Nickel-dependent superoxide dismutases (NiSODs) represent a novel solution to controlling the deleterious effects of reactive oxygen species derived from superoxide in biology. The expression of recombinant Streptomyces coelicolor NiSOD and its in vitro processing and reconstitution to yield fully active enzyme is reported. The results of studies of NiSODs involving mutations in two putative nickel binding ligands are also reported. These studies show that mutation of M28, a strictly conserved residue and one of only three S-donor ligands in the enzyme, has no measurable effect on the spectroscopic or catalytic properties of the enzyme. In contrast, mutation of the strictly conserved N-terminal H residue has dramatic effects on both the spectroscopic and catalytic properties. These results provide insights into structural and mechanistic aspects of the novel nickel-containing reactive site.

We report here the cloning of the HAP2 gene, a locus required for the expression of many cytochromes and respiratory functions in Saccharomyces cerevisiae. The cloned sequences were found to direct integration of a marked vector to the chromosomal HAP2 locus, and derivatives of these sequences were shown to yield chromosomal disruptions with a Hap2-phenotype. The gene maps 18 centimorgans centromere proximal to ade5 on the left arm of chromosome VII, distinguishing it from any other previously characterized nuclear petite locus. The HAP2 locus encodes a 1.3-kilobase transcript which is present at extremely low levels and which is derepressed in cells grown in media containing nonfermentable carbon sources. Levels of HAP2 mRNA are not reduced in strains bearing a mutation at the HAP3 locus, which is also required for expression of respiratory functions. Models outlining possible interactions of the products of the HAP2 and HAP3 genes are presented.

Activation of the CYCi upstream activation site (UAS2) and other Saccharomyces cerevisiae genes encoding respiratory functions requires the products of the regulatory loci HAP2 and HAP3. We present here the DNA sequence of the yeast HAP2 gene and an initial investigation into the function of its product. The DNA sequence indicated that HAP2 encoded a 265-amino-acid protein whose carboxyl third was highly basic. Also found in the sequence was a polyglutamine tract spanning residues 120 to 133. Several experiments described herein suggest that HAP2 encodes a direct activator of transcription. First, a bifunctional HAP2-0-galactosidase fusion gene was localized to the yeast nucleus. Second, a lexA-HAP2 fusion gene was capable of activating transcription when bound to a lexA operator site. The additional requirement for the HAP3 product in activation is discussed.Yeast upstream activator sites (UASs) and mammalian enhancers are sites that act at a distance to turn on transcription in response to specific physiological signals (for reviews, see references 7 and 9). In several instances single enhancers have been shown to contain several elements that respond to different trans-acting factors. For example, the long terminal repeat of the mouse mammary tumor virus contains separate elements that respond independently to an activation factor found in murine cells and a glucocortocoid receptor (5, 18). In Saccharomyces cerevisiae, the participation of multiple factors in regulation at specific sites can be inferred in several systems. For example, many amino acid biosynthesis genes are expressed at the basal level by as yet undefined regulatory mechanisms and are derepressed during amino acid starvation by the binding of the GCN4 gene product to specific promoter sites (13,14). In the mating type system, expression of oa-specific genes requires the products of both the MATaJ and STE12 genes. The role of STE12 is not to permit MATTaJ expression because expression of MATaJ from a heterologous promoter does not overcome an stel2 defect (6).We have recently found that activation of the upstream activation sites, UAS2, of the yeast CYCI gene requires the product of the HAP2 locus (23). Activation of the other CYCI upstream activation site, UAS1, is unaffected in hap2 mutants (19). HAP2 was shown to reside on chromosome VII, 18 centimorgans centromere-proximal to ADE5, and to encode a nonabundant transcript of 1.3 kilobases (kb).Activation of UAS2 also requires the product of a second locus, HAP3 (S. Hahn, J. Pinkham, and L. Guarente, manuscript in preparation). Mutations in either HAP2 or HAP3 abolish the activity of UAS2. In this report we begin to dissect the roles of the HAP gene products in the activation of UAS2. We present the sequence of the HAP2 locus and evidence that the gene product is a yeast nuclear protein and is an activator of transcription. MATERIALS AND METHODSStrains and general genetic methods. S. cerevisiae strains BWG1-7A (MATa leu2-3 leu2-112 his4-519 adel-100 ura3-52), LGW1 (MATa, leu2-3 leu2-112 his4...

SPKI was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. MATERIALS AND METHODS Plasmids. pSK9-15 and pSK15-9 are Bluescript plasmids (Stratagene Cloning Systems) carrying a 3.6-kbp EcoRI fragment including SPK1 coding and upstream sequences (52); pNB187 was obtained from Peter Novick (Yale University School of Medicine); clnl::URA3 and pH2AB1 were obtained from Fred Cross (Rockefeller University); pRS303, pRS305, pRS315, and pRS316 (51) were obtained from Aaron Mitchell (College of Physicians and Surgeons, Columbia University); and pJM229 (6) was obtained from Steve Brill (Rutgers University).pNB187-SPKI. The 2,764-bp HindIll fragment delineated by the single HindIII site upstream from the SPK1 coding sequences and the Bluescript polylinker (downstream from the SPK1 insert) in pSK9-15 was cloned into the BamHI site of pNB187 by using BamHI-HindIII adaptors. In the resulting plasmid, pNB187-SPK1, SPK1 coding sequences follow the GALl promoter and are flanked by functional BamHI, but not HindIII, cleavage sites. pNB187-spklK227A. pSK9-15 was used as a template for four-primer polymerase chain reaction mutagenesis to mutate nucleotides 712 to 717 (original numbering system [52]) from AAG (Lys) ATT (Ile) to GCA (Ala) ATC (Ile). First, two reactions were run in parallel to produce overlapping DNA fragments with one mutation as follows: reaction A, wild-type forward primer (5') CCCAGCCTGTGACTATCA 'Tl r7AGG (3') and mutagenic reverse primer (5') GCGTTTA ClGTATGATTGCCACC (3'); reaction B, mutagenic forward 5829