表达序列标签在寄生虫功能基因组学研究中的应用

随着后基因组时代的到来，基因组学已从结构基因组学向功能基因组学领域拓展。表达序列标签（expressed sequence tags，EST）是一种快捷、高效地揭示基因组功能信息的方法。本文就EST在寄生虫功能基因组学研究中的应用作一综述。     

Using RNA interference to identify genes required for RNA interference

RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) silences endogenous gene expression. By injecting pools of dsRNAs into Caenorhabditis elegans, we identified a dsRNA that acts as a potent suppressor of the RNAi mechanism. We have used coinjection of dsRNAs to identify four additional candidates for genes involved in the RNAi mechanism in C. elegans. Three of the genes are C. elegans mes genes, some of which encode homologs of the Drosophila chromatin-binding Polycomb-group proteins. We have used loss-of-function mutants to confirm a role for mes-3, -4, and -6 in RNAi. Interestingly, introducing very low levels of dsRNA can bypass a requirement for these genes in RNAi. The finding that genes predicted to encode proteins that associate with chromatin are involved in RNAi in C. elegans raises the possibility that chromatin may play a role in RNAi in animals, as it does in plants.     

应用基因芯片技术筛选大鼠再生肝中差异表达基因

目的应用基因芯片技术筛选大鼠再生肝中差异表达基因,为探讨急性肝功能衰竭的发病机制提供基础。方法雄性SD大鼠行95％肝部分切除术,用含1176个基因的大鼠尼龙膜微阵列筛选大鼠再生肝组织中差异表达的基因。RT—PCR随机验证若干差异表达基因在微阵列中的表达。结果再生肝组织有138个基因明显较对照组织表达高,它们主要是生长因子基因、核受体基因、核糖体蛋白基因、应急反应蛋白基因、细胞周期调节基因、神经递质基因等;下调基因共50个,主要为代谢酶基因、激素受体基因及表面抗原基因等。结论cDNA微阵列技术有助于研究急性肝功能衰竭的发病机制,并提供诊断和治疗的潜在靶点。     

日本血吸虫(大陆株)成虫表达序列标签的获取及电子延伸

目的获取日本血吸虫(Schistosoma japonicum，Sj)新基因。方法从Sj(大陆株)成虫cDNA文库中随机挑选单个重组克隆进行部分测序以获取表达序列标签(expressed sequence tag，EST)，用其提供的BLAST程序和Genebank数据库进行序列比较和同源性分析；建立电子拼接的方法，对所获EST进行电子延伸，并对Sj延伸后序列进行同源性分析。结果在Sj成虫cDNA文库中，随机挑选182个单个重组克隆，获取53个有价值EST序列，并在Genebank中登录，获得53个EST的延伸序列及分析结果。结论Sj表达基因EST测序、同源性分析及EST的电子延伸是获取Sj新基因的有效对策。     

乙型肝炎病毒序列数据库发展现状

乙型肝炎病毒序列数据库是集病毒序列收集和相关信息分析为一体的专用数据共享平台。目前，国际上已建立的数据库包括HBVRegDB、HVDB、SeqHepB和HepSeq。这4个数据库收录样本以国外患者为主，且都不能提供HBV基因组或基因片段克隆实物。因此，建立以中国患者为主体，且能提供基因实物的HBV序列数据库，对推动我国乙型肝炎的防治研究十分必要。     

High concentrations of long interspersed nuclear element sequence distinguish monoallelically expressed genes

Genes subject to monoallelic expression are expressed from only one of the two alleles either selected at random (random monoallelic genes) or in a parent-of-origin specific manner (imprinted genes). Because high densities of long interspersed nuclear element (LINE)-1 transposon sequence have been implicated in X-inactivation, we asked whether monoallelically expressed autosomal genes are also flanked by high densities of LINE-1 sequence. A statistical analysis of repeat content in the regions surrounding monoallelically and biallelically expressed genes revealed that random monoallelic genes were flanked by significantly higher densities of LINE-1 sequence, evolutionarily more recent and less truncated LINE-1 elements, fewer CpG islands, and fewer base-pairs of short interspersed nuclear elements (SINEs) sequence than biallelically expressed genes. Random monoallelic and imprinted genes were pooled and subjected to a clustering analysis algorithm, which found two clusters on the basis of aforementioned sequence characteristics. Interestingly, these clusters did not follow the random monoallelic vs. imprinted classifications. We infer that chromosomal sequence context plays a role in monoallelic gene expression and may involve the recognition of long repeats or other features. The sequence characteristics that distinguished the high-LINE-1 category were used to identify more than 1,000 additional genes from the human and mouse genomes as candidate genes for monoallelic expression.     

Gene-trap expression screening to identify endothelial-specific genes

The endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene-trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. In a pilot screen, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor-beta type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene-trap expression screening can thus be used to identify early endothelial-specific genes and analyze their function in mice.     

Use of in vivo-induced antigen technology (IVIAT) to identify genes uniquely expressed during human infection with Vibrio cholerae

In vivo-induced antigen technology is a method to identify proteins expressed by pathogenic bacteria during human infection. Sera from 10 patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor Vibrio cholerae O1, and used to probe a genomic expression library in Escherichia coli constructed from El Tor V. cholerae O1 strain N16961. We identified 38 positive clones in the screen, encoding pili (PilA and TcpA), cell membrane proteins (PilQ, MshO, MshP, and CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA and CheR), a quorum-sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual patients demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. These results also demonstrate substantial immune responses to TcpA in patients infected with El Tor V. cholerae O1. In vivo-induced antigen technology provides a simple method for identifying microbial proteins expressed during human infection, but not during in vitro growth.     

Using paramedics to identify at-risk elderly.

OBJECTIVES: To evaluate paramedics' ability to identify elderly at risk and refer them for assessment and service. DESIGN: A prospective nonrandomized open trial. SETTING: Akron, Ohio, a midsize city with a well-developed advanced life support emergency medical services system. TYPE OF PARTICIPANTS: One hundred thirty firefighter paramedics evaluated 6,000 elderly patients. Assessments were performed by trained geriatric assessors. INTERVENTION: Regardless of the reason for the call, paramedics screened all emergency medical services users age 60 and older for medical, mental health, social, and environmental problems. Identified cases were referred to the Area Agency on Aging for assessment and follow-up. MAIN RESULTS: Paramedics identified 197 people with possible problems, 124 of whom received an assessment. The remainder could not be assessed due to death, moving, referral, or transfer to a long-term care facility. Assessors confirmed the presence of a problem in 121 of 124 assessed cases, a positive predictive value of 98%. The program was useful for 94 people, 48% of those identified and assessed. CONCLUSION: Paramedics can serve as case finders for at-risk elderly, and effective linkage to service agencies can occur.     

Using computerized tomography to identify neurologic problems

Computerized tomography (CT) scanning accurately identifies neurologic abnormalities in many elderly patients, often making it possible to differentiate symptomatic neurologic changes of normal aging from treatable pathologic states such as occult masses and cerebral infarction producing much the same symptoms. The scan also singles out patients in whom further diagnostic measures are necessary. The advantages of CT--low morbidity, noninvasiveness, and high sensitivity--far outweigh its limitations. Concomitant cerebral atrophy and metabolic imbalance do not significantly affect diagnostic accuracy. Risks are minimal, related chiefly to contrast allergy, and occasionally to anesthetics for patients who cannot remain motionless during the procedure.     

A catalogue of genes in the cardiovascular system as identified by expressed sequence tags.

The heart, which is composed of all the cellular components of the circulatory system, is a representative organ for obtaining genes expressed in the cardiovascular system in normal and disease states. We used partial sequences of cDNA clones, or expressed sequence tags, to identify and tag genes expressed in this organ. More than 3500 partial sequences representing > 3000 cDNA clones have been obtained from either the 5' or 3' end of inserts derived from human heart cDNA libraries. Of 3132 cDNA clones analyzed by sequence similarity searching against the GenBank/EMBL data bases, 1485 (47.4%) were found to represent additional, previously undiscovered genes, whereas 267 clones were matched to human brain expressed sequence tags. Clones matching to known genes were catalogued according to their putative structural and cellular functions. cDNA probes from reverse-transcribed mRNAs of fetal and adult hearts were used to study differential expression of selected clones in cardiac development. Cataloguing genes expressed in the heart may provide insight into the genes involved in health and cardiovascular disease.