CDNA CLONES CONTAIN AUTONOMOUS REPLICATION ACTIVITY

We have undertaken to investigate transcription as a regulatory event in mammalian DNA replication. Subpopulations of transcripts represente d in a cDNA library of human embryo lung fibroblasts (IMR90) were exam ined for their ability to support autonomous replication after transfe ction into human cells (HeLa). Two of three cDNA clones (343, 363) con taining 'O'-family repetitive sequences, after subcloning into pBR322 and transfection into HeLa cells, were capable of autonomous replicati on. One of these cDNA clones, 343, is enriched by selection for poly(A )+ RNA. In contrast, none of five Alu-containing transcripts was capab le of autonomous replication in human cells. However, six out of ten c DNA clones contained neither 'O'-family or Alu homologous sequences an d were as efficient as the cDNA clones containing 'O'-family sequences in replicating autonomously in human cells. cDNA clones, from an olig o-d(T)-primed library of human poly(A)+ enriched RNA, contain a signif icant proportion of independent clones that can also support autonomou s replication of bacterial plasmids in human cells. cDNA clone 343 was observed to contain in a 448 bp EcoRI-HincII fragment, yeast ARS cons ensus, SAR consensus, IRs, bent DNA and a DUE, all sequence and struct ural characteristics often associated with many prokaryotic, viral and eukaryotic origins. Sequence analysis of seven other cDNA clones (fro m non-'O'-family, non-Alu homologous sequences, NOA) showed that five contained some of the same consensus sequences. Two NOA clones (NOA4 a nd -5) did not contain any representations of ARS and SAR consensus se quences, suggesting that these two features may not be essential for a utonomous replication activity in mammalian cells.