Inhibition of Translation of Preformed lac Messenger Ribonucleic Acid by T4 Bacteriophage

Cells infected with bacteriophage T4 are unable to translate beta-galactosidase messenger ribonucleic acid which exists in the cell before infection, unless translation is underway at the time of infection.     

Inhibition of Ribonucleic Acid Bacteriophage Release from Its Host by Rifampin

Rifampin, in addition to interfering with intracellular growth of the ribonucleic acid-containing phage MS2, also inhibits the release of mature phage particles from Escherichia coli cells.     

Superinfection with Bacteriophage T4: Inverse Relationship Between Genetic Exclusion and Membrane Association of Deoxyribonucleic Acid of Secondary Bacteriophage

The majority of the deoxyribonucleic acid (DNA) of superinfecting T4 bacteriophage which is injected and not hydrolyzed does not attach to host cell membrane. Low levels of association of secondary phage DNA with membrane appear to be related to temporal genetic exclusion.     

Synthesis of Messenger Ribonucleic Acid After Bacteriophage T1 Infection

Synthesis of host-specific and phage-specific messenger ribonucleic acid (mRNA) was studied in bacteria infected by unmodified (T1 . B) or modified [T1 . B(P1)] bacteriophage T1. In a "standard" infection of Escherichia coli B by T1 . B (no host-controlled modification involved), the rate and amount of T1 mRNA synthesis was intermediate between those values reported for infections by a virulent phage such as T4 or a temperate phage such as lambda. The initial rate of mRNA synthesis was slightly increased after T1 . B(P1) infection of E. coli B in comparison with T1 . B infection of the same host. Little or no phage mRNA synthesis could be detected in T1 . B infection of E. coli B(P1). Phage mRNA synthesis in T1 . B(P1)-infected E. coli B(P1) cells was approximately the same in amount as that seen in T1 . B(P1) infection of E. coli B. Synthesis of host-specific mRNA continued throughout the latent period in all infections studied. However, the enzyme beta-galactosidase could not be induced, except after T1 . B infection of E. coli B(P1). In an attempt to understand the apparent differences in mRNA synthesis after infection of E. coli B by phages T1 . B or T1 . B(P1), the effect of altered T1 deoxyribonucleic acid (DNA) methylation on mRNA synthesis was studied. Methyl-deficient T1 DNA, made in cells infected with ultraviolet-irradiated phage T3, inhibited (14)C-uridine incorporation more strongly than normal T1. One passage of methyl-deficient T1 through E. coli B restored uracil incorporation rates to those seen with ordinary T1. This suggests that methylation of T1 DNA can influence the rate of phage mRNA synthesis. However, attempts to relate the difference in mRNA synthesis seen between T1 . B and T1 . B(P1) in E. coli B to the activity of the P1 modification gene were not conclusive.     

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.     

Replication of Bacteriophage Ribonucleic Acid: Some Properties of Native and Denatured Replicative Intermediate

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli cells infected with the ribonucleic acid (RNA) bacteriophage R17 were denatured with dimethyl sulfoxide at 37 C or in aqueous solvents of low ionic strength at 97 C. Denaturation was demonstrated for RF and RI by an increase in specific infectivity and a striking change in the hyperchromicity curves after treatment. RI denaturation was also demonstrated by a shift in the buoyant density in Cs(2)SO(4) from 1.619 to the buoyant density of single-stranded R17 RNA (1.627). Analysis of the denatured RI hyperchromicity curves and the equilibrium distributions of denatured RI in Cs(2)SO(4) gradients revealed, however, a residual double-stranded component. Velocity sedimentation of denatured RI was performed, and the weight distribution of S values was calculated. From the known relation between molecular weight and S values, it was possible to transform the weight distribution into a number distribution of chain lengths. This distribution was compared with that predicted from the steady-state hypothesis for RI. Deviations from the predicted distribution may be due to the residual double-stranded component.     

Non-Continuous Translation of Coat Protein and Ribonucleic Acid Polymerase Cistrons in MS2 Bacteriophage Ribonucleic Acid

In an MS2 phage ribonucleic acid (RNA)-directed in vitro protein-synthesizing system, the coat protein cistron and the adjacent RNA polymerase cistron are translated non-continuously. The ribosomes which have completed the synthesis of coat protein dissociate from the MS2 RNA and do not read through the intercistronic gap. Translation of the adjacent RNA polymerase cistron requires ribosomes other than those translating the coat protein cistron.     

Induction of P1 Prophage and Superinfection by Bacteriophage T1

Induction of the resident prophage did not permit restricted phage T1 to replicate freely in P1-lysogenic hosts. A few induced cells did become infectible.     

Inhibition of Host Deoxyribonucleic Acid Synthesis by T4 Bacteriophage in the Absence of Protein Synthesis

The requirement for phage protein synthesis for the inhibition of host deoxyribonucleic acid synthesis has been investigated by using a phage mutant unable to catalyze the production of any phage deoxyribonucleic acid. It has been concluded that the major pathway whereby phage inhibit host syntheses requires protein synthesis. The inhibition of host syntheses by phage ghosts is not affected by inhibitors of protein synthesis.     

Lysis Inhibition in Escherichia coli Infected with Bacteriophage T4

A technique of continuous filtration of T4-infected Escherichia coli has been devised to study the phenomenon of lysis inhibition. Studies using this technique revealed that the length of the lysis delay caused by superinfection can attain only certain discrete values, which for low average multiplicity of superinfection is thought to be a reflection of the actual number of superinfecting particles per cell. The time interval between primary and superinfection had little effect on the length of lysis delay. With increasing rate of superinfection, the length of lysis delay decreased. In superinfected cells, the concentration of endolysin exceeded the final concentration in nonsuperinfected cells. Superinfection of a lysing culture induced lysis inhibition immediately. Temperature-shift experiments, with cells primarily infected by a temperature-sensitive endolysin mutant, revealed that after the normal latent period superinfection was unable to induce lysis inhibition. Amber-restrictive cells, which were primarily infected by an endolysin negative amber mutant, released adenosine triphosphate (ATP) at the end of the normal latent period although lysis did not occur. Superinfection reduced the loss of ATP markedly. The hypothetical role of the cytoplasmic membrane in lysis inhibition is discussed.     

Replication and Recombination in Ligase-Deficient rII Bacteriophage T4D

Deoxyribonucleic acid replication and genetic recombination were investigated after infection of Escherichia coli with ligase-deficient rII bacteriophage T4D. The major observations are: (i) deoxyribonucleic acid synthesis is discontinuous, (ii) the discontinuities are more slowly repaired than in wild-type infection, (iii) host ligase is required for viability, and (iv) genetic recombination is increased.     

Role of Ribonucleic Acid Synthesis in Replication of Deoxyribonucleic Acid

An experiment previously interpreted to show a ribonucleic acid requirement for propagation of deoxyribonucleic replication is reexamined and the earlier interpretation is shown to be incorrect.     

Cytoplasmic Fractions Associated with Semliki Forest Virus Ribonucleic Acid Replication

When actinomycin D-treated chick fibroblasts were labeled with (3)H-uridine for varying periods during the log phase of Semliki Forest virus infection, radioactivity was found associated with different cytoplasmic fractions. After a 1-min period of labeling, it appeared in a large cytoplasmic structure which was seen in electron micrographs of infected cells. Sediments of sucrose density gradients of cytoplasmic extracts of these cells also contained these structures. Three forms of viral ribonucleic acid (RNA) were associated with this cytoplasmic structure: a ribonuclease-sensitive 42S form identical to the RNA of the mature virus, a ribonuclease-sensitive 26S form, and a ribonuclease-resistant 20S form. After a 5- to 10-min labeling period, radioactivity was associated with a ribonuclease-sensitive 65S cytoplasmic fraction which contained only the 26S RNA form. Finally, after a 1-hr labeling period, a 140S ribonuclease-resistant particle was the most prominent radioactive structure in the cytoplasm. This particle contained only 42S viral RNA. Negative-contrast electron micrographs of the 140S particle and the virion demonstrated structural differences between them. The base compositions of the 42S and 26S viral RNA forms were not significantly different. The base composition of the 20S form differed significantly from that of the other two viral RNA forms, but the values obtained for the mole fractions of the bases present in the 20S form differed, and depended on the period during the virus growth cycle in which (32)P was present. These results suggested that viral RNA originated in the large cytoplasmic body. The 20S RNA appeared to be a structure engaged in viral RNA replication and the 140S particle appeared to be a virus precursor.     

Inhibition of Ribonucleic Acid Synthesis by Nalidixic Acid in Escherichia coli

The effect of low concentrations of nalidixic acid on ribonucleic acid (RNA) synthesis in Escherichia coli was examined. It was observed that RNA synthesis in exponentially growing cells was not significantly affected, in harmony with previous studies. However, RNA synthesis was markedly depressed by nalidixic acid during starvation for an amino acid or during chloramphenicol treatment. This effect was not caused by increased killing or inhibition of nucleoside triphosphate synthesis by nalidixic acid. The pattern of radioactive uracil incorporation into transfer RNA or ribosomes was not changed by the drug. The sensitivity of RNA synthesis to nalidixic acid in the absence of protein production may be useful in probing the amino acid control of RNA synthesis.     

Early Intracellular Events in the Replication of Bacteriophage T4 Deoxyribonucleic Acid: V. Further Studies on the T4 Protein-Deoxyribonucleic Acid Complex 1

Soon after infection parental deoxyribonucleic acid (DNA) enters a structure sedimenting fast to the bottom of a sucrose gradient. The addition of chloramphenicol (CM) prevents formation of this structure, whereas treatment with Pronase releases DNA which sediments thereafter with the speed characteristic of phenol-extracted replicative DNA. It is assumed therefore that the structure responsible for fast sedimentation of replicative DNA is a newly synthesized protein. Those fast-sedimenting complexes contain preferentially the replicative form of parental DNA; this was proven by density labeling experiments. Progeny DNA labeled with (3)H-thymidine added after infection can also be detected preferentially in this fast-sedimenting moiety. The association of the DNA with the complexing protein is of a colinear or quasi-colinear type. This was proven by introducing double-strand scissions into DNA embedded in the replicative complex; double-strand scissions do not liberate DNA from the fast-sedimenting complex. Despite the apparent intimate relation between protein and DNA, DNA residing in complexes is fully sensitive to the action of nucleases. Shortly prior to the appearance of the fast-sedimenting complex, parental DNA displays still another characteristic: at about 3 min after infection, it sediments faster than reference, but sizeably slower than the complex which appears at roughly 4 to 5 min after infection. The transition between these two fast-sedimenting types of moieties is not continuous. This fast-sedimenting intermediate, which appears at 3 min after infection, cannot be inhibited by the addition of CM either at the moment or prior to infection. Fast-sedimenting intermediate can be destroyed by sodium dodecyl sulfate, Pronase, or phenol extraction. The progeny DNA labeled with (3)H-thymidine between 3 and 3.5 min after infection can be recovered in fast-sedimenting intermediate. The contribution of newly synthesized progeny DNA is so small that it cannot be detected as a shift of the parental density in a density labeling experiment. Small fragments of progeny DNA recovered in fast-sedimenting intermediate are not covalentlv attached to parental molecules and represent both strands of T4 DNA.     

Early Intracellular Events in the Replication of Bacteriophage T4 Deoxyribonucleic Acid: VI. Newly Synthesized Proteins in the T4 Protein-Deoxyribonucleic Acid Complex 1

Shortly after infection of Escherichia coli B with T4 phage, the phage deoxyribonucleic acid (DNA) can be isolated as a fast-sedimenting, proteinaceous complex. Formation of the complex is inhibited by the addition of chloramphenicol between 3 and 4 min after infection, suggesting that phage-coded proteins are necessary to form the complex and may contribute to its structure. To determine whether the phage DNA is associated with a random collection of proteins after infection or whether the complex contained a specific set of proteins, total protein from phage-infected cell lysates was compared to complex protein isolated from similar lysates by gel acrylamide electrophoresis. The proteins obtained from complexes exhibited a distinctly different pattern of separation, indicating that the complex contained a specific set of those proteins newly synthesized after infection. The proteins of the complex appear to be associated directly with the DNA rather than with some other component which could impart the characteristic of fast sedimentation to the complex. Fast-sedimenting complexes were isolated from a (3)H-leucine-labeled cell lysate. Part of this material was treated with pancreatic deoxyribonuclease. Deoxyribonuclease-treated and untreated complexes were resedimented in sucrose gradients. Virtually all the untreated complex remained fast-sedimenting, whereas most of the (3)H-leucine label of the deoxyribonuclease-treated material was located toward the top of the gradient. These data suggest a direct association of DNA and protein in the complex.     

Saint Louis Encephalitis Viral Ribonucleic Acid Replication Complex

Pulse-labeled Saint Louis encephalitis viral ribonucleic acid (RNA) is found in the cytoplasm of infected cells associated with a membranous structure which sediments with an average value of 250S. The integrity of the complex is destroyed by detergents and ribonuclease; however, it is stable in ethylenediaminetetraacetic acid (EDTA) which differentiates this structure from cellular polyribosomes. With cultures in which cellular RNA was highly labeled prior to infection, ribosomal RNA could not be demonstrated in the complex isolated from EDTA-sucrose gradients. Single-stranded 43S and the 26S and 20S forms of viral RNA were found in the complex. Viral RNA polymerase activity in sucrose-gradient fractions sedimented in the same region as the fractions which contained the pulse-labeled viral RNA. The polymerase incorporated (3)H-guanosine triphosphate into acid-precipitable material in the absence of added template. It was also found that the replication complex contains viral-specific proteins.     

Bacteriophage T4 Inhibits Colicin E2-Induced Degradation of Escherichia coli Deoxyribonucleic Acid I. Protein Synthesis-Dependent Inhibition

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we show that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with bacteriophage T4. At least two modes of inhibition may be differentiated on the basis of their sensitivity to chloramphenicol. The following observations on the inhibition of E2 by phage T4 in the absence of chloramphenicol are described: (i) Simultaneous addition to E. coli B of E2 and a phage mutated in genes 42, 46, and 47 results in a virtually complete block of the DNA solubilization normally induced by E2; the mutation in gene 42 prevents phage DNA synthesis, and the mutations in genes 46 and 47 block a late stage of phage-induced solubilization of host DNA. (ii) This triple mutant inhibits equally well when added at any time during the E2-induced solubilization. (iii) Simultaneous addition to E. coli B of E2 and a phage mutated only in gene 42 results in extensive DNA solubilization, but the amount of residual acid-insoluble DNA (20 to 25%) is more characteristic of phage infection than of E2 addition (5% or less). (iv) denA mutants of phage T4 are blocked in an early stage (endonuclease II) of degradation of host DNA; when E2 and a phage mutated in both genes 42 and denA are added to E. coli B, extensive solubilization of DNA occurs with a pattern identical to that observed upon simultaneous addition of E2 and the gene 42 mutant. (v) However, delaying E2 addition for 10 min after infection by this double mutant allows the phage to develop considerable inhibition of E2. (vi) Adsorption of E2 to E. coli B is not impaired by infection with phage mutated in genes 42, 46, and 47. In the presence of chloramphenicol, the inhibition of E2 by the triple-mutant (genes 42, 46, and 47) still occurs, but to a lesser extent.