Point mutations implicate repeated sequences as essential elements of the CYC7 negative upstream site in Saccharomyces cerevisiae.

Abstract

The transcription of the CYC7 gene of Saccharomyces cerevisiae, encoding the iso-2-cytochrome c protein, is controlled by two upstream regulatory elements, a positive element and a negative element. The nature of the DNA sequences in the negative element were investigated in a two-part approach. The first involved the construction of a CYC7-galK fusion gene which placed the coding sequence of the Escherichia coli galactokinase gene under the regulation of the CYC7 upstream sequences. This fusion allowed the quantitation by galactokinase enzyme assays of the effects on gene expression of a variety of previously isolated deletion mutations within the negative site. The results suggested that the negative site contained three related sequences. This hypothesis was tested in the second part of these studies, the selection of point mutations within the region of the negative site which led to increased CYC7 expression. Point mutations were introduced by a technique which induced mutations within a localized region at high efficiency. All but one of the mutations involved more than a single base-pair change. The mutations followed the pattern that multiple base-pair changes occurred in one repeat or single base-pair changes occurred in two repeats, with the exception of one mutant, which had a single base-pair change in one repeat. This pattern of mutations and the base pairs that were altered strongly supported the hypothesis that the repeats are integral elements of the negative site.Transcriptional regulation of genes in Sac charomyces cerevisiae is mediated through sequences located one hundred to several hundred base pairs 5' to the coding sequence (6, 10-12, 15-18, 29, 31, 32, 34; J. L. Weiss, C. V. Lowry, and R. S. Zitomer, submitted for publication). In all cases reported, there is a sequence which stimulates transcription and is often the site of positive regulation or activation; such sites have been designated upstream activation sites. In addition, in a relatively rare number of cases, there is also a site for repression or negative regulation (32, 34). The CYC7 gene, encoding the iso-2-cytochrome c protein, contains both the positive site and the rarer negative site and, therefore, offers the opportunity to study the nature of both types of sites. Fortunately, the CYC7 gene also offers an excellent genetic system that can be exploited in such studies.S. cerevisiae has two unlinked nuclear genes encoding the cytochrome c protein; CYCI and CYC7 encode the iso-1 and iso-2 proteins, respectively (7, 28). Although the proteins are functionally indistinguishable, the genes are expressed at different levels and differ in their regulation. Transcription of the CYCJ gene is induced by oxygen through two upstream activation site sequences (10), and the resulting protein constitutes 95% of the cytochrome c in aerobically grown cells. Transcription of the CYC7 gene, on the other hand, occurs at a low level which is only marginally sensitive to oxygen (Weiss et al., submitted). The low level of expression is apparentl...

Elements involved in oxygen regulation of the Saccharomyces cerevisiae CYC7 gene.

“…Of 173 down mutants, 4 were identified as mitochondrial mutations by their inability to complement [rho-] mutations, and only 4 proved to contain mutant plasmids as judged by the ability of plasmids derived from these cells to retransform cells to the down phenotype. The reason for the large number of falsepositives in this screen is unclear; the same basic scheme was much more efficient for the isolation of up mutations in the CYC7 negative site carried out previously (47).…”

“…This plasmid contains a 42-bp deletion from -274 to -315, thus lacking the three repeated elements that compose the negative site (47), but retains the positive site and the rest of the CYC7 upstream sequences intact. The CYC7 coding sequence in this plasmid was replaced by the CYC7-lacZ fusion.…”

“…To determine the precise sequences which compose the positive site, point mutations were generated in the region around -240 by using a variation of the sodium bisulfite mutagenesis of Peden and Nathans (32,47). In this scheme, heteroduplexes were formed between a wild-type plasmid containing the CYC7 gene and a similar plasmid containing a deletion in the upstream region.…”

Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences.

“…In * Corresponding author. addition to having elements that activate transcription, some yeast genes have negative regulatory regions which act by repressing transcription (25,52,53). To date, however, the precise role of upstream positive and negative regulatory regions in promoter function remains unclear.…”

Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation.

“…Studies of the CYC7 regulatory region by Zitomer and colleagues have led to the proposal that the region contains separate sites that mediate positive and negative control (28,29). Further evidence for positive control comes from the existence of a dominant mutation, CYPI-18, that is unlinked to the CYC7 locus and substantially increases expression of that locus (3) acting via CYC7 upstream regulatory sequences (14).…”