Use of gene fusions to study the expression of PYK1 in Saccharomyces cerevisiae
This study examined the role of PYKI coding sequences in the expression of PYK1::lacZ gene fusions in Saccharomyces cerevisiae. Further aims were to examine the effects of the vector system upon the mRNA levels from these gene fusions the effect that these gene fusions have upon the yeast cell in general. Analysis of the PYK1::lacZ gene fusions revealed that PYK1 coding sequences were responsible for elevating mRNA levels. This elevation was not due to a single element within the coding region of the PYK1 gene as had been previously proposed (Purvis et al., 1987a; Lithgow, 1989). Models for the stimulatory action of the PYK1 coding region upon the transcription of the PYK1::lacZ gene fusion were presented. PYKI coding region fragments in the PYK1::lacZ gene fusions stabilized the mRNA, but the data presented here were not consistent with a stability element within the PYK1 coding region. An alternative model was presented whereby the translation rate of the mRNA can influence its decay. The effect of expression of these gene fusions upon the yeast cell in general was monitored by examining the mRNA level of two chromosomal loci, PYK1 and PGK1, and by measuring the generation time. In contrast to previous findings, PYK1 and PGK1 mRNA levels were found not to change and so it was concluded that expression of these gene fusions had no general effect upon transcription or mRNA decay. However expression of these gene fusions did lead to an increase in generation time, and it was proposed that this might be due to a general effect upon translation brought about by a reduction in the intracellular pools of tRNAs for non-preferred codons.