CDNA clones contain autonomous replication activity

Abstract

We have undertaken to investigate transcription as a regulatory event in mammalian DNA replication. Subpopulations of transcripts represented in a cDNA library of human embryo lung fibroblasts (IMR90) were examined for their ability to support autonomous replication after transfection into human cells (HeLa). Two of three cDNA clones (343, 363) containing ‘O’-family repetitive sequences, after subcloning into pBR322 and transfection into HeLa cells, were capable of autonomous replication. One of these cDNA clones, 343, is enriched by selection for poly(A) + RNA. In contrast, none of five Alu-containing transcripts was capable of autonomous replication in human cells. However, six out of ten cDNA clones contained neither ‘O’-family or Alu homologous sequences and were as efficient as the cDNA clones containing ‘O’-family sequences in replicating autonomously in human cells. cDNA clones, from an oligo-d(T)-primed library of human poly(A) + enriched RNA, contain a significant proportion of independent clones that can also support autonomous replication of bacterial plasmids in human cells. cDNA clone 343 was observed to contain in a 448 bp EcoRI- HincII fragment, yeast ARS consensus, SAR consensus, IRs, bent DNA and a DUE, all sequence and structural characteristics often associated with many prokaryotic, viral and eukaryotic origins. Sequence analysis of seven other cDNA clones (from non-‘O’-family, non-Alu homologous sequences, NOA) showed that five contained some of the same consensus sequences. Two NOA clones (NOA4 and -5) did not contain any representations of ARS and SAR consensus sequences, suggesting that these two features may not be essential for autonomous replication activity in mammalian cells.