Novel mutations in an otherwise strictly conserved domain of CuZn superoxide dismutase.

All mutations in the human gene for CuZn superoxide dismutase (CuZnSOD) reported to date are associated with the disease amyotrophic lateral sclerosis (ALS). These mutations, mostly of a familial nature (ALS 1, MIM 105400), span all of the coding region of this enzyme except for a highly conserved centrally located domain that includes all of exon III. We describe the identification and characterization of two mutations in this region, both found in mice. One mutation, a glutamate to lysine amino acid substitution was found in position 77 (E77K) of the strain SOD1/Ei distributed by the Jackson Laboratory. The other mutation, a lysine to glutamate substitution at position 70 (K70E) of a human transgene, was discovered in mouse line TgHS/SF-155. Enzyme activity measurements and heterodimer analysis of the CuZn SOD variant in SOD1/Ei suggest a mild loss of activity, which differs from the enzyme activity losses detected in patients with autosomal dominant ALS 1. Similarly, the presence of the mutant transgene in TgHS/SF 155 does not produce any phenotypic manifestations.

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.

Purification of the heteromeric protein binding to the URS1 transcriptional repression site in Saccharomyces cerevisiae.

The protein that binds to the URS1 site situated upstream of many genes in Saccharomyces cerevisiae is a central element responsible for global negative control of transcription in this organism. Among the genes whose expression is regulated by this protein are those that participate in nitrogen metabolism, carbon metabolism, electron transport, inositol metabolism, heat shock response, meiosis, and sporulation. This factor, binding URS1 factor (BUF), has been purified and shown to be a heteromeric protein composed of 37.5- and 73.5-kDa monomers. The heteromeric form of BUF is stably maintained both in solution and bound to its DNA target site.

The yeast UME6 gene product is required for transcriptional repression mediated by the CAR1 URS1 repressor binding site.

URS1 is known to be a repressor binding site in Saccharomyces cerevisiae that negatively regulates expression of many genes including CAR1 (arginase), several required for sporulation, mating type switching, inositol metabolism, and oxidative carbon metabolism. In addition to the proteins previously shown to directly bind to the URS1 site, we show here that the UME6 gene product is required for URS1 to mediate repression of gene expression in the absence of inducer. We also show that mutations in the CAR80 (CARGRI) gene are allelic to those in UME6.

Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion.

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.

A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene.

Induction of the arginase (CAR1) gene expression in Saccharomyces cerevisiae has previously been shown to require participation of a cis-dominantly regulated upstream repression sequence (URS). Deletion of this element results in high-level expression of the CAR1 gene without inducer. To determine the structure of the CAR1 URS element, we performed a saturation mutagenesis. Results of the mutagenic analysis indicated that the CAR1 URS was a 9-base-pair palindromic sequence, 5'-AGCCGCCGA-3'. A DNA fragment containing this sequence was shown to bind one or more proteins by a gel shift assay. DNA fragments containing point mutations that completely eliminated URS function were not effective competitors in this assay, whereas those which supported URS function were effective competitors. Sequences in the 5'-flanking regions of 14 other genes were found to be homologous to the CAR1 URS. These sequences were shown to support varying degrees of URS function in the expression vector assay, to bind protein as demonstrated by the gel shift assay, and to compete with a DNA fragment containing the CAR1 URS for protein binding. These results indicate that the CAR1 URS element possesses the characteristics of a repressor binding site. Further, they are consistent with the suggestion that sites homologous to the CAR1 URS may be situated in the 5'-flanking regions of multiple unrelated yeast genes. The widespread occurrence of this element raises the possibility that it is the target site for one or more negatively acting general transcription factors.