High‐throughput cloning and expression library creation for functional proteomics

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single‐gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high‐throughput proteomics platforms, such as protein microarrays and cell‐based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high‐throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator^TM DNA Cloning System) and compare them side‐by‐side. We also report an example of high‐throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).     

hunchback and Ikaros-like zinc finger genes control reproductive system development in Caenorhabditis elegans

Here we provide evidence for a C2H2 zinc finger gene family with similarity to Ikaros and hunchback. The founding member of this family is Caenorhabditis elegans ehn-3, which has important and poorly understood functions in somatic gonad development. We examined the expression and function of four additional hunchback/Ikaros-like (HIL) genes in C. elegans reproductive system development. Two genes, ehn-3 and R08E3.4, are expressed in somatic gonadal precursors (SGPs) and have overlapping functions in their development. In ehn-3; R08E3.4 double mutants, we find defects in the generation of distal tip cells, anchor cells, and spermatheca; three of the five tissues derived from the SGPs. We provide in vivo evidence that C. elegans HIL proteins have functionally distinct zinc finger domains, with specificity residing in the N-terminal set of four zinc fingers and a likely protein-protein interaction domain provided by the C-terminal pair of zinc fingers. In addition, we find that a chimeric human Ikaros protein containing the N-terminal zinc fingers of EHN-3 functions in C. elegans. Together, these results lend support to the idea that the C. elegans HIL genes and Ikaros have similar functional domains. We propose that hunchback, Ikaros, and the HIL genes arose from a common ancestor that was present prior to the divergence of protostomes and deuterostomes.     

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.     

新疆维吾尔族妇女宫颈癌组织中乳头瘤病毒16型E6基因的克隆和序列分析

为了分析新疆南部地区维吾尔族妇女宫颈癌组织中HPV16型E6基因结构特点，从中国新疆南部地区维吾尔族妇女宫颈癌活检组织标本中提取DNA，以宫颈癌活检组织标本DNA为模板进行PCR扩增，获得HPV16 E6基因，将其克隆到pUCm-T载体上，并对其进行基因全序列分析.PCR检测结果显示宫颈癌组织中HPV16 E6阳性率为82.35%（14/17）；测序结果显示，新疆株HPV16 E6基因全长456 bp，大小与德国标准株一致.E6基因的第247位碱基发生T→G突变，并由此引起所编码的氨基酸亦发生改变.上述结果表明，中国新疆南部地区维吾尔族妇女宫颈癌患者组织中HPV16 E6的基因结构与德国标准株HPV16 E6基因之间存在差异.     

应用siRNA干扰胃癌的研究进展

胃癌是消化系统最常见的恶性肿瘤,而我国是胃癌高发区,其发病率和死亡率均高于世界平均水平。在我国大多数患者明确诊断时已进入进展期,所以大多数患者再行手术切除后还需放化疗治疗。近来随着对胃癌的研究深入,可通过对胃癌肿瘤标本行分子检测给予患者药物靶向治疗,实现个体化治疗。RNA干涉(RNAi)技术被广泛用于基因功能的研究,并且在哺乳动物研究中得到飞速发展.si RNA是在RNAi中起中心作用,其可抑制特定m RNA,以调节不同蛋白在肿瘤发生时的异常表达。对si RNA的研究将为胃癌的基因治疗供更广阔的空间。     

Identification of stress-induced genes from the drought tolerant semi-arid legume crop horsegram (Macrotyloma uniflorum (Lam.) Verdc.) through analysis of subtracted expressed sequence tags

Abiotic stresses adversely affect the agricultural productivity worldwide. Horsegram (Macrotyloma uniflorum (Lam.) Verdc.) is a legume crop that can tolerate severe adverse environmental conditions such as drought, salinity and heavy metal contamination. As a first step towards characterization of genes that contribute to combating abiotic stresses, construction and analysis of subtracted cDNA library is reported here. Using this strategy a total of 1050 ESTs were isolated, sequenced, 959 high quality ESTs were obtained and clustered. Further, our analysis revealed that of these 531 sequences are unique and 30% of these have no homology to known proteins in the database. This observation has great relevance since horsegram is a stress-adapted legume crop. Further, to validate the identified differentially expressed genes, expression profiles of selected clones were analyzed using reverse-northern, northern blot analysis and we show that indeed these clones are differentially expressed under various abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance also discussed.     

植物重金属胁迫耐受机制

重金属是一类会对植物产生毒害作用的污染物,植物在长期进化过程中演变出耐受重金属胁迫的相关机制。以植物重金属耐受性为基础,对近几年来国内外植物响应重金属胁迫的耐受机制研究作一简要综述。主要概述了重金属对植物的胁迫影响及植物抗氧化系统,脯氨酸、可溶性糖、可溶性蛋白等渗透调节物质和不同类型基因家族等方面对植物耐受重金属胁迫机制的研究进展。以期为提高植物耐重金属胁迫能力及研究植物修复重金属污染土壤的应用奠定一定的基础。     

鸡基因组差异表达基因翻译起始密码子上下游序列的比较研究

翻译起始调控是基因表达调控的一个关键步骤之一。本文以鸡为研究材料,比较研究了鸡基因组高表达基因和低表达基因翻译起始密码子上下游的碱基序列差异,旨在寻找影响鸡基因表达水平的特异性调控位点。全部3 020个单剪接基因完整的mRNA序列及有详细注释的5＇UTRs序列从Ensembl下载。编写计算机程序,读取每个基因mRNA起始密码子上下游各位点的碱基。研究发现,起始密码子上游-3、-2位点可能是鸡基因组基因表达起始密码子正确识别的关键位点。起始密码子上下游的碱基组成分析发现,高表达基因和低表达基因起始密码子的上游均倾向使用（G＋C）,高表达基因的使用偏倚尤为强烈。序列差异比较发现,高表达基因在-9、-6、-3、＋4位点显著偏向G,在-1、-2、-4、-5位点显著偏向C。低表达基因起始密码子上游使用A、U的频率显著高于低表达基因。在-19位点强烈偏向A,在＋1、＋11、＋14位点强烈偏向U。