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PubMedDate not supplied

[Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12].

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed.

Chromosome Deletion↗
PubMed1986

[Behavior of autonomous plasmids and plasmids integrated into the chromosome during the sporulation of Bacillus subtilis cells].

The behaviour of plasmids in free and integrated states was studied upon sporulation of Bacillus subtilis cells. Autonomous plasmids pBD12 and pGG10 were shown to be either transmitted into spores in small copy numbers or completely eliminated from the sporulating cell. However, insertion of the autonomous plasmid into the host chromosome may occur with a certain degree of probability (about 10(-3)) during sporulation. When in the integrated state, pBD12 plasmid may either excise from the host chromosome or amplify within the genome with the probability 1.8-2.10(-3) in the course of sporulation. The pGG102 plasmid carrying the fragment of wheat DNA and integrated by this fragment into the chromosome was shown to enter spores without whichever intragenome rearrangements.

Bacillus subtilis↗
PubMed1987

[Genetic study of plasmid integration into yeast chromosomes. V. Mapping of integration sites for the plasmid pYF91].

The data on mapping the episomal plasmid integration sites in yeast chromosomes I, III, IV, V, VII, XV are presented. In addition to the integration site at leu2 of chromosome III localized earlier, 6 more loci containing apparently the homologous yeast transposons, with a copy in a plasmid, were defined. The fact of plasmid integration was proved by colony hybridization technique with the pBR322 probe. The plasmid DNA segregation (the ratio 2:2) and its linkage to pLEU2 plasmid marker gene were observed in hybrids of all integrants studied.

Chromosome Mapping↗
PubMed1987

[Genetic study of plasmid integration into yeast chromosomes. IV. Integration of the plasmid pYF91 into different yeast chromosomes].

Integration of the episomic chimeric plasmid pYF91 into yeast chromosomes has been studied. Plasmid insertion into the chromosomes was observed to occur with the frequency of 4 X 10(-8). 379 integrants were selected from the highly unstable (cir0) transformants. The fact of plasmid integration into particular chromosomes was confirmed for 318 integrants. Genetic analysis showed that the plasmid can integrate into the region of LEU2 gene or into another arm of chromosome III (227 integrants), and also into other chromosomes: I, II, IV, V, VI, VII, VIII, IX, XII, XV (91 integrants). It is suggested that integration is the result of recombination between yeast chromosomes and homologous plasmid regions carrying LEU2 gene or Ty element and "delta" sequence.

DNA, Fungal↗
PubMed1985

[Induced mutagenesis of plasmid as well as chromosomal genes inserted into plasmid DNA. II. The mutagenic action of chemical factors].

To proceed the works on induced mutagenesis in plasmids, mutagenic effects of chemicals on the DNA of RSF2124 plasmid mediating colicine E1 biosynthesis and resistance to ampicillin, were studied. After exposure to mutagens, plasmid DNA was used to transform Escherichia coli C600 rk-mk-cells. The lethal effect was estimated from inactivation of the ampicillin marker, the mutagenic effect being measured by the appearance of mutants unable to synthesize colicine (Col-). The reaction of the plasmid DNA with a mutagen was stopped by 10-fold dilutions of aliquots in TEN buffer, followed by dialysis in 10 mH CaCl2 for 24 h. To select the most efficient mutagens for plasmid DNA, the compounds were predominantly tested which are known to be effective in other systems (transforming and transfecting DNA, microbial viruses). An a result, all chemicals tested by their activity were classified into 4 groups: inducing more than 100 fold increase (hydroxylamine, O-methylhydroxylamine); inducing 10 fold increase (UV-irradiation, lambda = 254 nm; W-mutagenesis, gamma-irradiation, nitrous acid, mitomycin C); inducing less than 10fold increase (indirect UV-mutagenesis, nitrous acid, beta-chloroethyldiethylamine hydrochloride, nitrosoguanidine); no mutagenic effect (acridine orange, ethyl methane sulfonate, sodium azide, O-beta-diethylhydroxylamine).

Chromosomes, Bacterial↗
PubMed1981

[Plasmid pKM101 sensitization of Escherichia coli strains to the action of ionizing radiation: the effect of the plasmid on survivability and induced mutagenesis].

The wild type Escherichia coli K-12 has been shown to be sensitized to inactivation by gamma-irradiation by the plasmid pKM101. The dnaA strains of E. coli are more sensitive to gamma-rays killing effect, as compared with the wild type E. coli, pKM101 plasmid showing only slight sensitizing effect. "Cis" or "trans" position of the plasmid in relation to the chromosome plays no role in sensitization, while the plasmid effect on UV-induced killing and mutability depends on "trans" position of the plasmid before irradiation. gamma-Rays induced mutability to prototrophy is completely dependent on the presence of pKM101 in "trans" in wild type and dnaA strains before irradiation.

Chromosomes, Bacterial↗
PubMed1994

ScreenMax plasmid mini-prep: super rapid plasmid DNA extraction method.

An ultra-quick method for plasmid DNA extraction using the ScreenMax Plasmid Mini-prep Kit was optimized. Since the method has fewer steps than current methods, the entire process takes only 12 min for two sets of plasmid DNA extractions. DNA quality was excellent for further analyses, including DNA sequencing. The kit's new medium, MMB, provided 5 times the viable cell count and 7 times the dry cell weight compared with the conventional LB medium. The yield of plasmid DNA was 5 times better than from LB medium. From 200 microL of culture, the maximum yield was 7.5 micrograms. The ScreenMax Plasmid Mini-Prep is not only simple and economical but also safe because of its mild reagents. This new procedure is both reliable and reproducible.

DNA↗
PubMed1996

Structure of a Gelasinospora linear plasmid closely related to the kalilo plasmid of Neurospora intermedia.

We have determined the complete nucleotide sequence of a linear mitochondrial plasmid from a natural isolate of a homothallic species of Gelasinospora. The plasmid genome is 8231 bp long. It carries terminal inverted repeats of 1137 bp. Extending inwards from the terminal repeats are two long open reading frames coding for putative proteins with similarity to DNA and RNA polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of the Neurospora intermedia senescence-plasmid kalilo. Overall the two plasmids have a similar genetic organization and are clearly homologous at the sequence level. The main differences are in the intergenic region and in the terminal repeats.

Amino Acid Sequence↗
PubMed1997

Enhancement of cell-mediated immunity against HIV-1 induced by coinnoculation of plasmid-encoded HIV-1 antigen with plasmid expressing IL-12.

Previous investigations have demonstrated that CTL may play an important role in suppressing the disease progression of HIV infection. In this study, we inoculated mice with IL-12 expression plasmid together with plasmid-encoding HIV-1 envelope to enhance CTL activity by activating a Th1-type response. The results of delayed-type hypersensitivity using the footpad swelling response and of CTL activity clearly showed that HIV-1-specific cell-mediated immunity was enhanced by inoculation of the IL-12 expression plasmid. Quantitation of cytokine in the sera of IL-12-inoculated mice revealed that IFN-gamma significantly increased. The enhanced cell-mediated immunity responses were abrogated by combined administration of the IL-12 expression plasmid and neutralizing anti-IFN-gamma Ab. Together, these results suggest that enhanced virus-specific cell-mediated immunity occurred via an endogenously produced IFN-gamma by inoculation of IL-12 expression plasmid.

AIDS Vaccines↗
PubMed1987

Identification and mapping of regions that confer plasmid functions and of sites for excisive recombination of plasmid pMMC7105.

Strain PP808 of Pseudomonas syringae pv. phaseolicola contains pEXC8080 (34.6 kb), the smallest of several plasmids that originated by partial excision of the cryptic plasmid, pMMC7105 (150 kb), from the host chromosome. This excision plasmid is derived entirely of sequences from pMMC7105 and contains a 24 kb region referred to as common DNA, which is present in each of the other excision plasmids. A six enzyme restriction endonuclease map was constructed of pEXC8080. The replication region was mapped by identifying small restriction fragments that conferred replication properties to pMB1 plasmids that otherwise fail to replicate in Pseudomonas. This region is located within the common DNA and is 0.8-3.8 kb in size. Sequences from pEXC8080 failed to stabilize pMB1 derivatives in Pseudomonas in the absence of antibiotic selection, but stability functions were mapped to a region of pMMC7105 that presumably remains integrated in the chromosome of strain PP808. An incompatibility region was mapped to a 7.3 kb region on pEXC8080 that is closely linked to, but not included within, the replication region. The recombination site was mapped to a 1.2 kb region of the fusion fragment that was formed upon excision of pEXC8080. RS-I, a repetitive sequence found on pMMC7105 was present in the fusion fragment at the site of recombination. RS-I was also mapped to BamHI fragments that recombined upon excision of pEXC8080 and suggest that it provides sites for homologous recombination.

Anti-Bacterial Agents↗
PubMed1981

Regulation of plasmid DNA synthesis: isolation and characterization of copy number mutants of miniR6-5 and miniF plasmids.

Copy number (Cop) mutants of miniR6-5 and miniF plasmid derivatives containing a beta-lactamase gene were isolated by selection for increased ampicillin-resistance. Mutants which exhibited an increased copy number and a reduced incompatibility response as compared to the respective parent mini-plasmid were obtained from both miniR6-5 and miniF. Characterization of these mutant plasmids has provided the first description of the replicative properties of miniF Cop mutants, and has also facilitated a comparison of plasmids representing the IncFI and IncFII incompatibility groups. Cop mutants from these groups differed in several respects: (i) MiniF Cop mutants were considerably more difficult to obtain and showed a markedly lower transforming efficiency than the corresponding miniR6-5 mutants. (ii) MiniR6-5 Cop mutants were stably maintained in a polA1 strain without selective pressure, whereas miniF Cop mutants severely reduced the viability of this host. (iii) MiniR6-5 replication stopped within a few minutes after inhibition of protein synthesis, whereas miniF replication continued at a declining rate for about one hour in the presence of chloramphenicol. (iv) In contrast to miniR6-5 replication, miniF DNA synthesis was blocked faster by rifampicin than by chloramphenicol. (v) The copy number of miniR6-5 plasmids (but not of miniF) was reduced by about 50% in an rnc strain deficient in RNAase III.

Ampicillin↗
PubMed1983

The repA2 gene of the plasmid R100.1 encodes a repressor of plasmid replication.

We have constructed two miniplasmids, derived from the resistance plasmid R100.1. In one of these plasmids 400 bp of R100.1 DNA have been replaced by DNA from the transposon Tn1000 (gamma-delta). This substitution removes the amino-terminal end of the repA2 coding sequence of R100.1 and results in an increased copy number of the plasmid carrying the substitution. The copy number of the substituted plasmid is reduced to normal levels in the presence of R100.1. The repA2 gene thus encodes a trans-acting repressor function involved in the control of plasmid replication.

DNA Replication↗
PubMed1986

Dispersal of a plasmid-borne chloramphenicol resistance gene in streptococcal and enterococcal plasmids.

Plasmids coding for chloramphenicol resistance, five isolated from streptococci of groups A, B, and G, ten from enterococci (Enterococcus faecalis, Enterococcus faecium), and two from staphylococci, were tested for sequence homology with the chloramphenicol resistance gene of pIP501, a 30-kb plasmid originally isolated from a group B Streptococcus. The 6.3-kb HindIII fragment of pIP501, known to carry the chloramphenicol resistance gene, was cloned into pBR322. A 1.6-kb portion of the cloned fragment, which included most of the chloramphenicol resistance gene, was used as probe in DNA-DNA hybridization experiments. Sequence homology was detected between the probe and four of the streptococcal, seven of the enterococcal, and one of the staphylococcal plasmids. The absence of hybridization between this probe and one plasmid isolated from a group B Streptococcus, as well as three isolated from E. faecalis, indicated that there are at least two different plasmid-borne chloramphenicol resistance determinants in the streptococci and in the enterococci.

Chloramphenicol↗
PubMed1984

Conjugation of plasmids of Neisseria gonorrhoeae to other Neisseria species: potential reservoirs for the beta-lactamase plasmid.

The discovery that penicillinase production in Neisseria gonorrhoeae was plasmid mediated and the spread of the beta-lactamase encoding plasmids in gonococcal isolates since 1976, raise the possibility that a nonpathogenic indigenous bacterium could serve as a reservoir for these plasmids. We initiated studies to define the ability of commensal Neisseria species and Branhamella catarrhalis strains, as well as strains of the pathogen Neisseria meningitidis, to serve as recipients in conjugation with Neisseria gonorrhoeae. We found that with N. gonorrhoeae as the donor, 3 of 5 Neisseria cinerea, 2 of 5 Neisseria flava, 0 of 1 Neisseria flavescens, 1 of 3 Neisseria subflava, 0 of 6 B. catarrhalis, 0 of 7 Neisseria lactamica, 1 of 5 Neisseria mucosa, 1 of 7 Neisseria perflava/sicca, and 0 of 13 N. meningitidis strains gave detectable conjugation frequencies (greater than 10(-8). N. cinerea was the only species found to maintain the gonococcal conjugal plasmid (pLE2451). A N. cinerea transconjugant containing pLE2451 was observed to transfer both the beta-lactamase plasmid and pLE2451 to N. gonorrhoeae at high frequency.

Conjugation, Genetic↗
PubMed1980

Parameters controlling interbacterial plasmid spreading in a gnotoxenic chicken gut system: influence of plasmid and bacterial mutations.

Conjugative transfer of R plasmids R64 and R64drd-11 has been compared in vitro and in vivo without selective pressure by antibiotics in a simplified experimental system; the ecosystem was the bowel of germfree chickens, with the host bacteria almost isogenic, and the plasmids differing only in their conjugative transfer frequency. The spread of repressed and derepressed (drd) R plasmids in recipient bacterial populations was very extensive. The repressed phenotype had only a transient effect during the first 4 h. The level of implantation of the donor bacterial population seems to be of minor importance. Only with a poor recipient (con strain) could the spread of R plasmids be reduced and a steady state with a predominantly sensitive bacterial population be established. It is suggested that this steady state results from an equilibrium between the frequencies of R plasmid transfer and loss.

Animals↗
PubMed1989

Resistance against Industrial Bacteriophages Conferred on Lactococci by Plasmid pAJ1106 and Related Plasmids.

Plasmid pAJ1106 and its deletion derivative, plasmid pAJ2074, conferred lactose-fermenting ability (Lac) and bacteriophage resistance (Hsp) at 30 degrees C to Lac proteinase (Prt)-negative Lactococcus lactis subsp. lactis and L. lactis subsp. lactis var. diacetylactis recipient strains. An additional plasmid, pAJ331, isolated from the original source strain of pAJ1106, retained Hsp and conjugative ability without Lac. pAJ331 was conjugally transferred to two L. lactis subsp. lactis and one L. lactis subsp. cremoris starter strains. The transconjugants from such crosses acquired resistance to the phages which propagated on the parent recipient strains. Of 10 transconjugant strains carrying pAJ1106 or one of the related plasmids, 8 remained insensitive to phages through five activity test cycles in which cultures were exposed to a large number of industrial phages at incubation temperatures used in lactic casein manufacture. Three of ten strains remained phage insensitive through five cycles of a cheesemaking activity test in which cultures were exposed to approximately 80 different phages through cheesemaking temperatures. Three phages which propagated on transconjugant strains during cheesemaking activity tests were studied in detail. Two were similar (prolate) in morphology and by DNA homology to phages which were shown to be sensitive to the plasmid-encoded phage resistance mechanism. The third phage was a long-tailed, small isometric phage of a type rarely found in New Zealand cheese wheys. The phage resistance mechanism was partially inactivated in most strains at 37 degrees C.

Journal Article↗
PubMed1977

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.

Ampicillin↗
PubMed1989

Comparison of tellurite resistance determinants from the IncP alpha plasmid RP4Ter and the IncHII plasmid pHH1508a.

The tellurite resistance (Ter) determinants of the IncHII plasmid pHH1508a and the broad host range IncP alpha plasmid RP4Ter were cloned into pUC8, creating plasmids pDT1364 and pDT1558, respectively. The Ter region of pDT1364 was localized to a 1.25-kilobase region by using Tn1000 insertion mutagenesis. Insertions of Tn1000 into pDT1558 which resulted in tellurite sensitivity spanned 1.75 kilobases of DNA. No similarity between the restriction maps of these two plasmids was observed, and no homology could be detected by DNA-DNA hybridization. Expression in an in vitro transcription-translation system showed that pDT1364 encoded two polypeptides with molecular masses of 23 and 12 kilodaltons (kDa) which were not expressed by pUC8. Some of the Tn1000 insertion mutants did not express the 23-kDa protein. pDT1558 encoded a 40-kDa polypeptide which was not expressed by pUC8. Both Ter determinants were expressed constitutively. Our findings suggest that the mechanisms of Ter encoded by these two plasmids are different.

Bacterial Proteins↗