Cloning a cDNA for the pro-alpha 2 chain of human type I collagen.
AUTOR(ES)
Myers, J C
RESUMO
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 S1 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 HB101. 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 EcoRII digestion. One clone with an insert of 2.2 kilobases was shown to contain sequences encoding for the pro-alpha 2 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 alpha 2 chain to the middle of the COOH-terminal propeptide.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=319600Documentos Relacionados
- An RFLP in the gene for the human pro-alpha 2 chain of type V collagen (COL5A2).
- Analysis of cDNA and genomic clones coding for the pro alpha 1 chain of calf type II collagen.
- Endonuclease S1-sensitive site in chicken pro-alpha 2(I) collagen 5' flanking gene region.
- Human pro alpha 1(I) collagen: cDNA sequence for the C-propeptide domain.
- Marfan syndrome: abnormal alpha 2 chain in type I collagen.