Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.     

Genes expressed in sugarcane maturing internodal tissue

To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.     

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.     

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.     

Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.

As part of the ongoing human and mouse genome projects, the aim of this study was to isolate novel, previously uncharacterized, genes from mouse testis. Two approaches were compared for their effectiveness in isolating novel genes: random, vs differential, complementary DNA (cDNA) cloning methods. In the differential approach, only the cDNA clones containing rare sequences (as determined by preliminary clone hybridization) are further analyzed; in the random approach, cDNA clones are isolated at random from the cDNA library. More than two hundred cDNA clones altogether were analyzed, using a PCR-mediated amplification and sequencing strategy. A comparison of these sequences to nucleic acid and protein sequence databases, revealed that 84% of the isolated rare cDNA clones represented new, previously uncharacterized mouse genes. In contrast, less than 63% of the cDNA clones isolated at random from cDNA libraries, contained novel genes. Thus, the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries can be markedly improved by focusing efforts on clones containing rare sequences.     

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 Sequences from Conidial, Mycelial, and Sexual Stages ofNeurospora crassa

In theNeurosporaGenome Project at the University of New Mexico, expressed sequence tags (ESTs) corresponding to three stages of the life cycle of the filamentous fungusNeurospora crassaare being analyzed. The results of a pilot project to identify expressed genes and determine their patterns of expression are presented. 1,865 partial complementary DNA (cDNA) sequences for 1,409 clones were determined using single-pass sequencing. Contig analysis allowed the identification of 838 unique ESTs and 156 ESTs present in multiple cDNA clones. For about 34% of the sequences, highly or moderately significant matches to sequences (of known and unknown function) in the NCBI database were detected. Approximately 56% of the ESTs showed no similarity to previously identified genes. Among genes with assigned function, about 43.3% were involved in metabolism, 32.9% in protein synthesis and 8.4% in RNA synthesis. Fewer were involved in defense (6%), cell signalling (3.4%), cell structure (3.4%) and cell division (2.6%).     

ES cell neural differentiation reveals a substantial number of novel ESTs

We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease. Electronic Publication     

47个早期人胚胎低丰度表达基因ESTs筛选及结果分析

构建高质量ｃＤＮＡ文库在基因克隆、ｍＲＮＡ差异展示、表达序列标签测序和基因定位等研究中具有十分重要的作用。为了从早期胚胎中分离人类新基因,构建了受精后３周龄的人ｃＤＮＡ文库,用标记的一链ｃＤＮＡ探针对该文库的６５０８个克隆子进行菌落原位杂交,得到１６７７个无任何杂交信号的低丰度表达克隆子,从中随机挑选了４７个进行５′端部分测序,将测序结果与三大基因库进行序列同源性比较,发现１８个克隆（３８．３％）