Construction of random transposition mutagenesis system in Rhodococcuserythropolis using IS1415

Recent studies on the metabolic activities of genus Rhodococcus have shown rhodococci to be of important use in industrial, pharmaceutical and environmental biotechnology. The increasing economic significance of Rhodococcus encourages renewed efforts to characterize their genetic systems, as Rhodococcus genetics are still poorly understood. The goal of this study is to adapt a transposon system for use in creating random mutagenesis in Rhodococcus erythropolis. A plasmid carrying IS1415, a member of IS21 family identified from Rerythropolis, has been constructed and designated as pTNR. pTNR is a non-replicating transposon tool introduced into target cells by electroporation. During its transposition, the transposable-marker gene is separated from the open reading frames (istAB) of IS1415, which should avoid secondary transposition. Transposition of pTNR into wild-type R. erythropolis created mutagenesis with a high efficiency of 1.23x10(6)mutants per microgram plasmid DNA. However, it could also be transposed into other Rhodococcus spp. at lower frequencies in comparison with that of R. erythropolis. It has been indicated by Southern hybridization that the generated kanamycin-resistant mutants were resulted from single transposition event of pTNR. The results also revealed that the transposable-marker gene of pTNR was randomly inserted into the chromosomal DNA of R. erythropolis. The affected DNA regions carrying the transposed DNA element could be conveniently recovered for further characterization using a plasmid rescue procedure. Sequence data of the insertion sites of 40 random mutants analyzed indicated that transposition of pTNR generated 6-bp direct target duplications in 36 cases, while in the remaining four mutants; it generated 5- or 7-bp target duplications (two cases each). This study concluded that pTNR could be served as an efficient genetic tool for construction of random mutagenesis system in Rhodococcus species.     

Exercise restores coronary vascular function independent of myogenic tone or hyperglycemic status in db/db mice

Regulation of coronary function in diabetic hearts is an important component in preventing ischemic cardiac events but remains poorly studied. Exercise is recommended in the management of diabetes, but its effects on diabetic coronary function are relatively unknown. We investigated coronary artery myogenic tone and endothelial function, essential elements in maintaining vascular fluid dynamics in the myocardium. We hypothesized that exercise reduces pressure-induced myogenic constriction of coronary arteries while improving endothelial function in db/db mice, a model of type 2 diabetes. We used pressurized mouse coronary arteries isolated from hearts of control and db/db mice that were sedentary or exercised for 1 h/day on a motorized exercise-wheel system (set at 5.2 m/day, 5 days/wk). Exercise caused a approximately 10% weight loss in db/db mice and decreased whole body oxidative stress, as measured by plasma 8-isoprostane levels, but failed to improve hyperglycemia or plasma insulin levels. Exercise did not alter myogenic regulation of arterial diameter stimulated by increased transmural pressure, nor did it alter smooth muscle responses to U-46619 (a thromboxane agonist) or sodium nitroprusside (an endothelium-independent dilator). Moderate levels of exercise restored ACh-simulated, endothelium-dependent coronary artery vasodilation in db/db mice and increased expression of Mn SOD and decreased nitrotyrosine levels in hearts of db/db mice. We conclude that the vascular benefits of moderate levels of exercise were independent of changes in myogenic tone or hyperglycemic status and primarily involved increased nitric oxide bioavailability in the coronary microcirculation.     

<Emphasis Type="Italic">Pichia stipitis</Emphasis> xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)

Background  

Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21.     

Intraspecific and Interspecific Competition Between Cotesia flavipes and Cotesia sesamiae (Hymenoptera: Braconidae), Gregarious Larval Endoparasitoids of Lepidopteran Stemborers

Super and multiple parasitism of Chilo partellus (Lepidoptera: Pyralidae) and Sesamia calamistis (Lepidoptera: Noctuidae) by Cotesia flavipes Cameron and Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) were investigated in the laboratory. Progeny production of Co. flavipes increased as a result of increasing the number of ovipositions, from one to three per one Ch. partellus host larva, then decreased as a result of four and five ovipositions per larva. Cocoon weight, sex ratio and emergence of the parasitoid progeny were not affected by superparasitism. Low progeny production of Co. sesamiae and poor survival of Ch. partellus host larvae were found as a result of superparasitism. When S. calamistis was the host, the duration of immature stages of Co. flavipes , parasitoid emergence, progeny production and sex ratio were not affected by superparasitism, but cocoon weight, adult longevity and the potential fecundity of adult females decreased. Superparasitism of S. calamistis by Co. sesamiae did not affect emergence, longevity or sex ratio of adult progeny of the parasitoid, but prolonged immature development, lowered cocoon weight and decreased potential fecundity of adult female progeny. Co. flavipes out-competed Co. sesamiae when Ch. partellus was parasitized by both species. The potential for local displacement of Co. sesamiae by Co. flavipes in areas dominated by Ch. partellus in East Africa is discussed.     

Dispersal of the exotic parasitoid Cotesia flavipes in a new ecosystem

A study on the dispersal of the exotic larval endoparasitoid, Cotesia flavipes Cameron (Hymenoptera: Braconidae), was conducted in a maize field in the northern Kilifi District in the coastal area of Kenya. Because C. flavipes did not previously occur in the release area, it was possible to use a unique indirect method to estimate dispersal by examining the distribution of parasitised hosts. Parasitoids released in the centre of the field moved as far as 64 meters during their life span, and dispersal was dependent on wind direction. The level of parasitism was influenced by the location of hosts in plants. The majority of parasitised stemborers (88.4%) were found inside the plant (stems and tassel stems), where 74.3% of the suitable hosts were found, which indicates that female parasitoids were not searching randomly for hosts. Aggregation of parasitoids in response to plants with different host densities was not detected. Implications of the release of C. flavipes on stemborers population in the agroecosystem of East Africa are discussed.     

Reduced natural selection associated with low recombination in Drosophila melanogaster

Synonymous codons are not used equally in many organisms, and the extent of codon bias varies among loci. Earlier studies have suggested that more highly expressed loci in Drosophila melanogaster are more biased, consistent with findings from several prokaryotes and unicellular eukaryotes that codon bias is partly due to natural selection for translational efficiency. We link this model of varying selection intensity to the population-genetics prediction that the effectiveness of natural selection is decreased under reduced recombination. In analyses of 385 D. melanogaster loci, we find that codon bias is reduced in regions of low recombination (i.e., near centromeres and telomeres and on the fourth chromosome). The effect does not appear to be a linear function of recombination rate; rather, it seems limited to regions with the very lowest levels of recombination. The large majority of the genome apparently experiences recombination at a sufficiently high rate for effective natural selection against suboptimal codons. These findings support models of the Hill-Robertson effect and genetic hitchhiking and are largely consistent with multiple reports of low levels of DNA sequence variation in regions of low recombination.      

Kappa-chain constant-region gene sequences in genus Rattus: coding regions are diverging more rapidly than noncoding regions

We have determined the nucleotide sequence of a 1,200-base pair (bp) genomic fragment that includes the kappa-chain constant-region gene (C kappa) from two species of native Australian rodents, Rattus leucopus cooktownensis and Rattus colletti. Comparison of these sequences with each other and with other rodent C kappa genes shows three surprising features. First, the coding regions are diverging at a rate severalfold higher than that of the nearby noncoding regions. Second, replacement changes within the coding region are accumulating at a rate at least as great as that of silent changes. Third, most of the amino acid replacements are localized in one region of the C kappa domain--namely, the carboxy-terminal "bends" in the alpha-carbon backbone. These three features have previously been described from comparisons of the two allelic forms of C kappa genes in R. norvegicus. These data imply the existence of considerable evolutionary constraints on the noncoding regions (based on as yet undetermined functions) or powerful positive selection to diversify a portion of the constant-region domain (whose physiological significance is not known). These surprising features of C kappa evolution appear to be characteristic only of closely related C kappa genes, since comparison of rodent with human sequences shows the expected greater conservation of coding regions, as well as a predominance of silent nucleotide substitutions within the coding regions.      

Synthetic and spectroscopic studies of 2-C-methyl-erythritol and 2-C-methyl-threito

The synthesis of 2-C-methyl-d,l-threitol and two isopropylidene derivatives of 2-C-methyl-d-erythritol is described. ¹H and ¹³C NMR spectra of these and several related compounds are discussed.