Stable propagation of ‘selfish’ genetic elements
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Extrachromosomal DNA
Genetic Fitness
Selfishness
Extrachromosomal DNA
Bovine papillomavirus
Autonomously replicating sequence
Shuttle vector
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Abstract Plasmids are extrachromosomal replicons found in gram‐negative and gram‐positive bacteria as well as in some lower eukaryotic organisms. They are present in bacterial cells replicating at a specific number of copies per cell. Their size varies from a few to several hundred kilobase pairs and bacterial cells can harbor more than one plasmid species. The medical importance of plasmids that code for antibiotic resistance and those that contribute directly to microbial pathogenicity is well‐documented, is the role played by plasmids in bacteria of importance in agriculture and industry. These extrachromosomal elements are of equal importance, however, for the study of the structure and function of DNA. Plasmids utilize a wide variety of strategies to initiate and regulate their replication. In this chapter we examine the replication of plasmids ColE1, whose replication system is part of most plasmids vectors; R6K, an iteron‐containing plasmid that possesses three replica origins; plasmids belonging to the RepABC family found in a‐proteobacteria; and of pT181, a group of plasmids found in gram‐positive bacteria. In addition,we also discuss linear plasmids.
Extrachromosomal DNA
Replicon
ColE1
Rolling circle replication
T-DNA Binary system
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We previously found that a plasmid bearing a replication initiation region efficiently initiates gene amplification in mammalian cells and that it generates extrachromosomal double minutes and/or chromosomal homogeneously staining regions. During analysis of the underlying mechanism, we serendipitously found that hairpin-capped linear DNA was stably maintained as numerous extrachromosomal tiny episomes for more than a few months in a human cancer cell line. Generation of such episomes depended on the presence of the replication initiation region in the original plasmid. Despite extrachromosomal maintenance, episomal gene expression was epigenetically suppressed. The Southern blot analysis of the DNA of cloned cells revealed that the region around the hairpin end was diversified between the clones. Furthermore, the bisulfite-modified PCR and the sequencing analyses revealed that the palindrome sequence that derived from the original hairpin end or its end-resected structure were well preserved during clonal long term growth. From these data, we propose a model that explains the formation and maintenance of these episomes, in which replication of the hairpin-capped DNA and cruciform formation and its resolution play central roles. Our findings may be relevant for the dissection of mammalian replicator sequences.
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Initial experiments with the endogenous 12.3 kb Dictyostelium discoideum plasmid Ddp1 led to the generation of a large shuttle vector, Ddp1–20. In addition to Ddp1, this vector contains pBR322 and a gene fusion that confers G418 resistance in Dicyostelium cells. We have shown that Ddp1–20 replicates extrachromo somally in Diclyostelium cells and can be grown in Escherichia coli cells (1). We have now examined deletions within this vector to identify the elements essential for extrachromosomal replication and stable maintenance of the plasmid. We find that a 2.2 kb fragment is sufficient to confer stable, extrachromosomal replication with a reduction in copy number from about 40 to ∼10–15 copies per cell. Vectors containing additional Ddp1 sequences have a higher copy number. The 2.2 kb region contains none of the complete, previously identified transcription units on Ddp1 expressed during vegetative growth or development. These results suggest that gene products expressed by Ddp1 are not essential for replication, stability, or partitioning of the plasmid between daughter cells. Vectors carrying only the 2.2 kb fragment plus the gene fusion conferring G418 resistance transform Dictyostelium cells with high efficiency using either calcium phosphate mediated transformation or electroporation. Finally, we have examined the relative levels of expression of actin promoters driving neo R genes when in extrachromosomal or integrating vectors.
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A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.
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Replicon
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Cultures of Tetrahymena thermophila were deprived of nutrients and later refed with enriched medium to obtain partial synchrony of DNA replication. Preferential replication of the extrachromosomal, macronuclear ribosomal RNA genes (rDNA) was found to occur at 40-80 min after refeeding. The rDNA accounted for one half of the label incorporated into cellular DNA during this period. Electron microscopy of the purified rDNA showed 1% replicative intermediates. Their structure was that expected for bidirectional replication of the linear rDNA from an origin or origins located in the central non-transcribed region of the palindromic molecule. Similar forms had previously been observed for the rDNA of a related species, Tetrahymena pyriformis. The electron microscopic data was consistent with an origin of replication located approximately 600 base pairs from the center of the rDNA of T. thermophila, in contrast to a more central location in the rDNA of T. pyriformis. One implication of an off-center origin of replication is that there are two such sequences per palindromic molecule.
Extrachromosomal DNA
Tetrahymena pyriformis
Macronucleus
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A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.
Extrachromosomal DNA
Shuttle vector
Replicon
ColE1
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Extrachromosomal DNA
Inverted repeat
Direct repeat
Autonomously replicating sequence
Scaffold/matrix attachment region
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We have used density shift analysis to monitor the autonomous replicating sequence (ARS) activity of plasmids containing various DNA fragments from the 5′-flanking region of the human c-myc gene. The ARS activity of certain of these plasmids implied that structures in the c-myc DNA could be recognized for the initiation of replication in the absence of chromosomal integration. The plasmid pNeo.Myc-2.4 contains 2.4 kb of c-myc 5′-flanking DNA, and replicated semiconservatively as a circular extrachromosomal element. Deletion derivatives of pNeo.Myc-2.4 containing either of two nonoverlapping regions of c-myc DNA semiconservatively incorporated bromodeoxyuridine into discrete populations of heavy-light supercoiled molecules to roughly the same extent as the chromosomal DNA in the same cultures. Some constructs displayed lower ARS activity, implying that distinct cis-acting sequences in the c-myc 5′-flanking DNA may independently affect DNA replication. The ARS activity of two separate c-myc sequences suggests that replication initiation signals are redundant in the c-myc origin. The smallest c-myc insert that displayed substantial ARS activity was 930 bp long and contained three 10/11 matches to the yeast ARS consensus and several additional features found in eukaryotic replication origins.
Extrachromosomal DNA
Autonomously replicating sequence
Origin recognition complex
Replication factor C
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Extrachromosomal DNA
Replicon
Rolling circle replication
Sequence (biology)
T-DNA Binary system
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