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PubMed1996

Identification of 4370 expressed sequence tags from a 3'-end-specific cDNA library of human skeletal muscle by DNA sequencing and filter hybridization.

A systematic study on the mRNA species expressed in the human skeletal muscle is presented in this paper. To carry on this study, a new method has been developed for the construction of unbiased cDNA libraries specially designed for the production of ESTs corresponding to the 3'-end portion of the mRNAs. The method has been applied to human skeletal muscle, where the analysis of the transcription profile is particularly difficult for the presence of several very abundant transcripts. To detect and quantify high-level mRNAs, the first 1054 ESTs were obtained from randomly selected clones. The 10 most abundant transcripts accounted for > 45% of the clones. Subsequently, these transcripts were identified by filter hybridization, thus making DNA sequencing more productive. Overall, 4370 clones were identified: 3372 by DNA sequencing and 998 by filter hybridization. The number of groups of sequences identifying individual transcripts was relatively low compared with other tissues, resulting in a total of 934 groups out of 4370 ESTs. Of these, 719 groups were represented by only one sequence.

Cloning, Molecular↗
PubMed1995

Regional assignment by hybrid mapping of 36 expressed sequence tags (ESTs) on human chromosome 6.

We have determined the regional chromosome assignment of 36 cDNAs from infant brain libraries by assessing the concordant segregation of PCR products using a human-rodent hybrid mapping panel that subdivides chromosome 6 into 15 regions. These mapped sequences serve as markers for the physical and expression maps of chromosome 6, as well as candidate genes for various disease loci. Sequence analysis has identified putative functions and motifs for some of these genes.

Animals↗
PubMed1997

Genetic mapping of 20 novel expressed sequence tags from midgestation mouse embryos suggests chromosomal clustering.

Current knowledge of genes that regulate pattern formation and differentiation processes during mammalian embryonic development is limited. In an effort to isolate developmentally relevant genes, 20 novel, end-sequenced cDNAs selected from a Day 10.5 postcoitum mouse embryo library were genetically mapped in intersubspecific backcross mice. Eleven of 20 cDNA clones mapped to three mouse autosomes (Chr 5, 11, and 14), a result that was unlikely (P < 0.03) if the distribution of genes expressed in embryos is random within the mouse genome. Several clones were candidates for mouse developmental mutations by virtue of genetic colocalization and concordance of embryonic expression patterns with the distribution of defects in mutant mice: Estm11 was a candidate for the mouse mutation wabbler-lethal (wl), since Estm11 mapped in the vicinity of wl on mouse Chr 14 and was expressed in those regions of embryonic brain that exhibit axonal degeneration in wl. End-sequence analysis, genetic mapping, and in situ hybridization appeared to be an effective combination of methods for identification and characterization of genes with potential regulatory functions during mammalian embryogenesis.

Animals↗
PubMed1998

Characterization of a novel gene encoding zinc finger domains identified from expressed sequence tags (ESTs) of a human heart cDNA database.

Seven types of zinc finger protein (ZFP) genes based on the combinations of cysteine and histidine residues were found in a human heart cDNA database. Here we report the isolation of 360 cDNA clones encoding putative ZFPs. Of these, 154 (42.8%) represent C2H2-type ZFPs, 101 (28.1%) represent C2C2-type, five (1.4%) represent C2HC-type, 71 (19.7%) represent C2HC4C(HD)-type, three (0.8%) represent C3H-type, eight (2.2%) represent C3HC4-type and 18 (0.5%) represent combination type (genes containing more than one type of zinc finger). Among these 360 ZFPs, a novel ZFP cDNA named HFHZ (human fetal heart ZFP) with sequence homology to a Kruppel-associated box (KRAB) was identified. Sequencing the full-length of this cDNA clone identified an open reading frame of 711 bp that encodes a 237 amino acid protein with a predicted molecular weight of 27.7 kDa. Sequence analysis indicated that HFHZ contained a truncated KRAB box at the N-terminus and two C2H2 zinc fingers at the C-terminus. The transcript of HFHZ is highly expressed in fetal heart and moderately expressed in fetal brain but not expressed in fetal liver as revealed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis suggesting that HFHZ is not expressed ubiquitously. The 3.3-fold higher expression in the fetal heart than in the adult heart suggests that HFHZ mRNA is downregulated in the process of development. In addition, the relatively high expression (1.9-fold) of HFHZ observed in the hypertrophic as compared to the normal adult heart suggests that this fetal gene is reactivated in response to hypertrophic stimuli. Chromosomal localization by in situ hybridization revealed that this gene is in 19q13.1, a region containing genes involved in both cell cycle and developmental regulation.

Adult↗
PubMed1997

Expansion of the pig comparative map by expressed sequence tags (EST) mapping.

We have used a PCR-based approach for the genetical and physical mapping of 34 transcripts isolated from a porcine small intestine cDNA library. All but one gene were regionally localized by using a somatic pig-rodent cell hybrid panel, and 12 genes were mapped by linkage analysis of single-stranded conformational polymorphisms developed in 3' untranslated regions of transcripts. For 20 of the transcripts, the human homolog has already been mapped. This study thus represents a significant contribution to the pig comparative map. Some important findings were that we could clarify the extent of a previously identified inversion event in a region of conserved synteny between SSC6q and HSA1p, that SSC14q does contain a region homologous to HSA1, a situation not clear from earlier ZOO-FISH studies, and that the homology between SSC17 and HSA20 includes the p-arm of HSA20.

Animals↗
PubMed1996

Glucose-stimulated expressed sequence tags from rat pancreatic islets.

Available evidence suggests that glucose, the most potent physiologic insulin secretagogue, capacitates glucose-induced insulin secretion by stimulating synthesis of various proteins in the beta cell. To obtain more clues about proteins that might be involved in insulin secretion, rat pancreatic cDNA libraries were screened by differential hybridization for non-preproinsulin transcripts that were increased when pancreatic islets were cultured for 1 day at a high (20 mM) versus a low concentration (1 mM) of glucose. More than 100,000 pfu were initially screened. After repeated rescreening, 33 transcripts were 1-3-fold higher in the presence of the high glucose. For comparison, preproinsulin transcripts were 4-8-fold higher at the high concentration of glucose. The sequences of 12 clones were > or = 85% similar to published sequences. These included annexin, calbindin, protein kinase C receptor, the G protein beta subunit, the guanyl cyclase A/atrial natriuretic peptide receptor and the serotonin 5HT-2 receptor. As previously reported, ferritin H chain transcripts were discovered to be 3-6-fold higher in the presence of the high glucose (MacDonald et al., FASEB J. 8, 777-781, 1994). Unidentified glucose responsive clones have been assigned GenBank accession numbers N55606-N55636, N65938 and N65939. The results implicate the proteins encoded by these mRNAs in insulin secretion.

Animals↗
PubMed1998

Comparative analysis of 82 expressed sequence tags from a cattle ovary cDNA library.

In total, 82 ESTs were generated from 51 unique clones randomly selected from a cattle ovary cDNA library. Among these clones, 22 (42.1%) had 5' and/or 3' ends that matched with known human or other mammalian coding sequences, 18 (35.3%) matched human or other ESTs, and 11 (21.6%) represented novel transcripts with no significant match to any sequence in the databases. The relatively high frequency of ESTs with no matches in GenBank or dbEST indicates that bovine ovary may be a source of novel candidate genes for loci affecting cattle reproduction traits. Primers were designed for 11 ESTs that had human orthologs in GenBank. These ESTs were mapped to 10 bovine autosomes by PCR screening of a somatic cell hybrid panel. Among these 11 ESTs, 4 corresponded to genes previously mapped in humans and had chromosome assignments on the bovine map that were consistent with available comparative mapping data. Although the human orthologs of the remaining 7 mapped bovine ESTs have not been mapped, the human map location could be predicted on the basis of existing comparative mapping data. Because of the general utility of our approach for comparative genome analysis, we have termed it comparative mapping by annotation and sequence similarity (COMPASS). With the cost of large-scale EST sequencing becoming more affordable, and the rapid expansion of DNA databases, it is likely that COMPASS will be a preferred strategy for high throughput comparative mapping.

Animals↗
PubMed1999

Winter Flounder Expressed Sequence Tags: Establishment of an EST Database and Identification of Novel Fish Genes.

: An EST database of more than 900 sequences has been constructed from complementary DNAs from six different tissues (stomach, intestine, pyloric cecum, liver, spleen, and ovary) of the winter flounder Pleuronectes americanus. Template preparation and automated sequencing were optimized to generate high-quality information in an economic fashion. Using computer scripts developed in our laboratory, the sequences were automatically compared with sequences in the databases via a Web-browser interface, and significant returns were recorded and organized on user-friendly HTML pages. Half (453) of the ESTs had significant matches to database sequences of known function, 33 matched ESTs from other organisms, 34 matched ribosomal RNAs, and 24 matched hypothetical open reading frames of unknown function. Forty-one percent (374) of the ESTs had no matches to sequences in the database and presumably represent previously unidentified cDNAs. Several sequences are the first isolated from teleost fish, and should be of interest for gene mapping and studies of developmental biology.

Journal Article↗