Template properties of bacteriophage T4 vegetative DNA. I. Isolation and characterization of two template fractions from gently lysed T4-infected bacteria.

The synthesis and template properties of T4 vegetative DNA were studied. The DNA-containing material in lysates of cells taken 20 min past T4 infection sediments in sucrose gradients as two major components. Both fractions function as templates for amino acid incorporation in a DNA-dependent in vitro system (coupled transcription-translation). The slower sedimenting activity is not present in uninfected cells and appears in wild type T4-infected cells only after 12 min at 30 degrees, shortly after DNA synthesis starts. It is dependent for its activity on an added S-30 fraction from either uninfected or T4-infected cells and is completely inhibited by deoxyribonuclease or rifampin. On a weight basis the slower sedimenting template is about 30 to 70% as active as mature T4 DNA when supplemented with S-30 extracts from uninfected cells. The spectrum of proteins synthesized in response to the slower sedimenting template is different from that produced in response to mature T4 DNA. In contrast to mature DNA, this template is capable of directing the synthesis of material that precipitates with antiserum directed against whole T4 particles. Thus, it appears capable of directing the synthesis of mRNA for phage structural proteins, i.e. late proteins. The faster sedimenting component is about 8-fold less active for stimulating amino acid incorporation than mature DNA. Significant amounts of RNA polymerase are associated with this DNA in active transcription complexes, yet polyacrylamide gel electrophoresis of the proteins synthesized in response to this fraction show a pattern that resembles the early proteins made from mature T4 DNA in extracts from uninfected cells.

The synthesis and template properties of T4 vegetative DNA were studied. The DNA-containing material in lysates of cells taken 20 min past T4 infection sediments in sucrose gradients as two major components. Both fractions function as templates for amino acid incorporation in a DNA-dependent in vitro system (coupled transcription-translation). The slower sedimenting activity is not present in uninfected cells and appears in wild type T4-infected cells only after 12 min at 30", shortly after DNA synthesis starts. It is dependent for its activity on an added S-30 fraction from either uninfected or T4-infected cells and is completely inhibited by deoxyribonuclease or rifampin. On a weight basis the slower sedimenting template is about 30 to 70% as active as mature T4 DNA when supplemented with S-30 extracts from uninfected cells. The spectrum of proteins synthesized in response to the slower sedimenting template is different from that produced in response to mature T4 DNA. In contrast to mature DNA, this template is capable of directing the synthesis of material that precipitates with antiserum directed against whole T4 particles. Thus, it appears capable of directing the synthesis of mRNA for phage structural proteins, i.e. late proteins.
The faster sedimenting component is about &fold less active for stimulating amino acid incorporation than mature DNA. Significant amounts of RNA polymerase are associated with this DNA in active transcription complexes, yet polyacrylamide gel electrophoresis of the proteins synthesized in response to this fraction show a pattern that resembles the early proteins made from mature T4 DNA in extracts from uninfected cells.
The cell-free biosynthesis of several bacteriophage T4 enzymes has been accomplished using amino acid-incorporating systems programmed by DNA isolated from mature phage particles. For example, Gold and Schweiger have described the synthesis of a-and @-glucosyltransferase (1) and deoxycytidylate deaminase (2), three enzymes made early after infection. Lysozyme, an enzyme usually made at later times, has also been synthesized in oitro in a DNA-dependent system (3). The same enzymes, as well as deoxynucleotide kinase and several phage structural proteins, have been made using mRNA or polyribosome fractions isolated from T4-infected cells (4-9).
However, only a few studies have examined the template properties of vegetative T4 DNA (10-12) and all of these dealt only with the process of transcription.
It is clear that DNA isolated from the phage particle (mature T4 DNA) is capable of transcribing in uitro mRNA for early proteins (13,14). Similarily, the Escherichia coli RNA polymerase transcribes only early RNAs from purified preparations of T4 vegetative DNA *This work was supported by grants from the American Cancer Society (Iowa Division) and the National Institutes of Health, AI11301. (intracellular DNA) (11). Thus, the report by Snyder and Geiduschek (15) that late mRNA could be transcribed from a crude DNA template isolated from gently lysed T4-infected cells suggested that such extracts might be useful to further characterize this template activity in a coupled in vitro system in which translation as well as transcription could be studied. It was of particular interest to determine whether these preparations could direct the synthesis of proteins different from those produced in cell-free extracts stimulated by mature T4 DNA. T4-infected Cells-Initial attempts to incorporate amino acids with extracts prepared according to Snyder and Geiduschek (15) were unsuccessful. It was found that virtually ail of the template activity was removed by a low speed centrifugation (20,000 x g for 15 min) used to clear cellular debris from the lysate before analyzing it on a sucrose gradient (Table I, Experiment 1). However, by using a slower centrifugation (Table I, Experiment 2) it was possible to leave at least some of the template activity in solution.
The modified procedure consists of a lysozyme-freeze-thaw treatment of cells harvested 18 to 20 min after infection at 30", followed by centrifugation of the lysate at 5,000 x g for 5 min to remove cellular debris and unlysed cells, instead of the 20,000 x g centrifugation used by Snyder and Geiduschek (15). The supernatant fraction is then dialyzed as described under "Experimental Procedure" and used as template in the transcription-translation system ( Table I, Experiment 2). This 5,000 x g supernatant fraction will be designated as the S-5 fraction.
To determine if the template activity in the S-5 fraction was phage-specific or also existed in extracts from uninfected cells, similar lysates were prepared from uninfected cells and assayed for amino acid incorporation. Table II shows that the activity from T4-infected cultures is over 30-fold higher than that from uninfected cells. The lack of activity in extracts of the uninfected cells is in agreement with the observation that, unlike T4 DNA, Escherichia coli DNA does not serve as an efficient DNA template in the coupled in vitro system (26,30).
To further clarify the nature of the template component in the S-5 and to determine whether the activity was a consequence of DNA or mRNA species present in the extracts, DNase and rifampin (an inhibitor of E. coli DNA-dependent RNA polymerase initiation) were added to in vitro reaction

Polyacrylamide Gel Electrophoresis
The 'C-labeled proteins synthesized in uitro were analyzed in two different electrophoresis systems. The first system was that of Davis (25) (25), with the exception that samples were layered onto the stacking gel in 10% sucrose instead of being polymerized in a sample gel. Electrophoresis was carried out toward the anode at 3 ma/tube through separating gels of 7% acrylamide. When the tracking dye (bromphenol'blue) had moved 6.5 cm, gels were removed from the tubes, placed in 7% acetic acid, and left overnight at room temperature. Gels were frozen and sliced laterally into 1.5-mm sections with a wire jig. The sections were placed in. scintillation vials with 0.2 ml of 30% hydrogen peroxide and heated in a water bath at 50" until solubilization was complete (16 hours). Samples were counted in a scintillation mixture containing toluene and Triton X-100 (27). The second method used was the Na dodecyl-SO,-polyacrylamide gel system of Laemmli (28). In this case incubations were stopped by adding 0.100 ml of a solution containing 0.1 M Tris-Cl (pH 6.8), 8% (w/v) Na dodecyl-SO,, 20% (v/v) 2-mercaptoethanol, and 40% (v/v) glycerol to in vitro reaction mixtures that were scaled up 2-fold (0.300 ml). &tracts from infected cells were treated similarly. Samples were heated in boiling water for 90 s just before analysis.
The final concentrations in the stacking ael were:    Table III. Pancreatic deoxyribonuclease (10 rg) or rifampin (1 rg) inhibited ["Cllysine incorporation for both templates to the background level obtained with no added template.
Since S-5 preparations from uninfected cells showed no stimulatory activity in the in vitro assay (Table II), the kinetics of template formation after T4 infection was examined. Fig. 1 shows that template activity starts to appear about 12 min after infection at 30", shortly after T4 DNA replication starts. The appearance of the DNA template corresponds roughly to the increase in DNA synthesis following infection suggesting that at least part of the template activity in the S-5 extract resides in the newly replicated T4 DNA. This vegetative DNA appears about 10 min after infection and increases at a rate of about 2 %Y phage eq/cell/min at 30".Z Thus, at 20 min past infection the average cell contains about 20 phage eq of DNA not in infective particles.

Conditions for Extraction
of Vegetatiue Template-Preliminary experiments were designed to determine whether the extraction procedure used to release DNA from the infected cells influenced template activity in the coupled in vitro system. It was found that most of the conditions tested, including the addition to the lysates of 0.2% Na dodecyl-SO,, 0.2% deoxycholate, 0.5% Brij 58, 0.5% Sarkosyl, or 1% Triton X-100, released about 10 to 20% of the total cellular DNA into the 5000 x g supernatant.
The same amount is released when no additions are made subsequent to the freeze-thaw step (see "Experimental Procedure"). However, a 2-or 3-fold increase in the release of DNA could be achieved by increasing the Na dodecyl-SO, to a final concentration of 1%; by heating at 55" in the presence of 0.2% Na dodecyl-SO,; or by increasing the ionic strength (  or unsheared S-5 and compared to control mixtures which contained mature or S-5 DNA but no sonicate. There was no apparent inhibition under these conditions. However, it should also be mentioned that some of the extracts prepared with Na dodecyl-SO, were maximally active at concentrations comparable to those for mature DNA. The stimulation of amino acid incorporation in response to the pad fraction was also examined. As seen in Fig. 5, this fast sedimenting fraction is less active than mature DNA, but the response was linear with respect to the amount of DNA added, even at 16 pg of DNA/reaction mixture. This is in sharp contrast to the response produced by mature DNA and S-5 DNA where saturation is reached at lower concentrations ( Fig.  4). Incorporation is completely sensitive to DNase under the assay conditions.
The kinetics of protein synthesis in response to S-5 and mature DNA are presented in Fig. 6. As seen, reaction mixtures supplemented with either DNA incorporate lysine linearly for about 20 min. The rate of incorporation with mature DNA, however, is about four times greater than that with the S-5 extract and the extent of incorporation is about 2-fold higher. Because amino acid incorporation in this system is dependent on the synthesis of messenger RNA, the rate of RNA synthesis using the different DNA templates was compared. The data in Fig. 7 shows that the rate and extent of GTP incorporation is lower in reaction mixtures primed with DNA present in the pad fraction than in those primed with mature DNA. and core RNA polymerase in addition to DNA, prompted the experiment illustrated in Fig. 8. A lysate from infected cells was centrifuged through a sucrose gradient and fractionated as described under "Experimental Procedure." Then an aliquot of each fraction was assayed for its ability to incorporate ["CIGTP into acid-insoluble material in the absence of either added DNA or RNA polymerase. A duplicate assay was carried out in the presence of 2.5 pg of rifampin, a drug which inhibits initiation of RNA synthesis by the E. coli DNAdependent RNA polymerase but not elongation of RNA chains started before its addition (35). The results show RNA polymerase activity in the absence of the drug at both the S-5 and pad positions in the gradient (compare to Fig. 3). However, the RNA polymerase activity associated with the pad fraction is about 85% resistant to the action of rifampin whereas that associated with the top fractions is completely inhibited by the drug. This result is in agreement with data presented earlier showing that S-5 DNA is rifampin-sensitive (Table III). When the salt concentrationof the gradient was raised from 0.1 to 1.0 M KCl, the RNA polymerase activity in the pad fraction was still 50% resistant to the action of rifampin. This resistance at a high salt concentration strongly suggests that the polymerase is not only bound to the DNA in these fractions but has initiated RNA chains (36, 37). It is suspected that the polymerase lacks the E. coli sigma subunit because this factor dissociates from the core polymerase upon initiation (38) and appears to be lost as a result of T4 infection (39,40).

RNA
The kinetics of RNA synthesis by DNA-dependent RNA polymerase in the pad fraction is shown in Fig. 7. Incorporation is substantial with the pad fraction alone, but can be stimulated 3-to 4-fold by the addition of S-145 protein.
Protein Products Synthesized in Vitro in Response to Vegetatiue DNA-The nature of the polypeptide products synthesized in response to the vegetative DNA templates was of considerable interest since Brody et al. (11) have shown that ton and Pettijohn (34), which contained nascent RNA chains the E. coli DNA-dependent RNA polymerase will transcribe only early cistrons on both mature and highly purified vegetative T4 DNA. However, since the crude  extracts had been shown to be competent for late messenger RNA transcription, it seemed possible that the vegetative templates might direct the synthesis of late proteins in the coupled cell-free system.
The in vitro labeled proteins synthesized in response to mature or S-5 T4 DNA were analyzed by polyacrylamide gel electrophoresis. Fig. 9A shows that several labeled polypeptides from the two reaction mixtures migrate with the same mobilities but that at least four differences are apparent. Two polypeptides (Peaks 24 and 33) specified by the mature DNA (solid line) are missing from the profile elicited by the S-5 template. On the other hand, two proteins (Peaks 18 and 38) synthesized in response to S-5 DNA (dashed line) are absent in the reaction mixture programmed with mature T4 DNA. The polypeptides synthesized in response to mature DNA, S-5 template, and the pad fraction were also compared by electrophoresis in Na dodecyl-SO,-polyacrylamide gels. Autoradiograms of these gels are presented in Fig. 9B. The S-5 and mature DNA patterns have differences that are readily apparent, whereas proteins labeled in response to DNA contained in the pad fraction resemble the pattern obtained using mature T4 DNA.
Since many of the phage late proteins are phage structural components, antiserum against phage particles should react specifically with any late proteins synthesized in uitro. Table V shows the results of an experiment in which mature T4 DNA and S-5 template were used to stimulate [Y!]lysine incorporation by S-30 fractions from uninfected cells. After incubation, the fraction of the total radioactivity which could be precipitated with T4 antiserum was determined.
The labeled extracts from uninfected and T4-infected cells served as controls. Both the in vitro reaction primed with mature T4 DNA and the labeled uninfected extract contained some cross-reacting material, although a 2-fold increase in radioactivity in the immune precipitate was seen with the reaction primed with S-5 DNA. The radioactive proteins from the extract labeled in uiuo late after infection reacted almost completely with the T4 antiserum in this experiment.
Since it has been shown that mature T4 DNA does not direct the synthesis of late proteins in vitro (13,26), it is assumed that the cross-reacting material observed in these tubes is due to nonspecific binding. Other experiments have shown smaller percentages of antibodyprecipitable material (11 to 19%) synthesized in response to S-5 preparations, but in all cases S-5 template yielded 2-fold more radioactive protein capable of reacting with T4 antisera than proteins whose synthesis was stimulated by mature DNA (5 to 10%). In these experiments only 30 to 45% of the extract labeled in uiuo late after infection reacted with antibody to form a precipitate. DISCUSSION A large body of evidence suggests that the early events in phage infection are directed by the expression of genes of the infecting parental DNA while the late functions are the result of the expression of genes in the newly synthesized progeny DNA. In particular, the experiments of Sechaud and Streisinger (41) showed that infection with two different tail fiber T4 mutants allowed tail fiber protein to be formed as a result of recombination, which was presumed to occur entirely in a pool of replicating DNA. Bolle et al. (42) and Riva et al. (43) showed by DNA-RNA hybridization-competition experiments that late mRNA species were not synthesized in cells infected with T4 mutants unable to synthesize DNA. This requirement of DNA replication for late messenger RNA synthesis could reflect a coupling of late transcription with DNA synthesis or the necessity to Gel Slice Number (100 pCi/pmol), 6.0 mg of S-30 protein, and 40.0 pg of mature T4 DNA or 14.9 rg of S-5 DNA, all in a volume of 0.60 ml. Incubation was at 37" for 40 min after which 100 pg of chloramphenicol were added to each tube. Samples were then treated for electrophoresis (24,25). Radioactivity applied to gels was 8,860 cpm for the mature T4 (0-O) and 7,600 cpm for the S-5 (0---0) DNAs. The gels were sliced, dissolved, and counted as described Procedure." Incubation was for 20 min at 37" in a total reaction volume of 0.30 ml. Reactions were terminated and electrophoresis carried out as described by Laemmli (28). Radioactivity applied to gels: A, 135,000 cpm, B, 130,000 cpm; C, 130,006 cpm. Autoradiography was for 8 days.  Hall et al. (12) showed that the core polymerase from either uninfected or T4-infected cells was capable of transcribing late cistrons of denatured T4 DNA at higher frequencies than native DNA. The transcription of late regions of intracellular T4 DNA by the T4 polymerase was also found to be increased. Hall et al. (12) suggested that the core polymerase responded in qualitatively different ways to mature, nonglucosylated, and nicked T4 DNA. In the experiments presented here, it was found that both the S-5 (slow sedimenting) and the pad (fast sedimenting) fractions contain RNA polymerase that is active on endogenous vegetative DNA (Fig. 8). Likely the T4 polymerase evokes late mRNA synthesis on vegetative DNA in view of the results presented in Table V, showing the synthesis of phage structural proteins.
However, replicative DNA complexed with RNA polymerase is not sufficient explanation for the difference in the spectrum of proteins synthesized in response to the S-5 DNA since the pad fraction, which also has polymerase associated with it, stimulated the synthesis of a set of proteins like those programmed by mature DNA (Fig. 9B). It is possible that the RNA polymerase itself is different, perhaps because of a change in the binding properties of one or more of the T4-polymerase associated proteins (45). Although we did not specifically investigate the possibility, the presence of an endonuclease in the S-5 extracts might also account for the differences in its template properties. The fact that DNA may be removed from a membrane replication complex by endonucleolytic breakage (46) and that formation of a late transcription-competent template probably results from endonucleolytic action on newly replicated DNA (44) are in accord with this possibility. In this regard, we have observed that extracts prepared from infected cells that had been stored overnight at 0" contained higher DNA concentrations in the S-5 fraction and that this DNA saturated the RNA polymerase in S-30 extracts at lower template concentrations than S-5 fractions prepared immediately from infected cells, a result possibly reflecting nuclease activity (see below). Although a nuclease with this specificity has not been reported in cell-free preparations, endonuclease activity associated with the product of gene 49 appears to be responsible for the formation of 200 S vegetative DNA (47).
The S-5 DNA template was found to saturate the amino acid incorporation activity of the S-30 fraction at a lower concentration than mature T4 DNA (Fig. 4). Double reciprocal plots showed an apparent K, for S-5 DNA about 2-to lo-times lower than that for mature T4 DNA. Maruschige and Bonner (48) suggest that the amount of DNA template required to achieve half-maximal velocity is dependent on the concentration of RNA polymerase and that template response curves are essentially titrations of the amount of polymerase present. On the other hand, Hurwitz et al. (49) have shown that the affinity constant (K,) of E. coli RNA polymerase for DNA varies with the DNA preparation.
They found that the K, determined for single-stranded DNA was about lo-times lower than that for double-stranded DNA and that for heat-denatured DNA was about 6-times lower than that for native DNA. Thus, the polymerase binds more tightly to single-stranded and denatured DNA than to the native duplex DNA. The saturation curves observed with fixed concentrations of the RNA polymerase in the S-30 fraction are probably also influenced by single-stranded regions or nicks in the S-5 DNA. In this regard, analysis of data presented in the following paper (50) shows that the S-5 DNA from cells infected with a T4 DNA ligase mutant (gene 30, am H39X) saturates the S-30 extract at a lower DNA concentration than that from the wild type infection. Shah and Berger (51) and Shalitin and Naot (46) have proposed the existence of membrane-bound phage DNA in infected cells. It is possible that the activity of the pad fraction is a complex of phage replicating DNA and the bacterial cell membrane.
In experiments not shown here, it was found that chloramphenicol (50 &ml) added at 10 min after infection inhibited the release of DNA from this fraction and incubation of a lysate with Pronase (1 mg/ml for 20 min at 30") caused a 40% reduction in DNA sedimenting with the pad material. Others have observed that the infecting parental T4 DNA enters into a fast sedimenting complex, containing both host and phage-induced proteins, shortly after infection (52-55) Also, the experiments by Earhart et al (56,57) (Fig. 6), as are condensed structures (32, 34) since some S-5 extracts and pad prepara-28 2g' ' tions seemed less viscous than solutions of mature DNA at comparable concentrations, possibly reflecting a compactness 30. of the DNA. n.