Cloning a cDNA for the pro-alpha 2 chain of human type I collagen.

Abstract

Poly(A)-RNA enriched for type I procollagen sequences was isolated from normal human fibroblasts and used as template to synthesize double-stranded cDNA with avian myeloblastosis virus (AMV) reverse transcriptase. After the ends had been blunted with nuclease SI and dGMP tails had been added with terminal deoxynucleotidyltransferase, the double-stranded cDNA was annealed with pBR322 DNA that had previously been cleaved with EcoRI, blunted with AMV reverse transcriptase, and dCMP-tailed with terminal deoxynucleotidyltransferase. The chimeric molecule was used to transform Escherichia coli strain HB1O1. Ninety-five recombinant clones were obtained and screened by dot hybridization analysis using 32P-labeled cDNA synthesized from the original poly(A)-RNA collagen-enriched population. Three positive clones were isolated and further characterized by blot hybridization techniques and by EcoRil digestion. One clone with an insert of 2.2 kilobases was shown to contain sequences encoding for the pro-a2 chain of human type I procollagen. DNA sequence analysis of a 172-nucleotide fragment demonstrated that the cloned cDNA extends from amino acid position 450 of the a2 chain to the middle of the COOH-terminal propeptide.Collagen represents a major class of proteins in mammals and plays a fundamental role in the structure and function of most connective tissues (1, 2). Collagen is synthesized from procollagen, a precursor composed ofthree pro-a chains. At least five genetically and biochemically distinct types of collagen are found in different tissues, and they have a characteristic developmental distribution (1-3). Type I collagen, [al(I)]2a2, is present in bones, skin, tendons, and lungs, and it is believed to be directly or indirectly involved in inherited diseases of the connective tissue in man such as some forms of osteogenesis imperfecta (1). Although a great deal of information is available about posttranslational events leading to the maturation of the human procollagen chains, very little is known about the structure, the genetics, and the mode of expression of this family of genes in man.In the past few years recombinant DNA techniques have proved to be a powerful tool for enhancing our understanding ofthe structure ofeukaryotic genes and their altered phenotypic expression, as exemplified by the human globin genes (4). Recently cloned cDNAs for type I procollagen from chicken have been prepared (5-7), and the entire chicken pro-a2 gene (8)(9)(10) MATERIALS AND METHODS Materials. Reverse transcriptase (RNA-dependent DNA polymerase) from avian myeloblastosis virus (AMV) was purified (13) from plasma kindly supplied by Joseph Beard (Life Sciences, Gulfport, FL). Nuclease S1 was purchased from Miles. Calf thymus terminal deoxynucleotidyltransferase was a generous gift from Sidney Pestka (Roche Institute of Molecular Biology). Rabbit reticulocyte lysate was purchased from Amersham. Restriction enzymes were purchased from New England BioLabs and were used according to the supplier's recommendations. Nitrocel...

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Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-β involves functional cooperation with p300/CBP transcriptional coactivators

“…Equal amounts of total RNA (15 mg) were separated in 1% formaldehyde agarose gels and transferred to nitrocellulose ®lters. Filters were hybridized with radiolabeled human COL1A2 (Myers et al, 1981), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probes, washed and exposed for 1 day.…”

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