oriGNAI3: A narrow zone of preferential replication initiation in mammalian cells identified by 2D gel and competitive PCR replicon mapping techniques

Abstract

The nature of mammalian origins of DNA replication remains controversial and this is primarily because two-dimensional gel replicon mapping techniques have identified broad zones of replication initiation whereas several other techniques, such as quantitative PCR, have disclosed more discrete sites of initiation at the same chromosomal loci. In this report we analyze the replication of an amplified genomic region encompassing the 3'-end of the GNAI3 gene, the entire GNAT2 gene and the intergenic region between them in exponentially growing Chinese hamster fibroblasts. These cells express GNAI3 but not GNAT2 . The replication pattern was first analyzed by two-dimensional neutral-alkaline gel electrophoresis. Surprisingly, the results revealed a small preferential zone of replication initiation, of at most 1.7 kb, located in a limited part of the GNAI3 - GNAT2 intergenic region. Mapping of this initiation zone was then confirmed by quantitative PCR. The agreement between the two techniques exploited here strengthens the hypothesis that preferred sites of replication initiation do exist in mammalian genomes.

Replication origins oriGNAI3 and oriB of the mammalian AMPD2 locus nested in a region of straight DNA flanked by intrinsically bent DNA sites

“…The oriGNAI3 was identified in the intergenic region between the GNAI3 and GNAT2 genes and oriB in the AMPD2 3' segment (13,23). The p4.4 kb plasmid (Y08232.1) that contains the intergenic region GNAI3-GNAT2 and the p2.5 kb (X96547) plasmid that contains the 3' segment of the AMPD2 gene were purified by the CTAB method (24) and cleaved with restriction endonucleases according to the manufacturer's instructions.…”

Molecular combing in the analysis of developmentally regulated amplified segments of Bradysia hygida

“…Some examples of this phenomenon include DNA segments that form DNA puffs in Sciaridae and the chorionic gene-containing chromosome segment in Drosophila melanogaster (Claycomb and Orr-Weaver, 2005). Examples of DNA amplification induced in vitro in cultured mammalian cells include the dihydrofolate reductase gene amplification in ovarian cells (Dijkwell et al, 1991;Altman and Fanning, 2004) and the adenosine monophosphate deaminase 2 gene amplification in Chinese hamster lung cells (Toledo et al, 1998;Anglana et al, 2003;Courbet et al, 2008). Gene amplification has also been reported to occur during the course of tumorigenesis in distinct chromosomal regions (Myllykangas and Knuutila, 2006).…”

Sequence-Directed DNA Curvature in Replication Origins Segments

“…The nature of the information required for activation of replication origins in multicellular organisms, where multiple initiation sites of replication occur in cells with differential gene expression, has made it difficult to associate function to a specific replication origin sequence. Analyses of replication intermediates using alternative methods indicated that the replication origin lacked a nucleotide consensus sequence (Biamonti et al, 2003;DePamphilis, 1999;Toledo et al, 1998;Tower, 2004). The activity of the initiation site for replication in metazoans, which lacks a consensus sequence, can be influenced by chromatin structure and/or selected by epigenetic factors (Anglana et al, 2003;Balani et al, 2010;Courbet et al, 2008;Debatisse et al, 2004;Fiorini et al, 2006a;Gimenes et al, 2009;Stehle et al, 2003).…”