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.

Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor.

Abstract: Human granulocyte/macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is essential for the in vitro proliferation and differentiation of precursor cells into mature granulocytes and macrophages. In this report we have used a mouse GM-CSF cDNA clone to isolate human GM-CSF clones from libraries made from HUT-102 messenger RNA and mitogen-stimulated T-lymphocyte messenger RNA. The human cDNA clones contained a single open-reading frame encoding a protein of 144 amino acids with a predicted molecular mass of 16,293 daltons and showed 69% nucleotide homology and 54% amino acid homology to mouse GM-CSF. One of these cDNA clones was shown to direct the synthesis of biologically active GM-CSF using a yeast expression system. The gene for human GM-CSF appears to exist as a single-copy gene.

Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA.

Abstract: We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were produced In the case of the TMV-CAT chimeras, however, the lesions were small compared to those of wild-type TMV and those produced by transcript derived from the coatless construct Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA The TMV-CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host CAT activity increased at least until 2 weeks post-inoculation and was approximately 01 units/mg of tissue at 10 days post-inoculation Thus, TMV-RNA may be utilized as a new plant expression vector

The nucleotide sequence of the gene for human protein C.

Abstract: A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping lambda Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity.

Abstract: Objective To determine the existence of mutant and variant CYP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression. Methods A bacterial artificial chromosome that contains the complete CYP3A4 gene was isolated and the exons and surrounding introns were directly sequenced to develop primers to polymerase chain reaction (PCR) amplify and sequence the gene from lymphocyte DNA. DNA samples from Chinese, black, and white subjects were screened. Mutating the affected amino acid in the wild-type cDNA and expressing the variant enzyme with use of the baculovirus system was used to functionally evaluate the variant allele having a missense mutation. Results To investigate the existence of mutant and variant CYP3A4 alleles in humans, all 13 exons and the 5′-flanking region of the human CYP3A4 gene in three racial groups were sequenced and four alleles were identified. An AG point mutation in the 5′-flanking region of the human CYP3A4 gene, designated CYP3A4*1B, was found in the three different racial groups. The frequency of this allele in a white population was 4.2%, whereas it was 66.7% in black subjects. The CYP3A4*1B allele was not found in Chinese subjects. A second variant allele, designated CYP3A4*2, having a Ser222Pro change, was found at a frequency of 2.7% in the white population and was absent in the black subjects and Chinese subjects analyzed. Baculovirus-directed cDNA expression revealed that the CYP3A4*2 P450 had a lower intrinsic clearance for the CYP3A4 substrate nifedipine compared with the wild-type enzyme but was not significantly different from the wild-type enzyme for testosterone 6β-hydroxylation. Another rare allele, designated CYP3A4*3, was found in a single Chinese subject who had a Met445Thr change in the conserved heme-binding region of the P450. Conclusions These are the first examples of potential function polymorphisms resulting from missense mutations in the CYP3A4 gene. The CYP3A4*2 allele was found to encode a P450 with substratedependent altered kinetics compared with the wild-type P450. Clinical Pharmacology & Therapeutics (2000) 67, 48–56; doi: 10.1067/mcp.2000.104391

Induction of endothelin-1 gene by angiotensin and vasopressin in endothelial cells.

Abstract: To elucidate the cellular mechanism of endothelin-1 biosynthesis induced by angiotensin and vasopressin, we first cloned and sequenced full-length bovine preproendothelin-1 complementary DNA (cDNA) from a cultured bovine carotid artery endothelial cell cDNA library. The predicted bovine preproendothelin-1 consists of 202 amino acid residues and has a high percentage of homology to human, porcine, and rat preproendothelin-1 (70%, 81%, and 77%, respectively). Big endothelin-1, an intermediate form, consists of 39 residues differing only at position Val28 from porcine (Ile28) and His27 from rat (Arg27). The predicted 21-residue mature endothelin-1 is identical to human, porcine, rat, canine, and mouse endothelin-1. Northern blot analysis with the cloned cDNA as a probe demonstrated that a single 2.3-kb preproendothelin-1 messenger RNA (mRNA) is expressed not only in endothelial cells, but also in various bovine tissues, including lung, brain, heart, intestine, kidney, ovary, and urinary bladder. Angiotensin II and arginine vasopressin immediately and dose-dependently induced expression of preproendothelin-1 mRNA, whose effects were abolished by specific receptor antagonists. These findings suggest that stimulation of endothelin-1 secretion from endothelial cells by both agonists may be principally due to induction of preproendothelin-1 mRNA.