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At least 1,621 records · Page 90Linked to original sources
PubMed1983

A novel type of E. coli mutants with increased chromosomal copy number.

We have isolated E. coli mutants which can grow at 30 degrees C but not at 42 degrees C and are able to harbor the oriC plasmid (minichromosome) at a higher copy number than the parental wild-type strain at the permissive temperature. The mutants were found to contain higher amounts of chromosomal DNA per mg protein than the wild-type, whether or not they harbor the plasmid. Experimental results suggest that the higher amount of chromosomal DNA is due to a higher copy number of chromosomes and not to a larger amount of DNA per chromosome. These properties in each of the mutants are caused by a single mutation at the rpoB or rpoC gene that code for the beta or beta' subunit of RNA polymerase, respectively. The mutations are thought to affect the regulation of replication of oriC-bearing replicons, that is, the E. coli chromosome and oriC plasmids, but not the miniF plasmid.

Bacterial Proteins↗
PubMed1982

Recombination between two TnA transposon sequences oriented as inverse repeats is found less frequently than between direct repeats.

Inverse repeats of the transposon Tn2660 in either a ColE1 or an R6K replicon, with or without inversions of the parental DNA sequences between the repeats, show no detectable (less than 2%) evidence of recombination between the repeats after 60 generations of growth in either recA or RecA+ hosts. In contrast, attempts made to construct plasmids which carry two direct repeats by in vitro cleavage and ligation in the recA host were unsuccessful, although homologous plasmids with inverse repeats could be constructed, and other plasmids were found consistent with products of recombination between the direct repeats of the transient intermediate structure. It is concluded that in recA or recA+ hosts recombination between direct repeats of a transposon is frequent, whereas recombination between inverse repeats of a homologous structure has not been observed. A model to explain this difference depends upon a mechanisms that produces a nick in only one of the pair of strands at the internal resolution site (IRS) sequence of the transposon.

Cloning, Molecular↗
PubMed1987

Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid.

We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA- and ParB- miniplasmid of R1 pKN1562, without increasing its copy number. The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid. Two proteins whose expression is coordinated are components of the system. The sequence of the PstI-EcoRI fragment was obtained. The wild-type ParD system determines in cis a basal but detectable stability.

Amino Acid Sequence↗
PubMed1981

The requirement for both DNA polymerase and 5' to 3' exonuclease activities of DNA polymerase I during Tn5 transposition.

By assaying transposition of Tn5 from lambda b221 cI857 rex::Tn5 (Berg 1977) in polA-proficient and deficient cells, both the polymerase activity and 5' to 3' exonuclease activity of DNA polymerase I have been shown to be required for transposition. This requirement could not be observed in three other systems in which the transposon donor replicon had existed in the PolA-proficient and deficient cells before the transposition event to be assayed occurred. By analogy to Tn3, this may indicate that the repressor encoded by Tn5 has already been expressed and hence become rate-limiting in the overall transposition process, even PolA-deficient cells still possessing a residual activity. One PolA mutant was found among more than 50 transposition deficient (tnp) mutants isolated by the use of lambda b221 cI857 rex::Tn5.

DNA Polymerase I↗
PubMed1981

Copy number control and incompatibility of plasmid R1: identification of a protein that seems to be involved in both processes.

Investigations into the genetic determinants for incompatibility of miniplasmids and hybrid replicons constructed from wide type and mutant R1 revealed the presence of an incompatibility function at the junction f two small PstI fragments. These two fragments were not distinguished in earlier experiments since they have the same mobility on agarose gels. This incompatibility function is distinct from other inc-determinants of R1 (Kollek and Goebel 1979; Molin and Nordström, 1980) and independent of R1-type replication. By means of specific deletions and subcloning of DNA fragments, the location of this new inc-determinant could be determined further. After deletion of this inc-determinant from inc-determinant from miniplasmids, a 5-fold increase in copy number was observed which could then be reduced to a copy number of about 1 plasmid per cell by complementation with hybrid plasmids having this function. Incompatibility of miniplasmids deleted in this determinant is not reduced, whereas analogous deletions introduced into recombinant plasmids nearly abolished their incompatibility. This determinant seems to exert strong incompatibility only when cloned on pBR322. Therefore, its main function is plasmid R1 is probably restricted to copy control. The appearance of low copy numbers of of miniplasmids carrying this determinant and of trans-acting copy control and strong incompatibility exerted by hybrid plasmids is consistently correlated with the presence of a protein of 11,000 molecular weight, synthesized in relatively large amounts in Escherichia coli minicells.

Bacterial Proteins↗
PubMed1992

Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine.

The nuclear matrix was prepared in situ from Swiss 3T3 cells, which were synchronized by contact inhibition and serum starvation and pulse-labelled for very short periods of time with 5-bromodeoxyuridine (5-BrdU). For the first time 5-BrdU has been employed to demonstrate the association of newly synthesized DNA with a nucleoskeleton. Immunofluorescence analysis using a monoclonal antibody to 5-BrdU revealed five different intranuclear staining patterns at different stages of the S phase. These patterns were observed also in intact cells and did not change during the matrix preparation steps which involve extraction with 2 M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of fluorescence, which was evaluated by cytofluorometry, increased to reach a maximum during mid-S phase and then decreased. Because no significant difference was found in the time to label residual DNA of different 5-BrdU staining patterns, this strongly suggests that a different number of replicons is activated at different stages of the S phase. These results strengthen the hypothesis that eukaryotic DNA replication occurs in close association with an insoluble protein nuclear skeleton, which determines the three-dimensional spatial organization of chromosome duplication.

3T3 Cells↗
PubMedDate not supplied

Genetics of lactobacilli: plasmids and gene expression.

This paper reviews the present knowledge of the structure and properties of small (< 5 kb) plasmids present in Lactobacillus spp. The data show that plasmids from Lactobacillus spp., like many plasmids from other Gram-positive bacteria, display a modular organization and replicate by a mechanism of rolling circle replication. Structurally, plasmids from lactobacilli are closely related to plasmids from other Gram-positive bacteria. They contain elements (plus- and minus origin of replication, element(s) for control of plasmid replication, mobilization function) showing extensive similarity to analogous elements in plasmids from these other organisms. It is believed that lactobacilli have acquired such elements by intra- and/or intergenic transfer mechanisms. The first part of the review is concluded with a description of plasmid vectors with a Lactobacillus replicon and integrative vectors, including data concerning their structural and segregational stability. In the second part of this review we describe the progress that has been made during the last few years in identifying and characterizing elements that control expression of genetic information in lactobacilli. Based on the sequence of eleven identified and twenty presumed promoters, some preliminary conclusions can be drawn regarding the structure of Lactobacillus promoters. A typical Lactobacillus promoter shows significant similarity to promoters from E. coli and B. subtilis. An analysis of published sequences of seventy genes indicates that the region encompassing the translation start codon AUG also shows extensive similarity to that of E. coli and B. subtilis. Codon usage of Lactobacillus genes is not random and shows interspecies as well as intraspecies heterogeneity. Interspecies differences may, in part, be explained by differences in G+C content of different lactobacilli. Differences in gene expression levels can, to a large extent, account for intraspecies differences of codon usage bias. Finally, we review the knowledge that has become available concerning protein secretion and heterologous gene expression in lactobacilli. This part is concluded with a compilation of data on the expression in Lactobacillus of heterologous genes under the control of their own promoter or under control of a Lactobacillus promoter.

Bacillus subtilis↗
PubMed1992

High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors in Pseudomonas stutzeri.

A number of Escherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained in Pseudomonas stutzeri ATCC 17588. A restrictionless mutant of P. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With the E. coli cloning vector pHSG298, transformation frequencies of up to 2 x 10(7) transformants/micrograms DNA were achieved. This frequency is comparable to that obtained for CaCl2-mediated transformation of E. coli; thus, direct cloning of DNA into P. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G + C genera in cases in which E. coli is not a suitable heterologous cloning host.

Bacteriocin Plasmids↗
PubMed1996

Identification of a partition and replication region in the Alcaligenes eutrophus megaplasmid pMOL28.

A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 of Alcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed. Three genes involved in plasmid maintenance were identified: parA28 and parB28 are involved in plasmid partitioning and stability, while repA28 encodes a protein required for replication. In addition to the par AB28 genes, a third locus, parS28, required in cis active partitioning was identified. The parABS28 locus of pMOL28 shows strong similarity in organization to the sop, par and rep regions, respectively, of the Escherichia coli F-factor, the E.coli P1 and P7 prophages and the Agrobacterium pTiB6S3 and pRiA4b plasmids. The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of the Pseudomonas putida and Bacillus subtilis chromosomes. The functionality of the pMOL28 par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850 which contain the 4.64 EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate in A. eutrophus. The RepA2 8 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of this E. coli phage. The replication origin of pMOL28, oriV28, seems to be located within the repA28 coding region, and pMOL28 replication may depend on transcriptional activation of oriV28.

Alcaligenes↗
PubMed1996

Cloning of sucrase operon with mini-Mu and plasmid-mediated metabolism of sucrose.

The in vivo cloning system based on mini-Mu derivatives was used for cloning the sucrase operon. We constructed several recombinant plasmids pJT21, pMM2324, pMM2325 with a complete sucrase operon. Two strains of Escherichia coli containing this plasmid replicon were able to grow on different concentrations of sucrose. The stability of recombinant plasmids was determined after cultivation under nonselective conditions. The stability after 5-d cultivation was higher than 75%.

Bacteriophage mu↗
PubMed1990

Construction of a Co1E1 plasmid bearing inducible high-copy-number phenotype.

In order to construct plasmids bearing inducible high-copy-number phenotype, the cloning plasmid pBR322 was modified as follows: a DNA fragment containing a strong synthetic promoter (P1), synthetic lac operator (O1), DNA sequence corresponding to the RNAI/RNAII region of the Co1E1 replicon and the CAT gene transcription terminator was substituted for the 29 bp EcoRI/HindIII DNA fragment. Two types of plasmids were constructed in this way, differing in the orientation of the RNAI/RNAII fragment. Depending on the orientation these plasmids coded for RNA molecules representing either RNAI or RNAII domains. It was found that when RNAII molecules were overproduced the plasmid copy number was about 4 times higher than that of pBR322 and only negligible change in the plasmid copy-number value was observed upon overproduction of RNAI molecules.

Bacteriocin Plasmids↗
PubMed1991

Genetic transformation of xylose-fermenting yeast Pichia stipitis. Scientific note.

A plasmid-mediated transformation system has been developed for the xylose-fermenting yeast Pichia stipitis. We found that plasmid vectors containing the Saccharomyces cerevisiae 2 mu replicon and the kanamycin resistance gene (KmR) could be introduced into the Pichia cells and maintained as extrachromosomal elements. Pichia transformants containing such vectors will be resistant to the antibiotic geneticin that can be inactivated by the protein product of KmR. Plasmids identical to those used for transformation can be recovered from the Pichia transformants. Protocols for transformation of P. stipitis by the CaCl2-polyethylene glycol-protoplast process or by direct electroporation of intact Pichia cells have both been developed.

Escherichia coli↗
PubMed2001

Inheritance of the replication complex: a unique or common phenomenon in the control of DNA replication?

Early models of the regulation of initiation of DNA replication by protein complexes predicted that binding of a replication initiator protein to a replicator region is required for initiation of each DNA replication round, since after the initiation event the replication initiator should dissociate from DNA. It was, therefore, assumed that binding of the replication initiator is a signal for triggering DNA replication. However, more recent investigations have revealed that in many replicons this is not the case. Studies on the regulation of the replication of plasmids derived from bacteriophage lambda demonstrated that, once assembled, the replication complex can be inherited by one of the two daughter plasmid copies after each replication round and may function in subsequent replication rounds. Since this DNA-bound protein complex bears information about specific initiation of DNA replication, this phenomenon has been called "protein inheritance." A similar phenomenon has recently been reported for oriJ-based plasmids. Moreover, the current model of the initiation of DNA replication in the yeast Saccharomyces cerevisiae proposes that the origin recognition complex (ORC) remains bound to one copy of the ori sequence (the ARS region) after initiation of DNA replication. Thus, it seems plausible that protein inheritance is not unique for lambda plasmids, but may be a common phenomenon in the control of DNA replication, at least in microbes.

Bacteriophage lambda↗
PubMed2003

Host-vector system for phenol-degrading Rhodococcus erythropolis based on Corynebacterium plasmids.

The strain Rhodococcus erythropolis CCM2595, which was shown to degrade phenol, was chosen for genetic studies. To facilitate strain improvement using the methods of gene manipulation, the technique of genetic transfer was introduced and cloning vectors were constructed. Using the plasmid pFAJ2574, an electrotransformation procedure yielding up to 7x10(4) transformants/microg DNA was optimized. Escherichia coli- R. erythropolis shuttle vectors were constructed using the replicons pSR1 and pGA1 from Corynebacterium glutamicum. The small vector pSRK21 (5.8 kb) provides six unique cloning sites and selection of recombinant clones using alpha-complementation of beta-galactosidase in E. coli. This vector, exhibiting high segregational stability under non-selective conditions in R. erythropolis CCM2595, was applied to cloning and efficient expression of the gene coding for green fluorescent protein (gfpuv).

Base Sequence↗
PubMed2005

Occurrence of pS86/pEF47-related plasmids in gram-positive cocci.

A small, low-copy-number cryptic 5.5-kb plasmid, designated pEF47, was isolated from a rumen bacterium Enterococcus faecalis 47 and partially characterized. Comparisons by restriction and partial sequence analysis of pEF47 demonstrated high homology to pS86 plasmid, recently isolated from a human clinical strain E. faecalis S-86. PCR followed by sequence analysis and Southern hybridization showed that pS86/pEF47-related replicons are regularly encountered in plasmids from gram-positive cocci.

Animals↗
PubMed2003

Ionizing irradiation effects on S-phase in checkpoint mutants of the yeast Saccharomyces cerevisiae.

In mammalian cells, gamma-irradiation activates checkpoint controls to delay entry into, or passage through S-phase, while chronic exposure to methyl methanesulfonate or hydroxyurea causes a similar delay in yeast. In yeast, at least five genes are involved: RAD9, RAD17, RAD24, RAD53 and MEC1, a homologue of ATM. Here, using flow cytometry analysis and alkaline sucrose gradient centrifugation of labeled, newly made DNA, we demonstrate, in synchronized RAD wild-type Saccharomyces cerevisiae cells, that: (1) gamma-irradiation at START delays entry into S-phase, (2) irradiation shortly before or during early S-phase delays completion of S-phase and (3) the latter response is largely a consequence of replicon initiation inhibition. The delay produced by irradiation during early S-phase depends on the function of the checkpoint genes RAD9, RAD17, RAD24, RAD53, MEC1 and MEC3. However, at least four, RAD17, RAD53, MEC1, MEC3, are not needed to delay S-phase progression when cells are irradiated shortly before S-phase begins.

DNA↗
PubMed2006

The temporal program of DNA replication: new insights into old questions.

During the last decades it has been shown that the replication timing program in metazoans is related to chromosome structure, the nuclear positioning and AT/GC content of chromosomal loci, their patterns of histone modifications, and their transcriptional regulation. Here, the current state of knowledge concerning these relationships is reviewed. An integrated view on structure-function relationships in the nucleus is provided and the determination and functional role of the replication timing program is discussed in this context. A corresponding comprehensive model is developed and a key aspect of this model is the suggestion that mammalian chromosomes are organized into stable units equivalent to replicon clusters. It is proposed that the nuclear positions of these units would depend on their histone modifications and determine the replication timing of the whole unit. It is furthermore predicted that replication timing is only indirectly linked to transcriptional regulation and contributes to the maintenance of gene expression patterns. These clear predictions, and the fact that the tools are at hand now to further test them, open an avenue towards solving the long standing problem on how replication timing is determined in metazoan cells.

Animals↗
PubMed1997

Molecular analysis of the replication region of the theta-replicating plasmid pUCL287 from Tetragenococcus (Pediococcus) halophilus ATCC33315.

The complete nucleotide sequence of the 8.7-kb theta-replicating plasmid pUCL287 from Tetragenococcus halophilus (formerly Pediococcus halophilus) ATCC33315 has been determined. The replication region was identified and analyzed. Its nucleotide sequence contains an untranslated region, the replication origin, followed by two open reading frames (ORFs) encoding two proteins of 311 (RepA287) and 168 (RepB287) amino acids, respectively. Evidence is presented to show that RepA287 represents the plasmid replication protein. RepB287, which is non-essential for replication, is involved in the plasmid copy-number control and segregational stability. The roles of lactococcal proteins homologous to RepB287 have not been defined so far. Nevertheless, the structural organization of the pUCL287 replication region is remarkably similar to those of well known theta-replicating lactococcal plasmids despite the absence of homology of the replication origin and of the replication protein, and this suggests that pUCL287 uses the same mechanism of replication. Nucleotide sequence comparisons show that pSMB74, a pediococcal plasmid encoding bacteriocin production, is a member of the pUCL287 replicon family.

Amino Acid Sequence↗