PubMed Health⌕ Search

Biomedical subjects

K Mizuuchi

Publications and source records attributed to K Mizuuchi.

83 records · Page 5Linked to original sources
PubMed1980

Integrative recombination of bacteriophage lambda: extent of the DNA sequence involved in attachment site function.

We have investigated the minimum extent of DNA sequence required for the attachment site of bacteriophage lambda to function in integrative recombination. A DNA fragment carrying the phage attachment site (attP) of bacteriophage lambda was trimmed, recloned, and tested for recombination proficiency. In order to recombine with the bacterial attachment site (attB), the phage attachment site must retain about 250 base pairs of its original sequence. On the left side, the essential sequence extends beyond 106 base pairs from the center of the 15-base-pair common core sequence but not beyond 152 base pairs. On the right side the required sequence extends beyond 68 base pairs but not beyond 99 base pairs from the center of the core. A trimmed site that has lost part of the sequence mentioned above cannot function as the phage attachment site. However, depending on which part of the sequence is present, such a site can still act in reactions normally requiring one of the prophage attachment sites or the bacterial attachment site. The results also suggest that the essential suquence of the bacterial attachment site consists only of the sequence common to the phage and bacterial attachment sites.

Attachment Sites, Microbiological↗
PubMed1978

DNA gyrase: subunit structure and ATPase activity of the purified enzyme.

DNA gyrase has been purified to near homogeneity from Escherichia coli. The enzyme consists of two subunits of molecular weights 90,000 and 100,000 present in roughly equimolar amounts. The subunits can be identified as the products of two genes, determining resistance to coumermycin A1 and novobiocin (cou) and to nalidixic acid and oxolinic acid (nalA), respectively. These antibiotics were previously shown to be specific inhibitors of DNA gyrase. The ATPase activity of DNA gyrase is stimulated by double-stranded DNA and strongly inhibited by novobiocin but is relatively insensitive to oxolinic acid. Covalent attachment of an ATP derivative to the smaller (coumermycin-specific) subunit is also inhibited by novobiocin, suggesting that this drug interferes with the energy-coupling aspect of the DNA supercoiling reaction by blocking the access of ATP to the enzyme.

Adenosine Triphosphatases↗
PubMed1977

Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity.

ATP-dependent DNA supercoiling catalyzed by Escherichia coli DNA gyrase was inhibited by oxolinic acid, a compound similar to but more potent than nalidixic acid and a known inhibitor of DNA replication in E. coli. The supercoiling activity of DNA gyrase purified from nalidixic acid-resistant mutant (nalA(R)) bacteria was resistant to oxolinic acid. Thus, the nalA locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (cou). Supercoiling of lambda DNA in E. coli cells was likewise inhibited by oxolinic acid, but was resistant in the nalA(R) mutant. The inhibition by oxolinic acid of colicin E1 plasmid DNA synthesis in a cell-free system was largely relieved by adding resistant DNA gyrase. In the absence of ATP, DNA gyrase preparations relaxed supercoiled DNA; this activity was also inhibited by oxolinic acid, but not by novobiocin. It appears that the oxolinic acid-sensitive component of DNA gyrase is involved in the nicking-closing activity required in the supercoiling reaction. In the presence of oxolinic acid, DNA gyrase forms a complex with DNA, which can be activated by later treatment with sodium dodecyl sulfate and a protease to produce double-strand breaks in the DNA. This process has some similarities to the known properties of relaxation complexes.

DNA, Bacterial↗
PubMed1976

Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.

A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.

Chromosome Mapping↗
PubMed1976

DNA gyrase: an enzyme that introduces superhelical turns into DNA.

Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase. This enzyme has been purified from Escherichia coli cells. The reaction requires ATP and Mg++ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin E1, phage lambda, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA.

Adenosine Triphosphate↗