PubMed Health⌕ Search

Biomedical subjects

R H Rownd

Publications and source records attributed to R H Rownd.

At least 19 recordsLinked to original sources
PubMed1992

Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide.

Examination of a group of mutants of plasmid NR1 that had lost the expression of IncFII plasmid incompatibility (Inc-) revealed a group that had also lost replication proficiency (Rep-). These mutants were obtained from plasmids in which the NR1 replication control region was present in a cointegrate with plasmid pBR322. Whereas the wild-type parental cointegrate plasmid was capable of replicating in a polA host owing to the PolA independence of NR1 replication, the mutants were not able to transform a polA host. Losses of both expression of IncFII plasmid incompatibility and replication proficiency were found to result from the same single base-pair substitution in four independently isolated Inc- Rep- mutants. The mutation inactivates promoter PE for the transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. Although the loss of RNA-E synthesis had been expected to increase the expression of repA1, the efficiency of translation of repA1 mRNA from these mutants was at least 100-fold lower than that from the wild type, as revealed by repA1-lacZ translational fusions. The PE mutation introduced a stop codon into a 24-amino-acid reading frame that precedes the repA1 gene and terminates just 2 bp downstream from the repA1 start codon. This putative leader peptide was also expressed in a lacZ translational fusion, and its expression was reduced by a factor of 10(4) by the PE mutation. The expression of the leader peptide and the expression of repA1 were regulated by RNA-E. These results suggest that the expression of repA1 is coupled to the translation of the leader peptide and that the repression of repA1 translation by RNA-E may occur via inhibition of the translation of the leader peptide.

Amino Acid Sequence↗
PubMed1992

Autoregulation of the stability operon of IncFII plasmid NR1.

The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB. Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated. A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold. The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid. StbB protein by itself had autorepressor activity. Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone. Plasmids with a mutation in stbA had reduced repressor activity. One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity. Repressor activity of the mutant StbB protein was effectively enhanced by stbA. These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein.

DNA Transposable Elements↗
PubMed1991

Transcription of the stability operon of IncFII plasmid NR1.

The stability (stb) locus of IncFII plasmid NR1 is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins. The stb locus was found to be transcribed from a promoter site just upstream from the first gene, stbA. This promoter was active for transcription both in vivo and in vitro and was located within the region that includes the essential cis-acting site. Transcripts initiated from this site were approximately 1,500 to 1,600 nucleotides in length. Northern (RNA) blot analysis indicated that the transcripts traversed both stbA and the downstream gene, stbB. Mutants from which the promoter had been deleted failed to produce detectable transcripts from either stbA or stbB. Transcription of a third open reading frame, stbC, which is contained within the stbB gene in the opposite DNA strand, could not be detected. For a mutant in which a transposon had been inserted in stbA, no transcription of stbB was detected. After deletion of most of the transposon, which left behind a 35-bp frameshift insertion in stbA, transcription of stbB was restored, although the insertion still had a polar effect on stbB function. The rate of in vivo transcription of the stb locus was measured by pulse-labeling of RNA followed by quantitative RNA-DNA hybridization. Mutants deleted of stbB had an approximately 10-fold increase in the rate of transcription, whereas those deleted of the promoter region had at least a 10-fold reduction in transcription rate. The half-life of stb mRNA was approximately 2 min. These data suggest that stbA and stbB are cotranscribed as an operon that may be autoregulated.

Blotting, Northern↗
PubMed1989

DnaA protein is not essential for replication of IncFII plasmid NR1.

By transformation of dnaA null mutant host cells that are suppressed either by an rnh mutation or by chromosomal integration of a mini-R1 plasmid, it was shown that replication of miniplasmids composed of the NR1 minimal replicon had no absolute dependence upon DnaA protein. In addition, the suppression of the dnaA null mutation by the integrated mini-R1, which is an IncFII relative of NR1, was found to be sensitive to the expression of IncFII-specific plasmid incompatibility. This suggests that the integrative suppression by mini-R1 is under the control of the normal IncFII plasmid replication circuitry. Although NR1 replication had no absolute requirement for DnaA, the copy numbers of NR1-derived miniplasmids were lower in dnaA null mutants, and the plasmids exhibited a much reduced stability of inheritance during subculture in the absence of selection. This suggests that DnaA protein may participate in IncFII plasmid replication in some auxiliary way, such as by increasing the efficiency of formation of an open initiation complex at the plasmid replication origin. Such an auxiliary role for DnaA in IncFII replication would be different from that for replication of most other plasmids examined, for which DnaA has been found to be either essential or unimportant.

Bacterial Proteins↗
PubMed1989

DNA bending near the replication origin of IncFII plasmid NR1.

The DNA replication origin of plasmid NR1 is located approximately 190 base pairs downstream from the 3' end of the repA1 gene, which encodes the essential initiation protein for replication of the plasmid. Restriction endonuclease fragments that contain the NR1 replication origin and its flanking sequences at circularly permuted positions were obtained by digesting oligomers of ori-containing DNA fragments with sets of enzymes that each cut only once in every ori fragment. Polyacrylamide gel electrophoresis of these permuted restriction fragments showed anomalous mobilities, indicating the presence of a DNA bending locus. Through analysis of the relative mobility plots of these permuted fragments, we found one or two possible DNA bending sites located in the intervening region between the repA1 gene and the replication origin of NR1. It seems possible that DNA bending in this region might help to orient the replication origin alongside the repA1 gene, which could contribute to the cis-acting character of the RepA1 initiation protein.

Amino Acid Sequence↗
PubMed1988

Complementation of mutants of the stability locus of IncFII plasmid NR1. Essential functions of the trans-acting stbA and stbB gene products.

A series of unstable mutants of the stability (stb) locus of IncFII plasmid NR1 was subjected to a complementation analysis. The mutant collection included plasmids with point, insertion and deletion mutations in stb. These mutations affected the tandem genes stbA and stbB, which encode stability proteins StbA and StbB, or the PAB transcription promoter, which is located upstream from stbA in a region that contains an essential cis-acting site. Deletion mutants that lacked the region containing promoter PAB could not be complemented (stabilized) by providing StbA and StbB in trans. Deletion mutants that lacked stbA and stbB but retained the PAB region were complemented in trans but required both StbA and StbB, indicating that both proteins were essential for stable inheritance. stbA- point mutants were complemented in trans by either wild-type or stbA+ stbB- clones of the stability region. However, mutants with insertions in stbA were complemented only by wild-type clones, which suggested the insertions were polar on expression of the downstream stbB gene. A plasmid with a stbB- point mutation was complemented in trans by wild-type but not by stbA- stbB+ clones. In addition, plasmid clones that expressed StbB in the absence of StbA caused destabilization of (were incompatible with) stb+ derivatives of NR1 in trans, whereas clones that expressed only wild-type StbA or both StbA plus StbB did not. Plasmid clones that contained only the essential cis-acting PAB region did not cause destabilization of stb+ plasmids in trans. These results suggest that an excess of StbB protein provided in trans may cause a depletion of the essential StbA protein. Therefore, these results may be consistent with the hypothesis that StbB is an autorepressor of the stbAB operon.

Bacterial Proteins↗
PubMed1988

In-vivo studies on the cis-acting replication initiator protein of IncFII plasmid NR1.

Using segment-directed mutagenesis, a temperature-sensitive mutant of the gene that encodes the cis-acting RepA1 initiation protein of the IncFII plasmid NR1 was isolated. The mutant protein was unable to promote initiation of plasmid replication in vivo at 42 degrees C. Both the wild-type and the mutant repA1 genes were cloned separately into the high-expression vector plasmid pAS1. In these pAS1-repA1 derivatives, the transcription of the repA1 gene was under the control of the lambda PL promoter, which was regulated by the temperature-sensitive lambda cI857 repressor protein. The translation initiation of the repA1 mRNA from these derivatives was mediated by the lambda cII Shine-Dalgarno sequence and initiation codon. The yield of 33,000 Mr RepA1 protein detected on SDS/polyacrylamide gels from Escherichia coli cells containing the pAS1-repA1 derivatives was dependent upon whether the newly synthesized RepA1 was capable of interacting in cis with the downstream NR1 replication origin on the cloned DNA fragment. Mutations in the repA1 gene or deletions of the cis origin region dramatically increased the detectable yield of RepA1 protein. Deletion of the NR1 origin region from the pAS1 derivative containing the wild-type repA1 gene enabled the cis-acting RepA1 protein to complement partially the temperature-sensitive repA1 mutant in trans, to increase the copy number in trans of plasmids that contained the NR1 replicon, and to help NR1 derivatives overcome plasmid incompatibility. The trans effects of RepA1 provided by the pAS1-repA1 derivatives that retained the origin in cis were much less significant. RepA1 provided in trans also stimulated the replication of plasmids carrying cloned copies of the NR1 replication origin region regardless of whether the origin was transcribed from an upstream promoter.

Amino Acid Sequence↗
PubMed1988

Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1.

The stability (stb) locus of IncFII plasmid NR1 was mapped to a 1700 base-pair NaeI-TaqI restriction fragment. A series of unstable plasmids that contained insertion, deletion, and point mutations that inactivated the stability function was isolated. The unstable point mutants examined were all stabilized (complemented) in trans by a copy of the wild-type stb locus, suggesting that the mutations had inactivated diffusible gene products. The nucleotide sequence of the stb locus contained two tandem open reading frames, designated stbA and stbB, that encoded essential trans-acting protein products with predicted sizes of 36,000 Mr and 13,000 Mr, respectively. A third open reading frame, stbC, that could encode a peptide of 8000 Mr was contained within stbB in the complementary DNA strand. Plasmid-encoded proteins of 36,000 Mr and 13,000 Mr were identified in minicell experiments as the products of stbA and stbB, respectively. Unstable deletion mutants that retained the promoter proximal region of the stb locus upstream from stbA but had deleted both stbA and stbB were stabilized in trans by plasmids that could supply StbA and StbB. In contrast, deletion mutants that had lost the stbAB promoter region were not complemented in trans, indicating that this region contained an essential cis-acting site (or sites). Unlike some other loci that mediate stable plasmid inheritance, cloned copies of the wild-type stb locus of NR1 did not exert strong incompatibility (i.e. trans destabilization) against other stb+ derivatives of plasmid NR1 present in the same cell.

Bacterial Proteins↗
PubMed1987

Regulation of mini-F plasmid DNA replication. A quantitative model for control of plasmid mini-F replication in the bacterial cell division cycle.

A quantitative model for the regulation of replication of plasmid mini-F in the Escherichia coli cell division cycle has been developed. The essential repE gene of mini-F encodes a polypeptide that serves both as a positive replication initiation protein and as a regulatory repressor protein. The mini-F regulatory processes include the interaction of repressor with an operator site in the autogenous control of transcription of the repE gene, and the binding of initiator to repeated DNA sequences located both downstream from the repE gene and at the replication origin. 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 events of the cell cycle such as 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 for mini-F. Simulations of deviations from regular segregation of plasmid copies at cell division and of premature or delayed initiation of plasmid replication suggest that mini-F replication control responds rapidly and precisely to these perturbations. The simulations also accurately mimic the response of plasmid mini-F to various plasmid copy number mutations and to various mini-F regulatory elements provided in trans. These simulations predict a stable pattern of inheritance for plasmid mini-F despite its low plasmid copy number, in agreement with experimental observation.

Cell Division↗
PubMed1987

Transcriptional pausing in a region important for plasmid NR1 replication control.

The results of in vitro single-round transcription experiments indicated that RNA polymerase pauses during transcription of the leader region that precedes the repA1 gene of IncFII plasmid NR1. Transcription initiated at either of the two transcription promoter sites of the repA1 gene, which encodes the essential replication initiation protein of NR1, was observed to pause in this region. Pausing was specifically enhanced by addition of NusA protein, an Escherichia coli transcription accessory factor. Northern blot RNA-DNA hybridization analysis of repA1 mRNA synthesized in vivo revealed RNA species that had lengths equivalent to those of the in vitro-paused intermediates. The steady-state rate of in vivo repA1 mRNA transcription downstream from the pause sites (measured by quantitative hybridization of pulse-labeled RNA to DNA probes complementary to different segments of repA1 mRNA) was not appreciably affected, which suggests that the pause sites do not promote premature termination of transcription. The pause sites were located between the target sequence within the leader region of the mRNA that interacts with a 91-base countertranscript and the beginning of the repA1 coding sequence. Because the countertranscript is an inhibitor of translation of repA1 mRNA, transcriptional pausing in this region may be an important feature of the regulation of RepA1 synthesis, which is the mechanism by which plasmid NR1 controls its replication.

Base Sequence↗
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

Drug resistance gene amplification of plasmid NR1 derivatives with various amounts of resistance determinant DNA.

Drug resistance gene amplification of derivatives of plasmid NR1 having various amounts of resistance (r) determinant DNA was examined with two types of NR1 derivatives. The first was an NR1 derivative that carried two tandem copies of the r determinant component which was isolated as an intermediate in the amplification process. The plating efficiency of host cells and restriction endonuclease analysis of the plasmid DNA indicate that plasmids with two tandem copies of the r determinant undergo spontaneous amplification to a more highly amplified state at a frequency 150-fold higher than that of wild-type NR1. The second class of derivatives consisted of plasmids in which different regions of the r determinant component had been deleted. The relationship between spontaneous amplification frequency and r determinant size was examined with these plasmids. Plating efficiency of host cells indicated that plasmids with a smaller r determinant undergo spontaneous amplification at a lower frequency than do plasmids with a larger r determinant. These results suggest that there is an ordered sequence of events in the amplification of the r determinant of NR1.

Acetyltransferases↗