Roberta Sumrada

Saccharomyces cerevisiae BUF protein binds to sequences participating in DNA replication in addition to those mediating transcriptional repression (URS1) and activation.

The heteromeric BUF protein was originally shown to bind to URS1 elements which are situated upstream of many genes in Saccharomyces cerevisiae and mediate negative control of their transcription. Among the genes regulated through the URS1 site and the proteins interacting with it are those participating in carbon, nitrogen, and inositol metabolism; electron transport; meiosis; sporulation; and mating-type switching. We show here that pure BUF protein, in addition to binding to the negatively acting URS1 site, also binds to CAR1 sequences supporting transcriptional activation (upstream activation sequences). To determine the BUF protein structure, we cloned and sequenced the BUF1 and BUF2 genes and found them to be identical to the RF-A (RP-A) gene whose products participate in yeast DNA replication as single-stranded DNA binding proteins. These data argue that BUF protein-binding sites serve multiple roles in transcription and replication.

Multiple positive and negative cis-acting elements mediate induced arginase (CAR1) gene expression in Saccharomyces cerevisiae.

Expression of the arginase (CAR1) gene in Saccharomyces cerevisiae is induced by arginine or its analog homoarginine. Induction has been previously shown to require a negatively acting upstream repression sequence, which maintains expression of the gene at a low level in the absence of inducer. The objective of this work was to identify the cis-acting elements responsible for CAR1 transcriptional activation and response to inducer. We identified three upstream activation sequences (UASs) that support transcriptional activation in a heterologous expression vector. Two of these UAS elements function in the absence of inducer, whereas the third functions only when inducer is present. One of the inducer-independent UAS elements exhibits significant homology to the Sp1 factor-binding sites identified in simian virus 40 and various mammalian genes.

Basic amino acid inhibition of cell division and macromolecular synthesis in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae on poor nitrogen sources such as allantoin or proline was totally inhibited by addition of a non-degradable basic amino acid to the medium. Cells treated with lysine contained greatly reduced quantities of histidine and arginine. Conversely, lysine and histidine were severely reduced in arginase-deficient cells treated with arginine. When all three basic amino acids were present in the culture medium, growth was normal suggesting that synthesis of all three basic amino acids was decreased by an excess of any one of them. Inhibition of growth was accompanied by a fivefold increase in the observed ratio of budded to unbudded cells. These morphological changes suggested that DNA synthesis was inhibited. Consistent with this suggestion, addition of a basic amino acid to the culture medium substantially reduced the ability of the cells to incorporate [14C]uracil into alkali-resistant, trichloroacetic acid-precipitable material. RNA and protein synthesis, although decreased, were less sensitive to the effects of such additions.