Investigation of messenger RNA stability in the yeast Saccharomyces cerevisiae

Genetics, Biochemistry

Santiago, Thairiyam Chinnappan

University:University of Glasgow

关键词: Genetics, Biochemistry;   

Others  :  http://theses.gla.ac.uk/76621/1/10948114.pdf

来源: University of Glasgow

【 摘 要 】

Factors which influence mRNA stability in the yeast, Saccharomvces cerevisiae have been investigated, in particular the influence of mRNA length and translation. The properties of 13 mRNAs of unknown function have been analysed using yeast cDNA clones as molecular probes. The pyruvate kinase mRNA was also analysed using a genomic clone. Preliminary characterization of each cDNA clone by northern blotting, Southern blotting and cross hybridization experiments demonstrated that each was derived from a different, single copy gene (with the possible exception of cDNA10). A new and convenient technique has been developed for the measurement of mRNA half-life in yeast. The method involves the quantitation of specific mRNAs by dot-blotting after inhibition of transcription using phenanthroline. The chemical half-lives for the 14 mRNAs ranged from 6.6 +/- 0.67 to over 100 minutes, relative to the half-life of the 18S rRNA control. Comparison of mRNA length and half-life revealed that two populations of mRNAs containing relatively stable or unstable mRNAs were present. Within each population there was an inverse relationship between mRNA length and half-life. The distribution of each mRNA across sucrose density gradients of yeast polysomes was analysed. There was no obvious correlation between ribosome loading' and mRNA half-life. Therefore mRNA length, but not ribosome loading, would appear to be an important factor in the determination of the stability of an mRNA in S. cerevisiae. To account for the division of yeast mRNAs into two clear populations on the basis of mRNA length and half-life, it is suggested that at least one other factor must have an influence.