Tightly regulated, beta-estradiol dose-dependent expression system for yeast.

We have refined the regulated expression of UASGAL1, 10-driven genes in yeast by modifying a vector encoding the beta-estradiol inducible activator, GAL4.ER.VP16 (GEV). The expression of GEV was placed under the regulation of the low-level, constitutive MRP7 promoter, and beta-estradiol-regulated expression was monitored by the expression of an integrated UASGAL10-lacZ reporter and by immunoblot analysis of a UASGAL1-regulated gene product. Target gene expression regulated by low levels of GEV has several advantages over the standard galactose-inducible expression systems. (i) Most importantly, the target gene expression is undetectable in the absence of hormone; (ii) target gene expression is beta-estradiol dose-dependent, and variable levels of target gene expression from low to several hundred-fold induction can be achieved; and (iii) induction or depletion studies can be conducted independent of carbon source in gal4 delta strains. In addition, any UASGAL1,10 expression construct can be used without modification of the target gene or many gal4 delta host strains, and GEV vectors are compatible with other inducible yeast expression systems. This method may be useful to researchers investigating the functions of essential genes, dominant negative mutants, mitochondrial genes, and viral, plant, and mammalian genes in yeast assay systems.

Heme regulates SOD2 transcription by activation and repression in Saccharomyces cerevisiae.

SOD2 encodes the Saccharomyces cerevisiae manganese superoxide dismutase (MnSOD), a mitochondrial matrix protein. Heme regulates SOD2 transcription during both fermentative and respiratory growth by three mechanisms. First, SOD2 transcription is activated 10-fold by growth on a non-fermentable carbon source via the heme activator protein (Hap) 2-3-4-5 complex. Second, SOD2 transcription is repressed 90% in the absence of heme. Finally, Hap1p, a heme-binding, DNA-binding transcription activator, is required for full SOD2 expression during growth on glucose. Extensive mutational analysis of the SOD2 promoter reveals three cis elements that mediate heme-dependent regulation. Interestingly, the DNA sequences necessary for repression in the absence of heme overlap those that mediate Hap1p activation. Genetic results suggest a novel role for Hap1p in the regulation of repression in the absence of heme.

T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.

An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression.

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase.

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Role of a key cysteine residue in the gating of the acetylcholine receptor.

We have examined changes in single-channel behavior that result from conservative amino acid substitutions at the Cys230 residue in the putative first transmembrane region (M1) of the murine nicotinic acetylcholine receptor. Mutations made in the gamma subunit altered the energy barrier for a single closing rate constant in proportion to the size of the substituted side chain. One of these substitutions, when made in the alpha subunits, had no effect on gating. No mutations altered permeation. We conclude that the region surrounding the M1 Cys is involved in the gating of the nicotinic acetylcholine receptor and that the gamma subunit contributes significantly to the control of channel closure.

Influence of the gamma subunit and expression system on acetylcholine receptor gating.

We have developed a partial kinetic theory for the gating of murine nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes and have used this theory to characterize the role of the gamma subunit in single-channel behavior. Permeation and gating were found to be largely unaffected in AChRs produced in oocytes when the gamma subunit transcript was omitted from microinjections of AChR subunit RNAs. In contrast, marked changes in gating kinetics resulted when even very conservative single amino acid substitutions were introduced into the gamma subunit, indicating that the gamma subunit can have a large effect on AChR gating. We also found that channel openings were much prolonged when murine AChRs were expressed in BC3H-1 cells.

Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator.

Activation of the CYC1 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-beta-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.

Cloning and molecular analysis of the HAP2 locus: a global regulator of respiratory genes in Saccharomyces cerevisiae.

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.

The nucleotide sequence of the rho gene of E. coli K-12.

We have determined the nucleotide sequence of the rho gene which encodes the E. coli K-12 transcription termination factor. The structural gene was located on a cloned 3.6 kilobase BglII-HindIII restriction fragment by the introduction of the insertion element gamma delta and analysis of the recombinant plasmids by restriction analysis and in maxicells. The coding region consists of 1260 nucleotides directing the synthesis of a polypeptide 419 amino acids in length with a calculated molecular weight of 46,094. The deduced amino acid composition, amino-terminal protein sequence and calculated molecular weight are consistent with the data from the analysis of purified rho protein (16). We have shown that the rho genes from E. coli K-12, B and C strains are located on PvuII-HindIII fragments of the same size by hybridization to the rho (K-12) coding sequences.