Helicobacter pylori genes involved in avoidance of mutations induced by 8-oxoguanine

Chromosomal rearrangements and base substitutions contribute to the large intraspecies genetic diversity of Helicobacter pylori. Here we explored the base excision repair pathway for the highly mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), a ubiquitous form of oxidized guanine. In most organisms, 8-oxoG is removed by a specific DNA glycosylase (Fpg in bacteria or OGG1 in eukaryotes). In the case where replication of the lesion yields an A/8-oxoG base pair, a second DNA glycosylase (MutY) can excise the adenine and thus avoid the fixation of the mutation in the next round of replication. In a genetic screen for H. pylori genes complementing the hypermutator phenotype of an Escherichia coli fpg mutY strain, open reading frame HP0142, a putative MutY coding gene, was isolated. Besides its capacity to complement E. coli mutY strains, HP0142 expression resulted in a strong adenine DNA glycosylase activity in E. coli mutY extracts. Consistently, the purified protein also exhibited such an activity. Inactivation of HP0142 in H. pylori resulted in an increase in spontaneous mutation frequencies. An Mg-dependent AP (abasic site) endonuclease activity, potentially allowing the processing of the abasic site resulting from H. pylori MutY activity, was detected in H. pylori cell extracts. Disruption of HP1526, a putative xth homolog, confirmed that this gene is responsible for the AP endonuclease activity. The lack of evidence for an Fpg/OGG1 functional homolog is also discussed.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Consequences of concurrent Ascaridia galli and Escherichia coli infections in chickens

   

Three experiments were carried out to examine the consequences of concurrent infections with Ascaridia galli and Escherichia coli in chickens raised for table egg production. Characteristic pathological lesions including airsacculitis, peritonitis and/or polyserositis were seen in all groups infected with E. coli. Furthermore, a trend for increased mortality rates was observed in groups infected with both organisms which, however, could not be confirmed statistically. The mean worm burden was significantly lower in combined infection groups compared to groups infected only with A. galli. It was also shown that combined infections of E. coli and A. galli had an added significant negative impact on weight gain.     

A new AluI satellite DNA in the root-knot nematode Meloidogyne fallax: relationships with satellites from the sympatric species M. hapla and M. chitwoodi

A highly abundant satellite DNA comprising 20% of the Meloidogyne fallax (Nematoda, Tylenchida) genome was cloned and sequenced. The satellite monomer is 173 bp long and has a high A + T content of 72.3%, with frequent runs of A's and T's. The sequence variability of the monomers is 2.7%, mainly due to random distribution of single-point mutations. A search for evidence of internal repeated subunits in the monomer sequence revealed a 6-bp motif (AAATTT) for which five degenerated repeats, differing by just a single base pair, could be identified. Pairwise comparison of the M. fallax satellite with those from the sympatric species Meloidogyne chitwoodi and Meloidogyne hapla revealed a high sequence similarity (68.39%) with one satellite DNA subfamily in M. chitwoodi, which indicated an unexpected close relationship between them. Given the high copy number and the extreme sequence homogeneity among monomeric units, it may be assumed that the satellite DNA of M. fallax could have evolved through some recent and extensive amplification burst in the nematode genome. In this case, its relatively short life would not yet have allowed the accumulation of random mutations in independent amplified repeats. Considering the morphological resemblance between the two species and their ability to produce interspecific fertile hybrids under controlled conditions, these results indicate that M. fallax may share a common ancestor with M. chitwoodi, from which it could have diverged recently. All these data suggest that M. fallax could be the result of a recent speciation process and show that Meloidogyne satellite DNAs may be of interest to resolve phylogenetic relationships among closely related species from this genus.      

Excision repair of 8-hydroxyguanine in mammalian cells: the mouse Ogg1 protein as a model

8-Hydroxyguanine (8-OH-Gua) is a major mutagenic lesion produced on DNA by the oxidative stress induced by either the endogen metabolism or the exposure to external agents. In bacteria and yeast this modified base can be removed by specific DNA glycosylases. Recently a human gene coding for an 8-OH-Gua DNA glycosylase/AP lyase has been identified by its homology to the yeast OGG1. This gene is located in human chromosome 3p25, a region commonly rearranged in various cancers, specially in lung tumor cells. We report here the cloning, by sequence homology to the yeast OGG1, of a mouse cDNA coding for a 8-OH-Gua DNA glycosylase with 84% and 38% identity to the human and yeast relevant proteins, respectively. The Ogg1 gene is localized to the mouse chromosome 6E. The mouse Ogg1 cDNA, when expressed in Escherichia coli, is capable of suppressing the spontaneous mutator phenotype of a DNA repair deficient fpg mutY strain. The mouse Ogg1 protein acts efficiently on duplexes in which the 8-OH-Gua is paired with a cytosine but is inactive on 8-OH-Gua:Ade pair, consistently with its proposed biological role in the avoidance of mutations. A comparison of the mouse enzyme with other eukaryotic Ogg1 enzymes is also presented. The isolation of this gene will allow the development of an animal model to study the effects of oxidative stress on carcinogenesis and degenerative diseases.     

Can Zipf's law be adapted to normalize microarrays?

Background  

Normalization is the process of removing non-biological sources of variation between array experiments. Recent investigations of data in gene expression databases for varying organisms and tissues have shown that the majority of expressed genes exhibit a power-law distribution with an exponent close to -1 (i.e. obey Zipf's law). Based on the observation that our single channel and two channel microarray data sets also followed a power-law distribution, we were motivated to develop a normalization method based on this law, and examine how it compares with existing published techniques. A computationally simple and intuitively appealing technique based on this observation is presented.