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J. Biol. Chem., Vol. 265, Issue 15, 8377-8381, May, 1990

Efficient expression and utilization of mutant 5 S rRNA in Saccharomyces cerevisiae

DI Van Ryk, Y Lee and RN Nazar
Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.

The expression of mutant 5 S rRNA genes in vivo is examined as a basis for further studies on the control, structure, and function of the ribosomal 5 S RNA. Specific single base substitutions (e.g. positions 98 or 99) or short insertions can result in substantial structural changes that can easily be detected by gel electrophoresis and permit the assay of mutant RNA synthesis and utilization. In addition, the use of high and low copy shuttle vectors as well as alternate growth conditions permits the adjustment of mutant RNA levels in vivo. Despite the high genomic copy number for the 5 S rRNA gene, under optimized conditions as much as 80% of the cellular 5 S RNA can be mutant, and RNA structure analyses indicate that some of these RNAs can readily be assembled into the ribosome structure resulting in an in vivo ribosome population which is also approximately 80% mutant. The results indicate that plasmid integrated 5 S rRNA genes are preferentially expressed and suggest that additional features of the chromosome structure regulate 5 S rRNA gene expression in vivo.
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