Fast sedimenting bacteriophage T7 DNA from T7-infected Escherichia coli

VIROLOGY

69,

Fast

70438

(1974)

Sedimenting

Bacteriophage

17

Escherichia PHILIP Department

of BioEogicaE

Chemistry,

from

T7-infected

coli

SERWER’

Harvard Accepted

DNA

Medical January

School, Boston,

Massachusetts

03116

7, 1974

The sedimentation rate of bacteriophage T7 DNA from m-infected Escherichia coli has been determined after lysis with the nonionic detergent Brij 58. After 8.5 min of infection at 37’, most of newly replicated T7 DNA sedimented more rapidly than 100 S(lO0 S+ DNA). Some of the T7 100 S+ DNA sedimented in a broad peak between 100 Sand 600 S; the remainder sedimented at speeds greater than 600 S. In the electron microscope 100 S+ T7 DNA appeared to be a massive DNA complex containing up to 30 phage equivalents of DNA in a single structure. In these complexes densely packed cores of DNA were surrounded by more loosely packed DNA. Kinetic labeling experiments strongly suggest that 100 S+ DNA is a precursor for DNA in progeny phage. It was also shown that: (i) Non-DNA substances, possibly membrane fragments and/or proteins, are bound to 100 S+ T7 DNA, but are probably not the primary cause of the rapid sedimentation of this DNA. (ii) Single-chain interruptions exist in the predominantly duplex structure of T7 100 S+ DNA. (iii) When cells are lysed with the ionic detergent, Sarkosyl, the 100 S+ DNA is obtained, though in reduced yield. Phenol extraction eliminates virtually all 100 S+ DNA. (iv) Some of the 100 S+ T7 DNA is more shear-resistant than linear, duplex DNA. INTRODUCTION

Analysis of bacteriophage DNA replication, recombination, and packaging requires the isolation of intermediates in these processes.The bacteriophage T7 system offers two advantages for the study of such intermediates, particularly intermediates that are DNA-protein complexes. First, host DNA synthesis ceases and host DNA is degraded soon after infection with T7 (Kelly and Thomas, 1969; Sadowski and Kerr, 1970; Schlegel and Thomas, 1972). This simplifies the recognition of phage DNA and DNA-protein complexes in the presence of host DNA. Second, the T7 genome is smaller than the genomesof most duplex DNA phages. This simplifies the isolation of intact mature T7 DNA and its concatemer. It also facilitates a systematic 1 Present address: Division fornia Institute of Technology, fornia 91109.

of Biology, Pasadena,

CaliCali70

Copyright All rights

0 1974 by Academic Press, of reproduction in any form

Inc. reserved.

identification of T7 gene products. About 30 phage-encoded proteins have been identified (Studier and Maizel, 1969; Studier, 1972). These proteins account for almost all of the coding capacity of T7. Mutations have been obtained in 25 T7 genes (Hausman and Gomez, 1967; Studier, 1969,197Z). T7 codes for its own DNA polymerase, ligase, endonuclease and exonuclease, all of which appear to be involved in T7 DNA synthesis (reviewed in Studier, 1972). During the first few rounds, T7 DNA replication appears to occur on a linear (as opposed to circular) template (Wolfson et al., 1971; Dressler et al., 1972). A concatemeric T7 DNA intermediate has also been isolated (Kelly and Thomas, 1969; Schlegel and Thomas, 1972). It has been proposed that T7 concatemers are made by the annealing of 3’OH single-chain terminals of mature-size T7 DNA molecules produced by the linear replicative forms (Watson, 1972).

FAST

SEDIMENTING

During the eclipse period, replicating T7 DNA can be isolated in a fast-sedimenting form which is thought to be membrane bound (Center, 1972). With the exception of the latter study, all previous investigations of vegetative T7 DNA have employed an ionic detergent, and sometimes phenol, to extract the DNA. In the present experiments the sedimentation rate of DNA from T7-infected E. coli has been examined after lysis at low temperature with the nonionic detergent, Brij 58. This was done for two reasons. First, cells lyse more slowly in Brij than in the presence of ionic detergents, and the rate of lysis decreases with decreasing temperature (Godson and Sinsheimer, 1967). This suggests that damage to fragile DNA forms from shear forces occurring during lysis might be minimized by lysing with Brij at low temperature. Second, Brij is less damaging to many proteins than ionic detergents (Godson and Sinsheimer, 1967; Fuchs and Hanawalt, 1970). Thus, the use of Brij lysis should increase the probability that proteins or other non-DNA substances involved in T7 DNA replication, recombination or packaging will remain complexed to intracellular DNA after lysis. The analysis of such DNAprotein complexes might yield considerably more information than analysis of DNA or protein separately. Using a Brij lysis procedure I have isolated and characterized another fast sedimenting replicative form of T7 DNA. This form contains as much as 30 phage equivalents of DNA and seems to be similar to a DNA form found during bacteriophage T4 infections (Frankel, 1966; Huberman, 1968; Altman and Lerman, 1970; Frankel et al., 1971). Although proteins and possibly other non-DNA substances appear to be attached to this DNA the primary causes of the rapid sedimentation are probably large size and compactness, not the attachment of large non-DNA structures such as membranes. Evidence is presented suggesting that this complex contains T7 concatemers. MATERIALS

AND

METHODS

Phage and bacterial strains. Wild-type T7 phage and T7 amber mutants were obtained from Dr. F. W. Studier. E. coli BB or E. coli

T7

DNA

71

BB, thy- was the host for wild-type T7 and was the nonpermissive host for amber mutants. E. coli O-11’ was the permissive host for amber mutants Media. All phage assays were done with top and bottom agar containing T broth: 10 g Bacto tryptone, 5 g NaCl, 1 liter HzO. Phage stocks were grown in a TCG medium previously described (Thomas and Abelson, 1966). Bu$ers. Phage were stored in T7 buffer: 0.5 M NaCl, 0.01 M Tris, 0.001 M MgSO+ 0.1% gelatin, pH 7.4. DNA was stored in standard buffer: 0.15 M NaCl, 0.05 M Tris, 0.005 M EDTA, pH 7.4. Preparation of phage stocks; extraction of phage DNA. Immediately after lysis 1 g of NaCl was added per 20 ml of lysate. Following two low-speed centrifugations the phage were pelleted in the Sorvall SS-34 rotor (17 K, 100 min, 4’) and were resuspendedin T7 buffer. Unlabeled phage to be used for the T7 infections of the Results were clarified with two low speedspins and not purified further. Radioactively labeled phage and unlabeled phage to be used in preparing DNA for hybridization filters were purified by banding-in a CsCl step gradient (Thomas and Abelson, 1966). For the preparation of T7 DNA, T7 phage were twice extracted with phenol (Kelly and Thomas, 1969). The DNA was dialyzed against standard buffer. To prepare phage with 3H or 32P-labeled DNA either 10 $Zi/ml of thymidine-methyl3H or 32P-tarrier free orthophosphate was added to the medium 1.5 hour before infection. Stocks of amber mutants were grown in T broth. Reversion frequencies were always lessthan 2 X 10-5. Infection and artificial lysis of bacteria. An overnight culture of E. coli BB in TCG was diluted 1: 100 into fresh TCG and was grown to 2 X log/m1 at 37”. The cells were then infected with T7 phage at an m.o.i. of 10. Lysis with Brij 58 was by a modification of the method of Godson and Sinsheimer (1967): A sample of the phage-infected culture was mixed with an equal volume of ice cold buffer containing 0.3 M NaCI, 0.01 M EDTA, 0.008 M KCN, 50 % sucrose,pH 7.4. The chilled lysate was pelleted at low speed

72

HERWER

and then resuspendedin 100 ~1of buffer containing 0.15 &’ NaCl, 0.05 nil Tris, 0.005 M EDTA, 0.004 M KCN, 25 % sucrose, pH 7.4 at 4”. Five microliters of lysozyme (1 mg/ml) in standard buffer was added and incubated at 4’ for 20 min. This was followed by the addition of 5 ~1of 10 % Brij 58. After further incubation at 4” for 30 min, lysis was completed by incubation at room temperature (25 f 3”) for 1 hour. Less than 0.5% of the bacteria were unlysed by this time, judged by observation in a phase contrast microscope. After lysis 5 ~1 of boiled pancreatic RNase (1 mg/ml) in 0.05 Jd Tris, pH 7.4, was added and incubated an additional 15 min at room temperature. Lysate, 100 ~1, was added t)o 500 ~1 of standard buffer and was allowed to soak for 15 min before being mixed with gentle rocking. All lysates were sedimented immediately after completion of this procedure. Wide-bore, disposable pipettes were used to handle lysates. When present, marker s2P-mature T7 DNA was added before the addition of lysozyme. These lysis conditions do not affect the infectivity of T7 phage. Radioactivity assays. Method I. Acid-preeipitable radioactivity in DNA was assayed by adding 5-100 ~1 of sample to 200 ~1 of a 50 pg/ml solution of carrier DNA and then precipitating with 1 ml of 10% TCA at 4’ for 30 min. The precipitate was collected on a Millipore filter (HAWP 02500, 0.45 pm); the filter was washed three times with 0.01 M HCl and dried. Filters were covered with toluene fluor and counted in a scintillation spectrometer. Method II. Sampleswere mixed with 10 ml of toluene fluor containing Triton X-100 and then counted in a scintillation spectrometer. Zone sedimentation. Sedimentation was done in linear 5-25 % sucrose gradients at 18-20”. Neutral sucrose gradients contained standard buffer; alkaline sucrose gradients contained 0.3 M NaOH, 0.7 M NaCl. A shelf of 62% sucrose or cesium chloride, 1.75 g/ml, was used to stop DNA at the bottom of sucrosegradients. Gradient volume was 4.8 ml when the SW 50.1 rotor was used and 11.0 ml when the SW 41 rotor was used. Shelf volumes were 0.3-0.4 ml and 0.8 ml, respectively. To block sticking of DNA to centrifuge tubes, the tubes were incubated for 12-16 hr at 4” with 1 mg/ml bovine serum

albumin. The albumin solution was decanted and the tubes were dried at 65” for 30 min. With this treatment of centrifuge tubes recovery of DNA after sedimentation was generally better than 90 %. DNA/DNA membranefilter hybridization. To determine the percentage of radioactivity in T7 DNA the membrane filter hybridization technique of Denhardt (1966) with the modifications of Kelly and Thomas (1969) was used. Electron microscopy. A version of the protein monolayer technique of Kleinschmidt and Zahn (1959) was used. To 100 11of sample was added 10 ~1of 1 mg/ml cytochrome c and 10 ~1 of 6 M ammonium acetate. The entire amount of this mixture was spread on a clean surface of 0.2 M ammonium acetate. The surface was touched lightly with a 200mesh copper grid covered with a carbon film. The grid was immersed in 95 % ethanol for 30 set and then in isopentane for 10 sec. It was then air dried. Specimens were examined in a Hitachi HS-7S electron microscope. Chemicals and enzymes. Radioactively labeled compounds were purchased from New England Nuclear Corp. Lysozyme and RNase were purchased from Worthington Biochemicals. Brij 58 was purchased from Atlas Chemical Industries, Inc. and Sarkosyl NL 97 was from Geigy Industrial Chemicals. Pronase, B grade, was purchased from CalBiochem. Ne~rosporu crassa endonuclease was a gift of Dr. M. Fraser; exonuclease I was a gift of Dr. Y. Masamune. Cesium chloride, optical grade, was purchased from Harshaw Chemical Company. RESULTS

A Fast Sedimenting Form of T?’ DNA A 5 ml culture of E. coli BB was pulse labeled with 3H-thymidine between 8 and 8.5 min after infection. The entire culture was lysed with Brij 58 and a 200+1 sample of the lysate was sedimented through a neutral sucrose gradient at low speed (SW 50.1 rotor, 20 min, 28,000 rpm) and at high speed (SW 50.1 rotor, 50 min, 50,000 rpm). Roughly 5 Hg of DNA were layered on each gradient. A sample of each fraction of both sedimentations was acid-precipitated and counted by Method I. T7 infectivity assays

FAST

SEDIMENTING

were made on each of the first twenty fractions of the low speed sedimentation. T7 phage, which sediment at 453 S (Dubin el al., 1970), served as a marker for the low speed sedimentation. 32P-T7 DNA, which sediments at 32 S (reviewed in Freifelder, 1970), was the marker in the high speed sedimentation. The sedimentation profiles are shown in Fig. IA, B. Samples of fractions from both sedimentations were pooled and analyzed by the filter hybridization technique to determine the percentage of 3H in T7 DNA. The results of the filter hybridizations are shown in the legend to Fig. 1. In the low speed sedimentation 69% of the acid-precipitable 3H sedimented at speeds greater than 100 S (100 S+ DNA). Forty-one percent was in a broad peak extending between 100 and 600 S with a peak fraction close to 250 S. At the bottom of the gradient was 28 % of the 3H (600 S+ DNA). The hybridizations revealed that almost all 3H in 100 S+ DNA was in T7 experisequences. In seven independent ments 63-69 % of the acid-precipitable 3H sedimented more rapidly than 100 S. At the bottom of the high speed gradient was 67 % of the acid-precipitable BH. This DNA has an s value of 75 S or greater. Thus, the high and low speed gradients have roughly the same amount of 100 S+ DNA. Less than 6% of the 32P-marlrer DNA went to the bottom of the high speed gradient. This suggests that post-lysis DNA aggregation is not responsible for the rapid sedimentation of 100 S+ T7 DNA. Denatured mature T7 DNA is also not entrapped by the 100 S+ intracellular DNA. Selected fractions from Fig. IA were pooled, diluted in standard buffer, and then resedimented with 32P-T7 phage (Fig. 2). The ratio of the distance traveled by the peak fraction of the DNA to the distance traveled by 32P-phage is called K. About 74% of the 3H in fractions 19, 20 of Fig. 1B resedimented in a peak with a K of 0.55 (Fig. 2A). The average K of these fractions in Fig. 1B was 0.53. The corresponding K values for fractions 14, 15 are 0.80 and 0.79. DNA from fractions 8, 9, and 24 of Fig. 1B underwent a substantially greater loss in sedimentation rate when resedimented; only 14% of the 3H resedimented t’o the

T7

73

DNA

._

__

FRACTION

NUMBER

FIG. 1. Sedimentation of newly replicated n DNA in neutral sucrose gradients after Brij lysie. A 5-ml culture of Escherichia coli BB was infected with T7. At 8.0 min after infection ZOO &i of 3Hthymidine was added. At 8.5 min after infection the culture was chilled and lysed with Brij. A 0.2 ml sample of the lysate was sedimented at high speed through a neutral sucrose gradient in the SW 50.1 rotor (50 K, 50 min). Another 0.2-ml sample was sedimented at low speed through a neutral sucrose gradient (28 K, 20 min). Ten ~1 of each fraction of the low speed gradient and 50~1 of each fraction of the high speed gradient were acid-precipitated and counted by Method I. Ten ~1 samples of fractions in the low speed gradient were taken for an infectivity assay. Samples of the fractions were pooled as indicated below and the percentage of 3H in pooled fractions which was in T7 DNA was determined by the filter hybridization technique. (A) High speed sedimentation; recovery of 3H = 98%, total 3H = 97,120 cpm. (B) Low speed sedimentation; recovery of 3H = 98yc, total 3H = 21,GOO cpm -@-, y0 aH cpm; -o-, % 32P cpm; -o--, % T7 infectivity. (A)

9-20 21-34

tB)

14 g7 8, Q lo-13 14, 15 16-18 19, 20 21-24 25-27

81.2 83.3 91.3 90.0 80.3 103.2 98.0 96.2 95.6 96.4 94.8

74

SERWER

0

IO FRACTION

20

0

NUMBER

10 FRACTION

20

?A

NUMBER

FIG. 2. Resedimentation of T7 100 S+ DNA. Equal amounts of fractions 19 and 20 of Fig. 1B were pooled and diluted 1:5 in standard buffer. Fractions 14,15; 8,9, and 24 were likewise pooled and diluted. To a portion of each dilution was added 32P-T7 phage. The diluted samples were sedimented through neutral sucrose gradients in the SW 50.1 rotor (28 N, 20 min). Radioactivity was assayed by Method II. (A) Fractions 19,20; recovery of 3H = 98%, total 3H = 1,767 cpm. (B) Fractions 14,15; recovery of 3H = 97%, total 3H = 1984 cpm. (C) Fractions 8,Q; recovery of 3H = 93y0, total 3H = 494 cpm. (D) Fractions 2-4; recovery of 3H = lOO?$,, total 3H = 1918 cpm. -a--, y0 3H cpm; -0--, y0 azP cpm.

bottom of the gradient in the latter case (Fig. 2C, D). The reason for the greater instability of the DNA with a K above 0.8 is not known. Low speed sedimentation profiles similar to the one in Fig. 1B have been obtained when the amount of DNA sedimented was 5 % of the amount in Fig. 1B (see Fig. 3A). Thus, it is unlikely that DNA at the bottom of the gradient in Fig. 1B is an artifact due to the large amount of DNA sedimented. In view of the instability of 100 S+ DNA with a K greater than 0.8, it is possible that some or all of the 100-600 S DNA in Fig. 1B was broken from a more rapidly sedimenting form. Kinetic

Labeling Experiment

A T7-infected cuhure was pulse labeled with 3H-thymidine from 8.0 to 8.5 min after infection. At 8.5 min a sample of the culture was chilled and to the remainder of the culture was added a chase of cold thymidine. Three further samples were chilled at later times. All four samples were lysed with Brij 58 and sedimented at low speed (Fig.

3A-D). Under these conditions T7-infected cells lyse at 13-15 min after infection yielding 50-100 phage per bacterium. The percentage of 3H in phage, 100 S+ DNA, 600 S+ DNA and 100-600 S DNA is plotted in Fig. 4 as a function of time after termination of the pulse. The percentage of 3H in 100 S+, 600 S+, and 100-600 S DNA all decreased as a function of time while the percentage of 3H in phage increased. This suggests a precursor-product relationship for 100 S+ DNA and DNA in progeny phage. Some parental T7 DNA is encapsulated in progeny phage (Summers, 1968; Miller, 1968). Thus, parental T7 DNA should also become incorporated into 100 S+ DNA. It was indeed observed that at 8-9 min after infection up to 50% of parental T7 DNA sediments more rapidly than 100 S (Serwer, 1973). Sensitivity to Pronase Suljate (SDS)

and Sodium

Dodecyl

To test for the attachment of proteins or membrane fragments to 100 S+ T7 DNA,

FAST

FRACTION

SEDIMENTING

NUMBER

T7

0

75

DNA

10

FRACTION

20

so

0

NUMBER

FIG. 3. Kinetic labeling experiment. A lo-ml culture of Escherichia coli BB was infected with T7. At 8.0 min after infection 250 pCi of 3H-thymidine was added to the culture. At 8.5 min after infection a Z-ml sample of the culture was chilled. Simultaneously 2 ml of a solution of unlabeled thymidine (29 mg/ml) in TCG was added to the culture. Further 2-ml samples were chilled at 9.25, 10.5, and 12.25 min after infection. The four chilled samples were lysed with Brij, and 25 pl of each lysate was sedimented through a neutral sucrose gradient in the SW 50.1 rotor (28 K, 20 min). Ten microliters were removed from each fraction for an infectivity assay. The remainder of each fraction was acid-precipitated and counted by Method I. (A) 8.5 min (pulse); recovery of 3H = 90%, total 3H = 16,906 cpm. (B) 9.25 min; recovery of 3H = 9370, total 3H = 27,708 cpm. (C) 10.5 min; recovery of 3H = 93%; total 3H = 19,967 cpm. (D) 12.25 min; recovery of 3H = 94%; total 3H = 21,572 cpm. -O-, ye $H cpm; -0--, c/;; 32P cpm; --O-, ‘% T7 infectivity. Total incorporation = 0.56ye of input 3H. PFU per bacterium in (b, = 17.5.

DNA from fractions 19, 20 of the low speed sedimentation in Fig. 1B was resedimented after treatment with Pronase (Fig. 5B) and after treatment with the anionic detergent sodium dodecyl sulfate (SDS) (Fig. 5C). The sedimentation profile of an untreated control is in Fig. 5A. Treatment with Pronase (1 mg/ml, 37”, 4 hr) reduced K from 0.53 to 0.47, a one fraction change. Treatment with SDS (3.3%, 37”, 1 hr) had a virtually identical effect. Because SDS reduces the drop size of aqueous solutions, the top of the sucrose gradient in Fig. 5C is distorted. Therefore K had to be measured after determining the correct number of fractions at the top of the gradient from the position of T7 phage and the known distance of sedimentation of the phage. The small reduction in K after treatment with Pronase and SDS suggests that non-DNA substances, possibly membrane fragments or proteins, are attached to 100 Sf T7 DNA; however, it also suggests that the attachment of sub-

stances sensitive to Pronase or SDS at 37” (most proteins and membranes) is not the major reason for the rapid sedimentation of this DNA. Pronase and SDS also have very little effect on the resedimentation profile of 600 S+ T7 DNA (Serwer, 1973). Furthermore, when DNA with a K of 0.53 was heated in standard buffer to temperatures up to 90” (the melting temperature of T7 DNA in this buffer), a sedimentation profile virtually identical to the one in Fig. SB was obtained (Serwer, 1973). This further suggests that the attachment of proteins or membranes is not the major reason for the rapid sedimentation of 100 Sf T7 DNA. Buoyant Density in Cesium Chloride; Newly Replicated DNA at a Later Time To further test for the binding of nonDNA substances to 100 Sf T7 DNA, this DNA was banded in cesium chloride. In Fig. 6A is the low speed sedimentation profile of a Brij lysate obtained as in Fig. 1,

76

SERWER

I I

1

MINUTES

3

AFTER

4

PULSE

FIG. 4. Time course of 100 S+, 600 SC, 100-600 S Ti’ DNA and T7 phage. The percentage of 3H in T7 phage was determined at each time in Fig. 3 by summing the 3H in all fractions which were part of the phage peak and then subtracting a background from each fraction. The background was determined by linear extrapolation from the percentage of aH in fractions on either side of the phage peak. The fractions which were part of the phage peak were determined from the infectivity profile. The percentage of 3H in phage was subtracted from the total percentage of 3H sedimenting fast,er than 160 S to determine the percentage of 3H in 100 Sf T7 DNA. The percentage of SH in 600 Sf DNA was obtained from the amount of 3H at the bottom and was subtracted from the percentage of 3H in 100 S+ DNA to obtain the percentage of 3H in 100600 S T7 DNA. These are plotted as a function of time after termination of the pulse. -0--, phage; -C-, 600 S’ DNA; -@--, 10&600 S DNA; -w, 100 S+ DNA.

except that the culture was labeled between 10.25 and 10.75 min after infection. In this experiment 83 % of the acid-precipitable 3H sedimented more rapidly than 100 S. This is significantly more 3H in 100 S+ DNA than at 8.5 min after infection. However, this increase in the amount of pulse label in 100 S+ DNA at later times is not always reproducible. DhTA from pooled fractians 15, 16 of Fig.

FRACTION

NUMBER

FIG. 5. Pronase and sodium dodecyl sulfate treatment of T7 100 S+ DNA. To a IV9~1 sample of diluted fractions 19,20 of Fig. 1B was added 5 ~1 of 20 mgjml Pronase in standard buffer. (The Pronase was preincubated for 1.5 hours at 37”.) Digestion was continued for 4 hours at 37”. To another lOO-~1 sample was added 50 ~1 of 10% sodium dodecyl sulfate in standard buffer and incubation was at 37” for 1 hour. The above samples and an untreated sample were sedimented at low speed through neutral sucrose gradients in the SW 50.1 rotor (28 K, 20 min). Radioactivity was assayed by Method II. Marker a2P-phage was present in all samples. It was added to the control sample and the sample treated with Pronase prior to incubation. The marker was added to the SDS-treated sample just prior to sedimentation. The Pronase and SDS treatment received by the azP-phage do not affect its infectivity. (A) Control; recovery of 3H = 98%; total 3H = 911 cpm. (B) Pronase treated ;recovery of”H = 9970, total3H = 975cpm. (C) Sodium dodecyl sulfate treated; recovery of 3H = 98y0, total 3H = 908 cpm. -@-, 70 aH cpm; -0--, y. 32P cpm.

6A was resedimented (Fig. 6B). Although these fractions originally had a K of 0.72, they resedrmented with a K of 0.56. A 200-~1 sample of pooled fractions 15, 16

FAST

SEDIMENTING

T7

DNA

FRACTION FRACTION

NUMBER

FIG. 6. Sedimentation

of newly replicated T7 DNA at a later time. A 5.ml culture of Escherichia coli BB was labeled with 200 &i of 3H-thymidine from 10.25 to 10.75 min after infection. After Brij lysis a 0.2-ml sample of the lysate was sedimented through a neutral sucrose gradient in the SW 50.1 rotor (28 K, 20 min). Twenty-microliter samples from each fraction were acid-precipitated and counted by Method I. Ten microliters from each fraction were taken for an infectivity assay. Fractions 15, 16 of the above sucrose gradient were pooled, diluted in standard buffer, mixed with 3eP-T7 phage and resedimented in the SW 50.1 rotor (28 K, 20 min). Radioactivity was assayed by Method II. (A) Pulse-labeled lysate; recovery of 3H = 99%, total 3H = 64,500 cpm. (B) Resedimentation; recovery of 3H = 99%, total 3H = 2,531 cpm. -O-, ‘% 3H cpm; -0-, y0 32P cpm; -O--, y0 T7 infectivity.

of Fig. 6A was layered on a cesium chloride step gradient and spun at 40,000 rpm for 18 hr, a time sufficient to bring mature T7 DNA to its equilibrium position (Fig. 7A).2 2 In cesium chloride solutions 100 S+ T7 DNA tends to stick to centrifuge tubes. To reduce the time of centrifugation necessary to obtain a steep cesium chloride gradient the DNA w&9 sedimented through a preformed gradient for a time sufficient to bring mature T7 DNA to its equilibrium position. In sedimentations done for shorter lengths of time, the 100 S+ DNA was closer to its equilibrium position than was mature T7 DNA. Thus, the 100 S+ DNA is also at equilibrium in Fig. 7A.

NUMBER

FIG. 7. Isopycnic banding of 109 SC T7 DNA in cesium chloride. Cesium chloride was dissolved in 0.01 M Tria, 0.001 M EDTA, pH 7.4 and was used to make a step gradient containing 2.0 ml, p = 1.7 g/ml; 1.0 ml, p = 1.5 g/ml; 2.0 ml, p = 1.3 g/ml. Two hundred microliters of pooled fractions 15, 16 of Fig. 6A were layered on the step gradient which was spun at 10-12” in the SW 50.1 rotor (40 K, 18 hr). A 20-~1 sample of each fraction was counted by Method II. The buoyant density of selected fractions was determined by measuring their refractive index (Szybalski and Szybalski, 1971). Fraction 7 of the step gradient was dialyzed for 45 min into standard buffer (recovery of 3H after dialysis = 90%). A sample of dialyzed fraction 7 was then sedimented through a neutral sucrose gradient in the SW 50.1 rotor (28 K, 20 min). (A) Step gradient; recovery of 3H = 73y0, total 3H = 4854 cpm. (B) Resedimentation; recovery of 3H = 99%, total 3H = 2,531 cpm. -a---, To 3H cpm; -0--, y0 32P cpm; -O-, buoyant density.

The 3H peaked at the position of mature T7 DNA, but was skewed to the lessdense side of the marker. This indicates that non-DNA substances were in fact attached to the 100 S+ DNA. Fraction 7 (the peak fraction) was dialyzed against standard buffer, was tied with 32P-T7 phage and was then resedimented at low speed (Fig. 7B). About 39 % of the 3H resedimented as 100 Sf DNA with a peak having a K of 0.54. If one assumesa density of 1.3 for protein in CsCl the material in fraction 8 of the cesium chloride

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SERWER

FIG. 8. Continuous label of 100 S+ T7 DNA. An overnight culture of Escherichia coli BB thywas grown in TCG supplemented with 40 pg/ml of unlabeled thymidine. A 0.2-ml sample of the overnight culture was diluted into 20 ml of TCG containing 4 pg/ml of methylJ4C-thymidine (54.7 mCi/mmole), and the culture was grown to 2 X 108/ml. The bacteria were infected with T7, were chilled at 10.75 min after infection and were lysed with Brij 58. Lysate, 0.4 ml, was sedimented through a neutral sucrose gradient in the SW 41 rotor (23 K, 45 min). Ten microliters of each fraction was counted by Method I. Another 10 ~1 was taken for an infectivity assay. Samples of the fractions indicated below were pooled and the percentage of “C in T7 DNA was determined by the filter hybridization technique. Recovery of 14C = 99%; total 14C = 22,500 cpm. -@--, y0 l*C cpm; -a--, ‘% T7 infectivity. Entire

lysate l-4 5-19 20-22 23-27

45 72 75 81 70

gradient was on the average 4.5 % protein.3 In fraction 7 the percentage of protein would be less than this. Thus these data support the idea that little or no protein is required for the rapid sedimentation of 100 S+ T7 DNA. Electron Microscopy It is possible that the rapid sedimentation of 100 S+ T7 DNA is caused by a large 3 The

formula

was used to calculate Kp, the number of grams protein per gram of DNA. 0n = density of DNA 1.71; e, = 1.3 and e, = density of the complex, fraction 8 = 1.69.

of = in

amount of DNA and/or a compact DNA configuration. Both of these possible features of 100 S+ DNA can be investigated by electron microscopy. E. coli BB thy- was grown for three generations in the presence of 14C-thyymidine and was then infected with T7. At 10.75 min after infection the entire culture was chilled and lysed mth Brij 58. The low speed sedimentation profile of this lysate is in Fig. 8. The 100 S+ DNA in this sucrose gradient was 70-80% T7 DNA as determined by the filter hybridization technique (seelegend to Fig. 8). DNA in fraction 3 and fraction 21 of Fig. 8 was observed in the electron microscope. Large complexes of duplex DNA were seen in both fraction 3 and fraction 21. These DNA complexes had central cores in which DNA was densely packed surrounded by DNA which was less densely packed. An electron micrograph of a 100 S+ DNA complex from fraction 3 is in Plate 1A; an electron micrograph of a complex from fraction 21 is in Plate 1B. The number of complexes per grid square was less than one. Therefore, fortuitous overlap of DNA from different complexes is improbable. There was also some smaller linear DNA on these grids, but no attempt was made to quantitate the amount. Such large complexes are not seenwhen the 40 S region of a high speed sucrose gradient is examined by electron microscopy (Serwer, 1973). A precise estimate of the total length of the DNA in a 100 Sf T7 DNA complex was not possible because of the tangling of the DNA. However, a minimum of 30 phage equivalents of DNA has been traced for both complexes in Plate 1. Spherical and ellipsoidal “objects” appeared to be bound to the 100 S+ DNA complexes. The diameter of these “objects” ranged between 200 A and 1500 8. When viewed under these same conditions a To7 phage capsid has a diameter of 600-1000 A (Serwer, 1974). Thus, if these “objects” are made of protein they are probably complexes of large numbers of protein molecules. Some of the “objects” may be T7 capsids. A few of the ‘(objects” appear to be bound to several strands of DNA, as many as 14. Thirty-five 100 S DNA complexes were

FAST

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T7 DNA

79

screened for the unambiguous presence of DNA strands longer than mature T7 DNA. No such long strands were found. However, the tangling of DNA strands in the complexes may be obscuring the presence of DNA strands longer than mature T7 size. Long stretches of single-chain interruptions in the duplex DNA of 100 S+ complexes should appear as amorphous puddles of DNA (Huberman, 1968). No such puddles were observed in electron micrographs of 35 complexes. The presence of single-chain regions interrupting the duplex structure of the 100 S+ T7 DNA complexes was, however, shown using enzymes specific for single-chain DNA. Interruptirma in the Duplex Structure Alkaline sucrose sedimentations of DNA from pooled fractions 24 of Fig. lB, and fraction 21 of Fig. 1B are shown in Fig. 9A, B. In both casesmost of the single chains are shorter than mature T7 single chains. However, S-10% of the 3H does sediment more rapidly than mature T7 DNA. Neurospora craSsa endonuclease is an enzyme which digests denatured DNA, but does not digest duplex DNA (Rabin et al., 1968). A sample of pooled fractions 17, 18 of Fig. 1B was mixed with 32P-mature T7 DNA and was then digested with 0.0, 1.2 and 5.4 units/ml of Neurospora crassa endonuclease for 30 min at 37”. The protocol is essentially that of Schlegel and Thomas (1972). The three digestion mixtures were sedimented at high speed (Fig. lOA-C). In the control 56% of the 3H went to the bottom of the gradient (Fig. 1OA). After digestion with increasing amounts of the Neurospora enzyme, progressively less 3H sedimented to the bottom of the gradient (Fig. lOB, C). DNA that was released from the bottom sedimented in a very broad peak centered at mature T7 DNA. The sedimentation of the 32P-marker DNA was not altered by this digestion. Thus, it was concluded that single-chain regions exist within 100 S+ DNA and that some of these singlechain regions are located between segments of duplex DNA. To solubilize single-chain regions in 100 Sf T7 DNA a combination of Neurospora crassa endonuclease and exonuclease I was

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FIG. 9. Alkaline sucrose sedimentation of 109 Sf T7 DNA. A sample of pooled, diluted fractions 2-4 and a sample of pooled, .diluted fraction 21 of Fig. 1B were mixed with 3eP-mature T7 DNA and an equal volume of 1 N NaOH. Denaturation was allowed to proceed for 15 min at room temperature. Both samples were then sedimented through alkaline sucrose gradients in the SW 50.1 rotor (50 K, 90 min). Radioactivity was assayed by Method II. (A) Fraction 21; recovery of 3H = 79oj,, total 3H = 1981 cpm. (B) Fractions 2-4; recovery of 3H = 78oj,, total 3H = 1709 cpm. -a--, y0 3H cpm; -O--, y. seP cpm.

used. The protocol is essentially that of Schlegel and Thomas (1972). In Fig. 11 is shown the time course of the solubilization of 3H in DNA taken from fractions 17, 18 of Fig. 1B. The reaction appeared to level off with 4.6 % of the 3H solubilized. No solubilization (co.5 %) of admixed 32Pmature T7 DNA was detected. This is further evidence of single-chain regions in 100 S+ T7 DNA. The percentage of 3H solubilized by the above treatment is roughly twice that solubilized from T7 concatemers using the same digestion protocol (Schlegel and Thomas, 1972). The concatemers had been labeled with 3H-thymidine for 1 min. Sarkosyl Lysis, Phenol Extract&m, and Shear Sensitivity It is probable that the DNA extraction procedures used by some investigators for

PLATE 1. Electron microscopy of 100 S+ T7 DNA. A sample of fraction 3, Fig. 8 was diluted 1:3 in standard buffer; a sample of fraction 21, Fig. 8 was diluted 1:5 in standard buffer. The diluted DNA was prepared for electron microscopy using the aqueous monolayer technique. The bars represent 1 pm. Plate A shows DNA from fraction 3. Plate B shows DNA from fraction 21. 80

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FIG. 10. Effect of Neurospora crassa endonuclease on 100 S+ T7 DNA. To a buffer containing 0.1 M NaCl, 0.1 M Tris, 0.01 M MgClr, 60 pg/ml sRNA was added a l/10 volume of fractions 17, 18 of Fig. 1B and a2P-mature T7 DNA. Samples of this mixture were then incubated at 37.0’ for 30 min with 0.0 unit, 1.2 units, and 5.4 units per milliliter of Neurospora crassa endonuclease. The digestions were terminated by adding a l/5 volume of 0.1 M EDTA, pH 7.4 and chilling on ice. The incubated samples were sedimented through neutral sucrose gradients in the SW 50.1 rotor (50 K, 50 min). (A) Control, (B) 1.2 units/ml, (C) 5.4 units/ml. -a-, ‘% $H cpm; -C--, v. 32P cpm.

intracellular T7 DNA have damaged or lost the 100 S+ DNA complexes. Ionic detergent, which is often used for lysis, can be used to obtain 100 S+ T7 DNA. However, the yield of 100 S+ DNA is less than with Brij. A T7-infected culture was labeled with 3H-thymidine between 8.0 and 10.75 min after infection and intracellular DNA was sedimented after lysis with Brij 58 (Fig. 12A) and Sarkosyl NL 97 (Fig. 12B). The amount of 3H in 100 S+ DNA was 57 % and 41%,

respectively. The amount of 3H in phage was 7 % and 6 %, respectively. Virtually all 100 S+ DNA is lost during phenol extraction. In Fig. 13A, B are high speed sedimentation profiles of DNA from fractions 17, 18 of Fig. lB, before and after phenol-chloroform extraction. The recovery of 3H in the aqueous phase after extraction was only 24%. Virtually all 100 S+ DNA was eliminated by this extraction. The sedimentation profile of the DNA that re-

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FIG. 11. Solubilization of 100 S+ T7 DNA with Neurospora craasa endonuclease and exonuclease I. Pooled fractions 17, 18 of Fig. 1B were mixed with 32P-mature T7 DNA and were then diluted 1:lO into a buffer containing 0.1 M NaCl, 0.1 M Tris, 0.01 M MgC12, 0.001 M p-mercaptoethanol, 60 pg/ml sRNA, pH 8.5. To the latter mixture was added Neurospora craaaa endonuclease and exonuclease I to a final concentration of 5.0 units/ml and 1.3 units/ml, respectively, and incubation proceeded at 37”. An additional 5.0 units/ml of the Neurospora enzyme and 1.3 units/ml of Exo I were added at 90 min. At the indicated times 50-~1 samples were withdrawn and pipetted into 0.25 ml of ice cold carrier DNA (2.5 mg/ml) in 0.001 M EDTA, pH 7.4. The samples were precipitated by adding 0.25 ml of ice cold 10% TCA and were kept at 0” for 15 min. The precipitate was pelleted at 10 K for 10 min and 0.4 ml of the supernatant was counted -by Method II. The percentage of 3H and 32P which was solubilized is plotted as a function of time. -@--, y0 3H solubilized; -0--, y. 32P solubilized.

mains in the aqueous phase after extraction is similar to the profile obtained when an entire pulse labeled lysate is sedimented after extraction with ionic detergent and phenol (Kelly and Thomas, 1969; Schlegel and Thomas, 1972). Pronase treatment of 100 S+ DNA prior to phenol-chloroform extraction doesnot prevent the lossof 100 S+ DNA. The shear sensitivity of 100 Sf T7 DNA was also tested. 3H-labeled T7 100 S+ DNA, with a K of 0.60, was prepared as in Fig. 1. A sample of this DNA was sheared 25 times through a 14 gauge needle at a volume rate of at least 0.5 ml/see. Other samples were similarly sheared through 16 and 22

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FIG. 12. Comparison of Brij and Sarkosyl lysis. A lo-ml culture of Esckerichia coli BB was infected with T7. At 8.0 min after infection, 100 PCi of 3Hthymidine was added to the culture. At 10.75 min after infection the culture was chilled, spun down, and digested with lysozyme. To a 50-/J sample of the lysozyme-digested bacteria was added 2.5 ~1 of 10% Brij ; another 50-J sample was lysed with 2.5 ~1 of 10% Sarkosyl. The preparation of the lysates was continued as described in the Methods section. Samples of the two lysates were sedimented through neutral sucrose gradients in the SW 50.1 rotor (28 K, 20 min). Ten microliters of each fraction was removed for an infectivity assay and the remainder was counted by Method I. (A) Brij lysate; recovery of $H = 99%, total 3H = 379,210 cpm. (B) Sarkosyl lysate; recovery of 3H = 99%, total 3H = 449,013. -a--, y. 3H cpm; -U--, 70 T7 infectivity.

gauge needles. The minimum shear rates were 18,000, 36,000, and 290,000 se&, respectively (Davison, 1959). In Fig. 14B-D are sedimentation profiles of the sheared DNA; in Fig. 14A is an unsheared control. In the control 72% of the 3H sedimented faster than 100 S. After shearing with the 14-, 16- and 22-gauge needles, the amount of 3H sedimenting faster than 100 S was 46 %, 46 %, and 13 %, respectively. Linear, duplex DNA with an s value greater than 100 would be greater than 3.5 times as long as mature bacteriophage T4

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Thomas (1972) have s values between 32 S and 70 S. Brij lysates of wild T7-infected E. c&i contain a DNA-protein complex which sediments at 40 S, and this makes the observation of concatemers in such lysates very difficult (Serwer, 1974). It will be shown below that this 40 S complex is not produced by amber mutants in genes 14-16 grown in a nonpermissive host. Therefore, the use of these mutants facilitates the observation of concatemers in Brij lysates. The products of genes 14-16 are minor components of the completed phage (Studier, 1972). Gene 14-16 mutants synthesize DNA in nonpermissive infections (Hausman and LaRue, 1969), but DNA-containing phage heads are not found in nonpermissive lysates (Studier and Maize], 1969). It has been reported that these mutants are partially capable of producing mature T7 DNA from FRACTION NUMBER fast sedimenting intermediates (Hausman Fro. 13. Phenol extraction of 100 S+ DNA. A and LaRue, 1969). sample of fractions 17, 18 of Fig. 1B was phenol A kinetic labeling experiment was perextracted. Phenol was removed from the aqueous formed with a gene 14 amber mutant in a phase by chloroform extraction. Recovery of 3H nonpermissive host. The labeling protocol in the aqueous phase was 24y0. Both phenol-exwas similar to the one used in Fig. 3 except tracted DNA and an untreated control were sedithat only one sample was taken after unmented, along with 3*P-mature l7 DNA through neutral sucrose gradients in the SW 50.1 rotor labeled thymidine was added. High speed (50 K, 50 min). Radioactivity was assayed by sedimentation profiles from this experiment Method II. (A) Control; recovery of 3H = 83y0, are shown in Fig. 15. Sixty-two percent of t.otal 3H = 2,251 cpm. (B) Phenol extracted; rethe 3H in the pulse was in 75 S+ DNA (Fig. covery of 3H = 98%, total 3H = 970 cpm. -a-, 15A). During the chase about 20% of the To 3H cpm; -0-, y0 seP cpm. 3H was released from the bottom of the sucrose gradient to form a broad peak beDNA.4 The shear fragility of linear DNA tween 30 and 50 S (Fig. 15B). The sediis an increasing function of DNA lengih mentation rate of 3H in this peak is not and mature T4 DNA is broken by shear sensitive to phenol. Thus, the 40 S DNArates smaller than those employed in Fig. protein complex mentioned above is not l4B-C (Davison, 1959). Thus, it is likely present. Since most 30-50 S intracellular that the T7 DNA which sediments faster T7 DNA is linear at 10-12 min after inthan 100 S after shearing has a more com- fection with wild T7 phage (Kelly and pact configuration than linear random coil Thomas, 1969; Schlegel and Thomas, 1972), DNA. The shear-resistant 100 S+ T7 DNA it is likely that the 30-50 S DNA in Fig. may be the densely packed DNA cores ob- l5B is T7 concatemers. A possible explanaserved in the electron microscope. tion for the release of concatemer-sedimenting DNA during the chaseis in the results of More about Ctmcatemer8 Huberman (1968) using a bacteriophage The T7 concatemers observed by Kelly T4 DNA complex which is in many ways and Thomas (1969) and Schlegel and similar to the 100 Sf T7 DNA complexes. It was demonstrated by autoradiography 4 The equation of Burgi and Hershey (1963) that 3H-thymidine was initially incorporated relating the molecular weight of duplex DNA to primarily in the dense, central DNA cores sedimentation coefficient was used, The sedimentation coefficient of T4 DNA was assumed to be of the T4 complex and that the 3H sub62 S [reviewed in Freifelder (1970)]. sequently went to the periphery of the

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sensitivity of 100 S+ T7 DNA. 100 S+ T7 DNA, with a K of 0.60, was sheared 25 times through a 14-gauge needle, a 16-gauge needle, and a 22-gauge needle. The sheared DNAs and an unsheared control were mixed with azP-T7 phage and were sedimented through neutral sucrose gradients in the SW 50.1 rotor (31 K, 20 min). Radioactivity was assayed by Method II. (A) Control; recovery of aH = 96%, total aH = 970 cpm. (B) 14-Gauge needle; recovery of 3H = 98$&, total 3H = 1,078cpm. (C) 16.Gauge needle; recovery of 3H = 97%, total aH = 732 cpm. (D) 22-Gauge needle; recovery of aH = 99%, total aH = 931 cpm. -•--, To 3H cpm; -C--, y0 azP cpm.

complex. If after replication T7 DNA moved to the periphery of the 100 S+ complexes in a concatemeric form, the lower density of DNA at the periphery might render these concatemers susceptible to mechanical breakage from the complex. Thus, the 3&50 S peak of Fig. 15 B may consist of concatemers broken from 100 S+ T7 DNA complexes. Similar sedimentation profiles were obtained during pulse-chase experiments with gene 15 and 16 amber mutants grown in a nonpermissive host (Serwer, 1973). These results indicate that little or no DNA of mature size is produced during nonpermissive infections with gene 14, 15, and 16 mutants. This is in contradiction to the data of Hausman and LaRue (1969), who found partial DNA maturation in these mutants. However, it should be noted that (a) the mutants and hosts in the two studies were different, (b) Hausman and LaRue’s host was UV-irradiated before use. For electron microscopy a Brij lysate was prepared at 14.5 min after nonpermissive infection with a T7 gene 16 amber mutant.

DNA from the bottom of a high speed sedimentation appeared in the electron microscope to be primarily in the form of massive DNA complexes similar to those seen with wild T7 phage (Plate 1). Of 25 complexes screened, one complex was observed to have extremely long strands emanating from it. This complex was photographed. Present in the complex were four strands which could be unambiguously traced and were greater than 1.5 times the length of mature T7 DNA. The longest of these strands was 2.8 times as long as mature T7 DNA. This suggeststhe presence of concatemers within the 100 S+ T7 DNA complexes. DISCUSSION

The 100 Sf T7 DNA complexes resemble a 200 S DNA complex isolated from T4infected E. coli (Frankel, 1966; Huberman, 1968; Altman and Lerman, 1970; Frankel et al., 1971). The points of resemblance are (a) the rapid sedimentation, (b) the large amount of DNA per complex which is organized into a dense core of DNA sur-

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FIG. 15. Kinetic labeling experiment with a gene 14 amber mutant. A 12-ml culture of Escherichia coli BB was infected with T7 140, an amber mutant in gene 14 (Studier, 1969). At 8.0 min after infection 120 pCi of 3H-thymidine were added to the culture. Thirty seconds later a 5-ml sample of the culture wsz~ chilled, and simultaneously 1.0 ml of a 10 mg/ml solution of unlabeled thymidine in TCG was added to the culture. A second 5-ml sample was taken at 12.5 min after infection. The samples were lysed with Brij and portions of both lysates were sedimented through neutral sucrose gradients in the SW 50.1 rotor (50 K, 50 min). Radioactivity in the entire amount of each fraction was assayed by Method I. (A) Pulse; recovery of 3H = 95%; total 8H = 71,273 cpm. (B) Chase; recovery of 3H = 970/,, total 3H = 91,645 cpm. -a--, y. 3H cpm; -C-, y0 QP cpm.

rounded by more loosely packed DNA, (c) the insensitivity to Pronase and ionic detergent, (d) the presence of single-chain interruptions of the duplex structure. The topological arrangement of DNA in these T4 and T7 DNA complexes is not understood. The fact that a large percentage of a pulse of 3H-thymidine is incorporated into 100 Sf T7 DNA complexes suggests that DNA replication occurs in these complexes in viva. Therefore, it is likely that branch points exist within the 100 S+ T7 DNA complexes. The linear T7 DNA replicative structures (Wolfson et al., 1971; Dressler et al., 1972)

are different from the T7 100 S+ DNA complexes in that they contain less DNA and do not have dense cores of compact DNA. It is possible that the linear structures are converted into 100 Sf DNA complexes in the latter stages of infection. The experiments in which the 100 S+ DNA complexes were discovered were all done late in or after the eclipse period. The linear T7 replicative structures never have DNA strands longer than mature T7 DNA. It seemsguite possible that concatemerization of T7 DNA and the production of 100 S+ DNA complexes are related events and that T7 concatemers are often part of a larger 100 S+ DNA complex. The T7 concatemers which have previously been observed (Kelly and Thomas, 1969; Schlegel and Thomas, 1972) may have been broken from the 100 S+ DNA complexes. A fast sedimenting ‘Lmembrane” bound form of T7 DNA has been isolated from T7-infected E. coli in the eclipse period (Center, 1972). DNA in this structure subsequently went into a slower sedimenting form and then back into a fast sedimenting form. The return to fast sedimentation was interpreted to signify reattachment to the cell membrane. However, it seems likely that the return to fast sedimentation was the result of the formation of 100 S+ T7 DNA complexes which owe their fast sedimentation more to their large amount of DNA and compactness than to attachment of a massive non-DNA structure like a cell membrane. Fast sedimenting T7 DNA (70 S+) has also been observed by Stratling et al. (1973). It is likely that this DNA is also the same as the 100 S+ T7 DNA complexes characterized in the present communication. The characterization of the 100 Sf T7 DNA complexes is not yet complete enough to draw conclusions about the mechanisms of T7 DNA replication, recombination and packaging in which these complexes may be involved. Further characterization of this DNA will involve analysis of the proteins bound to it. The buoyant density profile of 100 S+ T7 DNA in cesium chloride suggests that some proteins remain bound to 100 S+ DNA during banding in cesium chloride. Cesium chloride banding will greatly aid in

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discriminating these proteins from non-DNA bound proteins which cosediment with 100 S+ T7 DNA complexes. Complexes between T7 capsids and T7 DNA, in which the DNA is outside of the capsid, have been isolated (Serwer, 1974), and these complexes can be banded in cesium chloride. This, coupled with the observation in the electron microscope of capsidlike objects on T7 100 S+ DNA, suggests that T7 capsids are bound to 100 S+ T7 DNA. Direct analysis of proteins bound to 100 S+ T7 DNA is in progress. ACKNOWLEDGMENTS I thank my advisor, Dr. Charles A. Thomas, Jr., and Dr. Lawrence Okun for discussions and criticism during the course of this work. I also thank Dr. C. S. Lee for critical reading of this manuscript. This work is included in a thesis submitted in partial fulfillment of the requirements for the degree of Ph.D. in Biophysics at Harvard University. Support was received from an NIH predoctoral fellowship and NSF grant GB 3118-X1. REFERENCES

ALTMAN, S., and LERMAN, L. S. (1970).

Kinetics and intermediates in the intracellular synthesis of bacteriophage T4 deoxyribonucleic acid. J. Mol. Biol. 50, 235-261. BURGI, E., and HERSHEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309321. CENTER, M. S. (1972). Replicative intermediates of bacteriophage ‘I7 deoxyribonucleic acid. J. Virol. 10, 115-123. DAVISON, P. F. (1959). The effect of hydrodynamic shear on the deoxyribonucleic acid from T2 and T4 bacteriophages. Proc. Nat. Acad. Sci. U.S. 45, 1560-1568. DENHARDT, D, T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-648. DRESSLER, D., WOLFSON, J., and MAGAZIN, M. (1972). Initiation and reinitiation of DNA synthesis during replication of bacteriophage T7. Proc. Nat. Acad. Sci. U.S. 69, 99&1002. DUBIN,~. B.,BENEDEK, G.B., BANCROFT, F. C., and FREIFELDER, D. (1970). Molecular weights of coliphages and coliphage DNA. II. Meazurement of diffusion coefficients using optical mixing spectroscopy, and measurement of sedimentation coefficints. J. Mol. Biol. 54,547-5.X?. FRANKEL, F. R. (1966). Studies on the nature of replicating DNA in T4-infected Eschetichia co&. J. Mol. Biol. 18, 127-143.

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FRANKEL, F. R., BATCHELER, M. L., and CLARK, C. K. (1971). The role of gene 49 in DNA replication and head morphogenesis in bacteriophage T4. J. Mol. Biol. 62, 439463. FREIFELDER, D. (1970). Molecular weights of coliphages and coliphage DNA. IV. Molecular weights of DNA from bacteriophages T4, T5, and T7 and the general problem of determination of M. J. Mol. Biol. 54, 567-577. Fuchs, E., and HANAWALT, P. (1970). Isolation and characterization of the DNA replication complex from Escherichia coli. J. Mol. Biol. 52, 301322. GODSON, G. N., and SINSHEIMER, R. L. (1967). Lysis of Escherichia coli with a neutral detergent. Biochim. Biophys. Acta 149,476488. HAUSMAN, R., and GOMEZ, B. (1967). Amber mutants of bacteriophages T3 and T7 defective in phage-directed deoxyribonucleic acid synthesis. J. Vi’irol. 1, 779-792. HAUSMAN, R., and LARUE, K. (1969). Variations in sedimentation patterns among deoxyribonucleic acids synthesized after infection of EscheTichia coli by different amber mutants of bacteriophage T7. J. Viral. 3, 278-281. HUBERMAN, J. A. (1968). Visualization of replicating mammalian and T4 bacteriophage DNA. Cold Spring Harbor Symp. Quant. Biol. 33, 509524. KELLY, T. J., and THOMAS, C. A. (1969). An intermediate in the replication of bacteriophage T7 DNA molecules. J. Mol. Biol. 44,459475.

KLEINSCHMIDT,

A. K., and ZAHN, R. K. (1959). Uber Desoxyribonucleinsiiure-Molekeln in Protein-Mischfilmen. 2. Naturforsch. B 14,770-779. MILLER, R. C., JR. (1968). Parental to progeny molecular recombination with bacteriophage T7. J. Vi’irol. 2, 157-159. RABIN, E. Z., MUSTARD, M., and FRASER, M. J. (1963). Specific inhibition by ATP and other properties of an endonuclease of Neurospora crassa. Can. J. Biochem. 46, 12351291. SADOWSKI, P. E., and KERR, C. (1970). Degradation of Escherichia coli B deoxyribonucleic acid after infection with deoxyribonucleic aciddefective amber mutants of bacteriophage T7. J. Viral. 6, 149-155. SCHLEGEL, R. A., and THOMAS, C. A. (1972). Some special features of intracellular bacteriophage T7 concatemers. J. Mol. Biol. 68,319-345.

SERWER, P. (1973).

Ph.D. Thesis, Harvard UniCambridge, Massachusetts. SERWER, P. (1974). Complexes between bacteriophage T7 capsids and T7 DNA. Virology 59, 89107. STRXTLING, W., KRAUSE, E., and KNIPPERS, R. (1973). Fast sedimenting deoxyribonucleic acid versity,

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in bacteriophage T7-infected cells. Virology 51, 109-119. STUDIER, F. W. (1969). The genetics and physiology of bacteriophage T7. virology 39,562-574. STUDIER, F. W. (1972). Bacteriophage T7. Science 176, 367-376. STUDIER, F. W., and MAIZEL, J. V. (1969). T7directed protein synthesis. Virology 39, 575-586. STUDIER, F. W., and HAUSMAN, R. (1969). Integration of two sets of T7 mutants. virology 39,587. SUMMERS, W. C. (1968). Equal transfer of both parental T7 DNA strands to progeny bacteriophage. Nature (London) 219, 159-160. SZYBALSKI, W., and SZYBALSKI, E. H. (1971). Equilibrium density gradient centrifugation.

In “Procedures in Nucleic Acid Research” (G. L. Cantoni and D. 1~. Davies, eds.), Vol. II, pp. 311-354. Harper & Row, New York. THOMAS, C. A., and ABELSON, J. (1966). The isolation and characterization of DNA from bacteriophage. In ‘ ‘Procedures in Nucleic Acid Research” (G. L. Cantoni and D. R. Davies, eds.), Vol. I, pp. 553-559. Harper & Row, New York. WATSON, J. D. (1972). Origin of concatemeric T7 DNA. Nature (London) New Biol. 239, 197-201. WOLFSON, J. D., DRESSLER, D., and MAGAZIN, M. (1971). Bacteriophage T7 DNA replication: A linear replicating intermediate. Proc. Nat. Acad. Sci. U.S. 69, 499-504.