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At least 109 records · Page 6Linked to original sources

PubMed1988

Cloning and partial sequencing of a new rat brain specific cDNA.

In order to find brain specific transcripts in a pUC13 cDNA library prepared from rat brain cytoplasmic poly(A)+ RNAs, the following steps were observed: 1. Randomly chosen cDNA clones from the brain library were screened by radiolabelled single stranded cDNAs (sscDNAs) prepared from liver, spleen, kidney and intestine mRNAs. 2. The brain clones containing genetic information shared with the peripheral organs were discarded. 3. After hybridization of the remaining clones with radiolabelled sscDNAs prepared from rat brain poly(A)+ RNAs, 210 possibly brain specific clones were selected. 4. Plasmids containing cDNA of each of these clones were purified and after estimation of their cDNA sizes, clones containing inserts of at least 500 base pairs (bp) were used as radiolabelled probes against rat brain, liver, spleen, kidney and intestine poly(A)+ RNAs spotted onto nitrocellulose filters (dot blot analysis). 5. By using this screening procedure, several brain specific clones were obtained. One of them (clone III 25) is presently subjected to sequencing in M13 phage. 6. A computer analysis of the partial sequence so far obtained gives no significant homology to the 15.000 genes contained in Genebank and EMBL data bank. It seems therefore that we have isolated a new brain specific cDNA coding for a not yet described peptide.

Animals↗

PubMed1999

Association of the glucocorticoid receptor alternatively-spliced transcript 1A with the presence of the high molecular weight membrane glucocorticoid receptor in mouse lymphoma cells.

Using the combination of a cDNA library prepared from membrane glucocorticoid (mGR)-enriched S-49 cells and a mouse leukocyte genomic library, we have cloned a 7.3 kb full-length glucocorticoid receptor 1A cDNA. Primer extension, 5'RACE, and long distance PCR identified the transcription start site as being located at 1026 bp from the ATG codon. The first 1,013 nucleotides (nts) of the full length sequence constitute 5' UTR sequence (exon 1), the next 2349 bp, the coding region, and the last 3,907 bp, the 3'UTR. The entire 5'UTR sequence is unique to transcript 1A. The 3'UTR sequence is approximately 88.5 % conserved with the rat 3'UTR. Western blot analysis compared the molecular weight of in vitro translation products from the cloned 1A cDNA with partially purified cellular mGR. Both preparations contained the novel 150 KD and the 94 KD classical GR peptides, suggesting that transcript 1A encodes both receptor forms. Transfection of mGR-less and glucocorticoid lysis-resistant AtT-20 and HL-60 cells with full-length GR 1A cDNA imparted both mGR expression and glucocorticoid lysis-sensitivity to these cells.

3' Untranslated Regions↗

PubMed1994

A novel human homeobox gene distantly related to proboscipedia is expressed in lymphoid and pancreatic tissues.

A novel human homeobox gene, HB9, was isolated from a cDNA library prepared from in vitro stimulated human tonsil B lymphocytes and from a human genomic library. The HB9 gene is composed of 3 exons spread over 6 kilobases of DNA. An open reading frame of 1206 nucleotides is in frame with a diverged homeodomain. The predicted HB9 protein has a molecular mass of 41 kilodaltons and is enriched for alanine, glycine, and leucine. The HB9 homeodomain is most similar to that of the Drosophila melanogaster homeobox gene proboscipedia. Northern blot analysis of poly(A) RNA purified from the human B cell line RPMI 8226 and from activated T cells revealed a major mRNA transcript of 2.2 kilobases. Similar analysis of poly(A) RNA from a variety of adult tissues demonstrated HB9 transcripts in pancreas, small intestine, and colon. Reverse transcriptase-polymerase chain reaction was used to examine HB9 RNA transcripts in hematopoietic cell lines. HB9 RNA transcripts were most prevalent in several human B cell lines and K562 cells. In addition, transcripts were detected in RNA prepared from tonsil B cells and in situ hybridization studies localized them in the germinal center region of adult tonsil. These findings suggest the involvement of HB9 in regulating gene transcription in lymphoid and pancreatic tissues.

Adult↗

PubMed2001

Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle.

An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.

Animals↗

PubMed1997

Mouse connexin40: gene structure and promoter analysis.

A family of related connexin genes encodes the subunit gap junction proteins that form intercellular channels in different tissues. Connexin40 (Cx40) is one of these proteins, and it exhibits limited expression only in a few cells of the cardiovascular system. To begin to analyze Cx40 expression, we isolated a 3.3-kb rat Cx40 cDNA by hybridization screening of a bacteriophage library prepared from BWEM cells and isolated corresponding mouse genomic clones from a bacterial artificial chromosome library. Restriction mapping, sequencing, and comparison to the rat cDNA showed that the mouse Cx40 gene contained a short first exon, an 11.4-kb intron, and a second exon containing the complete coding region and 3'-UTR. Exon I contained only 1 base that differed between rat and mouse. Primer extension experiments yielded a single band and confirmed the position of the transcriptional start site. We obtained 1.2 kb of sequence 5' of the transcriptional start site and 400 bp 3' of exon I. Exon I was closely preceded by a consensus TATA box. The flanking sequences contained a number of potential transcription factor binding sites (including AP-1, AP-2, SP1, TRE, and p53). To identify transcriptional regulatory elements in the Cx40 promoter region, a series of DNA deletion fragments flanking exon I was prepared, subcloned adjacent to a luciferase reporter gene, and used for transient transfections of BWEM, SHM, and N2A cells. The resulting luciferase activity determinations suggested that an area of 300 bp 5' of the transcription start site acted as a basal promoter for Cx40 and that there was a strong negative regulatory element in the region from +100 to +297.

Animals↗

PubMed1982

Isolation and characterization of a cDNA coding for human factor IX.

A cDNA library prepared from human liver has been screened for factor IX (Christmas factor), a clotting factor that participates in the middle phase of blood coagulation. The library was screened with a single-stranded DNA prepared from enriched mRNA for baboon factor IX and a synthetic oligonucleotide mixture. A plasmid was identified that contained a cDNA insert of 1,466 base pairs coding for human factor IX. The insert is flanked by G-C tails of 11 and 18 base pairs at the 5' and 3' ends, respectively. It also included 138 base pairs that code for an amino-terminal leader sequence, 1,248 base pairs that code for the mature protein, a stop codon, and 48 base pairs of noncoding sequence at the 3' end. The leader sequence contains 46 amino acid residues, and it is proposed that this sequence includes both a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 1,248 base pairs code for a polypeptide chain composed of 416 amino acids. The amino-terminal region for this protein contains 12 glutamic acid residues that are converted to gamma-carboxyglutamic acid in the mature protein. These glutamic acid residues are coded for by both GAA and GAG. The arginyl peptide bonds that are cleaved in the conversion of human factor IX to factor IXa by factor XIa were identified as Arg145-Ala146 and Arg180-Val181. The cleavage of these two internal peptide bonds results in the formation of an activation peptide (35 amino acids) and factor IXa, a serine protease composed of a light chain (145 amino acids) and a heavy chain (236 amino acids), and these two chains are held together by a disulfide bond(s). The active site residues including histidine, aspartate, and serine are located in the heavy chain at positions 221, 270, and 366, respectively. These amino acids are homologous with His57, Asp102, and Ser195 in the active site of chymotrypsin. Two potential carbohydrate binding sites (Asn-X-Thr) were identified in the activation peptide, and these were located at Asn157 and Asn167. The homology in the amino acid sequence between human and bovine factor IX was found to be 83%.

Amino Acid Sequence↗

PubMed1985

Isolation of cDNA for a human granulocyte-macrophage colony-stimulating factor by functional expression in mammalian cells.

A cDNA sequence coding for a human granulocyte-macrophage colony-stimulating factor has been isolated from cDNA libraries prepared from mRNA derived from concanavalin A-activated human T-cell clones. The libraries constructed in the pcD vector system were screened by transfecting COS-7 monkey cells with DNA pools to express the products encoded by full-length cDNA inserts. By assaying the cell supernatants, we identified clones encoding a factor that stimulates the formation of granulocyte and macrophage colonies from human progenitor cells. These results demonstrate that identification of full-length cDNAs for many colony-stimulating factors may be achieved entirely on the basis of detecting the functional polypeptide produced in mammalian cells.

Amino Acid Sequence↗

PubMed1996

Multiplex selection technique (MuST): an approach to clone transcription factor binding sites.

We have used a multiplex selection approach to construct a library of DNA-protein interaction sites recognized by many of the DNA-binding proteins present in a cell type. An estimated minimum of two-thirds of the binding sites present in a library prepared from activated Jurkat T cells represent authentic transcription factor binding sites. We used the library for isolation of "optimal" binding site probes that facilitated cloning of a factor and to identify binding activities induced within 2 hr of activation of Jurkat cells. Since a large fraction of the oligonucleotides obtained appear to represent "optimal" binding sites for sequence-specific DNA-binding proteins, it is feasible to construct a catalog of consensus binding sites for DNA-binding proteins in a given cell type. Qualitative and quantitative comparisons of the catalogs of binding site sequences from various cell types could provide valuable insights into the process of differentiation acting at the level of transcriptional control.

Amino Acid Sequence↗

PubMed1985

Use of a cDNA expression vector for isolation of mouse interleukin 2 cDNA clones: expression of T-cell growth-factor activity after transfection of monkey cells.

A cDNA sequence coding for mouse interleukin 2 (IL-2) has been cloned from a cDNA library prepared from mRNA derived from a concanavalin A-activated mouse T-cell clone. The library was constructed by using the pcD vector system, which permits the expression of cDNA inserts in mammalian cells. Screening of the library was performed by transfecting COS-7 monkey cells with pools of cDNA clones in order to express the products encoded by full-length cDNA inserts. By assaying the supernatant fluid, IL-2 cDNA clones that express T-cell growth-factor (TCGF) activity were identified. The DNA sequence codes for a polypeptide of 169 amino acid residues including a putative signal peptide. The mouse IL-2 amino acid sequence deduced from the nucleotide sequence of its cDNA shares extensive homology with the human IL-2 amino acid sequence reported previously. These results demonstrate that identification of full-length cDNA clones for many lymphokines may be achieved entirely on the basis of detection of the functional polypeptides in mammalian cells.

Amino Acid Sequence↗

PubMed2001

Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens.

Molecular diversity of rumen archaea was analyzed by PCR amplification and sequencing of two 16S rRNA clone libraries prepared from the bovine rumen fluid using two different archaea-specific primer sets. The first library of 19 clones which was generated with primers D30 and D33, produced essentially two groups of sequences, one affiliated with Methanomicrobium mobile (21% of clones) and the other -- with the uncultured archaeal sequences from anaerobic digester, which are distantly associated with Thermoplasma (79% of clones). The second library of 25 clones, which was generated with primers 0025e Forward and 1492 Reverse, produced a higher degree of diversity: in addition to the previous two groups, with the M. mobile- (56%) and Thermoplasma-associated sequences (20%), four clones (16%) were identified as Methanobrevibacter spp. The remaining two sequences were associated with unidentified archaeal sequences from the rumen and swine waste. Phylogenetic placement of eight almost complete 16S rRNA sequences revealed the existence of a novel cluster of the rumen Euryarchaeota, which is not affiliated with the known methanogenic archaea.

Animals↗

PubMed1997

A novel delta-subdivision proteobacterial lineage from the lower ocean surface layer.

A small-subunit ribosomal RNA (16S rRNA) gene lineage (SAR324) affiliated with the delta-subdivision of the class Proteobacteria (DP) was discovered in a 16S rRNA gene clone library prepared from a water sample collected from 250 m in the western Sargasso Sea. This clone library of nearly full-length amplicons of bacterial 16S rRNA genes has been the subject of previous studies aimed at identifying bacteria that inhibit the lower ocean surface layer. The novel lineage was identified by randomly sequencing clones that did not hybridize to oligonucleotide probes specific for several abundant bacterioplankton groups identified in previous studies. Phylogenetic analysis indicated that SAR324 was most closely affiliated with the DP, although it showed no specific relationship to any DP 16S rRNA genes in databases. Eight of the clones in the library of 148 clones were identified as members of the SAR324 lineage by hybridization to an oligonucleotide probe specific for SAR324. Subsequent hybridizations showed that the SAR324 group is stratified in the lower surface layer of both the Atlantic and Pacific Oceans, with maxima between 160 and 500 m. The repeated discovery of sequences belonging to different gene clusters with similar distributions in this region of the water column suggests that microbial communities in the lower surface layer may be functionally specialized.

Atlantic Ocean↗

PubMed1999

Negative selection: a method for obtaining low-abundance cDNAs using high-density cDNA clone arrays.

The identification of the entire complement of genes expressed in a cell, tissue, or organism provides a framework for understanding biological properties and establishes a tool set for subsequent functional studies. The large-scale sequencing of randomly selected clones from cDNA libraries has been successfully employed as a method for identifying a large fraction of these expressed genes. However, this approach is limited by the inherent redundancy of cellular transcripts reflecting widely variant levels of gene transcription. As a result, a high percentage of transcript duplications are encountered as the number of sequenced clones accrues. To address this problem, we have developed a negative hybridization selection method that employs the hybridization of complex cDNA probes to high-density arrays of cDNA clones and the subsequent selection of clones with a null or low hybridization signal. This approach was applied to a cDNA library constructed from normal human prostate tissue and resulted in the reduction of highly expressed prostate cDNAs from 6.8 to 0.57% with an overall decline in clone redundancy from 33 to 11%. The selected clones also reflected a more diverse cDNA population, with 89% of the clones representing distinctly different cDNAs compared with 67% of the randomly selected clones. This method compares favorably with cDNA library re-association normalization approaches and offers several distinct advantages, including the flexibility to use previously prepared libraries, and the ability to employ an iterative screening approach for continued accrual of cDNAs representing rare transcripts.

DNA, Complementary↗

PubMed1989

Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.

Animals↗

PubMed1985

Isolation of mouse N-CAM-related cDNA: detection and cloning using monoclonal antibodies.

Clones coding for the mouse neural cell adhesion molecule (N-CAM) were isolated from a cDNA library prepared in the expression vector lambda gt 11 from mRNA extracted from a mouse neuroblastoma cell line. This library was screened with two anti-N-CAM monoclonal antibodies directed against different sites on the molecule and with rabbit anti-N-CAM serum. Two clones were identified with the first monoclonal antibody, three with the second one, none reacted with both. The relevance of these cDNA clones to N-CAM was confirmed by several observations. First, cDNA sequences detected with one monoclonal antibody cross-hybridized with those identified by the other antibody. Second, the different fusion proteins all bound the rabbit serum in addition to one monoclonal antibody. Finally, the probes hybridized to discrete mRNA species of sufficient lengths to code for the very large N-CAM polypeptides in RNA preparations from N-CAM-expressing, but not from N-CAM-negative cells. An additional mRNA species not seen in embryonic brain was expressed in adult mouse brain. Genomic blot experiments indicated that sequences corresponding to one of our probes are present only a few times in the mouse genome.

Animals↗

PubMedDate not supplied

Solution phase synthesis of libraries of polycyclic natural product analogues by cascade radical annulation: synthesis of a 64-member library of mappicine analogues and a 48-member library of mappicine ketone analogues.

An improved cascade radical annulation route to (+/-)-mappicine, (S)-mappicine, and mappicine ketone is reported. The route is used to prepare libraries of mappicine and mappicine ketone analogues in a semiautomated fashion. Key diversity generating steps include the addition of an aldehyde to a Grignard reagent derived from a D-ring iodopyridine, N-propargylation of a subsequently derived iodopyridone, and cascade radical annulation with an isonitrile to form a mappicine analogue. Parallel oxidation of mappicine analogues produced mappicine ketones. The route is general and flexible and could be used to make very large libraries. It is also illustrative of how late stage cascade reactions can be employed strategically to generate libraries of polycyclic natural product analogues.

Alkaloids↗

PubMed1998

Recombinant cDNA clones for immunodiagnosis of strongyloidiasis.

Because diagnosis of strongyloidiasis by stool examination is unreliable and because of the potential for serious disease in Strongyloides infections, there is need for improved diagnostic aids to facilitate recognition and treatment of this parasitic infection. Serologic testing, when available, requires antigen preparation from infected primates or dogs that can be difficult to maintain. Several recombinant clones from a cDNA library prepared from the infective stage of Strongyloides stercoralis were characterized. Serologic results indicate that the recombinant proteins were equally or more reactive than the larval somatic antigen. No cross-reactivity with recombinant antigen 5a was found with sera from patients with filarial or intestinal nematode infections. Recombinant antigens 5a and 12a detected parasite-specific IgE and IgG4 antibodies in Strongyloides-infected patients. Sequence analysis showed these antigens to be rich in proline and charged amino acids. Lack of homology from database searches suggests that the antigens are unique. These recombinant antigens should be useful in diagnostic and epidemiologic studies of strongyloidiasis.

Amino Acid Sequence↗

PubMed2000

Aristolochene synthase: purification, molecular cloning, high-level expression in Escherichia coli, and characterization of the Aspergillus terreus cyclase.

Aristolochene synthase catalyzes the cyclization of farnesyl diphosphate (6) to (+)-aristolochene (1). The Aspergillus terreus enzyme has been purified 75-fold to homogeneity in six steps. Based on the sequence of 3 internal peptides obtained by Lys-C digestion of the native protein, a set of degenerate PCR primers was used to amplify a 550-bp segment of cDNA corresponding to a portion of the aristolochene synthase transcript. A second round of PCR using specific primers was used to prepare a (32)P-labeled 180-bp segment, which was used to screen an A. terreus cDNA library prepared using lambdaZapII, resulting in the identification and sequencing of the A. terreus aristolochene synthase cDNA. Aristolochene synthase was encoded by an open reading frame (ORF) of 960 bp, corresponding to a protein of 320 amino acids with a predicted M(D) of 36,480. Comparison of the A. terreus ORF with the sequence of the previously described aristolochene synthase from Penicillium roqueforti revealed a 66% of identity at the nucleic acid level and a 70% identity at the deduced amino acid level between the aristolochene synthases from the two different fungal sources. PCR was used to insert the A. terreus aristolochene synthase gene into the T7lac expression vector pET11a. Cloning of the resultant construct into Escherichia coli XL1-Blue and subcloning into the expression host E. coli BL21(DE3)/pLysS gave, after induction with IPTG, soluble aristolochene synthase as 5-10% of total protein. The recombinant aristolochene synthase, which was purified 13-fold to homogeneity, appeared to be identical in all respects with the native A. terreus enzyme, displaying essentially the same steady-state kinetic parameters, with a K(m) of 15 nM and k(cat) 0.015 s(-1). Using PCR to amplify the aristolochene synthase gene (Aril) from A. terreus genomic DNA revealed the presence of 2 introns, identical in relative location but different in both sequence and length compared to the corresponding Ari1 gene of P. roqueforti.

Amino Acid Sequence↗

PubMed2001

Cloning and expression of a down-regulated gene (TrEnodDR1) of white clover responded by the nod genes derived from Rhizobium leguminosarum bv. trifolii strain 4S.

The nodulation genes of Rhizobium leguminosarum bv. trifolii 4S (strain 4S) were cloned into cosmid vector pLAFR1 named pC4S8 which was contained nodNMLFEDABCIJ and a part of nodT as an insert. The pC4S8 was transferred to strain H1, Sym plasmid (pRt4Sa) cured strain of strain 4S, and isolated as Tc resistant and nodulation restored mutant, strain H1(pC4S8). During infection process of this strain, visible symbiotic features, such as root hair curling (Hac), root hair deformation (Had) and infection thread formation (Inf) were also restored. The nodule forming ability of strain H1(pC4S8) was increased 3-4 times in nodule number than that of strain 4S. Then, to investigate the effect of Rhizobium nod genes on the host plant (Trifolium repens L.) gene expression, cDNAs which were responded to the inoculation of rhizobia were differentially screened based on the presence or absence of nod genes treated with strains H1(pC4S8) or H1, respectively. The cDNA, TrEnodDR1 (Trifolium repens early nodulin down regulation 1) gene was isolated from cDNA library prepared from white clover seedlings treated with nod- strain H1, but didn't exhibit in nod+ treated cDNA library, as a down-regulated gene. Expression analysis of TrEnodDR1 was performed in various tissues of white clover, it is suppressed in root nodule and also strongly suppressed by the inoculation of rhizobia in the seedlings. It is discussed that TrEnodDR1 gene is suppressed when the white clover comes into symbiosis with rhizobia.

Amino Acid Sequence↗