Laboratory of Yeast Molecular Genetics

Intro

The spectrum of genetic events that occur in the nucleus could not be understood without possessing a deep knowledge on the structural packaging of  DNA. In the cell nucleus DNA is compacted with proteins in a complicated DNA-protein complex called chromatin. Although it is discovered long time ago and thousands of papers were published on this subject, until now, how exactly chromatin is organized especially in higher-order structures and how it is connected with the regulation of  gene activity remains mostly obscure. The methods which were developed recently have led to accumulation of new data  better explaining how the chromatin is working. Chromatin is the basic nuclear structure in which DNA is organized in the cell nucleus. Activity of the genes relays not only on the upstream/downstream regulating sequences and transcription factors but also is strongly affected by the way in which DNA is packaged with the chromatin proteins. Chromatin is composed predominantly of DNA and four core histones H2, H2B, H3 and H4. All four histones through their post-translational modifications play an important role in the regulation of genome function. The fifth histone, H1 is involved in the organizing the higher-order chromatin structures and presumably also in the fine regulation of gene activity. However, how and where in the cell cycle this role is accomplished remains mostly obscure.

Laboratory objectives

The main aim of the Laboratory Of Yeast Molecular Genetics is centered on molecular biology and genetics of yeasts. However, the research is performed not only on yeast cells but also includes mammalian culture cells and laboratory animals (mice and rats). The research is mostly centered on the chromatin and epigenetic signals in the gene regulation.

Research projects

The main research projects of the laboratory are in the following areas:

1) Investigation the roles of the chromatin structure and linker histones in the transcription and life cycle of yeast and mammals; 2) Application of the Single Cell Gel Electrophoresis (SCGE) or Comet assay in chromatin research;

3) Protein glycosulation and secretion in Kluyveromyces lactis: cloning and characterization of KlSEC53, KlPSA1 and KlPCL1 genes;

4) Development of a more sensitive test for detection of mutagens, cancirogenes, genotoxins basis on the Yeast Comet Assay (YCA);

1)     In order to elucidate the role/s of linker histones we expressed linker

histones and parts from them in yeast Saccharomyces cerevisiae strains with and without endogenous and exogenous histone H1. The results are promising in that that histone H1 is involved in the completion of higher order chromatin structures and, as was shown by others, in the fine regulation not only of transcription but also in very important processes as apoptosis, recombination, repair etc.

2)     Comet assay is a method initially developed for assessing DNA damages. We succeeded to design a protocol in which Comet assay could be used for revealing the differences in chromatin structure. Initially, we applied the method for mammalian cells. The budding yeast Saccharomyces cerevisiae are single eukaryotes with most of the features of higher eukaryotes. That is why it is often used for fundamental research. The chromatin of Saccharomyces cerevisiae, however, shows some differences with that of higher eukaryotes. In searching for the reasons of these defferences we modified the classical Comet assay by applying it on yeast and developing the Yeast Comet Assay (YCA). Application of YCA demonstrated that it could be used as a tool for chromatin research in yeast, revealing new and unexpected contacts of chromatin proteins with nuclear matrix.

3)     Because of its outstanding secretory capacity and its food grade status as GRAS organism the dairy yeast Kluyveromyces lactis found broad industrial applications during the last years. K. lactis has been utilized for many years as a source of ß-galactosidase and recently it has been successfully used for high-level production of secreted heterologous proteins such as calf prochymosin, human serum albumin and human interleukin-1γ. However, the glycosylation and secretion systems of K. lactis are not so far characterized in details. In order to gain insight into the processes of protein glycosylation and secretion we isolated, sequenced and partially characterized two genes encoding enzymes involved in the early steps of glycosylation, i.e. KlSEC53 and KlPSA1. In addition, the cloning of a gene coding for a protein passing through the secretory pathway is in process. These are the genes encoding phosphomannomutase, GDP-mannose pyrophosphorylase and carboxypeptidase Y, respectively. The construction and analysis of mutant alleles as well as the interaction between the three genes will be further studied.

4)     During last decade Single Cell Gel Electrophoresis (SCGE) also known as Comet assay has become an inevitable tool for detecting damages of DNA. The alkali version of the method could reveal double strand breaks, nicks, base modifications and other changes in the DNA molecule caused by different chemical or physical agents. Initially Comet assay has been used on mammalian and plant cells. We were first who applied Comet assay on yeast and called it Yeast Comet Assay (YCA). According to our data yeast DNA appears to be 100 times more sensitive than DNA of mammalian cells to the action of certain chemicals. We are attempting to improve the YCA method by applying it on a S. cerevisiae cell wall mutant strain. The preliminary results showed that YCA could be used for development of a test system. Such a test system, based on S. cerevisiae mutant could be up to 1000 times more sensitive to DNA damaging agents presented in the environment.