Rates of nucleotide substitution in Cornaceae (Cornales)-Pattern of variation and underlying causal factors

Identifying causes of genetic divergence is a central goal in evolutionary biology. Although rates of nucleotide substitution vary among taxa and among genes, the causes of this variation tend to be poorly understood. In the present study, we examined the rate and pattern of molecular evolution for five DNA regions over a phylogeny of Cornus, the single genus of Cornaceae. To identify evolutionary mechanisms underlying the molecular variation, we employed Bayesian methods to estimate divergence times and to infer how absolute rates of synonymous and nonsynonymous substitutions and their ratios change over time. We found that the rates vary among genes, lineages, and through time, and differences in mutation rates, selection type and intensity, and possibly genetic drift all contributed to the variation of substitution rates observed among the major lineages of Cornus. We applied independent contrast analysis to explore whether speciation rates are linked to rates of molecular evolution. The results showed no relationships for individual genes, but suggested a possible localized link between species richness and rate of nonsynonymous nucleotide substitution for the combined cpDNA regions. Furthermore, we detected a positive correlation between rates of molecular evolution and morphological change in Cornus. This was particularly pronounced in the dwarf dogwood lineage, in which genome-wide acceleration in both molecular and morphological evolution has likely occurred.     

Effects of drill cuttings on biogeochemical fluxes and macrobenthos of marine sediments

Experimental work was performed on drill cuttings sampled from off-shore drilling operations. The cuttings contained remnants of two different types of drilling muds: a water-ilmenite based mud and an olefin-barite based mud. In a pilot experiment, a gradient of up to 65 mm thick layers of water-ilmenite based cuttings were added to 78 cm² core samples. In another set-up, 3 mm layers of water-ilmenite and olefin-barite based cuttings were added to replicate 1000 cm² box-core samples with natural benthic communities transferred from the Oslofjord, S.E. Norway. Boxes with no addition and addition of 3 mm “clean” sediment were used for control. Increased consumption of oxygen and nitrate indicated the presence of degradable organic phases in both mud systems. The release of silicate showed a general decrease with increasing thickness of the cuttings layer, but maximum rates in the 3.1 mm treatment indicated increased bioturbation at low and moderate doses. The initial (field) composition of the macrobenthic communities was maintained throughout the 3 months experimental period, and at community level, no significant difference was observed between the four treatments at the end of the exposure period. However, after grouping the treatments into “clean” (untreated control and 3 mm clean sediment) and “cuttings” (3 mm water-ilmenite and 3 mm olefin-barite based cuttings), multivariate statistical analyses revealed a significant difference in community composition between clean and cuttings treatments, and three taxa showed significantly reduced abundances in the cuttings treatments. Chemical toxicity of mud components is assumed to be small, and the observed effect was more likely a result of physical properties such as the size or shape of cuttings particles.     

Structural relatedness between mosquitocidal endotoxins of Bacillus thuringiensis subsp. israelensis

A mosquitocidal toxin gene, cloned from Bacillus thuringiensis subsp. israelensis, was introduced into mutant crystal-negative B. thuringiensis subsp. israelensis cells. Partial toxicity to mosquitos was restored. The 58-kilodalton cloned gene product is a minor protein component of B. thuringiensis subsp. israelensis crystals and is structurally related to a major, 135-kilodalton crystal toxin.     

Chromosomal mapping of Bacillus thuringiensis by transduction.

Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.     

Development and Evaluation of Methods To Detect Nucleopolyhedroviruses in Larvae of the Douglas-Fir Tussock Moth, Orgyia pseudotsugata (McDunnough)

Various molecular methods are used to detect pathogenic microorganisms and viruses within their hosts, but these methods are rarely validated by direct comparison. Southern hybridization, enzyme-linked immunosorbent assay (ELISA), and a novel DNA extraction/PCR assay were used to detect Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) in Douglas-fir tussock moth larvae. PCR was more sensitive than Southern hybridization and ELISA at detecting semipurified virus. ELISA, however, was the most accurate method for detecting virus within larvae, given that Southern hybridization and PCR produced false-negative results (31% and 2.5%, respectively). ELISA may be preferable in some applications because virus infections can be quantified (r² = 0.995). These results may be applicable to both applied and academic research that seeks to accurately identify the incidence of viruses and microorganisms that regulate insect populations.     

Prediction of putative protein interactions through evolutionary analysis of osmotic stress response in the model yeast Saccharomyces cerevisae

The osmotic stress response signalling pathway of the model yeast Saccharomyces cerevisae is crucial for the survival of cells under osmotic stress, and is preserved to varying degrees in other related fungal species. We apply a method for inference of ancestral states of characteristics over a phylogeny to 17 fungal species to infer the maximum likelihood estimate of presence or absence in ancestral genomes of genes involved in osmotic stress response. The same method allows us furthermore to perform a statistical test for correlated evolution between genes. Where such correlations exist within the osmotic stress response pathway of S. cerevisae, we have used this in order to predict and subsequently test for the presence of physical protein-protein interactions in an attempt to detect novel interactions. Finally we assess the relevance of observed evolutionary correlations in predicting protein interactions in light of the experimental results. We do find that correlated evolution provides some useful information for the prediction of protein-protein interactions, but that these alone are not sufficient to explain detectable patterns of correlated evolution.     

Brucella abortus strain RB51 vaccination in elk. I. Efficacy of reduced dosage

Bovine brucellosis is a serious zoonotic disease affecting some populations of Rocky Mountain elk (Cervus elaphus nelsoni) and bison (Bison bison) in the Greater Yellowstone Area, USA. The fear that elk and/or bison may spread Brucella abortus to livestock has prompted efforts to reduce or eliminate the disease in wildlife. Brucella abortus strain RB51 (RB51) vaccine has recently been approved for use in cattle. Unlike strain 19 vaccine, RB51 does not cause false positive reactions on standard brucellosis serologic tests. If effective, it may become the vaccine of choice for wildlife. In February 1995, 45 serologically negative female elk calves were trapped and taken to the Sybille Wildlife Research and Conservation Education Unit near Wheatland, Wyoming, USA. In May 1995, 16 of these elk calves were hand-vaccinated with 1 x 10(9) colony forming units (CFU) of RB51, 16 were vaccinated with 1 x 10(8) CFU RB51 by biobullet, and 13 were given a saline placebo. The elk were bred in fall of 1996 and they were challenged with 1 x 10(7) CFU of B. abortus strain 2308 by intraconjunctival inoculation in March 1997. Thirteen (100%) control elk aborted, 14 (88%) hand-vaccinated elk aborted, and 12 (75%) biobullet vaccinated elk aborted or produced nonviable calves. These results suggest that a single dose of 1 x 10(8) to 1 x 10(9) CFU RB51 does not provide significant protection against B. abortus induced abortion in elk. However, the vaccine appears to be safe at this dose and additional study may reveal a more effective RB51 vaccine regimen for elk.