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 of a Novel Membrane-type Matrix Metalloproteinase from a Human Breast Carcinoma

Abstract: A new member of the matrix metalloproteinase (MMP) family of enzymes has been cloned from a human breast carcinoma cDNA library. The isolated cDNA contains on open reading frame 1554 bp long, encoding a polypeptide of 518 amino acids. The predicted amino acid sequence displays a similar domain organization as the remaining MMPs, including a prodomain with the activation locus, the zinc-binding site, and the hemopexin domain. In addition, it contains a C-terminal extension, rich in hydrophobic residues and similar in size to those present in the different membrane-type MMPs (MT-MMPs) identified to date. On the basis of these structural characteristics, this novel MMP has been tentatively called MT4-MMP, because it represents the fourth member of this subclass of MMPs characterized mainly by the occurrence of putative transmembrane domain in their amino acid sequences. MT4-MMP also contains a nine-residue insertion between the propeptide and the catalytic domain, which is a common feature of MT-MMPs and stromelysin-3. This amino acid sequence insertion ends with the consensus sequences R-X-R/K-R, which seems to be essential in the activation of these proteinases by furin. Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that the MT4-MMP gene ( MMP-17 ) is expressed mainly in the brain, leukocytes, the colon, the ovary, and the testis. The expression of MT4-MMP in leukocytes together with its putative membrane localization suggest that this enzyme could be involved in the activation of membrane-bound precursors of growth factors or inflammatory mediators such as tumor necrosis factor-α. In addition, MT4-MMP transcripts were detected in all breast carcinomas, as well as in all breast cancer cell lines analyzed in the present work. On the basis of these expression data in breast tumors, a potential role for human MT4-MMP in the tumoral process is also suggested.

Expression and characterization of the cystic fibrosis transmembrane conductance regulator.

Abstract: Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.