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Author Contact: cwhite01@wesleyan.edu
Local Identifier: 365
PID: ir:2822"},{"label":"Author-supplied Keywords","value":["RRB","Agc","S. cerevisiae"]},{"label":"Degree","value":["Master of Arts"]},{"label":"Description","value":"

Organisms regulate gene expression at the transcriptional level to meet the variable needs of the cell. These needs change in response to environmental stimuli and developmental needs, and one of the biggest and most complex pathways that microbial cells need to control is that of ribosome biosynthesis. Ribosomal biogenesis in yeast requires hundreds of gene products to produce upwards of 2,000 ribosomes per minute. This tightly regulated metabolic process depends on the synthesis of rRNAs and ribosomal proteins, in conjunction with a coregulated set of genes constituting the rRNA and ribosomes biogenesis regulon (RRB). The RRB regulon of S. cerevisiae consists of over 200 genes including those that process and assemble the ribosomes. Interestingly, a significant fraction of these RRB genes (28) occur as adjacent pairs on the chromosome. Gene expression analysis of the coregulated MPP10-MRX12 RRB gene pair has shown that the promoter of MPP10 regulates the transcription rate of MRX12. This phenomenon has come to be known as adjacent gene coregulation (AGC). Typically, mRNA expression is monitored through qRT-PCR analysis. However, this technique can be laborious and a visual assay for measuring gene expression would prove to be a useful alternative. Here we investigate the effectiveness of a two-color assay for monitoring AGC. Through Gibson assembly, we created a plasmid that replaced the adjacent gene pair of MPP10-MRX12 with yemVenus- mRuby2 open reading frames. Fluorescent microscopy and flow cytometry were then utilized to analyze the fluorescent intensity produced by yemVenus (green) and mRuby2 (red), and this was used as a proxy for mRNA expression. The strain carrying the reporter plasmid was subjected to heat shock and the mRNA levels of both reporters were repressed similar to the expression of the chromosomal MPP10-MRX12. However, only yemVenus produced a fluorescent signal that was visible above background, and it was repressed throughout a heat shock. We then screened through five alternative fluorescent genes to replace mRuby2, all of which were repressed transcriptionally, but did not produce a visible fluorescent signal. In tandem, our reporter system was transformed into various trans- and cis-factor mutated strains known to disrupt AGC. However, these factors only abrogate AGC on the chromosome and do not affect the co-repression of adjacent genes on the plasmid. These results suggest that proper AGC regulation may depend on structure that is unique to the chromosomal context. Ultimately, we may integrate these reporters into the chromosome in order to shed more light on the mechanisms behind the regulation of paired genes in coregulated metabolic pathways.

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