VIROLOGY
93, 578-581
Three Different Oncogenic
(1979)
Classes of Human Adenovirus Transforming DNA Sequences: Highly Subgroup A-, Weakly Oncogenic Subgroup B-, and Subgroup C-Specific Transforming DNA Sequences
KEI FUJINAGA,’ Department
of Molecular
Biology,
YUKIHARU
SAWADA, AND KENJI SEKIKAWA
Cancer Research Institute, Accepted November
Sapporo Medical
College, Sapporo 060, Japan
8, 1978
DNA-DNA homology measurements showed the presence of three different classes of human adenovirus transforming gene sequences: one specific for highly oncogenic subgroup A (types 12, 18, and 31), the second specific for weakly oncogenic subgroup B (types 3, 7, and 16), and the third specific for transforming subgroup C (types 2, 5, and 6). Adenovirus transforming DNA sequences with molecular weights of 1.6-1.8 x lo6 are in common or almost identical among members within the same subgroup. Less than one-third of the transforming DNA sequences are shared among members of different subgroups. All of the DNA fragments containing transforming DNA sequences investigated are located at the end of the molecule.
Recent studies have revealed that the transforming activity of adenoviruses (Ad) resides in a small left-hand region of the viral DNA molecule, the transforming segment. Cells can be transformed by isolated transforming segments, HindIII-G of Ad2 and Ad5 (l), HindIII-1. J of Ad7 (Z), and HindIII-G of Ad12 (3). These transforming segments have molecular weights of 1.6-1.8 x lo6 and coding capacities for only one or two kinds of proteins which are required for cell transformation (4). It is important to examine the homology or heterology of the DNA sequences of the transforming segments of adenoviruses which may code proteins required for transformation of cells. We recently showed that most, if not all, of the nucleotide sequencesof the Ad12 transforming segment, HindIII-G, were also present in viral DNAs of Ad18 and Ad31, other members of the highly oncogenic subgroup A (5). In this study, we carried out further detailed DNA-DNA homology measurements using labeled Ad12 HindIII-G (subgroup A), Ad7 HindIII-I* J (subgroup B), and Ad5 HindIII-G (subgroup C) to compare transforming gene sequences among members of highly onco1 To whom reprint requests should be addressed. 0042~6822/79/040578-04$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
578
genie subgroup A, weakly oncogenic subgroup B, and transforming subgroup C. Filter and liquid hybridization experiments using whole viral DNA (4) showed that viral genomes within each group are closely related, sharing 55-100% of their base sequences, and those of different groups share only 5-28% of their sequences. Heteroduplex analysis of different adenovirus DNAs (6) led to a conclusion similar to the above. However, no direct comparison among transforming gene sequences of adenovirus had been carried out. Viral DNAs from Ad12 (Huie), Ad18 (D.C.), Ad31 (1315), Ad3 (G.B.), Ad7 (Grider), Ad16 (ch 79), Ad2 (38-2), Ad5 (Adenoid 75), and Ad6 (Ton 99) were prepared as described (7, 8). Seed virus cultures were obtained from Dr. M. Green (Ad12, Ad7, Ad5, and Ad2) and from Dr. Hiroto Shimojo (Ad18, Ad31, Ad3, Ad16, and Ad6). Transforming DNA segments of Ad12 (HindIII-G), Ad7 (HindIII-1. J), and Ad5 (HindIII-G) were isolated and purified from viral DNAs, labeled in vitro by nick translation as described (9, 10). Hybridization experiments using membrane-immobilized DNA were performed by the procedure described previously (11) with minor modifications. Alkali-denatured DNA in
SHORT COMMUNICATIONS
4x SSC (SSC: 0.15M NaCl + 0.015 M sodium citrate) was passed three times through a nitrocellulose membrane filter (Millipore HA) and dried at room temperature for 4 hr or overnight and for 2 hr at 80” in a vacuum oven. Membrane-bound DNA was hybridized with labeled DNA in 4x SSC containing 0.1% SDS for 40 hr at 60”. After hybridization, membrane filters were washed with 3 r&f Tris-HCl buffer, pH 9.2, repeatedly by shaking. Filters were dried and radioactivity was determined in a scintillation counter. It has previously been shown in our experiments that labeled Ad12 HindIII-G hybridized with DNAs from Ad18 and Ad31, members of the highly oncogenic subgroup A, as efficiently as with Ad12 DNA (5). On the other hand, as shown in Table 1, only a small portion (ll-20%) of the labeled Ad12 HindIII-G hybridized with excess amounts of DNAs from members of other subgroups, Ad3, Ad7, Ad16, Ad2, Ad5, and Ad6. Similar results were obtained with the hybridization experiments between labeled
579
Ad7 HindIII-I *J or labeled Ad5 HindIII-G and DNAs from different adenoviruses. The above results show the presence of the common and unique transforming DNA sequence specific for each of the three subgroups, highly oncogenic subgroup A, weakly oncogenic subgroup B, and transforming subgroup C, respectively. Only one-third or less of the transforming DNA sequences are shared by members of different subgroups. In order to find the portions of various adenovirus DNAs homologous to the transforming DNA sequences of Ad12, Ad’7, or Ad5, hybridization experiments using the Southern technique were carried out with minor modifications. Viral DNA was digested with restriction endonuclease Hind111 and viral DNA fragments were separated by 1% agarose gel electrophoresis for 11 hr at 40 V as described (12). After staining DNA bands with 0.5 pg/ml of ethidium bromide, the gel was soaked in a denaturing solution containing 0.5 1MNaOH and 1.5 M NaCl for 30 min at room temperature to denature
TABLE 1 HYBRIDIZATION OF ADENOVIRUS TRANSFORMING DNA SEGMENTS WITH VARIOUS ADENOVIRUS DNAP
Labeled transforming DNA segment6 Viral DNA (1 pg/filter)
Ad12 HindIII-G
Subgroup A Ad12 Ad18 Ad31
100” 103’ 97”
Subgroup B Ad 3 Ad 7 Ad16 Subgroup C Ad 2 Ad 5 Ad 6
Ad7 HindIII-1.J
Ad5 HindIII-G
16 16 -
9 16 9
20 12 15
112 100 96
23 28
11 12 14
29 32 -
98 100 103
DAverage of duplicate hybridization experiments. Background (3-5% of the input) was subtracted, and binding to homologous viral DNA was normalized to 100%. Hybridization reaction was carried out in 4x SSC, 0.1% SDS for 40 hr at 60” as described in the text. * Transforming DNA segments were labeled with 32Pby nick translation. Input radioactivities of Ad12 HindIII-G, Ad7 HindIII-I-J, and Ad5 HindIII-G were 1000 cpm (1.37 x 10’ cpm/pg), 806 cpm (8.7 x lo6 cpmlpg), and 800 cpm (4.2 x lo6 cpm/pg), respectively. e Data from Ref. (5).
580
SHORTCOMMUNICATIONS
DNA fragments. For neutralization, the gel was soaked in a neutralizing solution containing 3 M NaCl and 0.5 M Tris-HCl, pH 7.2, for 15 min at 0”. The denatured DNA fragments were transferred to a nitrocellulose membrane filter (Millipore HAWP) by the Southern technique (13). The DNA membrane filter containing DNA was dried for 2 hr or overnight (or for 3 min in a vacuum oven at SO’), folded in a cylindrical shape, and placed in a siliconized test tube. After heating at 80” in a vacuum oven, the DNA membrane was immersed in a hybridization solution containing 0.6 M NaCl, 0.2 M Tris-HCl, pH 7.9, 0.02 M EDTA, 0.5% SDS, 50% formamide (14), and labeled DNA probe, and incubated at 37” for 40-48 hr in a circular rotating machine. The filter was washed four times with 2 x SSC + 50% formamide for 15 min in a shaker, washed two times with 2x SSC, dried, and autoradiographed using Kodak XRPl X-O mat film with or without intensifying screen (DuPont Chronex Lightning-plus). The results of the Southern hybridization using Ad12 HindIII-G, Ad7 HindIII-I* J, and Ad5 HindIII-G as probes are shown in Fig. 1. Labeled Ad12 HindIII-G hybridized with the HindIII-C fragment of Ad18 (10% at the end; Sawada and Fujinaga, unpublished) and with the HindIII-G fragment of Ad31 (7% at the end; Sawada and Fujinaga, unpublished). Labeled Ad7 HindIII-1. J hybridized with the HindIII-1.J fragment of Ad3 located on the left 8% of the molecule (15). Labeled Ad5 HindIII-G hybridized with the HindIII-G fragment of Ad2. No significant hybridizations were detected between the above transforming DNA segments and the other DNA fragments on the filter. The above results show that all of the transforming gene sequences locate within a region at the end 10% of the viral DNA molecule. We detected only a small region of the transforming DNA sequences shared in common by viral DNAs from members of three different subgroups. These results suggest that different virus-coded transforming proteins are involved in carcinogenesis by these three subgroups of human adenoviruses. Recently, however, a weak
Subgroup
A
Ad 12 HinduI-G Ad I8 Hind III-C Ad31 Hirdlll-G subgroy,
B
Ad 3 Ad
Hindlll-
7
I.J
Hind Ill- I, J
subgroup c Ad 2 Hindlll-G
Ad 5 Subgroup
Hindlll-G A Ad 12 Ad18 Ad31
Subgrwp
B Ad 3 Ad7
Subgroup
C Ad 2
Ad 5
FIG. 1. Hybridization of labeled transforming DNA segments with Hind111 fragments from different viral DNAs by the Southern technique. HinIII fragments of viral DNAs were resolved by electrophoresis in 1.0% agarose gels, denatured in situ, and transferred to nitrocellulose membrane filters by the technique of Southern (13) as described in the text. The filters, containing 2 pg of each viral DNA, were incubated with lo5 cpm of nick-translated [32P]DNA fragments in a hybridization solution containing 50% formamide for 40 hr at 37”, washed, and exposed to X-ray Film as described in the text (A). A photograph of DNA fragment bands stained with ethidium bromide, as observed in the gel prior to transfer, is presented also (B). Viral DNAs from Ad12, Ad18, and Ad31 were hybridized with the labeled HindIII-G fragment of Adl2. Viral DNAs from Ad3 and Ad7 were hybridized with the labeled HindIII-1. J of Ad7. Viral DNAs from Ad2 and Ad5 were hybridized with the labeled HindIII-G of Ad5.
common antigenicity was found between T antigens of Ad7 and Ad12 (Shiroki et al., in press), and we should note that a distant relationship of DNA sequences does not always reflect distantly related proteins coded. Direct sequencing of the transforming DNA segments and detailed investigation of the proteins coded by them will provide definitive conclusions about the
SHORT COMMUNICATIONS
virus-coded genetic information involved in cell transformation by these three different subgroups of human adenoviruses. ACKNOWLEDGMENTS We thank Ms. Setsuko Ojima and Ms. Yoshiko Uemlzu for their excellent technical assistance. This work was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. GRAHAM, F. L., ABRAHAMS, P. J., MULDER, C., HEIJNEKER, H. L., WARNAAR, S. O., DE VRIES, F. A. J., FIERS, W., and VAN DER EB, A. J., Cold Spring Harbor Symp. Quant. Biol. 39, 637-650 (1974). 2. SHIROKI, K., HANDA, H., SHIMOJO, H., YANO, S., OJIMA, S., and FUJINAGA, K., Virology 82, 462-471 (1977). 3. SEKIKAWA, K., SHIROKI, K., SHIMOJO, H., OJIMA, S., and FUJINAGA, K., Virology 87, 446-452 (1978).
581
4. WOLD, W. S. M., GREEN, M., and BUTPNER, W., In “The Molecular Biology of Animal Viruses” (D. P. Nayak, ed.), pp. 673-768, Marcel Dekker, New York, (19’78). 5. FUJINAGA, K., OJIMA, S., YANO, S., and SHINAGAWA, M., Proc. Japan. Acad. 53, 152-155 (1977). 6. GARON, C. F., BERRY, K. W., HIERHOLZER, J. C., and ROSE, J. A., Virology 54, 414-426 (1973). 7. GREEN, M., and PINA, M., Virology 20, 199-207 (1963). 8. GREEN, M., and PINA, M., Proc. Nat. Acad. Sci. USA 51, 1251-1259 (1964). 9. RIGBY, P. J. W., DIECKMANN, M., RHODES, C., and BERG, P., J. Mol. Biol. 113,237-251(1977). 10. MACKEY, J. K., BRACKMANN, K. H., GREEN, M. R., and GREEN, M., Biochemistry 16, 4478-4483 (1977). II. FUJINAGA, K., PINA, M., and GREEN, M., Proc. Nat. Acad. Sci. USA 64, 255-262 (1969). 12. YANO, S., OJIMA, S., FUJINAGA, K., SHIROKI, K., and SHIMOJO, H., Virology 82,214-220 (1977). 13. SOUTHERN, E. M., J. Mol. Biol. 98563-517 (1975). 11. DAWID, I. B., Rio&m. Biophys. Acta 477, 191194 (1977). 15. TIBBETT, C., J. Viral. 24, 564-579 (1977).
93, 578-581
Three Different Oncogenic
(1979)
Classes of Human Adenovirus Transforming DNA Sequences: Highly Subgroup A-, Weakly Oncogenic Subgroup B-, and Subgroup C-Specific Transforming DNA Sequences
KEI FUJINAGA,’ Department
of Molecular
Biology,
YUKIHARU
SAWADA, AND KENJI SEKIKAWA
Cancer Research Institute, Accepted November
Sapporo Medical
College, Sapporo 060, Japan
8, 1978
DNA-DNA homology measurements showed the presence of three different classes of human adenovirus transforming gene sequences: one specific for highly oncogenic subgroup A (types 12, 18, and 31), the second specific for weakly oncogenic subgroup B (types 3, 7, and 16), and the third specific for transforming subgroup C (types 2, 5, and 6). Adenovirus transforming DNA sequences with molecular weights of 1.6-1.8 x lo6 are in common or almost identical among members within the same subgroup. Less than one-third of the transforming DNA sequences are shared among members of different subgroups. All of the DNA fragments containing transforming DNA sequences investigated are located at the end of the molecule.
Recent studies have revealed that the transforming activity of adenoviruses (Ad) resides in a small left-hand region of the viral DNA molecule, the transforming segment. Cells can be transformed by isolated transforming segments, HindIII-G of Ad2 and Ad5 (l), HindIII-1. J of Ad7 (Z), and HindIII-G of Ad12 (3). These transforming segments have molecular weights of 1.6-1.8 x lo6 and coding capacities for only one or two kinds of proteins which are required for cell transformation (4). It is important to examine the homology or heterology of the DNA sequences of the transforming segments of adenoviruses which may code proteins required for transformation of cells. We recently showed that most, if not all, of the nucleotide sequencesof the Ad12 transforming segment, HindIII-G, were also present in viral DNAs of Ad18 and Ad31, other members of the highly oncogenic subgroup A (5). In this study, we carried out further detailed DNA-DNA homology measurements using labeled Ad12 HindIII-G (subgroup A), Ad7 HindIII-I* J (subgroup B), and Ad5 HindIII-G (subgroup C) to compare transforming gene sequences among members of highly onco1 To whom reprint requests should be addressed. 0042~6822/79/040578-04$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
578
genie subgroup A, weakly oncogenic subgroup B, and transforming subgroup C. Filter and liquid hybridization experiments using whole viral DNA (4) showed that viral genomes within each group are closely related, sharing 55-100% of their base sequences, and those of different groups share only 5-28% of their sequences. Heteroduplex analysis of different adenovirus DNAs (6) led to a conclusion similar to the above. However, no direct comparison among transforming gene sequences of adenovirus had been carried out. Viral DNAs from Ad12 (Huie), Ad18 (D.C.), Ad31 (1315), Ad3 (G.B.), Ad7 (Grider), Ad16 (ch 79), Ad2 (38-2), Ad5 (Adenoid 75), and Ad6 (Ton 99) were prepared as described (7, 8). Seed virus cultures were obtained from Dr. M. Green (Ad12, Ad7, Ad5, and Ad2) and from Dr. Hiroto Shimojo (Ad18, Ad31, Ad3, Ad16, and Ad6). Transforming DNA segments of Ad12 (HindIII-G), Ad7 (HindIII-1. J), and Ad5 (HindIII-G) were isolated and purified from viral DNAs, labeled in vitro by nick translation as described (9, 10). Hybridization experiments using membrane-immobilized DNA were performed by the procedure described previously (11) with minor modifications. Alkali-denatured DNA in
SHORT COMMUNICATIONS
4x SSC (SSC: 0.15M NaCl + 0.015 M sodium citrate) was passed three times through a nitrocellulose membrane filter (Millipore HA) and dried at room temperature for 4 hr or overnight and for 2 hr at 80” in a vacuum oven. Membrane-bound DNA was hybridized with labeled DNA in 4x SSC containing 0.1% SDS for 40 hr at 60”. After hybridization, membrane filters were washed with 3 r&f Tris-HCl buffer, pH 9.2, repeatedly by shaking. Filters were dried and radioactivity was determined in a scintillation counter. It has previously been shown in our experiments that labeled Ad12 HindIII-G hybridized with DNAs from Ad18 and Ad31, members of the highly oncogenic subgroup A, as efficiently as with Ad12 DNA (5). On the other hand, as shown in Table 1, only a small portion (ll-20%) of the labeled Ad12 HindIII-G hybridized with excess amounts of DNAs from members of other subgroups, Ad3, Ad7, Ad16, Ad2, Ad5, and Ad6. Similar results were obtained with the hybridization experiments between labeled
579
Ad7 HindIII-I *J or labeled Ad5 HindIII-G and DNAs from different adenoviruses. The above results show the presence of the common and unique transforming DNA sequence specific for each of the three subgroups, highly oncogenic subgroup A, weakly oncogenic subgroup B, and transforming subgroup C, respectively. Only one-third or less of the transforming DNA sequences are shared by members of different subgroups. In order to find the portions of various adenovirus DNAs homologous to the transforming DNA sequences of Ad12, Ad’7, or Ad5, hybridization experiments using the Southern technique were carried out with minor modifications. Viral DNA was digested with restriction endonuclease Hind111 and viral DNA fragments were separated by 1% agarose gel electrophoresis for 11 hr at 40 V as described (12). After staining DNA bands with 0.5 pg/ml of ethidium bromide, the gel was soaked in a denaturing solution containing 0.5 1MNaOH and 1.5 M NaCl for 30 min at room temperature to denature
TABLE 1 HYBRIDIZATION OF ADENOVIRUS TRANSFORMING DNA SEGMENTS WITH VARIOUS ADENOVIRUS DNAP
Labeled transforming DNA segment6 Viral DNA (1 pg/filter)
Ad12 HindIII-G
Subgroup A Ad12 Ad18 Ad31
100” 103’ 97”
Subgroup B Ad 3 Ad 7 Ad16 Subgroup C Ad 2 Ad 5 Ad 6
Ad7 HindIII-1.J
Ad5 HindIII-G
16 16 -
9 16 9
20 12 15
112 100 96
23 28
11 12 14
29 32 -
98 100 103
DAverage of duplicate hybridization experiments. Background (3-5% of the input) was subtracted, and binding to homologous viral DNA was normalized to 100%. Hybridization reaction was carried out in 4x SSC, 0.1% SDS for 40 hr at 60” as described in the text. * Transforming DNA segments were labeled with 32Pby nick translation. Input radioactivities of Ad12 HindIII-G, Ad7 HindIII-I-J, and Ad5 HindIII-G were 1000 cpm (1.37 x 10’ cpm/pg), 806 cpm (8.7 x lo6 cpmlpg), and 800 cpm (4.2 x lo6 cpm/pg), respectively. e Data from Ref. (5).
580
SHORTCOMMUNICATIONS
DNA fragments. For neutralization, the gel was soaked in a neutralizing solution containing 3 M NaCl and 0.5 M Tris-HCl, pH 7.2, for 15 min at 0”. The denatured DNA fragments were transferred to a nitrocellulose membrane filter (Millipore HAWP) by the Southern technique (13). The DNA membrane filter containing DNA was dried for 2 hr or overnight (or for 3 min in a vacuum oven at SO’), folded in a cylindrical shape, and placed in a siliconized test tube. After heating at 80” in a vacuum oven, the DNA membrane was immersed in a hybridization solution containing 0.6 M NaCl, 0.2 M Tris-HCl, pH 7.9, 0.02 M EDTA, 0.5% SDS, 50% formamide (14), and labeled DNA probe, and incubated at 37” for 40-48 hr in a circular rotating machine. The filter was washed four times with 2 x SSC + 50% formamide for 15 min in a shaker, washed two times with 2x SSC, dried, and autoradiographed using Kodak XRPl X-O mat film with or without intensifying screen (DuPont Chronex Lightning-plus). The results of the Southern hybridization using Ad12 HindIII-G, Ad7 HindIII-I* J, and Ad5 HindIII-G as probes are shown in Fig. 1. Labeled Ad12 HindIII-G hybridized with the HindIII-C fragment of Ad18 (10% at the end; Sawada and Fujinaga, unpublished) and with the HindIII-G fragment of Ad31 (7% at the end; Sawada and Fujinaga, unpublished). Labeled Ad7 HindIII-1. J hybridized with the HindIII-1.J fragment of Ad3 located on the left 8% of the molecule (15). Labeled Ad5 HindIII-G hybridized with the HindIII-G fragment of Ad2. No significant hybridizations were detected between the above transforming DNA segments and the other DNA fragments on the filter. The above results show that all of the transforming gene sequences locate within a region at the end 10% of the viral DNA molecule. We detected only a small region of the transforming DNA sequences shared in common by viral DNAs from members of three different subgroups. These results suggest that different virus-coded transforming proteins are involved in carcinogenesis by these three subgroups of human adenoviruses. Recently, however, a weak
Subgroup
A
Ad 12 HinduI-G Ad I8 Hind III-C Ad31 Hirdlll-G subgroy,
B
Ad 3 Ad
Hindlll-
7
I.J
Hind Ill- I, J
subgroup c Ad 2 Hindlll-G
Ad 5 Subgroup
Hindlll-G A Ad 12 Ad18 Ad31
Subgrwp
B Ad 3 Ad7
Subgroup
C Ad 2
Ad 5
FIG. 1. Hybridization of labeled transforming DNA segments with Hind111 fragments from different viral DNAs by the Southern technique. HinIII fragments of viral DNAs were resolved by electrophoresis in 1.0% agarose gels, denatured in situ, and transferred to nitrocellulose membrane filters by the technique of Southern (13) as described in the text. The filters, containing 2 pg of each viral DNA, were incubated with lo5 cpm of nick-translated [32P]DNA fragments in a hybridization solution containing 50% formamide for 40 hr at 37”, washed, and exposed to X-ray Film as described in the text (A). A photograph of DNA fragment bands stained with ethidium bromide, as observed in the gel prior to transfer, is presented also (B). Viral DNAs from Ad12, Ad18, and Ad31 were hybridized with the labeled HindIII-G fragment of Adl2. Viral DNAs from Ad3 and Ad7 were hybridized with the labeled HindIII-1. J of Ad7. Viral DNAs from Ad2 and Ad5 were hybridized with the labeled HindIII-G of Ad5.
common antigenicity was found between T antigens of Ad7 and Ad12 (Shiroki et al., in press), and we should note that a distant relationship of DNA sequences does not always reflect distantly related proteins coded. Direct sequencing of the transforming DNA segments and detailed investigation of the proteins coded by them will provide definitive conclusions about the
SHORT COMMUNICATIONS
virus-coded genetic information involved in cell transformation by these three different subgroups of human adenoviruses. ACKNOWLEDGMENTS We thank Ms. Setsuko Ojima and Ms. Yoshiko Uemlzu for their excellent technical assistance. This work was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. GRAHAM, F. L., ABRAHAMS, P. J., MULDER, C., HEIJNEKER, H. L., WARNAAR, S. O., DE VRIES, F. A. J., FIERS, W., and VAN DER EB, A. J., Cold Spring Harbor Symp. Quant. Biol. 39, 637-650 (1974). 2. SHIROKI, K., HANDA, H., SHIMOJO, H., YANO, S., OJIMA, S., and FUJINAGA, K., Virology 82, 462-471 (1977). 3. SEKIKAWA, K., SHIROKI, K., SHIMOJO, H., OJIMA, S., and FUJINAGA, K., Virology 87, 446-452 (1978).
581
4. WOLD, W. S. M., GREEN, M., and BUTPNER, W., In “The Molecular Biology of Animal Viruses” (D. P. Nayak, ed.), pp. 673-768, Marcel Dekker, New York, (19’78). 5. FUJINAGA, K., OJIMA, S., YANO, S., and SHINAGAWA, M., Proc. Japan. Acad. 53, 152-155 (1977). 6. GARON, C. F., BERRY, K. W., HIERHOLZER, J. C., and ROSE, J. A., Virology 54, 414-426 (1973). 7. GREEN, M., and PINA, M., Virology 20, 199-207 (1963). 8. GREEN, M., and PINA, M., Proc. Nat. Acad. Sci. USA 51, 1251-1259 (1964). 9. RIGBY, P. J. W., DIECKMANN, M., RHODES, C., and BERG, P., J. Mol. Biol. 113,237-251(1977). 10. MACKEY, J. K., BRACKMANN, K. H., GREEN, M. R., and GREEN, M., Biochemistry 16, 4478-4483 (1977). II. FUJINAGA, K., PINA, M., and GREEN, M., Proc. Nat. Acad. Sci. USA 64, 255-262 (1969). 12. YANO, S., OJIMA, S., FUJINAGA, K., SHIROKI, K., and SHIMOJO, H., Virology 82,214-220 (1977). 13. SOUTHERN, E. M., J. Mol. Biol. 98563-517 (1975). 11. DAWID, I. B., Rio&m. Biophys. Acta 477, 191194 (1977). 15. TIBBETT, C., J. Viral. 24, 564-579 (1977).
