CLONING AND CHARACTERIZATION OF THE FULL-LENGTH CDNA-ENCODING ALPHA-2TYPE-I COLLAGEN OF BULLFROG RANA-CATESBEIANA

The present study determined nucleotide sequences of the full-length c DNA of alpha 2 chain of bullfrog type I collagen. Hybridization of a b ullfrog cDNA library with human alpha 1 type I collagen cDNA yielded a clone named 6A-1 which was 3449 bp long and lacked a 5' region of the gene. A 5' region containing the translation initiation site was ampl ified by the reverse transcription polymerase chain reaction using pol y(A)(+) RNA from tadpole tail tissues as template, and oligonucleotide s encoding the translation initiation region of mammalian fibrillar co llagens and the Gly-X-Y repeat region of clone 6A-1 as primers. As a r esult we obtained a 1518 bp long clone Y31. A 355 bp long clone Y31-9 was produced by extending clone Y31 from its ATG codon to a 127 bp ups tream region. Combining these three clones, the complete nucleotide se quence of the full-length cDNA was determined which contained 4692 bp as a whole and 4065 bp in the open reading frame. The comparison of it s structure with known collagen cDNAs of various vertebrates showed th at the cDNA obtained codes for alpha 2(I) chain of bullfrog. Its deduc ed amino acid sequence revealed the complete conservation of seven cys teine residues in the C-propeptide and three lysine residues in the N- telopeptide through the helical domain. Northern blot analysis reveale d that the thyroid hormone regulated the expression of alpha 2(I) coll agen in an organ-dependent manner: intense up-regulation in the back s kin and intestine, weak and transient up-regulation in the liver, and initial down-regulation, but later up-regulation in the tail. Prolacti n increased its expression in both the back skin and tail. These resul ts suggested that the expression of bullfrog alpha 2(I) collagen is co operatively regulated by these two metamorphosis-regulating hormones. (C) 1997 Elsevier Science B.V.