Elton T. Young

Vegetative bacteriophage λ DNA. II. Physical characterization and replication

The structure and replication of the fast-sedimenting form of λ DNA found after infection have been examined. The sedimentation rate at neutral and alkaline (strand-separating) pH, shear resistance, and buoyant density in alkaline CsCl are consistent with the interpretation that the rapidly sedimenting DNA is a covalently-closed, circular duplex wound in a superhelix. Mitomycin C has been used to inhibit bacterial DNA synthesis in Escherichia coli hcr− cells infected with λ. An analysis of the λ DNA synthesized under these conditions reveals that before intracellular phage are produced, synthesis of DNA with the sedimentation properties of the closed circular form predominates. At the end of the phage eclipse period, net synthesis of circular DNA stops and synthesis of DNA with the sedimentation properties of mature viral DNA begins. If bacteria are infected with λ in the presence of 100 μg of chloramphenicol/ml. the conversion of the injected phage DNA to the fast-sedimenting form occurs, but no replication ensues. In the presence of 30 μg of chloramphenicol/ml. (added 15 minutes before infection), the circular DNA is able to replicate, albeit more slowly than in the absence of chloramphenicol. The synthesis of DNA with the sedimentation properties of mature viral λ DNA is completely inhibited in the presence of 30 μg of chloramphenicol/ml. The replication of the circular DNA has been studied using density and radioactivily labeled phage particles to initiate the infection. The circular DNA of parental origin consists of both unreplicated and semi-conservatively replicated molecules. If the infection is performed in medium containing [3H]thymine, the progeny circular DNA has either a hybrid density or a fully light density. The ratio of progeny to parental DNA rises to at least 20 by the end of the phage eclipse period. Most closed-circular DNA molecules containing parental label replicate more than once semi-conservatively. The DNA of hybrid density derived from the fast-sedimenting component on a sucrose gradient is resistant to strand separation in alkali. The formation of parental and progeny circular DNA explains the eclipse of infectivity measured in the helper assay (Dove & Weigle, 1965).

Vegetative bacteriophage λ DNA: I. Infectiv in a spheroplast assay☆

The conditions for infection of spheroplasts of several strains of Escherichia coli by λ DNA have been studied. An assay has been developed and used to compare the capacity of λ DNA, at various stages of infection, to infect either spheroplasts or helper-phage-infected whole bacteria. In contrast to the results obtained with the helper assay, λ DNA extracted from an immune lysogen after superinfection is able to infect spheroplasts. The component from the infected cell which is responsible for most of the infectivity in the spheroplast assay is the twisted, circular form of λ DNA. This component is non-infective in the helper assay. Similarly, after infection of a sensitive bacterium by λ, there is no loss of DNA infectivity, measured in the spheroplast assay, although the capacity of the extracted DNA to infect helper-infected bacteria decreases 85%. During infection, synthesis of λ DNA which is able to infect spheroplasts precedes the synthesis of λ DNA which is able to infect helper-infected bacteria by at least 20 minutes at 30 °C. The conclusion suggested by these and experiments presented in the following paper is that synthesis of the twisted, circular form of λ DNA occurs until the time of phage maturation, at which time linear phage DNA, containing cohesive ends, is synthesized and encapsulated. The “notched” circular form of λ DNA, prepared in vitro, is also able to infect spheroplasts, although it has little infectivity in the helper assay.

Bacteriophage T4 gene transcription studied by hybridization to cloned restriction fragments.

Cloned restriction fragments of bacteriophage T4 DNA (Mattson et al. , 1977; Selzer et al. , 1978) have been used as hybridization probes to study T4 transcription during infection of Escherichia coli . Eleven early genes and about 35 late genes have been studied. The time at which the genes are active, and their rate of transcription, have been measured by quantitative hybridization to excess DNA immobilized on nitrocellulose filters. The early genes studied include representatives of the three pre-replicative classes previously defined: immediate early (IE), delayed early (DE), and quasi-late (QL). Transcription of genes known or inferred to be in the IE class (genes 30, 39, 52, 40 and/or 41 and 42 ) are transcribed at the highest relative rate during the first four minutes of infection, and are transcribed in the presence of chloramphenicol. Transcription of DE genes rIIA, rIIB and 43 is highest four to eight minutes after infection and does not occur in the presence of chloramphenicol. Transcription of rIIB can be detected prior to transcription of rIIA , confirming previous data (Schmidt et al. , 1970) on rII transcription. The only genes studied which exhibit quasi-late behavior are the transfer RNA genes. They also have IE characteristics. Both l - and r -strand transcripts have been detected from the late region of the chromosome. Some of the l -strand (early) transcription detected from the late region may be anti-late RNA, but it is also possible that there are early genes in one of these regions (between genes 24 and 25 ). Differences in the kinetics of late gene transcription have been observed. A comparison of the earliest times at which late gene transcriptions can be detected shows that initiation of late gene transcription is not entirely synchronous. For example, transcription of genes 50 to 6 is detected by six minutes but transcription of genes 12 to 16 is not detected until 10 minutes. For all of the late genes studied, transcription, once begun, continues until at least 24 minutes. However, some genes ( 50 to 6 ) are transcribed at their maximal relative rate by 12 minutes whereas the relative rate of transcription of other genes ( 12 to 16, 23 ) continues to increase until at least 20 minutes. The relative rates of synthesis of late gene transcripts vary much less than the relative rates of synthesis of late proteins, suggesting that differential synthetic rates of late proteins are not controlled predominantly by messenger RNA synthetic rates.

Purification and properties of Intracellular Lamba DNA Rings

Intracellular lambda DNA has been purified from bacteria infected with lambda phage. The prediction from sedimentation properties that one intracellular form of lambda DNA is a twisted, closed-circular molecule has been confirmed by observation in the electron microscope. Two unique forms of lambda DNA are present in infected cells: twisted, closed-circular molecules (component I) and open-circular molecules (component II). One single-strand scission is sufficient to convert component I to component II. Both components I and II, either native or denatured, are able to infect spheroplasts. The infective entity present after denaturation of component I is a molecule in which strand separation has not occurred. That present after denaturation of component II is a single-strand ring; single-stranded linear molecules are not infective. The ionic strength dependence of the sedimentation rate of components I and II and phage lambda DNA has been studied. For comparative purposes, similar studies have been made with φX174 replicative form. A new consequence of DNA circularity is evident from these studies.

Control of synthesis of glucosyl transferase and lysozyme messengers after T4 infection

Abstract Messenger RNA from T4-infected cells is able to direct the cell-free synthesis of the early phage enzyme, α-glucosyl transferase (Young, 1970) and the late enzyme, T4 lysozyme (Salser, Gesteland & Bolle, 1967). We have used in vitro protein synthesis programmed by RNA from T4-infected cells as an assay to study the in vivo synthesis of the messengers for these enzymes. α-Glucosyl transferase messenger RNA which is functional in vitro can first be detected in vivo at about four minutes after infection at 30 °C. The amount of messenger increases rapidly until about ten minutes after infection and then decays with a half-life of about three minutes. This latter observation suggests that transferase messenger RNA synthesis is repressed at ten minutes. During infection of the non-permissive host with a DNA negative amber mutant, amN122 , transferase messenger RNA synthesis is prolonged several minutes so that more messenger accumulates than during infection of the permissive host or during an infection with wild type T4. The increased messenger level can account for the hyper-production of T4 early enzymes observed after infection with DNA-negative mutants. During infection with a mutant defective in gene 55 (necessary for maturation) both the synthesis and the degradation of the transferase messenger proceed normally, indicating that neither the gene 55 product nor any of the late T4 proteins that are absent in gene 55-infected cells are necessary for the repression of transcription of the transferase cistron. Neither mutant makes a detectable amount of functional lysozyme messenger RNA in the non-permissive host.

Vegetative λ DNA: III. Pulse-labeled Components

The structures of λ DNA labeled during short pulses of [^3H]thymine at various times after infection have been studied by ultracentrifugal analysis. Circular DNAs, both the supercoiled form and an open form, are the main DNA components synthesized in the first half of the latent period. The open circular form is the most rapidly synthesized and may be a precursor to the supercoiled DNA. During the later period of progeny DNA accumulation the open circular form and a heterogeneous, more rapidly sedimenting component(s) are synthesized during such pulses. These are apparently involved in the generation of the linear, viral DNA.

Regulation of synthesis of bacteriophage T7 lysozyme mRNA

An analysis of the kinetics of synthesis of lysozyme and lysozyme mRNA after T7 infection reveals a lag between the appearance of lysozyme mRNA and the appearance of lysozyme activity. This lag is not due to post-translational modification of the protein or to the presence of a lysozyme inhibitor in early extracts. The lysozyme mRNA does not appear to be unusually stable as judged by the decline in functional mRNA concentration late in infection. The lysozyme mRNA is transcribed in vivo only by the phage-coded RNA polymerase, whereas in vitro the E. coli RNA polymerase can transcribe the lysozyme gene. However, transcription of the lysozyme gene in vitro by E. coli RNA polymerase is prevented by the host termination factor, rho. Thus, the lysozyme gene is clearly one of the late genes of T7 rather than an early gene, and its messenger appears to be under some form of transient translational control.

Translational discrimination against bacteriophage T7 gene 0·3 messenger RNA

Abstract Based on genetic manipulation of T7 late messenger RNA levels in vivo , we previously hypothesized that wild-type T7 infection of Escherichia coli develops in mRNA excess and that there is translational discrimination against T7 gene 0·3 mRNA (Strome & Young, 1978) . The results presented here support our hypothesis. The discrimination against 0·3 mRNA translation observed in vivo can be mimicked in a cell-free system by increasing the concentration of T7 RNA beyond the level needed to saturate the translational machinery or by translating T7 RNA with a low concentration of ribosomes. This discrimination can be overcome by adding ribosomes to the cell-free system (increasing the ribosome to mRNA ratio) or by slowing the rate of polypeptide chain elongation. In addition 0·3 mRNA activity as well as a substantial fraction of T7 late mRNA activity is found to be shifted off of polysomes late in T7 infection. Our results are indicative of a low initiation rate constant for 0·3 mRNA compared to T7 late mRNAs.

Cell-free synthesis of bacteriophage T4 glucosyl transferase☆☆☆

Abstract Either messenger RNA from infected cells or T4 DNA itself directs the cell-free synthesis of phage glucosyl transferase. The appearance of enzymic activity in vitro depends on protein synthesis and also on RNA synthesis in the coupled system. A product of the reaction catalyzed by the in vitro -synthesized transferase has been identified as α-glucosylated hydroxymethylcytosine, demonstrating the de novo synthesis of α-glucosyl transferase in the cell-free system described.

Translational control of the expression of bacteriophage T7 gene 0.3

Abstract When Escherichia coli are infected at 43 °C with a bacteriophage T7 mutant that produces a temperature-sensitive RNA polymerase (ts342), the rate of transcription of the T7 late genes is reduced three- to fourfold below the rate of transcription in cells infected with wild-type T7. The reduction in T7 late mRNA concentration in cells infected with ts342 is accompanied by the over-production at late times of at least one T7 early protein, the gene 0.3 protein. Despite the difference in T7 late mRNA concentration in cells infected with wild-type T7 and ts342 at 43 °C, T7 late proteins are synthesized at the same rate in the two infected-cell cultures. These findings support an hypothesis of discrimination against 0.3 mRNA translation in favor of translating T7 late messages when mRNA is in excess of the protein synthetic machinery of the cell.