Expression of the yeast CYC genes and CYC1/GalK fusion genes on yeast plasmids.
We have presented the results of our studies of the expression of the CYC genes from plasmids. All our data indicate that the levels of expression and the regulation of expression are very similar for the plasmid-borne genes and the chromosomal genes when care is taken to construct the appropriate plasmids. The usefulness of these plasmids has been demonstrated: mutations affecting regulatory sites adjacent to genes of interest have been constructed [such as the Xho I deletion and inversion in the YCpCYC1(2.4) plasmid] and selected [as in the case of the IS1 insertion into the YCpCYC7(2) plasmid], and these mutations have led us to some tentative conclusions about the location and nature of the regulatory sites of these genes. Furthermore, transformation with plasmids containing modified genes or fusions has permitted isolation of genomic regulatory mutants, as in the selection of lac+ suppressors of the lac- CYC1 1/x inversion carried on the YCpCYC1(2.4) 1/x plasmid. Although we cannot rule out the possibility that use of plasmids might cause us to miss a class of regulatory effects that can be propagated only along a chromosomal structure, we believe that the regulatory effects that we do observe can be more quickly and completely defined by working with plasmids. If any regulatory effects occur only on chromosomes, they can be studied more easily once the basic regulatory phenomena have been analyzed. The regulatory regions of the CYC1, CYC7, and TR2 genes that we have crudely mapped so far all exert their effects 100-300 bp away from the putative transcriptional starting sites. How the information in these regions is transmitted along the DNA is an intriguing question. We are engaged in a mutational analysis of these sites to locate them more precisely, to map second-site mutations that moderate the effects of the original mutations, to obtain genomic mutations that define the genes whose products interact with these sites, and to test combinations of genomic and plasmid mutations to define the sites with which regulatory elements interact. This approach should aid our understanding of the spatial relationships between yeast regulatory sites and transcriptional signals. Ultimately, obtaining mutations in regulatory genes, such as the mutations described here for the anaerobic regulation of TR2, will allow the cloning of these genes by complementation. This will lead to the isolation of the protein encoded and ultimately to an approach to the molecular mechanism of regulation through study of protein-DNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002503:Centromere; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004926:Escherichia coli / Q000235:genetics; D005686:Galactokinase / Q000096:biosynthesis; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000502:physiology; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D005686:Galactokinase; D010100:Oxygen