Promiscuous initiation on mammalian chromosomal DNA templates and its possible suppression by transcription.

Abstract:

:The Chinese hamster dihydrofolate reductase (DHFR) origin consists of many inefficient initiation sites scattered throughout a 55-kb intergenic spacer, with the ori-beta, ori-beta', and ori-gamma subregions being preferred. To test for the presence of genetic replicators within these subregions, fragments containing ori-beta, ori-beta', or, as a negative control, a fragment from the DHFR gene that never initiates in loco were placed at ectopic chromosomal positions. Two-dimensional gel analysis demonstrates that initiation occurs in all three fragments, including the fragment from the gene, and appears to occur at different positions within each fragment in different cells in the population. However, initiation is not detectable in the adjacent, transcribed, neomycin-resistance marker. When a cosmid containing an active DHFR gene was inserted into an ectopic chromosomal site, initiation could no longer be observed in the body of the gene, but was clearly evident in the transcriptionally-silent bacterial vector sequences. In the thirteen independent chromosomal positions into which the four different constructs were inserted, initiation occurred in early S-phase. Since these loci necessarily had to be permissive for transcription of the neo(r) marker or DHFR gene used to select transfectants, it is possible that early-firing origin activity and local transcription are somehow linked. In total, these data suggest that potential initiation sites are distributed at very frequent intervals in the mammalian genome, with usage being regulated both positively and negatively by local transcription.

journal_name

Exp Cell Res

authors

Lin HB,Dijkwel PA,Hamlin JL

doi

10.1016/j.yexcr.2005.04.012

keywords:

subject

Has Abstract

pub_date

2005-08-01 00:00:00

pages

53-64

issue

1

eissn

0014-4827

issn

1090-2422

pii

S0014-4827(05)00188-6

journal_volume

308

pub_type

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