Biomedical subjects

R K Hamatake

Publications and source records attributed to R K Hamatake.

25 records · Page 2Linked to original sources

PubMed1988

The J gene of phi X174: isolation and characterization of a J gene mutant.

The J gene protein of bacteriophage phi X174 is a component of the mature phage. The previous lack of J gene mutants has prevented an in vivo analysis of J protein functions. A phi X174 mutant was constructed by inserting an 11 nucleotide sequence into the J gene. This mutant, designated insJ, was viable only in the presence of a wild-type J gene carried on a plasmid that could provide J protein. An analysis of DNA synthesis during insJ mutant infection under non-permissive conditions confirmed that the J protein is not required for viral DNA synthesis.

Amino Acid Sequence↗

PubMed1985

The J gene of bacteriophage phi X174: in vitro analysis of J protein function.

The J protein of phi X174 is a small, highly basic protein and is a component of the phage capsid. We have investigated the role of J protein during single-stranded viral DNA synthesis and phage morphogenesis by using an in vitro system composed of purified viral and host components (Aoyama et al., Proc. Natl. Acad. Sci. U.S.A. 80:4195-4199, 1983). The characterization of the products made in the presence and absence of J protein shows that J protein is not required for viral DNA synthesis, but is required for the packaging of DNA into infectious phage. The ability of J protein to bind to double-stranded DNA as well as single-stranded DNA and other interactions with DNA suggest a model in which J protein binds to double-stranded, replicative form DNA and enters the phage prohead by remaining bound to viral DNA as it is encapsidated.

Adenosine Triphosphatases↗

PubMed1983

Purification of phi X174 gene C protein.

The product of gene C of bacteriophage phi X174 is required for the replication of phiX174 single-stranded DNA in Escherichia coli cells infected with phi X174. The protein has been purified to homogeneity using an in vitro complementation system. The protein exhibits a molecular weight of 5800 under denaturing conditions. The NH2-terminal amino acid sequence of the protein is (NH2)-Met-Arg-Lys, which is consistent with the sequence predicted from the nucleotide sequence of gene C. The protein has an affinity for single-stranded DNA but less for double-stranded DNA.

Bacteriophage phi X 174↗

PubMed1983

In vitro synthesis of bacteriophage phi X174 by purified components.

An in vitro system capable of synthesizing infectious phi X174 phage particles was reconstituted from purified components. The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA.

Bacteriophage phi X 174↗

PubMed1981

Morphogenesis of phi X174: in vitro synthesis of infectious phage from purified viral components.

An in vitro system that synthesizes infectious phage phi X174 was developed. The synthesis depended on phi X174 supercoiled replicative form DNA, purified phi X174 gene A protein, gene C protein, gene J protein, prohead (phage head precursor composed of gene F, G, H, B, and D proteins), and uninfected host crude extract. The infectious phage synthesis was coupled with DNA synthesis. De novo initiation, elongation, and termination of phi X174 single-stranded DNA was observed. The phage synthesized in vitro cosedimented with in vivo phage in sucrose gradients and had the same buoyant density as in vivo phage in a CsCl gradient. Our results indicate that the in vitro system mimics the in vivo phi X174 assembly process.

Bacteriophage phi X 174↗

PubMed1981

Role of DNA gyrase subunits in synthesis of bacteriophage phi X174 viral DNA.

The role of Escherichia coli DNA gyrase subunit A and subunit B during phi X174 viral DNA synthesis was investigated. Addition of nalidixic acid (an inhibitor of gyrase subunit A) and novobiocin (an inhibitor of gyrase subunit B) to an in vitro system capable of synthesizing phi X174 viral DNA inhibited DNA synthesis. The inhibition caused by novobiocin, however, was not due specifically to an inhibition of gyrase subunit B because DNA synthesis in an in vitro system composed of an extract containing novobiocin-resistant gyrase subunit B was also inhibited by novobiocin. The requirement for gyrase subunit A and the dispensability of gyrase subunit B during viral strand synthesis was confirmed in vivo by examining phi X174 viral DNA synthesis in host bacteria containing temperature-sensitive gyrase subunits.

Bacteriophage phi X 174↗

PubMed1979

Isolation and identification of bacteriophage phi X174 prohead.

The morphogenesis of bacteriophage phi X174 has been investigated by using an in vitro DNA synthesizing system. An extract of a B-mutant-infected cell is capable of synthesizing infectious phage in vitro when the B gene function is provided by the addition of an ammonium sulfate fraction of a C-mutant-infected-cell extract. This fraction contains the omega complex, a complex of phage-coded proteins with S = 108; the B-mutant extract does not. The purified omega complex, isolated from the C-mutant extract, caused the synthesis and encapsidation of viral DNA when added to B-mutant extract. The omega complex contains the B protein but it is the intact omega complex that functioned in the in vitro complementation of the B-mutant extract because other fractions containing B protein but no omega complex had little or no complementing activity. The results indicate that the omega complex is the phi X174 prohead. The B protein is not found in either the 132S or 114S phage particles but is an essential component of the prohead. This suggests that it may have a scaffolding function.

Bacteriophage phi X 174↗