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N P Higgins

Publications and source records attributed to N P Higgins.

At least 37 records · Page 2Linked to original sources
PubMed1991

Mutations altering chromosomal protein H-NS induce mini-Mu transposition.

Bacteriophage Mu is one of the most efficient transposons known, capable of moving a hundred viral copies to new positions in the bacterial chromosome in an hour. Mu also forms stable lysogens. In bacteria lysogenic for the defective protein fusion-forming phage MudII1681, which can transpose and replicate but does not encode genes for DNA packaging and cell lysis, the frequency of transposition changes as colonies age. To find host genes that alter the spontaneous Mu transposition frequency, we used a genetic screen with mini-MudlacZ fusion formation as an assay. H-NS (also called H1a and B1) is an abundant nonspecific DNA-binding protein localized to the bacterial chromosome. H-NS has an unusual structure of interspersed patches of acidic and basic residues reminiscent of eukaryotic HMG proteins. Mutations in hns caused an increase in Mu-specific transcription and a dramatic increase in MudII1681 transposition rates when cells were put under certain growth conditions. Purified H-NS stabilized Mu repressor-DNA complexes in vitro, suggesting that H-NS contributes to the organization of transcriptionally inactive DNA in vivo.

Amino Acid Sequence↗
PubMed1990

A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition.

We have discovered a centrally located site that is required for efficient replication of bacteriophage Mu DNA and identified it as a strong DNA gyrase-binding site. Incubation of Mu DNA with gyrase and enoxacin revealed a cleavage site 18.1 kilobases from the left end of the 37.2-kilobase genome. Two observations indicate a role for the site in Mu replication: mutants of Mu, able to grow on an Escherichia coli gyrB host that does not allow growth of wild-type Mu, were found to possess single-base changes resulting in more efficient gyrase binding and cleavage at the site. Introduction of a 147-base-pair deletion that eliminated the site from a prophage inhibited the onset of Mu replication for greater than 1 hr after induction.

Bacteriophage mu↗
PubMed1990

Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient in supercoiling by 10 to 15% compared with wild-type cells, and the topoisomer distribution was significantly more heterogeneous than in wild-type or single-mutant strains. Other systems altered by HU inactivation included flagellar phase variation and phage Mu transposition. However, Mu transposition rates were only about fourfold lower in Salmonella HU double mutants. One reason that Salmonella HU double mutants may be less defective for Mu transposition than E. coli is the synthesis in double mutants of a new, small, basic heat-stable protein, which might partially compensate for the loss of HU. The results indicate that although either HU-1 or HU-2 subunit alone may accommodate the cellular need for general chromosomal organization, the selective pressure to conserve HU-1 and HU-2 structure during evolution could involve specialized roles of the individual subunits.

Bacterial Proteins↗
PubMed1989

Supercoiling and integration host factor change the DNA conformation and alter the flow of convergent transcription in phage Mu.

Transcription of bacteriophage Mu is modulated by its repressor, by negative supercoiling, and by the Escherichia coli protein integration host factor (IHF). Two converging Mu promoters regulate lytic and lysogenic development. The influence of IHF on these convergent promoters depended on the DNA conformation. When Mu operator DNA changed from the relaxed to the negative supercoiled form, IHF changed from a stimulatory factor to an inhibitor of the repressor promoter, and the ratio of the lytic transcript relative to the repressor transcript increased by 40-fold. Flexibility in Mu operator DNA was demonstrated by an unusual supercoil-induced DNA conformation, which was detectable by chemical modification with bromoacetaldehyde or digestion with P1 nuclease. IHF binding adjacent to this site induced dramatic bending of Mu DNA. A topological model we call a superloop is proposed to explain the effect of IHF on Mu transcription in vitro and on the lytic-lysogeny decision of the virus grown in IHF and gyrase mutants.

Bacterial Proteins↗
PubMed1989

Differential activity of a transposable element in Escherichia coli colonies.

In Escherichia coli colonies, patterns of differential gene expression can be visualized by the use of Mu d(lac) fusion elements. Here we report that patterned beta-galactosidase expression in colonies of strain MS1534 resulted from a novel mechanism, spatially localized replication of the Mu dII1681 element causing lacZ transposition to active expression sites. Mu dII1681 replication did not occur constitutively with a fixed probability but was dependent on the growth history of the bacterial population. The bacteria in which Mu dII1681 replication and lacZ transposition had occurred could no longer form colonies. These results lead to several interesting conclusions about cellular differentiation during colony development and the influence of bacterial growth history on gene expression and genetic change.

Blotting, Southern↗
PubMed1988

Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages.

Hybrid genetic elements, Mud-P and Mud-Q (collectively, Mud-P22s), have been constructed that carry two-thirds of the temperate Salmonella phage P22 genome sandwiched between the ends of transposon Mu. Insertions of these elements in the Salmonella chromosome generate locked-in P22 prophages that cannot excise. Upon induction (as a consequence of the inactivation of P22 c2 repressor), a locked-in prophage replicates its DNA in situ, resulting in the amplification of neighboring regions of the chromosome and the processive packaging of three contiguous headsful of adjacent DNA in one direction from the P22 packaging site, pac. Phage particles in an induced lysate of a Mud-P22 lysogen contain DNA molecules corresponding to several minutes of chromosomal DNA adjacent to the site of prophage insertion and transduce nearby genetic markers with high efficiencies. Mud-P22 prophages have been introduced into an F' episome by transposition; resident Mud insertions on the Salmonella chromosome may be converted to Mud-P22 insertions by homologous recombination in P22-mediated transductional crosses.

Chromosomes, Bacterial↗
PubMed1988

Primary structure and mapping of the hupA gene of Salmonella typhimurium.

In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. Comparison of hupA of E. coli and S. typhimurium revealed that the HU-2 proteins were identical and that there was high conservation of nucleotide sequences outside the coding frames of the genes. A 300-member genomic library of S. typhimurium was constructed by using random transposition of MudP, a specialized chimeric P22-Mu phage that packages chromosomal DNA unidirectionally from its insertion point. Oligonucleotide hybridization against the library identified one MudP insertion that lies within 28 kilobases of hupA; the MudP was 12% linked to purH at 90.5 min on the standard map. Plasmids expressing HU-2 had a surprising phenotype; they caused growth arrest when they were introduced into E. coli strains bearing a himA or hip mutation. These results suggest that IHF and HU have interactive roles in bacteria.

Amino Acid Sequence↗
PubMed1986

Purification of the gam gene-product of bacteriophage Mu and determination of the nucleotide sequence of the gam gene.

The gam gene of bacteriophage Mu encodes a protein which protects linear double stranded DNA from exonuclease degradation in vitro and in vivo. We purified the Mu gam gene product to apparent homogeneity from cells in which it is over-produced from a plasmid clone. The purified protein is a dimer of identical subunits of 18.9 kd. It can aggregate DNA into large, rapidly sedimenting complexes and is a potent exonuclease inhibitor when bound to DNA. The N-terminal amino acid sequence of the purified protein was determined by automated degradation and the nucleotide sequence of the Mu gam gene is presented to accurately map its position in the Mu genome.

Amino Acid Sequence↗
PubMed1986

Positive and negative regulation of the Mu operator by Mu repressor and Escherichia coli integration host factor.

Bacteriophage Mu utilizes two converging promoters to regulate the lytic and lysogenic pathways. Messenger RNA encoding the repressor gene is synthesized leftward from a promoter (PcM) located 1066 base pairs from the Mu left end. This transcript overlaps and is complementary to RNA synthesized rightward from the early promoter (PE) for transposase and replication proteins which initiates transcription at base pair 1028. Purified Mu repressor binds to three distinct operator sites (O1, O2, and O3); repressor binding at O2 blocks RNA polymerase binding at PE and repressor binding at O3 blocks RNA polymerase binding at PcM. O1 and O2 have higher affinity for repressor than O3, and transcription from PE is blocked at repressor concentrations that do not affect PcM. Thus, maintenance of the lysogenic state and autoregulation of the repressor gene is achieved by RNA polymerase transcription through DNA-repressor ensembles at O1 and O2. Integration host factor (IHF) encoded by the Escherichia coli him A and him D genes binds to Mu operator DNA between sites O1 and O2. IHF enhances transcription from PE 3-5-fold on supercoiled plasmid substrates in vitro and in Mu monolysogens in vivo. In vitro, IHF simultaneously decreases transcription from PcM 5-10-fold which results in a 25-fold change in lytic transcription relative to repressor transcription. A model for regulating the Mu lysis-lysogeny decision is presented.

Bacterial Proteins↗
PubMed1984

Analysis of mu DNA replicated on cellophane discs.

We have presented restriction analyses which indicate that the first transposition event after induction can be carried out and analyzed in vitro using cellophane disc lysates. This would be a first step in describing in molecular terms a "wiring diagram" of replicative transposition.

Bacteriophage mu↗
PubMed1984

Mu DNA replication in vitro: criteria for initiation.

An in vitro system for investigating Mu replication nd transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates. Two conditions reported to specifically decrease Mu DNA replication in vivo ( Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.

Bacteriophage mu↗
PubMed1983

The early promoter of bacteriophage Mu: definition of the site of transcript initiation.

The early promoter of bacteriophage Mu has been identified and characterized by a nitrocellulose filter binding assay and analysis of RNA products transcribed in vitro and in vivo. A tight, heparin resistant, RNA polymerase-DNA binding site overlaps the left Mu Hind III site. In vitro [gamma 32P] ATP initiated transcription begins approximately 25 nucleotides to the right of the Hind III restriction site. Dinucleotide initiation with ApA and S1 mapping of in vitro and in vivo transcripts shows that transcription is initiated 1028 base pairs from the Mu genetic left end.

Base Sequence↗
PubMed1983

Poly(ADP-ribosylation) of a DNA topoisomerase.

A DNA topoisomerase activity, copurifying with poly(ADP-ribose) synthetase from calf thymus, is greater than 95% inhibited if extensive poly(ADP-ribosylation) is allowed to occur. The inhibited DNA topoisomerase, which has drastically different elution properties on hydroxylapatite, can be reactivated by mild alkaline treatment. These results are consistent with a poly(ADP-ribosylation) of the DNA topoisomerase and covalent attachment of the poly(ADP-ribose) moieties to the topoisomerase by alkali-labile bonds.

Animals↗
PubMed1983

Bacteriophage Mu DNA replication in vitro.

An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.

Coliphages↗
PubMed1983

Bacteriophage Mu: a transposing replicon.

Mu DNA replication has been carried out in vitro on cellophane discs in the presence of dBUTP. If the DNA is sheared to 80 kb pieces, the Mu replicas band anomalously in CsCl gradients between hybrid and light DNA density positions. The intermediate density DNA comprises semiconservatively replicated Mu sequences, flanked by unreplicated light DNA. This and previous data are consistent with replication occurring within Mu boundaries. Both the synthesis of Mu sequences and the intermediate density DNA are abolished by protein synthesis inhibition in vivo just prior to lysis on cellophane discs. These observations indicate that at least some steps in bona fide Mu transposition-replication are being observed in vitro.

Bacteriophage mu↗
PubMed1982

The binding of gyrase to DNA: analysis by retention by nitrocellulose filters.

Three distinct Escherichia coli DNA gyrase complexes with DNA can be identified using a nitrocellulose filter-binding assay. One complex consists of an ensemble of two subunit A and two subunit B protomers bound noncovalently to specific sequences of DNA. High levels of each subunit alone are inactive but a single gyrase molecule binds DNA to a filter. At 23 degrees, the complex has a dissociation constant of approximately 10(-10) M and a half-time of decay of about 60 h. It is sufficiently stable that it can be purified by gel filtration and retain full supercoiling activity. Gyrase binds preferentially to relaxed DNA over supercoiled DNA by a factor of about 10. On addition of oxolinic acid, a second complex is formed that is distinguished by its stability in high ionic strength solutions and by efficient conversion to a third form upon addition of protein denaturants. The first and second complexes require Mg++ for optimal formation. The third form has been shown previously to contain denatured A protomers covalently linked to DNA that is broken at the site of attachment.

Collodion↗
PubMed1980

DNA gyrase subunit stoichiometry and the covalent attachment of subunit A to DNA during DNA cleavage.

Escherichia coli DNA gyrase contains a 1:1 ratio of protomers coded by the genes gyrA and gyrB. This along with previous results shows that the enzyme has two copies of each protomer and thus a molecular weight of 400,000. Abortion of the gyrase reaction results in double-strand breakage of the DNA and covalent attachment of both gyrA protomers to the 5'-cut ends. We conclude that the gyrA protomer contains a critical part of the active site for the concerted breakage and reunion reaction of gyrase, the topoisomerase activity of the enzyme.

Binding Sites↗