Absence of alpha 2(1) procollagen synthesis in a clone of SV40-transformed WI-38 human fibroblasts.
Normal diploid human embryonic lung fibroblasts (WI-38) produce type I collagen of chain composition [alpha 1(1)]2.alpha 2(1) together with small amounts of type III collagen. We have examined the synthesis and secretion of type I collagen in a clone of SV40-transformed WI-38 fibroblasts (SVWI-38). These cells produced only 20-25% of the total collagen synthesized by their normal counterparts, with no detectable synthesis of alpha 2(1) chains and deposited a type I trimer consisting of overmodified alpha 1(1) chains. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cyanogen bromide peptides of collagens isolated from cells cultured either in the presence or absence of alpha,alpha-dipyridyl revealed that this overmodification occurred along the entire length of the alpha 1(1) chains. Analysis of poly(A+) RNA by Northern blot analysis and total RNA by slot blot analysis using cloned type I procollagen cDNA probes revealed that no alpha 2(1) mRNA was present in the SVWI-38 cells. Primer extension of the RNA confirmed this finding. The SVWI-38 cells had a normal chromosome number, but contained 28 normal and 18 abnormal and marker chromosomes. Restriction mapping of the entire alpha 2(1) procollagen gene did not reveal any gross deletions or insertions within this gene, nor was the gene hypermethylated in the transformed cells, when compared with their normal counterparts. One interesting feature, however, was the fact that certain CpG dinucleotides in the alpha 2(1) gene were methylated in the normal as well as the transformed cells. These SV40-transformed WI-38 fibroblasts therefore do not produce any alpha 2(1) collagen chains due to transcriptional inactivation of their genes.
Parker
M I
MI
Department of Medical Biochemistry, University of Cape Town Medical School, Observatory, South Africa.
Smith
A A
AA
Gevers
W
W
D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D005347:Fibroblasts / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D007621:Karyotyping; D008745:Methylation; D010446:Peptide Fragments / Q000032:analysis; D011347:Procollagen / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D013539:Simian virus 40
D010446:Peptide Fragments; D011347:Procollagen; D012333:RNA, Messenger