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Biomedical subjects

A Taketo

Publications and source records attributed to A Taketo.

At least 55 records · Page 3Linked to original sources
PubMed1988

DNA transfection of Escherichia coli by electroporation.

Electroporation was applied to transfection and transformation of Escherichia coli. Efficient transfer of DNA was achieved by a single voltage pulse at 2.5 kV (initial electric field strength = 6.25 kV/cm), with a 25 microF capacitor. As the recipient for transfecting DNA in the electroporation, spheroplasts, EDTA-treated cells and osmotically shocked bacteria were inferior to intact E. coli. Various parameters affecting the transfection efficiency were defined including growth phase of recipient cells, concentrations of DNA and cells, temperature and additions. In most strains tested, electroporation was far more efficient than Ca2+-dependent transfection (transformation). Various aspects of the electroporation-mediated DNA uptake are discussed.

Cell Membrane Permeability↗
PubMed1987

Reactivation of streptolysin S by oligonucleotide.

Oligonucleotide-streptolysin S complex inactivated by alkali treatment remains nonhemolytic, even after acidification and mixing with intact carrier oligonucleotide rich in guanyl residue. Upon dehydration, however, the inactive streptolysin S-oligonucleotide mixture turned to be hemolytic, and this reactivation of the hemolysin was promoted by treatment with guanidine hydrochloride. After alkaline hydrolysis, streptolysin S was freed from nucleotide moiety, by gel filtration through a Sephadex G-50 column. From this nonhemolytic apotoxin as well, active streptolysin S complex was reconstructed upon dehydration with the carrier.

Bacterial Proteins↗
PubMed1986

Activation of streptolysin S in vitro by oligonucleotides.

When washed streptococci were incubated in a phosphate buffer containing MgSO4 and maltose (Bernheimer's basal medium) and centrifuged, no hemolytic activity was detected in the supernatant. Although incubation of the spent medium with carrier oligonucleotide did not yield active hemolysin, the mixture turned to be significantly hemolytic, upon ethanol precipitation and dehydration. Oxygen stability, sensitivity to trypan blue, absence in the spent medium from strain C203U, as well as chromatographic properties demonstrated that the hemolytic activity was due to streptolysin S-oligonucleotide complex. The latent streptolysin S was detected in the streptococcal culture supernatant as well. These results indicate that, even in the absence of exogenous carrier, streptolysin S is produced extracellularly by hemolytic streptococci but suffers rapid denaturation, and that the carrier oligonucleotide serves as an effector for the toxin peptide to assume active conformation, through noncovalent interaction. Effects of several protein denaturants were investigated on the toxin activation in vitro.

Bacterial Proteins↗
PubMed1985

Function and structure of microvirid phage alpha 3 genome. DNA sequence of H gene and properties of missense H mutant.

The nucleotide sequence of wild-type alpha 3 H gene and its surrounding region was determined and compared with those of phi X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.

Bacteriophage phi X 174↗
PubMed1984

Function and structure of microvirid phage alpha 3 genome. II. Isolation and properties of various mutants of alpha 3.

Various mutants were isolated from a microvirid (isometric single-stranded DNA) phage alpha 3, by mutagenesis with hydroxylamine or nitrous acid. They were divided into eight complementation groups, and mainly by genetic crosses the gene alignment was determined as -A-B-C'-D-J'-F-G-H-. Except for groups C' and J', each defective gene product was clearly discerned in electropherograms of proteins extracted from the phage-infected suppressor-negative (Su-) Escherichia coli. Only gene A mutants abolished synthesis of the progeny replicative-form DNA (RF), whereas mutants belonging to groups B, C', D, E, F and J' affected RF replication at late stage, as well as synthesis of the single-stranded DNA (SS). Additional properties of several mutants are also discussed.

Bacteriophage phi X 174↗
PubMed1984

Isolation of human erythrocyte membranes in glucose solution.

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.

Cell Fractionation↗
PubMed1984

Isolation and some properties of bacteriophage alpha3 gene J mutant.

To elucidate the in vivo function of the J gene of microvirid (isometric) phages, we isolated several strains carrying a double mutation in J and H genes from phage alpha3 and then constructed single mutants each having an amber codon in the J gene. The J mutants could not multiply in suppressor-less hosts and were deficient in single-stranded progeny DNA synthesis. Nucleotide sequences of the wild-type and mutant alpha# J genes were analyzed to determine the mutation sites. The amino acid sequence of the J gene was also deduced from the nucleotide sequence and compared with those of phiX174 and G4.

Amino Acid Sequence↗
PubMed1981

Effect of the dnaN mutation on the growth of small DNA phages.

The effect of the dnaN mutation on the growth of single-stranded DNA phages was studied by burst experiments. In HC138 dnaN cells exposed to 42.5 degrees C at 5 min before infection, growth of spherical (microvirid or isometric) phages such as alpha 3, phi Kh-1 and phi X174 was partially reduced at the nonpermissive temperature. When infection was performed at 30 min after temperature shift-up, viral replication was completely inhibited at 42.5 degrees C in the dnaN strain but not in a dna+ revertant. At 41 degrees C, multiplication of filamentous (inovirid) phages M13 and fd was restricted specifically in HC138 F+ dnaN bacteria. When dnaN cells lysogenic for lambda i21 were grown at 42.5 degrees C for 60 min and then shifted down to 33 degrees C, a burst of lambda i21 occurred with concomitant cellular lysis, manifesting induction of the prophage development.

Bacteriophage lambda↗
PubMed1978

Replication of bacteriophage G13 DNA in dna mutants of Escherichia coli.

Host functions required for replication of microvirid phage G13 DNA were investigated in vivo, using thermosensitive dna mutants of Escherichia coli. In dna+ bacteria, conversion of viral single-stranded DNA into double-stranded replicative form (stage I synthesis) was resistant to 150 microgram/ml of chloramphenicol or 200 microgram/ml of rifampicin. Although multiplication of G13 phage was severely inhibited at 42--43 degrees C even in dna+ host, considerable amount of parental replicative form was synthesized at 43 degrees C in dna+, dnaA or dnaE bacteria. In dnaB and dnaG mutants, however, synthesis of parental replicative form was severely inhibited at the restrictive temperature. Interestingly enough, stage I replication of G13 DNA was, unlike that of phiX174, dependent on host dnaC(D) function. Moreover, the stage I synthesis of G13 DNA in dnaZ was thermosensitive in nutrient broth but not in Tris/casamino acids/glucose medium. In contrast with the stage I replication, synthesis of G13 progeny replicative form was remarkably thermosensitive even in dna+ or dnA cells.

Coliphages↗
PubMed1978

Growth of single-stranded DNA phages in replication mutants of Escherichia coli.

Host capacity for growth of single-stranded DNA phages was investigated with several replication mutants of E. coli. In dnaL708, dnam709 and dnaS707 mutants, multiplication of phiK was not restricted even at 42 degree C. In dnaM710 cells, however, growth of phiK was severely effected at 42 degree C but not at 33 degree C. Upon infection of phiK, parental replicative form was synthesized at the restrictive temperature, whereas subsequent step (replication of progeny replicative form) was blocked in the dnaZ strain. Growth of phiX174 and alpha3, as tested by transfection, was also thermosensitive in the dnaM710 mutant but not in the dnaL708, dnaM709 and dnaS707 strains. In contrast with lambda, microvirid phages could grow in E. coli cells bearing the groPC259, groPC756 or seg-2 mutation.

Coliphages↗
PubMed1978

Synthesis of alpha3 phage DNA in replication mutants of Escherichia coli.

Host functions for DNA replication of bacteriophage alpha3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna+ cells, conversion of phage alpha3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30--150 microgram/ml of chloramphenicol, 200 microgram/ml of rifampicin, 50 microgram/ml of nalidixic acid, or 200 microgram/ml of novobiocin. At 43 degrees C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage alpha3 progeny RF was prevented by 50 microgram/ml of mitomycin C (in hcr+ bacteria), 50 microgram/ml of nalidixic acid or 200 microgram/ml of novoviocin, but neither by 30 microgram/ml of chloramphenicol nor by 200 microgram/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for progeny RF synthesis. Host factor dependence of alpha3 was relatively simple and, in contrast with phages phiX174 and G4, alpha3 did not require dnaB and dnaC(D) activities.

Anti-Bacterial Agents↗
PubMed1978

Replication of G4 progeny double-stranded DNA in dna mutants of Escherichia coli.

Host functions required for replication of progeny double-stranded DNA of bacteriophage G4 were examined by using metabolic inhibitors and Escherichia coli dna mutants. In dna+ bacteria, synthesis of the progeny replicative form (RF) was relatively resistant to 30 microgram/ml of chloramphenicol, but considerably sensitive to 200 microgram/ml of rifampicin. The RF replication was severely inhibited by 50 microgram/ml of mitomycin C, 50 microgram/ml of nalidixic acid, or 200 microgram/ml of novobiocin. At 41 degrees C, synthesis of G4 progeny RF was distinctly affected in a dnaC(D) mutant and in a dnaG host. The progeny RF replication was prevented at 42 degrees C in a dnaE strain as well as in a dnaB mutant. In a dnaZ strain, the synthetic rate of the progeny RF was markedly reduced at 42 degrees C. At 43 degrees C, the rate of G4 progeny RF synthesis was reduced even in dna+ or dnaA bacteria, but significant amounts of the progeny RF were still synthesized in these hosts at the high temperature. In addition to five dna gene products, host rep function was essential for the RF replication.

Anti-Bacterial Agents↗
PubMed1978

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.

Coliphages↗
PubMed1977

Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.

Coliphages↗
PubMed1976

Host factor requirements and some properties of phiXtB. An evolutionary aspect of phiX-type phages.

Replication of phiXtB, a capsid mutant of bacteriophage phiX174, depends on the host functions directed by the E.coli genes dnaE, dnaF, dnaG, dnaZ, lig and rep. The cellular products of dnaA, dnaB, dnaC(D), dnaI, dnaP, polA, polB and xth genes are, however, dispensable for the viral growth. In these host factor requirements, phiXtB resembles phages phiK and St-1 rather than phiX174. Host ranges of phiXtB, St-1 and phiK overlap considerably, and growth temperature of the three phages is somewhat higher than that of phiX174. Furthermore, phiXtB is, like phiK, inactivated by antiserum against St-1. phiXtB may thus fill an evolutionary gap between the phiX174 group and the St-1 group.

Biological Evolution↗
PubMed1976

Host genes involved in the replication of single-stranded DNA phage phiK.

Using various replication mutants of E. coli, the host genes that participate in the replication of some K12-specific single-stranded DNA phages have been determined. Functional products of dnaE, -F, -G and -Z genes are required for the multiplication of phiK, whereas dnaA, -B, -C(D), H, -I and -P are dispensable for viral replication. In contrast with polB, recA, B, C, or xth functions, host rep activity is essential for phiK. At the restrictive temperature, the yield of phiK was markedly reduced in the ligts7 mutant and partially decreased in a polAts strain. The phage phiK is thus less dependent on the host cells than phiX174 and phiA which require additionally the dnaB, -C(D) and -H functions. Replication of phage St-1 depends on dnaG and -Z gene products, but not on dnaP function. Although not much affected in polAts host, growth of St-1 was significantly diminished in dnaF or ligts7 mutants.

Coliphages↗
PubMed1975

Replication of phiA and phiX174 in Escherichia coli mutants thermosensitive in DNA synthesis.

Thermosensitive mutants of E. coli defective in DNA replication were tested for their capacity to support multiplication of phiA and phiX174. At the restrictive temperature, the viral growth was markedly affected in dnaH, dnaZ, or ligts7 mutants. Even when these strains were transfected with RF1 molecules, the virus yield was still very low. The dnaI function was, however, dispensable for replication of phiA and phiX174. In addition, these viruses could multiply in dnaP or polAts mutants at the high temperature.

Coliphages↗