Development of a Yeast Transcription System Using a Saccharomyces Cerevisiae Enzyme Extract and a Drosophila Melanogaster Histidine tRNA Gene

Abstract

This research problem is directed toward the isolation of an enzyme from the yeast Saccharomyces pombe which endonucleolytically processes the 3' terminus of transfer ribonucleic acids. Plasmids containing tRNA genes were a gift from Dieter Soll, Yale University Department of Molecular Biophysics and Biochemistry. In order to provide increasing amounts of the cloned gene, the bacterium Escherichia coli HB101 was transformed using standard transformation procedures and methods. The transformed bacteria were cultured, and the plasmids amplified using chloramphenicol. These bacteria were then lysed and cesium chloride-ethidium bromide gradients were run to isolate plasmids containing the desired genes. The plasmids were then used in a transcription assay following the procedure of Klekamp and Weil (1). The purpose of this transcription was to synthesize precursor tRNA genes which would serve as substrates for the detection of S. pombe processing nucleases. The synthesized [? – 32p] labeled precursor tRNAs were purified using polyacrylamide gel electrophoresis. Autoradiographic techniques were employed to identify the location of the precursor on the gel. The precursor was then eluted from the gel. The initial goal of this thesis project is to obtain sufficient quantities of precursor tRNAs to use in the detection of 3' processing nucleases. Following detection, the processing nucleases will be purified by standard enzyme purification techniques.