Transcriptional Regulation of an Extrachromosomal Gal7 Gene in Saccharomyces Cerevisiae (Yeast Plasmids, Regulatory Mutants, Over Expression)
Baker, Steven Morris
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https://hdl.handle.net/2142/70545
Description
Title
Transcriptional Regulation of an Extrachromosomal Gal7 Gene in Saccharomyces Cerevisiae (Yeast Plasmids, Regulatory Mutants, Over Expression)
Author(s)
Baker, Steven Morris
Issue Date
1985
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Abstract
A combination of cloned DNA fragments encoding the yeast GAL7 gene, yeast plasmid vectors, chromosomal gal7 deletion strains, and GAL regulatory mutants were used to characterize the in vitro transcription of the GAL7 gene on autonomously replicating plasmids. Normal expression of the plasmid born GAL7 gene could occur in the absence of DNA encoding the functional genes of the GAL cluster. The single copy centromeric and chromosomal locations of GAL7 were indistinguishable in their response to induction by galactose, repression by glucose or control by positive (GAL4) or negative (GAL80) regulatory factors. Increasing the gene dosage to more than 200 copies per cell increased the galactose induced accumulation of GAL7 mRNA more than 10 fold, reduced galactose induced expression of the single copy chromosomal GAL10 gene, and resulted in constitutive expression of both genes. To determine if the gene dosage effect was due to limiting amounts of GAL4 or GAL80 regulatory factors, the transcription of high copy and single copy genes was characterized in GAL regulatory mutants constructed for this purpose. These results show that a completely functional GAL4 gene product is not required for basal level transcription. In strains containing the GAL4 gene fused to the high expression ADH I promoter, glucose can replace galactose to induce high levels of transcription. Maximum levels of accumulation of mRNA from single copy and high copy genes is elevated two fold. Disruption of GAL80 in combination with the ADH I-GAL4 fusion results in another two fold increase. In this strain, galactose induced transcription of the high copy GAL7 gene results in a greater than 50 fold increase in the levels of GAL7 mRNA, representing 30%-50% of the total cellular mRNA, however, transcription of GAL10 remains subject to the gene dosage effect. These results are consistent with a cooperative effect of saturation of GAL4 DNA binding sites and with a limiting factor, in addition to GAL4, that is required for transcription of the GAL genes. Preliminary results indicate that crude plasmid chromatin fractions contain actively transcribed GAL7 genes.
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