On the origin of oligomer forms of polyoma DNA

J. Mol. Biol. (1973) 7’7, 197-206

On the Origin of Oligomer Forms of Polyoma DNA PIERRE BOURGAUX

D&artement de Microbiologic, Centre Hospitalier Universitaire Universitd de Sherbrooke, P.Q., Canada (Received 4 December 1972, and in revised form 21 March 1973) Polyoma virus DNA synthesized in the presence of puromycin or cycloheximide contains an increased proportion of oligomers and molecules having a reduced number of tertiary turns. When doses of inhibitors producing a submaximal effect are used, molecules with an anomalous tertiary structure are more frequent among the oligomers than among the monomers. It is suggested that protein synthesis inhibitors modify the balance of factors controlling the topological constraints imposed on replicating molecules. Hence, final closure of the chains upon completion of replication would result in the formation of a mature product with an altered tertiary structure which sometimes fails to segregate into two daughter molecules.

1. Introduction While first identified in preparations of polyoma virus DNA (Vinograd et al., 1964, molecules with a covalently closed double-stranded structure have since been observed in a number of instances (Bauer $ Vinograd, 1971). In many of the cells containing such structures, oligomer or complex forms were found in addition to the basic monomeric unit (Bauer & Vinograd, 1971). Although explicable by various mechanisms (Hudson et al., 1968), the formation of these oligomers generally has been envisaged in terms of recombination (Hudson & Vinograd, 1967; Hudson et al., 1968). Recent observations, however, suggest that oligomers are formed during replication of bacterial plasmid (Goebel & Helinski, 1968; Goebel, 1970,1971), mitochondrial (Ness, 1969,1970), viral (Meinke & Goldstein, 1971; Jaenisch & Levine, 1971) and protozoan (Brack et al., 1972) DNAs. Inhibition of protein synthesis has been shown to enhance the formation of oligomers of circular DNA in bacteria (Goebel t Helinski, 1968), mitochondria (NEW, 1969,197O) and virus-infected nuclei (Jaenisch & Levine, 1972). In another respect, we have demonstrated that polyoma DNA synthesized in the presence of puromycin has a low superhelix density, i.e. the monomeric viral DNA formed during inhibition of protein synthesis contains a reduced number of tertiary turns (Bourgaux & Bourgaux-Ramoisy, 1972a). We report here the results of further work on the effect of puromycin and cycloheximide on polyoma DNA synthesis. These experiments were undertaken with the aim of investigating a possible relation between the emergence of oligomer forms and the topological properties of a given circular DNA, subject to active replioation. 197

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2. Materials and Methods (a) Marker viral DNA The methods used to grow polyoma virus, label and extract the viral DNA from productively infected mouse embryo cells, as well as most of the methods used for characterizing the viral DNA, have already been described (Bourgaux et al., 1971; Bourgaux & Bourgaux-Ramoisy, 1971,1972a). Marker viral DNA was selectively extracted using sodium deoxycholate from cells labelled continuously with [2-14C]thymidine (0.05 pCi/ ml; spec. act. 59.35 mCi/mmol) from 18 to 40 h after infection; it was deproteinized using sodium sarcosinate and pronase (Bourgeux et al., 197 1). Over 75% of the acid-precipitable radioactivity in such marker preparations sedimented in neutral and alkaline sucrose solutions, and banded after dye-buoyant density-gradient centrifugation (Radloff et al., 1967) 88 DNA I, i.e. the intact covalently closed form of polyoma DNA extracted frorn virions. With aging, an increasing proportion of DNA II, i.e. the nicked form of the viral DNA, was of course detected in those preparations. (b) Isolation of labelled oligomers Cultures of mouse embryo cells, at 26 h after infection, were covered with medium containing either puromycin at concentrations ranging from 10 to 200 pg/ml, or cycloheximide (10 @/ml), or no inhibitor. After 1 h of incubation at 37”C, [3H]thymidine (10 &/ml; spec. a&. 14.1 Ci/mmol) was added to the cultures, which were incubated for another 4 h. Viral DNA was then selectively extracted and deproteinized as described for marker DNA (Bourgaux et al., 1971). The covalently closed dimers were then separated from the monomers by a procedure ,inspired by that used by Jaenisch & Levine (1972). The DNA solution was layered on to a 5 % to 20% sucrose gmdient, the sucrose solutions in 05 M-N&~, 0.001 M-EDTA, 0.01 m-Tris.HCl being adjusted to pH 12.1 with NaOH immediately before forming the gradient. It was then spun for 108 mm at 40,000 revs/min in the SB283 rotor. At the end of the run, fractions (-&0.35 ml) were collected from the bottom of the centrifuge tube and neutralized by the addition of 0.05 ml of N-HCI followed by O-1 ml of 0.5 M-TriseHCl buffer, pH 6.8. After determination of the radioactive content of each f&&ion, those presumed to contain dimers were pooled and dialysed against 0.015 x-N&l, @OOl5M-SOdiUm citrate. (c) Ultracentifu@ion ultracentrifuge, using the All sedimentations were done at 20°C in a B60 International SBCOS rotor unless otherwise specified. Neutral and alkaline (& pH 124 sucrose solutions contained 0.6 M-N&~, 0.001 M-EDTA and either 0.01 M-Tris*HCl (pH 8.6) or N-NaOH.

3. Results (a) Velocity sedimentation of polym DNA replicated dut$ng inhibition protein synthesis A molecule consisting of two complementary DNA, may be defined by the relation r=a--j3,

of

cyclic chains, like that of polyoma (1)

where a is the topological winding number, /3 the duplex winding number, and 7 the superhelix win&q nuder (Vinograd et al., 1968). Polyoma virus DNA consisting of some 5000 nucleotide pairs has a /I value of about 600 (Vinograd & Lebowitz, 1966). In the covalently closed form (DNA I), a is smaller than p and the molecule thus contains negative superhelical turns (T = -16; Gray et al., 1971). Hence DNA I sediments more rapidly at neutral pH than DNA II which, being nicked, has no superhehcal turns. Polyoma DNA synthesized in the presence of 220 pg puromycin/ml

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appears to consist mostly of covalently closed molecules of virion DNA size containing three to five negative superhelical turns only (Bourgaux & Bourgaux-Ramoisy, 1972a). Hence, this DNA sediments at neutral pH with an s-value between those of markers DNA I and II. Also, it sediments at alkaline pH with a sedimentation coefficient dependent on cc,as expected for a molecule with cyclic complementary chains (Vinograd et al., 1968; Bourgaux & Bourgaux-Ramoisy, 1972b). Since its a value should increase by 2’$$, only 7 being reduced fourfold, this means that the DNA synthesized in the presence of 220 pg puromycin/ml co-sediments with marker DNA I in alkaline sucrose solution. Under the conditions used in this series of experiments (see Materials and Methods), puromycin was found to reduce from three- t,o eightfold the incorporation of [3H]thymidine into polyoma DNA. For concentrations of puromycin ranging from 10 to 200 pg/ml, the reduction in incorporation was not dependent upon inhibitor concentration, in agreement with previous observations (Bourgaux-Ramoisy & Bourgaux, unpublished results). The properties of the DNA synthesized, however, were affected by the drug concentration. As observed previously (Bourgaux & Bourgaux-Ramoisy, 1972a), most of the viral DNA synthesized in the presence of high concentrations of puromycin sedimented more slowly than marker DNA I (at approximately 17 S) in neutral sucrose solution (Fig. 1). In contrast, the DNA synthesized at low concentration of inhibitor formed a peak co-sedimenting with marker DNA I, although a

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FIG. 1. Velocity sedimentation of selectively extracted virel DNA in neutral suorose solution. Polyoma vim1 DNA was selectively extracted from infected mouse embryo cells which had been labelled for 4 h with [3H]thymidine in the presence of 0, 10 and 200 G of puromycin/ml (see Materials and Methods). After being deproteinized using sodium saroosinate and pronase, a sample of the pulse-labelled DNA ww mixed with W%labelled mature vim1 DNA used as B marker, and centrifuged for 108 min through a SoJ0to 20% neutral sucrose gmdient (see Materials end Methods). The radioactivity of the fractions collected from the bottom of the tube was determined. Since identical sedimentation patterns were observed for the marker in the three gradients, the patterns regietered for the SH-labelled DNAs were superimposed on the same graph. -@-a--, 3HWelled DNA, no puromycin; --n--o--, 3H-labelled DNA, 10 pg of puromycin/ml; -m--m--, 3H-l&elled DNA, 200 pg of puromycin/ml. The arrow indicates the position of DNA I (20 S) in such gradients.

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shoulder was clearly present in the 17 S position. Over 85% of the radioactive material in all preparations sedimented at 53 S in alkaline sucrose solution, as expected for covalently closed molecules of the size of virion DNA (not shown). Covalently closed dimers of polyoma DNA are expected to sediment 1.35 times more rapidly than covalently closed monomers at neutral pH (Cuzin et al., 1970). Material sedimenting at 1.35 times the rate of the major molecular species was estimated to represent 6, 11 and 19% of the radioactive DNA synthesized in the presence of 0, 10 and 200 pg of puromycin/ml, respectively. Although fractions containing covalently closed dimers were expected to be contaminated with monomers and with nicked or linear molecules of cellular and viral origin, these figures nevertheless suggested that the frequency of dimers might increase with increasing doses of puromycin. Indeed a discrete radioactive peak was visible at the expected position for dimers in the sedimentation pattern registered for the DNA synthesized at high drug concentration (Fig. 1). (b) Isolation of covalently closed oligomers Quite clearly, the quantitation and characterization of the covalently closed oligomers required a satisfactory method for separating those expected to be the most numerous, i.e. the dimers. Velocity sedimentation at neutral pH obviously was inadequate since (i) it would not allow a satisfactory separation of the dimers from the monomers, nor the removal of nicked molecules, as mentioned above; (ii) it would separate cyclic double-stranded molecules on the basis of both molecular weight and number of superhelical turns per 10 base pairs, or superhelix density, u. (Bauer & Vinograd, 1968). Therefore, dimers having a u. of -0.008, similar to that of monomers synthesized in the presence of 220 pg puromycin/ml (Bourgaux & BourgauxRamoisy, 1972a), were not expected to separate satisfactorily from normal monomers with a (r. of -0.033 (Gray et al., 1971).

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Fraction no Pm. 2. Fractionation of viral DNA at pH 12.1. The [3H]thymidine-labelled preparations analysed in Fig. 1 were fractionated by sedimentation through a 6% to 20% sucrose gradient buffered at pH 12.1, as described in Materials and Methods. After neutralization of the fractions and determination of their radioactive content (-*-a-, sH-labelled DNA, no puromycin; --O--O--, 3H-labelled DNA, 10 LIP of nuromvcinlml: -m-m--, 3H-labelled DNA. 200 M of puromycin/ml), those expected ‘to con&n c&&l& olosed dimers of polyoma DNA (see horizontal bar) were pooled.

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Viral DNA was thus fractionated after sedimentation at pH 12.1 (see Materials and Methods). Under these conditions, covalently closed oligomers were expected to sediment well ahead of monomers and of most nicked molecules, with a sedimentation coeflicient dependent on CL,which varies appreciably with molecular weight only (see above). Since pH 12.1 is well below the value known to result in the complete denaturation of DNA I, covalently closed molecules were expected to renature instantaneously upon neutralization of the solution (Vinograd & Lebowitz, 1966). The fractions presumed to contain covalently closed dimers (see Fig. 2) were thus pooled and immediately neutralized. These fractions contained 3.2, 4.6 and 8*8o/o of the acidinsoluble radioactivity detected in the gradients corresponding, respectively, to the 0, 10 and 200 pg/ml puromycin samples. The value of 3.2% is very close to the observed frequency of polyoma dimers in several lytic systems (D. Blangy, personal communication). These data suggest that puromycin enhances the formation of oligomers of polyoma DNA.

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3. Velooity sedimentation of sH-labelled DNA fractionated at pH 12.1. Samples from the pools constituted as described in Fig. 2 were mixed with 14C-labelled mature viral DNA and centrifuged for 1 h at 45,000 revs/min through 6% to 20% alkaline (&-pH 12.6) sucrose solutions (see Materials and Methods). The distribution of the activities (-a--a-, 3H; --n--n--, 14C) through the gmdients was determined at the end of the run. (a) No puromycin; (b) 10 ag of puromyoin/ml; (a) 200 M of puromycin/ml. The arrow indicates the position of denatured DNA I (53 S) in the gradients. Pm.

In order to ascertain the nature of the material present in the three pools, samples were taken from these snd subjected to velocity sedimentation at pH 12.5. In all three profiles shown in Figure 3, four different radioactive bands are readily discernible. The slowest band corresponds to nicked material. The next slowest band shows that some covalently closed monomers were not excluded after sedimentation at pH 12.1. As expected from the relative amounts of monomers and oligomers, this band is least apparent in the 200 pg puromycin/ml sample. Not surprisingly, the largest band

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is found immediately ahead of the marker, at the expected position for polyoma DNA dimers (Cuzin et al., 1970). The fastest band is at the expected position for trimers and shows a shoulder on its faster side, probably indicating the presence of higher oligomers. Trimers of polyoma DNA and mitochondrial DNA have similar molecular weights. It is unlikely, however, that mitochondrial DNA contributed significantly to the trimer band, since this DNA would probably have been nicked after two successive sedimentations at alkaline pH (Jaenisch & Levine, 1972). For each gradient, we calculated the total radioactivity we presumed would correspond to covalently closed molecules of polyoma DNA. From these totals, the dimers amounted to 46%, 38% and 43%, and higher oligomers to 23%, 30% and 39% for the 0, 10 and 200 pg puromycin/ml samples, respectively. This suggests that puromycin favours the formation of all oligomers, rather than that of dimers only. (c) Superhelix density of oligomers The oligomers isolated after sedimentation at pH 12.1, as well as the corresponding unfractionated DNA preparations, were subjected to dye-buoyant density-gradient centrifugation (Radloff et al., 1967) in propidium di-iodide/caesium chloride solutions (Hudson et al., 1969). Both the unfractionated DNA and the oligomers synthesized at high inhibitor concentration formed a symmetrical band at a greater density than marker DNA I (Fig. 4(a) and (b) bottom). This indicated a difference in superhelix density (Au,) between the two DNAs of 0.026, as calculated using the equation of Eason & Vinograd (1971). This value is similar to that already obtained in another set of experiments (Bourgaux & Bourgaux-Ramoisy, 1972a). In contrast, the DNA formed at low inhibitor concentration was heterogeneous in nature, consisting of a mixture of molecules with either a normal or a low superhelix density (Fig, 4(a) and (b) centre). The latter however were 2.5 times more frequent amongst the oligomers than in the unfractionated DNA preparation. About 5% of the intact viral DNA synthesized in the absence of puromycin showed a deficiency in superhelix density (Fig. 4(a) top). Quite remarkably, these molecules were eight times more frequent in the population of oligomers (Fig. 4(b) top). Finally, two additional aspects of the results shown in Figure 4 deserve comment. First, in all preparations the population of defective molecules appeared rather homogeneous with a calculated A u0 of 0~026-&0~001. Assuming that the structure of polyoma DNA is controlled in the cell by a protein binding to it (Bourgaux BEBourgaux-Ramoisy, 1972a), this might mean that the regulation of DNA structure requires a specific amount of bound protein : for instance, one molecule of protein per molecule of DNA. Second, radioactive material banding at a position intermediate between those of intact and nicked DNAs is discernible in all preparations of oligomers. The density of this material suggests that it might consist of covalently closed template duplexes released after partial denaturation of replicating molecules (Bourgaux & Bourgaux-Ramoisy, 1972b). This is not unlikely, for such duplexes should indeed sediment faster than mature monomers, that is, almost co-sediment with mature dimers at pH 12.1. Alternatively, this material could represent catenated oligomers consisting of interlocked covalently closed and relaxed rings. The experiments reported above were repeated using 10 pg of cycloheximide/ml instead of puromycin. Results similar to those observed with 200 pg of puromycin/ml were obtained.

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FIG. 4. Dye-buoyant density-gradient centrifugation of fractionated and unfraationated DNA. Samples of the unfraotionatad preparations analysed in Fig. 1 and of the pools oonstituted aa described in Fig. 2 were mixed with “C-labelled mature viral DNA. The mixtures were dissolved in 3 ml of a ceesium ohloride solution (0.02 M-EDTA, 0.02 M-Tris.HCl, pH 8.6) of density 1.63 g/ml containing 600 ~18of propidium di-iodide/ml (Eaton & Vinograd, 1971), and centrifuged for 90 h et 22,000 revs/min and 20°C in the SB283 rotor. The distribution of the 3H (-O-O-) and l&C (--n--A--) activities through the gradients was then determined. (a) Unfractionated viral DNA; (b) oligomeric DNA. Top, no puromycin; centre, 10 pg of puromycin/ml; bottom, 200 a of puromycin/ml.

4. Discussion Our results indicate that puromycin and cycloheximide, in addition to decreasing the rate of viral DNA replication in mouse embryo cells, do induce two qualitative changes in the mature polyoma DNA which is being produced. The formation of oligomers and of molecules with an anomalous tertiary structure is enhanced in such cells, to an extent which at least in the case of puromycin, is dependent upon the dose of inhibitor used. Under circumstances such that only some of the molecules

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synthesized exhibit a low superhelix density, these molecules clearly are more frequent among the oligomers. This was also found to be the case for the DNA synthesized in the absence of inhibitor, a finding which was verified on several occasions. These observations suggest a possible relation between the occurrence of abnormalities in tertiary structures and the formation of oligomers. Using Mobius-like strips as models, Douthart (1972aJ) has shown that, with a cyclic double-stranded monomer as initial template, it is possible to account for the formation of all complex circular forms of DNA, assuming variations in the topological constraints imposed on replicating molecules. We wish to argue that in our system, viral DNA is replicating under the sort of conditions which, according to the topological model, should result in the formation of complex circular forms. Replicating polyoma-as well as SV40 (Sebring et al., 1971)-DNA is generally extracted from the cells as molecules containing two cyclic template chains (Bourgaux & Bourgaux-Ramoisy, 19723). The template duplex of such molecules is thus subjected to the kind of topological constraints which are imposed on intact mature viral DNA. For the purpose of this discussion however, it is quite pointless to take into consideration such parameters as duplex winding number (/?), superhelix winding number (T) or superhelix density (a,), since we know little about the exact secondary and tertiary structures assumed by DNA inside the cell. The topological winding number (u) of interwound chains, on the contrary, cannot vary with the environmental conditions. In addition, we have evidence suggesting that as replication of polyoma monomeric DNA proceeds, the ccof the template duplex progressively goes from a value of about 480, characteristic of mature DNA (Vinograd BcLebowitz, 1966), down to about zero, as required by the semi-conservative nature of the process (Bourgaux & BourgauxRamoisy, 1972b). Presumably, this is made possible by repeated nicking and sealing of the template chains, an operation which provides the intermittent swivel required to remove topological restraints to replication. An additional parameter is worth considering in the case of replicating molecules, namely the number of times the two replicating double-stranded branches are wrapped around each other, i.e. p (Douthart, 1972a). Actually, in the absence of a nick in the template duplex, invariance of the cc of the template chains implies that replication through one turn of the template should twist the two replicating branches one turn around one another, or less likely, be compensated for by the introduction of an additional turn in the unreplicated portion of the molecule. In spite of the fact that nicking of template chains will occur intermittently to alter these parameters, there is nothing to suggest that p should consistently be nil upon completion of replication. Figure 5 shows how the formation of oligomers could then be envisaged. Puromycin treatment causes an increase in the cc(see equation (1)) of the mature DNA being produced, presumably by altering the balance of various factors regulating the structure of replicating DNA (Bourgaux & Bourgaux-Ramoisy, 1972a). It seems likely that conditions which would increase the a of the template duplex would also tend to increase 1-1,and thereby enhance oligomer formation. Finally, I would like to comment on the molecules with a low u,, which I detected in the DNA synthesized in the absence of inhibitor. First, it should be noted that the frequency of these molecules was very high in the oligomer population as compared with their frequency-in the unfractionated population. This may be due to the fact that molecules with a low u,, may have a high probability of going through several cycles of replication in these cultures, while this is not the case in drug-treated cultures

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where DNA synthesis is strongly depressed. Second, these molecules were comparatively rare in marker DNA (see Fig. 4 top left) which had been obtained, using a similar procedure, from infected cells subjected to a different labelling regime (see Materials and Methods). This might reflect a different pattern of viral DNA synthesis at different times after infection, a possibility which is currently being investigated. Third, that some of the oligomers detected in untreated cultures had a normal uO

B

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FIQ. 5. Topological model for the formation of mature monomers, catenated dimers and circular dimers from replicating monomers. (E) and (b) Molecules 8re shown immediately before (replicated molecules) and after (mature molecules) final chain closure. Replicated molecules thus consist exclusively of two replicated double-stranded branches (A and B) which were formed by two growing points (indicated by arrows) travelling from the origin of replication (0) to 8 termination point (T). The latter is encased by an interrupted line on replicated molecules and indicated by a dot on mature molecules. (a) p is either 0 or an even multiple of 180’. Upon final chain closure, two monomers are produced in the fist instance, while in the second, the topological bond is transferred to the mature product which then consists of a catenated dimer. (b) ~1is an odd multiple of 180’. Upon final chain olosure, branches A and B are linked together by covalent bonds (see below), and a circular dimer is formed. Note that this is analogous to the partition of a circular strip with half 8 twist giving rise to a double-length strip with a full twist (Mijbius strip). (c) and (d) Detail of termixmtion points (T) at the time of chain closure. Each replicated branch consists of one template chain (1 or 2) and one growing chain (3 or 4) having opposing polarities. It is presumed that each growing point included one moving nick in one of the template chains (-I I-). Chain closure occurs as indicated by arrows. As shown in (d), the form8tion of 8 circuler dimer involves each template chain becoming connected with the daughter chain complementary to the other template chain. The formation of oligomers of higher order can also be understood on the basis of such a topological model (Douthart, 19728,b). 14

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does not rule out the topological model as a means of accounting for all complex circular forms which would be produced through a replicative process. Indeed, there is now good evidence indicating that : (1) non-replicating cyclic double-stranded DNA is often subjected in the cell to repeated nicking and sealing, resulting in changes in u,, (Eason t Vinograd, 1971; White t Eason, 1973); (2) oligomers in particular are extensively processed after synthesis (Jaenisch t Levine, 1973). I thank Mrs Monique D’Allaire for excellent technical assistance, Dr David Baltimore, Dr Franpois Lamy and Dr Claude Lech&ne for useful suggestions and discussions, Dr Jean-Paul Thirion and Dr David Gibson for critical reading of the manuscript. This work was supported by a grant from the Medical Research Council of Canada. REFERENCES Bauer, W. & Vinograd, J. (1968). J. Mol. Bid. 33, 141. Bauer, W. & Vinograd, J. (1971). In Propem in Molecular and Subcellular Biology, vol. 2, p. 181, Springer-Verlag, Berlin. Bourgaux, P. & Bourgaux-Ramoisy, D. (1971). J. MoZ. Biol. 62, 513. Bourgaux, P. & Bourgaux-Ramoisy, D. (1972a). Nature, 235, 105. Bourgaux, P. & Bourgaux-Ramoisy, D. (197%). J. Mol. Biol. 70, 399. Bourgaux, P., Bourgaux-Ramoisy, D. & Seiler, P. (1971). J. Mol. Biol. 59, 195. Brack, C., Delain, E. & Riou, G. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 1642. Cuzin, F., Vogt, M., Dieckmann, M. & Berg, P. (1970). J. Mol. BioZ. 47, 317. Douthart, R. J. (19’72a). J. Theoret. BioZ. 35, 315. Douthart, R. J. (19726). J. Theoyet. BioZ. 35, 337. Eason, R. & Vinograd, J. (1971). J. ViroZ. 7, 1. Goebel, W. (1970). Eur. J. Biochem. 15, 311. Goebel, W. (1971). B&him. Biophys. Acta, 232, 32. Goebel, W. BEH&n&i, D. R. (1968). Proc. Nat. Acd Sci., U.S.A. 61, 1406. Gray, H. B., Jr, Upholt, W. B. & Vinograd, J. (1971). J. Mol. BioZ. 62, 1. Hudson, B. & Vinograd, J. (1967). Nature, 216, 647. Hudson, B., Clayton, D. A. & Vinograd, J. (1968). CoZd Sp. Harb. Symp. Quant. B&Z. 33, 435.

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Jaenisch, R. & Levine, A. (1971). Virology, 44, 480. Jaenisch, R. & Levine, A. (1972). Virology, 48, 373. Jaenisch, R. & Levine, A. (1973). J. Mol. BioZ. 73, 199. Meinke, W. & Goldstein, D. A. (1971). J. Mol. BioZ. 61, 643. Naas, M. M. K. (1969). Nature, 223, 1124. Nass, M. M. K. (1970). Proc. Nat. Acad. Sk, U.S.A. 67, 1926. Radloff, R., Bauer, W. & Vinograd, J. (1967). Proc. Nat. Acud. Sci., U.S.A. 57, 1514. Sebring, E. D., Kelly, T. J., Jr, Thoren, M. M. & Salzman, N. P. (1971). J. Viral. 8, 478. Vinograd, J. & Lebowitz, J. (1966). J. Gen. Phyaiol. 49, 103. Vinograd, J., Lebowitz, J., Radloff, R., Watson, R. & Laipis, P. (1965). Proc. Nut. Ad. Sci., U.S.A. 53, 1104. Vinograd, J., Lebowitz, J. & Watson, R. (1968). J. Mol. BioZ. 33, 173. White, M. & Eason, R. (1973). Na;ture New Biol. 241, 46.