title: Structure of cDNAs encoding the triple-helical domain of murine alpha 2 (VI) collagen chain and comparison to human and chick homologues. Use of polymerase chain reaction and partially degenerate oligonucleotide for generation of novel cDNA clones.
abstract: Type VI collagen cDNAs of human and avian origin were recently obtained and characterized by screening cDNA libraries in lambda phage. Based on the published sequences of these cDNAs, we constructed partially degenerate oligonucleotide primers that we used in polymerase chain reactions for the generation of alpha 2(VI) collagen clones of murine origin. As template, we used cDNA derived from murine total RNA. We amplified, cloned and sequenced a 1043-bp fragment that contains the coding sequence for the entire triple-helical domain except for the first two amino acids that are Gly-Pro in both man and chicken. Comparison of the nucleotide and derived amino acid sequences revealed 84.6% and 92.5% identity between mouse and man at the DNA and protein levels, respectively. Comparison with chicken sequences showed 72% and 79.1% identity. The third base usage showed a distinct preference for A in the glycine codons for the three species; whereas, U is preferred in all human fibrillar collagen genes previously defined. The preference for third base codon in Y position prolines is U for the alpha 2(VI) collagen as it is for the human fibrillar collagen genes. A single cysteine at position 89, two Arg-Gly-Asp sequences, and one triple helix-interruption are conserved in mouse, man and chicken. Comparison of hydropathy plots showed great similarity between those of murine and human alpha 2(VI) collagen chains and to a lesser extent between murine and chick. Northern blot hybridization of murine poly A+ RNA with a nick-translated radiolabeled alpha 2(VI) collagen probe detected one major transcript of 3.7 kb.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Medicine Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
authors.forename: C D
authors.initials: CD
authors.lastname: Constantinou
authors.forename: S A
authors.initials: SA
authors.lastname: Jimenez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D003094:Collagen / Q000235:genetics*; D004247:DNA / Q000235:genetics; D005347:Fibroblasts / Q000502:physiology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012879:Skin Physiological Phenomena
chemical_list: D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D003094:Collagen; D004247:DNA