In silico analysis of promoter regions and regulatory elements (motifs and CpG islands) of the genes encoding for alcohol production in Saccharomyces cerevisiaea S288C and Schizosaccharomyces pombe 972h.

BACKGROUND: The crucial factor in the production of bio-fuels is the choice of potent microorganisms used in fermentation processes. Despite the evolving trend of using bacteria, yeast is still the primary choice for fermentation. Molecular characterization of many genes from baker's yeast (Saccharomyces cerevisiaea), and fission yeast (Schizosaccharomyces pombe), have improved our understanding in gene structure and the regulation of its expression. This in silico study was done with the aim of analyzing the promoter regions, transcription start site (TSS), and CpG islands of genes encoding for alcohol production in S. cerevisiaea S288C and S. pombe 972h-. RESULTS: The analysis revealed the highest promoter prediction scores (1.0) were obtained in five sequences (AAD4, SFA1, GRE3, YKL071W, and YPR127W) for S. cerevisiaea S288C TSS while the lowest (0.8) were found in three sequences (AAD6, ADH5, and BDH2). Similarly, in S. pombe 972h-, the highest (0.99) and lowest (0.88) prediction scores were obtained in five (Adh1, SPBC8E4.04, SPBC215.11c, SPAP32A8.02, and SPAC19G12.09) and one (erg27) sequences, respectively. Determination of common motifs revealed that S. cerevisiaea S288C had 100% coverage at MSc1 with an E value of 3.7e-007 while S. pombe 972h- had 95.23% at MSp1 with an E value of 2.6e+002. Furthermore, comparison of identified transcription factor proteins indicated that 88.88% of MSp1 were exactly similar to MSc1. It also revealed that only 21.73% in S. cerevisiaea S288C and 28% in S. pombe 972h- of the gene body regions had CpG islands. A combined phylogenetic analysis indicated that all sequences from both S. cerevisiaea S288C and S. pombe 972h- were divided into four subgroups (I, II, III, and IV). The four clades are respectively colored in blue, red, green, and violet. CONCLUSION: This in silico analysis of gene promoter regions and transcription factors through the actions of regulatory structure such as motifs and CpG islands of genes encoding alcohol production could be used to predict gene expression profiles in yeast species.

A non-radioactive DNA synthesis assay demonstrates that elements of the Sigma 1278b Mip1 mitochondrial DNA polymerase domain and C-terminal extension facilitate robust enzyme activity.

The yeast DNA polymerase gamma, Mip1, is a useful tool to investigate the impact of orthologous human disease variants on mitochondrial DNA (mtDNA) replication. However, Mip1 is characterized by a C-terminal extension (CTE) that is not found on orthologous metazoan DNA polymerases, and the CTE is required for robust enzymatic activity. Two MIP1 alleles exist in standard yeast strains, encoding Mip1[S] or Mip1[Σ]. Mip1[S] is associated with reduced mtDNA stability and increased error rates in vivo. Although the Mip1[S] allele was initially identified in S288c, the Mip1[Σ] allele is widely present among available yeast genome sequences, suggesting that it is the wild-type (WT) allele. We developed a novel non-radioactive polymerase gamma assay to assess Mip1 functioning at its intracellular location, the mitochondrial membrane. Membrane fractions were isolated from yeast cells expressing full-length or CTE truncation variants of Mip1[S] or a chimeric Mip1[S] isoform harboring the Mip1[Σ]-specific T661 residue (cMip1 T661). Relative incorporation of digoxigenin (DIG)-11-deoxyuridine monophosphate (DIG-dUMP) by cMip1 T661 was higher than that by Mip1[S]. A cMip1 T661variant lacking 175 C-terminal residues maintained WT levels of DIG-dUMP incorporation, whereas the C-terminal variant lacking 205 residues displayed a significant decrease in incorporation. Newly synthesized DIG-labeled DNA decreased during later phases of reactions carried out at 37°C, suggesting temperature-sensitive destabilization of the polymerase domain and/or increased shuttling of the nascent DNA into the exonuclease domain. Comparative analysis of Mip1 enzyme functions using our novel assay has further demonstrated the importance of the CTE and T661 encoded by MIP1[Σ] in yeast mtDNA replication.

The reference genome sequence of Saccharomyces cerevisiae: then and now.

The genome of the budding yeast Saccharomyces cerevisiae was the first completely sequenced from a eukaryote. It was released in 1996 as the work of a worldwide effort of hundreds of researchers. In the time since, the yeast genome has been intensively studied by geneticists, molecular biologists, and computational scientists all over the world. Maintenance and annotation of the genome sequence have long been provided by the Saccharomyces Genome Database, one of the original model organism databases. To deepen our understanding of the eukaryotic genome, the S. cerevisiae strain S288C reference genome sequence was updated recently in its first major update since 1996. The new version, called "S288C 2010," was determined from a single yeast colony using modern sequencing technologies and serves as the anchor for further innovations in yeast genomic science.