Carbon source responsive elements and gene regulation by CAT8 and SIP4 in the yeast Kluyveromyces lactis [Elektronische Ressource] / von Jorrit-Jan Krijger

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2002
• Nombre de lectures: 18
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Carbon Source-Responsive Elements and gene regulation
by CAT8 and SIP4 in the yeast Kluyveromyces lactis

DISSERTATION


zur Erlangung des akademischen Grades
doktor rerum naturalium (Dr. rer. nat.)

vorgelegt der

Mathematisch-Naturwissenschaftich-Technischen Fakultät
(mathematisch-naturwissenschaftlicher Bereich)
der Martin-Luther-Universität Halle-Wittenberg


von
Jorrit-Jan Krijger
geb. am 28.06.1970 in Rotterdam


Gutachterin bzw. Gutachter:
1. Prof. Dr. K.D. Breunig, Institut für Genetik, Martin-Luther-Universität
Halle-Wittenberg
2. Prof. Dr. J.J. Heinisch, Institut für Lebensmitteltechnologie, Universität Hohenheim
3. Dr. Ir. H.Y. Steensma, Instituut Moleculaire Plantkunde, Universiteit Leiden,
Niederlande

Halle (Saale), den 30.03.2002

Die Arbeit wurde im Rahmen eines ordentlichen Promotionsverfahrens am 12. Juli
2002 verteidigt.
urn:nbn:de:gbv:3-000005080
[ http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000005080 ]
Contents

1 Introduction 7
1.1 Regulation of genes: both end and means 7
1.1.1 Three classes of genes, transcribed by different RNA polymerases 7
1.1.2 Promoter elements of RNA polymerase II 8
1.1.3 Trans-acting factors of RNA polymerase II 8
1.1.4 The general transcription machinery 9
1.1.5 General transcriptional coactivators and repressors 13
1.1.6 Specific transcriptional activators and repressors 16
1.2 Regulation of transcription by carbon source in yeast 22
1.2.1 Glucose repression 23
1.2.2 Galactose induction 26
1.2.3 Derepression on poor carbon sources 27
1.3 Aim of the PhD thesis 29

2 Materials and Methods 31
2.1 Yeast strains and growth media 31
2.2 Escherichia coli strains and growth media 33
2.3 Plasmids 33
2.4 Oligonucleotides 34
2.4.1 Double-stranded oligonucleotide probes for
electrophoretic mobility shift assay DNA binding studies 34
2.4.2 Primers for construction of the ∆Klsip4 construct 36
2.4.3 RT-PCR primer pairs 36
2.5 Transformation procedures 36
2.5.1 Transformation of E. coli 36
2.5.2 Transformation of K. lactis 37
2.6 Preparation and manipulation of DNA 37
2.6.1 Plasmid isolation from E. coli 37
2.6.2 Plasmid rescue from K. lactis
2.6.3 Isolation of chromosomal DNA from K. lactis 37
2.6.4 Two-step gene disruption of KlSIP4 38
2.6.5 DNA sequencing 40
2.6.6 General enzymatic manipulation of DNA 40
2.7 Preparation and manipulation of RNA 40
2.7.1 Extraction of RNA from K. lactis 40
2.7.2 Transcript analysis by RT-PCR 40
2.8 Preparation and manipulation of proteins 41
2.8.1 Extraction of proteins from K. lactis 41
2.8.2 Determination of protein concentration 42
2.8.3 Electrophoretic mobility shift assay 42
2.8.4 Determination of enzyme activity 42
2.8.4.1 β-Galactosidase activity measurement 42
2.8.4.2 Isocitrate lyase activity measur 43

3 Results 45
3.1 Analysis of the CSRE LAC4
3.1.1 Competition of the CSRE for Kdf1-binding 45
3.1.2 Binding of Kdf1 to the CSRE is not influenced by the LAC4
adjacent putative Adr1p binding site but depends on the carbon source 46
3.1.3 The CSRE alone is sufficient to replace the Basal Control Region 48 LAC4
3.1.4 The quantity of Kdf1-CSRE complex formed LAC4
depends not only on carbon source but also on growth medium 49
3.1.5 Glucose leads to loss of Kdf1
complex-formation in the presence of glycerol 50
3.2 Analysis of regulatory gene KlCAT8 51
3.2.1 Identification of KlCAT8
3.2.2 The influence of KlCAT8 on Kdf1-binding to the CSRE 52 LAC4
3.2.3 KlCat8p is not the factor binding to the CSRE 53 LAC4
3.2.4 The influence of deletion of KlCAT8 on LAC4 expression 55
3.3 Analysis of regulatory gene KlSIP4 56
3.3.1 Identification of KlSIP4
3.3.2 Deletion of KlSIP4 62
3.3.3 In a Klsip4 deletion strain Kdf1-binding is severely impaired 63
3.3.3 The effect of Klcat8 and Klsip4 deletions on β-galactosidase activity 64
3.4 Analysis of regulation mediated by KlSIP4 66
3.4.1 Identification of KlSIP4 target genes 66
3.4.2 The KlSIP4 promoter contains two CSREs that bind Kdf1 67
3.4.3 Regulation of KlSIP4 gene expression 69
3.4.4 Multicopy KlCAT8 does not suppress
the growth defect of the Klsip4 deletion 70
3.4.4 The KlCAT8 promoter contains no carbon source-responsive element 71
3.4.6 The effect of KlSIP4 on isocitrate lyase 72
3.4.7 The KlICL1 promoter contains a low-affinity CSRE 73
3.4.8 The effect of KlSIP4 on malate synthase 74
3.4.9 Acetyl-CoA synthetase is not regulated through a CSRE 76
3.5 Galactose repression of Kdf1-CSRE binding 77 LAC4
3.5.1 The influence of galactose on Kdf1-binding to the CSRElac4
3.5.2 The role of galactokinase KlGal1p
in regulation of CSRE -binding of Kdf1 78 LAC4
3.5.3 Influence of Klgal1 on regulation
of Kdf1-binding in a Klgal80 background 79
3.5.4 Influence of galactose phosphorylation on regulation of Kdf1-binding 81

4 Discussion 83
4.1 The LAC4 Carbon Source-Responsive Element 83
4.2 The role of KlCAT8 in growth on poor carbon sources 86
4.3 The role of KlSIP4 in grow carces 88
4.4 Target genes of SIP4 in K. lactis 91
4.5 Galactose repression of Kdf1-CSRE complex formation 96 LAC4

5.1 Abstract 99

5.2 Zusammenfassung 101

6 References 03

7 Abbreviations 121

1 Introduction

1.1 Regulation of genes: both end and means

In the five years since the complete genome sequence of baker's yeast
Saccharomyces cerevisiae (Goffeau et al, 1996; http://genome-www.stanford.edu/
Saccharomyces/) became available, the genome sequences of other eukaryotic
model organisms have been completed and made public: the nematode
Caenorhabditis elegans (The C. elegans Sequencing Consortium, 1998; http://www.
sanger.ac.uk/Projects/C_elegans/), the fruit fly Drosophila melanogaster (Adams et
al, 2000; http://www.fruitfly.org/), the flowering plant Arabidopsis thaliana (The
Arabidopsis Genome Initiative, 2000; http://www.arabidopsis.org/) and, finally, the
human genome (Lander et al, 2001; Venter et al, 2001; http://www.ncbi.nlm.nih.gov/
genome/guide/human/). Comparison of these sequences has taught us that the
increase in complexity, particularly from worm to fly to man, is not reflected in the
number of genes to the extent it was thought to be previously. The estimates of
30,000 to 40,000 genes in man amount to roughly double to triple the number of C.
elegans (~14,300) or D. melanogaster (~13,600) genes and five to six times as many
genes as the unicellular baker's yeast (~6,300). This means that the observed
diversity of proteins is created mostly though modifications such as alternative
splicing of primary transcripts to mature mRNAs during transcription of a gene.
Additionally, co- or post-translational modification of nascent polypeptides, such as
cleavage of pre- or preproproteins, methylation or myristoylation to name only a few,
serve to form the mature protein. Finally the biological activity of mature proteins can
be regulated short-term by phosphorylation/dephosphorylation, differential complex
formation and differential localization. Regulated degradation of mRNAs and proteins
plays an additional role in determining protein abundance and regulatory response
timing.
Even so, the first and prerequisite step leading to production and activity of a given
protein is the transcription of the gene that encodes it. As it turns out transcriptional
activity is subject to regulation, involving many autoregulatory loops, that is no less
complex and intricate than any of the other regulatory mechanisms mentioned above.

1.1.1 Three classes of genes, transcribed by different RNA polymerases

Enzymes of the family of DNA-dependent RNA polymerases carry out the process of
transcription, that is the synthesis of a molecule of RNA mirroring the information
encoded on the template DNA. Eukaryotes have three different RNA polymerases,
RNA pol I, II and III, which transcribe different, specific sets of genes. RNA
7
polymerase I is responsible for the synthesis of the 18S, 25S and 5.8S ribosomal
RNAs, which are cleaved from single precursor RNAs encoded by the highly
transcribed rRNA genes (RDN1 on Chr. XII in S. cerevisiae) that are clustered on the
chromosomes as tandem repeats. RNA polymerase III synthesizes the 5S ribosomal
RNA, the tRNAs and other small, non-protein-encoding RNA molecules (reviewed in
Paule an