Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase.
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, alpha and beta. 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 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta 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 beta-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 beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.
Kao
W W
WW
Department of Ophthalmology, University of Cincinnati College of Medicine, Ohio 45267.
Nakazawa
M
M
Aida
T
T
Everson
W V
WV
Kao
C W
CW
Seyer
J M
JM
Hughes
S H
SH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005786:Gene Expression Regulation; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D013045:Species Specificity
D007106:Immune Sera; D012333:RNA, Messenger; D004247:DNA; D011393:Procollagen-Proline Dioxygenase