IN-VIVO ACTIVITY FOR INITIATION OF DNA-REPLICATION RESIDES IN A TRANSCRIBED REGION OF THE HUMAN GENOME

The potential for autonomous replicating activity has been demonstrated, in the preceding paper, to exist in abundance in those sequences which are transcribed from the genome of human embryonic lung fibroblasts (IMR90). In this paper we demonstrate for one sequence present in an 'O'-family homologous cDNA clone (clone 343), the likelihood of its in vivo activity by analysis of nascent replicated strands. Analysis of seven independent sources of human DNA with three different restriction enzyme digests failed to reveal any significant polymorphisms of the cDNA 343 homologous sequence. Homologous sequences were found in human, cow, mouse and monkey DNA, but not in rat, dog, rabbit, chicken or yeast DNA. An approx. 18 kbp fragment obtained as a clone from a genomic library of human embryonic lung fibroblasts was found to contain the sequence present in cDNA 343 in a 2.2 kbp EcoRI fragment. Sequence analysis of the clone provided three regions suitable for use as oligonucleotide primers in a PCR method of mapping the in vivo site of initiation of DNA replication. The initiation zone and a chromosomal origin for DNA replication are mapped to a region of approximately 1.6 kbp and were inclusive of the sequence detected in cDNA 343.