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M Chandler

Publications and source records attributed to M Chandler.

At least 109 records · Page 6Linked to original sources
PubMed1985

Preferential binding of bacteriophage Mu repressor to supercoiled Mu DNA.

It was shown, using a relatively simple assay, that Mu repressor, cI, binds specifically to a region which spans the leftmost HindIII cleavage site on the phage genome. This extends the observations of Kwoh and Zipser [Nature (London) 277, 489-491 (1979)], who were able to define a binding region to the left of this site. These results provide support for the idea that the eight blocks of repeated DNA sequences, which also span the HindIII cleavage site, are involved in repressor binding. These results also indicate that cI repressor has a marked preference for supercoiled DNA.

Bacteriophage mu↗
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

Cloning and expression of the phage Mu A gene.

We have cloned the phage Mu A gene, with and without the gene ner, under the control of the pL promoter of phage lambda in a multicopy plasmid vector. We demonstrate that plasmid-carrying cells are able to support growth of superinfecting Mu A am phages in a temperature-dependent fashion in a host strain carrying a defective lambda prophage which specifies the cI857-coded lambda repressor. In addition, we show that the presence of the ner gene reduces the efficiency of plating of the superinfecting phage. Analysis of proteins specified by the cloned Mu fragments indicates that two proteins, 70 and 33 kDal, are synthesized. The level of synthesis, compared to that of the vector-encoded beta-lactamase, was found to increase with temperature. This indicates that their transcription is driven by the pL promoter. The Mr of the 70-kDal protein is identical to that previously observed for pA.

Bacteriophage lambda↗
PubMed1983

Cointegrate formation mediated by Tn9. II. Activity of IS1 is modulated by external DNA sequences.

We have studied the effect on the transposition activity of IS1 of the position and orientation of the element in the donor replicon. Previous studies have shown that the IS1s in the compound transposon Tn9 have different activities in forming plasmid cointegrates. In this study, designed to investigate the sources of this differential activity, we have cloned Tn9 (together with 132 base-pairs of flanking DNA) into several sites in pBR322 and created various derivatives of these plasmids to be used as donor molecules in plasmid fusion experiments. These pBR322-Tn9 plasmids were allowed to form cointegrates with a conjugative, F-derived plasmid, and a large collection of independently formed cointegrates was isolated. The relative activities of the two IS1 elements of Tn9 were estimated by examining the structures of the cointegrates for the frequency of usage of each of the IS1s in cointegrate formation. The results suggest that IS1 activity is modulated by the transcription activity of adjacent DNA sequences in the donor plasmids. Transcription directed into an IS1 inhibits its activity. Confirmatory evidence for this hypothesis is provided by the observation that deletion of adjacent promoters of transcription relieves the inhibitory effect on IS1 activity.

Acetyltransferases↗
PubMed1983

IS1-mediated tandem duplication of plasmid pBR322. Dependence on recA and on DNA polymerase I.

Transposable-element-mediated fusion of the conjugal plasmid pOX38::Tn9 with pBR322 results in the appearance of cointegrates composed of a single copy of each plasmid, and cointegrates which carry a single copy of pOX38 but multiple tandem copies of pBR322. These plasmids are separated by directly repeated copies of the transposable element. We demonstrate here that such multimers can be generated from monomeric cointegrates, probably by unequal crossing over between the flanking Tn9(IS1) elements. Their appearance is thus not necessarily associated with the original transposition (fusion) event. Our study demonstrates that the process of duplication is strongly dependent on the homologous recombination system of Escherichia coli, since it is undetectable by our methods in recA- strains. It is also strongly dependent on the presence of a functional DNA polymerase I in the cell. The major pathway(s) for this duplication thus appears to rely on both the homologous recombination system and the replication of the duplicated segment.

Bacterial Proteins↗
PubMed1982

Cointegrate formation by Tn5, but not transposition, is dependent on recA.

We have studied the effect of the recA-dependent homologous recombination system of Escherichia coli on both Tn5-mediated cointegrate formation and Tn5 transposition. We demonstrate here that, whereas transposition of Tn5 is independent of the recA gene product (as has been shown by other workers), Tn5-mediated cointegrate formation is strongly dependent on recA. The structures of both the simple transposition products and the cointegrates formed in a recA- background seem to be the same as those produced in a recA+ background. These results provide strong evidence that Tn5 does not transpose via an obligate cointegrate intermediate and suggest that the recA effect on cointegrate formation is exerted during the process of transposition.

Bacterial Proteins↗
PubMed1982

Isolation of the kanamycin resistance region (Tn2350) of plasmid R1drd-19 as an autonomous replicon.

We have isolated a circular form of Tn2350, an IS1-flanked kanamycin resistance transposon forming part of the plasmid R1drd-19. This circle (pTn2350::9.6 kilobases) contains a single IS1 element and probably arises by recombination between the two directly repeated Is1 sequences of Tn2350. It can be used to transform Escherichia coli to kanamycin resistance. It is capable of autonomous replication but is not maintained stably in dividing cells and segregates under nonselective conditions. Cloning of a segment of pTn2350 on a conditional plasmid vector allowed us to assign the replication functions of this plasmid to a 1.6-kilobase restriction fragment. The plasmid R1drd-19 can thus be considered as a cointegrate between two replicons separated by IS1 sequences.

Base Sequence↗
PubMed1981

The structure of R1drd19: a revised physical map of the plasmid.

We have analyzed derivatives of the plasmid R1drd19 carrying the transposon Tn10 by electron microscopy following denaturation and renaturation of the molecules, and by digestion with various restriction enzymes, gel electrophoresis and Southern blotting. We show: 1) that the published restriction map of R1drd19 is inconsistent with our results. We present a modified map which is consistent with our data. 2) that R1drd19 carries a single resident copy of the element IS10 which is normally associated with Tn10 as an inverted repeat, and 3) that R1drd19 carries three copies of the insertion element IS1 in the resistance determinant region.

DNA Restriction Enzymes↗
PubMed1981

The initiation of chromosome replication in a dnaAts46 and a dnaA+ strain at various temperatures.

The regulation of chromosome replication initiation was studied at various temperatures with an E. coli dnaA46 strain and its dnaA+ parent. We find that, in both strains, the "initiation mass" varies depending upon growth temperature while the replication time remains constant relative to the cell doubling time. In the permissive temperature range, the initiation mass of the dnaA46 mutant strain is larger by a constant factor than that for a dnaA+ strain. We conclude that, even at temperatures permissive for growth of the dnaA46 strain, the activity of the dnaA46 product is lower than that of the wild-type protein. The dnaA gene product, therefore, plays an important role in regulating initiation.

DNA Replication↗
PubMed1981

On the molecular mechanisms of transposition.

We present a model for transposition that allows a choice between cointegrate formation (replicon fusion) and direct transposition. We propose that initiation of the process occurs by invasion of the target DNA by a single-stranded end of the transposable element. This leads to nicking of one of the DNA strands of the target molecule and ligation of this strand to that of the invading transposon. Transposition then occurs in a processive way by replication of the element from the invading end into the target site in a looped rolling-circle mode similar to replication of phage phi X174 replicative form to viral strand. The choice between cointegrate formation and direct transposition occurs at the nick-ligation step, which terminates the process. We suggest that the choice is determined by the topology of the transposition enzymes and could be related to whether the element generates five- or nine-base-pair repeats in the target DNA on insertion.

Base Sequence↗
PubMed1980

Isolation of an IS1 flanked kanamycin resistance transposon from R1drd19.

We have isolated and identified an IS1-flanked transposon from the plasmid R1drd19. This transposon specifies resistance to kanamycin and is 10.4 kg long. It exhibits a frequency of transposition two orders of magnitude lower than that of the smaller, IS1-flanked transposon Tn9. We have named it Tn2350.

DNA Transposable Elements↗