Complementary DNA

About: Complementary DNA is a research topic. Over the lifetime, 55301 publications have been published within this topic receiving 2752650 citations. The topic is also known as: cDNA & DNA, Complementary.

Molecular cloning and characterization of an insulin-regulatable glucose transporter.

Abstract: A MAJOR mechanism by which insulin stimulates glucose transport in muscle and fat is the translocation of glucose transporters from an intracellular membrane pool to the cell surface1–5. The existence of a distinct insulin-regulatable glucose transporter was suggested by the poor cross-reactivity between antibodies specific for either the HepG2 or rat brain glucose transporters and the rat adipocyte glucose transporter6,7. More direct evidence was provided by the production of a monoclonal antibody (mAb 1F8) specific for the rat adipocyte glucose transporter that immunolabels a species of relative molecular mass 43,000 (43K) present only in tissues that exhibit insulin-dependent glucose transport8, suggesting that this protein may be encoded by a different gene from the previously described mammalian glucose transporters9–13. This antibody has been used to immunoprecipitate a 43K protein that was photoaffinity-labelled with cytochalasin B in a glucose displaceable way, and to immunolabel a protein in the plasma membrane of rat adipocytes, whose concentration was increased at least fivefold after cellular insulin exposure. Here we describe the cloning and sequencing of cDNAs isolated from both rat adipocyte and heart libraries that encode a protein recognized by mAb 1F8, and which has 65% sequence identity to the human HepG2 glucose transporter9. This cDNA hybridizes to an mRNA present only in skeletal muscle, heart and adipose tissue. Our data indicate that this cDNA encodes a membrane protein with the characteristics of the translocatable glucose transporter expressed in insulin-responsive tissues.

Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification

Abstract: Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine methylation to methionine. MTHFR deficiency, an autosomal recessive disorder, results in homocysteinemia. Using degenerate oligonucleotides based on porcine peptide sequence data, we isolated a 90-bp cDNA by PCR from pig liver RNA. This cDNA was used to isolate a human cDNA, the predicted amino acid sequence of which shows strong homology to porcine MTHFR and to bacterial metF genes. The human gene has been localized to chromosome 1p36.3. Two mutations were identified in MTHFR-deficient patients: a missense mutation (Arg to Gln), in a residue conserved in bacterial enzymes, and a nonsense mutation (Arg to Ter).

Existence of distinct sodium channel messenger RNAs in rat brain

Abstract: The sodium channel is a voltage-gated ionic channel essential for the generation of action potentials1–3. It has been reported that the sodium channels purified from the electric organ of Electrophorus electricus (electric eel)4,5 and from chick cardiac muscle6 consist of a single polypeptide of relative molecular mass (Mr) ∼260,000 (260K), whereas those purified from rat brain7 and skeletal muscle8 contain, in addition to the large polypeptide, two or three smaller polypeptides of Mr 37–45K. Recently, we have elucidated the primary structure of the Electrophorus sodium channel by cloning and sequencing the DNA complementary to its messenger RNA9. Despite the apparent homogeneity of the purified sodium channel preparations, several types of tetrodotoxin (or saxitoxin) binding sites or sodium currents have been observed in many excitable membranes10–19. The occurrence of distinguishable populations of sodium channels may be attributable to different states of the same channel protein or to distinct channel proteins. We have now isolated complementary DNA clones derived from two distinct rat brain mRNAs encoding sodium channel large polypeptides and present here the complete amino-acid sequences of the two polypeptides (designated sodium channels I and II), as deduced from the cDNA sequences. A partial DNA sequence complementary to a third homologous mRNA from rat brain has also been cloned.