Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Generation of expressed sequence tags of random root cDNA clones of Brassica napus by single-run partial sequencing.

Two hundred thirty-seven expressed sequence tags (ESTs) of Brassica napus were generated by single-run partial sequencing of 197 random root cDNA clones. A computer search of these root ESTs revealed that 21 ESTs show significant similarity to the protein-coding sequences in the existing data bases, including five stress- or defense-related genes and four clones related to the genes from other kingdoms. Northern blot analysis of the 10 data base-matched cDNA clones revealed that many of the clones are expressed most abundantly in root but less abundantly in other organs. However, two clones were highly root specific. The results show that generation of the root ESTs by partial sequencing of random cDNA clones along with the expression analysis is an efficient approach to isolate genes that are functional in plant root in a large scale. We also discuss the results of the examination of cDNA libraries and sequencing methods suitable for this approach.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

Expressed Sequence Tag Analysis of Kidney and Gill Tissues from Rainbow Trout (Oncorhynchus mykiss) Infected with Infectious Hematopoietic Necrosis Virus

Expressed sequence tags (ESTs) were obtained from the kidney and gill tissues of rainbow trout, Oncorhynchus mykiss, infected with infectious hematopoietic necrosis virus (IHNV). The results of single-pass sequencing of ESTs from 198 clones (AU081027–AU081192) from kidney complementary DNA and 45 clones (AU081193–AU081236) from gill cDNA are reported herein. Sequences of the cDNA clones were compared with sequences in the GenBank database. Fourteen clones (20%) appeared to be completely unknown and may represent newly described genes, whereas 158 clones (80%) were identified on the basis of matches to sequences in the database. Three of the unidentified sequences were isolated from both the kidney and the gill cDNA libraries. However, no sequences were identical between kidney and gill clones. Received December 7, 1999; accepted April 28, 2000.     

The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.     

A survey of genes in the Atlantic salmon (Salmo salar) as identified by expressed sequence tags

We describe the construction and quality analysis of six cDNA libraries from the liver, ovary, testis, brain, spleen and muscle tissues of adult Atlantic salmon. The cDNA libraries were then screened with total cDNA probes to catalogue clones representing the abundant and rare mRNA populations in each tissue. Subsequently, the 5'-terminal DNA sequences of 1152 cDNA clones, composed of 96 clones from each of the abundant and rare mRNA populations in the six tissues, were determined. Bioinformatic analysis revealed that 510 (50%) of the salmon expressed sequence tags (ESTs) of sufficient length showed significant homology to previously identified genes from salmonid and other species, while 517 (50%) of salmon ESTs were unidentified or novel. After accounting for multi-EST redundancy, the 510 identified ESTs provided DNA sequence markers for 178 salmon genes which are listed in terms of tissue of origin and mRNA abundance class.     

Expressed Sequence Tags from Developing Castor Seeds

To expand the availability of genes encoding enzymes and structural proteins associated with storage lipid synthesis and deposition, partial nucleotide sequences, or expressed sequence tags (ESTs), were obtained for 743 cDNA clones derived from developing seeds of castor (Ricinus communis L.). Enrichment for seed-specific cDNA clones was obtained by selecting clones that did not detectably hybridize to first-strand cDNA from leaf mRNA. Similarly, clones that hybridized to storage proteins or other highly abundant mRNA species from developing seeds were selected against. To enrich for endomembrane-associated proteins, some clones were selected for sequencing by immunological screening with antibodies prepared against partially purified endoplasmic reticulum membranes. Comparison of the deduced amino acid sequences of the ESTs with the public data bases resulted in the assignment of putative identities of 49% of the clones selected by differential hybridization and 71% of the clones selected by immunological screening. Open reading frames in 100 of the ESTs exhibited higher homology to 78 different nonplant gene products than to any previously known plant gene product.     

Comparative analysis of sequences expressed during the liquid-cultured mycelia and fruit body stages of Pleurotus ostreatus

To characterize genes involved in fruit body development, two complementary DNA (cDNA) libraries were constructed from RNA isolated from liquid-cultured mycelia and fruit bodies of Pleurotus ostreatus. Using single-pass sequencing of cDNA clones, 952 and 1069 expressed sequence tags (ESTs) were generated from liquid-cultured mycelia and fruit body cDNA library, respectively. A BLASTX search revealed that 390 of the liquid-cultured mycelia ESTs (41%) and 531 of the fruit body ESTs (50%) showed significant similarity to protein sequences described in the nonredudant database (E values < or =1 x 10(-5)). When liquid-cultured mycelia and fruit body ESTs were compared by the SeqMan II program, among the total of 2021 ESTs, 1256 ESTs were unigenes, and 66 unigenes (5.3%) were commonly expressed during both stages. The functional catalogs of the ESTs were made by comparison with functionally identified Saccharomyces cerevisiae genes. Liquid-cultured mycelium ESTs were compared with fruit body ESTs and changes of the expressed genes during fruit body development were analyzed.     

日本七鳃鳗(Lampetra japonica)口腔腺表达序列标签(EST)分析

以日本七鳃鳗口腔腺为材料,构建库容量为2.1×106pfu/mL的cDNA文库。通过对文库中克隆子的序列测定和生物信息学初步分析,得到1323条有效EST序列。经BlastX及BlastN软件进行同源对比分析,653条(49.36%)EST可在蛋白质或核苷酸水平上找到同源序列,其中328条与七鳃鳗科物种同源。同源序列功能分类大致分为11类,与蛋白质合成有关的蛋白所占比例最大。1323条EST进行片段重叠群分析(contig analysis)获得包括547条序列在内的162组片段重叠群并确定了8条全长cDNA。日本七鳃鳗口腔腺cDNA文库以及EST文库的成功构建,为研究日本七鳃鳗口腔腺的功能基因和蛋白质组学奠定了基础。     

白粉病菌诱导的小麦表达序列标签(EST)研究(英)

白粉病是我国小麦的主要病害之一。尝试用表达序列标签 (expressedsequencetags,EST)技术 ,研究了经白粉病菌诱导后的小麦基因表达。从构建的普通cDNA文库中随机挑取约 15 0 0个阳性克隆并进行测序 ,获不重复ESTs序列 387条。不重复序列均获GenBank的存储号。其中 4 9.4 %的序列与已知基因同源 ,196条序列功能未知 ,84条序列为新ESTs。将不重复序列制备成高密度点阵膜 ,用差示杂交法筛选到几个抗病相关序列。     

Cloning and characterization of chitin synthase gene fragments from Penicillium chrysogenum

Abstract We constructed a 3'-directed cDNA library of cleistothecia and Hülle cells of Aspergillus nidulans to examine gene expression patterns of the sexual structures and to have probes necessary to isolate sexual structure-specific genes. Sequencing of 360 randomly selected cDNA clones yielded 272 expressed sequence tags (ESTs), most of which probably represent frequently or less expressed genes in sexual structures of A. nidulans . Among the 272 ESTs, 33 ESTs (87 cDNA clones) appeared more than once and 2 ESTs appeared 6 times; 9 ESTs matched GenBank entries. When compared with sequences obtained from a mycelial 3'-directed cDNA library of A. nidulans , 28 out of 33 ESTs seem to be sexual structure-specific. Northern blot analyses of 20 ESTs showed that 17 are sexual structure-specific. The remaining three ESTs also hybridized with RNA isolated from vegetative mycelia. These results suggest that analyses of ESTs from different cell types or tissues can readily demonstrate gene expression patterns of specific cell types and identify cell type-specific cDNA probes.     

Identification of preferentially expressed mRNAs in retina and cochlea

To search for genes that could be involved in genetic disorders primarily involving the retina and the cochlea, we tried to identify mRNAs preferentially expressed in retina and cochlea and to establish their chromosomal localization. Two approaches were employed. First, a mouse subtracted library (retina + cochlea against liver + brain) was generated. Randomly selected cDNA clones were sequenced and compared to databases. Tissue expression of some of them was analyzed by RT-PCR. Using radiation hybrid cell lines, the mouse chromosomal localization was determined for those showing the highest level in the retina and the cochlea. Second, human Expressed Sequence Tags (ESTs) with preferential expression in the retina and the cochlea were searched for in databases, and chromosomal localization was also established. From 171 sequenced clones, 73 were classified as known genes (with 17 clones coding for 6 genes), 86 were homologous to ESTs, and 12 were unidentified. Of 108 selected clones, 22 (18.5%) had the highest level of expression in the retina and/or the cochlea, while expression was higher in another tissue or ubiquitous for 60 (55.5%) and 22 (20.4%) of them, respectively. By RT-PCR, one clone similar to the mouse Asic3 cDNA (proton-gated channel) was found mainly in the retina and cochlea, but its human ortholog was widely expressed. We selected 17 ESTs from the UniGene database with restricted expression including in the retina and cochlea. We mapped 10 of these ESTs as well as four mouse clones from the subtracted library. Some of them localized to morbid intervals. The combined information from expression analysis and chromosomal localization allowed for the identification of potential candidate genes for retinal diseases (CORD8, CORD9) and syndromic blindness/deafness/renal defects.     

Expressed sequence tags of Trichinella spiralis muscle stage larvae

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.