Über infektiöse substrukturen aus Escherichia coli bakteriophagen

J. Mol. Biol. (1966) 21, 501-516

iiber infektib’se Substrukturen aus Escherichia cob Bakteriophagent

VIII. On the Tertiary Structure and Biological Properties of $X174 Replicative Form?

R. JAENISCH, P. H. HOESCHNEIDER AND ANNELIESE F’REUS~

Max-Plan&-Institut fiir Biochemie, Goethe&awe 31, Munich, Germany

(Received 12 May 1966, and in revised form 25 July 1966)

As already known, +X174 replicative-form DNA is separated into two components when sedimented in a sucrose gradient. The faster component, I, is not denatured by heat treatment, whereas the slower component, II, is converted t,o single strands. Only the infectivity of component II increases after heat denaturation.

A quantitative comparison of data obtained by band centrifugation and by electron microscopy shows that the tertiary structure of component I is a highly twisted circle and that of component II an extended circle. By treatment with DNase, component I (as observed in band centrifugation) and twisted rings (as observed by electron microscopy) are converted at the same rate to component II and to extended circles and linear molecules.

The plating efficiency of replicative-form DNA, in contrast to denatured component II replicative form and to +X174 single-stranded DNA, is diminished by the addition of Eecherichia coli DNA to the adsorption medium. The inhibition by E. coli DNA at concentrations above 0.5 pglml. is of tist order. The lower the relative plating efficiency of native replicative-form DNA, the greater is the increase of infectivity for the denatured component II. The importance of these findings with respect to normal test conditions is discussed. Some observations suggest that under ideal test conditions the relative plating efficiency of the replicative-form DNA may rise to that of $X174 single-stranded DNA.

1. Introduction When cells of Escherichia c&i C are infected with +X174 in the presence of chlor- amphenicol, the single-stranded DNA of this phage is converted to a double-stranded “replicative form” (Sinsheimer, 1961; Hofschneider, 1961; Sinsheimer, Starman, Nagler & Guthrie, 1962; Hayashi, Hayashi & Spiegelman, 1963; Matsubara, Taketo & Takagi, 1963). The infectious #X174 replicative-form DNA is a ring-shaped molecule, as can be shown by its resistance to exonuclease action (Burton & Sins- heimer, 1963) and by electron microscopy (Kleinschmidt, Burton $ Sinsheimer, 1963; Chandler, Hayashi, Hayashi & Spiegelman, 1964).

Ultracentrifugal analysis shows that preparations of +X174 RFf contain a mixture of at least two components in variable amounts (Burton t Sinsheimer, 1965; Sins-

t Paper VII of this series appeared in J. Mol. Bid. (1966) 16, 544. f Abbreviations used: RF, replicative-form DNA; MAK, methylated albumin-kieselguhr;

m.o.i., multiplicity of infection; p.f.u., plaque-forming units; DIP-trypsin, diisopropyl-phosphoryl trypsin.

33 501

502 R. JAENISCH, P. H. HOFSCHNEIDER AND A. PREUSS

heimer, Lawrence & Nagler, 1965; Jansz & Pouwels, 1965). These components can be distinguished by their different sedimentation coefficients and by their behaviour after denaturation.

Component I (21 s) represents the intact, ring-shaped double-stranded DNA, whereas component II (16 S) is a double strand with at least one single-strand break. After denaturation by heat (Jansz & Pouwels, 1965) or by alkali (Burton & Sinsheimer, 1965), component II segregates into the two single-stranded DNA molecules, whereas the two strands in component I do not come apart. By DNase treatment, component I is converted to component II (Jansz & Pouwels, 1965). Similar results were obtained with Ml3 phage (Ray, Preuss & Hofschneider, 1966).

These results correspond to the earlier findings on polyoma DNA (Vinograd, Lebowitz, Radloff, Watson & Laipis, 1965), where two circular forms were found. Based mainly on sedimentation data, it was concluded that the faster sedimenting component I (20 S) has the tertiary structure of a twisted ring, whereas the slower sedimenting component II (16 s) is an extended circular molecule. This conculsion was supported qualitatively by electron microscopic observations. Under conditions which convert component I into II, the slight twist of the electron-microscopically visible molecules disappears. However, no quantitative electron microscopic studies have been performed with polyoma DNA. Furthermore, it has not yet been shown whether the electron microscopical observations on polyoma DNA hold for +X RF. In this paper it will be demonstrated that #X RF can exist in a highly twisted form. Morphological appearance and physical and biological properties of the two RF components are compared quantitatively, showing that component I corresponds to twisted circles and component II to untwisted circles.

The relative biological plating eiliciency of $X RF is about 5% of that of $X single strand as described by Denhardt & Sinsheimer (1965) and Sinsheimer et al. (1965). RF shows an increase in infectivity after denaturation either by heat (Hofschneider, 1961; Benzinger & Hofschneider, 1963; Pouwels & Jansz, 1964) or by alkali (Burton & Sinsheimer, 1965). The differences in the relative plating efficiencies observed for single-stranded and double-stranded $X DNA could be explained exclusively by their different secondary structures. Biological assays, however, as presented in this paper show that the degree to which their infectivity differs depends also on the test conditions.

2. Materials and Methods (a) Media

Standard dilution buffer: 0.005 M-phosphate buffer (pH 7.2), 2 x 10m4 M-EDTA. Sus- pension buffer: 0.1 M-NaCl, 0.01 M-Tris (pH 8.5). Minimal medium for preparation of 3H-labelled 4X RF: synthetic medium of Maalee 8: Hanawalt (1961) supplemented with 2 pg/ml. thymidine, 1 mg/ml. vitamin-free Casamino acids, 1% glucose.

(b) Bacteria and bacteriophage

The bacterial strains used in the bioassay are described by Benzinger, Delius, Jaenisch & Hofschneider (1966, manuscript in preparation).

For preparation of 3H-labelled #X174 RF, an E. coli C Thy- mutant was used, which was kindly supplied by Dr Okada.

4X174 phage was prepared according to Rueckert, Zillig & Huber (1962).

(c) Preparation of rvucleic acids

E. coli C DNA was prepared according to Marmur (1961). +X174 phage DNA was prepared according to Benzinger et aZ. (1966, manuscript in

preparation), as described here: purified +X174 (biological plaque t,itre loll/ml.) was

TERTIARY STRUCTURE OF +X174 RF 503

suspended in borat+EDTA buffer (O-03 M-sodium tetraborate, pH 9, 0.001 M-EDTA). Sodium dodecyl sulphate (20%) was added to a final concentration of 1 o/o and the suspension was left at room temperature for 10 min. Then an equal volume of water-saturated phenol was added and the suspension shaken by hand for 10 min. The two layers were separated by low-speed centrifugation. The aqueous phase was removed, fresh phenol added and t.he procedure was repeated twice. Finally, the aqueous phase cont.aining the nucleic acids was dialysed against dilution buffer.

Ml3 DNA was a gift from H. Bscheider. Tritium-lebelled RF was prepared in the following manner: 500 ml. of minimal medium were inoculated with E. coli C Thy- mutant with aeration

at 37°C to 2 to 3 x 10s cells/ml. “d”

oramphenicol was added to a concentration of 30 pg/ml. After 10 min, 4X174 was a ded to a m.o.i. of 4, followed by addition of 2 PC/ml. 3H-labelled thymidine (specil% activity: 3.0 c/m-mole; from The Radiochemical Centre, Amersham, England). After 50 min the cells were harvested by collecting on a membrane filter MF 50 (Membrarullter-Gesellschaft, Gottingen). The filter was washed 3 times with 50 ml. of suspension buffer and then the bacteria were resuspended in 100 ml. of suspension buffer. The solution was centrifuged for 10 min at 5000 rev./min and the pellet resuspended in 45 ml. of suspension buffer. EDTA was added to a final concentration of 10e2 M, lysozyme was added at 100 pg/ml. and the solution was allowed to stand at 37°C for 10 min. After addition of 1% of SDS, lysis occurred immediately. After another 30 min at 37”C, an equal volume of water-saturated phenol was added to the solution. To reduce shearing of E. coli DNA to a minimum, the mixture was very gently shaken by hand. The phenol and water phases were separated by centrifugation and the aqueous phase was removed by pipetting gently through a large-bore pipette.

The phenol extraction was repeated 4 times, after which the still very viscous solution was dialysed overnight against suspension buffer plus 0.01 M-EDTA. Then 20 pg/ml. of DNase- free RNase, prepared by the method of Marmur (1961), was added and the solution allowed to stand at 37°C for 30 min. The enzyme was removed by another phenol extraction.

After dialysis against column buffer, the nucleic acids were chromatographed on a MAK column of standard size (Mandell & Hershey, 1960). The RF was eluted before the bulk of E. coli DNA. After collection of 100 fractions of about 6 ml., four infectious fractions were pooled and rechromatographed on an MAK column of half the standard size.

From the first cohunn, about 50% of the trichloroacetic acid-precipitable input radioactive material was recovered. The recovery of biological activity was not estimated, since RF infectivity is inhibited by high E. coli DNA concentrations present in the crude lysate (see section 3(d)).

Hayashi et al. (1963) reported that after one rechromatography, RF is free of contaminating E. coli DNA. This was tested by electron microscopy of the infectious RF fractions. Of 1000 RF molecules counted, not more than 12 molecules were found with a length of 1.5 to S-fold that of ring-shaped RF. Thus, on a weight basis, contamination by E. coli DNA was less than 3%.

The cohunn fractions were dialyzed against dilution buffer prior to further experiments.

(d) DNme treatment of replicutive-form DNA For each enzyme digestion, pancreatic DNase (twice recrystallized, 90,000 dornase

units from Nutritional Bioohemicals Corp.) was freshly dissolved in 0.05 m-MgCl,. The enzyme was diluted in 0.05 M-Mgcl, to a concentration of 0.02 pg/ml. At zero time, l/20 of the volume of the RF fraction was added, so that the final DNase concentration was 0.001 pg/ml. and the final MgCI, concentration 10e3 M. Digestion was performed at 37’C. To stop the enzyme action, 0.01 M-sodium citrate was added and the samples placed in an ice-water bath.

(e) Heat-denaturation of replicative-form DNA For heat-denaturation of RF, the method of Benzinger & Hofschneider (1963) was

slightly modified. The salt concentration of the RF fraction was always that of dilution buffer (0.005 aa-phosphate buffer, pH = 7.2, 2 x lo-* M-EDTA). The samples were heated to 92°C for 2 min and then quickly cooled in an ice-water bath.


(f) Preparative zone stxbime~ion in sucrose gradients

The sucrose was autoclaved in dilution buffer (0.005 M-phosphate buffer, 2 x 10m4 M-

EDTA) to destroy any contaminating nucleases. Gradients from 20% to 4% (w/v) sucrose were prepared for the SW25/2 rotor of the Spinco model L2 centrifuge (50 ml. total volume). A l-ml. sample of the RF to be sedimented was layered on the sucrose gradient. The samples were then centrifuged in a Spinco model L2 at 4°C at 25,000 rev./mm for 18 or 22 hr before drop collection.

(g) Assay of nucleic acids for infectivity

The biological tests for infectious $X single-stranded DNA and RF were performed according to Benzinger et al. (1966, manuscript in preparation) as described here.

(i) Materials for infectious DNA assays

Growth medium: 1% liver digest (Oxoid), 0.08 M-NaCl. Resuspending medium: 5 vol. of 0.1 M-Tris (pH 8*5), 3 vol. of 1 M-sucrose. Bovine serum albumin: 30% solution of “Povite” bovine serum albumin from “Biotest” Serum Institute, Frankfurt, Germany. Adsorption medium: 0.1 M-NaCl, 0.1 M-sucrose, 0.005 m-MgSO,, 0.5 M-Povite albumin. Top-layer agar: 0.7% Difco Bacto agar, 1% Difco nutrient broth; 0.38 M-sucrose, 0.07 ivr-NaCl; 0.001 M-CaCI,; 0.005 M-MgSC4. Bottom-layer agar: 1000 ml. water, 15 g agar (Merck), 10 g peptone (Merck), 5 g of NaCl, neutralized with NaOH after autoclaving.

(ii) Biological assays for infectious $X single-stranded DNA and replicative form DNA

This method represents a modification of the procedure of Guthrie & Sinsheimer (1963). 400 ml. of growth medium in a flat-bottomed shake-flask were inoculated with E. coli K12 W3350 from an overnight slant. The flask was shaken at 60 strokes/min at 35 to 37%. When the cells had reached a titre of 5 x 108/ml., they were harvested by low-speed sedimentation at room temperature. The pellet was taken up in 30 ml. of resuspending medium. Then 0.8 ml. of 0.11 M-EDTA, 0.5 ml. of 30% Povite albumin and 1 ml. of lysozyme (2mg/ml.) were added. After 4 to 5 mm, 0.4 ml. of 0.5 M-MgSC, was added to stop lysozyme action. The spheroplasts were stored at 4°C for at least 24 hr and then tested in the following manner: 0.05 ml. of a suitable dilution of +X DNA in dilution buffer was added to 0.45 ml. of adsorption medium in a test tube at 37°C. Two drops of spheroplasts, which were always kept on ice, were added to each tube. A suitable volume of melted top agar was added to a cylinder in a 42°C bath. Ten mm later, 1 ml. of E. coli C indicator (grown in the medium of Fraser & Jerrel (1953) to saturation) was added for every 30 ml. of top agar. Twelve mm after the addition of spheroplasts, 3 ml. of the top-agar indicator mixture was added to each test tube. The tubes were immediately poured on to bottom-layer agar plates. No plates were poured later than 15 mm after addition of spheroplasts. The plates were incubated at 37°C for 4 to 5 hr and then scored for plaques.

Ml3 DNA was tested according to Ray, Bscheider & Hofschneider (1966). Tests, with inhibition by E. coli DNA, were carried out as normal, with the difference

that purified E. coli DNA was added to the adsorption medium in different amounts.

(h) Electron microscopy

DNA samples were prepared for electron microscopy by the protein monolayer tech- nique of Kleinschmidt & Zahn (1959). Slight modifications were introduced. Instead of cytochrome c, “DIP-treated trypsin” (Worthington Biochemical Corp.) was used as described by Stoeckenius (1963). Up to 3 pg DNA/ m . in 0.005 ;M-phosphate buffer, 0.0002 1 M-EDTA and 0.05 M-ammonium acetate were spread on distilled wat’er containing O.OO5o/0 ammonium hydroxide. The shadowed specimens were examined in a Siemens Elmiskop I at an original magnification of SOOO-fold.

(i) Radioactive assay The incorporated tritium was assayed by adding a small sample to 5 ml. ice-cold 5%

trichloroacetic acid, collecting by suction on a MF50 membrane filter (Membranfllter- Gesellschaft, Gottingen), and rinsing with two 5-ml. portions of distilled water. After

TERTIARY STRUCTURE OF $X174 RF 505

drying at 50°C, the filters were added to counting vials containing 5 ml. toluene, 18 mg 2,5-diphenyloxazole (PPO) and 0.45 mg 1,4-bis-2-(4-methyl-&phenyloxazolyl-benzene (POPOP) and assayed in a Packard TriCarb scintillation spectrometer.

3. Results With the intention of correlating RF components I and II with the tertiary struc-

ture of the molecules, band sedimentation and electron microscopy were performed with the same RF fractions. Furthermore, the kinetics of the conversion of component I into component II were compared with the kinetics of change in tertiary structure (conversion of twisted into untwisted circles). Since it has been already shown that DNase treatment converts component I into component II, the DNase digestion of RF was used to study the kinetics of the conversion.

RF was treated for different lengths of time with DNase, and portions of the same samples were prepared in parallel for sucrose gradient sedimentation and electron microscopy. In sections (a) and (b), the sedimentation and electron microscopical experiments are described and in section (c) a quantitative comparison of the results obtained by the two independent methods is made.

Section (d) describes the influence of E. coli DNA on the infectivity of native and denatured RF.

Fraction no.

FIG. 1. Sucrose gradient sedimentation of 4X and Ml3 phage DNA’s

A l-ml. sample containing purified 4X174 (1 x 10” phage equiv.) and Ml3 DNA (3 x 1012 phage equiv.) was layered on a linear sucrose gradient (50 ml., 4 to 20% 0.005 aa-phosphate buffer, 0.002 M-EDTA) and centrifuged for 22 hr at 25,000 rev./min in a Spinco model L2, SW 25/Z rotor at 4°C. 70 fractions were collected and tested for infectivity.

If not otherwise stated, all sucrose-gradient sedimentations presented in this paper were centrifuged under the same conditions.

(-X-X -), 4X DNA plaque-forming units/ml.; (.-A-.-A-.), Ml3 DNA plaque-forming units/ml.


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FIG. 2. Separation of RF components by sucrose gradient sedimentation.

Tritium-labelled RF was purified by MAK column chromatography as described under Materials and Methods. A l-ml. sample of RF and of 0.1 ml. of Ml3 DNA (3 x 10la phage equiv.) were mixed, layered on a linear sucrose gradient and centrifuged for 18 hr. The 35 fractions collected were tested for radioactivity and infectivity of +X and Ml3 DNA’& 4X DNA was tested before and after heat denaturation. The eamplas to be heated were diluted tenfold in dilution buffer (0.005 M-phosphate buffer, 2 X 10eh M-EDTA) and heated for 2 min at 92% and then quickly cooled in an ice bath.

The upper part of the Figure shows the infectivity of RF before and after heating as tested by the addition of 7 pg/ml. of E. c&i DNA to the adsorption medium (see Results, section (d)).

(-A-A-), Radioactivity, cts/min/ml.; (--@--a--), 4X single-strand equiv./ml. after heat treatment; (a. X . . X . n), 4X single-strand equiv./ml. before heat treatment; (.-A-*-A-.), Ml3 DNA plaque-forming units/ml.

TERTIARY STRUCTURE OF 4x174 RF 507

(a) Separatkn of the two RF wmponetis by sucrose gradient centrifugation

Tritium-labelled RF and infectious Ml3 DNA (as a sedimentation marker) were sedimented together in a sucrose gradient. Under the conditions described, +X single- stranded DNA sediments slightly faster than Ml3 DNA (Fig. 1). Sedimentation of purified RF shows two peaks of radioactivity (Fig. 2), in good agreement with data obtained by Jansz & Pouwels (1965). The fast sedimenting peak (I) contains approximately 80% of the radioactive material. The slower sedimenting peak (II) contains approximately 20% of the radioactive material and is found closer to the posit,ion of the marker DNA.

RF from the same preparation was subjected to DNase treatment for different lengths of time (0.001 pgg/ml. DNase 37°C 4 and 15 minutes). The material wus centrifuged before (Figs 3(a) and 4(a)) and after heat denaturation (Figs 3(b) and 4(b)). A comparison of the distribution of radioactivity in the unheated samples (Figs 2, 3(a) and 4(a)) shows that, with increasing time of DNase treatment, the amount of radioactivity of component II increases at the same degree as component I decreases. Table 1 gives a quantitative comparison of the total radioactivity of component I and II after different times of DNase treatment. The ratio of II t’o I increases with increasing time of DNase treatment.

TABLE 1

Conversion of component I into component II by DNase treatment

Time of DNase treatment

(mb)

o/0 of radioactivity O/, of radioactivity due to component I due to component II

0 83 17 4 62 38

10 32 68 15 21 79

RF was treated for different lengths of time with DNase as described under Materials and Methods and subsequently sedimented in a sucrose gradient. The Table shows the percentage of radioactivity found in component I and component II peaks (radioactivity of both peaks = 100%).

After heat denaturation (two minutes at 92°C then quickly cooled), neither the amount nor the position of component I was affected, whereas component II shifted to the position of the marker Ml3 DNA. These results suggest that RF of form II denatures to single strands under the conditions described, whereas the sedimentation properties of RF of component I are not affected by the denaturation procedure.

The assumption that RF in component II forms single strands after heat denaturation is supported by the biological properties of that component, as measured before and after heat treatment. Each fraction, collected from the gradients recorded in Figs 2 to 4, was tested biologically before and after heat denaturation. Tests with E. wli DNA inhibition (upper part) are discussed later (section (d)). Whereas the infectivity of component I was not affected by heat treatment, component II, as obtained by centrifugation of unheated RF (Figs 2, 3(a) and 4(a)), showed an increase of infectivity after heat treatment by a factor of 2 to 3. The infectivity of

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denatured component II (as obtained by centrifugation of heat-denatured RF (Figs 3(b) and 4(b)) at the position of single-stranded Ml3 DNA) was not affected by a second heat treatment. These biological observations agree with the assumption that heat denaturation converts component II, but not component I, into single strands; i.e. component II in Figs 3(b) and 4(b) has the same sedimentation properties as Ml3 single strands and the same biological properties as $X single strands (no increase of infectivity after heat denaturation, no inhibition of infectivity by E. coli DNA see section (d)).

In Figs 3(b) and 4(b), the material of the maximum of the infectivity curve of component II RF, denatured before band sedimentation, is found to sediment slight’ly in front of its radioactivity maximum. This is in agreement with the observa- Con that only circular phage DNA molecules are infective, but not linear molecules; melting of component II gives rise to at least one linear DNA molecule, and these uninfective molecules sediment more slowly than infective circular molecules (Fiers & Sinsheimer, 1962; Burton & Sinsheimer, 1965).

(b) Differentiation of two replicutive form DNA’s by electron microscopy

Electron microscopy w&s used to study the morphology of the two RF components. Plate I shows an electron micrograph of the same material as that used for sucrose gradient sedimentation described in Fig. 2 (no DNase). Only a few ring-shaped RF molecules are visible. Most of the material has the appearance of condensed aggregates. The thickness of the strands of these aggregates is clearly greater than that of circular RF rings. That they do not represent spreading artifacts is demonstrated by the two micrographs in Plates II and III. They show the same RF material as used for experiments described in Figs 3 and 4 (4 and 15 minutes DNase treatment). It can be seen that the aggregates are replaced with increasing time of DNase treatment by ring-shaped RF molecules. It is concluded that the branched, not circular- looking molecules in Fig. 5(a) represent highly twisted RF rings. The thin loops, seen mainly at the ends of the branches (see arrows in Plate I) represent untwisted double strands, while the aggregates themselves are constituted of two DNA double strands twisted around each other. Sfter prolonged DNase treatment (15 minutes DNase) rod-shaped forms are also found of the same length as closed untwisted rings. One of these molecules is visible in Plate III.

FIG. 3. Separation of RF components by sucrose-gradient sedimentation subsequent to 4 min of DNase treatment.

(a) Tritium-labelled RF was treated for 4 min with pancreatic DNase at 37°C (incubation mixture: 1Om3 M-MgCl,, 0.001 pg/ml. DNase). To stop enzyme action, 10Va M-sodium citrate was added after 4 min. A l-ml. sample was layered on a sucrose gradient and centrifuged as described in Fig. 2. The 34 fractions collected were tested for radioactivity and for infectivity as described in Fig. 2. The upper part of the Figure shows the infectivity of 4X RF as tested by the addition of 7 pg/ml. of E. coli DNA to the adsorption medium (see Results, section (d)).

(b) Part of the sample used for sedimentation in Fig. 3(a) was heat denatured before sedimentation. The RF was heated in 0.005 M-phosphate buffer and 2 x 10e4 M-EDTA to 92°C for 2 min and then quickly chilled.

Sedimentation and test conditions as described in Fig. 2. (-A- A-) Radioactivity, cts/min/ml.; (--O--O--) +X single-strand equiv./ml. after

heat treatment; (.n x . . x ..) 4X single-strand equiv./ml. before heat treatment; (.-&-.--A --.) Ml3 DNA plaque-forming units/ml.

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TERTIARY STRUCTURE OF 4x174 RF

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FIG. 5. Kinetics of the conversion of one RF form into the other by DNase treatment.

RF was treated for different lengths of time with DNase as described under Materials and Methods. The samples were then sedimented in a sucrose gradient or prepared for electron microscopy. In some experiments (0, 4 and 16 min DNase) sucrose gradient sedimentation and electron microscopy were performed in parallel from the same sample. The Figure shows the percentage of radioactivity in component I (-x - x -), calculated from sedimentation experiments, and the percentage of twisted rings (-•--a-), calculated from electron micrographs, remaining in the RF preparation after different times of DNase treatment. The data are taken from Tables 1 and 2.

For quantitative comparison of these two morphologically distinguishable forms (twisted and untwisted rings), more than 350 molecules were counted after different times of DNase treatment. Molecules of which the entire countour length could be followed were considered to be untwisted, even though they might contain several crossing points; i.e. molecules which were obviously circular were counted as being untwisted. The strands of the twisted molecules are clearly thicker and it is not obvious by visual inspection that these moleoules are circular. The percentages of twisted, untwisted and opened molecules were determined. The results are shown in Table 2. The Table records two other experiments (l-5 and 6 minutes DNase), for which no parallel sucrose gradient centrifugations were performed.

Whereas in the untreated preparation no linear RF molecules and 20% of untwisted circles are found, after four minutes of DNase treatment 4% of the RF molecules are linear and 40% untwisted rings. After 15 minutes only 19% of the RF remain twisted, whereas 60% have an untwisted structure and 20% are in the linear conformation.

FIG. 4. Separation of RF components by sucrose gradient sedimentation subsequent to 15 min of DNase treatment.

(a) Tritium-labelled RF was treated for 16 min with DNase as described in Fig. 3. A l-ml. sample was layered on a sucrose gradient and centrifuged for 22 hr at 25,000 rev./min. The 59 fractions collected were tested for radioactivity and infectivity as described in Fig. 2.

(b) Part of this sample was heat denatured as described in Fig. 3 (b) and centrifuged for 22 hours. The 61 fractions collected were tested for radioactivity and infectivity as described in Fig. 2.

(-A-A-) Radioactivity, cts/min/ml.; (-O---O-) 4X single-strand equiv./ml. after heat treatment ; (. x . . x .) +X single-strand equiv./ml. before heat treatment; (-A-m- A-.) Ml3 DNA plaque-forming units/ml.


TABLE 2

Conversion of twisted into untwisted rings by DNa.se treatment

Time of Actual DNase number of

treatment rings (min) counted

“/; of twisted o/0 of untwisted y0 of linear rings rings molecules

0 377 80 20 - 1.5 133 65 35 -

4 419 56 40 4 6 116 42 52 6

15 911 19 61 20

RF was treated for different lengths of time with DNase as described under Materials and Methods and prepared for electron microscopy. A number of RF molecules were counted on randomly taken micrographs (second column) and the percentage twisted, untwisted and opened rings of the total number counted, was calculated. Data after 0, 4 and 15 min DNase treatment were obtained from the same RF fractions as used for gradient sedimentation shown in Figs 2 to 4.

(c) Quantitative cowqarison of data obtained by sucrose gradient centrifugation and electron microscopy

Sucrose gradient centrifugation of RF (Results section (a)) demonstrated that the degree to which component I is converted into component II depends on the time of DNase treatment. Electron microscopy also shows a DNase-dependent conversion of twisted into untwisted and opened forms.

If component I corresponds to twisted rings and II to untwisted and opened rings, the percentage of radioactivity in component I should decrease in parallel to the percentage of twisted rings as a function of the time of DNase treatment.

In Fig. 5 the percentage of component I (from Table 1) and the percentage of twisted rings (from Table 2) are plotted against the time of DNase treatment. Both agree well and decrease in parallel with increasing time of DNase treatment. From this result it is concluded that component I corresponds to twisted rings and component II to untwisted and to opened molecules.

(d) Specific inhibition of relplicative form infectivity by E. coli DNA

The increase of the infectivity of RF preparations due to heat denaturation was used to characterize RF biologically (Hofschneider, 1961; Benzinger & Hofschneider, 1963; Benzinger, Jaenisch & Hofschneider, 1966). The experiments shown in Results section (a) suggest that the increase of infectivity after heat denaturation of RF is due only to component II present in the RF preparation. Only component II can be denatured to single strands, the relative biological plating efficiency of which is 20 times as high as that of native RF (Denhardt t Sinsheimer, 1965; Sinsheimer et al., 1965).

Different increases of infectivity of RF after denaturation were foumd by different authors. Benzinger & Hofschneider (1963), using osmotic-shock spheroplasts as test cells, found a 6 to 300-fold increase. Burton $ Sinsheimer (1965) observed after alkaline denaturation a 25-fold increase, Pouwels & Jansz (1964) reported a lo- to loo-fold increase of infectivity after heat denaturation of component II; and in the experiments reported here the increase is found to be to two- to threefold. This

TERTIARY STRUCTURE OF 4X174 RF 513

considerable difference in the increases of infectivity after denaturation reported by different authors could be explained by the different denaturation procedures (heat or alkali) or by the different RF fractions used (heat denaturation of unfractionated RF, Benzinger & Hofschneider, 1963; alkaline denaturation of component I, Burton & Sisheimer, 1965; heat denaturation of component II, Jansz & Pouwels, 1965; and in experiments reported here). In addition to this, different methods for preparing spheroplasts were used, which could also explain the different increases. However, even in the experiments reported here using the same method of preparing spheroplasts each time, the increase of infectivity of the same fraction of component II RF (isolated by sucrose sedimentation) varied from one spheroplast preparation to another (zero to threefold). This observation suggests that the increase of infectivity after denaturation is influenced by properties of the test system.

It is known that after bacteria have been converted to spheroplasts, cell contents are released into the medium by some of the cells. The amounts of intracellular components (as measured for ,Cgalactosidase, Benzinger et al., 1966, manuscript in preparation) found in the medium increase with the time after conversion and also depend on the conversion conditions.

A possible explanation for the increase of infectivity of RF after heat denaturation could therefore be that double-stranded E. coli DNA is released by the test cells into the medium and specifically inhibits the infectivity of native RF and not that of denatured RF and 4X single strands. An inhibition of RF infectivity by E. coli DNA has been observed already by Burton t Sinsheimer (1963,1965).

Therefore, the concentration dependence of the inhibition by E. coli DNA of RF- infectivity was investigated. Native RF, denatured component II and $X single strands were tested for infectivity with different concentrations of E. coli DNA added to the adsorption medium. The result is shown in Fig. 6. The infectivity of native RF

Concentration of E.co/i DNA in pg/ml.

FIG. 6. Inhibition of the infectivity of 4X RF and single-stranded 4X DNA by E. COG DNA. Native RF (-O-O-), denatured RF of component II (- X--X -) and 4X single-stranded

DNA (-•---~--) were tested biologically. Different concentrations of E. coli DNA were added to the adsorption medium. The data are presented aa the percentage of the infectivity of the control tested without addition of E. wli DNA.


decreases with increasing concentrations of E. coli DNA. This inhibitory effect starts at concentrations of E. coli DNA less than 0.1 pg/m.l. and at concentrations greater than 0.5 pg/ml. the remaining RF infectivity (plaque-forming units/ml.) is a linear function of the E. coli DNA concentration. This linear dependence suggests that the process of infection with native RF is competitively inhibited by E’. coli DNA.

The infectivity of single-stranded $X DNA and denatured component II is not affected by E. coli DNA concentrations less than 10 pg/ml. At this concentration the infectivity of native RF is already reduced loo-fold. The highest concentration of E. coli DNA tested also reduced the infectivity of +X single-stranded DNA and denatured RF, but only to 30%.

Using these results, the presence of component II in a RF preparation can be detected biologically without separation of the two components. The more the relative plating etliciency of native RF is decreased, the higher should be the increase of infectivity after denaturation of component II.

The upper parts of Figs 2 to 4 show the RF infectivity of fractions collected from the sucrose gradients, assayed after the addition of 7.0 pg E. coli DNA/ml. to the adsorption medium. The infectivity of native RF (component I as well as II) is greatly reduced, whereas the infectivity of denatured component II is not affected, as can be seen by a comparison of the upper and lower parts of Figs 2 to 4. Both these effects result in a relative infectivity increase of the order of 30-fold for component II after denaturation. This relative increase of infectivity was reproducible for component II even when using different spheroplast preparations if 7 pg E. coli DNA/ml. was added each time.

The fact that component I also shows a slight increase of infectivity after addition of E. coli DNA (upper parts of the curves) may be explained by random single-strand breaks in some of the twisted rings, either acquired after sucrose gradient sedimentation or by scission of phosphodiester bonds during heating or by incomplete re- naturation of some RF molecules.

4. Discussion

RF was purified by repeated chromatography on MAK columns. The contamination by E. coli DNA, as revealed by electron microscopy (see Materials and Methods), was not more than 3% on a mass basis. The RF material isolated was composed of 80% component I and 20% component II (Fig. 2).

The infectivity properties of the two components are consistent with the physico- chemical data reported earlier by others (Burton & Sinsheimer, 1965; Jansz & Pouwels, 1965). Only component II shows an increase of infectivity when heat denaturation followed the band sedimentation. When the RF is heat denatured before band sedimentation, no increase of infectivity is found in any fraction of the gradient after further heating (Figs 3(b), 4(b)), th us indicating on a biological basis that component II is converted completely to single strands. The infectivity of component I, which cannot be heat denatured, is not affected by this brief heat treatment.

From sedimentation data, Burton & Sinsheimer (1965) and Jansz & Pouwels (1965) deduced a twisted tertiary structure for component I. However, no electron microscopical evidence was provided. Electron microscopical studies reported here reveal that in RF containing 80% component I and 20% component II, 80% of the molecules are highly twisted and 20% have the tertiary structure of extended circles.


This indicates that component I corresponds to twisted rings and component II to untwisted rings. This assumption was confirmed by comparing quantitatively the kinetics of the conversion of component I into II with the kinetics of the conversion of twisted into untwisted molecules. The rate of morphological change was found to be in good agreement with the change in sedimentation properties (Fig. 5), showing the identity of component I with twisted rings and that of component II with untwisted and opened rings.

This result is in good agreement with data obtained by Vinograd et al. (1965), who demonstrated qualitatively by electron microscopy that twisted polyoma DNA rings are identical with component I. Electron micrographs of +X RF (Plate I) and of Ml3 RF, as demonstrated by experiments carried out in parallel to those described here (Ray, Bscheider & Hofschneider, 1966; Ray, Preuss & Hofschneider, 1966) reveal, in comparison to polyoma DNA, a considerably higher degree of twisting. It is not known whether this different morphology is caused by structural differences between polyoma DNA and RF of $X174 or Ml3 or by differences in the preparation of the nucleic acids for electron microscopy.

As already mentioned, native RF was observed to be one twentieth as infective as single-stranded phage DNA (Denhardt & Sinsheimer, 1965; Sinsheimer et al., 1965). It has been demonstrated in this paper that E. coli DNA added to the adsorption medium inhibits the infectivity of native RF in a first-order reaction at concentrations of E. coli DNA above 0.5 ,ug/ml. (component I and native component II were inhibited to the same degree). In this concentration range the inhibition appears to be competitive. The infectivity of denatured component II RF or 4X single-stranded DNA is inhibited by addition of E. coli DNA to a much lesser degree. The lower the relative plating efficiency of native RF, the higher is the increase of infectivity of component II after denaturation (compare tests with and without inhibition by E. coli DNA in Figs 2 to 4). In contrast to these results, the infectivity of native polyoma DNA is not affected by E. coli DNA concentrations up to 300 pg/ml. (Weil, 1963). It is not known if the results reported here also hold for alkaline denatured component I (Burton & Sinsheimer, 1965), where the two strands cannot separate.

Spheroplasts, as used for the DNA tests, release cell contents into the transforma- tion medium to differing degrees (Benzinger et al., 1966). Therefore, even uuder normal test conditions (no E. coli DNA added), the variable concentrations of free bacterial DNA may become high enough to influence the relative plating efficiency of native RF, thus causing an increase of infectivity of component II after denaturation. This assumption was not examined systematically. It is however supported by the observation that purified component II gives no increase of infectivity after heat denaturation as tested with one spheroplast preparation (spheroplasts perhaps releasing little E. coli DNA into the medium), but shows an increase of up to threefold when tested with another preparation (spheroplasts perhaps releasing more E. coli DNA into the medium). Therefore, assuming that all RF molecules have the same potential infectivity, RF might have the same infectivity per molecule as single- stranded phage DNA, when tested under ideal conditions (no E. coli DNA present).

The comparatively small increase of infectivity of component II after denaturation (maximum threefold without addition of E. coli DNA) indicates that in the spheroplast system used here, the relative plating efficiency of native RF is higher than in other systems which show an increase of infectivity between lo- to 300-fold. The experiments performed under inhibition with E. wli DNA show that the infectivity


of single-stranded phage DNA and denatured RF is practically independent of the state of the test cells, whereas the infectivity of native RF varies considerably. Therefore, results obtained by calculations based on the relative plating efficiency of native RF cannot easily be generalized or compared with results obtained in another biological test system. It was demonstrated that the increase of infectivity of component II after denaturation depends on the test conditions (the greater the amount of E. coli DNA present, the greater the increase). Therefore, the magnitude of this increase represents a criterion of the degree to which the infectivity of native RF is inhibited and could be used to correlate results obtained with different test systems.

Note added in proof: The results of Jansz & Pouwels (1965) have recently been published in more detail. Pouwels, P. H., Jansz, H. S., van Rotterdam, J. & Cohen, J. A. (1966). Biochim. biophys. Acta, 119, 289.

We thank Professor A. Butenandt for his continuous interest in this work, Dr D. S. Ray for advice in ultracentrifugal techniques and valuable criticism while performing this study and writing the manuscript. Thanks are due to Dr R. Benzinger and Dr H. Delius for allowing us to publish the method of infectious DNA assays prior to a more detailed publication concerning nucleic acid assays, and to Dr R. Benzinger for suggesting that E. coli DNA released by test cells could inhibit RF infectivity. We also thank Dr J. Ammann for helpful discussions and Miss H. Riesemann for her able technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft.

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