Identification of regions essential for extrachromosomal replication and maintenance of an endogenous plasmid inDiclyosrelium
32
Citation
38
Reference
10
Related Paper
Citation Trend
Abstract:
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.Keywords:
Extrachromosomal DNA
Shuttle vector
Extrachromosomal DNA
Bovine papillomavirus
Autonomously replicating sequence
Shuttle vector
Cite
Citations (11)
Shuttle vector
Mutation frequency
Restriction map
Cite
Citations (22)
pBR 322와 pBD9을 이용하여 Staphylococcus aureus에서 분리된 chloramphenicol 저항성(Cmr) plasmid인 pSBK 203상의 ori 부위를 cloning하였다. 또한 E. coli 내에서도 발현하는 pSBK 203상의 Cm 저항성 부위 및 cloning 된 ori 부위를 pBR 322에 재조합시켜 E. coli와 그람양성균인 Bacillus subtilis 양쪽 모두에서 복제되고 또 항생물질에 대한 저항성도 각각 발현되는 shuttle vector 구성을 시도하였다. 【The replication region of the chloramphenical resistance plasmid pSBK203 of Staphylococcus aureus was cloned using pBR322 and pBD9 as vectors. Cloned replication tegion and chloramphenicol resistance gene were recombined to pBR322. The reconstructed vector behaved as a shuttle vector for E. coli and B. subtilis.】
Shuttle vector
Cloning (programming)
Cloning vector
Cite
Citations (0)
Shuttle vector
Multiple cloning site
Cloning (programming)
Cloning vector
Replication
Autonomously replicating sequence
Cite
Citations (18)
Extrachromosomal DNA
Southern blot
Dot blot
genomic DNA
Cite
Citations (5)
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
Cite
Citations (21)
Abstract The Saccharomyces cerevisiae ( Sacch. cerevisiae ) strain RXII, like many others, harbours plasmid DNAs. and one category of them is homologous to the 2 μ plasmid of yeast. DNA‐DNA hybridization experiments indicated altered structures of this species as regards the number and distribution of the restriction sites. The efforts made to clone either the whole plasmid in pBR328 or its fragments in pBR322 vectors remained unsuccessful, since deleted plasmids were isolated without insert DNA, and even the loss of vector sequences was observed. The data suggest, that the 2 μ derivative plasmid in strain RXII represent an unique category of this extrachromosomal genetic element.
Extrachromosomal DNA
Insert (composites)
Strain (injury)
Plasmid preparation
T-DNA Binary system
Cite
Citations (0)
With one exception, all spiroplasma strains examined contained extrachromosomal DNA, most of which was in the form of covalently closed circular plasmids. One plasmid, pIJ2000, carried by Spiroplasma citri strain ASP-1, was purified and characterized and used to probe for related plasmids in other strains. Unsuccessful attempts were made to clone pIJ2000 into Escherichia coli using the vectors pAT153 and pBR322. However, spiroplasma chromosomal DNA fragments could be cloned without difficulty.
Spiroplasma
Extrachromosomal DNA
clone (Java method)
Cite
Citations (8)
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
Cite
Citations (7)
Extrachromosomal DNA
Cite
Citations (11)