Tandemly Repeated 147 bp Elements Cause Structural and Functional Variation in Divergent MAL Promoters of Saccharomyces cerevisiae

We have studied four novel MAL promoters isolated from a single strain of bakers' yeast. Within these promoters we have identified up to five tandem 147 bp repeats located between the MAL UAS region and the MALT TATA box. These repeats strongly reduce MALT (maltose permease) gene expression but only weakly reduce MALS (maltase) gene expression. Insertion of the 147 bp elements into the heterologous CYC1 promoter reduced expression when located between the CYC1 UAS and the TATA box, but not when located upstream of the UAS. We propose that these naturally occurring repeats have evolved as a mechanism to lower the level of MALT expression relative to MALS expression, thus avoiding possible toxic effects associated with over-expression from multiple copies of the permease gene. Accession numbers are: WIG1, U86359; WIG3, U86360; WIG4, U86361; WIG5, U86362. © 1997 by John Wiley & Sons, Ltd.     

The role of reactive oxygen species in the induction of Ty1 retrotransposition in Saccharomyces cerevisiae

Here we provide evidence for a dependence between the increased production of reactive oxygen species and the activation of Ty1 retrotransposition. We have found that the strong activator of Ty1 mobility, methylmethane sulphonate, can not induce Ty1 retrotransposition in cells with compromised mitochondrial oxidative phosphorylation (rho^?; sco1Δ), which is the major source for production of reactive oxygen species (ROS) in Saccharomyces cerevisiae. The quantitative estimation of superoxide anions in living cells showed that rho⁺ cells exposed to methylmethane sulphonate increase Ty1 retrotransposition and superoxide levels. The increase of superoxide anions by the superoxide generator menadione is accompanied by induction of Ty1 mobility without any treatment with a DNA‐damaging agent. Higher frequencies of retrotransposition were found in rho⁺ and rho^? cells treated with exogenously added hydrogen peroxide or in cells with disrupted YAP1 gene characterized by increased intracellular levels of hydrogen peroxide. These data indicate that increased levels of ROS may have an independent and key role in the induction of Ty1 retrotransposition. Copyright © 2010 John Wiley & Sons, Ltd.     

XVI. Yeast sequencing reports. A new essential gene GCR4 located in the upstream region of GCR1

A new essential gene of Saccharomyces cerevisiae was found upstream of GCR1. Its cloning and sequencing predict a 280 amino acid protein (32 577 Da). The predicted protein is fairly hydrophobic, and a search of the database did not identify any homologous proteins. A LEU2 disruption at codon 104 was lethal, but disruption at codon 221 showed a temperature-sensitive conditional growth phenotype. Abnormalities were observed in some glycolytic enzyme levels. The sequence has been submitted to GenBank-EMBL-DDBJ under Accession Number D29645.     

DNA-binding properties of the yeast transcriptional activator,Gcr1p

In Saccharomyces cerevisiae the GCR1 gene product is required for high-level expression of genes encoding glycolytic enzymes. In this communication, we extend our analysis of the DNA binding properties of Gcr1p. The DNA-binding domain of Gcr1p binds DNA with high affinity. The apparent dissociation constant of the Gcr1p DNA-binding domain for one of its specific binding sites (TTTCAGCTTCCTCTAT) is 2·9×10⁻¹⁰ M. However, competition experiments showed that Gcr1p binds this site in vitro with a low degree of specificity. We measured a 33-fold difference between the ability of specific competitor and DNA of random sequence to inhibit the formation of nucleoprotein complexes between Gcr1p and a radiolabeled DNA probe containing its binding site. DNA band-shift experiments, utilizing probes of constant length in which the positions of Gcr1p-binding sites are varied relative to the ends, indicated that Gcr1p–DNA nucleoprotein complexes contain bent DNA. The implications of these findings in terms of the combinatorial interactions that occur at the upstream activating sequence elements of genes encoding glycolytic enzymes are discussed.     

XIV. Yeast sequencing reports. A 21·7 kb DNA segment on the left arm of yeast chromosome XIV carries WHI3, GCR2, SPX18, SPX19, an homologue to the heat shock gene SSB1 and 8 new open reading frames of unknown function

We report the amino acid sequence of 13 open reading frames (ORF > 299 bp) located on a 21·7 kb DNA segment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Five open reading frames had been entirely or partially sequenced previously: WHI3, GCR2, SPX19, SPX18 and a heat shock gene similar to SSB1. The products of 8 other ORFs are new putative proteins among which N1394 is probably a membrane protein. N1346 contains a leucine zipper pattern and the corresponding ORF presents an HAP (global regulator of respiratory genes) upstream activating sequence in the promoting region. N1386 shares homologies with the DNA structure-specific recognition protein family SSRPs and the corresponding ORF is preceded by an MCB (MluI cell cycle box) upstream activating factor. The sequence has been deposited in the EMBL data library under Accession Number X78898.     

Improved gap‐repair cloning method that uses oligonucleotides to target cognate sequences

In vivo or gap‐repair cloning in yeast has been widely recognized as one of the most efficient means for error‐free construction of plasmids. A protocol is described here that allows easy and efficient gap‐repair cloning that is based on two major modifications. Instead of subcloning, the targeting plasmids are constructed using oligonucleotides from sequences derived from the upstream and downstream sequences of the fragment to be cloned. These sequences are selected so that they can lead to the generation of recognition sites for restriction enzymes that produce blunt ends. Accordingly, this procedure can be applied to any DNA fragment, regardless of whether these include unique restriction sites to generate the targeting ends. With the strategy described, ～50 bp upstream and downstream targeting ends are generated that allow efficient cloning. Further, to allow easy identification of the positive clones, the annealed oligonucleotides are cloned in frame with the lacZ fragment present in the plasmid. Accordingly, these plasmids produce blue Escherichia coli colonies on media containing X‐Gal. On the other hand, plasmids rescued from yeast that have acquired the respective cognate sequences produce white colonies. To demonstrate the efficiency of the method, this report includes the cloning of fragments harbouring the CDC28, CAK1, CIN5 and CLB2 genes. We found that 30–100% of the analysed plasmids carried the expected inserts. Copyright © 2009 John Wiley & Sons, Ltd.