RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1.
RNA fingerprinting using arbitrarily primed PCR (RAP) samples an RNA population and allows the detection of differentially expressed genes between two or more populations. This method was applied to mink lung epithelial cells, which respond to treatment with transforming growth factor beta (TGF-beta) by undergoing cell cycle arrest at or near the G1/S-phase boundary. The steady-state abundances of approximately 200 RNAs were surveyed, a few of which displayed differential regulation in response to TGF-beta 1. Three products were isolated, cloned, and sequenced. One differentially regulated RNA corresponded to cyclin A, a gene known to be required for the progression of mammalian fibroblasts through S phase. Northern blot analysis confirmed that the cyclin A mRNA steady-state abundance decreased dramatically in response to a 24-hr TGF-beta 1 treatment and also in response to cell cycle arrest caused by contact inhibition. A second RAP product corresponded to a previously unknown 7.5-kb mRNA, the level of which decreased dramatically in response to TGF-beta 1 treatment. Unlike the cyclin A mRNA, the abundance of this transcript did not decrease in response to growth arrest induced by contact inhibition. A third RAP product corresponded to the mRNA for osteonectin, an extracellular matrix protein. The abundance of this mRNA increased at least 2-fold during TGF-beta 1 treatment. This observation is consistent with other reports of increases in extracellular matrix proteins during TGF-beta treatment. RAP should be able to identify many of the genes that change in steady-state expression during the cell cycle.
Ralph
D
D
California Institute of Biological Research, La Jolla 92037.
McClelland
M
M
Welsh
J
J
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016213:Cyclins / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression / Q000187:drug effects; D066298:In Vitro Techniques; D008907:Mink; D008969:Molecular Sequence Data; D015676:Osteonectin / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D016213:Cyclins; D017931:DNA Primers; D015676:Osteonectin; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta