Abstract
Chicken embryo fibroblasts infected with a strain of Rous sarcoma virus containing a temperature-sensitive mutation in the gene coding for pp60src, a protein kinase, undergo changes in collagen synthesis within 4 h after a temperature shift. Cells shifted from the restrictive to the permissive temperature for transformation show decreasing levels of collagen synthesis and increasing levels of kinase activity; the reverse occurs when infected cells are shifted from the permissive to the restrictive temperature. Levels of type I procollagen mRNAs coding for pro alpha 1 and pro alpha 2 chains, measured by hybridization to nick-translated cloned alpha 1 and alpha 2 cDNA, decreased simultaneously soon after a reduction in temperature and reached a new steady state at about 50 h after the shift. In order to test for regulation at the transcriptional level, nuclei were isolated from normal and Rous sarcoma virus-transformed chicken embryo fibroblasts and allowed to transcribe in the presence of [alpha-32P]UTP. Procollagen mRNA sequences in newly synthesized and in total RNA from transformed cell preparations were both about 5-fold lower than the levels in normal cell preparations. We conclude that the coordinate decrease in procollagen mRNAs observed in Rous sarcoma virus-transformed chicken embryo fibroblasts is caused primarily by a decrease in the transcription of the type I procollagen genes, a decrease which is directly or indirectly mediated by the pp60src protein kinase.
Highlights
Background was determined in hybridizations withEscherichia coli DNA linked to cellulose and subtracted.c The al probe was a 1100-base-pair proal fragment from pCg54.d The a2 probe was a 2500-base-pair proa2 fragment from pCg45. .0 P ERNA, H9g PER ASSAY (O, C11) and RSV-transformed (, *) CEF
DNA linkage was carried out in the same manner except that plasmid DNA was heated in a boiling water bath for 8 min to nick and denature the DNA, and the DOM cellulose was washed once with 20 mM sodium phosphate, pH 5.5, to reduce the salt concentration
Since this would create an unacceptable background in solution hybridization, and greatly reduce the sensitivity of measurements which could be mode, we linked the RNA to cellulose so that only the DNA probe hybridized to RNA would be recovered
Summary
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 256, No 10, Issue of May 25. pp. 5022-5028, 1981 Printed in U.S.A. Chicken embryo fibroblasts infected with a strain of Rous sarcoma virus containing a temperature-sensitive mutation in the gene coding for pp608'", a protein kinase, undergo changes in collagen synthesis within 4 h after a temperature shift. We conclude that the coordinate decrease in procollagen mRNAs observed in Rous sarcoma virus-transformed chicken embryo fibroblasts is caused primarily by a decrease in the transcription of the type I procollagen genes, a decrease which is directly or indirectly mediated by the pp6081' protein kinase. Chicken embryo fibroblasts infected with RSV' undergo a variety of changes in cellular biochemistry, including alterations in glycolipid structure [1], increased hyaluronate synthesis [2] and sugar transport [3], reduced cAMP levels [4], decreased amounts of surface fibronectin [5], and decreased collagen synthesis [6,7,8] and procollagen mRNA levels [9,10,11,12,13]. Our results show that there is an inverse correlation between pp60src kinase activity and the levels of type I mRNA and collagen synthesis and that the reduced mRNA levels can be accounted for by a reduction in the transcription of al and a2 procollagen genes
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