Sequence comparison of three mammalian type-X collagen promoters and preliminary functional analysis of the human promoter.
The mechanism(s) controlling the specific expression of the type-X collagen (COL10A1)-encoding gene in the growth plate of developing long bones is not known. In preparation for identifying and characterizing the 5'-regulatory sequences and transcription factors which control mammalian Col10a1 gene expression, we have isolated and sequenced the first exon and 5' flanking promoter regions of bovine Col10a1. Sequence comparisons, including those previously published for mouse Col10a1, highlighted a number of conserved domains within the promoter and upstream elements. Reporter cat gene (encoding chloramphenicol acetyltransferase, CAT) constructs containing 5'-regulatory sequences of human COL10a1 (hCOL10a1) were transfected into primary cultures of foetal bovine growth plate chondrocytes producing COL10A1 and non-producing epiphyseal cartilage chondrocytes. Constructs containing up to 900 bp of promoter sequence exhibited low levels of CAT production in expressing cells and non-expressing cells. Addition of a further 1.5 kb of upstream sequence resulted in a dramatic increase in CAT production in expressing cells only. The results demonstrate the presence of enhancer-like elements between 900 bp and 2.4 kb upstream of the transcription start point(s) of hCOL10a1, which is distinctly different from that reported for the chick.
Thomas
J T
JT
School of Biological Sciences, University of Manchester, UK.
Sweetman
W A
WA
Cresswell
C J
CJ
Wallis
G A
GA
Grant
M E
ME
Boot-Handford
R P
RP
D000818:Animals; D001483:Base Sequence; D002356:Cartilage / Q000378:metabolism / Q000473:pathology; D002417:Cattle / Q000235:genetics; D002478:Cells, Cultured; D003094:Collagen / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004838:Epiphyses / Q000166:cytology; D005091:Exons / Q000235:genetics; D005796:Genes; D017930:Genes, Reporter; D006132:Growth Plate / Q000166:cytology; D006801:Humans; D006984:Hypertrophy; D051379:Mice / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D018076:DNA, Complementary; D003094:Collagen