Isolation of cDNA clones and genomic dna clones of β-subunit of chicken prolyl 4-hydroxylase

Abstract

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, α and β. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8– and pUC9. Antibodies identified twenty-five clones among the approximately 2 × 10⁵ clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against β subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9–10B encodes a portion of β-subunit. The cDNA from CPH 9–10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH₂-terminus of β-subunit and two CNBr peptides derived from β-subunit. The cDNA of CPH 9–10B was also used to screen a genomic DNA library constructed with λ EMBL-3. Two overlapping genomic clones λgCPH β-22 and β-50 were isolated and characterized by restriction enzyme analysis. The results indicate that λgCPH β-22 contains the portion of the β-subunit gene that is transcribed into the 5’ portion of β-subunit mRNA, whereas λgCPH β-50 contains the 3’ portion.

Key Words:

• Prolylhydroxylase
• cDNA
• collagen biosynthesis
• hydroxylation genomic DNA sequences