Similarities in Restriction Fragment Patterns of Mitochondrial DNAs of PhytophthoraSpecies Strongly Depend on the Restriction Enzyme Used Due to Heterogeneous Base Distribution and Sequence Conservation

Mitochondrial DNAs of six morphologically different Phytophthora species were digested with 15 restriction enzymes. The numbers of restriction fragments obtained differed considerably from those theoretically expected for random base distribution. Enzymes with relatively many G and C in their recognition sequences produced significantly larger numbers of fragments. Moreover, fragments generated by most of these enzymes were more often shared by two or more species than those from enzymes with more A and T in their recognition sequence. It is concluded that base distribution in mitochondrial DNA of Phytophthora is heterogeneous,AT-rich stretches occurring scattered over the mitochondrial genome and GC-rich regions present in conserved sequences, presumably genes. A practical consequence for taxonomic RFLP studies is that optimal enzymes can be selected, depending on the desired level of resolution.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Evaluation of the <Emphasis Type="Italic">OPTC</Emphasis> gene in primary open angle glaucoma: functional significance of a silent change

Background  

We investigated the molecular basis of primary open-angle glaucoma (POAG) using Opticin (OPTC) as a candidate gene on the basis of its expression in the trabecular meshwork cells involved in the disease pathogenesis. Two hundred POAG patients and 100 controls were enrolled in this study. The coding sequence of OPTC was amplified by PCR from genomic DNA of POAG patients, followed by SSCP, DHPLC and DNA sequencing. Subsequent bioinformatic analysis, site-directed mutagenesis, quantitative RT-PCR and western blot experiments were performed to address the functional significance of a 'silent' change in the OPTC coding region while screening for mutations in POAG patients.     

XRCC3 ATPase activity is required for normal XRCC3-Rad51C complex dynamics and homologous recombination

Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, cross-links, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, we generated mammalian expression vectors that produce human wild-type XRCC3 or mutant XRCC3 with either a nonconservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF Chinese hamster ovary cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, whereas ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to that in wild-type-complemented clones. Because of dysfunction of the mutants, we propose that ATP binding and hydrolyzing activities of XRCC3 are essential. We tested in vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C upon co-expression in bacteria, nickel-affinity purification, and Western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, whereas the K113R mutant did not and was predominantly insoluble. The addition of 5 mm ATP but not ADP also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 probably regulates the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis with both processes being essential for the ability of the complex to participate in homologous recombinational repair.     

Untersuchungen zur Tiergesundheit,Leistung und zum Rückstandsverhalten beim Schwein bei gleichzeitiger Aufnahme der Mykotoxine Ochratoxin A,Deoxynivalenol, Zearalenon über das Futter im 90-Tage-Test

The effect of the administration of the mycotoxins OTA, ZEA and DON alone resp. in combination on animal health and the residue behavior of pigs from 50 – 60 kg living weight over 90 days was investigated in 4 separate studies. Due to its fast metabolisation the administration of 1000 µg DON resp. 250 µg ZEA per kg feed alone or in combination with other mycotoxins does not lead to detectable residues of these mycotoxins in organs and tissues. Therefore these mycotoxins should not be relevant to the consumer.There is an effect of the simultaneous administration of ZEA resp. DON on the metabolisation resp. secretion of OTA. OTA is of relevance from the point of view of residue toxicology.     

An anchored restriction-mapping approach applied to the genetic analysis of the Anopheles gambiae malaria vector complex 1

We introduce here a simple approach for rapidly determining restriction maps for a number of regions of a genome; this involves "anchoring" a map with a rare restriction site (in this case the seldom-cutting EagI) followed by partial digestion of a frequent-cutting enzyme (e.g., Sau 3A). We applied this technology to five species of the Anopheles gambiae complex. In a single Southern blot we obtained about a 15-kb restriction map each for the mtDNA, rRNA gene, and a scnDNA region for each of five species. Phylogenetic analyses of these regions yield trees at odds with the more traditional chromosome inversion-based trees. The value of the approach for systematic purposes is the ease with which several large, independent regions of the genome can be quickly assayed for molecular variation.      

A Comparison of the Use of In Vitro-Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment

Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.