PubMed Health⌕ Search

Biomedical subjects

A L Abeles

Publications and source records attributed to A L Abeles.

13 recordsLinked to original sources
PubMed1991

Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

Bacterial Proteins↗
PubMed1990

A single DnaA box is sufficient for initiation from the P1 plasmid origin.

The P1 plasmid replication origin requires the host DnaA protein for function. Two DnaA-binding boxes lie in tandem within the previously defined minimal origin, constituting its left boundary. Three more boxes lie 200 base pairs to the right of these, in the leader region for the P1 repA gene. We show that either set alone is active for origin function. One of the two origin boxes is relatively inactive. Constructs with just one of the five boxes are active for specific origin function as long as the box conforms exactly to the published consensus. This single consensus box is functional when placed either to the left or right of the core origin sequences. The flexibility shown by this system suggests that the boxes play a role different from those in the host oriC origin, where the number and position of boxes are critical.

Bacterial Proteins↗
PubMed1989

Protein-DNA interactions in regulation of P1 plasmid replication.

The P1 RepA protein appears to play three roles in P1 plasmid replication: acting at the origin both as a specific initiator and as a repressor of transcription, and interacting with the copy-control locus incA to bring about a negative control of initiation. We have used the DNase I footprinting technique to show that RepA binds specifically to repeat units of a 19-base-pair consensus sequence present in both the origin and incA control regions. RNA polymerase was shown to bind to two specific regions within the origin repeats. One of these constitutes the known promoter sequence for the repA gene. We show evidence that the polymerase can be efficiently displaced from the promoter by subsequent RepA binding, thus providing a direct mechanism for RepA autoregulation. Under the conditions used, there were no obvious differences in the affinities of individual repeat sequences for the purified protein.

Bacterial Proteins↗
PubMed1987

P1 plasmid replication requires methylated DNA.

Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.

Base Sequence↗
PubMed1987

P1 plasmid replication: measurement of initiator protein concentration in vivo.

To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.

Cloning, Molecular↗
PubMed1986

P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA.

The minimal P1 replicon encompasses an open reading frame for the essential replication protein, RepA, bracketed by two sets of multiple 19-base pair repeated sequences, incA and incC. This study focused on the interaction of RepA with the incC and incA repeated sequences because earlier studies suggested that incA might control P1 copy number by titrating limiting amounts of RepA and because the incC repeats, which are part of the origin of replication, contain the promoter for repA. RepA is essential for origin function, autoregulates its own synthesis from the promoter, and, when overproduced, blocks origin function. In this study, RepA was overproduced from an expression vector and purified to 90% homogeneity. The binding of RepA to the DNA encompassing repeat sequences was assayed by monitoring the mobility of protein-DNA complexes on polyacrylamide gels. Distinct species of retarded bands were seen with the maximum number of bands corresponding to the number of repeats present in the target fragment. No evidence was found for RepA binding to fragments not containing the repeats. This suggests that the specific binding of RepA to the repeats may be involved in each of the diverse activities of RepA.

Bacteriophages↗
PubMed1985

Partition of unit-copy miniplasmids to daughter cells. III. The DNA sequence and functional organization of the P1 partition region.

The boundaries of the P1 par (plasmid partition) region of the unit-copy plasmid P1 were defined to within 2.7 X 10(3) base-pairs of DNA. The DNA sequence of the region revealed two large open reading frames that could encode proteins of Mr 44,000 and Mr 38,000. Both would be read in the same direction. The first open reading frame corresponds to the par A gene, the Mr 44,000 protein product of which was shown to be trans acting and essential for partition. The second open reading frame (parB) follows closely and may be cotranscribed with par A. The codon usage frequency for parB is consistent with its producing a protein product. The ParB protein was identified in cell extracts as a product with an apparent Mr of 45,000, suggesting that it behaves anomolously on gel electrophoresis. Following parB is the incB region, an incompatibility determinant thought to be the cis acting site that constitutes the putative attachment point on the DNA for the cellular partition apparatus. Subcloning of this site showed it to consist of a maximum of 174 base-pairs. The incB sequence is highly A + T-rich and contains a 20 base-pair inverted repeat. Another A + T-rich inverted repeat of similar size but different sequence is found between the putative parA promoter and the ribosome initiation sequence at the start of the parA open reading frame and may be involved in the autoregulation of ParA synthesis. The par region appears to contain a functional analog of the centromere of eukaryotic chromosomes. It is responsible for ensuring that newly replicated plasmids are properly distributed to daughter cells during cell division of its Escherichia coli host.

Bacterial Proteins↗
PubMed1985

Trans- and cis-acting elements for the replication of P1 miniplasmids.

Replication-deficient mutants of the unit-copy miniplasmid lambda-P1:5R were isolated after hydroxylamine mutagenesis. Complementation tests showed that the majority of these mutants are defective in the production of the repA protein product. Two of these mutants have suppressible nonsense (amber) mutations. The DNA sequence of one of these, repA103, has been determined. The lesion lies within the repA open reading frame, showing that the repA product is essential for plasmid replication. Complementation of deletion mutants of lambda-P1:5R by repA protein showed that the origin of replication lies to the left of repA and that this 300-base-pair origin region is the only portion of the DNA essential for plasmid replication if repA protein is supplied in trans. Six of the 21 hydroxylamine-induced mutants were not complemented by repA. Replication of three of these could be restored by introduction into the plasmid of a wild-type origin region, suggesting that they were origin-defective. The DNA sequence of two mutants was determined. Mutant rep-11 has a 43-base-pair deletion within the incC sequence (incC is a series of five direct repeats of a 19-base-pair sequence known to be involved in the regulation of plasmid replication). The deletion appears to have been generated by homologous recombination between two repeats. Mutant rep-30 has a single base substitution in a region just to the left of incC that destroys one of five G-A-T-C (dam methylation) sites in this region. As lambda-P1:5R is unable to establish itself as a plasmid in a methylase-defective (dam-) strain, it seems probable that methylation of the G-A-T-C sequences is important for origin function. The incC region and the sequences to its left appear to constitute an essential part of the origin of replication.

Bacteriophages↗
PubMed1985

P1 plasmid replication: multiple functions of RepA protein at the origin.

Replication functions of a bacteriophage P1 miniplasmid are carried on a 1.2-kilobase pair (kb) segment that can be subdivided into a 245-base pair (bp) replication origin and a 959-bp region that encodes a protein required for replication (RepA). The origin region contains five 19-bp direct repeats. By using primer extension and gene-fusion assays, we mapped the promoter of the repA gene within the repeated sequences and showed that the promoter is repressed by RepA. Regulation of RepA synthesis is apparently achieved by the binding of RepA to the repeat sequences. This regulation might be a key step in the replication-control circuit, as we found that overproduction of RepA (from a foreign promoter) inhibits replication. Thus, in addition to being an autoregulated activator of replication, the protein also can have a negative regulatory role.

Bacteriophages↗
PubMed1969

Structure-activity relationships of several cardiotonic steroids with respect to inhibition of ion transport in frog muscle.

Cesium uptake by sodium-loaded frog sartorius muscles was inhibited 100% by 10(-6)M ouabain and 10(-6)M cymarin. The doses for 50% inhibition of cesium uptake by five cardiotonic aglycones were 1.5 x 10(-6)M for strophanthidin, 2 x 10(-7)M for telocinobufagin, 1.6 x 10(-6) for digitoxigenin, 2.4 x 10(-6)M for periplogenin, and 6.3 x 10(-6)M for uzarigenin. Because of the limited solubility of sarmentogenin the maximum concentration studied was 2 x 10(-6)M which inhibited cesium uptake about 36%. Inhibition of cesium uptake by cymarin was not reversed during a 3.5 hr incubation in fresh solution while the muscles treated with ouabain and strophanthidin recovered partly during this time. Cymarin was a more potent inhibitor of sodium efflux than strophanthidin and periplogenin was less potent. Increased cesium ion concentration in the external solution decreased the strophanthidin inhibition of cesium uptake but 25 mM cesium did not overcome the inhibition by 10(-8)-10(-6)M strophanthidin. Increased potassium ion concentration in the external solution decreased but did not completely overcome inhibition of sodium efflux by strophanthidin. It is concluded that potassium or cesium ions do not compete with these drugs for a particular site on the ion transport complex. The same structural features of the drugs are necessary for inhibition of ion transport in frog muscle as are required for inhibition of ion transport in other tissues, inhibition of sodium-potassium-stimulated ATPases, and toxicity to animals.

Animals↗
PubMedDate not supplied

Unique sequence requirements for the P1 plasmid replication origin.

We have carried out a detailed genetic analysis of the P1 plasmid replication origin and shown that it has four major structural requirements: the DnaA box, a series of five 7-base pair (bp) repeats, a GC-rich spacer and five 19-bp repeats that bind the P1 RepA protein. The origin requires the DnaA protein and its recognition sequence (the DnaA box). However, although five boxes are present in two separate blocks in the wild type, just one, placed either to the left or right of the core origin sequences, is sufficient for function as long as the box conforms exactly to the proposed consensus. Each of the five 7-bp repeats that constitute the core of the origin is required; mutations within any of the first six base pairs can block origin function. The required bases include, but are not limited to, those constituting dam methylation sites. Also essential is a 39-bp GC-rich sequence. We show this to be a spacer of critical length that separates the 7-bp repeats from the last required region; a series of 19-bp repeats that bind the P1 RepA initiator protein.

Bacterial Proteins↗