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Protein synthesis by Escherichia coli infected with bacteriophage T4D
Abstract
Proteins made by E. coli cells infected with bacteriophage T4 were analyzed by a method which combined disc electrophoresis and autoradiography. The precursorproduct relationship between subunits and larger components (head, tail) was studied by taking advantage of the fact that the larger components cannot penetrate into the gel used in disc electrophoresis.These studies have shown that the early proteins are not controlled as a single homogeneous class all members of which are synthesized during the same time periods; instead, they start being formed and are shut off at various times during the early part of the infection process.Amber mutants in gene 30 (polynucleotide ligase-defective) synthesized a small amount of DNA, which was later degraded to a fraction soluble in trichloroacetic acid. The ligase-defective mutants were capable of synthesizing an almost normal amount of late proteins, whereas all other DNA-negative mutants, including a deoxycytidine triphosphatase(dCTPase)-defective mutant and maturation-defective mutants could not induce late protein synthesis. The dCTPase-defective mutant, which also synthesizes a small amount of unstable DNA, did not induce late protein synthesis even when the degradation of DNA was prevented.
... During a normal T4 developmental program, accumulation of most prereplicative RNAs stops at about the same time that late transcription starts (4, Gen. Virol., in press). When nonpermissive cells are infected with amber-defective viruses that display either the DNA-negative (DO) or maturation-defective (MD) phenotypes, turnoff of prereplicative RNA accumulation is delayed (1,34,35,50,71,72,73,76,77; Witmer et al., in press) and the corresponding enzymes overaccumulate (7,9,13,29,38,64,69,70,77). ...
... The genes of bacteriophage T4 can be split into two large groups. Early (prereplicative) genes are first expressed prior to the onset of viral DNA synthesis (1,4,5,11,23,25,29,31,33,43,44). Late gene expression begins several minutes after the onset of DNA replication, and abundant expression of these genes requires (i) continuous DNA replication as well as (ii) continuous presence of the products of genes 33 and 55 (1, 6, 7, 12-14, 23, 25, 29, 37, 43, 47-49, 73). ...
... During a normal T4 developmental program, accumulation of most prereplicative RNAs stops at about the same time that late transcription starts (4, 6, 7-9, 33, 50, 51, 62, 71, 72, 76, 77 Gen. Virol., in press). When nonpermissive cells are infected with amber-defective viruses that display either the DNA-negative (DO) or maturation-defective (MD) phenotypes, turnoff of prereplicative RNA accumulation is delayed (1,34,35,50,71,72,73,76,77;Witmer et al.,in press) and the corresponding enzymes overaccumulate (7,9,13,29,38,64,69,70,77). ...
A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior synthesis of a phage-coded protein synthesis may not be required for the turnoff of reductase messenger production.
... The role of each head geine has been inferred from the stage of morphological arrest in the various mutants (20,7,18,6,30,23,31). More recently the problem has been approached biochemically, and the proteins from lysates of mutant-infected cells have been examined by acrylamide gel electrophoresis (21,2,14,15,3,8,17,19). From these observations, capsid morphogenesis can be divided into four stages-head formation, DNA packaging, and two completion steps-but these processes are poorly understood. ...
... The observation that DNA made later becomes detached from the M band more quickly may be explained by the increase in the pool of phage head precursors at later times (14), i.e., greater amounts of those gene products required for detachment. DNA released from the M band becomes resistant to DNase more quickly later in infection when structural precursors are more abundant. ...
We have presented a new approach to studying bacteriophage T4 head maturation. Using a modified M-band technique, we have shown that progeny deoxyribonucleic acid (DNA) was synthesized on the host cell membrane throughout infection. This DNA was released from the membrane later in infection as the result of formation of the phage head; detachment of the DNA required the action of gene products 20, 21, 22, 23, 24, 31, 16, 17 and 49, known to be necessary for normal head formation. Gene products 2, 4, 50, 64, 65, 13 and 14, also involved in head morphogenesis were not required to detach progeny DNA from the membrane; the presence of the phage tail and tail fibers also was not required. DNA was released in the form of immature heads and initially was sensitive to deoxyribonuclease (DNase). Conversion to DNase resistance followed rapidly. The amount of phage precursors present at the time of DNA synthesis determined the time of onset and detachment rate of DNA from the M band as well as the kinetics by which the detached DNA become DNase resistant.
... Some new enzymes are produced in the bacterium infected by phage with 5hmC modified DNA (Flaks and Cohen, 1959;Kornberg et al., 1959;Koerner et al., 1960). Substitution of C for 5hmC in vegetative T4 DNA depresses the synthesis of late proteins (Hosoda and Levinthal, 1968;Kutter and Wiberg, 1969), moreover, transcription of some late genes can only occur from 5hmC-containing, but not from C-containing DNA (Kutter and Wiberg, 1969). ...
Dynamic DNA modifications, such as methylation/demethylation on cytosine, are major epigenetic mechanisms to modulate gene expression in both eukaryotes and prokaryotes. In addition to the common methylation on the 5th position of the pyrimidine ring of cytosine (5mC), other types of modifications at the same position, such as 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC), are also important. Recently, 5hmC, a product of 5mC demethylation by the Ten-Eleven Translocation family proteins, was shown to regulate many cellular and developmental processes, including the pluripotency of embryonic stem cells, neuron development, and tumorigenesis in mammals. Here, we review recent advances on the generation, distribution, and function of 5hmC modification in mammals and discuss its potential roles in plants.
The antibiotic rifampicin has been used in an investigation of the temporal regulation of gene expression during bacteriophage T4 infection of Escherichia coli. We have asked at what times after infection the transcription of specific cistrons becomes rifampicin insensitive. Gene expression has been studied using analyses of proteins on sodium dodecyl sulfate-acrylamide gels; thus translation has been used to assay for the appearance of specific transcripts.
Two types of promoters account for most prereplicative transcription; early promoters are recognized immediately after infection, whereas quasi-late promoters are recognized only after a delay of 1frac12; min. Those transcripts previously defined as immediate early and delayed early RNAs initially appear to be under the control of early promoters; however, the quasi-late promoters may provide a second mode of regulation for several of those transcripts.
Reinfection of Escherichia coli with the bacterial virus T4 causes modifications of the properties of the host cell envelope during the preeplicative phase of the lytic cycle. These changes include altered densities cell enveloped and their subfractions, morphological modifications of membrane vesicles, and association of newly synthesized proteins with the host cell envelope. Polypeptide analysis by high resolution electrophoresis on polyacrylamide slab gels in dodecyl sulfate revealed that most of some 30 prereplicative phage-coded polypeptides are attached to this structure. Different means of cell disruption and selective extraction procedures, such as variations of ionic strength, removal of divalent cations, and the addition of chaotropic agents or detergents were used to study the characteristics of these attachments. Many proteins appeared to be artifactually absorbed or weakly bound to the envelope, Separation of cell walls from plasma membranes showed that all of the tightly bound proteins were associated withthe cell membrane fraction. The partitioning of phage proteins between the different fractions was monitored using 12 polypeptides which were identified as products of distinct phage genes. Of these, 8 were eliminated as potential membrane markers. Four polypeptides, the products of genes rIIA, rIIB, 39, and 52 were operationally defined as membrane proteins. The number of molecules of the 12 identified phage gene products, synthesized during a single lytic cycle, was determined. The results allowed the estimation of the concentration in the membrane of those proteins which were found to be quantitatively associated with that structure. Association of phage proteins with the cell envelope was found to be unaffected by mutations in any of the identified phage polypeptides.
A nuclease has been isolated from T4-infected Escherichia coli which attacks single- or double-stranded DNA from a variety of sources, including T4. The enzyme appears late during the T4 development, and its action is dependent on the presence of —SH reagents and Mg⁺⁺ or Mn⁺⁺ ions. The rate and extent of the reaction on single-stranded DNA is about 10-fold higher in the presence of Mn⁺⁺ than in the presence of Mg⁺⁺. Single and double-strand breakage of native DNA was observed in reaction mixtures containing Mn⁺⁺ while no reaction with native DNA was detected in the presence of Mg⁺⁺. The products of the enzymatic reaction include oligonucleotides which contain 3′-OH and 5′-phosphate ends. While all four nucleotides are found at the 5′ end, a preference toward A and T was noted.
T4 mutants in genes 49, 16, or 17 which lead to defects in phage maturation induce wild type levels of the nuclease. In contrast, T4 mutants in gene 43 (T4 DNA polymerase) do not induce late functions and for this reason do not induce the Mn⁺⁺-activated nuclease.
This chapter presents the methods for the study of mRNA synthesis in bacteriophage-infected E. coli. T-even phages and their host Escherichia coli are model systems for genetic and biochemical studies of transcription and translation of phage genome. Messenger RNA (mRNA) in E. coli infected by DNA phages is assayed at the translational level by measuring the activities of various phage-specific enzymes and antigenic properties of phage structural proteins in the infected cell extracts. Meaningful analysis of mRNA depends on the RNA preparations assayed. This chapter also describes a number of methods in detail with brief comments on modifications of the methods and nature of the preparations. Two or more methods that measure different properties of mRNA are used sequentially to assay RNA preparations. This chapter also reviews the analytical methods available; useful modifications, limitations of the methods, and applications that yield more fundamental results in studies of phage development.
The structural proteins of mature LPP-1 particles and the patterns of protein synthesis after LPP-1 infection have been examined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Structural proteins account for 35% of the LPP-1 genome, and proteins that would require about 65% of the total coding capacity have been detected after infection. The major head proteins have molecular weights of 39,000 and 13,000, whereas the major tail protein is an 80,000-molecular-weight species. Host protein synthesis is depressed soon after infection and appears to be entirely shut off by 5 hr. Three classes of viral proteins are distinguished in infected cells, based on their time course of synthesis and their presence in mature virions.
The lytic process of coliphage T4 begins with adsorption of the virion to the bacterial cell wall and subsequent injection of the viral DNA into the host (COHEN, 1963, 1968; MATHEWS, 1971). Synthesis of bacterial proteins stops within seconds (BENZER, 1953; BILEZIKIAN, KAEMPFER, and MAGASANIK, 1967; HAYWARD and GREEN, 1965; KAEMPFER and MAGASANIK, 1967; KENNELL, 1970; LEVIN and BURTON, 1961; ROUVIERE et al., 1968; SHER and MALLETTE, 1954). Bacterial DNA and RNA continue to be synthesized for several minutes, but at much-reduced rates (ADESNIK and LEVINTHAL, 1970; COHEN, 1963, 1968; KENNELL, 1968; LANDY and SPIEGELMAN, 1968; MATHEWS, 1971; NOMURA, HALL, and SPIEGELMAN, 1960; VOLKIN and ASTRACHAN, 1956). Transcription of the viral DNA begins immediately (OLESON, PISPA, and BUCHANAN, 1969) and the first viral proteins are completed less than one minute after infection (HOSODA and LEVINTHAL, 1968). Replication of the viral chromosome starts 5 min after infection but viral DNA replication does not reach its maximum rate until 10 min (COHEN, 1963, 1968; MATHEWS, 1971). Intracellular progeny viruses first appear 20 or so min after infection and their number increases steadily until 30 to 35 min, at which time the infected cell lyses (COHEN, 1963, 1968; MATHEWS, 1971).
The interactions between bacteriophage P 2 and its satellite phage P 4 comprise a promising model system for studying the control of transcription because the two phage have relatively small and therefore tractable genomes and yet exhibit a surprisingly complex set of effects upon one another. Each phage possesses the ability to drastically alter the expression of the other’s genome.
This chapter describes polyacrylamide gel electrophoresis of viral proteins. Viral structural and nonstructural proteins are agents of expression for the information carried in viral nucleic acid genomes. An understanding of the behavior of a virus requires its proteins be characterized. For this purpose and for the characterization of nucleic acids, electrophoresis in gels is the most sensitive and versatile method. The genetic content and number of proteins of most viruses is relatively small compared even to simple cells. Two aspects of gels as supporting media for zonal electrophoresis experiments are responsible for the dramatic separations. First, as simple anti-convection media gels, they are more homogeneous and stable than other commonly used materials. Second, with the correct choice of gel concentrations, the spaces in the gel matrix are of similar dimensions to macromolecules so that selective sieving occurs during the electrophoretic migration. Gels composed of polyacrylamide are extensively used at the present time.
In this chapter, we shall analyze the ways in which gene activity is regulated in the development of the larger DNA viruses of bacteria. The kind of analysis that we write about has now been carried out for about 20 years, with increasing sophistication and insight as the understanding of genetic regulation has improved and as the methods of genetics, molecular biology, and enzymology have developed. The immediate goal of this search is to understand, in the molecular terms of protein-nucleic acid interaction, all the strategies of genetic regulation that are encompassed in the development of the phages. That goal is far from realization, although great strides toward it have been taken during the last few years. In fact, we originally hoped to organize our presentation of this subject around a classification of the mechanisms of action of regulatory elements and, in this way, to draw together the common principles of phage development. We eventually decided against the scheme because, with the notable exceptions that we shall emphasize, the mechanisms of gene regulation in phage development are not sufficiently worked out. We have instead chosen several prototypes of lytic phage development in order to analyze the diversity of regulatory mechanisms while avoiding excessive repetition of quasiequivalent detail. However, one should stress that, although regulatory schemes are wondrously diverse, the general features of the development of the lytic phages fit a common general pattern.
Assembly of complex bacterial viruses has been investigated intensively during the past decade, revealing, in rich detail, the explicit steps from the information stored in genes to the formation of three-dimensional structure. Research in this area has been motivated by curiosity about virus assembly as an important aspect of virus multiplication, as well as by the conviction that knowledge of the genetic control of viral morphogenesis would provide a foundation for approaching the more difficult problems of the assembly of organelles in higher organisms. Although basic questions remain to be answered about bacteriophage assembly, a great deal already has been learned, primarily because of the convenience of bacteriophages as genetically, biochemically, and structurally accessible experimental systems.
By far the best-known representative of the large virulent bacteriophages is coliphage T4, which has been an object of intense biochemical, genetic, and morphological investigation since its original description some three decades ago (Demerec and Fano, 1945). A major reason for the popularity of T4, particularly when compared with its cousins T2 and T6, is the early availability of a relatively complete genetic map (Epstein et al., 1963). Thus, at a time when biochemists were becoming aware of the value of phages as tools for study of many biological problems, a large catalogue of mutants defective in essential viral functions became available. Analysis of the molecular functions controlled by each gene product led to a productive relationship between those of primarily biochemical orientation and those of genetic persuasion. Biochemical analysis of the defect associated with a particular mutant would often suggest the existence of new types of mutants, which could then be isolated and analyzed in turn. Thus, although T4 is a large and complex virus, the ultimate goal of mapping each gene and determining the function of its product is within range. The large number of active laboratories working on T4 and the great extent to which they cooperate and communicate with one another have combined to make this a satisfying branch of science in which to work.
RNA-directed protein synthesis in vitro can be a powerful tool for the characterization of messenger RNA populations. This chapter describes and analyzes the preparations of RNA from E. coli cells infected with bacteriophage T4 that directs, in vitro protein synthesis, and shows that certain of these gene products can be formed in vitro. There are a number of other gene products for which no biological assays are known, but which can be characterized as bands on polyacrylamide gels after electrophoresis. For comparison with the in vitro products, the proteins made in vivo by cells infected with T4 are also analyzed on gels. The chapter presents characterization of the products of T4 mRNA-directed in vitro protein synthesis by an electrophoresis procedure, and concludes that the products of T4 genes 57 and 22 are synthesized in vitro under the direction of the mRNA from infected cells.
T7 messenger RNA appears to be stable. Kinetic experiments show that fulllength messenger RNA molecules accumulate in the infected cell. Actinomycin and rifampicin decay experiments show that T7 mRNA is long-lived with an average lifetime of more than 20 minutes. Control experiments with the same strain showed that Escherichia coli and T4 RNA are unstable. In T7-infected cells, however, E. coli mRNA is stabilized. This suggests that T7 infection interferes in some general way with mRNA breakdown.
Purified preparations of complete T4 bacteriophage, tail fiberless particles, whole tail fibers and four tail fiber precursors were dissociated by heating briefly at 100 °C in 1% sodium dodecyl sulfate containing 1% mercaptoethanol. Analysis of the dissociated structures by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed two high molecular weight (150,000 and 123,000 daltons) polypeptides as major tail fiber components. These two components could be easily identified by autoradiography of sodium dodecyl sulfate gels of radioactively labeled infected cell extracts. The larger of the two was missing from extracts of cells infected with gene 34 amber mutants, and the smaller from extracts of cells infected with gene 37 amber mutants. It is concluded that the two components represent the products of genes 34 and 37 (P34 and P37), respectively. Molecular weight calculations indicate that two copies of each polypeptide are present in each complete tail fiber. Amber mutations in genes 38 and 57 were found to affect the apparent solubility of P34 and P37 and their resistance to dissociation in cold sodium dodecyl sulfate, but not their synthesis. Based on these results, the previously reported pathway of tail fiber assembly (King & Wood, 1969) has been reformulated in more detail.
A total of 23 phage specific proteins (including four head and six tail proteins) could be identified after SDS polyacrylamide gel electrophoresis of extracts from phage SPP1 infected Bacillus subtilis cells. The total molecular weight of the proteins amounts to approximately 1.9×106 daltons, equivalent to the majority of the coding capacity of SPP1 DNA. It can thus be assumed that almost all SPP1 coded proteins have been identified. Protein assignments to phage cistrons were made by analysis of extracts from nonpermissive cells infected with sus-mutants. The SPP1 specified proteins can be subdivided into three groups on the basis of the time of their synthesis during the latent period. Host protein synthesis is not significantly affected by SPP1 infection. Normal expression of host genes appears to be essential for SPP1 growth.
T4 deoxyribonucleic acid(DNA)madebydeoxycytidine triphosphatase (dCTPase) ambermutantsisextensively degraded, andthat nucleases controlled bygenes46and 47participate inthis process.Inthis paper,we examine other consequencesofa defective dCTPase. Included arestudies ofDNA synthesis andphageproduction, andofthecontrol ofbothearly andlate protein synthesis after infection ofEscher- ichia coli B withvarious T4mutantsdefective ingenes56(dCTPase), 42(dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43(DNA polymerase), 30(poly- nucleotide ligase), 46and47(DNA breakdown) ore(lysozyme). Byvarying the temperature ofinfection witha temperature-sensitive dCTPasemutant, we have beenabletocontrol intracellular dCTPaseactivity, andthusvarythecytosine content ofthephageDNA. We haveproduced andcharacterized viable T4phagein whichcytosine replaces 20%ofthe5-hydroxymethylcytosine (HMC)intheDNA. We present evidence whichsuggests thatintact, cytosine-containing T4DNA is muchless efficient thanisnormalT4DNA indirecting thesynthesis oftail-fiber antigen. Lysozyme production ismuchlessaffected byprogressively decreasing dCTPaseactivity; however, complete substitution ofcytosine iscorrelated witha depression oflysozyme synthesis greater thanexpected fromthedefective synthesis ofDNA.Lowbutsignificant lysozyme synthesis isobserved late after infection of E.coliB withT4 ambermutantsdefective ina numberofgenescontrolling DNA synthesis. The"20%cytosine" T4phage, onceproduced, can initiate an ap- parently normal infection atpermissive temperatures; thesynthesis ofearly enzymes, DNA,andphage doesnotappeartobeimpaired. Tworoles forHMC inT4DNA havebeenindicated previously: (i) involvement inhost-controlled restriction ofthe phage, inwhichglucosylation ofthehydroxymethyl groupplays a crucial role(16, 29,53,58), and(ii) protection ofvegetative DNA against phage-controlled nucleases, aprotection notdependent on glucosylation (41, 66,67). A third roleissuggested by our present results: transcription ofatleast some lategenescan occuronlyfrom HMC-containing DNA andnotfromcytosine-containing DNA.
Unter der Transkription der genetischen Information versteht man die Übertragung der genetischen Information von der DNA auf mRNA, die von der DNA-abhängigen RNA-Polymerase katalysiert wird. Die Transkription umfaßt die Bindung der RNA-Polymerase an die DNA, die Initiation, die Elongation und die Termination. Am Beispiel von Bakterien und Bakteriophagen wird gezeigt, daß die Transkription durch ein Wechselspiel von positiven und negativen Kontrollelementen reguliert wird.
The expression of late SPP1 genes depends on preceding SPP1 DNA replication. This is shown in nonpermissive infection with a mutant defective in DNA replication and after inhibition of DNA synthesis by HPUra. The potential for host gene expression is not significantly influenced by SPP1 infection, as evidenced by the continuation of host protein synthesis and the inducibility of glycerolphosphate dehydrogenase after infection. The involvement of a positive control element in the regulation of SPP1 gene expression is deduced from the observation that chloramphenicol prevents the synthesis of the only class of mRNA which is transcribed from the L-strand.
Phenotypic reversion by 5-fluorouracil of amber mutations in phage T4 was studied. The rescued functions of the early genes 1 (deoxynucleotide kinase), 42 (deoxycytidylate hydroxymethylase) and 56 (deoxycytidine triphosphatase) were investigated by direct measurements of the rescued enzyme activities. Addition of 5-fluorouracil early (2 min after infection) but not late (16 min) was effective in rescuing enzyme activities corresponding to the mutated genes. The time course of formation of the rescued enzymes was similar to that of other early enzymes in nonpermissive bacteria after DNA-negative mutant infection: Enzyme activity increased for 10–15 min beyond the time of shut-off in wild-type T4-infected cells. These results indicate that transcription of the mutated early genes, rather than the translation of early messengers, is restricted in the late period of infection with early amber mutants.
The final activities of the rescued enzymes were 1–3% of the wild-type-induced activities. One of the rescued enzymes (deoxycytidylate hydroxymethylase) showed increased temperature sensitivity compared to the wild-type enzyme.
Phage production after 5-fluorouracil rescue of early amber mutants is slow compared to the efficiency of rescue of DNA synthesis. This is not due to a disturbance of the regulatory mechanisms, switching on late protein synthesis, since determinations of late RNA synthesis and of the late enzyme, endolysin, showed that these late functions were restored to a high extent after FU rescue of early amber mutations.
The rates of DNA elongation by wild-type phage T4 and a gene 52 DNA-delay am mutant were estimated by pulse-labeling infected cells with tritiated thymidine and visualizing the gently extracted DNA by autoradiography. The estimated rate of chain elongation of wild-type DNA was 749 nucleotides/second early in synthesis and 516 to 581 nucleotides/second at a later time. The rate of DNA elongation by the am mutant was measured to be 693, 758 and 829 nucleotides/second during successive stages of synthesis, indicating that elongation was not slower than in wild-type. The kinetics of DNA increase after infection of host cells by wild-type phage T4 or by the gene 52 DNA-delay am mutant was followed using [methyl-3H]thymidine uptake into acid-insoluble material. It was found that DNA increase in both wild-type and am infections could be represented as exponential during early times and linear during late times of DNA synthesis. From the rates of DNA increase and the rates of DNA elongation we were able to estimate the number of growing points per chromosome equivalent of template DNA during the exponential and linear phases. Our estimates for wild-type phage were 0.55 and 0.71 to 0.80 growing points per chromosome equivalent of template DNA in the exponential and linear phases, respectively. For the am mutant we found 0.14 and 0.12 to 0.13 growing points per chromosome equivalent of template DNA during the exponential and linear phases, respectively. The apparent lower incidence of growing points in the am mutant infections suggests that the mutant may be defective in the initiation of growing points.
In this paper we have further analyzed the role of individual replication proteins in phage T4 late gene expression. One special objective has been to determine whether individual replication gene products affect late gene expression that has independently been uncoupled from replication. In the experiments which achieve this objective, T4 poltspost−ligampost−exofampost−amX phage have been constructed, with mutations X in the various replication genes. Viral RNA and proteins have been analyzed by hybridization-competition and by acrylamide gel electrophoresis respectively. Mutations in the replication genes have been found to produce two kinds of effects: (1) a mutation in gene 45 almost completely abolishes late gene expression. Only the gene 45 mutation does this. Gene 45 protein is continuously required for late transcription. (2) Amber mutations in the other replication genes increase the temperature sensitivity of replication-independent late T4 transcription to varying degrees. The product of gene 45 is thus identified as a third essential component of late transcription, together with the previously identified gene 33 and 55 proteins.The endonucleolytic cleavage of parental DNA in the absence of phage DNA replication has also been examined. The distribution of single-strand breaks between the complementary DNA strands is not appreciably asymmetric. Breaks are introduced into the parental DNA of all phage mutants examined, in which no DNA replication occurs, including a mutant in gene 45.In the closing discussion we attempt to analyze the processing of T4 DNA for selective gene expression. The possibility that this processing and late transcription involve a complex of many replication proteins is examined.
The rates of synthesis were determined for those early mRNA species which code for early enzymes involved in phage-specific DNA synthesis. Evidence was found for a regulation mechanism which specifically restricts the transcription of early genes.The formation of early mRNA representing certain early genes was measured at different times after infection by phenotypic suppression by 5-fluorouracil of amber mutations in the early genes 1 (hydroxymethyl deoxycytidylate kinase), 42 (deoxycytidylate hydroxymethylase), and 56 (deoxycytidine triphosphatase), respectively. Suppression was measured by the determination of DNA synthesis and phage production. Since 5-fluorouracil suppression of amber mutations is effected by the incorporation of the analog into mRNA, the relative efficiency of suppression at different times after infection was interpreted to reflect the rate of mRNA formation at the time of fluorouracil addition.The regulation of early mRNA synthesis was also directly demonstrated by short-time labeling with uracil-3H and fluorouracil-3H after the infection of E. coli B with the mentioned phage mutants, which are DNA negative and produce no late mRNA.The uptake of 5-fluorouracil as an RNA precursor into the ribonucleotide pools of the infected cell was checked by electrophoretic analysis at different times after infection.
A temperature-sensitive mutation (ts553) in bacteriophage T4 gene 55, which codes for a positive control element of viral late transcription, has latent k properties (cf. Takahashi et al., 1975). It can be used to efficiently and specifically select a new class of Escherichia coli mutants (tabD). When a tabD mutant is infected with wild type T4, viral development proceeds almost normally; when tabD) is infected at 30 °C with ts553 late transcription is blocked. The tabD-generated defective phenotype is identical to that observed when tab+ is infected with an amber mutant in gene 55. A temperature-sensitive mutation (tsCB53) in T4 gene 45, which codes for a protein controlling late transcription and replication, also has latent k properties. It selects E. coli mutants, quite similar to those selected with ts553, which grow wild type T4 normally but fail to grow tsCB53 or ts553 at 30 °C; in the latter cases late transcription is blocked but not replication. The tab-generated deficiency is thus in striking contrast to that observed when tab+ is infected with an amber mutant in gene 45, characterized by a block in late transcription and replication. We argue that the products of T4 genes 55 and 45, and the bacterial protein/s identified by tabD mutants form a complex and discuss two alternative modes of interaction which may be relevant to late transcription. Since P55 and P45 bind to RNA polymerase (Ratner, 1974) one or more of the subunits of this enzyme are likely candidates for the tabD protein/s.
φX174 protein synthesis in ultraviolet-irradiated Escherichia coli C hcr− cells and. unirradiated cells was examined on sodium dodecyl sulfate-acrylamide gels. Eight viral proteins were identified and six of these assigned to cistrons. Viral protein synthesis in ultraviolet-irradiated cells is not normal. There is a large accumulation of coat protein and relatively little synthesis of cistron V (single-strand DNA synthesis) protein. The relative amount of the other φX gene products synthesized is also different from that observed during a normal infection of an unirradiated cell.
The nucleoside triphosphate of 5-(4',5'-dihydroxypentyl)uracil (DHPU) was detected in the acid-soluble extract from bacteriophage SP15-infected Bacillus subtilis W23. No uracil was found in the DNA of either replicating or mature phage. Labeled thymidine added during phage DNA synthesis was incorporated into phage DNA. The presence of DHPU as a nucleoside triphosphate in the acid-soluble pool and the incorporation of thymidine into phage DNA suggest that both DHPU and thymine are incorporated into SP15 DNA via their nucleoside triphosphates. 5-Fluorodeoxyuridine inhibited biosynthesis of SP15 DNA, and this inhibition was reversed by thymidine, resulting in the synthesis of a DNA containing reduced amounts of fully modified DHPU. It is proposed that 5-fluorodeoxyuridine, or its metabolic product, inhibits a step in the biosynthetic pathway to the nucleoside triphosphate of DHPU.
We have studied the in vivo effects of T4 endonucleases II and IV on the cytosine-containing T4 DNA made after infection of Escherichia coli B with dCTPase amber mutants of bacteriophage T4. Both nucleases are specific for cytosine-containing DNA; endonuclease II is a nickase, and endonuclease IV is specific for single-stranded stretches of DNA. The small amount of DNA made by dCTPase amber mutants is rapidly degraded; in contrast, the (dCTPase, endonuclease II, endonuclease IV) mutant makes normal amounts of DNA. Although this cytosine-containing T4 DNA is actually larger than that made by T4D+, as determined by both neutral and alkaline density-gradient sedimentation, no viable phage particles are produced.The relative importance of the two nucleases in the degradation process differs from that observed previously for host DNA degradation. Degradation of cytosine-containing T4 DNA is largely prevented by abolishing endonuclease IV alone but occurs normally in the absence of endonuclease II, whereas the reverse is true for host DNA degradation. Possible reasons for this disparity are discussed.To determine the reason for the non-viability of the (dcTPase, endonuclease II, endonuclease IV) triple mutant, we have studied the in vivo patterns of protein synthesis. Using polyacrylamide gel electrophoresis, we find that the synthesis of all late proteins we can analyze is almost totally abolished when cytosine replaces hydroxymethylcytosine in the progeny DNA, even though DNA degradation has been prevented by the nuclease mutations. In fact, substantially more late-protein synthesis is seen with T4 mutants totally defective in DNA synthesis than when the progeny DNA contains cytosine rather than hydroxymethyl-cytosine. Only with gene 45 or 55 mutants, both of which affect RNA polymerase alterations, is the block of late-protein synthesis more stringent.In contrast, the timing of shutoff of early-enzyme synthesis is almost normal, although there is some overproduction of most early enzymes. Those proteins synthesized by T4D+ throughout the infection cycle are produced in similar manner and amounts by our triple mutant, except that the gene 32 protein is grossly overproduced, as it is after infection with certain other mutants that make aberrant unencapsulated DNA.
It has previously been shown that the product of gene 22 (P22) disappears completely from lysates of T4-infected bacteria during head formation and is not found in the finished phage. We show here that P22, as part of a phage head precursor, is subject to proteolysis in vivo. The only identifiable surviving fragments of this proteolysis may be the internal peptides, which are found inside the finished phage head.We further show that in vitro, head-defective lysates contain a protease activity highly specific for P22. The activity is dependent on the presence of wild-type gene 21 protein (P21). The protease is itself inactivated during the protein cleavages that accomplish capsid formation. The proteolytic activity is found associated with the defective heads produced by temperature-sensitive mutants in gene 23, but not in finished normal capsids.We have characterized this P21-dependent protease activity as it is exhibited in vitro.
T1 infected bacteria exhibit a distinct pattern of gene expression. The control of this expression is accessible to biochemical analysis. T1 induces the synthesis of 31 proteins in E. coli. The virion contains 15 proteins. By means of T1 amber mutants, 10 gene products have been assigned to specific T1 genes. Three classes of T1 proteins are defined by the kinetics of their syntheses: early, early-late and late proteins. The regulation of protein synthesis involes at least three mechanisms: for cessation of host gene expression, for discontinuation of the early class during the late phase and for induction of the late T1 proteins. The positive control of late gene expression is not coupled to replication. The host RNA-polymerase transcribes the viral genome throughout the infectious cycle. No virus coded RNA-polymerase is induced.
Cold centrifugation of lysis-inhibited Escherichia coli B infected with wild-type T4D results in extensive lysis beginning around 20 min after infection at 37 degrees C. Infection with an e mutant, which fails to make lysozyme, prevents lysis, but does not prevent a marked loss of K+ and Mg3+. The t gene product, thought to disrupt the cytoplasmic membrane in natural lysis, is not required for this handling-induced cation loss or lysis. Three lines of evidence argue that late protein synthesis is required to develop this potential for cation loss; the potential does not develop in infections by: (i) mutants defective in DNA synthesis, (ii) mutants defective in gene 55, and (iii) wild-type T4 when chloramphenicol is added at 6 min after infection. All late mutants examined, which are blocked in the major pathways of morphogenesis, do not prevent development of the potential. The evidence argues for a new, late effect of T4 infection on the cytoplasmic membrane.
In this and the following paper the control of gene expression from different T4 DNA templates is examined. Absence of glucosylation did not grossly alter T4 early gene expression. Late gene expression, on the other hand, was altered extensively. The absence of late gene expression in DNA negative or maturation defective mutants was overcome to an appreciable extent if the infecting phage DNA lacked glucose as well. Fragmentation of the nonglucosylated genome by restriction cleavage had distinct effects on both early and late gene expression.
Some mutations in the structural gene for T4 DNA polymerase (gene 43) behave as suppressors of a deficiency in T4 dCMP-hydroxymethylase (gene 42). The suppression appears to involve a functional interaction between the two enzymes at the level of DNA replication. The hydroxymethylase deficiency caused DNA structural abnormalities in replication, and DNA polymerase lesions appeared to partially reverse these abnormalities. The results do not necessarily imply protein-protein interactions between the two enzymes, although both enzymes appear to play roles in controlling the fidelity of phage DNA replication.
The proteins specified by at least 18 bacteriophage T4 genes are needed for the formation of the complex hexagonal baseplate of the phage tail. At least 14 of these protein species are incorporated into the mature, structure of this organelle.Cells infected with amber mutants defective in any of five clustered baseplate genes (53, 6, 7, 8 and 10) are unable to form hexagonal structures.These mutantinfected infected cells accumulate structural protein complexes which are precursors to baseplates. They can be assembled into mature baseplates and thus viable phage, in vitro, in mixtures of extracts of mutant-infected cells.We have isolated the precursor proteins and protein complexes specified by the five genes, by gradient centrifugation. The proteins have been characterized by sodium dodecyl sulfate gel electrophoresis, and by their in vitro assembly activity. The results show that the major structural precursor of the baseplate is a 15 S complex containing six protein species. These interact in the following sequence:If any step in this pathway is blocked by mutation, the subsequent proteins remain soluble and unassembled. With the exception of the initiating proteins, reactive sites are not present on the soluble subunits, but are generated only upon incorporation of a subunit into the growing complex. Thus reactive sites are limited to growing structures, ensuring efficient assembly.
During development of bacteriophage T1 at least 28 viral proteins are synthesized. Depending on the time of appearance, these polypeptides have been divided into three classes termed I, II and III. Second and third class proteins include the 13 structural monomers that make up T1 virion. A few of these proteins are synthesized before the onset of DNA replication. The molecular weights of the structural polypeptides range from 79,000 to 14,000 daltons. Pulse-chase experiments have shown that the major structural protein is synthesized in the form of a larger precursor which is then cleaved to give a final product of 30,000 daltons.
The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and phage DNA biosynthesis.
The requirement for T4 gene 57 function, normally essential for tail fiber assembly and phage production, can be partially bypassed in mutants of host E. coli strains B and K12 (byp mutants). 57− Mutants plate with almost full efficiency on the byp hosts, but burst sizes in liquid culture are only 10% of normal, and tail fiber antigen production is also subnormal. The byp mutations are specifically permissive for 57− mutant phage; amber mutants defective in other genes do not grow on the byp hosts. Enhanced 57− phage growth reflects an increased rate of intracellular phage production rather than an extended latent period. The mutation in one byp strain, BP4, affects cell growth, cell morphology, and T4 rII plaque morphology in addition to the growth of 57− phage. Simultaneous reversion of all these mutant characteristics suggests a single mutational origin. The nature of host byp mutations is discussed in relation to the possible mechanism of gene 57 function in normal T4 development.
Functional half-life measurements of the bacteriophage T4 gene 32 messenger RNA indicate that this mRNA is extremely stable. Regulation of gene 32 expression at the transcriptional level cannot account for the rapidity with which P32 synthesis can be repressed. Furthermore, derepression of P32 synthesis occurs in the presence of rifampicin, a drug which inhibits transcriptional initiation. In addition, T4-infected cultures in which P32 expression is repressed possess almost as much gene 32 mRNA as derepressed cultures. We conclude that expression of the T4 gene 32 protein is regulated at the level of translation.
A total of 23 phage specific proteins (including four head and six tail proteins) could be identified after SDS polyacrylamide gel electrophoresis of extracts from phage SPP1 infected Bacillus subtilis cells. The total molecular weight of the proteins amounts to approximately 1.9 X 10(6) daltons, equivalent to the majority of the coding capacity of SPP1 DNA. It can thus be assumed that almost all SPP1 coded proteins have been identified. Protein assignments to phage cistrons were made by analysis of extracts from nonpermissive cells infected with sus-mutants. The SPP1 specified proteins can be subdivided into three groups on the basis of the time of their synthesis during the latent period. Host protein synthesis is not significantly affected by SPP1 infection. Normal expression of host genes appears to be essential for SPP1 growth.
The effects of immunization with Streptococcus mutans on the development of caries and the immune responses were investigated in 37 young rhesus monkeys (Macaca mulatta) during a period of up to 33 months. The monkeys were supplied a human type of carbohydrate-rich diet that contained about 15% sucrose. The monkeys were separated into seven groups, and the effects of two whole cell vaccines and an extracellular culture extract of S mutans in Freund's incomplete adjuvant were compared with a vaccine of a noncariogenic Streptococcus CHT, the adjuvant alone, and a sham immunized group. Sequential analysis of complement fixing, hemagglutinating and precipitating antibodies to the cell wall, and extracellular culture extract have shown that a significant reduction in smooth surface and fissure caries resulted from immunization with the S mutans vaccines, if antibodies reached an optimum level before caries development started. Protection was not elicited by the culture extract of S mutans or the noncariogenic Streptococcus CHT vaccines. A recently developed bacteriological sampling technique of crevicular fluid, plaque, and saliva showed that caries reduction in immunized animals was associated with a significantly decreased percentage of S mutans in crevicular fluid. Immunochemical studies showed IgG and IgM classes of antibodies in serum and secretory IgA antibodies in saliva, but it appears that reduction in caries was best associated with serum IgG antibodies to the culture extract of S mutans. The humoral and cellular mechanisms involved in the immunologic control of caries are discussed in terms of a central afferent mechanism required for antigen processing and cellular proliferation, and two peripheral effector mechanisms that function in the crevicular and salivary domains.
Excerpt
INTRODUCTION
Following infection of a sensitive bacterium with a phage, a characteristic series of intracellular events occur. In the case of the virulent phage T4, these events include both the cessation of synthesis of many macromolecular constituents characteristic of the growing bacterial cell, and the establishment of a new biosynthetic pattern directed toward the growth and reproduction of the phage. In this new pattern of events, one set of synthetic activities follows another in temporal sequence. For example, a series of enzymes concerned with the synthesis of phage-specific DNA are formed during the first ten minutes following infection while the protein components of the phage particles are synthesized later (see, for example, Kellenberger, 1961). These events are due to the introduction of the phage genome into the bacterial cell and it becomes, therefore, of basic interest to understand how the phage genome is implicated in these processes. This problem which...
Infection of E. coli with phage T4 causes a sudden inhibition of the synthesis of host DNA, host RNA, and host protein. Experiments were performed to test the hypothesis that the physical destruction of the host chromosome is a primary cause of this inhibition.
Physical destruction of host DNA after infection was studied using sucrose density gradient sedimentation and CsCl equilibrium density gradient centrifugation techniques. No destruction of host DNA was detected after 5 min. However, a definite destruction was observed after 10 min. The addition of Chloromycetin or streptomycin before infection prevented this destruction. Nevertheless, host DNA synthesis and host messenger RNA synthesis are inhibited under such conditions. Therefore, physical destruction of the host chromosome does not seem to be the primary cause of the inhibition.
Functional integrity of the host chromosome after infection was studied. During infection in the presence of streptomycin, its functional integrity was demonstrated by introducing the inhibited host chromosome into phage-resistant female cells and observing the subsequent expression of its function to synthesize β-galactosidase. Thus, the primary inhibitory process seems to be reversible in its inherent nature.
Some features of such an inhibitory mechanism are briefly discussed.
S ummary
The technique of disc electrophoresis has been presented, including a discussion of the technical variables with special reference to the separation of protein fractions of normal human serum.
During intracellular growth of T2 phage there is a production of rodshaped particles, which have the length of phage tail and which represent a structure related to the inner core of phage tail. They have little or no serum blocking power (SBP), but are to some extent adsorbable to bacteria. The intracellular appearance of rods and empty heads is studied in the presence and absence of proflavine. It is found that the production of both structures is not influenced by this substance, or by enrichment of the medium by amino acids. Proflavine depresses only the production of mature, active phage. The interpretation of these findings is discussed.
By applying analytical acrylamide electrophoresis to degraded purified capsid-related material, we found that (1) normal T4 capsids contain a major component, identified as a product of gene 23, plus at least two minor components, k and l, which we could not yet identify genetically; (2) capsids of the short-headed variant contain the same components as the normal ones; and (3) polyheads contain mainly the product of gene 23 and very little, if any, of the minor components k and l.The minor components can be extracted from capsids by treatment with 8 M urea at 45°. A residual capsid is left behind which, in the electron microscope, is not significantly different from the normal one.It is discussed why k and l are not likely to have a morphopoietic role, since they are identifiable neither with the product of genes 66 and 20, whose morphopoietic functions are known, nor with that of other genes known to be necessary for the production of stable heads.
The complex structure of bacteriophage T4 includes a variety of proteins which become assembled into mature particles during intracellular development of the virus. Some insight into the genetic control of this process has been provided by physiological studies with conditional lethal mutants, which show that over 40 phage genes are involved in T4 morphogenesis (Fig. 1). However, the mechanisms by which components are assembled have remained obscure, due in part to the lack of a suitable system for their study. In the experiments reported below, conditional
lethal mutants of strain T4D have been exploited to develop an in vitro system in which several of the steps in phage morphogenesis can be demonstrated.
Ornstein and Davis (1964) have introduced a method, called “disc electrophoresis”, which yields extremely high resolution of proteins electrophoresed in cylindrical columns of polyacrylamide gel. By means of disc electrophoresis, a large number of protein components in a complex mixture can be separated and detected in a single operation. Bands of C14-labeled proteins in disc electropherograms can be detected using techniques described by Heideman (1964); and Jovin, Chrambach, and Naughton (1964). This report presents an alternative procedure involving autoradiography of dried longitudinal gel slices. The method is relatively uncomplicated and can be used to develop the entire pattern of radioactivity in a gel without significant sacrifice of resolution.
Three acid-soluble components have been detected in E. coli infected with bacteriophage T4D. These components first appear in infected cells at 11 minutes after infection (at 37 °), and two of them are incorporated into mature phage particles from which they can be released by osmotic shock as well as by trichloroacetic acid extraction. The properties of these components indicate that they are polypeptides of several thousand molecular weight. They thus appear to correspond, at least in part, to the acid-soluble peptide fraction in T2H phage particles described earlier by Hershey (1957).Conditionally lethal (amber) mutants of T4D blocked in head formation fail to produce all three of the components in a nonpermissive host. This is true for mutants affected in any of six different genes. By contrast, mutants blocked in other kinds of assembly functions are able to produce the components. This finding suggests that the appearance of the components is associated with formation of the phage head.A delay of several minutes between the incorporation of a labeled amino acid into protein and the appearance of label in the acid-soluble components indicates that that these components arise from a precursor.These findings suggest that during phage maturation a protein is encapsulated with the phage DNA and is subsequently fragmented to yield the acid-soluble components which remain trapped inside the phage head. Some possibilities are discussed concerning the relation of this process to phage maturation.
DISK electrophoresis on small columns of polyacrylamide gels, a new method for the separation of serum proteins, has been developed by Ornstein and Davis1,2.
Disc electrophoresis basic proteins and theory
- Reisfeld
Electron microscopical studies of phage multiplication
- Kellenberger