Molecular biologic techniques applied to the microbial prospecting of oil and gas in the Ban 876 gas and oil field in China

Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth; however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively, were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area.     

Mechanics of DNA bridging by bacterial condensin MukBEF in vitro and in singulo

Structural maintenance of chromosome (SMC) proteins comprise the core of several specialized complexes that stabilize the global architecture of the chromosomes by dynamically linking distant DNA fragments. This reaction however remains poorly understood giving rise to numerous proposed mechanisms of the proteins. Using two novel assays, we investigated real‐time formation of DNA bridges by bacterial condensin MukBEF. We report that MukBEF can efficiently bridge two DNAs and that this reaction involves multiple steps. The reaction begins with the formation of a stable MukB–DNA complex, which can further capture another protein‐free DNA fragment. The initial tether is unstable but is quickly strengthened by additional MukBs. DNA bridging is modulated but is not strictly dependent on ATP and MukEF. The reaction revealed high preference for right‐handed DNA crossings indicating that bridging involves physical association of MukB with both DNAs. Our data establish a comprehensive view of DNA bridging by MukBEF, which could explain how SMCs establish both intra‐ and interchromosomal links inside the cell and indicate that DNA binding and bridging could be separately regulated.     

A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.     

Water dynamics clue to key residues in protein folding

A computational method independent of experimental protein structure information is proposed to recognize key residues in protein folding, from the study of hydration water dynamics. Based on all-atom molecular dynamics simulation, two key residues are recognized with distinct water dynamical behavior in a folding process of the Trp-cage protein. The identified key residues are shown to play an essential role in both 3D structure and hydrophobic-induced collapse. With observations on hydration water dynamics around key residues, a dynamical pathway of folding can be interpreted.     

围绕生物学核心概念的教学设计——以“遗传信息的传递”为例

基于生物学核心概念进行课堂教学,对提高学生的生物科学素养有着重要的意义。以“遗传信息的传递”为例,围绕核心概念设计教学活动,帮助学生获得核心概念。     

nm23-H1对全反式维甲酸诱导的人肝癌细胞凋亡及其相关信号转导通路的影响

为了解nm23-H1转染对全反式维甲酸诱导的人肝癌H7721细胞凋亡的影响,本研究通过质粒转染把nm23-H1导入人肝癌7721细胞,建立了nm23-H1过表达稳转细胞株。首先采用MTT法测定细胞生长曲线,再通过流式细胞术和丫啶橙染色法观察细胞凋亡,最后采用Western blot检测凋亡相关的信号通路分子bcl-2、PKB、PKB-Ser473、PKB-Thr308和p53的表达情况。研究发现,nm23-H1转染对人肝癌7721细胞的生长没有影响;但nm23-H1转染能明显促进全反式维甲酸诱导的细胞凋亡,nm23-H1转染细胞凋亡率为18.2%,而对照细胞凋亡率仅为1.0%;nm23-H1和对照细胞的过量表达对bcl-2的表达没有明显影响,但PKB的Ser473和Thr308位的磷酸化显著下调,抑癌基因p53的表达量上调。研究结果表明,nm23-H1的过量表达增加了人肝癌细胞对全反式维甲酸诱导的凋亡的敏感性,因此推测nm23-H1可作为肝癌治疗的有效靶点。     

Inhibition of dark‐induced stomatal closure by fusicoccin involves a removal of hydrogen peroxide in guard cells of Vicia faba

Fusicoccin (FC) treatment prevents dark‐induced stomatal closure, the mechanism of which is still obscure. By using pharmacological approaches and laser‐scanning confocal microscopy, the relationship between FC inhibition of dark‐induced stomatal closure and the hydrogen peroxide (H₂O₂) levels in guard cells in broad bean was studied. Like ascorbic acid (ASA), a scavenger of H₂O₂ and diphenylene iodonium (DPI), an inhibitor of H₂O₂‐generating enzyme NADPH oxidase, FC was found to inhibit stomatal closure and reduce H₂O₂ levels in guard cells in darkness, indicating that FC‐caused inhibition of dark‐induced stomatal closure is related to the reduction of H₂O₂ levels in guard cells. Furthermore, like ASA, FC not only suppressed H₂O₂‐induced stomatal closure and H₂O₂ levels in guard cells treated with H₂O₂ in light, but also reopened the stomata which had been closed by darkness and reduced the level of H₂O₂ that had been generated by darkness, showing that FC causes H₂O₂ removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomata treated with H₂O₂ and had been closed by dark, and on H₂O₂ levels in guard cells of stomata treated with H₂O₂ and had been closed by dark, and both FC and butyric acid reduced cytosol pH in guard cells of stomata treated with H₂O₂ and had been closed by dark, which demonstrates that cytosolic acidification mediates FC‐induced H₂O₂ removal. Taken together, our results provide evidence that FC causes cytosolic acidification, consequently induces H₂O₂ removal, and finally prevents dark‐induced stomatal closure.     

Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways

Background  

c-erbB2, a proto-oncogene coding epidermal growth factor receptor-like receptor, also as a chemosensitivity/prognosis marker for gynecologic cancer, may be involved in initiation of growth of rat primordial follicles. The aim of the present study is to investigate the role and signal pathway of c-erbB2 in onset of rat primordial follicle development.     

Microbiological and production characteristics of the high-temperature Kongdian bed revealed during field trial of biotechnology for the enhancement of oil recovery

Microbiological technology for the enhancement of oil recovery based on the activation of the stratal microflora was tested in the high-temperature horizons of the Kongdian bed (60 degrees C) of the Dagang oil field (China). This biotechnology consists in the pumping of a water-air mixture and nitrogen and phosphorus mineral salts into the oil stratum through injection wells in order to stimulate the activity of the stratal microflora which produce oil-releasing metabolites. Monitoring of the physicochemical, microbiological, and production characteristics of the test site has revealed large changes in the ecosystem as a result of the application of biotechnology. The cell numbers of thermophilic hydrocarbon-oxidizing, fermentative, sulfate-reducing, and methanogenic microorganisms increased 10-10 000-fold. The rates of methanogenesis and sulfate reduction increased in the near-bottom zone of the injection wells and of some production wells. The microbial oil transformation was accompanied by the accumulation of bicarbonate ions, volatile fatty acids, and biosurfactants in the formation waters, as well as of CH4 and CO2 both in the gas phase and in the oil. Microbial metabolites promoted the additional recovery of oil. As a result of the application of biotechnology, the water content in the production liquid from the test site decreased, and the oil content increased. This allowed the recovery of more than 14000 tons of additional oil over 3.5 years.     

Novel bacterial sulfur oxygenase reductases from bioreactors treating gold-bearing concentrates

The microbial community and sulfur oxygenase reductases of metagenomic DNA from bioreactors treating gold-bearing concentrates were studied by 16S rRNA library, real-time polymerase chain reaction (RT-PCR), conventional cultivation, and molecular cloning. Results indicated that major bacterial species were belonging to the genera Acidithiobacillus, Leptospirillum, Sulfobacillus, and Sphingomonas, accounting for 6.3, 66.7, 18.8, and 8.3%, respectively; the sole archaeal species was Ferroplasma sp. (100%). Quantitative RT-PCR revealed that the 16S rRNA gene copy numbers (per gram of concentrates) of bacteria and archaea were 4.59 × 10⁹ and 6.68 × 10⁵, respectively. Bacterial strains representing Acidithiobacillus, Leptospirillum, and Sulfobacillus were isolated from the bioreactors. To study sulfur oxidation in the reactors, pairs of new PCR primers were designed for the detection of sulfur oxygenase reductase (SOR) genes. Three sor-like genes, namely, sor _Fx, sor _SA, and sor _SB were identified from metagenomic DNAs of the bioreactors. The sor _Fx is an inactivated SOR gene and is identical to the pseudo-SOR gene of Ferroplasma acidarmanus. The sor _SA and sor _SB showed no significant identity to any genes in GenBank databases. The sor _SB was cloned and expressed in Escherichia coli, and SOR activity was determined. Quantitative RT-PCR determination of the gene densities of sor _SA and sor _SB were 1,000 times higher than archaeal 16S rRNA gene copy numbers, indicating that these genes were mostly impossible from archaea. Furthermore, with primers specific to the sor _SB gene, this gene was PCR-amplified from the newly isolated Acidithiobacillus sp. strain SM-1. So far as we know, this is the first time to determine SOR activity originating from bacteria and to document SOR gene in bioleaching reactors and Acidithiobacillus species.