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T Minagawa

Publications and source records attributed to T Minagawa.

At least 109 records · Page 6Linked to original sources
PubMed1987

Early events in DNA packaging in a defined in vitro system of bacteriophage T3.

We have developed a defined in vitro system for packaging phage T3 DNA which is composed of purified proheads and the noncapsid proteins gp18 and gp19, products of genes 18 and 19. The reaction requires Mg2+, ATP, and polyethylene glycol and is inhibited by a nonhydrolyzable ATP analog, adenosine-5'-O-(3'-thiotriphosphate) (ATP-gamma-S) (K. Hamada, H. Fujisawa, and T. Minagawa, 1986, Virology 151, 119-123). About 30% of added mature T3 DNA was packaged into heads in the defined system. A complex with a sedimentation coefficient of about 50 S (50 S complex) accumulated in the reaction mixture containing ATP-gamma-S. The 50 S complex was DNase sensitive and was converted to filled heads by a second reaction in the presence of ATP without addition of DNA, proheads, gp18, and gp19. These results indicate that during early stages of DNA packaging, formation of precursor complexes proceeds by an allosteric mechanism with ATP acting as effector. The movement of DNA into the head is driven by the energy released by hydrolysis of ATP. gp18 formed a complex with DNA without addition of ATP-gamma-S and gp19. gp18-DNA complex was DNase sensitive and did not bind gp19; it was converted to filled heads by way of a second reaction after addition of ATP, gp19, and proheads. gp19 formed a functional complex with prohead in the presence of ATP-gamma-S or ATP. The complex did not bind gp18 but was converted to filled heads by incubation with ATP, gp18, and DNA. In the absence of ATP-gamma-S, gp19 formed complexes with prohead that were abortive in DNA packaging. Formation of the 50 S complex occurred in a reaction mixture containing gp18-DNA and gp19-prohead complexes in the presence of ATP-gamma-S. From these results, we propose details of the molecular mechanism of DNA packaging in the defined in vitro system.

Adenosine Triphosphate↗
PubMed1987

Studies on the recombination genes of bacteriophage T4: suppression of uvsX and uvsY mutations by uvsW mutations.

Genes uvsW, uvsX and uvsY are dispensable for T4 growth but are implicated in recombination and in the repair of damaged DNA. We found that large-plaque mutants arose efficiently from small-plaque uvsX and uvsY mutants at 42 degrees and were pseudorevertants containing a new mutation in uvsW. Using reconstructed double mutants, we confirmed that a mutation in uvsW partially increases the burst size and UV resistance of uvsX and uvsY mutants. At 41 degrees the uvsW mutation completely restores the arrest in DNA synthesis caused by mutations in genes uvsX, uvsY and 46, but at 30 degrees it only partially restores DNA synthesis in a gene 46 mutant and does not restore DNA synthesis in uvsX and uvsY mutants. Restored DNA synthesis at 41 degrees was paralleled by the overproduction of single-stranded DNA and gene 32 protein. Based on these findings, we propose that the uvsW gene regulates the production of single-stranded DNA and we discuss the phenotype of uvsW mutants and their suppression of some uvsX and uvsY phenotypes. Infection of restrictive cells with am uvsW mutants revealed a defect in the synthesis of a protein of molecular weight 53,000 daltons, suggesting that this protein is the uvsW gene product.

DNA Repair↗
PubMed1986

Subunit arrangement of the tail fiber of bacteriophage T3.

A tail fiber of phage T3 is a trimer of the product of gene 17 (gp17). Treatment of T3 phage particles with chymotrypsin resulted in cleavage of only the tail fiber protein, at a site near the distal end of the fiber, causing a decrease of about 10% in the size of gp17 in the treated virion. The N-terminal amino acid sequences of intact and cleaved tail fiber proteins were identical and corresponded to that deduced from the nucleotide sequence of gene 17 except for the absence of the initiation Met residue. These results indicate that cleavage of the tail fiber occurred near the C terminus and suggest that gp17 polypeptides are oriented parallel to each other in the tail fiber. Association of tail fibers with the tail involves the N-terminal region of gp17. Under mild conditions of SDS-polyacrylamide gel electrophoresis, intact tail fibers dissociated from virions but cleaved ones did not. The nucleotide sequences indicate that T3 and T7 gp17 contain many sites that are potentially sensitive to chymotrypsin. In fact, free tail fibers, purified from T3-infected cells, were cleaved to many smaller fragments by chymotrypsin. These results suggest that the attachment of the tail fibers to the tail may induce a change(s) in the configuration and/or arrangement of gp17 to mask the sensitive sites from cleavage by chymotrypsin.

Amino Acid Sequence↗
PubMed1986

Cloning and sequencing of the genetic right end of bacteriophage T3 DNA.

The genetic right end of phage T3 DNA, from the beginning of gene 17, was cloned and sequenced. Genes 17, 18, and 19 were identified by comparing the sequence with the genetic map and by comparing the calculated and observed molecular weights of gene products. N-terminal amino acid sequence of the gene 17 product (gp17) predicted from the nucleotide sequence was consistent with the data from the analysis of purified gp17. Gene 17.5 was identified as the lysis gene on the basis of the presence of a nonsense codon within an open reading frame in the sequence of DNA from an amber mutant of lysis gene. In addition, five potential genes have been identified. Sequences corresponding to a promoter for phage T7 RNA polymerase (Rosa and Andrews, 1981) and to a class-III promoter for phage T3 RNA polymerase (Sarker et al., 1985) were found. The genomic organization and the nucleotide and deduced amino acid sequences of T3 were compared with those of T7. The genomic organizations of T3 and T7 were identical in this region. The sequence comparisons of T3 and T7 DNA point out the highly conserved sequences in all genes but also heavily varied regions in some genes. From these comparisons, possible implications with regard to structural and functional domains within several genes are discussed.

Amino Acid Sequence↗
PubMed1986

Overproduction and purification of the products of bacteriophage T3 genes 18 and 19, two genes involved in DNA packaging.

The products of gene 18 (gp18) and gene 19 (gp19) of bacteriophage T3 are noncapsid proteins involved in DNA packaging. A restriction fragment containing gene 18 or 19 was cloned into the plasmid vector pNT45 under the control of the inducible leftward promoter (PL) of phage lambda. Induction of transcription of gene 18 or 19 by derepression of the PL promoter led to the synthesis of a high level of gp18 or gp19. By using complementation of T3 DNA packaging in vitro as an assay, gp18 and gp19 were purified to near homogeneity. The overall yields of gp18 and gp19 were 1.4 mg and 0.35 mg, respectively, from 1 g wet wt cells. Addition of gp18 to the in vitro DNA packaging system resulted in increased phage production with increasing amounts of gp18 until 10% of the DNA was packaged into infectious phage particles. In contrast, addition of gp19 to the packaging system initially caused an increase in phage production, but increasing amounts of gp19 inhibited DNA packaging.

Cloning, Molecular↗
PubMed1986

A defined in vitro system for packaging of bacteriophage T3 DNA.

Using purified components, we have constructed an in vitro system for packaging of mature phage T3 DNA. In addition to mature T3 DNA, the system contained T3 proheads and the products of gene 18 (gp18) and gene 19 (gp19). The reaction required Mg2+, ATP, and polyvinyl alcohol. Spermidine was stimulatory but not absolutely required for the packaging reaction. Polyvinyl alcohol could be replaced by polyethylene glycol. The packaging efficiency decreased with decreasing molecular weight of the polymer, and low molecular weight polyols such as sucrose, sorbitol, and glycerol were inactive. The packaging reaction exhibited a sigmoidal relationship with respect to the concentration of ATP with the concentration for half maximal activity about 15 microM. A nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate), inhibited the packaging reaction.

Adenosine Triphosphate↗
PubMed1986

Impaired alpha-interferon production and natural killer activity in blood mononuclear cells in myelodysplastic syndromes.

The producibility of interferon (IFN)-alpha, which indicates one of the functions of large granular lymphocytes (LGL), was impaired at a high frequency in myelodysplastic syndrome (MDS) patients. However, IFN-alpha production in refractory anaemia, which is a subtype of MDS, was almost normal. In contrast, abnormality has not been observed in either proliferative response or the production of IFN-gamma of T-cells by stimulation with PHA. NK activity of peripheral blood mononuclear cells (PBMC) from MDS patients was generally low and was not augmented by IFN-alpha treatment. These results indicate that, in addition to the abnormality at the level of haematopoietic tissues, LGL among PBMC may be impaired in MDS patients.

Adult↗
PubMed1986

Activities of 2'-5'oligoadenylate synthetase in peripheral blood mononuclear cells of patients with viral hepatitis, and chronic type B hepatitis during interferon therapy.

The activities of the interferon-induced enzyme 2'-5' oligoadenylate synthetase in peripheral blood mononuclear cells were determined in patients with viral hepatitis. Increased levels of the enzyme were found in patients with acute type B hepatitis at the early phase and also in those with chronic type B hepatitis at the active stage. The levels of this enzyme activity were not significantly increased in patients with acute type A hepatitis or in those with acute type non A non B hepatitis. However, in two patients with acute type A hepatitis the levels of this enzyme were elevated shortly at the onset. These results support the hypothesis that an endogenous interferon response in patients with acute type A hepatitis and in those with acute type B hepatitis may be instrumental in the modulation of these types of hepatitis virus infections. Whereas hepatitis non A non B virus replication in patients with acute type non A non B hepatitis may be associated with poor interferon response. The activities of the enzyme in peripheral blood mononuclear cells were determined in patients with chronic type B hepatitis during interferon therapy. Increased levels of the enzyme were found in all patients during interferon therapy. This increase correlated well with the decreased DNA-polymerase activities. The data also showed that assay of this enzyme activity is useful to determine the optimal dosage and means of interferon therapy for chronic type B hepatitis.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

T4 phage gene uvsX product catalyzes homologous DNA pairing.

Gene uvsX of phage T4 controls genetic recombination and the repair of DNA damage. We have recently purified the gene product, and here describe its properties. The protein has a single-stranded DNA-dependent ATPase activity. It binds efficiently to single- and double-stranded DNAs at 0 degrees C in a cooperative manner. At 30 degree C the double-stranded DNA-protein complex was stable, but the single-stranded DNA-protein complex dissociated rapidly. The instability of the latter complex was reduced by ATP. The protein renatured heat-denatured double-stranded DNA, and assimilated linear single-stranded DNA into homologous superhelical duplexes to produce D-loops. The reaction is stimulated by gene 32 protein when the uvsX protein is limiting. With linear double-stranded DNA and homologous, circular single-stranded DNA, the protein catalyzed single-strand displacement in the 5' to 3' direction with the cooperation of gene 32 protein. All reactions required Mg2+, and all except DNA binding required ATP. We conclude that the uvsX protein is directly involved in strand exchange and is analogous to the recA protein of Escherichia coli. The differences between the uvsX protein and the recA protein, and the role of gene 32 protein in single-strand assimilation and single-strand displacement are briefly discussed.

Adenosine Triphosphatases↗
PubMed1985

Sequence of the T4 recombination gene, uvsX, and its comparison with that of the recA gene of Escherichia coli.

We have determined the nucleotide sequence of the uvsX gene of bacteriophage T4 which is involved in DNA recombination and damage repair, and whose product catalyzes in vitro reactions related to recombination process in analogous manners to E. coli recA gene product. The coding region consisted of 1170 nucleotides directing the synthesis of a polypeptide of 390 amino acids in length with a calculated molecular weight of 43,760. Amino acid composition, the sequence of seven NH2-terminal amino acids and molecular weight of the protein deduced from the nucleotide sequence were consistent with the data from the analysis of the purified uvsX protein. The nucleotide sequence and the deduced amino acid sequence were compared with those of the recA gene. Although a significant homology was not found in the nucleotide sequences, the amino acid sequences included 23% of identical and 15% of conservatively substituted residues.

Adenosine Triphosphatases↗
PubMed1985

Genetic analysis of subunit assembly of the tail fiber of bacteriophage T3.

Bacteriophage T3 virions have six tail fibers composed of the product of gene 17 (gp17). Each tail fiber is a trimer of gp17 polypeptide. To characterize the assembly process of the tail fiber, temperature-sensitive (ts) mutants of gene 17 (ts17) were analyzed by SDS-polyacrylamide gel electrophoresis and by extract complementation. Newly synthesized gp17 polypeptide chains matured to SDS-resistant native trimers with a half time of about 7.5 min at 30 degrees. Although all ts17 mutants had similar plating efficiencies at restrictive temperature (41.5 degrees or 42 degrees), they showed different phenotypes. tsNG75, whose mutation was located in the carboxyl-terminal region of gene 17, was defective in trimer assembly at 41.5 degrees. The ts tail fibers formed at 30 degrees lost the ability to attach to the tail upon treatment at 41.5 degrees. There was a change in temperature sensitivity of tsNG75 tail fibers upon attachment to the tail, suggesting that the tail fiber may change conformation after attachment to the tail. tsNG215 and tsNG169, whose mutation sites were located in the amino-terminal region of gene 17, were not defective in the trimer assembly and attachment to the tail at the restrictive temperature. tsNG215 tail fibers formed at 41.5 degrees appear to be aberrant because they were not active in extract complementation and their attachment to fiberless particles resulted in production of noninfectious phage. Tail fibers produced by cells infected with tsNG169 at the restrictive temperature were active in extract complementation. Phage particles were formed in tsNG169-infected cells at the restrictive temperature. These particles were infectious at the permissive temperature and the mutant was non-infectious only if infection was continued at the restrictive temperature. These phenotypic differences exhibited by different gene 17 mutants may indicate the regions within the gene 17 polypeptide that play a role(s) in the folding and assembly of gp17 and in the biological activity of the mature tail fiber.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Purification and characterization of gene 17 product of bacteriophage T3.

Tail fiber proteins of bacteriophage T3 are encoded by gene 17. By using in vitro complementation for phage assembly as an assay, the product of gene 17 (gp17) was purified to near homogeneity from cells infected with a double mutant of T3 defective in DNA synthesis and head assembly. The purified gp17 consists of a single polypeptide having a molecular weight of 67,000. Electron microscopy of the purified gp17 showed a fiber structure similar to the tail fiber in a virion. The subunit structure of the purified, native gp17 was analyzed by using a crosslinking agent, dimethyl suberimidate. The results indicate that native gp17 is a trimer of gp17 monomer.

Macromolecular Substances↗
PubMed1985

Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA-packaging specificity.

In vitro DNA-packaging systems of bacteriophages T3 and T7 packaged homologous DNA more efficiently than heterologous DNA. Packaging of phage DNA proceeds by way of concatemeric intermediates (H. Fujisawa, J. Miyazaki, and T. Minagawa (1978), Virology 87, 394-400). The conversion of mature homologous and heterologous DNAs to concatemers was efficient in both the T3- and T7-packaging systems. In vitro complementation experiments indicate that the gene 19 product (gp19) specifies which DNA enters the capsid. To identify DNA regions recognized by the packaging systems, T3/T7 hybrids were constructed and physical maps of the hybrid DNAs were determined by restriction enzyme analysis. By comparing restriction maps and in vitro packaging of hybrid DNAs, it is concluded that the sequence responsible for specificity of DNA packaging is confined within 5% of the ends of the T3 and T7 genomes.

Base Sequence↗
PubMed1985

Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvsX and uvsY.

The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination. Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Their relative molecular masses are 39 000 and 16 000, respectively. In the normal wild-type infection process they are produced early but not late in infection. Their synthesis continues for a longer period when DNA synthesis is blocked. We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye. The purification procedures suggest that these products may be membrane proteins. Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single-stranded fd DNA fragment into superhelical double-stranded fd DNA (RFI). The reaction requires ATP and Mg2+ besides substrate DNAs and uvsX protein. The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.

Adenosine Triphosphatases↗