Inverted Repeats and Genome Architecture Conversions of Terrestrial Isopods Mitochondrial DNA

Mitochondrial DNA (mtDNA) is usually depicted as a circular molecule, however, there is increasing evidence that linearization of mtDNA evolved independently many times in organisms such as fungi, unicellular eukaryotes, and animals. Recent observations in various models with linear mtDNA revealed the presence of conserved inverted repeats (IR) at both ends that, when they become single-stranded, may be able to fold on themselves to create telomeric-hairpins involved in genome architecture conversions. The atypical mtDNA of terrestrial isopods (Crustacea: Oniscidea) composed of linear monomers and circular dimers is an interesting model to study genome architecture conversions. Here, we present the mtDNA control region sequences of two species of the genus Armadillidium: A. vulgare and A. pelagicum. All features of arthropods mtDNA control regions are present (origin of replication, poly-T stretch, GA and TA-rich blocks and one variable domain), plus a conserved IR. This IR can potentially fold into a hairpin structure and is present in two different orientations among the A. vulgare populations: either in one sense or in its reverse complement. This polymorphism, also observed in a single individual (heteroplasmy), might be a signature of genome architecture conversions from linear to circular monomeric mtDNA via successive opening and closing of the molecules.     

Chromosome 17 abnormalities and lack of TP53 mutations in paediatric central nervous system tumours

Central nervous system (CNS) tumours are the most common solid tumours in children. Cytogenetic and molecular genetic studies of these neoplasms have previously shown abnormalities of chromosome 17, implicating genes on this autosome in tumorigenesis. To identify mutations in the TP53 tumour suppressor gene (17p13.1), we have sequenced the five highly conserved regions of this gene in 29 mixed paediatric CNS tumors. No mutations were detected by this analysis. In order to identify other candidate disease loci on chromosome 17, we have carried out a detailed deletion mapping analysis using 16 polymorphic DNA markers on 19 of the above tumours and an additional four cases. Abnormalities of chromosome 17 occurred in nine cases (39%), six of which were primitive neuroectodermal tumour (PNET)-medulloblastomas. These findings suggest that it is unlikely that the TP53 gene is directly involved in the development of common paediatric brain tumours. This is in contrast to findings from adult brain and other tumour types. Moreover, the frequency of chromosome 17 aberrations, especially in PNET-medulloblastomas, suggests that other genes on this chromosome contribute to tumourigenesis.     

Yeast genome-wide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.     

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.