Human proα1(I) collagen: cDNA sequence for the C-propeptide domain

We have previously constructed a cDNA clone pHCALl, covering most of the Cterminal propeptide domain of human proal(I) collagen mSNA, by inserting a 678 bp EcoRI-XhoI fragment of cDNA into pBR322 (1). Since the Xhol/Sall ligation prevented removal of the insert, we used the same strategy to obtain a similar clone in pUC8. RNA was isolated from fetal calvarial bones. The cDNA was digested with EcoRI and Xhol and fractionated on a 1 X agarose gel. Fragments of 650-700 bp were cloned in pUC8 at the polylinker site, which now permits easy removal of the insert (2). The new clone was named pHCALIU since the RNA was isolated from another individual. Sequencing of pHCALlU by the Sanger dideoxy method filled the two gaps (nucleotides 67 and 487) which remained in the previously published sequence (3). Three base differences were observed (shown above pHCALIU sequence): two of these bordered the missing nucleotide (positions 66 and 68). The third difference (a transversion at nucleotide 616) changes the codon from TCC (ser, ref. 3) to ACC (thr), and may represent individual variation. The corresponding chick sequence also contains ACC (4). The approach outlined here is useful for studies on individual variation which is important to recognize when searching for disease-related mutations in type I collagen.