A subtracted cDNA library from the zebrafish (Danio rerio) embryonic inner ear

A database was built that consists of 4694 sequence contigs of approximately 18,000 reads of cDNAs isolated from the microdissected otocysts of zebrafish embryos at 20-30 hour postfertilization, following subtraction with a pool of liver cDNAs from adult fish. These sequences were compared with those of public databanks. Significant similarity were recorded and organized in a relational database at http://www.genoscope.cns.fr/zie. A first group of 2067 sequences correspond to 1428 known zebrafish genes or ESTs present in the Danio rerio section of UniGene. A second group of 302 sequences encode putative proteins that showed significant similarity (50%-100%) with 302 nonzebrafish proteins in the nr databank, a public databank containing an exhaustive nonredundant collection of protein sequences from different species (ftp://ftp.ncbi.nlm.nih.gov/blast/db/nr). The remaining 2325 (49.5%) sequence contigs or singletons showed no significant similarity with sequences available in public databanks. Several genes known to be expressed in the developing inner ear were represented in the present database, in particular genes involved in hair cell differentiation or innervation The occurrence of these genes validates the outcome of this study as the first collection of ESTs preferentially expressed in the zebrafish inner ear during the period of hair cell differentiation and neuroblast delamination from the otic vesicle epithelium. Novel zebrafish genes also involved in these processes are thus likely to be represented among the sequences obtained herein, for which no homology was found in the D. rerio section of UniGene. [The sequence data from this study have been submitted to EMBL under accession nos. AL714032-AL731531].     

大鼠小脑部分表达序列标签的鉴定

目的:为了获取大鼠小脑特异表达的cDNA序列,筛选新的EST基因片段,以便进一步获取有意义的cDNA全基因。方法:以大鼠大脑、脑干mRNA逆转录cDNA作为driver(驱动)cDNA,大鼠小脑mRNA逆转录cDNA作为tester(测试)cDNA,经消减杂交法,消除与大脑及脑干相同的cDNA,并富集低丰度基因,从而获得大鼠小脑特异表达基因片段,以及低表达基因片段,克隆制备成大鼠小脑特异表达cDNA文库。结果:共挑选出32个阳性克隆质粒,测序得到34个不同基因片段的序列。最后用反Northern杂交去除假阳性,筛选出8个有意义的差异表达cDNA基因片段。同时,将测序结果与Genbank进行同源性比较,发现13个新的EST序列,并申请获得基因序列号(AW288461-AW288474)。结论:抑制消减杂交法是一种筛选特异表达基因的有效方法。本结果可为研究脑功能的分子机制提供有益的资料。     

人类纤维化心脏瓣膜噬菌体表达文库的构建及质量分析

目的: 各种病因引起的心脏瓣膜纤维化是导致心脏瓣膜功能丧失的主要原因,样本来源有限和不可重获性是其分子机制研究的瓶颈。本研究旨在成功获取患者纤维化瓣膜的遗传信息物质,为探明瓣膜病变的分子机制提供直接证据。方法: 心脏换瓣手术中取纤维化瓣膜组织,提取高质量RNA,用SMART技术合成cDNA第1链,再用长距离PCR合成双链cDNA,经 Sfi I酶切后过柱分级收集长片段cDNA,经T4 DNA连接酶催化连接入λTriplEx2载体,最后经λ噬菌体包装后构建成原始文库,用PCR、X-gal等方法进行文库质量鉴定。 结果: 成功构建了纤维化心脏瓣膜噬菌体表达文库,原始文库滴度为8× 10⁹ pfu/L,重组率为99%,外源基因片段大小在500到3 000 bp之间,其中81.25%片段大于1 000 bp。 结论: 成功构建了高质量的人类纤维化心脏瓣膜噬菌体表达文库,该文库的库容量足够代表人类基因的复杂性,高重组率能保障功能筛选的高效性,长片段更有助于全长基因的获得和表达。     

Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray

In the present study, the complex gene expression responses of Plutella xylostella to microbial challenges and injury were surveyed using a newly constructed expressed sequence tag (EST) clone collection and cDNA microarray analysis. A total of 1132 P. xylostella ESTs were cloned, annotated and categorized by their putative functions; these included proteases, protease inhibitors, recognition molecules and anti-microbial peptides. GeneOntology revealed that 4% of the P. xylostella ESTs corresponded to immunity-related genes potentially involved in innate immunity. We then used microarray analysis to identify 44 genes that were differentially expressed with at least a two-fold expression difference in P. xylostella before and after pathogen challenge. Together, our EST categorization and microarray profiling analyses allowed us to identify 70 genes that should be considered candidate immune response genes, providing important new insights into the molecular events that occur during the innate immune response in P. xylostella.     

Identification of candidate coding region single nucleotide polymorphisms in 165 human genes using assembled expressed sequence tags

Using assembled expressed sequence tags (ESTs) from 50 different cDNA libraries, we have identified contigs that represent the complete coding sequences of 850 known human genes, and have scanned these for high quality sequence substitutions. We report the identification and characteristics of 201 candidate single nucleotide polymorphisms found in the coding sequences (cSNPs) of 165 of these genes. Using a conservative calculation, coding region nucleotide diversity (the average number of differences between any pair of chromosomes) was found to be 3 per 10,000 bp based on this data. This analysis reveals that assembled ESTs from multiple libraries may provide a rich source of comparative sequences to search for cSNPs in the human genome.     

Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database

As an approach to characterizing all human ATP-binding cassette (ABC) superfamily genes, a search of the human expressed sequence tag (EST) database was performed using sequences from known ABC genes. A total of 105 clones, containing sequences of potential ABC genes, were identified, representing 21 distinct genes. This brings the total number of characterized human ABC genes from 12 to 33. The new ABC genes were mapped by PCR on somatic cell and radiation hybrid panels and yeast artificial chromosomes (YACs). The genes are located on human chromosomes 1, 2, 3, 4, 6, 7, 10, 12, 13, 14, 16, 17 and X; at locations distinct from previously mapped members of the superfamily. The characterized genes display extensive diversity in sequence and expression pattern and this information was utilized to determine potential structural, functional and evolutionary relationships to previously characterized members of the ABC superfamily.      

A novel PCR-based technique using expressed sequence tags and gene homology for murine genetic mapping: localization of the complement genes

The complement system is a cascade of serum proteins and receptors which forms a vital arm of innate immunity and enhances the adaptive immune response. This work establishes the chromosomal localization of four key genes of the murine complement system. Mapping was performed using a novel and rapid PCR restriction length polymorphism method which was developed to exploit the murine expressed sequence tag (EST) database. This technique circumvents the laborious cDNA or genomic cloning steps of other mapping methods by relying on EST data and the prediction of exon-intron boundaries. This method can be easily applied to the genes of other systems, ranging from the interests of the individual researcher to large-scale gene localization projects. Here the complement system, probably one of the most well-characterized areas of immunology, was used as a model system. It was shown that the C3a receptor C1r and C1s genes form an unexpected complement gene cluster towards the telomeric end of chromosome 6. The second mannose binding lectin-associated serine protease gene was mapped to the telomeric end of chromosome 4, which is distinct from other complement-activating serine proteases. These results provide new insights into the evolution of this group of proteins.     

人骨肉瘤9901细胞cDNA表达文库的构建和鉴定

目的:构建骨肉瘤9901细胞cDNA表达文库,为筛选骨肉瘤特异性抗原创造条件。方法:从9901细胞中提取总RNA,分离mRNA,反转录合成双链cDNA,末端削平,和EcoR I适配子连接。磷酸化EcoR I适配子5’端,过Sephacryl一S400柱除去小于400 bp的cDNA片段,与噬菌体λgt11(载体)连接,用包装蛋白体外包装后形成初级cDNA文库。取适量包装体系倍比稀释后感染E.coli Y1090,测定文库克隆数、重组率,用PCR法测定cDNA插入片段的大小。最后扩增cDNA文库。结果:建成含1.5×106个重组子的骨肉瘤9901细胞cDNA表达文库,重组率为94.3％。重组子中插入的外源片段不小于 0.5 kb,平均长约1.4 kb。结论:达到良好文库的质量标准,适合进一步筛选目的cDNA克隆。     

Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different variant surface glycoproteins (VSGs) were detected in the metacyclic library and 26 VSG EST clusters were found in the bloodstream library, 6 of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the Trypanosoma brucei and Trypanosoma cruzi genomes. Normalized cDNA libraries were constructed from the metacyclic and bloodstream stages, and found to be further enriched for T. congolense-specific ESTs. Given that cultured T. congolense offers an experimental advantage over other African trypanosome species, these ESTs provide a basis for further investigation of the molecular properties of these four developmental stages, especially the epimastigote and metacyclic stages for which it is difficult to obtain large quantities of organisms. The T. congolense EST databases are available at: http://www.sanger.ac.uk/Projects/T_congolense/EST_index.shtml.     

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development.

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes.     

Identification and characterization of a novel zebrafish semaphorin

The semaphorin gene family contains numerous secreted and transmembrane proteins. Some of them function as the repulsive and attractive axon guidance molecules during development. Herein, we report the cloning and characterization of a novel member of zebrafish semaphorin gene, semaphorin 6E (sema6E). Sema6E is expressed predominantly in the nervous system during embryogenesis. Results also show that Sema6E binds Plexin-A1, but not other Plexins. Sema6E chemorepels not only dorsal root ganglion axons but also sympathetic axons. Therefore, Sema6E might utilize Plexin-A1 as a receptor to repel axons of the specific types during development.     

Preparation of a recombinant cDNA library from poly(A+) RNA of the Alzheimer brain. Identification and characterization of a cDNA copy encoding a glial-specific protein

Studies were undertaken to assess the extent to which messenger RNA prepared from the postmortem Alzheimer's disease (AD) brain can be used for the successful preparation of a recombinant cDNA library. Initial experiments focused on the glial-specific marker glial fibrillary acidic protein (GFAP) since GFAP expression appeared to be a model for further studies on mRNAs that may continue to be expressed at high levels in the vicinity of lesioned sites in the AD brain. An AD cDNA library, prepared in the lambda gt11 expression vector system contained GFAP-specific recombinants. One of these was sequenced and the insert was shown to exhibit 88% homology with the similar sequence from mouse GFAP. As established by Northern blots, the size of the GFAP mRNA prepared from the routinely acquired postmortem AD cortex, approximately 2.7 kb, was the same as from a neurologically normal control brain. These results agree with earlier studies on GFAP mRNA from fresh mouse brain. The results demonstrate that in the postmortem AD brain, astroglial-specific mRNA remains sufficiently stable for molecular genetic analysis and may serve as a useful model for examining the genetic expression of mRNAs that may be related to the molecular pathogenesis and the etiology of AD.