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At least 145 records · Page 8Linked to original sources
PubMed1989

Isolation and characterization of human blood platelet mRNA and construction of a cDNA library in lambda gt11. Confirmation of the platelet derivation by identification of GPIb coding mRNA and cloning of a GPIb coding cDNA insert.

We have developed a purification method for the isolation of platelet-specific poly (A+) RNA and demonstrated that human blood platelets, despite the absence of a nucleus, contain stable mRNA. The poly (A+) RNA was used to construct a platelet-specific cDNA expression library in lambda gt11. The platelet derivation of the purified mRNA was confirmed by identification of membrane glycoprotein Ib (GPIb) message by immunoprecipitation of rabbit reticulocyte lysate translation products with poly- and monoclonal antibodies against GPIb alpha and by sequencing of a GPIb alpha cDNA clone.

Amino Acid Sequence↗
PubMed1993

Anchor-ligated cDNA libraries: a technique for generating a cDNA library for the immediate cloning of the 5' ends of mRNAs.

A new, efficient and simple method based on single-stranded anchor ligation has been developed that allows for the generation of an anchor-ligated library from which the 5' ends in many mRNAs can be cloned. Random hexamers are used to prime poly A+ RNA for the first strand synthesis of a cDNA library, and a modified single-stranded anchor is then ligated to the 3' end of the cDNA using T4 RNA ligase. The library can then be used to amplify the 5' end of any mRNA expressed in the tissue. The efficiency and utility of this method has been demonstrated by amplification of three human genes: transferrin receptor, tissue-type plasminogen activator and beta actin.

Actins↗
PubMed1996

[Expression of cDNA of human chorionic gonadotropin beta-subunit (beta-hCG) cDNA in insect cells and effect of expressed product on mouse lymphocytes in vitro].

Expression vector pVL 1393-hCG beta containing beta-hCG cDNA has been constructed using an unfused protein nuclear polyhedrosis virus (AcNPV) expression vector. The insect cells (Sf 9) were cotransfected by the expression vector and nuclear polyhedrosis virus genomic DNA, and recombinant virus AcNPV-hCG beta was screened out, beta-hCG cDNA was expressed in insect cells infected by recombinant virus and recombinant beta-hCG (r beta-hCG) was secreted into medium. The purity of r beta-hCG, purified by immuno-affinity chromatography, was about 90% and the molecular weight of r beta-hCG was 22,500 Da. Like hCG, r beta-hCG suppressed significantly proliferation of induced lymphocytes, as well as production of IL-2 to some extent, on a parallel with suppression of lymphoproliferation.

Animals↗
PubMed1998

cDNA of eight nuclear encoded subunits of NADH:ubiquinone oxidoreductase: human complex I cDNA characterization completed.

NADH:ubiquinone oxidoreductase (complex I) is an extremely complicated multiprotein complex located in the inner mitochondrial membrane. Its main function is the transport of electrons from NADH to ubiquinone, which is accompanied by translocation of protons from the mitochondrial matrix to the intermembrane space. Human complex I appears to consist of 41 subunits of which 34 are encoded by nDNA. Here we report the cDNA sequences of the hitherto uncharacterized 8 nuclear encoded subunits, all located within the hydrophobic protein (HP) fraction of complex I. Now all currently known 41 proteins of human NADH:ubiquinone oxidoreductase have been characterized and reported in literature, which enables more complete mutational analysis studies of isolated complex I-deficient patients.

Adult↗
PubMed1992

Isolation of cDNA sequences around the chromosomal breakpoint in a female with Lowe syndrome by direct screening of cDNA libraries with yeast artificial chromosomes.

The Lowe oculocerebrorenal syndrome (OCRL; McKusick 309000) is an X-linked disorder characterized by congenital cataracts, muscular hypotonia, mental retardation, and Fanconi syndrome of the renal tubules. A pair of yeast artificial chromosomes (YACs) that span the Xq25-q26 translocation breakpoint in a female with OCRL were used as probes to screen cDNA libraries made from bovine lens and human kidney. The methods used to prepare the YACs as probes and to screen the libraries are presented in detail. Two different transcripts were found that map to the region around the Xq25-q26 breakpoint. These transcripts are now being studied to determine whether one or the other is a candidate gene for OCRL.

Blotting, Southern↗
PubMed2000

Molecular cloning of a cysteine proteinase cDNA from adult Ancylostoma ceylanicum by the method of rapid amplification of cDNA ends using polymerase chain reaction.

The hookworm Ancylostoma ceylanicum is a parasite of great importance in human and veterinary medicine. The most promising vaccination trials against hookworm infections are based on antigens belonging to the proteinase family. The aim of the present research was to isolate a cysteine proteinase gene from A. ceylanicum. This was achieved by rapid amplification of cDNA ends using polymerase chain reaction (RACE-PCR). A set of consensus oligonucleotide primers was designed to anneal to the conserved coding regions of cysteine proteinase. The PCR products were cloned and sequenced. The novel sequence displayed a high degree of homology with genes of cysteine proteinases known from other hookworm species. In the coding region the nucleotide identity with accp-1, the cysteine proteinase gene of A. caninum, reaches 84.3%. Analysis of the expression of acey-1. the cysteine proteinase gene of A. ceylanicum, suggests that it is produced exclusively in the gland cells of either adult worms or blood-feeding stages of A. ceylanicum.

Amino Acid Sequence↗
PubMed1994

The Schizosaccharomyces pombe rad1 gene consists of three exons and the cDNA sequence is partially homologous to the Ustilago maydis REC1 cDNA.

We show that the rad1 gene of Schizosaccharomyces pombe is comprised of three exons and encodes a protein of 37 kDa. A cDNA clone containing these three exons complements the sensitivity of the rad1-1 mutant to ultraviolet and gamma-radiation and to hydroxyurea. The newly identified ORF of the rad1 gene was found to exhibit partial homology to the REC1 gene of Ustilago maydis. These two genes share putative functional similarities in their respective organisms.

Amino Acid Sequence↗
PubMedDate not supplied

Improvements in production of cDNA libraries in lambda gtll. Detection of ligation inhibitor and separation of cDNA from free linkers.

A ligation assay was designed to determine if a ligation inhibitor is present in a Bio-Gel A-50-m column, the traditional tool used for separation of cDNA from free linkers during the lambda gtll cloning procedure. A new and simple electrophoresis method is also presented for performing this separation, which is rapid, efficient, and reduces the chance of carrying along a ligation inhibitor.

Chromatography, Gel↗
PubMed1993

Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library.

A human infant brain cDNA library, made specifically for production of expressed sequence tags (ESTs) was evaluated by partial sequencing of over 1,600 clones. Advantages of this library, constructed for EST sequencing, include the use of directional cloning, size selection, very low numbers of mitochondrial and ribosomal transcripts, short polyA tails, few non-recombinants and a broad representation of transcripts. 37% of the clones were identified, based on matches to over 320 different genes in the public databases. Of these, two proteins similar to the Alzheimer's disease amyloid precursor protein were identified.

Amino Acid Sequence↗
PubMed1987

Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA.

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).

Amino Acid Sequence↗
PubMed1997

Characterization of cDNA clones in size-fractionated cDNA libraries from human brain.

To evaluate the size-fractionated cDNA libraries of human brain previously constructed (O. O'hara et al. DNA Research, 4, 53-59, 1997), the occurrence of chimeric clones and the content of clones with coding potentiality were analyzed using the randomly sampled clones with insert sizes of 5 to 7 kb. When the chromosomal location of 30 clones was determined by the radiation-hybrid mapping method, the map positions assigned from the 3'- and 5'-end sequences separately were coincident for 29 clones, suggesting that the occurrence of chimeric clones is at most 1/30. Using 91 clones mapped to chromosome 1, the content of clones that have the potentiality coding for proteins larger than 100 amino acid residues was estimated to be approximately 50% (46 out of 91 clones) on the basis of nucleotide sequence analysis and coding potentiality assay in vitro. No significant open reading frames were detected in the remaining clones. Although the clones coding for short peptides may not have been included in the above estimation, the libraries constructed from the whole brain mRNA fraction appear to contain a considerable amount of clones corresponding to the 5'-truncated transcripts in an unprocessed form and/or those with long 3'-untranslated regions.

Animals↗
PubMed2000

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.

Arabidopsis↗
PubMed1992

Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function.

Molecular events associated with the evolution of an enzyme for C4 photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4.1.1.31] are known: the C4-form, the C2-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I.P. & Osmond, C.B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C3-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C3-form and C4-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C4-form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C4-form and C3-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2 x 10(8) yr ago), though the divergence of maize (C4 plant) from wheat (C3 plant) is supposed to have occurred much later (6 x 10(7) yr ago).

Amino Acid Sequence↗
PubMed1996

Rescue of Sendai virus cDNA templates with cDNA clones expressing parainfluenza virus type 3 N, P and L proteins.

Several years ago, we reported that a Sendai virus (SeV) defective genome (DIH4UV) could be rescued in vivo with human parainfluenza virus type 1 (hPIV1) and bovine PIV3 but not by measles virus or vesicular stomatitis virus. It was concluded that the cis-acting RNA sequences were conserved within the SeV/PIV1/PIV3 group but that interactions between the polymerase complex (P-L) and the template protein N were unique for each virus. We have re-examined these conclusions using proteins expressed from cloned N, P and L genes for SeV and PIV3. The results demonstrate the specificity of the protein-protein interactions between polymerase and template, and confirm the prediction of the earlier work that PIV3 N, P and L proteins are capable of assembling and replicating SeV mini-genomes also expressed from a cDNA clone.

Animals↗
PubMed1993

A majority of Ig H chain cDNA of normal human adult blood lymphocytes resembles cDNA for fetal Ig and natural autoantibodies.

Certain Ig VH gene segments, with few or no mutations, recur frequently in natural autoantibodies, fetal antibodies, and products of B cell tumors. The goal of this study was to determine whether similar Ig gene segment usage occurs in normal human adult PBL. Extending previous analyses, 105 randomly picked H chain V region clones of representative cDNA libraries from PBL were sequenced. Clones were from: IgM and IgG libraries from one RNA sample of a normal adult; a second IgM library from the same subject 11 mo later; and one IgM library from a second subject. Although some clones had clear evidence of mutation, 48 of 77 IgM clones (62%) shared 99% or more identity with known germline VH segments, and most of these had no mutations in the CDR3 portion of the JH segment. Certain VH gene segments, expressed in autoantibodies and fetal antibodies, occurred at high frequency in these libraries. Fourteen of the clones with 99% identity to known VH segments had CDR3 segments identical to portions of known germline DH gene sequences; two such clones had no N nucleotides at the VHDH or DHJH junctions. IgG-encoding sequences had more mutations than IgM-encoding sequences. JH and DH usage was not random. The circulating B cell population may represent a distinct compartment, with a large proportion of cells similar to those of the fetal and natural autoantibody repertoire. Polyreactive Ig products of these circulating cells may serve a screening function, binding and delivering diverse Ag to secondary lymphoid tissues where more highly selective antibodies are formed to foreign or self-Ag.

Adult↗