Molecular & General Genetics : MGG

The SeqA protein acts as a regulator of chromosomal replication initiation in Escherichia coli by sequestering hemi-methylated oriC, effectively blocking methylation and therefore preventing rapid re-initiation. The level of SeqA protein is maximal at mid-log phase and decreases when cells enter late-log phase. In hup mutants that lack the HU protein, the maximal seqA expression is also seen at mid-log phase, but seqA expression, as well as SeqA levels and activity, is increased by up to four fold relative to that in the wild type...

In this study, the unicellular photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used as a model phototroph to study the contribution of enzymatic photoreactivation to the overall protection against UV irradiation. We have isolated genes encoding two DNA photolyase homologs, phrA and phrB, from Synechocystis 6803. phrA encodes an 8-hydroxy-5-deazariboflavin (HDF) type, Class I DNA photolyase. By complementing a photolyase-deficient mutant strain of Escherichia coli, we demonstrated that PhrA is a DNA photolyase...

Recently, we found that transcription of the yvyD gene of Bacillus sibtilis from a sigmaH-dependent promoter is induced in response to amino acid starvation. This induction occurred in the wild type, but not in the relA mutant, suggesting that ppGpp might be involved in the activa tion of sigmaH. In order to substantiate this finding we looked for other sigmaH-dependent genes which also required an active relA gene product for activation in amino acid-starved cells. Three additional transcription units, namely spo0A, spoVG and the ytxGHI operon (also known as the csb40 operon), were found to be under the dual control of RelA and sigmaH in amino acid-starved cells, but many other sigmaH-dependent genes were not activated under these conditions...

We identified two rpoH-related genes encoding sigma32-like proteins from Sinorhizobium meliloti, a nitrogen-fixing root-nodule symbiont of alfalfa. The genes, rpoH1 and rpoH2, are functionally similar to rpoH of Escherichia coli because they partially complemented an E. coli rpoH null mutant. We obtained evidence indicating that these genes are involved in the heat shock response in S. meliloti. Following an increase in temperature, synthesis of several putative heat shock proteins (Hsps) was induced in cultures of wild-type cells: the most prominent were 66- and 60-kDa proteins, both of which are suggested to represent GroEL species...

At elevated temperatures, the Neurospora crassa mutant colonial, temperature-sensitive 3 (cot-3) forms compact, highly branched colonies. Growth of the cot-3 strain under these conditions also results in the loss of the lower molecular weight (LMW) isoform of the Ser/Thr protein kinase encoded by the unlinked cot-1 gene, whose function is also involved in hyphal elongation. The unique cot-3 gene has been cloned by complementation and shown to encode translation elongation factor 2 (EF-2). As expected for a gene with a general role in protein synthesis, cot-3 mRNA is abundantly expressed throughout all asexual phases of the N...

The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1. We constructed a set of 26 Ste6 mutants using a random mutagenesis approach. Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6. Trafficking, therefore, does not seem to be affected in most of the mutants. To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N)...

Microsatellite DNA/simple-sequence-repeat (SSR) loci were identified, isolated and characterized in white spruce (Picea glauca) by screening both a non-enriched partial genomic library and a partial genomic library enriched for (AG/TC)n-containing clones. Inheritance and linkage of polymorphic SSR loci were determined in F1 progeny of four controlled crosses. We also assessed the compatibility and usefulness of the P. glauca microsatellite DNA markers in five other Picea species. Twenty-four microsatellites were identified by sequencing 32 clones selected from screens of 5,400 clones from the two libraries...

The chimeric transcriptional activator TGV mediates dexamethasone (dx)-inducible and tetracycline (tc)-repressible transgene expression in tobacco (dx-on/ tc-off system). The expression profiles of four different synthetic target promoters, comprising multiple TGV binding sites upstream of a core promoter, were characterised using the sensitive luciferase assay. Induction factors of over 1,000 were measured in roots and leaves of over 30% of the transgenic plants, irrespective of the promoter used. Promoters PTF and PTax, which carry the -48 to +1 region of the Cauliflower Mosaic Virus 35S promoter, showed higher expression levels in both the uninduced and induced states than PTop10 and PTFM, which harbour several point mutations in this region...

RNA polymerase I of Saccharomyces cerevisiae contains a small subunit, A12.2, encoded by RPA12, that was previously shown to be involved in the assembly and/or stabilization of the largest subunit, A190, of RNA polymerase I. To examine whether an equivalent subunit is present in another eukaryotic RNA polymerase I, we have cloned a Schizosaccahromyces pombe cDNA that is able to complement the rpa12 mutation in S. cerevisiae. The gene, named Sprpa12+, encodes a polypeptide of 119 amino acids that shows 55% identity to S...

Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively...

We examined whether strand identity with respect to DNA replication influences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis. The specificity of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that differed only in the orientation of supF with respect to a unique origin of replication. Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C-->T:A transversions (> 64%), while the majority in the mutT strain were base substitutions (> 92%), predominantly A:T-->C:G transversions (> 91%)...

Mithramycin is a glycosylated aromatic polyketide produced by Streptomyces argillaceus, and is used as an antitumor drug. Three genes (mtmV, mtmU and mtmC) from the mithramycin gene cluster have been cloned, and characterized by DNA sequencing and by analysis of the products that accumulate in nonproducing mutants, which were generated by insertional inactivation of these genes. The mtm V gene codes for a 2,3-dehydratase that catalyzes early and common steps in the biosynthesis of the three sugars found in mithramycin (D-olivose, D-oliose and D-mycarose); its inactivation caused the accumulation of the nonglycosylated intermediate premithramycinone...

PsaF is a nuclear gene for subunit III of the reaction center of photosystem I, and its expression is stimulated by cytokinins and light, when monitored at the mRNA level or at the level of GUS activity directed by chimeric promoter::uidA gene fusions in transgenic tobacco. These inductive effects can be mimicked by pertussis toxin, serotonin, phorbol acetate myristate or Ca2+, suggesting the involvement of heterotrimeric G proteins, phospholipids and Ca2+-dependent processes. Both breakdown products of the phosphatidylinositol cycle, inositol triphosphate (IP3) and diacylglycerol (or its homolog phorbol myristate acetate, PMA) appear to be involved...

In the course of the Bacillus subtilis genome sequencing project, an ORF called yshD was identified, and its product was classified as a mismatch repair protein. Further analysis of the YshD primary sequence showed that the protein belongs to the MutS2 protein family, sharing a high degree of identity with the Thermootoga Inaritima protein TM1278 (34%) and with the so-called MutS2 protein sl11772 of Synechocystis (32%). The COG1193 family of MutS-like proteins is made up of polypeptides that have been predicted from genomic sequencing data from various prokaryotes, but their biological role has not yet been analysed...

It has been shown that in Escherichia coli the chaperone DnaK is necessary for the late stages of 50S and 30S ribosomal subunit assembly in vivo. Here we focus on the roles of other HSPs (heat-shock proteins), including the chaperonin GroEL, in addition to DnaK, in ribosome biogenesis at high temperature. GroEL is shown to be required for the very late 45S-->50S step in the biogenesis of the large ribosome subunit, but not for 30S assembly. Interestingly, overproduction of GroES/GroEL can partially compensate for a lack of DnaK/DnaJ at 44 degrees C...

Although most eukaryotic cells are sensitive to the 80S ribosome inhibitor cycloheximide (CYH), naturally occurring CYH resistance is widespread amongst yeast species. The primary determinant of resistance appears to be a single residue within ribosomal protein L41; resistance is acquired by the substitution of a conserved proline (P56) by a glutamate residue. We have isolated the L41 gene (RPL41) from the green alga Chlamydomonas reinhardtii, and investigated the molecular basis of CYH resistance in various mutant strains...

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene...

The yeast ORF YPL060w/LPE10 encodes a homologue of the mitochondrial protein Mrs2p. These two proteins are 32% identical, and have two transmembrane domains in their C-terminal regions and a putative magnesium transporter signature, Y/F-G-M-N, at the end of one of these domains. Data presented here indicate that Lpe10p is inserted into the inner mitochondrial membrane with both termini oriented towards the matrix space. Disruption of the LPE10 gene results in a growth defect on non-fermentable substrates (petite phenotype) and a marked defect in group II intron splicing...

We previously described the isolation of yeast mutants (sex mutants) that secrete reduced amounts of mature alpha-factor when it is synthesized as part of a fusion with prosomatostatin. In the present study we show that the sex3-1 mutant displays pleiotropic phenotypes. These include an abnormal morphology, an osmoremediable caffeine sensitivity, reduced secretion of mature alpha-factor, a weakened cell wall and a marked deficiency in halotolerance. Cloning of the SEX3 gene revealed that it is identical to the RPB4 gene...

A 5.2-kb NotI DNA fragment isolated from a genomic library of Acremonium chrysogenum by hybridization with a probe internal to the Penicillium chrysogenum lys2 gene, was able to complement an alpha-aminoadipate reductase-deficient mutant of P. chrysogenum (lysine auxotroph L-G-). Enzyme assays showed that the alpha-aminoadipate reductase activity was restored in all the transformants tested. The lys2-encoded enzyme catalyzed both the activation and reduction of alpha-aminoadipic acid to its semialdehyde, as shown by reaction of the product with p-dimethylaminobenzaldehyde...