Gene Regulation in Sacchromyces yeast

All of the cells in the body contain the same set of genes, but different cells express different genes. Two possible ways to control gene expression are to prevent transcription of DNA or regulate the translation of RNA into protein. The binding of regulatory proteins to regulatory sites either inhibits or activates gene transcription. By changing the environment that cells are grown in, the binding of different proteins to their corresponding regulatory sites is influenced. In addition, the presence of regulatory sites is a determining factor as to whether a gene is expressed or not. I worked with the GAL1 gene of the Saccromyces genus of yeast. The GAL1 gene is present in numerous yeast species, but the regulatory sites can vary among different species. When comparing different species, the closest species to S.cerevisiae in sequence were S. paradoxus and S. bayanus; the regulatory sequences were conserved. Thus, the hypothesis is that species close in evolution (S. cerevisiae, S. paradoxus, and S.bayanus) would exhibit similar gene expression. My objective for this summer was to determine whether GAL1 was regulated in the same way in all of the species tested (S. bayanus, S. castellii, S.cerevisiae, S. kluyveri, S. mikatae, and S. paradoxus).

In the presence of galactose, the regulatory protein Gal4 binds to the Gal4 binding site, which activates GAL1 transcription. The GAL1 gene encodes galactokinase, which catalyzes the conversion of galactose into glucose. In addition, the GAL1 gene is also involved in a negative feedback mechanism. In the presence of glucose, the regulatory protein Mig1 binds to the corresponding Mig1 site on the promoter, which results in the repression of transcription of the GAL1 gene and galactose is not converted into glucose.
Since the amount of sugar cannot be measured, a reporter gene, lacZ, is needed as its activity is easy to measure. The first part of my project was to fuse the lacZ gene from E.coli bacteria to the GAL1 promoters of different yeast species. I used a substance called X-Gal as a substrate in the assay. The cells expressing lacZ would turn blue on an X-Gal plate, and the cells not expressing lacZ would remain white . However, this method only enabled me to collect qualitative observations. A different substrate, ONPG, was used in another set of assays to collect quantitative data. When lacZ is expressed, beta galactosidase catalyzes the conversion of ONPG into ONP and galactose. ONP is a yellow substance, and the intensity of the yellow color can be measured using a spectrophotometer. Higher values correspond to higher GAL1 expression levels, and the GAL1 expression levels are compared between cells grown in glucose and cells grown in galactose of the same species.

In both experiments, Saccromyces cerevisiae served as the positive control since it is the most commonly studied species in laboratories. It is expected to exhibit GAL1 expression in galactose, and it is not expected to exhibit GAL1 expression in glucose. My experiments showed this, and most of the species that I tested exhibited similar activity. However, my results showed that S. paradoxus appeared to be regulated differently than other species. While species of S. castellii, S. bayanus, S. mikatae, and S.kuyveri exhibited lacZ expression, S. paradoxus did not.

Since my data represented only a few experiments, the experiments must be repeated many times before the conclusion that S. paradoxus is regulated differently by galactose can be validated. Nonetheless, my research has shown that DNA sequence analysis is a viable method to predict gene regulation in most cases.