The replication of the ring-shaped DNA of polyoma virus

J. Mol. Biol. (1971) 59, 195-206

The Replication II.? Identifkation

of the Ring-shaped DNA of Polyoma Virus of Molecules at Various Stages of Replication

PIERRE BOUROAUX, DANIELLE

BOTJRGAUX-RAMOISY AND PAUL SEILER

DtGpartement de Hicrobiologie Centre Hospitulier U&emit&e Universitd de Sherbrooke P. Q., Canada (Received 14 September 1970, and in revised form 26 Janwry

1971)

Viral DNA was selectively extracted from mouse embryo cells which had been subjected to short pulses of radioactive thymidine during productive infection with polyoma virus. The replicetive intermediate of the viral DNA, component II*, was then isolated and characterized using dye buoyant density centrifugation, chromatography on benzoylated-naphthoylated DEAE cellulose and velocity sedimentation in sucrose solutions. The results of the sedimentations performed at both neutral and alkaline pH values suggested that component II* represented a collection of molecules at all stages of replication. This was further supported by the electron microscopic observation of purified preparations of component II*, which revealed circular molecules with two branch points.

1. Introduction Both from the biological and from the physical point of view, the intact mature form of polyoma virus DNA (component I) offers several interesting properties. This molecule has been shown to possess both the infectivity (Dulbecco & Vogt, 1963) and the transforming activity (Crawford, Dulbecco, Fried, Montagnier & Stoker, 1964; Bourgaux, Bourgaux-Ramoisy & Stoker, 1965) of the virus itself. These biological properties remain unchanged after the molecule has been exposed to conditions sufficient to denature it completely. This is due to the fact that, component I having a ring structure, its two complementary strands cannot come apart since they are entangled as a result of being separately continuous (Weil & Vinograd, 1963; Vinograd, Lebowitz, Radloff, Watson & Laipis, 1965). This structural lay-out allowed us to predict what should be a pre-requisite step for the replication of component I, i.e. nicking. Indeed, the introduction of at least one single-strand break in this covalently closed ring-shaped DNA is required to make possible first, the unwinding of the parental duplex which takes place during replication, and secondly the segregation of the parental strands which is implied by the semi-conservative nature of the process (Hirt, 1966). We recently identitled, and partially characterized, a replicative intermediate of polyoma virus DNA (Bourgaux, Bourgaux-Romoisy & Dulbecco, 1969). This newly observed molecular species of the viral DNA, referred to as II*, actually t Paper I in this series is Bourgaux,

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& Dulbecco,

1969.

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consists of largely double-stranded nicked molecules which, after denaturation, give rise to both circular and linear strands. When II* was labelled with short pulses of tritiated thymidine (5 to 15 min), after denaturation, essentially all the label was found in linear strands, which although variable in size, never appeared longer than the strands of mature viral DNA. These data suggested that replication of polyoma virus DNA proceeds as proposed by Cairns (1963), a conclusion which was consistent with Hirt’s interpretation of the electron microscopic appearance of replicating polyoma virus DNA (Hirt, 1969). Among others, the following questions, however, remained unanswered. Does II* represent replicating molecules as we suspected, or alternatively, an already replicated material, precursor of mature DNA ? Assuming II* consists of replicating molecules identical to those observed by Hirt (1969), are all stages, or rather some particular stages, of replication represented as observed in the case of SV40 DNA (Levine, Kang & Billheimer, 1970) 2Taking into account the known heterogeneity of component I with respect to molecular weight (Thorne, 1968; Blackstein, Stanners & Farmilo, 1969), would II* then represent, in addition to molecules of normal contour length, shorter replicating molecules! We report here the results of an investigation undertaken to answer these questions.

2. Materials and Methods (a) ViTu.3 Both the large-plaque (Vogt & Dulbecco, 1962) and small-plaque (Crawford, 1962) strains of polyoma virus were cultivated as already described (Borgaux, 1964; Bourgaux, et al., 1969) and were used during these experiments with identical results. (b) Cultures Secondary cultures of whole mouse embryos in resting state were used (Fried & Pitts, cultures were plated per 50-mm Petri dish in Dulbecco’s 1968). 2 x lo6 cells from primary modified Eagle’s medium (Grand Island Biological Co.) containing 0.5% calf serum. The cultures were used 4 days later. After infection with virus (1 x 10s plaque-forming units in 0.2 ml.) for 1 hr, the cultures were covered with fresh medium. When large amounts of material were needed, cells were grown in rotating cylindrical bottles (Bourgaux, 1964) under standard conditions. Four days before infection the growth medium was replaced by low serum medium (0.5% calf serum). (c) Labelling

of DNA

30 hr after infection, the cells were first incubated for 0.5 hr in the presence of Mluorodeoxyuridine (6 x 10m6 M) in order to deplete the thymidilic pool (Hirt, 1966). Tritiated thymidine (spec. act. 10 Ci/m-mole) was then added at a final concentration of 26 &X/ml. Each Petri dish then contained 1 ml. of medium, Dulbecco’s modifkd Eagle’s medium buffered with Hepes instead of bicarbonate (Williamson & Cox, 1968) and containing O.So/o calf serum, was used in most experiments. Satisfactory control of the pH was thus ensured without a CO,-enriched atmosphere. For pulses less than 5 min, the Petri dishes were floated on a water bath set at 37’0. For longer pulses an incubator was used. Pulses were terminated by placing the Petri dishes on ice. (d) Extraction

of DNA

Viral DNA was selectively extracted from the infected cells using sodium deoxycholate as aheady described (Bourgaux et al., 1969). After centrifugation of the cell lysate, however, the supernatant containing the deoxyoholate-extracted DNA was further treated with sodium sarcosinate (0.26%) and pronase (100 pg/ml.) for at least 12 hr at 37°C.

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(e) Dye-buoyant dewity-gradient centrifuga&on Covalently closed and nicked forms of polyoma virus DNA were separated after equilibrium density-gradient centrifugation in ethidium bromide-caesium ohloride solutions (Radloff, Bauer L Vinograd, 1967). After fractionation of the gradients, the material present in the “dense” (component I) and “ light ” (component II + component II* +cellular DNA) bands was dialysed for 48 hr against 0.01 M-NaC1-0~005 a6-EDTA-O-005 MTriseHCl, pH 8.6, to remove ethidium bromide. Although removal of the dye probably was not complete, this did not seem to affect the sedimentation properties of the DNA when these were compared with those of suitable markers. (f) Velocity seo?immtation in 8ucro8e aolutiona The DNA sample (up to 0.1 ml.) was layered on 4 ml. of a 6 to 20% linear sucrose gradient (in 0.5 a6-NaCl-0.001 M-EDTA containing either 0.01 M-Tris.HCl (pH 8.6) or 0.26 N-NaGH). It was then spun in neutral solution (for 2 hr when the centrifugation time is not specified) or in alkaline solution (for 1 to 4 hr) at 60,000 rev./min and 20°C At the end of the run, the gradient in the SB406 rotor of a B60 International ultracentrifuge. was fractionated by collecting drops from the bottom of the tube. (g) Chromatography on benzoylated-naphthoylated DEAE-cellulose As already done by Levine et al. (1970), for SV40 DNA, the method of Gillam et al. (1967) including the modifications introduced by Kiger & Sinsheimer (1969), was followed. Commercial BNDt-cellulose (Gallard-Schlesinger) was used throughout. (h) A88ay of radioactivity Radioactive samples were collected directly on to glass fibre disks (Whatman GF 83). These were washed either by the Bollum (1969) technique, or by first filtering through ice-cold 6% trichloroacetic acid and then ethyl alcohol. The disks were dried and immersed in a PPO-POPOP-toluene mixture for counting in a scintillation spectrometer. (i) Prepmation of the DNA for electron microscopy Viral DNA W&B extracted ae stated above, from approximately 3 x 10s infected cells which had been pulse&belled for 16 min with radioactive thymidine. It wss fractionated after dye-buoyant-density gradient centrifugation and the material present in the light band was subjected to a batch chromatography on BND-cellulose after extensive dialysis against the adsorption buffer. More than 90% of the radioactive DNA eluted with buffered saline only sedimented like component II in neutral sucrose solution. This fraction was not further studied. The DNA eluted in the caffein fraction (replicating DNA) was further fractionated by velocity sedimentation (see Results). Successive fractions were pooled, dialysed against 0.01 MaNaCl and concentrated by evaporation to a flnal volume of 0.06 ml. The DNA samples were diluted fourfold using 4 M-ammonium acetate (pH 7) and then mixed with oytochrome c to a final concentration of O*O5o/o.Approximately 0.04 ml. of the mixture was spread on a distilled water hypophase aa described by Kleinschmidt & zahn (1969). The DNA-cytochrome film was oolleoted on copper grids (previously covered with a Formvar-carbon film) and dried with alcohol. The grids were then rotary shadowed with platinum-iridium at an angle of 7”. Speoimens were scanned using a Philips EM300 electron miorosoope at a magnifloation of 17,200. Magnification was checked with a carbon grating replica (I&d, Inc.). All circular moleoules were photographed, magnified 200,000 times and measured with a calibrated map ruler.

3. Results (a) Sedimentatiim pq0ertie.s of II* in sucrose solutions Aa could be expected fkom the results obtained on SV4-0 DNA by Levine et al. (1970), II* sediments more rapidly than either I or II in a neutral suorose gradient. Under these conditions, II* forms a broad band, thus appearing to be heterogeneous t Abbreviation

used: BND, benzoylatad-naphthoylsted

DEAE.

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1. Sedimentation properties of II* in neutral solution. Infected mouse embryo oells were pulsed for 16 min with [3H]thymidine. Viral DNA was extracted using sodium deoxychohte and subjected to equilibrium density gradient centrifugation in an ethidium bromide-CsCl solution. After dialysis, the material recovered from the light band (which contains both II* and II) was mixed with 1%labelled marker DNA and analysed by centrifugation through a 5 to 20% neutral sucrose gradient as described in Materials and Methods. At the end of the run, the eH (-@-a-) and i4C (--O--O--) contents of the fractions colleoted from the bottom of the tubes were determined. The marker was a preparation of viral DNA labelled with [Wlthymidine which consisted of 82% component I (20 s) and 18% component II (10 8). FIQ.

(Fig. 1). The heterogeneous nature of II* was further tested by the following experiment: pulse-labelled viral DNA was subjected to dye-buoyant density-gradient centrifugation (see Materials and Methods), and the material present in the light band was fractionated after velocity sedimentation in neutral sucrose solution. Successive fractions were pooled and re-centrifuged in either neutral or alkaline sucrose solutions (Fig. 2). The centrifugations performed at neutral pH showed that the observed differences in X-values were indeed reproducible (Fig. 2 (b) and (c)). At alkaline pH, the material identified as II in neutral solution sedimented like a mixture of circular and linear single-strands (Fig. 2 (f)) of viral length (Vinograd et al., 1966; Thorne, 1968; Bourgaux et al., 1969). Under the same conditions, the radioactive DNA identified as II* migrated as a mixture of linear strands of various sixes, as already observed for unfractionated II* (Bourgaux et al., 1969). When the various pools made from that material were compared, it became obvious that the DNA which proved to be slower at neutral pH produced a higher proportion of small radioactive strands at alkaline pH, the large radioactive strands being, however, largely dominant in all pools studied (Fig. 2 (d) and (e)). These results suggest that the heterogeneous nature of II* observed in neutral solution reflects the existence of molecules which differ in molecular weight as a consequence of containing replicating chains of various lengths.

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FIQ. 2. Fraotionation of II*. Radioaebive viral DNA was prepared and subjeoted to equilibrium density gradient eentrifugation as stated in Fig. 1. The mixture of II* and II present in the light band ~8s then centrifuged through a neutral suorose gradient (see Materials and Methods). The distribution of the radioactive material in the gradient was determined on samples taken from the fractions collected from the bottom of the tube (a). Successive fractions were then mixed to constitute five pools (A, B, C, D, E). The material present in these pools was further oharaeterized by either a second eentrifugation under identical conditions in neutral suorose solution ((b) pool A; (0) pool C) or a centrifugation in alkaline suorose solution for 3.6 hr ((d) pool B; (e) pool D; (f) pool E).

(b) L&l&g

of viral DNA after short pulses

From kinetic data on the incorporation of radioactive thymidine into the various forms of the DNA, it was estimated that the duplication time of polyoma virus DNA is close to four minutes (Roman, Bourgaux & Dulbecco, unpublished results), during the phase when viral DNA replication proceeds at a constant rate. By duplication time we mean a temporal parameter which would include the actual replication time and possibly a pause taking place between successive rounds of replication. It was therefore, of

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FIQ. 3. Labelling of II* after pulses of various lengths. Viral DNA was selectively extra&ad from infested cells whioh had been subjeated to pulses of [sH]thymidine for various lengths of time. It was mixed with [Wlthymidine-lsbelled viral DNA (same preparation ss for Fig. l), used &s marker, and subjeoted to velocity sedimentation in 6 to 20% suorose gradients buffered at either neutral or alkaline pH. At the end of the runs the gradients were fractionated for determination of the SH (-@-a-) and 14C (--O--O--) distributions. Left panel: sedimentation st neutral pH: (a) 30-set pulse; (b) 2-min pulse; (a) 8-min pulse. The arrowa denote, within the II* bmd, the fraction with maximum radioactivity (1, 6 and 7 fractiona away from marker oomponent I in (a), (b) and (c), respectively). Right panel: sedimentation at alkaline pH for 4 hours: (d) 30-se0 pulse; (e) 2-min pulse;

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interest to investigate the sedimentation properties of radioactive II*, labelled after pulses which would be either shorter or longer than the calculated duplication time. Infected cells were accordingly subjected to 30-second, l-, 2-, 4-, 8- and 16minute pulses with radioactive thymidine, viral DNA was then extracted and analysed after velocity sedimentation. As shown in Figure 3, the radioactive profiles observed for II* are markedly dependent on the labelling regime. In neutral solution, although the mean &values for II* appear rather constant, a progressive shift of the maximum of radioactivity towards higher S-values is observed when the length of the pulse is increased (see arrows in Fig. 3(a), (b) and (c)). This may mean that maximum labelling of the most extensively replicated II* molecules may require a pulse which is at least two minutes long. The second maximum of radioactivity which appears close to the marker in the case of the eight-minute pulse (Fig. 3 (c)) is due to labelling of component I, which is negligible for the shorter pulses. Indeed, as estimated after a one-hour sedimentation in alkaline solution (which separates covalently closed molecules from the other forms of the viral DNA), radioactive component I represents 0, 2 and 14%, respectively, of the counts present in DNA in the 30-second, 2minute and g-minute pulse-labelled preparations. Since all pulses were relatively short, the radioactivity in II* sediments in alkaline solution as a mixture of linear strands of various sizes, as already described (Bourgaux et al., 1969). However, there is relatively more radioactivity in the larger strands after the longer pulses than after the shorter ones (Fig. 3 (d) to (f)). This shows that the replicating strands in II* are not uniformly labelled unless the r3H]thymidine pulse is several minutes long, actually two to four minutes (4-min sample not shown here). The results of both the neutral and alkaline runs, therefore, indicate first, that the more rapidly sedimenting II* molecules are the more extensively replicated, and secondly that the replication time of polyoma virus is not grossly different from the calculated duplication time. With respect to the possible implications of the use of fluorodeoxyuridine for the calculation of these parameters, it should be mentioned here that we performed several experiments in which this analogue was omitted during pulse-labelling of the DNA. Although incorporation of [3H]thymidine into viral DNA was markedly reduced (for an S-min pulse, this resulted in a threefold reduction in incorporation), the proportion of the radioactivity recovered in the various forms of the viral DNA did not appear to be altered at any given time. It is, therefore, unlikely that fluorodeoxyuridine significantly reduced (or increased) the rate of synthesis of the viral DNA, at least when the radioactive thymidine was present. It is worth pointing out that labelled component II was detected in all viral DNA preparations examined, even when the labelling conditions had been such that very little radioactivity was present in I, an observation which further substantiates the role proposed for II in polyoma virus DNA replication (Bourgaux et al., 1969).

FIG t.--contin~d (f) 3min pulse. The presence of both i4C and 3H counts in the first 6 fractions of the gradients is due to the variable recovery of labelled component I (53 s form) from the bottom of the tube. The 18 8 and 16 s peaks produced by the denatured i4C-lctbelled component II, although small, are clearly visible. The 3H counts floating on top of the gradients are due to redioaative thymidine which was present in the crude extracts analysed, and remsined on the filters after treatment by the Bollum (1969) teahnique. This is shown by the superimposed radioactive profiles (-- x -- x --) obtained when extra&e of similarly labelled uninfected oells were sedimented under identioal conditions.

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Fra. 4. Fractionation of II* for electron microscopy. Viral DNA was extracted from 3 x 1OBinfected cells which had been pulse-labelled for 15 min. Replicating DNA (II*) was separated from the other viral forms by a procedure involving equilibrium density-gradient oentrifugation in ethidium bromide-CsCl solution followed by ohromatography on BND-oellulose (see Materials and Methods). It was then subjected to a velocity sedimentation (60,000 rev./min for 108 min) through a 6 to 20% neutral sucrose solution. Samples of the fractions colleoted from the bottom of the tube were used for the determination of the radioactive protie (-@-a--). Suocessive fractions were then mixed to constitute 7 pools as indioated on the Figure. These were dialysed and concentrated before being examined under the electron miorosoope.

(G) Electron microscopy of puri@d II* Purified radioactive II* was obtained by a procedure involving dye-buoyant densitygradient centrifugation followed by a chromatography on BND-cellulose (seeMaterials and Methods). It was subjected to velocity sedimentation in neutral sucrose solution and all pools made from the fractions collected (see Fig. 4) were examined under the electron microscope. A minimum of 30 grids from each pool were scanned. All pools contained small amounts of contaminating fragments of cellular DNA, while only the pools corresponding to the radioactive peak (Pig. 4, pools 4,5 and 6) contained material which could be interpreted as replicating polyoma virus DNA. It consisted essentially of circular molecules with two branch points, three branches and no end (Plates I and II), as already described by Hirt (1969). We observed 97 such molecules and 23 simple circular molecules of polyoma virus DNA length. On the same grids, nine circular molecules with linear pieces of DNA attached at branch points (tails) were also found. Having a circumference corresponding to polyoma DNA and a tail never longer than the circumference, they were considered as broken replicating polyoma DNA and were not further studied. The circumference of each intact replicating molecule was obtained by adding the length of the unrepliaated part to the mean length of the replicated branches (Hirt,

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1969). The replicating branches were easily identified, since, out of 97 molecules, 80 had two branches which differed in length by less than 6%. Figure 5 shows the distribution of the circumferences of simple circular and replicating molecules. In both cases,a maximum number of molecules was found with a circumference of between l-74 and l-81 p. In addition, a second classof replicating moleculeswith an approximate circumference of 1.57 p was present. This heterogeneity is not due to a lack of standardization in the preparation or observation of the specimens,since the two types of replicating molecules could be observed on the samegrids. Four simple circular molecules, with circumferences between 2.3 and 3-2 CL,were also seen, but were not included in the calculations. The fraction of DNA which had beenduplicated, i.e. the ratio as a percentage of the mean length of the replicated branchesto the circumference, was also determined for each replicating molecule. As shown in Figure 6, t’he material which had sedimented more rapidly in the sucrosegradient contained a higher proportion of largely replicated molecules. As already suggested by the results of the sedimentations, these were, furthermore particularly numerous: out of a total of 81 molecules detected in pool 5 (which represented the fractions from II* with maximum radioaotivity and intermediate S-value), 28 had completed more than 90% of their replication, while the mean replicated length represented 75% of the circumference, Finally, three circular moleculeswith one cross-over and total contour lengths of 3.44, 3.30 and 3.04 p were observed in pool 5 and could not be classified, since it was not possible to decide whether they represented fully replicated monomers or mature

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6. Number of replicating moleo&s as a function of the percentage of DNA length duplicated. The mean replicated length represents 75% of the circumference for the 81 replicating moleonles observed in pool 6. The corresponding value for the 13 molecules in pool 6 is 28%. Three replicating molecules only were found in pool 4; they had completed 90, 97 and almost 100oJOof their replication. FIQ.

dimers of polyoma virus DNA. The finding that the extensively replicated molecules were more numerous than those which had just started their replication was still equivocal at this stage. Indeed, it could have been argued that the equilibrium sedimentation had resulted in selective loss of a particular classof replicating molecules (Levine, personal communication), or that recovery of the DNA from the various pools had been unequal. We therefore examined a preparation of II* obtained after a shortened purification technique avoiding both the equilibrium and the velocity sedimentation steps. In this unfractionated II* preparation 90 intact replicating molecules were observed, with a mean replicated length of 60%. Comparing the categories ranging from 20 to 50% and from 50 to 800/ replication, 22 and 36 molecules were found, respectively. Heterogeneity with respect to circumference length was again apparent, two thirds of the replicating molecules being found within two narrow ranges. While 33 moleculeshad circumferences between 1.70 and 1.86 CL,only 17 were longer and as many as 40 were shorter. From the latter, 25 had circumferences ranging from l-50 to 1.65 CL.

4. Discussion Our results show that as we proposed previously (Bourgaux et al., 1969), II* represents molecules of polyoma virus DNA at all stages of replication. Since these could be satisfactorily fractionated on neutral sucrosegradients, it was possible to establish the existence of a direct correlation between S-value, content in large replicating chains and total contour length of II* molecules. The short-pulse experiments suggest that polyoma DNA replication might require between two and four minutes. This seemsa rather long time, since the expected value would be lessthan one minute if polyoma DNA replicated at the samerate as mam-

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malien DNA (Hubermcm & Riggs, 1968). Although it is less marked than observed by Levine et al. (1970) for SV40 DNA, the predominance of largely replicated molecules observed with the electron microscope could still indicate that polyome DNA molecules are not replicated at a constant rate. This could contribute to an unusucllly long replication time. The unexpected observation that a significant proportion of the replicating population (about 6%) consisted of molecules which could be considered as almost 100% replicated (see for instance Plate II (r)), leads us, furthermore, to envisage a mechanism for the generation of polyoma DNA dimers (Cuzin, Vogt, Dieckmann & Berg, 1970). Indeed, the relative slowness of replication towards its end could result in a relatively high incidence of recombinrttions between DNA regions which, by definition, contain complementary base sequences. The existence of fully replicated and/or double-length molecules does suggest that this might actually occur at low frequencies during wild-type polyoma virus DNA replication. Another striking feature is the occurence of two classes of replicating molecules of different circumferences. This was not observed by FIirt (1969). It should be pointed out, however, that besides the fact that we studied a larger number of molecules, the cell system was different. Using, as we did, whole mouse embryo cells to grow polyoma virus, Thorne (1968) as well as Blackstein et al. (1969) described the presence of short defective molecules in the DNA extracted from the virus particles. Our results probably mean that viral DNA molecules of less than the normsl length nevertheless replicate. We thank Dr Jesse Summers for valuable suggestions and constructive criticism. skilful technical assistance of Mrs Monique D’Allaire is gratefully acknowledged. work was supported by a grant from the Medical Research Council of Canada.

The This

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AND

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P. (1965).

50, 730.

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Proc. Nat. Ad.