIVE COLONIES 
 
Yeast colonies, obtained from the transformation with YIplac211 plasmid 
containing the expression construct, were streaked on plates, which derepresses 
the ADH2 promoter thus enabling the detection of β-galactosidase activity resulting 
in positive colonies turning blue. Ethanol and galactose were used as carbon 
sources [6.7 g/l yeast nitrogen base without amino acids, 100 ml of 10X dropout 
solution without L-Uracil, 20 g galactose l-1 or 30 ml ethanol l-1, pH 7.0]. After 
autoclaving, 1M KH2PO4 (pH 7) to a final concentration of 50 mM (50 ml l-1) and 2 
ml l-1 of X-Gal (of a 20 mg ml-1 solution in DMFA) was added before pouring the 
plates. After streaking out the plates were incubated at 30 ºC for 2 days or until the 
blue colour appeared. The positive colonies were selected for preliminary β-
galactosidase assay.  
 
3.7. β-GALACTOSIDASE ASSAY 
 
The method of Rose and Botstein (1983) was used for β-galactosidase 
determinations. The culture was prepared by inoculating 100 μl of the preinoculum 
(yeast cells inoculated in YEPD broth and incubated for 12 h at 30 ºC) into YEP 
media containing three different carbon sources to compare the expression of β-
galactosidase under both repressing (glucose) and derepressing (galactose and 
ethanol) growth conditions. Cells were grown until an OD595 1-1.5 was reached, 
harvested by centrifugation and the supernatant was discarded. The cells were 
washed with 2 ml ice cold buffer Z (16.1 g Na2HPO47H2O l-1, 5.5 g NaH2PO4H2O 
l-1, 0.75 g KCl l-1 and 0.246 g MgSO47H2O l-1; pH 7.0), pelleted and resuspended 
in the same buffer. Cold glass beads were added up to the level just below the 
meniscus of the liquid and 12.5 μl of 40 mM phenylmethylsulfonyl fluoride (PMSF) 
was added, vortexed twice for 1 min with cooling on ice in between followed by the 
addition of 0.5 ml cold buffer Z. The liquid phase was transferred to a new 
microcentrifuge tube and centrifuged at 14 000 x g for 15 min at 4oC. The 
supernatant was transferred to another microcentrifuge tube for enzymatic 
 60 
CHAPTER3 
determinations. For the assay o-nitrophenyl-β-D-galactopyranoside (ONPG) was 
used as a substrate for β-galactosidase enzyme. 100 μl of crude extract was 
added to 900 μl of Z buffer and incubated for 5 min at 28 ºC. The reaction was 
started through addition of 200 μl ONPG and incubated at 28 ºC for 10 min. The 
reaction was stopped by adding 0.5 ml Na2CO3 and the OD was measured at A420. 
The Miller units were calculated using the following formula:
              
Units/ml =  (delta A420/min) (Vr) (D)
 0.0045 x extract volume x time 
 
 61 
CHAPTER3 
4. RESULTS AND DISCUSSIONS 
 
4.1. CONSTRUCTION OF THE EXPRESSION CASSETTE 
 
The gene encoding ß-galactosidase (LacZ) of E.coli has been widely used as a 
reporter gene in many different prokaryotic and eukaryotic organisms. In particular, 
this gene has proven useful for studying gene expression in the yeast S. 
cerevisiae. ß-galactosidase enzyme catalyzes the hydrolysis of lactose into 
glucose and galactose and many beta-D-galactopyranosides.  Advantages in using 
the LacZ gene include sensitive biochemical assays to detect β-galactosidase 
activity (Ausubel et al. 1987) and an associated blue-colour colony phenotype 
when cells are grown in the presence of the chromogenic substrate 5-bromo-4-
chloro-3-indolyl-β-D-galactoside (X-gal) (Kudla et al. 1988). β-galactosidase 
cleaves X-gal into galactose resulting in the formation of a blue insoluble product. 
In order to study the expression of ADH2 in S. cerevisiae, an expression cassette 
was constructed using the E. coli LacZ gene as a reporter gene. Detection of the 
blue-colour phenotype on colonies of S. cerevisiae is dependent on the pH of the 
growth medium, with optimal conditions at pH 7.0 (Rose et al. 1990). The blue 
colonies obtained after yeast transformation confirmed that the cassette was 
successfully constructed. 
 
4.2. AMPLIFICATION OF THE ADH2 FLANKING REGIONS AND LACZ GENE 
 
To construct the expression cassette, the genomic DNA of S. cerevisiae (W303) 
was isolated and used as a template for the amplification of the ADH2. This was 
carried out by amplifying the ORF with ~1000 bp on either side yielding a product 
of approximately 3000 bp (Fig. 2). To confirm successful amplification the PCR 
product was subjected to gel electrophoresis and a single band was observed (Fig. 
3). 
 62 
CHAPTER3 
 
 1 λIII 
21226 bp 
5148 bp 
~ 3000 bp 
2027 bp 
947 bp 
 
 
FIGURE 3. PCR product after amplification of ADH2 together with ~1000 bp region 
up – and downstream from the open reading frame, lane 1 and λIII 
molecular weight marker, lane 2. 
 
The PCR product was cloned into a pGEM-T-Easy vector and transformed into 
competent E. coli cells. Small scale plasmid isolation were performed on white 
colonies and digested with HindIII/EcoRI restriction enzymes to confirm whether 
the ADH2 was successfully cloned. The expected band sizes of approximately 
~3000 bp, ~1750 bp and ~1250 bp were obtained (Fig. 4). 
 63 
CHAPTER3 
  1       2  λIII 
21226 bp 
5148 bp 
~ 3000 bp 
~ 1750 bp 2027 bp 
~ 1250 bp 
947 bp 
 
 
FIGURE 4.  Lane 1 and 2 represents the expected bands from ADH2 in pGEM-T-
Easy after digestion with EcoRI and HindIII.  Lane 3 contains λIII DNA 
marker 
 
The pGEM-T-Easy plasmid containing the complete ADH2 gene together with the 
up and downstream regions was used as a template to amplify the upstream and 
downstream regions separately after numerous attempts to clone the LacZ 
between the flanking in the pGEM-T-Easy plasmid were unsuccessful. The primers 
used for the amplification of the upstream region where designed in such a way 
that they incorporated a PstI restriction site at the 5’ end and an XbaI site at the 3’ 
end and those used for the amplification of the downstream region incorporated an 
Acc65I site at the 5’ end and a SalI site at the 3’ end to facilitate cloning. The 
expected bands of ~991 bp and ~943 bp of both the upstream and downstream 
regions respectively were obtained (Fig. 5).  
 64 
CHAPTER3 
   1   2       λIII      3 
 
Upstream Region Downstream 
~ 991 bp Region ~943 bp 
 
 
FIGURE 5. Lane 1 and 3 represents the expected bands after amplification of the 
upstream and downstream regions of ADH2.  Lane 2 contains λIII DNA 
marker. 
 
Due to non specific amplicons obtained during amplification, the PCR product with 
the correct size were isolated from the gel and purified using GFXTM PCR DNA and 
Gel Band purification kit (Amersham) followed by cloning into pGEM-T-Easy. The 
cloned pGEM-T-Easy plasmid was transformed into Top 10 competent E.coli cells. 
White colonies were selected for plasmid isolation and purified plasmid DNA was 
digested with EcoRI for confirmation of the correct insert. The expected bands of 
~3000 bp representing the plasmid backbone and  ~991 bp (upstream region) and 
~943 bp (downstream region) were observed for both upstream and downstream 
region cloned in pGEM-T-Easy (Fig. 6 A & B respectively). 
 65 
CHAPTER3 
  A B 
     1      2     3  λIII 1     2     3   λIII 
  
pGEM-
pGEM- T-Easy 
T-Easy  
 
Downstream  
Region 
Upstream  
Region 
 
            
 
FIGURE 6. Lanes 1 to 3 in both A and B represents the correct band profile 
expected after cloning the upstream and downstream region. in pGEM-
T-Easy. A- upstream region and B-downstream region. 
 
The LacZ reporter gene was amplified from wild type E.coli strain using primers, 
which incorporated an XbaI restriction site and an Acc65I restriction site at the 3' 
end and 5' end of the gene respectively to facilitate ligation of LacZ with the ADH2 
flanking region. Numerous approaches were employed to directly ligate the 
upstream and downstream regions of ADH2 with the LacZ gene amplified from E. 
coli without any success. An alternative approach was attempted using a shuttle 
vector (YIp356R) generally used for expression studies to construct the expression 
cassette. 
  
4.3. CONSTRUCTION OF THE EXPRESSION CASSETTE USING YIp356R 
There are a number of shuttle vectors, which can be used for gene expression 
studies in yeasts. Most of these expression vectors utilise the initiation codon of 
the gene under study. It was therefore important to select a shuttle vector, which 
will enable in frame cloning of the upstream region of ADH2 with the reporter gene. 
YIp356R shuttle vector (Myers et al., 1986) was selected because when the 
 66 
CHAPTER3 
upstream region of ADH2 is fused to the 5' end of the LacZ gene in the shuttle 
vector using the incorporated XbaI restriction site, the correct reading frame of 
LacZ is maintained (Fig. 7).   
Downstream region  Upstream region  
 
FIGURE 7. Construction of the expression cassette from the YIP356R shuttle 
vector. The LacZ gene in this plasmid is not functional in both E.coli 
and S. cerevisiae, unless when a functional promoter is cloned at the 
5’ end of this gene in the plasmid. The expression cassette was 
constructed through restriction enzyme digestion with relevant 
restriction enzyme to clone the upstream and downstream region. 
 67 
CHAPTER3 
 
To achieve this, pGEM-T-Easy containing the upstream region and the YIp356R 
shuttle vector were both digested with XbaI/SalI. The digestion products were 
subjected to gel electrophoresis for selection of appropriate bands [~ 991 bp 
(Upstream region) and ~7086 bp (YIp356R plasmid with LacZ). The bands were 
isolated, purified, and ligated overnight followed by transformation into competent 
E.coli cells. Colonies were selected for plasmid isolation and confirmation was 
done by digesting the plasmid with BamHI/HindIII and the correct bands were 
observed (~7086 bp plasmid and ~991bp upstream region) (Fig. 8).  
 
1 2 3 4  5 6 λIII 
21226 bp 
5148 bp 
YIp356R 
2027 bp 
Upstream 947 bp 
Region 
 
 
FIGURE 8. Confirmation of cloning of the upstream region at the 5’ end of LacZ in 
YIp356R shuttle vector by digestion with BamHI/HindIII. Lane 1-3 
represents the correct band profile. 
 
Additional confirmation was obtained through amplification of the ADH2 upstream 
region and the upstream region fused to LacZ gene using YIp356R shuttle vector 
with the cloned upstream region as a template.  The expected bands were 
observed (~991 bp & ~4057 bp) (Fig. 9). 
 68 
CHAPTER3 
 
 1        2       3       λIII     5        4 
Upstream Region 
+LacZ (~4057 bp) 
Upstream Region 
(~991 bp) 
 
 
FIGURE 9. Confirmation of cloning of the upstream region at the 5’ end of LacZ in 
YIp356R shuttle vector by amplification of the upstream region (Lane 1 
to 3) and upstream region fused to LacZ (Lane 4).  
 
Before the downstream region of ADH2 was cloned in the YIp356R, containing the 
upstream region fused to LacZ, this shuttle vector was transformed into S. 
cerevisiae to confirm that the cloning of the ADH2 upstream region was in frame 
with the LacZ gene. The expression of LacZ gene in the yeast was qualitatively 
determined by detecting β-galactosidase presence using the X-Gal agarose 
overlay assay method. The colonies obtained were streaked out on a medium that 
allows for the detection of β-galactosidase activity, the media contains a non-
fermentable carbon source that allows the derepression of the ADH2 promoter 
(ethanol and galactose were used) and 5-bromo-4-chloro-3-indolyl-ß-D-galactoside 
(X-gal), which is a substrate for β-galactosidase. Therefore colonies with the 
functional LacZ expression construct should be blue in colour. The blue colour 
appeared after 2 h confirming that the upstream region was incorporated in frame 
with the LacZ resulting in the expression of β-galactosidase.  
 
To complete the expression construct, the downstream region of ADH2 was cloned 
in the shuttle vector already containing the upstream region at the 3’ end of LacZ. 
 69 
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To clone the downstream region in the shuttle vector both the shuttle vector and 
the pGEM-T-Easy containing the downstream region were digested with NcoI/NsiI. 
Two bands were expected from the pGEM-T-Easy digestion and two bands (~600 
bp &~ 6500 bp) from the shuttle vector. However the results obtained from the 
digestion of YIp356R with the upstream region showed additional bands indicating 
partial digestion occurred (Fig. 10).  
 
 1        2      λIII 
21226 bp 
~5300 bp 5148 bp 
2027 bp 
947 bp 
~600 bp 564bp 
 
 
FIGURE 10. Band profile obtained after the digestion of YIp356R+Upstream 
Region with NcoI/NsiI. Arrows indicate the expected bands after 
digestion. 
 
The digestion was repeated and the same profile was observed. To overcome this 
problem the band with the expected size of the YIp356R vector containing the 
upstream region fused to the LacZ gene (~6500 bp) was isolated from the gel. The 
1000 bp fragment consisting of the downstream was ligated into this linearised 
fragment. Ligation was performed and the ligation product was transformed into 
Top 10 E.coli competent cells. Since the positive clones expressed LacZ, blue 
colonies were selected for plasmid isolation. The plasmid was digested with SalI to 
select for positive clones. SalI was selected for confirmation and the presence of 
 70 
CHAPTER3 
two bands (~5300 bp and ~3170 bp) was an indication that the downstream region 
was fused to the 5’-side of LacZ (Fig. 11).  
 
 
 1         2         3          4       λIII   
 
21226 bp 
~5300 bp 5148 bp 
 
 ~3170 bp 
2027 bp 
947 bp 
 
 
FIGURE 11. Confirmation of the cloning of downstream region at the 3’end of LacZ 
in YIp356R shuttle vector. Lane 1 represents the expected band profile. 
 
To confirm that the complete construct was obtained, a PCR was performed to 
firstly, detect the downstream region (Fig. 12) and secondly, the LacZ gene flanked 
by the ADH2 up- and downstream regions (Fig. 13).  
 71 
CHAPTER3 
 
1 2  λIII 
21226 bp 
5148 bp 
2027 bp 
Downstream 
Region (943 bp) 947 bp 
 
 
FIGURE 12. Amplification of the downstream region from YIp356R after ligation of 
this region to the 3’-side of the LacZ gene. Lane 1-2 represents the 
amplified downstream region from two positive clones. 
 1 λIII 
21226 bp 
LacZ::ADH2  5148 bp 
 
2027 bp 
 
 
FIGURE 13. Amplification of the complete expression cassette containing the 
LacZ::ADH2 from YIp356R. Lane 1 represents the correct PCR 
product and lane 2 represents the molecular marker. 
 
 72 
CHAPTER3 
To further confirm that the PCR product obtained when the complete construct was 
amplified was correct, this amplicon was subjected to restriction analysis.  The 
PCR product was digested with EcoRV and SacI. The expected bands were 
observed in both digestions (~3067bp & ~2159bp for EcoRV digestion and ~296 
bp, ~2007 bp & ~2340 bp for SacI digestion) (Fig.14) confirming the complete 
construction of the expression cassette. 
 
 1 2 λIII 
21226 bp 
5148 bp 
2027 bp 
947 bp 
 
 
FIGURE 14. Digestion of the LacZ::ADH2 PCR product with with EcoRV (Lane 1) 
and SacI (Lane 2) The top band in lane 2 is the PCR product. 
 
4.4. CONFIRMATION OF THE EXPRESSION CASSETTE 
 
Before using the LacZ expression cassette to replace the URA3::ADH2 in the 
Saccharomyces cerevisiae genome, the expression construct was cloned into 
YEplac112 (Gietz and Sugino, 1988), an episomal plasmid shuttle vector with 
TRP1 as an auxotrophic marker. This was done to ensure that the LacZ 
 73 
CHAPTER3 
expression construct would be functional when transformed into yeast. The 
plasmid was digested with SmaI and a PCR product containing the LacZ flanked 
by the ADH2 up and downstream regions was ligated to the linearised plasmid and 
transformed into Top 10 E.coli competent cells. The plasmid isolated was digested 
with EcoRI for confirmation. Four bands expected (~4990 bp, ~3000 bp & 2X 
~1000 bp) were obtained (Fig.15).  
 
 1 2 λIII 4 
~4990 bp 
~3000 bp  
 
 
 
~1000 bp 
 
 
FIGURE 15.  Digestion of YEplac112 containing LacZ::ADH2 with EcoRI.  Lane 2 
represents the correct band profile.  
 
The positive plasmid was transformed into S. cerevisiae (W303) and grown on 
dropout media without tryptophan for selection of transformed colonies. The 
colonies obtained were streaked on a medium that allows for the detection of β-
galactosidase activity. However, after several attempts β-galactosidase activity 
could not be detected. To confirm that the colonies obtained from the yeast 
transformation contained the plasmid, the colonies were plated out on different 
dropout media and incubated for 4 days. The following dropout media was used: -
URA, -LEU and –TRP plates. The only colonies, which grew, were the ones in –
 74 
CHAPTER3 
TRP plates. This confirmed that the transformation was successful but there was 
something that prevented the expression of the LacZ gene. Since it was confirmed 
that the upstream region, when ligated to the LacZ in the YIp356R leads to the 
expression of the gene it was then concluded that the lack of expression was due 
to the YEplac112 that was used for cloning the expression construct. It was thus 
decided to clone the expression construct in another shuttle vector, YIplac211 
(Gietz and Sugino, 1988), an integrating plasmid with URA3 as an auxotrophic 
marker. To clone the expression construct, the plasmid was linearised by digesting 
with SmaI followed by the ligation of the complete expression construct into this 
vector. After transformation into E. coli Top10 cells, plasmid DNA was isolated and 
digested with EcoRI and the expected bands were observed (~3790 bp, ~3000 bp 
& 2X ~1000 bp) (Fig.16). 
 
 1 2 λIII 
 
21226 bp 
5148 bp 
~3700 bp 
~3000 bp   
 
 2027 bp 
 
 
 
 
~1000bp 
947 bp 
 
 
FIGURE 16. Digestion of YIplac211 cloned with LacZ::ADH2 with EcoRI.  Lanes 1 
and 2 represent the correct banding profile. 
 
The YIplac211 plasmid containing the expression cassette was linearised (to allow 
integration into the yeast genomic DNA at the URA3 locus) and transformed into S. 
cerevisiae followed by plating on URA– plates for selection of positive clones. The 
 75 
CHAPTER3 
colonies obtained were streaked out on X-gal plates, which allows for the detection 
of β-galactosidase activity. The detection of a blue colour in all the transformants 
screened confirmed that the expression construct was successfully constructed 
and integrated into the genome of S. cerevisiae (Fig.17).  
 
 
 
FIGURE 17. Growth of transformants on media containing X-gal. Blue color is 
indicative of active expression of β-galactosidase confirming the 
expression of this enzyme through regulation of the ADH2 promoter. 
 
4.5. PRELIMINARY Β-GALACTOSIDASE ACTIVITY ASSAY FROM THE 
TRANSFORMED YEAST 
 
The positive colonies were selected for preliminary assay of β-galactosidase 
activity. The enzyme assay was done under repressed and derepressed state of 
the ADH2 promotor. The enzyme assay was performed by selecting five colonies 
and was grown in media containing three different types of carbon sources. For the 
enzyme assay under repressed conditions, YEPD medium was used since it 
contains glucose as a carbon source. For the assay under derepressed conditions 
ethanol and galactose were used as carbon sources. The results obtained 
confirmed that the expression of LacZ is under glucose repression, the lowest 
 76 
CHAPTER3 
specific activity was obtained during growth on glucose after 14 h of growth and 
the lowest specific activity after 37 h of growth were obtained during growth on 
galactose, in both sampling times the highest activity was obtained during growth 
on ethanol (Table 2).  
 
 
Table 2. Enzyme activity after growth under repressing and derepressing growth 
conditions 
Carbon Source Specific Enzyme Specific Enzyme 
Activity (U/mg) (14h) Activity (U/mg) (37h) 
20 g glucose l-1 78.6 504 
20 g galactose l-1 361.4 414.5 
30 g ethanol l-1 1679.6 2120.8 
 
 
After this confirmation, the expression cassette in YIp356R shuttle vector was 
amplified and this product was transformed into S. cerevisiae (W303). The host 
strain used, contained a deletion of ADH2 where the ORF of ADH2 is replaced with 
the URA3 gene (adh2∆::URA3). The reasoning behind this was as follows: the 
expression cassette (ADH2::LacZ) gene needs to be targeted through homologous 
recombination to replace the native ADH2 gene; however, this construct does not 
contain any selective properties to enable the selection of the recombination event. 
To circumvent this problem, a strain containing an adh2∆::URA3 was used as host. 
Therefore, if the recombination event is successful this strain will revert to a uracil 
negative auxotroph. For selection purposes, 5-FOA was included in the media on 
which the transformed cells were plated. If 5-FOA is present, the gene product of 
URA3 (orotidine-5’-phosphate decarboxylase) converts 5-FOA to a toxic product 
that kills cells containing an active URA3 gene. Therefore the cells without the 
LacZ::ADH2 will not form colonies and the colonies obtained will be those with the 
expression construct. However transformations also yielded colonies on negative 
control plates, indicating that the level of 5-FOA toxicity was too low.  Therefore the 
 77 
CHAPTER3 
concentration of 5-FOA in the medium was increased to 2 g l-1 which lead to no 
colonies obtained on the negative control plate. The colonies obtained on the 
positive transformation plates were plated out on X-Gal plates for selection, 
however, no blue colonies were obtained. Further confirmation was obtained 
through amplification of a section of the LacZ::ADH2 cassette from the genomic 
DNA isolated from positive transformants. In all the colonies tested no PCR 
product could be obtained. After numerous attempts without any success it was 
clear that it is not possible to direct the expression cassette to the ADH2 locus. 
 
Because the expression cassette is functional in S. cerevisiae, seeing that β-
galactosidase activity was obtained when the expression cassette was transformed 
using the integrative plasmid YIplac211, it was concluded that the problem is not 
with the cassette itself but rather with the targeting of this construct to the ADH2 
locus.  Furthermore, the construction of the deletion of ADH2 was also extremely 
difficult and it was only successful after numerous attempts (O. De Smidt, personal 
communication), which indicated that the replacement of the deletion cassette with 
the expression cassette would also be problematic, as was proved in this case.  
 
As an alternative approach the YIplac211 containing the expression cassette 
integrated in the genome of S. cerevisiae (used to test the functionality of the 
expression cassette, see section 4.4), was used to study the expression of ADH2 
under different growth conditions.  
 
 78 
CHAPTER3 
5. REFERENCES 
 
Ausubel et al. Protein Expression In: Current Protocols in Molecular Biology. Ed: 
Ausubel et al John Wiley & Sons, Inc. New York (1987) Chapter 16. 
 
Ciriacy M. 1975. Genetics of alcohol dehydrogenases in Saccharomyces 
cerevisiae  II. Two loci controlling synthesis of the glucose-repressible ADHII. 
Mol. Gen. Genet. 138:157-154 
 
Ciriacy M. 1979. Isolation and characterization of further cis-and trans-acting 
regulatory elements involved in the synthesis of glucose-repressible alcohol 
dehydrogenase (ADHII) in Saccharomyces cerevisiae. Mol. Gen. Genet. 
176:427-431 
 
du Preez J.C., Maré J.E., Albertyn J. and Kilian S.G. 2001. Transcriptional 
repression of ADH2-regulated β-xylanase production by ethanol in 
recombinant strains of Saccharomyces cerevisiae. FEMS Yeast Res. 1:233-
240. 
 
Gietz R.D and Sugino A. 1988. New yeast-Escherichia coli shuttle vectors 
constructed with in vitro mutagenized yeast genes lacking six-base pair 
restrictions sites. Gene 74 (2):527-534 
 
Inoue H., Nojima H. and Okayama H. 1990 High efficiency transformation of 
Escherichia coli with plasmids. Gene 96:23–28 
 
Kudla B., Persuy M.A., Gaillardin C., Heslot H. 1988. Construction of an 
expression vector for the fission yeast Schizosaccharomyce pombe. Nucleic 
Acids Res. 16:8603–8617 
 
 79 
CHAPTER3 
Lutstorf U. and Megnet R. 1968. Multiple forms of alcohol dehydrogenase in 
Saccharomyces cerevisiae. I. Physiological control of ADH-2 and properties 
of ADH-2 and ADH-4. Arch. Biochem. Biophys. 126:933-944 
 
Myers A.M., Tzagoloff A., Kinney D.M. and Lusty C.J. 1986. Yeast shuttle and 
integrative vectors with multiple cloning sites suitable for construction of LacZ 
fusions. Gene 45:299-310 
 
Rose M. and Botstein D. 1983. Construction and use of gene fusion to LacZ 
(beta-galactosidase) that are expressed in yeast. Methods Enzymol. 101:167-
180 
 
Rose M.D., Winston F. and Hieter, P. 1990. Methods in yeast genetics: A 
laboratory course manual. Laboratory Press, Cold Spring Harbor, NY.  
 
Sambrook J., E.F. Fritsch and T. Maniatis. 1989. Molecular cloning:  A 
laboratory manual.  2nd edition.  Cold spring harbor laboratory press.  Cold 
Spring Harbor.NY. Vol 1, pp 1.25-1.28; Vol 3, p. A.1. 
 
 
 
 80 
 CHAPTER 4 
 
CHAPTER 4 
 
INVESTIGATION OF β-GALACTOSIDASE PRODUCTION 
UNDER THE CONTROL OF ADH2  
 
 
1. ABSTRACT 
To study the effect of ethanol on the expression of ADH2, Saccharomyces 
cerevisiae transformed with an integrative plasmid containing the ADH2::LacZ 
fusion, was grown on 20 g glucose l-1 as well as on three different ethanol 
concentrations (5, 20 and 30 g l-1). β-Galactosidase activity was measured as an 
indicator of ADH2 expression. The highest β-galactosidase specific activity was 
obtained during growth on 20 g ethanol l-1 (3 643 U mg-1) and the lowest during 
growth on 5 g ethanol l-1 (2 533 U mg-1). At ethanol concentrations above 24 g l-1 
low β-galactosidase activity were obtained that might have been due to the low 
growth rate of the yeast on 30 g ethanol l-1. The data suggested that ethanol do 
not repress the expression of ADH2. These results are contrary to previous 
findings (du Preez et al., 2001) that ethanol at high concentrations repressed 
ADH2 expression. 
 
2. INTRODUCTION 
 
The growth, metabolism and gene expression of Saccharomyces cerevisiae are 
highly regulated in response to carbon source availability (Carlson, 1998). This 
yeast prefers rapidly fermentable sugars namely glucose, mannose and fructose 
but it can also adapt to growth on non-fermentable carbon sources such as 
ethanol. S. cerevisiae has evolved a regulatory mechanism to cope with widely 
fluctuating levels of glucose. During the assimilation of glucose, various genes 
involved in the assimilation of alternative carbon sources are repressed including 
 81 
 CHAPTER 4 
ADH2, and are only derepressed during growth on a non-fermentable carbon 
source (Maeda et al., 1994). Ethanol, the by-product of fermentation, is re-
introduced into metabolism via its oxidation and the subsequent biosynthesis of 
acetyl-CoA. Adh2p, an alcohol dehydrogenase isozyme encoded by ADH2, is the 
major isozyme involved in the assimilation of ethanol (Lutstorf and Megnet, 1968; 
Ciriacy, 1979).  
 
Because ADH2 has an inducible promoter, this promoter has been widely used 
for the production of heterologous proteins (Hadfield et al., 1993). Inducible 
expression cassettes are employed for the regulated production of heterologous 
proteins. Since the ADH2 promoter is repressed over 100-fold by glucose, it can 
be used for the efficient expression of toxic proteins, such as the insulin-like 
growth factor (IGF-1) (Brierley, 1998). To maintain repression of ADH2, cells 
must be grown in the presence of excess glucose until induction, which is 
effected by changing to fresh medium containing a non-fermentable carbon 
source such as ethanol. Alternatively, ADH2 can be induced by culturing initially 
on a low concentration of glucose that is gradually depleted (Romanos et al., 
1992). Since this promoter is often utilized for heterologous protein production, it 
is necessary to understand its regulation, especially the effect of ethanol on the 
expression of ADH2.  
 
ADH2 transcription is known to be derepressed during growth on ethanol 
(Ciriacy, 1979), however du Preez et al. (2001) showed that 17.9 g ethanol l-1 
repressed the expression of ADH2 in batch cultures and that derepression 
occurred below 2.3 g ethanol l-1 in continuous cultures. Van den Berg et al. 
(1998) also reported that ADH2 expression was partially repressed when the 
yeast grew in the presence of ethanol. These authors performed a glucose pulse 
into a glucose-limited chemostat culture and analysed mRNA levels at different 
phases distinguished by the carbon source detectable during the growth. During 
growth on glucose, ethanol and acetate were produced and mRNA levels were 
analysed during the presence of glucose followed by ethanol, ethanol/acetate 
 82 
 CHAPTER 4 
and acetate in the medium. These authors found that ethanol had to disappear 
completely from the culture to obtain full transcription of ADH2 when the yeast 
was growing on acetate. This suggested that Adh3p, a mitochondrial alcohol 
dehydrogenase, might be an important isozyme catalyzing the initial step 
(ethanol conversion to acetaldehyde) during ethanol assimilation, since the 
transcription of ADH3 was elevated during growth on ethanol (Ciriacy, 1997; 
Bakker et al., 2000). 
 
Saccharomyces cerevisiae W303, transformed with YIplac211 (Gietz and 
Sugino, 1988), an integrative plasmid containing the ADH2::LacZ expression 
cassette, was grown on 20 g glucose l-1 and three different ethanol 
concentrations (5, 20 and 30 g l-1) as sole carbon sources. Whereas the results of 
du Preez et al. (2001) were obtained using a multicopy plasmid, in the work 
reported here an integrative plasmid was used to avoid transcriptional 
misregulation that might result from multicopy effects.  
 
 
 83 
 CHAPTER 4 
3. MATERIALS AND METHODS 
 
3.1 YEAST STRAIN 
 
Saccharomyces cerevisiae W303-1A strain (MATa leu2-3/112 ura3-1 trp1-1 his3-
11/15 ade2-1 can1-100 GAL SUC2) transformed with the YIplac211 vector 
containing a LacZ::ADH2 expression cassette, prepared as in chapter 3 and 
preserved in 50% glycerol at –80 oC was used. 
 
3.2 INOCULUM AND MEDIUM COMPOSITION 
 
Two yeast colonies from URA– selective plates were randomly selected to 
perform all the experiments in duplicate. To prepare the preculture, each was 
inoculated in a test tube containing 5 ml of a chemically defined medium. The 
medium contained the following per litre: C6H8O7.H2O, 0.5 g; (NH4)2SO4, 5 g; 
MgSO4.H2O, 0.4 g; CaCl2, 0.015 g; KH2PO4, 5 g; 10 ml trace elements, 10 ml 
vitamin solution, 10 ml of a 10 X amino acids solution, 1.6 ml leucine solution, 1 
ml TRP/HIS solution, glucose (20 g l-1) or ethanol (5, 20, 30 g l-1) as a carbon 
source and adjusted pH to 5.5. To maintain selection pressure, uracil was not 
added to the medium. The test tubes were then incubated at 30oC for 12 h before 
being used to inoculate a 100 ml shake flask with 20 ml of the same media to an 
OD600 of 0.1-0.2. The flaks were incubated for 10 h to inoculate the glucose 
containing medium. The medium with ethanol as a carbon source was inoculated 
after 15 h of incubation to ensure that all the glucose was depleted.  
 
The composition of the 10 X trace element solution was as follows: FeSO4.7H2O, 
35 g; MnSO4.H2O, 7 g; ZnSO4.7H2O, 11 g; CuSO4.5H2O, 1 g; CoCl2, 2 g; 
NaMoO4.2H2O, 1.3 g; H3BO3, 2 g; KI, 0.35 g; Al2(SO4)3, 0.5 g; dissolved in 950 
ml distilled water and 50 ml concentrated HCl. The composition of the vitamin 
solution was as follows: biotin, 0.005 g; calcium pantothenate, 0.1 g; folic acid, 
 84 
 CHAPTER 4 
0.005 g; niacin, 0.1 g; p-aminobenzoic acid, 0.02 g; pyridoxine HCl, 0.1 g; 
riboflavin, 0.1 g; thiamine HCl, 0.1 g; inositol, 2.5 g; dissolved in 20 ml of 1 M 
NaOH and distilled water up to 1 l.  
 
To prepare the 10 X amino acid solution, 0.5 g of each of the following amino 
acids was dissolved in a 100 ml distilled water; arginine, asparagine, aspartic 
acid, glutamic acid, glutamine, glycine, isoleucine, lysine, methionine, 
phenylalanine, proline, serine, threonine, tyrosine and valine.  A 1000 X TRP/HIS 
stock were prepared by dissolving tryptophan (2.5 g) and histidine (2.5 g) in 50 
ml distilled water. Cysteine and leucine were prepared separately, 1.25 g of 
cysteine was dissolved in 25 ml of 1 N HCl, 4 g of leucine was dissolved in 50 ml 
distilled water. NaOH pearls were used to dissolve the amino acids in water with 
a resulting pH of between 10-11. 
 
3.3. ANALYTICAL METHODS 
 
Samples for assays were taken at 2 to 10 h intervals, depending on the carbon 
source and growth phase of the yeast. The growth of the yeast cells was followed 
by measuring the optical density at 600 nm with an Eppendorf biophotometer 
(Eppendorf AG, Germany). The dry biomass of 10 ml samples from the late 
exponential phase were gravimetrically determined after centrifugation at 5 000 
rpm for 5 min at 4 oC, washed twice with distilled water and dryed at 105 oC for 
24 h. Optical densities were converted to cell dry weight according to a standard 
curve. The maximum specific growth rate was determined from the slope of the 
exponential phase of the growth curve.  
 
The culture supernatant was used for glucose and ethanol analysis. Glucose 
concentration was determined with a Sugar Analyser 1 HPLC equipped with a 
refractive index detector and a Sugar Pack 1 column (Waters Associates, Milford, 
MA, USA) operating at 85 oC with a deionised water flow rate of 0.5 ml min-1. 
 
 85 
 CHAPTER 4 
Ethanol was determined with a Hewlett-Packard 6 890 A gas chromatograph 
using 2 ml hydrogen carrier gas min-1 at an oven temperature of 70oC for 2 min 
followed by temperature programming at 10oC min-1 to 130oC and held for 1 min. 
 
The β-galactosidase activity was assayed according to the method of Rose and 
Botstein (1983), using 4 ml samples. Culture samples were harvested by 
centrifugation and washed with buffer Z (16.1 g Na2HPO47H2O l-1; 5.5 g 
NaH2PO4H2O l-1; 0.75 g KCl l-1 and 0.246 g MgSO47H2O l-1, adjusted pH to 7.0) 
and stored at –20 oC. Before being assayed the samples were thawed and 
resuspended in 200 μl ice cold buffer Z. Cell extracts were prepared by adding 
cold glass beads up to the level of the liquid suspension and 12.5 μl PMSF 
(phenylmethylsulfonyl fluoride) before vortexing for 2 min at 1 min intervals on 
ice. Cell debris was removed by centrifugation and the supernatant was used as 
the cell extract. This extract and the ONPG (o-nitrophenyl- β-D-
galactopyranoside) substrate were incubated for 15 min at 28oC and absorbance 
was measured at 420 nm using a spectrophotometer (Spectronic genesisTM, 
Miltonroy Co., USA). Protein concentrations were determined using the Bio-Rad 
protein reagent as indicated by the manufacturers (BioRad Laboratories, USA), 
based on the method of Bradford (1976) and measured at an absorbance of 595 
nm. One unit of β-galactosidase activity is the amount of β-galactosidase that 
hydrolyses 1 nmol of ONPG to ο-nitrophenol and D-galactose per min at 28ºC at 
pH 7. The specific rate of enzyme production was determined from the slope of 
the curve of specific enzyme activity (U protein mg -1) vs time. 
 
 86 
 CHAPTER 4 
4. RESULTS  
 
4.1 GROWTH PROFILE DURING GROWTH ON GLUCOSE AND ETHANOL AS 
RESPECTIVE CARBON SOURCES. 
 
To study the effect of ethanol on the expression of ADH2, S. cerevisiae  
transformed with an ADH2::LacZ expression cassette was grown on ethanol 
concentrations of 5, 20, and 30 g l-1. In addition, glucose (20 g l-1) was used to 
investigate ADH2 expression under repressed conditions. These experiments 
were performed in a chemically defined medium using two randomly selected 
recombinant S. cerevisiae W303 colonies and the presented results are the 
average obtained for the two transformants (Table 1). The cultivation parameters 
are shown in Table 1. 
 
Table 1. Specific growth rate, final biomass produced and rate of β-galactosidase 
production during growth on glucose and different concentrations of 
ethanol as carbon source 
 
Carbon µmax Final YX/S Enzyme Specific qpmax 
Source (h-1) Biomass (g g-1) Activity Enzyme (U mg-1h-1) 
(g l -1) (g l-1)  (U ml-1) Activity 
(U mg -1) 
Glucose, 20 0.47 3.4 0.17 947 532 46 
Ethanol, 5 0.24 2.9 0.54 4911 2533 128 
Ethanol, 20 0.18 2.7 0.24 6372 3643 294 
Ethanol, 30 0.13 1.4 0.118 5919 3496 93 
 
µmax,  maximum specific growth rate; qpmax, maximum specific rate of β-galactosidase production; 
YX/S, biomass yield. 
 
 87 
 CHAPTER 4 
4.1.1 GROWTH UNDER REPRESSING CONDITIONS ON GLUCOSE 
 
The yeast exhibited the highest growth rate (0.47 h-1) and the highest final 
biomass concentration on glucose (Table 1). After 14 h the yeast reached 
stationary phase with concomitant depletion of the glucose in the medium. 
Ethanol production was evident after 8 h of growth and reached the maximum 
concentration after 18 h (Fig. 1). After 55 h of growth the ethanol was depleted, 
indicating the slow assimilation of ethanol in comparison to the rapid utilization of 
glucose. 
 
10 35
30
1 25
20
15
0.1 10
5
0.01 0
0 10 20 30 40 50 60
Time (h)
 
 
FIGURE 1. Cultivation profile of the transformed yeast on 20 g l-1 glucose 
as carbon source. Symbols: Biomass (●), Glucose (▲) and 
ethanol (■) 
 
An increase in the β-galactosidase activity was only observed when glucose was 
almost depleted (Fig. 2). This, together with the fact that the ethanol 
concentration after 14 h was about 7 g l-1, suggested that glucose repressed the 
ADH2 promoter. This is in agreement with the existing literature that the activity 
 88 
Biomass (g l-1)
 Glucose and Ethanol (g l-1)
 CHAPTER 4 
of both Cat8p and Adr1p, the transcription activators of ADH2, was carbon 
source dependent (Walther and Schüller, 2001; Hedges et al., 1995; Young et 
al., 2002). 
 
4000 35
3500 30
3000 25
2500
20
2000
15
1500
1000 10
500 5
0 0
0 10 20 30 40 50 60
Time (h)
 
FIGURE 2. β-Galactosidase activity, glucose utilisation, ethanol production 
and utilisation by the yeast during growth on 20 g l-1 glucose. 
Symbols: Enzyme activity (♦), ethanol concentration (■) and 
glucose concentration (▲) 
 
This steady increase of β-galactosidase activity shows that derepression of the 
ADH2 promoter was due to activation of the ADH2 transcription activators in the 
absence of glucose when ethanol is assimilated by the yeast (Young et al. 2002). 
The maximum specific enzyme activity obtained during this cultivation was 532 U 
protein mg-1 (Table 1). β-Galactosidase production reached a maximum level 
after 30 h of growth with no decrease in activity during the following 20 h. During 
this time there was also a slow decrease in the ethanol concentration from a 
maximum of 7.2 g l-1 to 3.6 g l-1 after a 50 h cultivation period. 
These results indicate that when the ADH2 promoter is employed in the 
production of heterologous genes, it is beneficial to grow the yeast in glucose to 
 89 
Enzyme Activity (U mg-1)
Glucose and Ethanol (g l-1)
 CHAPTER 4 
obtain maximum biomass production prior to the induction of the ADH2 promoter 
using ethanol, so as to avoid the slow growth and low biomass production when 
ethanol is utilized. 
 
4.1.2 GROWTH UNDER DEREPRESSING CONDITIONS ON ETHANOL  
 
To determine the effect of ethanol concentration on the expression of ADH2, the 
yeast was grown on different ethanol concentrations (5, 20 and 30 g l-1) as a sole 
carbon source. The growth rate (Table 1) and biomass produced was higher 
during growth on 5 g ethanol l-1 in comparison to growth on either 20 or 30 g l-1. 
This growth rate was approximately two-fold less than on glucose. The ethanol in 
the medium was completely utilized after 40 h of cultivation (Fig. 3).  
 
10 35
30
25
1
20
15
0.1
10
5
0.01 0
0 10 20 30 40 50 60
Time (h)
 
 
FIGURE 3. Cultivation profile of the transformed yeast on 5 g ethanol l-1 as 
sole carbon source. Symbols: Biomass (●) and ethanol (■) 
 
The β-galactosidase activity was expected to be high during this cultivation in 
comparison to growth on the other two ethanol concentrations, according to the 
 90 
Biomass (g l-1)
Ethanol (g l-1)
 CHAPTER 4 
results obtained by du Preez et al. (2001), since high ethanol concentrations had 
a negative effect on the expression of ADH2. However, this was not the case as 
the lowest β-galactosidase activity was obtained during growth on 5 g ethanol l-1. 
A maximum specific activity of 2 533 U protein mg-1 was obtained (Fig. 4). This 
maximum specific activity was higher to that observed during growth on glucose, 
thus confirming derepression of the ADH2 promoter during growth on a non-
fermentable carbon source such as ethanol. 
 
4000 35
3500 30
3000 25
2500
20
2000
15
1500
1000 10
500 5
0 0
0 10 20 30 40 50 60
Time (h)
 
 
FIGURE 4. β-Galactosidase activity and ethanol utilization during growth 
on 5 g ethanol l-1. Symbols: Enzyme activity (♦) and ethanol 
concentration (■) 
 
The enzyme production rate of 128 U mg-1h-1 was observed, and it was higher 
than that observed during growth on glucose. The β-galactosidase production 
rate decreased after 24 h when the ethanol concentration decreased to below 2.5 
g l-1.  
 
The final biomass obtained during growth on 20 g ethanol l-1 was similar to that 
obtained during growth on 5 g ethanol l-1 (Table 1). However, the growth rate was 
 91 
Enzyme Activity (U mg-1)
Ethanol (g l-1)
 CHAPTER 4 
slower than during growth on 5 g ethanol l-1 (Table 1), indicating that ethanol has 
negative effect on the growth of the yeast cells (Walker-Caprioglio et al., 1985). 
Approximately 10 g ethanol l-1 was utilized after 55 h of growth (Fig. 5). At a 
cultivation time of 55 h approximately 9.5 g ethanol l-1 was still present (Fig.5).  
 
10 35
30
1 25
20
15
0.1 10
5
0.01 0
0 10 20 30 40 50 60
Time (h)
 
 
FIGURE 5. Cultivation profile on 20 g ethanol l-1. Symbols:  Biomass (●) 
and ethanol concentration (■) 
 
These results showed that 50% (10 g ethanol l-1) of the ethanol provided was 
utilized during this cultivation to produce the amount of biomass similar to that 
produced during growth on 5 g ethanol l-1. 
 
The β-galactosidase activity during this cultivation was higher than that obtained 
on 5 g ethanol l-1, with a maximum specific activity of 3 643 U protein mg-1 (Fig. 
6). The β-galactosidase production rate was also higher (Table 1). After 16 h a β-
galactosidase specific activity of about 2 780 U protein mg-1 was obtained as 
compared to 1 032 U protein mg-1 obtained during growth on 5 g ethanol l-1 after 
16 h (Fig.6). This indicated that a decrease in the ethanol concentration during 
this cultivation was not necessary to obtain high expression levels. The β-
galactosidase production ceased after 36 h. 
 
 92 
Biomass (g l-1)
Ethanol (g l-1)
 CHAPTER 4 
 
4000 35
3500 30
3000 25
2500
20
2000
15
1500
1000 10
500 5
0 0
0 10 20 30 40 50 60
Time (h)
 
FIGURE 6. β-galactosidase specific enzyme activity and ethanol utilization 
during growth on 20 g ethanol l-1. Symbols: Enzyme activity (♦) 
and ethanol concentration (■) 
 
About a 30% higher maximum specific β-galactosidase activity was observed 
during growth on 20 g ethanol l-1 than on 5 g ethanol l-1, suggesting that an 
increase in ethanol concentration enhanced the expression of ADH2. 
 
To verify that a high ethanol concentration had a negative effect on the 
expression of ADH2, the yeast was grown on 30 g ethanol l-1 as sole carbon 
source. Under these growth conditions the lowest biomass and yield was 
obtained (Fig. 7 and Table1).  
 
 93 
Enzyme Activity (U mg-1)
Ethanol (g l-1)
 CHAPTER 4 
10 35
30
1 25
20
15
0.1 10
5
0.01 0
0 10 20 30 40 50 60
Time (h)
 
 
FIGURE 7. Cultivation profile obtained during growth on 30 g ethanol l-1. 
Symbols: Biomass (●) and ethanol concentration (■) 
 
Most of the ethanol (20 g l-1) still remained after a cultivation time of 55 h. A final 
biomass concentration of 1.4 g l-1 of biomass was produced (Table 1) from the 10 
g ethanol l-1 utilized by the yeast during this cultivation, which was much less in 
comparison to the biomass obtained on 20 g ethanol l-1. Similar amounts of 
ethanol were utilized in both cultivations. Even though ethanol was still available 
at the end of the cultivation on 20 and 30 g ethanol l-1, in both cases the growth 
rate had decreased dramatically. This might have been due to the exhaustion of 
essential nutrients that may have occurred during growth on ethanol.  
 
Contrary to the results expected, the specific β-galactosidase activity obtained 
here was higher than that obtained during growth on 5 g ethanol l-1 (Fig. 8). 
 
 94 
Biomass (g l-1)
Ethanol (g l-1)
 CHAPTER 4 
4000 35
3500 30
3000 25
2500 20
2000
1500 15
1000 10
500 5
0 0
0 10 20 30 40 50 60
Time (h)
 
 
FIGURE 8. β-galactosidase specific activity and ethanol utilization during 
growth on 30 g ethanol l-1. Symbols: Enzyme activity (♦) and ethanol 
concentration (■). 
 
However, this maximum specific β-galactosidase activity was achieved after the 
level of the ethanol concentration decreased to about 24 g l-1 (Fig. 8). There was 
a slow steady increase in the β-galactosidase activity observed during this 
cultivation (Fig. 8), reaching a maximum of 3 496 U mg-1 protein when the 
ethanol concentration reached 24 g l-1 after 40 h. This slow production of β-
galactosidase observed here might have been due to the slow growth of the 
yeast during this cultivation. Although a maximum specific activity and the 
maximum volumetric enzyme activity obtained here were almost as high as that 
obtained during the cultivation on 20 g ethanol l-1, the final biomass produced 
during this cultivation was less by almost a half to that produced on 20 g ethanol 
l-1 (Table 1), this therefore indicated that β-galactosidase production was two fold 
higher. Therefore higher expression occurred when the ethanol concentration of 
30 g l-1 was used than on either 20 or 5 g l-1. 
 
 95 
Enzyme Activity (U/mg)
Etoh (g/l)
 CHAPTER 4 
The maximum specific enzyme activity values obtained under these three 
different ethanol concentration and 20 g glucose l-1 are represented in the Figure 
9. 
 
4000
3500
3000
2500
2000
1500
1000
500
0
 
 
FIGURE 9. The maximum specific enzyme activity obtained during growth 
on 20 g glucose l-1, 5, 20 and 30 g ethanol l-1.  
 
 96 
Enzyme Activ ity (U mg-1)
20 g glucose l-1
5  g ethanol l-1
20 g ethanol l-1
30 g ethanol l-1
 CHAPTER 4 
The maximum specific activity of 519 U mg-1 was obtained when the ethanol 
concentration reached 7 g l-1 during cultivation on 20 g glucose l-1 During 
cultivations under derepressing conditions with the different ethanol 
concentrations the maximum specific activity was 2 533, 3 496 and 3 643 U mg-1 
during growth on ethanol concentrations of 5, 30, and 20 g l-1, respectively. 
These maximum specific activities were obtained when the ethanol concentration 
in the medium was 2.2 g l-1, 24 g l-1 and 11 g l-1 during growth on 5, 30, and 20 g 
l-1, respectively. This suggested that ethanol did not repress the expression of the 
ADH2 gene. Cultivation of the yeast on 20 g l-1 glucose confirmed glucose 
repression on ADH2. The data obtained in this study suggested that ethanol 
enhanced the expression of ADH2 when its concentration was increased. 
However it was shown that above a certain concentration the production rate was 
slow. According to the above results, to produce heterologous proteins using the 
ADH2 promoter effectively, the yeast should be grown on glucose prior to 
derepression of the promoter through cultivation on ethanol and that a 20 g 
ethanol l-1 concentration should be used during derepression as the highest 
enzyme activity was achieved at this initial ethanol concentration. 
 97 
 CHAPTER 4 
5. DISCUSSION 
 
The yeast grew better on glucose as opposed to growth on ethanol and this may 
be attributed to the fact that ethanol at a high concentration is toxic to the yeast, 
the major targets for ethanol toxicity being the plasma membrane and the 
cytosolic enzymes: Ethanol alters the membrane organization and permeability 
and inactivates and unfolds globular cytosolic enzymes (Thomas and Rose, 
1979). Ethanol has also been found to induce a delay in the yeast cell cycle 
(Kubota et al., 2004). The highest growth rate and biomass yield on ethanol was 
achieved during growth on 5 g l-1, the lowest ethanol concentration used in this 
study. 
 
The above results are consistent with the existing literature in terms of the 
regulation of ADH2. Glucose repression was confirmed by the low β-
galactosidase activity obtained during cultivation on 20 g glucose l-1. Enzyme 
production was observed when glucose was almost depleted, relieving the 
transcription activators from glucose repression (Gancedo, 1998). The increase 
in β-galactosidase activity in the presence of ethanol supported the theory that 
the activity of Snf1p, which is at the centre of ADH2 derepression and is also 
essential for growth on non-fermentable carbon sources, was inhibited by 
glucose (Jiang and Carlson, 1996) and was only induced when a non-
fermentable carbon source is utilized, resulting in the derepression of ADH2 by 
the two transcription activators, Adr1p and Cat8p (Walther and Schüller, 2001). 
 
The results also indicated that the expression of ADH2 increased in the presence 
of ethanol. This was indicated by an increase in the maximum specific activity 
which correlated with an increased ethanol concentration in the medium. 
However to confirm the possible induction of ADH2 expression by ethanol, the 
yeast should be grown on another non-fermentable carbon source such as 
glycerol to compare the expression levels, since it has been reported that ethanol 
 98 
 CHAPTER 4 
does not induce ADH2 expression (Ciriacy, 1997). According to available 
literature, the increase in β-galactosidase activity during growth on ethanol was 
rather due to the absence of glucose repression and not due to ethanol induction, 
although an increase in ethanol may however lead to a high demand for Adh2p 
resulting in an increased ADH2 expression.  
 
It has been found that ethanol limits the growth of S. cerevisiae in a 
concentration dependent manner. An increase in ethanol concentration leads to 
an increase in the lag phase and a decreased growth rate (Walker-Caprioglio et 
al., 1985). Alexandre et al. (2001) have shown that high ethanol concentrations 
affect the expression of genes that are involved in protein biosynthesis, cell 
growth, RNA metabolism and cellular biogenesis and the genes up-regulated by 
ethanol are mainly involved in energetic metabolism, ionic homeostasis and 
stress response. One of the genes that was found to be induced by a high 
ethanol concentration is ALD4, a mitochondrial aldehyde dehydrogenase; the 
expression of this gene was ADR1 dependent (Young et al., 2003). This 
indicates that Adr1p is functional under high ethanol concentration conditions, 
suggesting that ADH2 is not repressed by high ethanol concentrations. The 
possibility of the presence of a repressor being involved in ADH2 regulation was 
excluded by Irani et al. (1987). Therefore, ethanol does not seem to act through 
any of the known repressors to repress ADH2. The low β-galactosidase activity 
obtained when the ethanol concentration in the medium was above 24 g l-1 might 
be a result of the slow growth of the yeast, at high ethanol concentrations 
(Kubota et al., 2004). Therefore, the apparent slow expression of the reporter 
gene might be due to the absence/low amounts of the transcription factors Adr1p 
and Cat8p as a result of the slowed growth during the adaptation of the yeast to 
the ethanol stress and not repression of the ADH2 promoter by high ethanol 
concentration. The results of the β-galactosidase production at 30 g ethanol l-1 
confirmed that the ADH2 promoter is not repressed by ethanol as similar 
maximum β-galactosidase specific activities were obtained to the maximum β-
 99 
 CHAPTER 4 
galactosidase specific activity obtained during growth on 20 g ethanol l-1 even 
though less biomass was produced on 30 g ethanol l-1. 
 
Previous findings by du Preez et al. (2001) indicated repression of ADH2 
expression at an ethanol concentration above 17.9 g l-1, which is contrary to the 
results obtained in this study. This suggested that the ethanol effect on the 
expression of ADH2 observed by du Preez et al. (2001) could be due to the 
existence of transcriptional misregulation since a multicopy expression system 
was used in their study or it might have been due to the presence of negative 
regulatory elements in the expression cassette used in the ADH2 promoter-XYN2 
construct used in their study. 
 100 
 CHAPTER 4 
6. REFERENCES 
 
Alexandre H., Ansanay-Galeote, Dequin S. and Blodin B. 2001. Global gene 
expression during short-term ethanol stress in Saccharomyces cerevisiae. 
FEBS Lett. 498:98-103 
 
Bakker B.M., Bro C., Kotter P., Luttik M.A., van Dijken J.P., Pronk J.T. 2000. 
The mitochondrial alcohol dehydrogenase Adh3p is involved in a redox 
shuttle in Saccharomyces cerevisiae. J. Bacteriol. 182:4730-4737. 
 
Bradford M.M. 1976. A rapid and sensitive methods for the quantification of 
microgram quantities of protein utilizing the principle of protein–dye binding. 
Anal. Biochem 72:248– 254.  
 
Brierley RA. 1998. Secretion of recombinant human insulin-like growth factor I 
(IGF-I). Methods Mol. Biol. 103:149-177 
 
Ciriacy M. 1979. Isolation and characterisation of further cis- and trans-acting 
regulatory elements involved in the synthesis of glucose-repressible alcohol 
dehydrogenase (ADHII) in Saccharomyces cerevisiae. Mol. Gen. Genet. 
176:427-431 
 
Ciriacy M. 1997. Alcohol dehydrogenases. p. 213-223. In F.K. Zimmermann and 
K.D. Entian (ed.), Yeast sugar metabolism. Technomic Publishing co., 
Lancaster, Pa. 
 
du Preez J.C., Maré J.E., Albertyn J. and Kilian S.G. 2001. Transcriptional 
repression of ADH2-regulated β-xylanase production by ethanol in 
recombinant strains of Saccharomyces cerevisiae. FEMS Yeast Res. 1:233-
240. 
 
 101 
 CHAPTER 4 
Gancedo, J.M. 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. 
Rev. 62:334-361 
 
Gietz R.D and Sugino A. 1988. New yeast-Escherichia coli shuttle vectors 
constructed with in vitro mutagenized yeast genes lacking six-base pair 
restriction sites. Gene 72:527-534 
 
Hadfield C. Raina K. K., Shashi-Menon K. and Mount R. C. 1993. The 
expression and performance of cloned genes in yeasts. Mycol. Res. 8:897-
944 
 
Hedges D., Proft M. and Entian K.D. 1995. CAT8, a new zinc cluster-encoding 
gene necessary for derepression of gluconeogenic enzymes in the yeast 
Saccharomyces cerevisiae. Mol. Cell. Biol. 15:1915-22. 
 
Irani M., Taylor W.E. and Young E.T. 1987. Transcription of ADH2 gene in 
Saccharomyces cerevisiae is limited by positive factors that bind 
competitively to its promoter region on multi copy plasmids. Mol. Cell. Biol. 
7:1233-1241 
 
Jiang R. and Carlson M. 1996. Glucose regulates protein interaction within the 
yeast SNF1 protein kinase complex. Genes Dev. 10:3105-3115 
 
Kubota S., Takeo I., Kume K., Kanai M., Shitamukai A., Mizunumi M. and 
Miyakawa T. 2004. Effect of ethanol on cell growth of budding yeast: genes 
that are important for cell growth in the presence of ethanol. Biosci. 
Biotechnol. Biochem. 68:968-972 
 
Lutstorf U. and Megnet R. 1968. Multiple forms of alcohol dehydrogenase in 
Saccharomyces cerevisiae. I. Physiological control of ADH-2 and properties 
of ADH-2 and ADH-4. Arch. Biochem. Biophys. 126:933-944. 
 102 
 CHAPTER 4 
 
Maeda T., Wurgler-Murphy S.M. and Santio H. 1994. A two component system 
that regulates an osmosensing cascade in yeast. Nature 369:242-245 
 
Romanos M.A., Scorer C.A. and Clare J.J. 1992. Foreign gene expression in 
yeast: a review. Yeast. 8:423-488. 
 
Rose, M. and Botstein, D. 1983. Construction and use of gene fusion to lacZ 
(beta-galactosidase) that are expressed in yeast. Methods Enzymol. 
101:167-180 
 
Thomas D.S. and Rose A.H. 1979. Inhibitory effect of ethanol on the growth and 
solute accumulation of Saccharomyces cerevisiae as affected by plasma-
membrane lipid composition. Arch. Microbiol. 122:49-55 
 
van den Berg M.A., de Jong-Gubbels P. and Steensma H.Y. 1998. Transient 
mRNA responses in chemostat cultures as a method of defining putative 
regulatory elements: application to genes involved in Saccharomyces 
cerevisiae acetyl-coenzyme A metabolism. Yeast. 14:1089-1104. 
 
Walker-Caprioglio H.M., Rodriguez R.J. and Parks L.W. 1985. Recovery of 
Saccharomyces cerevisiae from ethanol-induced growth inhibition. Appl. 
Environ. Microbiol. 50:685-689 
 
Walther K. and Schüller H.J. 2001. Adr1 and Cat8 synergistically activate the 
glucose-regulated alcohol dehydrogenase gene ADH2 of the yeast 
Saccharomyces cerevisiae. Microbiology 147:2037-2044. 
 
Young E.T., Kacherovsky N. and Van Riper K. 2002. Snf1 protein kinase 
regulates Adr1 binding to chromatin but not transcription activation. J. Biol. 
Chem. 277:38095-38103.  
 103 
 CHAPTER 4 
 
Young E.T., Dombek K.M., Tachibana C. and Ideker T. 2003. Multiple 
pathways are co-regulated by the protein kinase Snf1 and the transcription 
factors Adr1 and Cat8. J. Biol. Chem. 278:26146-26158. 
 
 
 
 
 104 
 CHAPTER 5 
 
CHAPTER 5 
 
GENERAL CONCLUSIONS 
 
 
The aim of the study was to confirm the negative effect of ethanol on the 
expression of ADH2 in the genomic copy of Saccharomyces cerevisiae. ADH2  
has been characterized as the major alcohol dehydrogenase isozyme required 
during ethanol utilization by S. cerevisiae (Ciriacy, 1975). Adh2p catalyses the 
conversion of ethanol to acetaldehyde and this is the initial reaction during 
ethanol assimilation. Therefore ethanol would not be expected to have inhibitory 
effect on the expression of this gene. The data obtained during this study is in 
agreement with this.  
 
To study ethanol repression on ADH2 expression, S. cerevisiae transformed with 
integrative plasmid cloned with ADH2::LacZ expression cassette was grown in 
different concentrations of ethanol (5, 20 and 30 g l-1). The data obtained during 
this cultivations was however different from that presented in the previous study 
(du Preez et al., 2001). They showed that ADH2 expression is repressed during 
growth at elevated ethanol concentration. They observed that during batch 
cultivation 17.9 g ethanol l-1 repressed the expression of ADH2. From the 
chemostat experiment they established that derepression of ADH2 occurs at 
ethanol concentration below 2.3 g l-1. In this study the lowest maximum β-
galactosidase specific activity under derepressing conditions was obtained during 
growth on 5 g l-1 with maximum specific activities of 2 533, 3 643 and 3 496 U 
protein mg-1 obtained during growth on 5, 20 and 30 g ethanol l-1 respectively 
(Chapter 4; Table 1, Fig. 4, 6 and 8). Although high maximum β-galactosidase 
specific activity was obtained during growth on 30 g l-1, the production rate (93 U 
mg-1h-1) of β-galactosidase was slow in comparison with the production rate 
observed during growth on 5 and 20 g ethanol l-1 concentrations, with the 
production rates of 128 and 294 U mg-1 h-1, respectively. The maximum specific 
 105 
 CHAPTER 5 
activity during growth on 30 g l-1 was only obtained when the ethanol 
concentration in the media was 24 g l-1. This can be attributed to the slow growth 
of the yeast on 30 g ethanol l-1. There was also a sudden increase in enzyme 
production on 20 g ethanol l-1 as compared to the enzyme production on 5 g 
ethanol l-1 and this may have indicated a possible ethanol induction of the ADH2 
gene. This was further supported by the high β-galactosidase production on 30 g 
ethanol l-1. However available literature does not support this. Therefore further 
studies are required to confirm this, whereby a non fermentable carbon source 
such as glycerol can be used and the results be compared with those obtained 
during growth on ethanol.  
 
The data obtained during glucose repression studies were as expected, as the 
lowest specific maximum β-galactosidase specific activity was achieved here. 
The β-galactosidase activity profile also showed that a decrease in glucose 
concentration was essential for the derepression of the promoter (Chapter 4; Fig. 
2). The data obtained in this study suggests that the presence of ethanol 
increased the expression of ADH2 and does not repress its expression. However 
since these results were obtained in batch cultivation experiments, a different 
picture might be observed in chemostat cultivations. Therefore further studies 
might be necessary to confirm ethanol effect on the expression of ADH2 in 
continuous cultivations. Even so, when comparing these results with the results 
of batch cultivation obtained by du Preez et al. (2001), ethanol represses the 
expression of ADH2 when the concentration is above 24 g l-1 as opposed to 
above 17.9 g ethanol l-1.  
 
This might suggest that the use of an integrative plasmid as opposed to a 
multicopy plasmid was more suitable for the expression study. Therefore these 
results might represent a true reflection of how ethanol might affect the 
expression of ADH2 in the genome of S. cerevisiae, or using ethanol as a sole 
carbon source may affect the regulation of ADH2 expression differently to when 
the ethanol is spiked into the medium. 
 106 
 CHAPTER 5 
 
REFERENCES 
 
Ciriacy M. 1975. Genetics of alcohol dehydrogenases in Saccharomyces 
cerevisiae  II. Two loci controlling synthesis of the glucose-repressible 
ADHII. Mol. Gen. Genet. 138:157-154 
 
du Preez J.C., Maré J.E., Albertyn J. and Kilian S.G. 2001. Transcriptional 
repression of ADH2-regulated β-xylanase production by ethanol in 
recombinant strains of Saccharomyces cerevisiae. FEMS Yeast Res. 1:233-
40. 
 
 107 
 CHAPTER 6 
 
CHAPTER 6 
 
SUMMARY 
 
 
Key terms 
Saccharomyces cerevisiae, alcohol dehydrogenase, glucose repression, β-
galactosidase, ethanol inhibition 
 
The aim of this study was to investigate the ability of ethanol to repress the 
expression of ADH2 in the genome of Saccharomyces cerevisiae. To achieve 
this, an expression cassette (ADH2::LacZ) using LacZ as a reporter gene was 
constructed using the YIp356R shuttle vector. A 1000 bp up and downstream 
region, flanking the open reading frame of ADH2, was fused to the 5’-end and 3’-
end of the LacZ gene in the YIp356R shuttle vector. Numerous attempts were 
made to transform the expression cassette (ADH2::LacZ) into S. cerevisiae 
(strain W303) containing a deleted ADH2 (adh2∆::URA3), to displace the deletion 
cassette through homologous recombination, thereby placing ADH2::LacZ in the 
place of the ADH2 in genome of S. cerevisiae. This was unfortunately not 
successful and it was decided to use an alternative approach.  In this case the 
expression cassette was cloned into the integrative vector YIplac211 and 
transformed into S. cerevisiae. For initial confirmation, the yeast transformants 
were grown on selective plates containing X-gal, which allows for the detection of 
β-galactosidase activity through the production of blue coloured colonies. The 
detection of the blue colour confirmed that the expression cassette was 
successfully constructed and integrated into the genome. 
Two randomly selected transformants were cultivated on 20 g glucose l-1 as sole 
carbon source, to study glucose repression and on three different ethanol 
concentrations to study the effect of ethanol on the expression of ADH2. 
Selection was maintained by growing the yeast in a URA– chemically defined 
media (pH 5.5) at 30ºC. Samples were taken at appropriate intervals to perform 
 108 
 CHAPTER 6 
β-galactosidase assay, assess utilization of substrate (ethanol and glucose), 
ethanol formation and biomass determination.  
During growth on 20 g glucose l-1 the production of β-galactosidase was apparent 
only when glucose concentrations were very low (2.3 g l-1), indicating that 
glucose levels have to decrease to a critical level before ADH2 expression can 
resume. The highest final biomass was produced during growth on 20 g glucose 
l-1. During growth on the three different ethanol concentrations the highest β-
galactosidase maximum specific activity was obtained during growth on 20 g 
ethanol l-1 (3 643 U mg-1) and the lowest during growth on 5 g ethanol l-1 (2 533 U 
mg-1). Although the maximum specific activity obtained during growth on 30 g 
ethanol l-1 were higher than that obtained during growth on 5 g l-1, the production 
rate was the lowest (93 U mg-1h-1) during growth on 30 g ethanol l-1, suggesting 
that 30 g ethanol l-1 concentration has negative effect on the expression of 
ADH2. However this slow production might have been due to the slow growth 
during this cultivation and not due to ethanol repression. The possible repression 
of ADH2 is further disputed by the high β-galactosidase production on 30 g 
ethanol l-1.  
 109 
 CHAPTER 7 
 
CHAPTER 7 
 
OPSOMMING 
 
 
Sleutelwoorde 
Saccharomyces cerevisiae, alkoholdehidrogenase, glukose onderdrukking,  β-
galaktosidase, etanol-inhibisie 
 
Hierdie studie was onderneem om vas te stel of etanol die uitdrukking van ADH2 
onderdruk.  Om dit te bepaal is ‘n uitdrukkings-kasset (ADH2::LacZ) waar LacZ 
gebruik is as verslaggewer-geen, gemaak deur gebruik te maak van die Yip356R 
pendelvektor. ‘n Eenduisend basispaar gebied, wat weerskante van die 
oopleesraam van ADH2 voorkom, is aan die onderskeie 5’- en 3’ kant van die 
LacZ geen geheg in die pendelvektor YIp356R. Verskeie pogings is aangewend 
om die uitdrukkings-kasset (ADH2::LacZ) in die gis Saccharomyces cerevisiae 
(stam W303) wat ‘n ADH2 delesie (adh2∆::URA3) bevat, te transformeer om te 
lei tot die vervanging van die delesie-kasset deur middel van homoloë 
rekombinasie. Dit was egter nie suksesvol nie en ‘n alternatiewe prosedure is 
gevolg.  In die geval is die uitdrukkings-kasset in die integrasievektor YIplac211 
geplaas en getransformeer in die gis S. cerevisiae. Bevestiging vir die uitdrukking 
van β-galaktosidase is verkry deur die gemodifiseerde gisselle te groei op 
selektiewe plate wat X-gal bevat het en sodoende toelaat dat die 
teenwoordigheid van die ensiem β-galaktosidase bevestig kan word deurdat 
kolonies blou verkleur. Die verkleuring van kolonies het bevestig dat die 
uitdrukkings-kasset korrek voltooi is en in die genoom van die gis geïntegreer 
was.  
Twee transformante wat lukraak gekies is, is gegroei in medium wat 20 g glukose 
l-1 as enigste koolstofbron bevat het, om die effek van glukose repressie te 
bestudeer. Om die effek te bestudeer wat etanol uitoefen op die uitdrukking van 
 110 
 CHAPTER 7 
ADH2, is drie verskillende konsentrasies etanol as enigste koolstof bron gebruik. 
Gisselle is gegroei in selektiewe URA- media (pH 5.5) by 300C.  Monsters is 
geneem met aanvaarbare tussenposes om sodoende β-galaktosidase aktiwiteit, 
verbruik van koolstofbron (glukose en etanol), etanol vorming en biomassa te 
bepaal. 
Gedurende groei in die teenwoordigheid van 20 g glukose l-1 is die produksie van 
β-galaktosidase alleenlik waargeneem indien die vlak van glukose daal na ‘n 
konsentrasie van 2.3 g glukose l-1.  Dit dui aan dat die vlak van glukose moet 
verlaag tot ‘n kritiese konsentrasie voordat ADH2 uitdrukking kan plaasvind. Die 
hoogste finale biomassa is geproduseer gedurende groei in die teenwoordigheid 
van 20 g glucose l-1. Gedurende groei op die drie verskillende konsentrasies 
etanol was die hoogste spesifieke aktiwiteit van β-galaktosidase waargeneem 
met groei op 20 g etanol l-1 (3 643 U mg-1) en die laagste vlak tydens groei op 5 g 
etanol l-1 (2 533 U mg-1). Alhoewel die hoogste spesifieke aktiwiteit van β-
galaktosidase, wat waargeneem is gedurende groei op 30 g etanol l-1, hoër was 
as die aktiwiteit wat waargeneem is tydens groei op 5 g etanol l-1 was die 
produksie tempo van eersgenoemde (93 U mg-1h-1) die laagste.  Dit mag 
moontlik aandui dat etanol teen ‘n konsentrasie van 30 gl-1 ‘n negatiewe effek het 
op die uitdrukking van ADH2. Dit is egter ook moontlik dat die laer produksie 
tempo die gevolg was van die verlaagde groei tempo en nie as gevolg van etanol 
onderdrukking nie.  Die verhoogde β-galaktosidase produksie wys egter teen die 
moontlikheid van etanol onderdrukking op die uitdrukking van ADH2 in die 
teenwoordigheid van 30 g etanol l-1.  
 111 
REGULATION OF ADH2 IN THE YEAST SACCHAROMYCES CEREVISIAE 
 
ABSTRACT 
ADH2 encodes for the main alcohol dehydrogenase isozyme that catalyses the 
conversion of ethanol to acetaldehyde during ethanol assimilation. Expression of 
this gene is known to be under glucose repression and is derepressed by growth 
on ethanol and other non-fermentable carbon sources. Results obtained by du 
Preez et al. (2001) showed, however, that this gene was repressed by growth on 
ethanol.  These results were obtained using a multicopy expression vector with a 
promoter-terminator expression cassette derived from the S. cerevisiae ADH2 
gene.  The aim of this study is the investigation of ethanol repression of ADH2 in 
the genome of S. cerevisiae. To achieve this, an expression cassette using LacZ 
as a reporter gene was constructed using the YIp356R shuttle vector. A 1000 bp 
up and downstream region, flanking the open reading frame of ADH2, was fused 
to the 5’-end and 3’-end of the LacZ gene. To study the effect of ethanol on the 
expression of ADH2, Saccharomyces cerevisiae transformed with an integrative 
plasmid containing the ADH2::LacZ fusion, was grown on 20 g glucose l-1 as well 
as on three different ethanol concentrations (5, 20 and 30 g l-1). β-Galactosidase 
activity was measured as an indicator of ADH2 expression. The highest β-
galactosidase specific activity was obtained during growth on 20 g ethanol l-1 (3 
643 U mg-1) and the lowest during growth on 5 g ethanol l-1 (2 533 U mg-1). At 
ethanol concentrations above 24 g l-1 low β-galactosidase activity were obtained 
that might have been due to the low growth rate of the yeast on 30 g ethanol l-1. 
The data suggested that ethanol do not repress the expression of ADH2. These 
results are contrary to previous findings (du Preez et al., 2001) that ethanol at 
high concentrations repressed ADH2 expression.