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L Caro

Publications and source records attributed to L Caro.

At least 19 recordsLinked to original sources
PubMed1992

Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA.

The replication of plasmid pSC101 requires the plasmid-encoded protein RepA. This protein has a double role: it binds to three directly repeated sequences in the pSC101 origin and promotes replication of the plasmid; it binds to two inversely repeated sequences in its promoter region and regulates its own transcription. A series of RepA protein derivatives carrying deletions of the C-terminal region were assayed for specific binding. We found that the last third of the protein is not needed for binding to the various specific sites. Truncated proteins that still bind can also form heterodimers with a wild-type protein. Analysis of band retardation assays conducted with wild-type and truncated proteins indicates that RepA binds to directly repeated sequences as a monomer and to inversely repeated sequences as a dimer.

Bacterial Proteins↗
PubMed1991

A copy-number mutant of plasmid pSC101.

Copy-number mutants of plasmid pSC101 were isolated by u.v. mutagenesis and selection for elevated expression of ampicillin resistance. Three independent mutations were identical and mapped in codon 93 of the initiation protein RepA. The mutated plasmids were maintained at a level four to five times higher than that of the wild type. For one of them, it was determined that: (i) the mRNA of the autoregulated repA gene, cloned onto a pUC19 plasmid under the control of its own promoter, was expressed at a level 1.7 times higher than that of the wild type; (ii) the RepA protein, under the same conditions, was expressed at a similarly higher level; (iii) the affinity of the mutated protein for three repeated sequences in the origin region of the plasmid was, on average, 3.4 times higher than that of the wild-type protein. We postulate that the copy-number effect is due to a combination of these two effects, i.e. higher protein concentration and increased affinity of the protein for the repeated sequences.

Ampicillin Resistance↗
PubMed1991

The replication of plasmid pSC101.

The origin of replication of plasmid pSC101 presents features reminiscent of those found in a number of plasmids. As for those plasmids, many details about the way it initiates its replication are beginning to be known, but the regulation of this process will not be easily understood.

Bacterial Proteins↗
PubMed1990

The par region of pSC101 affects plasmid copy number as well as stability.

The par locus is a segment of pSC101 that has been identified as a cis-acting determinant of plasmid stability. We show that par also determines copy number and must, therefore, play a role in plasmid replication. The segregation defect, but not the copy-number reduction, of par- replication origins is completely suppressed by a short sequence from the bacteriophage lambda gene O which is present in plasmid pKO-4. Thus, replication and segregation functions are separable from each other.

Ampicillin Resistance↗
PubMed1989

Transcription events in the origin of replication of plasmid pSC101.

Insertion mutations were isolated in the origin fragment of the plasmid pSC101 after random cleavage with DNase I. The replication properties of the resulting plasmids confirmed previous findings and extended the characterization of the essential regions. Using these plasmids, we analyzed by various methods the transcription events in the pSC101 origin. In addition to the mRNA of repA, a gene coding for the self-regulated RepA protein which is essential for replication of the plasmid, we characterized a transcript, which we called RNA Y, that runs in the opposite direction and that starts in the middle of the second repeated sequence in the origin region. Like the self-regulated repA mRNA, RNA Y is weakly expressed. It does not code for a complete protein within the origin fragment but may do so in the wild-type plasmid. We also found indications for one or, possibly, two small RNA species, called RNA X, which run in the same direction as RepA and which are partially complementary to RNA Y. We postulate that RNA Y and, possibly, RNA X are implicated in the initiation of replication of pSC101.

Base Sequence↗
PubMed1986

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF.

We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.

Amino Acid Sequence↗
PubMed1985

An essential replication gene, repA, of plasmid pSC101 is autoregulated.

Measurements of the rate of replication of a mutant pSC101 plasmid, cloned into a ColE1 vector, showed that insertions of the transposon Tn1000 into the repA gene of pSC101 abolished replication activity, but could be complemented in trans, albeit at a low level. The promoter of the repA gene was mapped by the construction of repA-lacZ gene fusions, and one of the fusions was used to demonstrate that repA protein, provided in trans, could repress expression of beta-galactosidase activity. This repression was primarily due to reduction of transcription of the repA-lacZ fusion. The sequence analysis of mutants of the repA-lacZ fusion gene which were no longer sensitive to the presence of repA protein showed that the site of action of repA was a 22 base-pair sequence, present as an inverted repeat, overlapping the repA promoter. The repA gene is thus autoregulated.

Bacterial Proteins↗
PubMed1984

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions.

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.

Chromosomes, Bacterial↗
PubMed1984

Replication functions encoded by the plasmid pSC101.

We describe the mapping of several genetic loci involved in the replication of the pSC101 plasmid. These include the origin of replication and a short segment of DNA that encodes a pSC101 incompatibility function. This short segment lies within the origin region. Flanking the incompatibility segment are two loci, repA and repB, which are required for replication. The product of the repA locus is shown to be trans-acting.

Bacterial Proteins↗
PubMed1984

Detection of displacement ("D") loops with the properties of a replicating intermediate synthesized by a DNA/membrane complex derived from the low-copy-number plasmid RK2.

A significant fraction of the plasmid DNA extracted from an RK2 miniplasmid DNA/membrane complex after incubation in vitro with appropriate substrates and cofactors contains "D" or displacement loops in one of at least three loci in the genome. These include the origin of replication and two transposon regions that code for kanamycin and tetracycline resistance. The relationship of these loops to early replicating intermediates for plasmid and transposon replication is discussed.

DNA Replication↗
PubMed1983

Plasmid pSC101 replication mutants generated by insertion of the transposon Tn1000.

A derivative of pSC101, pLC709, was constructed by ligation of the HincII-A fragment of pSC101 to the mini-colEI plasmid pVH51 and to a DNA fragment encoding resistance to the antibiotics streptomycin and spectinomycin. Insertions of the transposon Tn1000 (gamma-delta) into the pSC101 replication region of pLC709 were isolated following cotransfer of the plasmid with the sex factor F. The sites of insertion of the transposon were determined by restriction enzyme analysis and the replication and incompatibility properties of the insertion plasmids and DNA fragments cloned from them were analysed. The insertion mutations defined a locus, inc, of approximately 200 base-pairs that is responsible for pSC101-specific incompatibility. Two mutations adjacent to this region inactivate pSC101 replication but can be complemented in trans by a wild-type pSC101 plasmid, and thus define a trans-acting replication function, rep. The inc locus is within a larger region of some 450 base-pairs that is essential for pSC101 replication and that includes the origin of replication. This 450 base-pair segment can replicate in the presence of a helper plasmid that supplies the rep function in trans.

DNA Replication↗
PubMed1983

The nucleotide sequence of replication and maintenance functions encoded by plasmid pSC101.

The nucleotide sequence of 1100bp around the origin of replication of the pSC101 plasmid has been determined. This segment of DNA is capable of replication in the presence of a helper plasmid. The sequence data reveal similarities between pSC101 and several other replicons. The origin of replication contains three direct repeats of an 18bp sequence associated with a segment exceptionally rich in A-T base pairs. A promotor that probably directs transcription of a gene encoding an essential plasmid replication function is associated with a region of extensive potential secondary structure. The sequence presented here includes the sequence of the par region involved in partitioning of plasmids at cell division.

Base Composition↗
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↗
PubMed1983

Role of the sfiA-dependent cell division regulation system in Escherichia coli.

Several authors have suggested that the SOS-associated (sfiA-dependent) system of division inhibition, normally induced by perturbations of DNA replication, also regulates steady-state (unperturbed) cell division. The present work shows that mean cell mass is identical in sfiA+ and sfiA mutant cultures during steady-state growth, that mass adjustment is identical after shift up, that sfiA expression is not induced by shift up, and that a sfiA mutation does not cause aberrant chromosome segregation.

Bacterial Proteins↗