NDLI: Lagging-Strand, Early-Labelling, and Two-Dimensional Gel Assays Suggest Multiple Potential Initiation Sites in the Chinese Hamster Dihydrofolate Reductase Origin

Content Provider	PubMed Central
Author	Wang, Shuntai Dijkwel, Pieter A. Hamlin, Joyce L.
Copyright Year	1998
Abstract	There is general agreement that DNA synthesis in the single-copy and amplified dihydrofolate reductase (DHFR) loci of CHO cells initiates somewhere within the 55-kb spacer region between the DHFR and 2BE2121 genes. However, results of lagging-strand, early-labelling fragment hybridization (ELFH), and PCR-based nascent-strand abundance assays have been interpreted to suggest a very narrow zone of initiation centered at a single locus known as ori-β, while two-dimensional (2-D) gel analyses suggest that initiation can occur at any of a large number of potential sites scattered throughout the intergenic region. The results of a leading-strand assay and two intrinsic labelling techniques are compatible with a broad initiation zone in which ori-β and a second locus (ori-γ) are somewhat preferred. To determine how these differing views are shaped by differences in experimental manipulations unrelated to the biology itself, we have applied the lagging-strand, ELFH, neutral-neutral, and/or neutral-alkaline 2-D gel assays to CHOC 400 cell populations synchronized and manipulated in the same way. In our experiments, the lagging-strand assay failed to identify a template strand switch at ori-β; rather, we observed a gradual, undulating change in hybridization bias throughout the intergenic spacer, with hybridization to the two templates being approximately equal near a centered matrix attachment region. In the ELFH assay, all of the fragments in the 55-kb intergenic region were labelled in the first few minutes of the S phase, with the regions encompassing ori-β and ori-γ being somewhat preferred. Under the same conditions, neutral-neutral and neutral-alkaline 2-D gel analyses detected initiation sites at multiple locations in the intergenic spacer. Thus, the results of all existing replicon-mapping methods that have been applied to the amplified DHFR locus in CHOC 400 cells are consistent with a model in which two somewhat preferred subzones reside in a larger zone of multiple potential initiation sites in the intergenic region.
Starting Page	39
File Format	PDF
ISSN	10985549
e-ISSN	10985549
Journal	Molecular and Cellular Biology
Issue Number	1
Volume Number	18
Language	English
Publisher	American Society for Microbiology
Publisher Date	1998-01-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology

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The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites.

Initiation Sites Are Distributed at Frequent Intervals in the Chinese Hamster Dihydrofolate Reductase Origin of Replication but Are Used with Very Different Efficiencies

Identification of Primary Initiation Sites for DNA Replication in the Hamster Dihydrofolate Reductase Gene Initiation Zone

The Chinese Hamster Dihydrofolate Reductase Replication Origin Decision Point Follows Activation of Transcription and Suppresses Initiation of Replication within Transcription Units

A Potential Role for Mini-chromosome Maintenance (MCM) Proteins in Initiation at the Dihydrofolate Reductase Replication Origin

Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells.

Molecular cloning of Chinese hamster dihydrofolate reductase-specific cDNA and the identification of multiple dihydrofolate reductase mRNAs in antifolate-resistant Chinese hamster lung fibroblasts.

The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

The Chinese Hamster Dihydrofolate Reductase Replication Origin Beta Is Active at Multiple Ectopic Chromosomal Locations and Requires Specific DNA Sequence Elements for Activity

Lagging-Strand, Early-Labelling, and Two-Dimensional Gel Assays Suggest Multiple Potential Initiation Sites in the Chinese Hamster Dihydrofolate Reductase Origin

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