THE OVERALL AIM OF THE PROJECT IS TO INVESTIGATE THE MECHANISMS WHICH REGULATE THE FLOW OF CARBON THROUGH THE FERMENTATIVE AND RESPIRATORY PATHWAYS OF YEASTS, AND WHICH RESPOND TO CHANGES IN GLUCOSE OR OXYGEN CONCENTRATION. THE PARTICULAR INTEREST OF THE DUBLIN GROUP LIES IN THE GENETIC CONTROL SITES OF THE GENES WHICH MEDIATE SWITCHES IN CARBON METABOLISM, WHICH IS AN IMPORTANT POINT TO UNDERSTAND THE SHIFTS IN METABOLIC ACTIVITIES OCCURING IN YEASTS.
The yeast Saccharomyces cerevisiae is widely used as an efficient agent to ferment sugary liquids to alcoholic beverages and, more recently, to ethanol as a fuel for motor vehicles. The enzymes involved in the glycolysis account for 50% of the total cell protein.
Research was carried out in order to identify the genes coding for factors which are required to enhance the synthesis of elevated levels of those enzymes which increase when yeast cells are exposed to fermentable sugars. This involved the isolation and characterization of those genes and their products which sense the availability of sugars, transmit a signal to the regulatory circuits which specifically activate transcription, translation and maturation of glycolytic enzymes. Mutants were isolated, genes cloned, sequenced, deleted and overexpressed. Direct mutagenesis in vitro was used to identify control regions of such genes and binding proteins. Overexpression of genes coding for different glycolytic enzymes was used to identify possible catalytic bottlenecks in glycolysis.
Pyruvate decarboxylase was used as an indicator enzyme to study the regulation of glycolytic enzymes. A second structural gene PDC5 was found and shown to have great sequence similarity with PDC1 in the coding but not in the flanking 5 prime regions and 3 prime regions. PDC5 is only transcribed when PDC1 is deleted. PDC1 codes for all enzyme activity in wild type. When active, PDC5 promotes the formation of pyruvate decarboxylase at 80% the level of PDC1. Deletion analysis of the 5 prime region of PDC1 identified a sequence required for glucose induction of enzyme formation and a second region with repressing effects. Transcription activation factors were identified by protein deoxyribonucleic acid (DNA) binding assays. A new gene, PDC3, was identified as necessary for pyruvate decarboxylase formation at the posttranscriptional level. Induction of pyruvate decarboxylase requires the function of gene BYP1 involved in transducing a glucose triggered cyclic adenosine monophosphate (cAMP) signal.
THE REGULATION OF FLOW CARBON THROUGH THE FERMENTATIVE AND RESPIRATORY PATHWAYS OF YEAST INVOLVES CHANGES IN THE LEVEL OF EXPRESSION OF A NUMBER OF GENES WHICH DIRECTLY AFFECT THE METABOLISM OF PYRUVATE, INCLUDING PDCI (THE STRUCTURAL GENE FOR PYRUVATE DECARBOXYLASE), PDC2 AND PDC3 (REGULATORY GENES FOR PYRUVATE DECARBOXYLASE).
IN ORDER TO DETERMINE THE CONTROL SITES OF THE GENES WHICH MEDIATE SWITCHES IN CARBON METABOLISM, IN VITRO MUTAGENESIS WILL BE USED. THESE MUTANTS WILL BE USED TO :
1) TO IDENTIFY THE GENETIC CONTROL SITES OF THESE GENES.
2) TO INTRODUCE SPECIFIC MUTATIONS IN THE CONTROL SITES.
3) TO TEST THE EFFECTS OF THESE MUTATIONS ON THE EXPRESSION OF THE COGNATE GENE.
4) TO TEST THE EFFECTS OF PROCESSES.
5) TO USE THE MUTANTS GENERATED IN VITRO TO SELECT SUPPRESSOR MUTATIONS IN OTHER GENES OR CONTROL SITES OF THE NETWORK. THE EFFECTS OF THESE IN VITRO MUTATIONS WILL BE TESTED IN VIVO BOTH IN WILD TYPE AND MUTANT GENETIC BACKGROUNDS, USING THE MUTANT STRAINS CONSTRUCTED AT DARMSTADT AND THE NMR AND OTHER ANALYTICAL FACILITIES IN LONDON, THUS REVEALING DETAILS OF THE ROLE OF THE CONTROL SITES IN THE GENETIC CONTROL NETWORK.
Funding SchemeCSC - Cost-sharing contracts