Compensatory substitutions and the evolution of the mitochondrial 12S rRNA gene in mammals

12S ribosomal RNA (rRNA) gene sequences from a suite of mammalian taxa (13 placentals, 4 marsupials, 1 monotreme), for which phylogenetic relationships are well established based on independent criteria, were employed to study the evolution of this gene. Phylogenetic analysis of 12S sequences produces a phylogeny that agrees with expectations. Base composition provides evidence for directional symmetrical substitution pressure in loops; in stems, base composition is much more even. Rates of nucleotide substitution are lower in stems than loops. Patterns of nucleotide substitution show an overall preference for transitions over transversions, with this difference more profound in stems than loops. Among different transversion pathways, there is a wide range of transformation frequencies. An analysis of compensatory substitutions shows that there is strong evidence for their occurrence and that a weighting factor of 0.61 should be applied in phylogenetic analyses to account for the dependence of mutations at stem positions relative to positions where changes are independent. Among stem variables (i.e., stem length, interaction distance, substitution rates, G+C content, and the percentage of bases that are paired), several significant correlations were discovered, but stem length and interaction distance are uncorrelated with other variables.      

Sister-chromatid exchanges in lymphocytes of smokers in an experimental study

The frequency of sister-chromatid exchanges (SCEs) was studied in peripheral blood lymphocytes of 26 young male smokers and 10 non-smokers who had recently entered military service. The levels of SCEs were examined in 4 consecutive blood samples taken after short experimental periods of smoking only low-tar (LT) or medium-tar (MT) cigarettes. The incidence of SCEs was significantly higher in the the group of smokers than in the group of non-smokers. The SCE levels of the smokers were found to be associated with the personal smoking history; the observed increase in the SCE frequency correlated with the years of smoking measured as cumulative pack years. The difference in type of cigarette did not influence the SCE frequencies.     

Analysis of chromosomal aberrations,sister chromatid exchanges and micronuclei among power linesmen with long-term exposure to 50-Hz electromagnetic fields

Chromosomal aberrations, sister chromatid exchanges (SCEs), replication indices and micronuclei in peripheral blood lymphocytes were analysed among 27 nonsmoking power linesmen with considerable long-term exposure to 50-Hz electromagnetic (EM) fields, and among 27 nonsmoking telephone lines-men serving as a reference group, pairwise matched with the exposed workers for age and geographical region. Blood samples from the two groups were collected, cultured and analysed in parallel. No differences between the groups were observed on analysis of SCEs, replication indices or micronuclei. However, the mean rate of lymphocytes with chromatid-type breaks was higher among the power linesmen (0.96% gaps excluded, 1.41% gaps included) than among the reference group (0.44% and 0.70%, respectively). The excess of aberrant cells was concentrated among those power linesmen who had smoked earlier in their life. Although the interpretation is somewhat complicatedy the confounding effect of previous smoking, these results suggest that exposure to 50-Hz EM fields is associated with a slight increase in chromatid breaks.     

Contribution of Glucosinolate Transport to Arabidopsis Defense Responses

Accumulation of glucosinolates, a class of defense-related secondary metabolites found almost exclusively in the Capparales, is induced in response to a variety of biological stresses. It is often assumed that elevated glucosinolate levels result from de novo biosynthesis, but glucosinolate transport from other parts of the plant to the site of herbivory or pathogen infection can also contribute to the defense response. Several studies with Arabidopsis and other crucifers have demonstrated that glucosinolates from vegetative tissue are transported to developing seeds. Here we discuss evidence that long-chain aliphatic glucosinolates are transported to the site of herbivory in response to Myzus persicae (green peach aphid) feeding on Arabidopsis.Key Words: glucosinolate, transport, graft, Arabidopsis, Myzus persicae, aphid     

Effects of the number of markers per haplotype and clustering of haplotypes on the accuracy of QTL mapping and prediction of genomic breeding values

The aim of this paper was to compare the effect of haplotype definition on the precision of QTL-mapping and on the accuracy of predicted genomic breeding values. In a multiple QTL model using identity-by-descent (IBD) probabilities between haplotypes, various haplotype definitions were tested i.e. including 2, 6, 12 or 20 marker alleles and clustering base haplotypes related with an IBD probability of > 0.55, 0.75 or 0.95. Simulated data contained 1100 animals with known genotypes and phenotypes and 1000 animals with known genotypes and unknown phenotypes. Genomes comprising 3 Morgan were simulated and contained 74 polymorphic QTL and 383 polymorphic SNP markers with an average r² value of 0.14 between adjacent markers. The total number of haplotypes decreased up to 50% when the window size was increased from two to 20 markers and decreased by at least 50% when haplotypes related with an IBD probability of > 0.55 instead of > 0.95 were clustered. An intermediate window size led to more precise QTL mapping. Window size and clustering had a limited effect on the accuracy of predicted total breeding values, ranging from 0.79 to 0.81. Our conclusion is that different optimal window sizes should be used in QTL-mapping versus genome-wide breeding value prediction.     

Identification of novel virulence-associated loci in uropathogenic Escherichia coli by suppression subtractive hybridization

To identify novel virulence-associated genes in uropathogenic Escherichia coli (UPEC) strains, a suppression subtractive hybridization strategy was applied to genomic DNA of four clinical UPEC isolates from patients suffering from cystitis or pyelonephritis. The genomic DNA of four isolates (tester strains) was subtracted from the DNA of two different driver strains, the well characterized UPEC strain CFT073 and the non-pathogenic E. coli K-12 strain MG1655. We determined the sequence of 172 tester strain-specific DNA fragments, 86 of which revealed only low or no homology to nucleotide sequences of public databases. We further determined the virulence association of the 86 novel DNA fragments using each DNA fragment as a probe in Southern hybridizations of a reference strain collection consisting of 60 extraintestinal pathogenic E. coli isolates, and 40 non-virulent E. coli strains from stool samples. From this, 19 novel DNA fragments were demonstrated to be significantly associated with virulent strains and thus may represent new virulence traits. Our results support the idea of a considerable genetic variability among UPEC strains and suggest that novel genomic determinants might contribute to virulence of UPEC.     

Ultrastructure of regions containing homologous loci in polytene chromosomes of Drosophila melanogaster and Drosophila subobscura

We have used a new approach involving in situ hybridisation and electron microscopy to establish ultrastructural homologies between polytene chromosome regions of Drosophila melanogaster and Drosophila subobscura. Twelve probes were chosen to cover all the chromosomal elements: the myospheroid gene, the collagen type IV gene, the collagen-like gene, the w26 homeobox gene, the β3 tubulin gene, the kinesin heavy chain gene, the tryptophan hydrolase gene, the Hsp82, Hsp22–26 and Hsp23–28, Hsp68, Hsp70 genes and the β unit of the F₀–F₁ ATPase gene. Most of these loci were previously undescribed in D. subobscura and imprecisely located in D. melanogaster. We have demonstrated here, by an ultrastructural analysis of each chromosomal region, that homologous genetic loci tend to show a similar ultrastructure in the two species. With a few exceptions, the structural homology extends to the chromosomal regions surrounding the loci. In some cases, however, no structurally recognisable homology can be seen either in the locus or in its flanking regions. Received: 15 December 1996; in revised form: 15 October 1997 / Accepted: 28 January 1998     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

Application of a semi-automated SOS chromotest for measuring genotoxicities of complex environmental mixtures containing polycyclic aromatic hydrocarbons.

Soxhlet-extracted samples of standard reference materials (SRMs) 1649 (PAR1: urban dust/organics) and 1650 (PAR2: diesel particulate matter) from the U.S. Institute of Standards and Technology were tested for induction of SOS functions using a semi-automated version of the SOS chromotest with Escherichia coli PQ37. Concentrations of 10 polycyclic aromatic hydrocarbons in the extracts were determined using reversed-phase HPLC. Only the diesel particulate matter (PAR2) extracts expressed SOS induction activity, which decreased when metabolic activation was used. Mutagenic PAH compounds (e.g., chrysene) were found in higher concentrations in the PAR2 extracts than in the PAR1 extracts but this could not explain the genotoxicity while it was mainly exhibited without metabolic activation. The direct genotoxic activity of the diesel particulate matter sample PAR2 is probably caused by nitroaromatic compounds; this was also supported by parallel studies with the Ames/Salmonella assay.