cDNA clones contain autonomously replicating sequences

We have shown that nine cDNA clones, which were derived from an oligo (dT) primed cDNA library of human embryonic lung fibroblast (IMR90), are capable of supporting autonomous replication of bacterial plasmid in mammalian (HeLa) cells. Two of these cDNA clones contain a region homologous to "O"-family middle repetitive sequences; the other seven clone hybridize with neither "O"-family nor Alu family sequences and are called NOA clones. Oligo (dT) primed cDNA of IMR90 has been demonstrated to be an enriched source for autonomously replicating sequences. Two out of three "O"-family homologous cDNA clones and seven out of ten NOA clones were capable of autonomous replication. In contrast, none of the five clones, which contained Alu repetitive sequences, demonstrated replicative potential. The two autonomously replicating, "O"-family homologous cDNA, 343 and 363, were characterized in the greatest detail. In particular, sequence and structural features of a 448 bp fragment of cDNA 343, which was capable of supporting autonomous replication activity, were: extensive asymmetrical A/T-rich regions; 10/11 match to yeast ARS consensus; scaffold attachment region consensus of Drosophila; bent DNA; a DNA unwinding element and an in vitro matrix binding activity. We have used a PCR-based mapping strategy to identify an in vivo initiation zone located in a region of approximately 1.6 kb which is inclusive of cDNA 343. The 343 sequence has been localized to the long arm (6q22-6qter) of human chromosome 6 by both Southern analysis of hamster-human cell hybrids and in situ hybridization. Transcripts homologous to 343 have been detected in both poly(A$ sp+$) and non-poly(A$ sp+$) RNA fractions to be 1.3 kb and 5 kb, respectively; the 1.3 kb transcript is greatly enriched in poly(A$ sp+$) RNA fraction, and the 5 kb transcript is present predominantly in non-poly(A$ sp+$) RNA fraction. The detailed characterization of 343 genomic locus demonstrated that the 343 sequence is conser