Browsing School, Graduate by Title "Yeast RNA polymerase II largest subunit replaced in vivo with a mouse RNA polymerase II largest subunit"
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Yeast RNA polymerase II largest subunit replaced in vivo with a mouse RNA polymerase II largest subunitThe conservation of many features of eukaryotic RNA polymerase II (pol II) subunits has made the yeast enzyme an excellent model for studying the role of eukaryotic RNA pol II in the process of transcription. Yeast RNA pol II is a multisubunit protein composed of 12 polypeptides individually transcribed from separate chromosomes throughout the yeast genome. Eukaryotic pol II largest subunit (RPB1) contains an unusual carboxyl terminal domain (CTD) composed of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This sequence, which plays an essential role in transcription, is repeated 26-27 times in the yeast Saccharomyces cerevisiae RPB1, and 52 times in the mouse. Due to the high homology between the RPB1 of yeast and mouse, we proposed that functional domains are conserved between mouse and yeast. By using PCR amplification, the complete full length mouse RPB1 gene was cloned and sequenced. Transformation of S. cerevisiae and homologous recombination was used to completely replace the yeast RPB1 gene with the mouse RPB1 gene in vivo. Pulsed-field electrophoresis in conjunction with Southern and Northern blot analysis was used to confirm the integration of the mouse RPB1 gene within the yeast chromosome IV, the correct orientation of the gene downstream of the yeast RPB1 promoter sequence, and transcription of the mouse RPB1 gene while in the diploid state. Unique PCR primers were generated in order to amplify gene-connecting regions and rule out the presence of unwanted yeast RPB1 and HIS3 gene sequences. Substitution of the yeast gene with the mouse gene, resulted in the production of four viable spores of which two contained the full length mouse RPB1 gene as determined by a colony hybridization of the tetrad dissection using the mouse RPB1 gene as a probe. The identification of homologues of RNA pol II among eukaryotic species allows understanding of the universal transcription machinery and how it has evolved from species to species while still exhibiting basal transcription functions. This chimeric model may prove useful for identifying mammalian promoters and transcription factors not recognized by the innate yeast RPB1 gene.