Proc. Nat. Acad. Sci. USA Vol. 71, No. 9, pp. 3545-3549, September 1974

Packaging and Maturation of DNA of T7 In Vitro (morphogenesis/in vitro complementation) CAROL KERR AND PAUL D. SADOWSKI Departments of Pathology and Medical Genetics, University of Toronto, Toronto, Canada M5S1A8 Communicated by William B. Wood, June 28, 1974

ABSTRACT We have developed an in vitro complemen- relatively simple for which there is a great deal of in- tation assay to demonstrate packaging and maturation formation about its genetics and physiology (25). The DNA ofDNA ofphageT7. Cells ofEscherichia coli B infected with an appropriate T7 amber mutant are concentrated 200- of phage T7 is a unique, terminally redundant molecule with fold and iysed by freezing and thawing. Two extracts from a molecular weight of 2.6 X 107 (25). The DNA replicates as cells infected with different amber mutants are mixed and structures many times longer than this (14, 15, 21) and, hence, incubated at 300. Positive complementation results in a must be matured during packaging. Because the DNA is 100-fold increase in phage titer. Using this assay we have demonstrated the packaging of terminally redundant, this extra DNA must be preserved phage DNA from an extract that contains no phage heads during DNA maturation. Thus, the mechanism of matura- ( 9-, 10-), within head structures present in an ex- tion of T7 DNA might differ from that of a unique, "sticky- tract that contains no phage DNA (gene 5-). We have also ended" DNA such as that of phage lambda. demonstrated an activity in extracts that contain no phage DNA or heads (gene 5-,9, 10-), which complements The formation of the phage head requires the function of gene 19-infected cells. We have proven that this activity six located in two groups on the T7 map (Fig. 1). The is due to the gene-19 product by showing that the activity gene 10 protein is the major head protein and the products of is temperature-sensitive if the extract is made from cells genes 8, 14, 15, and 16 are also structural components of the infected with a mutant having a temperature-sensitive head. The gene 9 protein is present in unfinished head-like mutation in gene 19. This assay should be useful in eluci- dating the mechanism of packaging and maturation of structures ("proheads") but not in DNA-containing heads DNA of phage T7. (25). It may thus act as a scaffold around which the head is assembled (26). Mutants defective in genes 18 and 19 fail to In recent years there has been rapid progress in our under- mature their DNA, and yet the products of these genes do not standing of the mechanisms by which complex appear to be structural components of the head (25). These are assembled. Thus, for bacteriophage T4, we know a great gene products may thus play some catalytic role in DNA deal about the pathways of assembly of tail fibers (1-4), base- maturation. An endonuclease and an exonuclease, the prod- plates, sheaths, and cores of the tail (5-9) and assembly of the ucts of genes 3 and 6, respectively, have also been implicated head (10). These advances have been aided greatly by the in T7 DNA maturation (27). development by Edgar and Wood of methods for performing The synthesis of T7 phage structural proteins does not these reactions in cell-free extracts (11, 12). depend upon prior replication of the T7 DNA. Thus, head- The DNA of several bacteriophages (T4, T7, X, and P22) like structures are formed even in the absence of phage DNA has been shown to replicate as molecules that are several times replication (25). This observation suggests that it may be longer than the DNA found inside the head of the mature possible to uncouple head formation from DNA replication virus (13-17). It is also known that maturation of these long and to use such structures to study DNA packaging and chains of DNA to monomeric units is closely coupled to the maturation. formation of the phage head, since cells infected with mutants In this paper we report the development of an efficient that are defective in head formation accumulate these long in vitro complementation assay which demonstrates that molecules of immature DNA (18-21). packaging and maturation of T7 DNA can occur in a cell- However, our knowledge of the molecular mechanisms free system. whereby the immature phage DNA enters the head ("DNA packaging") and is cleaved to monomeric length ("DNA MATERIALS AND METHODS maturation") has been limited, in part, by the inability to Bacterial and Phage Strains. B and E. coli perform these reactions in vitro. Recently, however, assays 011' were used as the nonpermissive and permissive strains, have been developed to demonstrate packaging of phage respectively. The atnber mutants used in this study were all DNA in vitro. Kaiser and Masuda (22) showed that ex- from the collection of F. W. Studier (28) and include the ogenous lambda DNA could be packaged by extracts from following: gene 1-am193, gene 5-am28, gene 9-aml7, gene induced lysogens and Hohn and Hohn (23) have demon- 10-aml3, gene 19-amlO. Phage bearing multiple amber muta- strated that head-related particles containing no DNA tions were constructed by standard phage crosses and were ("petit lambda") can be filled with DNA in vitro. Finally, the identified by complementation tests (28). A temperature- experiments of Pruss, Goldstein, and Calendar (24) showed sensitive mutant in gene 19 (ts10) was obtained from F. W. that the size of the head of phage P2 is determined solely by Studier. Phage stocks were grown on E. coli 011', as de- the proteins involved and not by the phage DNA. scribed (29), and were concentrated by differential centrifuga- Bacteriophage T7 may be a convenient system in which tion. The phage lysates were not treated with chloroform, to study DNA packaging and maturation because it is a and phage were not concentrated with polyethylene glycol 3545 Downloaded by guest on September 23, 2021 3546 Biochemistry: Kerr and Sadowski Proc. Nat. Acad. Sci. USA 71 (1974)

Head proteins

Scaffolding protein I II | Major head protelni I I II I I I I T II|I 0.3 0.7 4 1.1 1.3 1.7 2 I) I 4 11 12 13 * 5®7(DbO 69.1.17 i 1 I I I 1 I Exo I I 1 I I I I 1 I I DNA I 1 I I polymerase : DNA Ligase 1 I I I ~ ~~~~I I maturation 1 I I DNA defective ' I 1 I I RNA 1 I I 1 I LyFseDzyme a I I polymerase 1 1 I 1 Endo Tail proteins DNA defective FIG. 1. Genetic map of phage T7. The "T" indicates the termination signal for early transcription and separates the early and late regions of the . The gene numbers that are circled indicate genes that have been implicated in DNA maturation. (30). Phage-infected cells that had been made with phage completed after 1 hr at 300. If was used, the reac- treated by either method gave very inefficient complementa- tion was supplemented with 20 mM MgSO4. tion in vitro. RESULTS Media. LB broth contains 10 g of bacto-tryptone, 5 g of In Vitro Complementation by Extracts of Mutant-Infected yeast extract, and 5 g of NaCl per liter, and 2 X LB broth Cells. We were interested in developing an in vitro complemen- has been described (29). T7 diluent contains 20 mM Trism HCl tation system capable of demonstrating the packaging of (pH 7.4), 10 mM MgSO4, 10 ug/ml of gelatin, and 0.1 M DNA and the maturation of DNA in bacteriophage T7. To NaCl. Concentrated phage stocks were stored in T7 diluent demonstrate DNA packaging, we measured infectious phage containing 1 M NaCl. formation when an extract from a nonpermissive host infected Preparation of Phage-Infected Cell Extracts. An overnight with a mutant that makes no heads (9-, 10-) but makes DNA, culture of E. coli B was grown in LB broth from a slant that was incubated with an extract prepared from nonpermissive was less than 1 month old and that had been stored at room cells infected with a mutant that makes no DNA (5-) but temperature. A 50-fold dilution of the culture was made into makes head structures normally. As shown in Table 1, a LB broth, and the cells were shaken at 300 until the cell 20-fold increase in phage titer was observed over that found density reached 5 X 108/ml. The cells were infected with the with the 5- extract or 9-, 10- extract alone. The genotypes of appropriate amber mutant at a multiplicity of infection of the phage produced during the complementation test in vitro 20; greater than 99% of the cells are killed at this multi- TABLE 1. Complementation between extracts of T7 mutant- plicity. Shaking was continued at 300, and 15-20 min after infected cells infection the culture was poured into a cold (-20°) flask and was then immediately centrifuged at 1500 X g,0. for 5 min. Extracts The cell pellet was resuspended in T7 diluent (1/200 the mixed Phage titer (X 10-') Genotype (%) original culture volume), and the cells were disrupted by freezing and thawing. The cells were frozen thoroughly in a 9-,10- + 5- 123 9-, 10- (100) dry ice-acetone bath, incubated 5 min at 00, thawed quickly 19- + 5- 208 19- (99); 5 (1) the 19- + 9-,10- 133 19- (75); 9-,10- (25) at 300, and stored at 00. In some experiments resuspended 1-+ 19- 6 cells were treated with 0.02 volume of lysozyme solution 1- 6 [Worthington; 1 mg/ml in 15 mM Tris * HC1 (pH 7.4), 7.5 mM 5- 2 MgSO4, 0.25 M EDTAJ. For convenience, an extract from 9-,10- 6 cells infected with a given amber mutant is referred to by the 19-. 2 number of the gene carrying the mutation; i.e., a "5- extract" refers to an extract prepared from cells infected with an E. coli B cells were infected with the appropriate phage, and amber mutant in gene 5. infected cells were harvested after 15 min at 300, concentrated 200-fold in T7 diluent, and disrupted by freezing and thawing. In Vitro Complementation. Complementation tests were Twenty-microliter volumes of each extract were mixed and performed by mixing equal volumes (20 ul each) of the ex- incubated at 300 for 1 hr. The assay was terminated by dilution tracts to be tested and incubating for 1 hr at 30°. The reac- and the phage titer was determined with E. coli 011'. The geno- tions were terminated by dilution with T7 diluent, and the types were determined on at least 100 plaques by complementa- phage titer was determined. Phage production is largely tion tests. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) T7 DNA Packaging and Maturation 3547

were determined by complementation tests. All the progeny for transcription of late T7 genes (25). This observation shows were of the 9-, 10- genotype, confirming that the DNA pres- that late protein synthesis is necessary for complementation ent in the 9-, 10- extracts had been packaged in the head to occur. Finally, in reconstruction experiments, uninfected structures in the 5- extracts. E. coli B cells were treated with lysozyme, followed by freeze- The gene 19 protein is involved in maturation of phage thawing, and then "infected" singly with a 19- phage, a 5-, DNA but is not a structural component of the phage head. 9-, 10- phage, or with both phages at multiplicities of either Gene 19- extracts contain concatameric DNA'(21) and a 1 phage or 10 phages per cell. There was no evidence of com- head-related particle that contains no DNA and is similar plementation, indicating that uninfected cells treated in this to particles present in T7+ or gene 5- extracts (unpublished way are inca,pable of supporting complementation by phage. observations). We therefore attempted to detect an activity in 5- extracts that would complement 19- extracts. As shown Further Characterization of the Gene 19 Complementing in Table 1, mixing of these two extracts resulted in a 100-fold Activity. We have studied several facets of the gene 19 com- increase in phage titer above that present in each extract alone plementing activity. We first investigated the kinetics of Furthermore, all of the progeny phage were determined to be appearance of the activity of the acceptor cells (19-) and of the 19- genotype, consistent with the fact that gene 19 of the gene 19 complementing activity present in the donor mutants produce phage DNA, whereas gene 5- mutants do cells (5-, 9-, 10-). The optimal acceptor activity of the 19- not. Similar results were Qbtained when 5 , 9- or 5, 9, infected cells was present 15-20 min after infection at 300, 10- extracts were used to complement gene 19- extracts, and its appearance was inhibited by addition of chloram- thus suggesting that the complementing extract is not donat- phenicol at the time of infection (Fig. 2a). These kinetics are ing some head-related structure in addition to the gene 19 consistent with the necessity for the synthesis of late proteins product. These results show that the gene 19 product can for the activity of the gene 19- acceptor cells (25). act in trans to promote packaging of DNA from another The maximal gene 19-complementing activity induced after extract. infection with a 5-, 9-, 10- mutant is present after 15-25 Positive complementation was also observed on mixing a min (Fig. 2b), and its appearance is also inhibited by chlor- 9-, 10- extract with a 19- extract (Table 1). The genotypes amphenicol. This result shows that the kinetics of appearance of the progeny were 75% 19- and 25% 9-, 10-. This result of the gene 19 complementing activity are those of a late pro- shows that the head-related particle in gene 19- extracts also tein as well (25). can act in trans to package the DNA from another extract It was important to demonstrate that the complementing in addition to the DNA present in the 19- extract. activity being observed was, in fact, due to the gene 19 pro- Positive complementation using this assay usually results tein. To this end the thermal stability of this activity in ex- tracts prepared from gene was in a phage titer in excess of 1011 per ml, and sometimes as high 19+-infected cells compared with that in extracts prepared from cells that had been in- as 1012 per ml. Since the concentration of the infected cells fected with a in gene 19. Both before freezing and thawing is 1011 per ml, this result indicates temperature-sensitive mutant phages also contained an amber mutation in gene 9. Each that an average of 1-10 phage are being produced per original infected cell. In order to gain some idea of the efficiency of the extract was incubated at 38° for various lengths of time and then chilled at 0°. The gene 19 complementing was assay, we infected cells with T7+ phage; harvested the cells activity 15 after infection at 300, then determined by mixing each extract with a 19- extract. min concentrated them 200-fold, The results (Fig. 3) and disrupted them by freezing and thawing. The cell ex- showed that the complementing activity from cells infected was more tract was incubated for 1 hr at 300, and the phage titer was with a l9ts mutant much thermo- then determined. A titer of 5 X 1012/ml was obtained, indi- labile than that from cells infected with a gene 19+ mutant. cating the production of about 50 phage per infected cell. Thus we concluded that the gene 19 complementing activity (Normally, if lysis is allowed to occur, T7 produces a burst was in fact due directly to the gene 19 protein. of from 100-300 phage per cell.) This finding shows that The requirements of the gene 19 complementing activity phage production by complementing cell extracts has an are shown in Table 2. There is an absolute requirement for efficiency of 2-2Q% of the phage production observed in ex- Mg++ ions, with an optimum of 20-30 mM. The reaction is tracts from T7+-infected cells. completely abolished by the presence of pancreatic DNase. This finding shows that the DNA present in the extract is Several experiments were done to confirm that the above results were indicative of true in vitro complementation and either not within a phage head or that the head is still perme- able to a not due to some artifact. The possibility that intact phage DNase. Ribonuclease had slight inhibitory effect, might be infecting residual intact cells and producing progeny the significance of which is uncertain. There is little effect by in vivo complenientation was excluded by finding that (i) of triphosphates, a sulfhydryl reagent, and spermidine in similar results were obtained when the assay was performed this reaction (Table 2), although we can not exclude the in the presence of chloramphenicol (100 ,ug/,ml) and '(ii) the possibility that many necessary factors are present within the frequency of wild-type recombinants among progeny pro- extracts in high enough concentration so that they need not duced during these reactions was very low (less than 0.5%), be added exogenously.. whereas dual infection of intact cells by the above mutants The acceptor activity of the 19-infected cells is stable for would give much higher recombination frequencies (28). We up to one month if the tells are stored as a cell pellet at -90° were also able to show that complementation was not simply or -170°. The gene 19 complementing activity present in due to the addition of nonspecific factors present in a cell gene 5-, 9--infected cells is stable for several months if the extract, since a gene 1--extract did not complement a gene cells are stored at -200. 9, 10-, or 5- extract (Table 1). Gene 1 is the structural Complementation with Three Cell Extracts. In order to dis- gene for a T7-specific RNA polymerase that is responsible sect the packaging and maturation reaction further, we at- Downloaded by guest on September 23, 2021 3548 Biochemistry: Kerr and-Sadowski Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 2. Requirements of the gene 19 complementing activity Units per ml of 5-, Reaction mixture 9 extract Complete 1640 0~~~~~~~~~~~~~~~~~~~~~01 Complete minus Mg++ 480 Complete minus Mg++ plus EDTA 0 Complete plus dithiothreitol 2350 0 U. Complete plus pancreatic DNase 0 Complete plus pancreatic RNase 516 Complete plus 4 deoxyribotriphosphates 1000 Complete plus 4 ribotriphosphates 1680 Complete plus ATP 570 0 10 +CAI20 0 10 CA',20 30 Complete plus spermidine 1220 -Minutes after Infection f+ FIG. 2. Appearance of gene 19 acceptor activity (a) and gene Gene 19--infected E. coli B cells, harvested 15 min after infec- 19 complementing activity (b) after infection. E. coli B cells were tion and stored at - 1700, were suspended in T7 diluent, and infected with gene 19- phage (a) or gene 5-, 9, 10- phage (b). 0.02 volume of lysozyme (1 mg/ml) was added. A 5 , 9 extract At various times after infection, 40-ml aliquots were chilled and was prepared in a similar manner. The complete reaction mixture centrifuged. The cells were suspended in T7 diluent, treated contained 20 ,d1 of 19- extract, 1 ,sl of 5- 9 extract, and 10 mM with lysozyme, and disrupted by freezing and thawing. To one MgSQ4. Where indicated, the following additions were made: portion of cells, 100.ug/nil of chloramphenicol (CA) was added 20 mM EDTA, 1 mM dithiothreitol, 8 ,ug/ml of DNase, 16 before addition of the phage. The gene 19 acceptor activity at jug/ml of RNase (preheated to 100° for 15 min), 50 ,uM four various times was determined by mixing the 19- extracts with an deoxyribotriphosphates, 50 MM four ribotriphosphates, 1 mM equal volume of 5-, 9P, lo- extract made from cells harvested ATP, 5 mM spermidine. The reaction mixtures were incubated for 15 min after infection (a). The gene 19 complementing activity 60 min at 300, and the phage titers were determined. Blank values at various times was determined by mixing the 5-, 9-, 10- were subtracted, and the activity was expressed as units/ml of extracts with a 19- extract made from -cells harvested 15 min 5, 9- extract added. One unit of activity is the production after infection (b). All reactions contained 20 mM MgSO4. of 109 plaque-forming units in the reaction after subtraction The results are expressed as the -fold increase in phage titer over of the blank. background level. The maximum phage titers were: (a) 3.4 X 101l/ml; (b) 9.5 X 1010/ml. but should contain the 19 protein; a 5-, 19- extract should contain no DNA or 19 protein but should contain a precursor head; and a 9-, 19- extract should contain neither heads nor tempted to obtain complementation among three extracts, the gene 19 protein but should contribute DNA. As can be each of which should contribute a to the unique component seen in Table 3, efficient complementation is observed when reaction. A 5-, 9- extract should contribute no DNA or heads the three extracts are mixed in the appropriate proportions. These results show that purification of some of the components of the packaging reaction (head precursor, DNA, and gene 19 protein) from the appropriate mutant-infected extract (or 100 wild type-infected cells).may be feasible. DISCUSSION

4C We have described an in vitro complementation system to demonstrate that packaging and maturation of T7 DNA can occur in cell-free extracts. The system is highly efficient in that the number of phage produced is at least 1% of that produced when cell-free extracts made from T7+-infected TABLE 3. In vitro complementation with a 5-, 9- extract, a 9, 19ts 5-, 19- extract, and a 9-, 19- extract

10. titer 0 10 20 30 Extracts added Phage per ml (X 10')

Minutes at 38 5, 9- + 5-, 19-, +9, 19- 320 FIG. 3. Temperature sensitivity of the gene 19 comple- 5-, 9- + 5-, 19- 1 menting activity in a l9ts extract. The extracts were prepared 5-, 9- + 9-, 19- 3.8 from infected cells, as described in the legend of Fig. 2. The 5-, 19- + 9-, 19- 3.9 5-, 9- 1.7 9-, 19+ and 9 , l9ts extracts were incubated at 380; aliquots were withdrawn after various times and chilled on ice. The gene 19 5-, 19- 2.8 complementing activity was determined as in the legend of Fig. 2 9 , 19- 8.7 in the presence of 20 mM MgSO4. The results are plotted as the percent maximum activity remaining (on a logarithmic scale) Phage-infected E. coli B were prepared as described in Ma- against time of incubation of the extract at 380. The 100% terials and Methods and disrupted by freezing and thawing. The activities present at 0 mm" were: 9, 19+, 2.3 X 1011 phage per ml; amounts of extracts added were 10 A of 5 , 9 , 10 Ml of 5 , 19-, 9, l9ts, 1.3 X 1011 phage per ml. and 5 A of 9 , 19-. Incubation was for 60 min at 300. Downloaded by guest on September 23, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) T7 DNA Packaging and Maturation 3549

cells are permitted to produce mature phage in vitro. The 2. Ward, S., Luftig, R. B., Wilson, J. H., Eddleman, H., Lyle, number of phage produced by two complementing. cell ex- H. & Wood, W. B. (1970) J. Mol. Biol. 54, 15-31. 3. King, J. & Laemmli, U. K. (1971) J. Mol. Biol. 62, 465-477. tracts in vitro is also about 1% of that produced by T7 phage 4. Ward, S. & Dickson, R. C. (1971) J. Mol. Biol. 62, 470-492. in vivo. The reaction requires added Mg++ ions and is in- 5. King, J. (1968) J. Mol. Biol. 32, 231-262. hibited by pancreatic DNase, but does not require the addi- 6. Meezan, E. & Wood, W. B. (1971) J. Mol. Biol. 58, 685-692. tion of ATP or spermidine, as does the packaging assay for 7. King, J. (1971) J. Mol. Biol.. 58, 693-709. 8. King, J. & Laemmli, U. K. (1973) J. Mol. Biol. 75, 315-337. exogenous lambda DNA developed by Kaiser and Masuda 9. King, J. & Mykolajewycz, N. (1973) J. Mol. Biol. 75, 339- (22). Determination of what additional soluble factors are 358. required for the reaction will be aided by the purification of 10. Laemmli, U. K. & Favre, M. (1973) J. Mol. Biol. 80, 575- the various active components of the system. 599. This assay may be exploited in several possible ways. It 11. Edgar, R.- S. & Wood, W. B. (1966) Proc. Nat. Acad. Sci. USA 55, 498-505. may be used to purify intracellular head precursor particles 12. Edgar, R. S. & Lielausis, I. (1968) J. Mol. Biol. 32, 263-276. ["proheads" (26)1, which are involved in the packaging re- 13. Frankel, F. R. (1968) Proc. Nat. Acad. Sci. USA 59, 131- action in vitro. Such particles are made in vivo when a non- 138. permissive host is infected with a mutant in which no DNA 14. Kelly, T. J. & Thomas, C. A., Jr. (1969) J. Mol. Biol. 44, 459-475. is synthesized (25), and we have begun to purify them from 15. Schlegel, R. A. & Thomas, C. A., Jr. (1972) J. Mol. Biol. 5- extracts using the in vitro complementation assay to moni- 68, 319-345. tor the recovery. 16. Enquist, L. W. & Skalka, A. (1973) J. Mol. Biol. 75, 185- The assay may- also be used to follow the purification of 212. proteins (e.g., nucleases or other factors) that are involved in 17. Botstein- D., Waddell, C. H. & King, J. (1973) J. Mol. Biol. 80, 669-695. the DNA maturation event. We have shown in this paper 18. MacKiinlay, A. G. & Kaiser, A. D. (1969) J. Mol. Biol. 39, that the assay can be used to detect the gene 19 protein, and 679-683. we are currently using the assay to follow the purification of 19. McClure, S. C. C., MacHattie, L. M. & Gold, M. (1973) this product. The assay may also be useful for purification of Virology 54, 1-18. 20. Frankel, F. R. (1968) Cold Spring Harbor Symp. Quant. other factors involved in the DNA maturation, for example, Biol. 33, 485-493. the gene 18 product. The experiment shown in Table 3 sug- 21. Hausman, R. & La Rue, K. (1969) J. Virol. 3, 278-281. - gests that the assay may be used to purify the forms of intra- 22. Kaiser, D. & Masuda, T. (1973) Proc. Nat. Acad. Sci. USA cellular phage DNA that are active in the packaging reaction. 70, 260-264. We have not attempted to show complementation 23. Hohn, B. & Hohn, T. (1974) Proc. Nat. Acad. Sci. USA 71, between 2372-2376. genes controlling head proteins (e.g., genes 8, 9, 10, 14, 15, 24. Pruss, G., Goldstein, R. N. & Calendar, R. (1974) Proc. 16) in vitro, although it is possible that the assay could be Nat. Acad. Sci. USA 71, 2367-2371. adapted to study the steps in head formation in T7. 25. Studier, F. W. (1972) Science 176, 367-376. 26. King, J., Lenk, E. V. & Botstein, D. (1973) J. Mol. Biol. 80, 697-731. This work was supported by a grant from the Medical Re- 27. StrAtfing, W., Krause, E. & Knippers, R. (1973) Virology search Council of Canada. P.S. is a Scholar of the Medical 51, 109-119; Research Council. We thank Drs. L. Siminovitch, M. Pearson, 28. Studier, F. W. (1969) Virology 39, 562-574. and A. Becker for their reading of the manuscript and Mary Jo 29. Sadowski, P. D., McGeer, A. & Becker, A. (1974) Can. J. Holland for technical assistance. Biochem. 52,. 525-535. 30. Yamamoto, K., Alberts, B. M., Benzinger, R., Lawhorne, 1. King, J. & Wood, W. B. (1969) J. Mol. Biol. 39, 583-601. L. & Treiber, G. (1970) Virology 30, 734-744. Downloaded by guest on September 23, 2021