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At least 685 records · Page 38Linked to original sources
PubMed1995

Segregational stability and copy number of the theta-type lactococcal replicon Rep22 in Lactococcus.

Rep22 is the replication region of the lactococcal theta replicating pUCL22 plasmid. The copy number of Rep22-based plasmids in Lactococcus was determined by using a chromosomal DNA fragment from Lactococcus lactis subsp. lactis MMS368 as reference. Segregational behavior appeared to be linked to copy number and therefore indicated random distribution of copies to daughter cells. Nevertheless, an active partitioning system was detected in the parental plasmid pUCL22. A pUCL22 138-bp DNA restriction fragment bearing a perfect 18-bp inverted repeat was involved in the improvement of Rep22-based plasmid segregational stability during discontinuous exponential growth.

Base Sequence↗
PubMed1982

Temperature sensitive replication plasmids are passively distributed during cell division at non-permissive temperature: a new model for replicon duplication and partitioning.

To see whether plasmid molecules in bacteria are equally partitioned or randomly distributed at cell division, the segregation properties of temperature sensitive replication mutants of the E. coli plasmid pSC101 were tested at non-permissive temperature. The results support the idea that at least unreplicated molecules are passively distributed and thus the Equipartition Model is unlikely even under physiological conditions if plasmids replicate randomly. Therefore, we developed a new model which involves the Random Replication Hypothesis and assumes that only the two products of the last plasmid replication event are actively partitioned into two daughter cells and the others are randomly distributed. Mathematical studies revealed that the incompatibility segregation rate predicted by this model fits the experimental data.

DNA Replication↗
PubMed1989

Structural and functional organization of ColE2 and ColE3 replicons.

The complete nucleotide sequences of the 1.5 kb regions of ColE2 and ColE3 plasmids containing the segments sufficient for autonomous replication have been determined. They are quite homologous (greater than 90%), indicating that these two plasmids share common mechanisms of initiation of replication and its regulation. An open reading frame with a coding capacity for a protein of about 300 amino acids is present in both ColE2 and ColE3 and it actually specifies the Rep (for replication) protein, which is the plasmid specific trans-acting factor required for autonomous replication. The amino acid sequences of the Rep proteins of ColE2 and ColE3 are quite homologous (greater than 90%). The cis-acting sites (origins) where replication initiates in the presence of the trans-acting factors consist of 32 bp for ColE2 and 33bp for ColE3. They are the smallest of all the prokaryotic replication origins so far reported. They are nonhomologous only at two positions, one of which, a deletion of a single nucleotide in ColE2 (or an insertion in ColE3), determines the plasmid specificity in interaction of the origins with the Rep proteins. Both plasmids carry a region with an identical nucleotide sequence and the one in ColE2, the IncA region, has been shown to express incompatibility against both ColE2 and ColE3. These results indicate that these plasmids share a common IncA determinant. A possibility that a small anti-sense RNA is involved in copy number control and incompatibility (IncA function) was suggested.

Bacteriocin Plasmids↗
PubMed1984

Plasmid formation in Streptomyces: excision and integration of the SLP1 replicon at a specific chromosomal site.

We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) originate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragments of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.

Base Sequence↗
PubMed1988

Comparison of mammalian cell expression vectors with and without an EBV-replicon.

We have characterized the properties of an Epstein-Barr virus vector (EBV-CMV) and compared its expression potential with a respective integrating vector (CMV). These vectors were used to express chloramphenicol acetyltransferase (CAT) gene in human HeLa, 293, monkey CV-1, dog MDCK, and hamster R 1610 cells. The EBV-CMV-cat DNA replicates extrachromosomally in HeLa, 293 and CV-1 cells, where also high expression of CAT gene was observed. The EBV-CMV vector integrated in MDCK and R 1610 cells and the CMV vector integrated in all cells tested. Integration yielded mostly clones with low CAT expression. In all cell lines, except HeLa cells, the existence of the extrachromosomal but not the integrated vector DNA is strictly dependent on the Hygromycin B selection pressure. The extrachromosomal state of the EBV vector is a prerequisite for good expression particularly in human and monkey cells.

Animals↗
PubMed1996

Use of viral replicons for the expression of genes in plants.

Autonomously replicating virus-based vectors have been investigated as a means of introducing heterologous genes into plants. This approach has a number of potential advantages over stable genetic transformation, particularly in terms of speed and levels of expression that can be obtained. Several groups of plant viruses, with genomes consisting of both DNA and RNA, have been investigated as possible gene vectors. In the case of DNA viruses, it has generally been possible to identify nonessential regions of the genome that can be replaced by foreign sequences. However, there appear to be limitations on the size of insert which can be tolerated. In the case of RNA viruses, replacement of viral sequences usually has a drastic effect on the viability. However, in several cases it has proved possible to substantially increase the size of the viral genome by the direct insertion of additional sequences while still retaining the ability of the viruses to multiply and spread in plants. These RNA virus-based systems appear to have the greatest potential as gene vectors.

Capsid↗
PubMed1998

Pulsed-field gel electrophoresis analysis of the genome of Rhodococcus fascians: genome size and linear and circular replicon composition in virulent and avirulent strains.

Total DNA of virulent and avirulent strains of Rhodococcus fascians was resolved by pulsed-field gel electrophoresis (PFGE) into a discrete number of fragments by digestion with the endonucleases AseI and DraI. Restriction endonucleases PacI, PmeI, and SwaI yielded no fragments upon digestion of R. fascians genome, and all the other tested endonucleases recognizing 6 bp released too many fragments. The genome size was 5.6 megabases for the type strain R. fascians DSM 20669, and 5.8 megabases for the virulent R. fascians D188 strain. However the genome size of R. fascians CECT 3001 (NRRL B15096) was 8.0 megabases. No linear chromosome in the megabase range was observed under pulse conditions in which Saccharomyces cerevisiae and Schizosaccharomyces pombe chromosomes were perfectly resolved, suggesting that the R. fascians chromosome is circular. A new linear plasmid pIRN640 of 640 kb was found in the avirulent R. fascians CECT 3001 that did not hybridize with a probe internal to the fas region of pFiD188 known to be involved in plant pathogenicity in the virulent strain R. fascians D188. Virulence was correlated in all strains tested with the presence of the fas region. The AseI and DraI bands corresponding to the extrachromosomal elements were identified providing the basis for a physical map of this organism.

DNA, Circular↗
PubMed1999

Respiratory syncytial virus G glycoprotein expressed using the Semliki Forest virus replicon is biologically active.

The respiratory syncytial virus (RSV) G glycoprotein mediates attachment of RSV to cells via an unknown receptor. To study G glycoprotein function we have cloned two variants of the RSV G gene into a Semliki Forest virus (SFV) expression vector, a full length (rG) and soluble (srG) G glycoprotein variant. By immunofluorescence microscopy, rG was found to be predominantly membrane associated, while srG was mostly cytoplasmic. The rG (80-85 kDa) and srG (75-80 kDa) constructs produced heavily glycosylated proteins, however they were slightly smaller than the G glycoprotein expressed in RSV infected HEp-2 cells (85-90 kDa). The biological activity of purified srG was tested by its ability to bind to RSV permissive cells. Purified srG bound to HEp-2 cells and the amount bound increased linearly with the quantity added. Binding was not saturable with the small quantities of protein available. Binding of srG to HEp-2 cells was inhibited (67-68%) by MAb 30 and neutralising anti-G MAb 29. Nonpermissive SF9 insect cells bound 20-50 times less srG than HEp-2 cells. SFV expressed recombinant RSV G glycoprotein should be useful for studying interactions between the RSV G glycoprotein and cells.

Cell Line↗
PubMed1983

Replicon initiation frequency and intracellular levels of ATP, ADP, AMP and of diadenosine 5',5'''-P1,P4-tetraphosphate in ehrlich ascites cells cultured aerobically and anaerobically.

When Ehrlich ascites cells were cultured for 2 h under oxygen-free atmosphere, a shut-down of initiation of new replication units was observed by chain length analysis of the nascent daughter strands and by DNA fibre autoradiography. The intracellular level of ATP, ADP and AMP remained virtually normal in the anaerobized cells, while that of diadenosine 5',5'''-P1,P4-tetraphosphate was found reduced by about two orders of magnitude. It is proposed that the ceasing of DNA synthesis after O2 removal is at actively controlled regulatory response of the cells in which diadenosine 5',5"'-P1,P4-tetraphosphate is probably involved.

Adenine Nucleotides↗
PubMed1987

Evidence for the involvement of a single major species of replicative complex in DNA synthesis from two diverse nuclear replicons in yeast.

The activity that replicates the 2-micron yeast DNA plasmid in vitro can be isolated as a high-molecular weight (approximately 2 X 10(6)) fraction, which possesses many of the features of a multiprotein replicative complex. This fraction also initiates DNA synthesis at the yeast chromosomal replicator ARS1 raising the question whether the preparations discriminate between origins. It was determined that the binding of replicative complex to plasmids containing either 2-microns or ARS1 origins of replication was indistinguishable. The preparations also showed no preference among them for replication. In addition, the DNAs competed with each other to the same extent for binding of replicative complex. These results suggest that these two origins share one major species of replicative complex.

Binding, Competitive↗
PubMed1989

Supercoiled loop organization of genomic DNA: a close relationship between loop domains, expression units, and replicon organization in rDNA from Xenopus laevis.

Analysis of the organization of a specific chromosomal gene, the gene for rRNA in Xenopus laevis, has evidenced a close relationship between loop organization, replication organization, and expression units. The nontranscribed spacer appears to be involved in all three levels of organization. Furthermore the replication origin region appears to be involved in nuclear matrix anchorage and is closely related to the 40 S transcription promoter. This organization suggests how expression domains may be regulated and how this functional organization may be transmitted to daughter cells after DNA replication, thus allowing selected expression patterns not to be lost during development.

Animals↗
PubMed1986

Expression of the cloned subunits of Escherichia coli transhydrogenase from separate replicons.

The pntA and pntB genes of Escherichia coli, encoding the alpha- and beta-subunits of the pyridine nucleotide transhydrogenase, were cloned individually in two different compatible plasmids into Escherichia coli mutants lacking transhydrogenase activity. Energy-linked and non-energy-linked transhydrogenase activities were produced only in cells carrying both plasmids thus showing that the products of both genes are required for the formation of an active enzyme. ATP-energized transhydrogenase activity was not increased in cells containing amplified levels of the transhydrogenase when the cell membrane ATPase was also amplified. It is suggested that the excess transhydrogenase is effectively uncoupled from the ATPase by compartmentalization in the cell.

Cell Membrane↗