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D D Womble

Publications and source records attributed to D D Womble.

32 records · Page 2Linked to original sources
PubMed1986

Regulation of IncFII plasmid DNA replication. A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle.

A quantitative model for the regulation of replication of the low copy number IncFII plasmid NR1 in the Escherichia coli cell division cycle has been developed. The initiation of NR1 replication requires a cis-acting initiator protein whose synthesis is regulated by several mechanisms. The NR1 regulatory processes include co-operative protein-protein interactions in the formation of an active transcription repressor, the interaction of repressor with a rightward operator site in the control of transcription of the initiator gene, and the interaction of an inhibitor RNA transcript with the initiator mRNA in the control of translation of the initiation protein. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to the events of the cell cycle, such as initiation of mRNA transcription and protein translation, and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and the intracellular concentrations of repressor protein and mRNA agreed with experimentally determined estimates. A number of different copy number mutants that have altered one or another of the regulatory processes were simulated by the model. The contributions of each of the regulatory processes toward the overall stability of inheritance of plasmid NR1 in a population of cells in culture were examined. These simulations predict a very stable pattern of inheritance for plasmid NR1 despite its low copy number, in agreement with experimental observation.

Cell Cycle↗
PubMed1986

Regulation of lambda dv plasmid DNA replication. A quantitative model for control of plasmid lambda dv replication in the bacterial cell division cycle.

A quantitative model for the regulation of replication of plasmid lambda dv in the Escherichia coli cell division cycle has been developed. The regulatory processes include the interactions of cro repressor proteins with the rightward operator DNA sites, the transcriptional activation of the lambda dv replication origin, and the interaction of initiation proteins with activated origins to form functional replication initiation complexes. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to the events of the cell cycle such as initiation of mRNA transcription and protein translation and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and repressor and initiator protein concentrations of the model agreed with experimentally determined estimates. Simulated deviations from regular segregation of the various components at cell division, such as plasmid copies and free and bound repressor proteins, suggest that lambda dv replication control responds only slowly to these perturbations. The consequence of this slow response to perturbations, which are expected at a random frequency, was simulated for a population of lambda dv-containing cells in a growing culture. This simulation predicts instability of inheritance of lambda dv plasmids in the population, despite the very high plasmid copy number, in agreement with experimental observation.

Bacteriophage lambda↗
PubMed1985

Transcription of the replication control region of the IncFII R-plasmid NR1 in vitro and in vivo.

The minimal replicon of the 90,000 base-pair IncFII R plasmid NR1 consists of a 2700 base-pair region of the DNA. Minireplicator plasmids consisting of the 2700 base-pair minimal replicon plus a 2200 base-pair region coding for chloramphenicol acetyltransferase (cat) were used as templates for in vitro transcription. Six RNA transcripts were synthesized from these templates in vitro. We have determined the directions of transcription and the approximate sites of initiation and termination of each of the in vitro RNA transcripts. One RNA transcript was synthesized from the cat gene, while the other five were transcribed from the minimal replicon. Four of the RNA transcripts also were identified by quantitative hybridization of RNA synthesized in vivo from these minireplicator plasmids. The strengths of the promoters for the RNA transcripts were estimated by the relative rates of transcription both in vitro and in vivo. Transcription from convergent promoters reduced the rate of RNA synthesis in vivo in both directions. In vivo, a significant fraction of the cat mRNA was extended past its in vitro termination point. Transcription of mutants that have altered plasmid copy number and/or incompatibility properties also were examined. The possible roles of each of the transcripts as mRNA and their involvement in regulation of DNA replication are discussed.

Base Sequence↗
PubMed1985

Incompatibility and IncFII plasmid replication control.

The DNA coding for replication control and incompatibility of the plasmid NR1 serves as a template in vivo and in vitro for RNA transcription in both directions. In the rightward direction, RNA synthesis begins from 2 different promoters, one of which is regulated and the other constitutive. In vivo, each of these transcripts is more than 1,000 nucleotides long, terminating near the estimated site for the origin of replication. These transcripts serve as messenger RNA for several proteins. One protein (repA1) is required for replication and another (repA2) serves as the repressor for the regulated rightward promoter. RNA synthesis in the leftward direction is constitutive and produces a single transcript of 91 nucleotides which is complementary in sequence to the rightward transcripts. This small transcript is the incompatibility product which regulates the replication of the plasmid. When the intracellular concentration of the small transcript is experimentally varied, the rate of translation of the rightward transcripts and the rate of initiation of replication (plasmid copy number) vary inversely to its concentration. The mode of action of this inhibitor RNA is likely to be formation of an RNA-RNA duplex with the rightward transcripts, thereby inhibiting the translation which would produce the required replication protein. The probability that a rightward transcript will escape interaction with the small RNA molecules and thus allow replication to initiate can be predicted from the laws of mass action based on base-stacking free energies for the likely sequences of initial contact. The estimated intracellular RNA concentrations, based on quantitative hybridization experiments, are agreement with those predicted from the calculated equilibrium constants for duplex formation.

Bacteriophage lambda↗
PubMed1985

Analysis of the individual regulatory components of the IncFII plasmid replication control system.

Replication of the IncFII plasmid NR1 is controlled by regulating the amount of synthesis of the repA1 initiator protein at both the transcriptional and translational levels. We have examined mutations which have altered each of these levels of regulation, resulting in different plasmid copy numbers. The genes which encode each of the individual wild-type or mutant regulatory components from the replication control region of NR1 have been cloned independently into pBR322 vectors, and their effects in trans, either individually or in various combinations, on plasmid incompatibility, stability, copy number, and repA1 gene expression have been defined.

Adenosine Triphosphatases↗
PubMed1985

Regulation of transcription of the repA1 gene in the replication control region of IncFII plasmid NR1 by gene dosage of the repA2 transcription repressor protein.

Transcription of the repA1 gene of the IncFII plasmid NR1 is initiated at two promoters in the replication control region. Transcription from the upstream promoter is constitutive at a low level, whereas transcription from the downstream promoter is regulated. The 5' end of the constitutively synthesized transcript also encodes the transcription repressor protein for the regulated downstream promoter. Therefore, the level of the repressor protein in the cell is gene dosage dependent. Using both lac gene fusions and quantitative hybridization methods, we have determined the in vivo relationship between the rate of transcription from the regulated promoter and the repressor protein concentration as a function of gene dosage. At the wild-type copy number of NR1, transcription from the regulated promoter is 96% repressed, but substantial derepression occurs when the copy number falls below the normal value. At or above the normal plasmid copy number, the basal level of repA1 mRNA is provided by transcription from the constitutive upstream promoter.

Adenosine Triphosphatases↗
PubMed1985

Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control.

DNA from the replication control region of plasmid NR1 or of the Inc- copy mutant pRR12 was cloned into a pBR322 vector plasmid. These pBR322 derivatives were mutagenized in vitro with hydroxylamine and transformed into Escherichia coli cells that harbored either NR1 or pRR12. After selection for the newly introduced pBR322 derivatives only, those cells which retained the unselected resident NR1 or pRR12 plasmids were examined further. By this process, 134 plasmids with Inc- mutations in the cloned NR1 or pRR12 DNA were obtained. These mutants fell into 11 classes. Two of the classes had plasmids with deletions or insertions in the NR1 DNA and were not examined further. Plasmids with apparent point mutations were classified by examining (i) their ability to reconstitute a functional NR1-derived replicon (Rep+ or Rep-), (ii) the copy numbers of the Rep+ reconstituted replicons, (iii) the cross-reactivity of incompatability among the various mutant classes and parental plasmids, and (iv) the trans effects of the mutants on the copy number and stable inheritance of a coresident plasmid.

Cloning, Molecular↗
PubMed1984

IncFII plasmid incompatibility product and its target are both RNA transcripts.

The region of DNA coding for incompatibility (inc) and copy number control (cop) of the IncFII plasmid NR1 is transcribed in both the rightward and leftward directions. The rightward transcripts serve as mRNA for the repA1 protein, which is required for replication. A small, 91-base leftward transcript is synthesized from the opposite DNA strand and is complementary to a portion of the rightward mRNA near its 5' end. A 262-base-pair Sau3A restriction fragment that encodes the small leftward transcript, but does not include the rightward transcription promoters, was cloned into the vector pBR322 or pUC8. The same fragment was cloned from an Inc- mutant of NR1 that does not make the small leftward transcript. Transcription through the cloned fragments in these derivatives was under control of the tetracycline resistance gene in pBR322 or the lac promoter-operator in pUC8. In one orientation of the inserted DNA, a hybrid transcript containing rightward NR1 RNA sequences was synthesized. In the other orientation, a hybrid transcript containing leftward NR1 RNA sequences was synthesized. These plasmids were used to vary the intracellular levels of the rightward or leftward NR1 RNA transcripts and to test their effects in trans on various coresident derivatives of NR1. An excess of rightward NR1 RNA in trans stimulated expression of the essential repA1 gene and caused an increase in the copy number of a coresident NR1 plasmid. An excess of leftward NR1 RNA in trans inhibited the expression of the repA1 gene and lowered the coresident NR1 copy number, thereby causing incompatibility. A pBR322 derivative with no transcription through the cloned NR1 DNA had no effect in trans. These results suggest that the small leftward transcript is the incompatibility inhibitor of NR1 and that its target is the complementary portion of the rightward mRNA.

Base Sequence↗
PubMed1978

Replication of plasmid DNA in Proteus mirabilis in limiting concentrations of thymine.

Replicating forms of the R plasmid pRR12 and the colicin E1 plasmid RSF2124 were isolated from Proteus mirabilis after growth in medium containing a limiting concentration of thymine. Both plasmids were replicated as partially supercoiled intermediates, which have densities between the values of covalently closed circular and nicked circular plasmid DNA in ethidium bromide-cesium chloride gradients. In addition, both plasmids had replication intermediates, which have densities lower than that of linear P. mirabilis chromosomal DNA. Some structural features of these replication intermediates were examined.

DNA Replication↗
PubMed1977

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.

Centrifugation, Density Gradient↗
PubMed1977

Properties of the relaxation complex of supercoiled deoxyribonucleic acid and protein of R plasmid NR1 in Escherichia coli.

Some properties of the supercoiled deoxyribonucleic acid (DNA)-protein relaxation complex of the R plasmid NR1, which contains more than one origin for DNA replication, were examined. The percentage of complexed NR1 molecules that can be converted to the relaxed (nicked) form appeared to be unaffected by the conditions under which the host cells were cultured. However, the percentage of supercoiled NR1 DNA that can be relaxed was highly dependent on the method used to prepare the DNA and the agents used to induce relaxation. Our data suggest that 100% of NR1 molecules may exist in situ as DNA-protein relaxation complexes. An RTF-Tc segregant of NR1, which has deleted the r-determinants component of the NR1 and therefore does not contain the two origins of replication located in the r-determinants, has indistinguishable relaxation properties in comparison with NR1 itself.

Bacterial Proteins↗