Recent experiments have shown that gene amplification can be mediated by submicroscopic, autonomously replicating, circular extrachromosomal molecules. We refer to those molecules as episomes (S. Carroll, P. Gaudray, M. L. DeRose, J. F. Emery, J. L. Meinkoth, E. Nakkim, M. Subler, D. D. Von Hoff, and G. M. Wahl, Mol. Cell. Biol. 7:1740-1750. The experiments reported in this paper explore the way episomes are formed and their fate in the cell over time. The data reveal that in our system the episomes are initially 250 kilobases, but gradually enlarge until they become double minute chromosomes. In addition, we show that episomes or double minute chromosomes can integrate into chromosomes. Our results also suggest that episomes can be produced by deletion of the corresponding sequences from the chromosome.
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.
N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modffication arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus suggested following the precise localization of m6A in Rous sarcoma virus (18). In that molecule, the modified residues were found bracketing the src splice acceptor site and therefore may influence in some way the accuracy and/or efficiency of processing. Methylation of internal adenosine residues of eucaryotic cell RNA also takes place in the nucleus, as evidenced by the demonstration of m6A in eucaryotic cell heterogeneous nuclear RNA (24). However, previous analysis indicated a conservation of m6A residues during processing and transport to the cytoplasm, suggesting that this modification occurs mainly within exon regions (19). It was therefore of great interest to determine whether m6A occurs in intron-specific regions of eucaryotic precursor RNA. This analysis would identify the temporal relationship between transcription, methylation, and nuclear processing and may assist in developing a concept for the role of this posttranscriptional modification in eucaryotic RNA biogenesis.Attempts have been made to determine the influence of m6A on processing of gene transcripts. Earlier studies with cycloleucine, an inhibitor of S-adenosylmethionine synthetase, suggested that m6A affects the splicing of avian sarcoma virus genomic RNA (34) and processing of CHO cell mRNA (2). This hypothesis is supported by the similarity between the m6A consensus sequences in both eucaryotic and viral RNA and the weakly conserved branch point sequence YNCU(G/A)AC used in intron removal (18,27,40). Camper et al. (5) used the mRNA methylation inhibitor S-tubercidinyl homocysteine to measure the effects of undermethylation on the half-life and cytoplasmic appearance of poly(A)+ RNA in HeLa cells. A significant effect on processing and/or transport of undermethylated mRNA occurred with a delay in its appearance in the cytoplasm, whereas no significant alteration in the cytoplasmic half-life of undermethylated mRNA was observed.
The selection of high-producing cell lines is usually time-consuming and labour-intensive. Following transfection, high-producing cells are selected using limiting dilution cloning to prevent non- and low-producing cells from outgrowing high-producing cells, a process that normally takes > 3 months. During this time, the cells have to be screened occasionally to ensure stability of the selected clone. Several new methods for selecting and screening cells using flow cytometry and cell sorting have recently been developed; these include gel microdrop technology, which encapsulates the cells in gelatine beads, and matrix-based secretion assays. This paper reviews these techniques for selecting high-producing cell lines and isolating rare cells.
Recent experiments have shown that gene amplification can be mediated by submicroscopic, autonomously replicating, circular extrachromosomal molecules. We refer to those molecules as episomes (S. Carroll, P. Gaudray, M. L. DeRose, J. F. Emery, J. L. Meinkoth, E. Nakkim, M. Subler, D. D. Von Hoff, and G. M. Wahl, Mol. Cell. Biol. 7:1740-1750, 1987). The experiments reported in this paper explore the way episomes are formed and their fate in the cell over time. The data reveal that in our system the episomes are initially 250 kilobases, but gradually enlarge until they become double minute chromosomes. In addition, we show that episomes or double minute chromosomes can integrate into chromosomes. Our results also suggest that episomes can be produced by deletion of the corresponding sequences from the chromosome.
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