Abstract
Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR)/origin/replicator and a nuclear matrix-attachment region (MAR) is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR). Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.
Highlights
Amplification of oncogenes or drug-resistance genes, resulting in overexpression of the encoded proteins, plays a pivotal role in malignant transformation of mammalian cells
We previously found that a plasmid with a mammalian replication-initiation region (IR)/origin/replicator and a nuclear matrix-attachment region (MAR) spontaneously initiates gene amplification and generates double minutes (DMs) or homogeneously staining region (HSR) de novo in transfected cells [1,2]
(AR1) with the hypothetical replication fork from the test sequence. Such an orientation efficiently generates HSRs [2]. We transfected this plasmid into COLO 320DM cells, selected for stable transfectants, and analyzed them by Fluorescence in situ hybridization (FISH) to detect HSRs composed of plasmid sequence
Summary
Amplification of oncogenes or drug-resistance genes, resulting in overexpression of the encoded proteins, plays a pivotal role in malignant transformation of mammalian cells. We previously found that a plasmid with a mammalian replication-initiation region (IR)/origin/replicator and a nuclear matrix-attachment region (MAR) spontaneously initiates gene amplification and generates DMs or HSR de novo in transfected cells [1,2]. The MAR sequence mediates the attachment to the nuclear matrix, which is required for the DNA replication initiation (for a recent review, see 3) We employed this “IR/MAR gene amplification” method to explore the intranuclear folding and replication of large HSR [4], the intracellular behavior of DMs [5], the specific binding of tramscription factor to the target sequence [6], and the transcription from either DMs [7] or HSR [8]. We employed the novel amplification method to efficiently produce recombinant protein [9,10,11,12], and to investigate the mechanisms of gene amplification [13,14]
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