Competence of membrane-bound T4rII DNA for in vitro "late" mRNA transcription.
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Carbon Radioisotopes↗
Biomedical subjects
C Shalitin
Publications and source records attributed to C Shalitin.
Role of gene 52 in bacteriophage T4 DNA synthesis.
In an attempt to elucidate the mechanism of delayed DNA synthesis in phage T4, Escherichia coli B cells were infected with H17 (an amber mutant defective in gene 52 possessing a "DNA-delay" phenotype). The fate of (14)C-labeled H17 parental DNA after infection was followed: we could show that this DNA sediments more slowly in neutral sucrose than wild-type DNA 3 min postinfection. In pulse-chase experiments progeny DNA was found to undergo detachment from the membrane at 12 min postinfection. Reattachment to the membrane was found to be related to an increase in rate of DNA synthesis. A nucleolytic activity that is absent from cells infected by wild-type phage and from uninfected cells could be detected in extracts prepared from mutant-infected cells. In contrast, degradation of host DNA was found to be less extensive in am H17 compared with wild-type infected cells. Addition of chloramphenicol to mutant-infected cells 10 min postinfection inhibited the appearance of a nuclease activity on one hand and suppressed the "DNA-delay" phenotype on the other hand. We conclude that the gene 52 product controls the activity of a nuclease in infected cells whose main function may be specific strand nicking in association with DNA replication. This gene product might directly attack both E. coli and phage T4 DNA, or indirectly determine their sensitivity to degradation by another nuclease.
Carbon Isotopes↗
Defective deoxyribonucleic acid replication of T4rII bacteriophage in lambda-lysogenic host cells.
Sedimentation of the replicative deoxyribonucleic acid through alkaline sucrose gradients showed that rII single chains reached the half-mature size at a time when wild-type molecules formed long chains (dimers and trimers of genome size). Long rII single chains could be observed on substitution of tris(hydroxymethyl)aminomethane buffer for Na(+)K(+) phosphate in the growth medium.
Buffers↗
Defective concatemer formation in cells infected with deoxyribonucleic acid-delay mutants of bacteriophage T4.
Nonpermissive host cells infected with phage T4 mutants in genes 52, 39, and 58 through 61 are shown to form short intracellular single-stranded deoxyribonucleic acid in contrast to wild-type infected cells, which form dimers and trimers of T4 genome size.
Carbon Isotopes↗
Role of gene 46 in bacteriophage T4 deoxyribonucleic acid synthesis.
In an attempt to establish whether Escherichia coli B infected with N130 (an amber mutant defective in gene 46) is recombination-deficient, the postinfection fate of (14)C-labeled N130 parental deoxyribonucleic acid (DNA) was followed, its amount in complex with the host cell membrane being determined in sucrose gradients after mild lysis of the infected cells. The parental DNA was found to undergo gradual detachment from the membrane during infection. Pulse-chase experiments similarly showed that newly synthesized DNA is normally attached to the host cell membrane and is detached by endonucleolytic breakage at a late stage of infection. The conclusion is that only attached DNA molecules are replicated by membrane-bound replicase, whereas those detached by endonucleolytic breakage are not. It thus seems that the gene 46 product controls the activity of a nuclease whose main function is recombination of DNA nicked by endonuclease, thereby attaching it to the host cell membrane. The rate of T4 DNA synthesis is apparently governed by the efficiency of recombination. Supporting evidence was found in experiments with the double mutant N130 x N134 (genes 46, 33).
Carbon Isotopes↗
Conversion of T4 gene 46 mutant deoxyribonucleic acid into nonviable bacteriophage particles.
The T4 amber mutant N130 in gene 46 produces, upon infection of a nonpermissive host, about 10 times more phage equivalent of deoxyribonucleic acid (DNA) than viable phage particles. Intracellular DNA is shorter than its mature phage counterpart and is converted into nonviable, light phage particles (287S) with about half of the normal DNA content, incapable either of adsorbing to or killing host bacteria. This DNA is highly unstable and breaks down, upon extraction, into subunits one-tenth the normal length of T4 DNA. Electron micrographs showed that the nonviable particles consist of normal-sized capsids of various degrees of fullness. Abnormal tail structures (naked cores with the sheaths missing) were also observed. Under conditions of arrested DNA synthesis, late messenger ribonucleic acid is produced, but some species are rare or missing, resulting in uneven transcription of the late genes. Our findings indicate that continuous DNA replication is necessary for normal transcription in T4.
Adsorption↗
Differential effect of polyamines on T4 morphogenesis.
The 5-fluorouracil (5 FU) technique for the phenotypic reversion of amber mutants was used to demonstrate that under certain circumstances, in the presence of putrescine or spermidine, early mutants have an enhanced response to 5 FU, whereas late mutants have a delayed response. Bacteria infected by T4D wild-type bacteriophage did not produce phage in the presence of high putrescine concentrations. Pulse treatments with putrescine showed that the production of lysozyme depends on a putrescine-sensitive process that begins immediately after infection at 26 C and ends at 36 min or even later. The addition of putrescine at any time during the critical period between 0 and 36 min led to a corresponding delay in lysozyme synthesis after the inhibitor was removed. Intracellular phage maturation was delayed by the addition of 100 mumoles of putrescine per ml. Early enzymes were not affected by the diamine, but the level of phage deoxyribonucleic acid was considerably decreased by the inhibitor. The putrescine-sensitive process that affects the timing of maturation is suggested to be the natural process controlling the T4 "clock."
Amines↗
Selective gene transcription in bacteriophage T4 by putrescine.
Messenger ribonucleic acid (mRNA) preparations from T4-infected cells formed in the presence or absence of putrescine have been characterized and compared. Hybrid competition experiments indicate that these mRNA molecules are derived from distinct genetic loci. The results are consistent with the hypothesis that putrescine might control differential transcription of the phage genome during morphogenesis. The data are also in accord with previously observed changes in the population of mRNA formed at different times after infection.
Butylamines↗
Additional evidence for two kinds of heterozygotes in phage T4.
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Coliphages↗
Cell cycle inhibition in human BE-13 T cell leukemia cells by haptoglobin-related (HPR) antisense cDNA.
We have recently cloned and sequenced a human haptoglobin-related cDNA. Hpr expression was found in various tumor cell lines. To determine whether the haptoglobin related protein (hpr) affects the growth of an established T-cell leukemia cell line, an Hpr antisense expression vector that specifically reduces hpr production was constructed. The vector was transfected into BE-13 cells, an established T-cell leukemia cell line in which Hpr is expressed. Three stable clones were isolated in which hpr protein expression was reduced. These established cell lines proliferated more slowly than vector transfected cells in proportion to Hpr antisense mRNA expression and the reduction in hpr protein production. Following a BrdU pulse, flow cytometric analysis was performed to estimate the fraction of cells in S phase. Hpr antisense transfected cells contained less cells in S phase compared to vector transfected cells. Also in soft agar, cells expressing the antisense cDNA insert, formed on average at least 7-fold fewer colonies than cells transfected with the vector alone. The data suggest that Hpr inhibitors might be of therapeutic value for T-cell leukemia.
Base Sequence↗