Construction of a full-length cDNA encoding human pro-alpha 2(I) collagen and its expression in pro-alpha 2(I)-deficient W8 rat cells.
We have constructed a cDNA encoding the entire human pro-alpha 2(I) collagen molecule. Sequence determination for 2196 base pairs at the 5' end of the cDNA clone, and comparison with previously characterized human alpha 2(I) sequences, identified a number of nucleotide and amino acid polymorphisms. Functionality of the cDNA clone, under control of the long terminal repeat of Rous sarcoma virus, was demonstrated by its introduction into the W8 cell line. The W8 line, a chemically transformed variant of K16 rat liver epithelial cells, has been previously shown to lack detectable levels of alpha 2(I) RNA, but to secrete alpha 1(I) homotrimers. Introduction of the human cDNA into W8 cells, resulted in secretion of chimeric type I collagen comprised of rat alpha 1(I) and human alpha 2(I) chains. Availability of a functional full-length clone of human alpha 2(I) cDNA, combined with the W8 cell line as expression system, will allow detailed analysis, through site-directed mutagenesis, of domains on the pro-alpha 2(I) molecule involved in assembly, transport, secretion, and fibrillogenesis.
Lee
S T
ST
Department of Pathology, University of Wisconsin, Madison 53706.
Smith
B D
BD
Greenspan
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D004247:DNA / Q000032:analysis*; D004720:Endonucleases / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D011347:Procollagen / Q000235:genetics*; D011392:Proline / Q000378:metabolism; D051381:Rats; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014162:Transfection
D011347:Procollagen; D004247:DNA; D011392:Proline; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases