枯草杆菌TrpRS的固定化及亲和RNA筛选

为研究 t RNATrp与色氨酰 - t RNA合成酶 ( Trp RS)的相互识别及其结构与功能的关系 ,纯化了枯草杆菌 Trp RS,并用溴化氰活化的 Sepharose4B将 Trp RS固定化 ,固定化 Trp RS的蛋白回收率为 95.5% ,活力回收率为 31 .3% .研究了固定化 Trp RS的酶学性质 ,其热稳定性和贮存稳定性方面均比液相 Trp RS有了较大的提高 ,最适温度、最适 p H均有一定程度的增大 ,工作稳定性良好 .以固定化 Trp RS为亲和层析介质 ,对含有 2 0个核苷酸随机序列 ,长度为 56个核苷酸的单链RNA随机库进行了三轮筛选 .实验结果表明 ,固定化 Trp RS可以作为 SELEX亲和层析介质 ,进行模拟 t RNATrp分子的 RNA随机库的 SELEX筛选 .     

CYP26B1 promotes male germ cell differentiation by suppressing STRA8-dependent meiotic and STRA8-independent mitotic pathways

Germ cell sex is defined by factors derived from somatic cells. CYP26B1 is known to be a male sex-promoting factor that inactivates retinoic acid (RA) in somatic cells. In CYP26B1-null XY gonads, germ cells are exposed to a higher level of RA than in normal XY gonads and this activates Stra8 to induce meiosis while male-specific gene expression is suppressed. However, it is unknown whether meiotic entry by an elevated level of RA is responsible for the suppression of male-type gene expression. To address this question, we have generated Cyp26b1/Stra8 double knockout (dKO) embryos. We successfully suppressed the induction of meiosis in CYP26B1-null XY germ cells by removing the Stra8 gene. Concomitantly, we found that the male genetic program represented by the expression of NANOS2 and DNMT3L was totally rescued in about half of dKO germ cells, indicating that meiotic entry causes the suppression of male differentiation. However, half of the germ cells still failed to enter the appropriate male pathway in the dKO condition. Using microarray analyses together with immunohistochemistry, we found that KIT expression was accompanied by mitotic activation, but was canceled by inhibition of the RA signaling pathway. Taken together, we conclude that inhibition of RA is one of the essential factors to promote male germ cell differentiation, and that CYP26B1 suppresses two distinct genetic programs induced by RA: a Stra8-dependent meiotic pathway, and a Stra8-independent mitotic pathway.     

Insights into the Microbial Degradation of Rubber and Gutta-Percha by Analysis of the Complete Genome of Nocardia nova SH22a

The complete genome sequence of Nocardia nova SH22a was determined in light of the remarkable ability of rubber and gutta-percha (GP) degradation of this strain. The genome consists of a circular chromosome of 8,348,532 bp with a G+C content of 67.77% and 7,583 predicted protein-encoding genes. Functions were assigned to 72.45% of the coding sequences. Among them, a large number of genes probably involved in the metabolism of xenobiotics and hardly degradable compounds, as well as genes that participate in the synthesis of polyketide- and/or nonribosomal peptide-type secondary metabolites, were detected. Based on in silico analyses and experimental studies, such as transposon mutagenesis and directed gene deletion studies, the pathways of rubber and GP degradation were proposed and the relationship between both pathways was unraveled. The genes involved include, inter alia, genes participating in cell envelope synthesis (long-chain-fatty-acid–AMP ligase and arabinofuranosyltransferase), β-oxidation (α-methylacyl-coenzyme A [α-methylacyl-CoA] racemase), propionate catabolism (acyl-CoA carboxylase), gluconeogenesis (phosphoenolpyruvate carboxykinase), and transmembrane substrate uptake (Mce [mammalian cell entry] transporter). This study not only improves our insights into the mechanism of microbial degradation of rubber and GP but also expands our knowledge of the genus Nocardia regarding metabolic diversity.     

Analysis of midgut gene expression profiles from different silkworm varieties after exposure to high temperature

The silkworm is a poikilothermic animal, whose growth and development is significantly influenced by environmental temperature. To identify genes and metabolic pathways involved in the heat-stress response, digital gene expression analysis was performed on the midgut of the thermotolerant silkworm variety ‘932’ and thermosensitive variety ‘HY’ after exposure to high temperature (932T and HYT). Deep sequencing yielded 6,211,484, 5,898,028, 5,870,395 and 6,088,303 reads for the 932, 932T, HY and HYT samples, respectively. The annotated genes associated with these tags numbered 4357, 4378, 4296 and 4658 for the 932, 932T, HY and HYT samples, respecti'vely. In the HY-vs-932, 932-vs-932T, and HY-vs-HYT comparisons, 561, 316 and 281 differentially expressed genes were identified, which could be assigned to 179, 140 and 123 biological pathways, respectively. It was found that some of the biological pathways, which included oxidative phosphorylation, related to glucose and lipid metabolism, are greatly affected by high temperature and may lead to a decrease in the ingestion of fresh mulberry. When subjected to an early period of continuous heat stress, HSP genes, such as HSP19.9, HSP23.7, HSP40-3, HSP70, HSP90 and HSP70 binding protein, are up-regulated but then reduced after 24 h and the thermotolerant ‘932’ strain has higher levels of mRNA of some HSPs, except HSP70, than the thermosensitive variety during continuous high temperature treatment. It is suggested that HSPs and the levels of their expression may play important roles in the resistance to high temperature stress among silkworm varieties. This study has generated important reference tools that can be used to further analyze the mechanisms that underlie thermotolerance differences among silkworm varieties.     

Comparative phylogenetic relationships and genetic structure of the caterpillar fungus Ophiocordyceps sinensis and its host insects inferred from multiple gene sequences

Ophiocordyceps sinensis (Ascomycota: Ophiocordycipitaceae) is a native fungal parasite of Hepialidae caterpillars and one of the most economically important medicinal caterpillar fungi in China. However, little is known about the phylogenetic and evolutionary relationships between O. sinensis and its host insects. In this study, nuclear ITS and β-tubulin sequences from O. sinensis and mitochondrial COI, COII, and Cytb sequences from its hosts were analyzed across 33 populations sampled from five regions in China. Phylogenetically, both O. sinensis and its hosts were divided into three geographically correlated clades, and their phylogenies were congruent. Analysis of molecular variance and calculated coefficients of genetic differentiation revealed significant genetic divergence among the clades within both O. sinensis (F_ST= 0.878, N_ST=0.842) and its hosts (F_ST=0.861, N_ST=0.816). Estimated gene flow was very low for O. sinensis (Nm=0.04) and the host insects (Nm=0.04) among these three clades. Mantel tests demonstrated a significant correlation (P<0.01) between the genetic distances for O. sinensis and its hosts, as well as a significant association (P<0.05) between geographic and genetic distances in both. The similar phylogenetic relationships, geographic distributions, and genetic structure and differentiation between O. sinensis and its hosts imply that they have coevolved.