Comparative analysis of promoter regions containing binding sites of the heterodimeric transcription factor Ino2/Ino4 involved in yeast phospholipid biosynthesis

The inositol/choline responsive element (ICRE) functions as a UAS element mediating coordinate expression of structural genes required for yeast phospholipid biosynthesis. However, ICRE motifs could be detected upstream of various genes apparently not involved in lipid metabolism. In this work we investigated the expression pattern of selected genes containing ICRE promoter motifs, as identified by in silico analysis (ARG4, ERG20, FAR8, GPD2, RSF1, URA8, VHT1 and YEL073C). It turned out that the presence of an ICRE upstream of a gene of unknown function indeed allows to conclude for regulation by phospholipid precursors, which is mediated by activators Ino2/Ino4 and the repressor Opi1. We also demonstrated in vitro binding of Ino2/Ino4 heterodimers to promoter regions. Thus, our analysis supports the view that identification of regulatory elements by a database search provides evidence for a specific pattern of gene expression. Activation by pathway-specific regulators may suggest a physiological function for as yet uncharacterized genes.     

Opi1 mediates repression of phospholipid biosynthesis by phosphate limitation in the yeast Saccharomyces cerevisiae

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are transcribed when precursor molecules inositol and choline (IC) are limiting. Gene expression is stimulated by the heterodimeric activator Ino2/Ino4, which binds to ICRE (inositol/choline‐responsive element) promoter sequences. Activation is prevented by repressor Opi1, counteracting Ino2 when high concentrations of IC are available. Here we show that ICRE‐dependent gene activation is repressed not only by an excess of IC but also under conditions of phosphate starvation. While PHO5 is activated by phosphate limitation, INO1 expression is repressed about 10‐fold. Repression of ICRE‐dependent genes by low phosphate is no longer observed in an opi1 mutant while repression is still effective in mutants of the PHO regulon (pho4, pho80, pho81 and pho85). In contrast, gene expression with high phosphate is reduced in the absence of pleiotropic sensor protein kinase Pho85. We could demonstrate that Pho85 binds to Opi1 in vitro and in vivo and that this interaction is increased in the presence of high concentrations of phosphate. Interestingly, Pho85 binds to two separate domains of Opi1 which have been previously shown to recruit pleiotropic corepressor Sin3 and activator Ino2, respectively. We postulate that Pho85 positively influences ICRE‐dependent gene expression by phosphorylation‐dependent weakening of Opi1 repressor, affecting its functional domains required for promoter recruitment and corepressor interaction. Copyright © 2016 John Wiley & Sons, Ltd.     

Negative regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by a Candida albicans orthologue of OPI1

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.     

Organization and localization of the dnaA and dnaK gene regions on the multichromosomal genome of Burkholderia multivorans ATCC 17616

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To reveal the distribution and organization of the genes for fundamental cell functions on the genome of this bacterium, the dnaA and dnaK gene regions of ATCC 17616 were cloned and characterized. The gene organization of the dnaA region was rnpA-rmpH-dnaA-dnaN-gyrB with a single consensus DnaA-binding box (TTATCCACA) between the rmpH and dnaA genes. This intergenic region, however, did not work as an autonomously replicating sequence in ATCC 17616. On the other hand, the gene organization of the dnaK region was grpE-orf1 (gene for thioredoxin homologue)-dnaK-dnaJ-pabB (gene for p-aminobenzoate synthetase component homologue). A putative heat-shock promoter that showed good homology to the sigma32-dependent promoter consensus sequence in Escherichia coli was found upstream of the grpE gene, suggesting that these five genes constitute an operon. In M9 succinate minimal medium the dnaJ mutant grew more slowly than the wild-type strain, indicating that this operon is functional. Pulsed-field gel electrophoresis and Southern blot analyses indicated that both the dnaA and dnaK gene regions exist as single copies on the 3.4 Mb chromosome.     

Cloning and expression analysis of two catalase genes from Aspergillus oryzae

Fungi contain distinct genes encoding the same class of enzyme that are differentially regulated according to conditions. We cloned two catalase genes, catA and catB, from Aspergillus oryzae. The catA gene predicts a 747-amino-acid polypeptide sharing 81% identity with Aspergillus fumigatus catalase (catA) and 77% with Aspergillus nidulans catalase (catA). The catB gene predicts a 725-amino-acid polypeptide sharing 82% identity with A. fumigatus catalase (catB) and 75% with A. nidulans catalase (catB). However, the catA and catB genes share little homology (41%) with one another, suggesting that each gene belongs to a distinct gene family. Overexpression studies demonstrated that both genes encode a functional catalase. Promoter assays indicated that the catA gene is developmentally regulated as it was preferentially expressed in solid-state cultures undergoing sporulation. However, its expression was not affected by hydrogen peroxide treatment. Conversely, the catB gene was highly expressed under all culture conditions tested, and it was induced by hydrogen peroxide treatment. These results suggest that the catB gene may be mainly used for detoxification of oxidative stress while the catA gene may have another role such as chaperoning proteins in the spore.     

Nucleotide sequences and characterization of genes encoding naphthalene upper pathway of pseudomonas aeruginosa PaK1 and Pseudomonas putida OUS82

A 12,808-nucleotide containing DNA fragment cloned from naphthalene-utilizing (Nah+) Pseudomonas aeruginosa PaK1 was analyzed and compared with the genes (pah(OUS)) of a 14,462-nucleotide DNA fragment from Pseudomonas putida OUS82. The DNA sequence analyses demonstrated that the naphthalene upper-pathway genes and their deduced enzymes were very similar between the two bacteria: nucleotide similarities, 83-93%; amino acid similarities, 79-95%. These genes were also similar to those of the nah operon of plasmid NAH7; in particular, the OUS82 genes were similar to the nah genes, whereas the PaK1 genes were almost identical to the dox genes of Pseudomonas sp. C18. A region homologous with the 84-bp repeated sequence that Eaton (J. Bacteriol., 176, 7757-7762, 1994) has found at a site upstream of he nah operon was found only in a region downstream of the pah(PaK) gene cluster in PaK1 and on both sides of the pah(OUS) gene cluster in OUS82. A PaK1 gene, corresponding to an unknown gene (nahQ) in the nah operon, is located between the 1,2-dihydroxynaphthalene dioxygenase gene and the trans-o-hydroxybenzylindenepyruvate (tHBP A) hydratase-aldolase gene (nahE), and was suggested to be involved in the conversion of naphthalene to salicylate. Just downstream of the pah(PaK) gene cluster, a portion of a region was identical to one-third of the transposase gene (tnpA) in a phenol-catabolic plasmid pEST1226.     

Bifidobacterium bifidum BF-1 suppresses Helicobacter pylori-induced genes in human epithelial cells

Helicobacter pylori infection alters gene expression in host cells. Specifically, inflammatory chemokines such as IL-8 are upregulated in the gastric mucosa during H. pylori infection. Although the mechanism by which H. pylori causes inflammation of the gastric mucosa is not yet understood, many studies have suggested that nuclear factor kappa B (NF-κB) plays a key regulatory role in host cells. We have shown that preincubation with Bifidobacterium bifidum strain BF-1, a probiotic strain known to improve H. pylori-associated gastritis, suppresses induction of IL-8 by the pathogen. To investigate how how BF-1 affects gene expression in H. pylori-infected cells, we performed microarray analysis to assess gene expression in epithelial cells, which had been preincubated with BF-1 and infected with H. pylori. We found that preincubation with BF-1 suppresses the expression of H. pylori-induced genes in human cells and that most of the affected genes are related to the NF-κB signaling pathways. These results suggest that BF-1 can affect the regulatory mechanism of the NF-κB signaling pathways.     

Frequency of DLK1 c.639C>T polymorphism and the analysis of MEG3/DLK1/PEG11 cluster expression in muscle of swine raised in Poland

DLK1 — (Drosophila like element 1) is a paternally expressed gene, associated with the callipyge phenotype in sheep. In a present study we designed a new real-time PCR alleleic discrimination assay for genotyping of a silent C/T mutation (c.639C>T) in DLK1 gene in swine. The DLK1 c.639C>T mutation was highly polymorphic in all breeds analyzed and C allele was predominant in Landrace and Duroc while T allele was more frequent in Pietrain and Pu?awska breed. Moreover, we analyzed mRNA expression of DLK1 and adjacent genes — MEG3 and PEG11 in muscles of swines of different breeds raised in Poland. We did not observe significantly different expression of DLK1, MEG3 or PEG11 mRNA in any of analyzed breeds. We also attempted to assess the effect of DLK1 (c.639C>T) on the expression of genes in callipyge locus but did not find significant differences between animals with alternate genotypes (C/C and T/T homozygotes).