DNA insertions which affect the expression of the yeast iso-2-cytochrome c gene

Summary

The plasmid YCpCYC7(2) was constructed containing the Saccharomyces cerevisiae CYC7 gene, encoding the iso-2-cytochrome c protein, replicative sequences and selective markers from both E. coli and yeast, and the centromere of yeast chromosome III. The expression of the plasmid-CYC7 gene in yeast was similar to the low level expression characteristic of the chromosomal CYC7 gene. A number of insertions into the sequences 5′ to the gene were constructed in vitro. The insertion at 142 by 5′ to the coding sequence of a 400 by fragment which lies 5′ to the CYC1 gene and is known to be essential for the high rates of CYC1 transcription increased transcription of the CYC7 gene to levels characteristic of CYC1 transcription. On the other hand, the insertion of random DNA fragments at the same position gave mostly decreased CYC7 transcription. In addition to these in vitro constructions, a mutant plasmid was selected which had increased CYC7 transcription. This mutation was caused by the insertion of the bacterial IS1 element 313 by 5′ to the CYC7 coding sequence. The significance of these results is discussed in terms of two alternative models for CYC7 gene expression.