Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin.

Abstract

In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307: [516][517][518][519][520] 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either (i) the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or (ii) specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.One mechanism by which a cell can accumulate large amounts of a specific protein or RNA is by amplification of the corresponding gene (for a review, see reference 47). Amplified genes are located either within expanded chromosomal regions (referred to as homogeneously staining or abnormally banding regions) or on extrachromosomal autonomously replicating elements (called minute or double minute chromosomes; 13) Molecular and cytogenetic analyses of many amplified regions in mammalian tumor cells have demonstrated that the sizes of amplified regions vary but are generally larger than the target genes and are often more than 200 kilobase pairs (kbp). One explanation for the presence of large and variable amounts of flanking sequence in amplified regions is that amplification is mediated by specific cis-acting elements present in the flanking sequences. For example, it has been proposed that amplification occurs in units of replication, in which case each amplification unit should contain an origin of DNA replication (10, 25) and any other sequences required for replication. Therefore, the repeated unit found within the amplified array may actually represent the chromosomal "domain" under the control of such cis-acting elements.We are interested in identifying and isolating cis-acting chromosomal elements which are involved in the process of gene amplification in mammalian cells.

NUP214-ABL1 amplification in t(5;14)/HOX11L2-positive ALL present with several forms and may have a prognostic significance

“…It is generally admitted that the first step of genomic amplification of genomic DNA results from episome formation. 12,13 In latter steps, the episomes may be included within double minute (dmin) chromosomes or homogeneously staining or abnormally staining regions (HSR or ABR). 14 Two mechanisms for the formation of amplicons have been proposed, conservative in which the original DNA sequence remains at its normal location onto the chromosome, and nonconservative in which the original sequence is extracted from its normal chromosomal location.…”

Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells.

“…Unidirectional High-Voltage Gel Electrophoresis and PFGE of High-Molecular-Weight DNA To detect circular DNA molecules, DNA (-10.0 Mg) corresponding to 1 X 106 cells (-1/4-1/5 of an agarose plug prepared using the Bio-Rad plug mold) were loaded directly onto 1% agarose gels (1X tris(hydroxymethyl)aminomethane-Borate buffer) and fractionated using high-voltage electrophoresis at 5.4 V/cm for 12-24 h at 4°C (Eckhardt, 1978;Carroll et al, 1987;Ruiz et al, 1989).…”

“…Pulsed-field gradient or field-inversion gel electrophoresis combined with electron microscopy studies were first used to identify these ©3 1992 by The American Society for Cell Biology structures in HeLa cells that contained amplified copies of the dihydrofolate reductase gene (Maurer et al, 1987). Since this initial study, submicroscopic circular DNAs have been detected in other mammalian cell lines containing amplified genes (Carroll et al, 1987;Von Hoff et al, 1988;Ruiz et al, 1989). These circular DNAs have been termed episomes by Carroll et al (1987) or amplisomes by Pauletti et al (1990).…”

“…Since this initial study, submicroscopic circular DNAs have been detected in other mammalian cell lines containing amplified genes (Carroll et al, 1987;Von Hoff et al, 1988;Ruiz et al, 1989). These circular DNAs have been termed episomes by Carroll et al (1987) or amplisomes by Pauletti et al (1990).…”

“…Several studies of cultured cell lines have provided a molecular chronology of events in which submicroscopic circular DNAs precede the appearance of cytogenetically detectable DM structures (Carroll et al, 1987(Carroll et al, , 1988. In addition, pulsed-field gel electrophoresis (PFGE) data from neuroblastoma biopsies imply that the multimerization of MYCN circles in vivo can generate DM structures .…”