Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.     

Short communication: Single tube allele specific (STAS) PCR for direct determination of the mutation in the porcine ryanodine receptor gene associated with malignant hyperthermia

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of “large offspring syndrome” were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.     

Polymorphisms of the Myostatin Gene and Its Relationship with Reproduction Traits in the Bian Chicken

Myostatin, or growth and differentiation factor 8, is a member of the transforming growth factor-β superfamily; it functions as a negative regulator of skeletal muscle development and growth in mammals. In this study, single nucleotide polymorphisms in the 5′ regulatory region and exon 1 of the myostatin gene were detected by PCR–SSCP in the Bian, Jinghai, Youxi, and Arbor Acre chickens, and the associations of the polymorphisms with reproduction traits were analyzed. Seven SNPs (A326G, C334G, C1346T, G1375A, A1473G, G1491A, and G2283A) were found in the myostatin gene. Association analysis showed that the G2283A were significantly associated with reproduction traits. Bian chickens of the GG genotype had a greater age at first egg than those of the GA and AA genotypes (P < 0.01). Correspondingly, Bian chickens of the GA and AA genotypes had larger egg number at 300 days than those of the GG genotype (P < 0.05 and P < 0.01, respectively). Bian chickens of the AA genotype had significantly higher body weight at 300 days than those of the GG genotype (P < 0.05). These results suggested that the myostatin gene may have certain effects on reproduction traits other than merely as a negative regulator of skeletal muscle development and growth in mammals previously reported.     

斑马鱼Fbxl5基因多克隆抗体的制备与检测

Fbxl5为F-box基因家族的一员,目前研究发现其与心脏的发育有关。为了在斑马鱼模型中进一步研究该基因的功能,有必要制备其多克隆抗体。通过桥式PCR扩增出亲水性和特异性均好的斑马鱼Fbxl5基因片段,将其克隆入表达载体pET-28a,转化至大肠杆菌Rosseta中。用IPTG诱导Fbxl5重组质粒得到His-Fbxl5的融合蛋白。这个融合蛋白用Ni-IDA凝胶柱亲和纯化,将纯化的His-Fbxl5融合蛋白免疫新西兰大白兔制备多克隆抗体。使用Western Blot检测,获得了Fbxl5原核表达重组融合蛋白及高效价的特异性兔抗Fbxl5多克隆抗体。随后检测了Fbxl5在斑马鱼胚胎和成体组织中蛋白的表达,通过基因芯片分析在Fbxl5-MO和Std胚胎样品的mRNA含量差异.所得结果显示获得了较高效价和特异性好的斑马鱼Fbxl5多克隆抗体,为Fbxl5功能的进一步研究奠定了基础。     

Gaussia分泌型萤光素酶的研究进展及其应用

Gaussia分泌型萤光素酶是近年发现的一种来源于海洋桡脚类动物Gaussia princeps的新型分泌型萤光素酶,是目前已知的可自然分泌的最小萤光素酶,因其分子小、灵敏度高、半衰期短和可高效分泌,而成为一种理想的报告基因,广泛应用于体内外研究。我们就Gaussia分泌型萤光素酶的发光原理、荧光特性及其应用等进行简要综述。     

基因表达谱中信号通路基因集分析方法进展

微阵列技术是生物技术变革的核心,允许研究者同时监测成千上万个基的表达水平,已广泛应用医学研究.如何挖掘海量基表达信息中的有用信息并进行生物学专业解释,是基表达谱数据分析领所面临的一个重要挑战.生物信号通路研究已成为基芯片中不同表型差异表达研究的主要方法,其是以整个信号通路作为一个整体作为研究对象,此得出的研究结果更加科学和准确.在本文中我们简要描述了近10年来信号通路基集富集分析方法的发展情况,将其分为三个阶段,对每个阶段方法的基础和特点做了一些简单的总结和阐述.     

人p53启动子萤光素酶报告基因的构建及其活性测定

目的：克隆p53基因的启动子,插入萤光素酶报告基因载体,并检测启动子活性。方法：采用PCR技术从人肝癌细胞系HepG2基因组中扩增人p53启动子,插入萤光素酶报告基因载体pGL4．0-empty,将重组质粒转染293T、ZR75-1、HepG2、A549细胞,测定p53启动子的转录活性。结果：构建了p53启动子的萤光素酶报告基因;通过测序及质粒酶切鉴定,所构建的p53启动子正确;活性实验表明,报告基因在多种细胞中显示构建的p53启动子活性,并呈现一定的剂量效应;转录因子USF能以剂量效应方式提高p53报告基因的转录活性。结论：克隆了人p53启动子,为进一步研究调控p53的转录因子奠定了基础。     

Phytochrome-interacting factors have both shared and distinct biological roles

Phytochromes are plant photoreceptors that perceive red and far-red light. Upon the perception of light in Arabidopsis, light-activated phytochromes enter the nucleus and act on a set of interacting proteins, modulating their activities and thereby altering the expression levels of ～10% of the organism’s entire gene complement. Phytochromeinteracting factors (PIFs) belonging to Arabidopsis basic helix-loop-helix (bHLH) subgroup 15 are key interacting proteins that play negative roles in light responses. Their activities are post-translationally countered by light-activated phytochromes, which promote the degradation of PIFs and directly or indirectly inhibit their binding to DNA. The PIFs share a high degree of similarity, but examinations of pif single and multiple mutants have indicated that they have shared and distinct functions in various developmental and physiological processes. These are believed to stem from differences in both intrinsic protein properties and their gene expression patterns. In an effort to clarify the basis of these shared and distinct functions, we compared recently published genome-wide ChIP data, developmental gene expression maps, and responses to various stimuli for the various PIFs. Based on our observations, we propose that the biological roles of PIFs stem from their shared and distinct DNA binding targets and specific gene expression patterns.     

Quantifying the effects of migration and mutation on adaptation and demography in spatially heterogeneous environments

How do mutation and gene flow influence population persistence, niche expansion and local adaptation in spatially heterogeneous environments? In this article, we analyse a demographic and evolutionary model of adaptation to an environment containing two habitats in equal frequencies, and we bridge the gap between different theoretical frameworks. Qualitatively, our model yields four qualitative types of outcomes: (i) global extinction of the population, (ii) adaptation to one habitat only, but also adaptation to both habitats with, (iii) specialized phenotypes or (iv) with generalized phenotypes, and we determine the conditions under which each equilibrium is reached. We derive new analytical approximations for the local densities and the distributions of traits in each habitat under a migration–selection–mutation balance, compute the equilibrium values of the means, variances and asymmetries of the local distributions of phenotypes, and contrast the effects of migration and mutation on the evolutionary outcome. We then check our analytical results by solving our model numerically, and also assess their robustness in the presence of demographic stochasticity. Although increased migration results in a decrease in local adaptation, mutation in our model does not influence the values of the local mean traits. Yet, both migration and mutation can have dramatic effects on population size and even lead to metapopulation extinction when selection is strong. Niche expansion, the ability for the population to adapt to both habitats, can also be prevented by small migration rates and a reduced evolutionary potential characterized by rare mutation events of small effects; however, niche expansion is otherwise the most likely outcome. Although our results are derived under the assumption of clonal reproduction, we finally show and discuss the links between our model and previous quantitative genetics models.     

Preparation of optically active 4-chlorophenylalanine from its racemate by deracemization technique using transformant Escherichia coli cells

We have developed a method for the preparation of l-4-chlorophenylalanine from its racemate with Escherichia coli cells expressing a single foreign gene. l-4-Chlorophenylalanine was obtained in a high optical yield by the inversion of configuration of its d-form via the tandem reactions catalyzed by d-amino acid dehydrogenase (DadA) and branched-chain amino acid aminotransferase (BCAAT). While we constructed a plasmid for BCAAT utilizing the gene from Sinorhizobium meliloti ATCC 51124, the first enzyme DadA was the dadA-gene product from E. coli host cell itself, which was activated by the addition of l-alanine in the growth medium.