- Email: [email protected]
Cleavage maps of human adenovirus type 7 DNA by restriction endonuclease HindIII and EcoRI
VIROLQGY
82, 509-512 (1977)
SHORT Cleavage
Maps of Human Adenovirus Endonuclease HindIll KENJI
Department
COMMUNICATIONS
of Molecular
Biology,
SEKIKAWA
AND
Type 7 DNA by Restriction and EcoRI KEI FUJINAGA’
Cancer Research Institute,
Sapporo Medical
College, Sapporo,
Japan
Accepted July 5,1977 Adenovirus type 7 DNA (Grider strain) was cleaved into ten and two specific fragments by the restriction endonuclease Hind111 and EcoRI, respectively. These specific fragments were mapped on the adenovirus type 7 genome by analyzing partial digestion products and by the double-digestion experiment.
Specific cleavages and physical mappings of DNA tumor virus genomes by restriction endonucleases provide powerful tools for analyzing viral genomes. Locations of early, late, and transforming genes (I, 2) along the viral genome, locations of ts mutations (31, and the portion of the viral genome present in viral-transformed cells (4, 5) were studied by these techniques. We previously reported that restriction endonuclease (Endo R) Hind cleaved the adenovirus type 7 (Ad 7) DNA into 23 unique fragments and these fragments were used to characterize viral DNA sequences in Ad 7 tumor cells (5). In this communication we analyzed partial-digestion and double-digestion products of EndoReHindIII and EndoR*EcoRI and constructed physical maps for further investigations of the structure and functions of the Ad 7 viral genome. Grider strain of Ad 7 was grown in KB cells, and virions were purified as described (6). Viral DNA was extracted from purified virions by the method of Green and Pina (6,7). 32P-Labeled Ad 7 was prepared from infected cells labeled with 50 &i/ml of carrier-free [32P]orthophosphate in phosphate-free Eagle’s minimum essential medium (MEM) supplemented with 5% dialyzed calf serum for 72 hr at 37”. The specific activity of Ad 7 [32P]DNA was l-2 x lo6 cpmlpg. EndoReHind was prepared 1 Author dressed.
to whom reprint
requests
should be ad-
from Haemophilus influenzae Rd strain by the procedure of Takanami (8) and EndoRsHindIII, one of the components of EndoR *Hind, was purified as described by Lai and Nathans (9). EndoRaEcoRI was generously provided by Dr. Yukio Sugino (Biological Research Laboratory, Takeda Chemical Industries Ltd. Osaka, Japan). One unit of the enzyme was defined as the minimum amount which could digest 1.0 pg of Ad 2 DNA in 60 min at 37”. EndoR digestion was carried out by incubating viral DNA in 20 to 50 ~1 of the reaction mixture at 37” for 2 hr with 1 unit/p1 of EcoRI, or for 4 hr with 1.7 unit/5 ~1 of HindIII. Buffer used for EcoRI digestion contained 100 mM Tris-HCl, pH 7.6, 10 n-&f MgC12, 50 mM NaCl, and that for Hind111 digestion contained 10 mM TrisHCl pH 7.6, 7 mM MgC12, 7 n&f KCl, 7 mM 2-mercaptoethanol. Ad 7 [32PlDNA was cleaved by Hind111 and resolved by 1.4% agarose slab gel electrophoresis. As shown in Fig 1B (left slot), ten distinct bands were observed. Molecular sizes of the ten fragments were estimated from the distribution of 32P radioactivity in each fragment and from electrophoretic mobility of each fragment using intact Ad 2 DNA and Ad 2 and Ad 12 DNA fragments produced by EcoRI digestion as size references (data not shown). Averages of the two determinations are listed in footnote b for Table 1. The plot of the relative 32Pradioactivity in each fragment versus each migration distance on a semiloga509
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
510
SHORT
COMMUNICATIONS TABLE
I
COMPLETE CLEAVAGE OF PARTIAL-DIGESTION PRODUCTS WITH Hind111 Partialdigestion product
Molecular weight” ‘“D”f#
b” FI ; f i j kc 1 m n 0
7.6 13.4 14.5 17.0 19.0 22.0 24.0 26.0 28.0 30.0 33.0 38.0 40.0 52.0 59.0
Complete
cleavage product
7
I, J F, H F, G E, I, J C, G B, H D, E D, E, J C, F, G B, F, H 2A, A, A, A A,
C, C, Q C, C,
D G E D, E D, F, G
Overlapping
Sum of product molecular weightb (% of Ad 7 DNA) 8.1 13.5 14.7 17.5 19.1 20.9 21.1 24.9 27.7 29.5 69.1 41.3 43.1 56.3 61.6
I
orders
J FH GF
I
JE CG HB ED JED CGF FHB DAC ACG EDA EDAC DACGF
IJEDACGFHB a Molecular weight was estimated from relative mobility of each fragment in agarose gel electrophoresis and percentage of viral genome was calculated from molecular weight of each fragment using 2.3 x 10’ for the molecular weight of whole Ad 7 DNA (14). b Molecular weight of each complete-digestion product by Hind111 was estimated as described in the text, and percentage of viral genome was calculated from molecular weight as described in footnote a. Values are as follows: A(22.2%), B(16.0%), C(13.081, D(I1.7%), E(9.4%), F(8.6%), G(6.1%), H(4.9%), I(4.381, J(3.8%). c Parital digestion product k is composed of two different DNA fragments, A-C and A-D.
rithmic scale showed that all fragments were present in equimolar amounts (data not shown). EcoRI digestion of Ad 7 yields two fragments, and their molecular weights are 19.9 x lo6 and 3.1 x 106, respectively. This result agrees with that of Ad 7 Cl 9 strain reported by Mulder et al. (10). Ad 7 DNA (Grider) has only one cleavage site and differs from Ad 7 DNA (Cl E46-LL) which has two cleavage sites for EcoRI (10). It was also shown that EcoRI cleaved the HindIII-B fragment (16.0%) into two fragments with molecular lengths of 13.4 and 2.6% of the whole viral genome. The order of Ad 7 Hind111 fragments along the viral genome was determined by the analysis of partial-digestion products. Partial digestion of Ad 7 [32P]DNA was carried out by incubating viral DNA with Hind111 for a short period. Products were separated by 0.9% agarose slab gel electrophoresis. As shown in Fig IA, several discrete bands other than those of the com-
plete-digestion product are present in partial-digestion products. The molecular size of each partial-digestion product was estimated from its electrophoretic mobility (Table 1). Fifteen partial-digestion products were eluted, purified by phenol extraction, and redigested with an excess amount of HindIII, followed by the separation on a 1.4% agarose gel electrophoresis. Typical results are presented in Fig 1B. The partial-digestion product rca,” for instance, yielded I and J; the partial “b” yielded F and H, the partial “c” yielded F and G, and the partial “d” yielded E, I, and J fragments, respectively. The film was scanned after autoradiography of the gel using a Gel Scanner (Varian M635 Gel Scanner). The area of each peak was measured, and the relative area of each peak was plotted on a logarithmic scale against its relative mobility. Thus, it was shown, for instance, that the partial ‘3” fragment yielded B, F, and H in an equimolar amount and the partial “k” fragment
SHORT
COMMUNICATIONS
511
FIG. 1. Partial digestion and redigestion of Ad 7 DNA by EndoR:HindIII. Ad 7 [32P]DNA (22.5 pg, 3.31 x 10’ cpm) in a 50-~1 reaction mixture was partially digested with 8.5 unit (25 ~1) ofHind at 37” for 15 min. The digestion products were electrophoresed in a 0.9% agarose slab gel (16 x 22 x 0.5 cm) in the buffer containing 36 mM Tris, 32 mM KH,PO,, 1 r&f EDTA for 24 hr at 50 V. Gels were stained with 0.5 pg/ml of ethidium bromide and visualized under uv light (II ) (A). Elution of DNA fragments from agarose gels was carried out by the freeze and squeeze method of Thuring et al. (12). Ethidium bromide was removed by passing through the Dowex 5OW-X8 resin (Bio-Rad, analytical grade) column (0.8 x 1 cm), and DNA fragments were purified by phenol extraction followed by dialysis against 0.1 x SSC (SSC: 0.15 M NaCl, 0.015 M Na citrate). Complete digestion of DNA fragments with Hind111 was carried out as described in the text, and digestion products were resolved by electrophoresis in a 1.4% agarose slab gel (16 x 22 x 0.2 cm) for 20 hr at 50 V. Gels were dried over steam heat as described by Maize1 (13). and then autoradiographed on Kodak blue X-ray films (B).
yielded 2A, C, and D. The result of the analysis of partial products are summarized in Table 1. As shown in the last column of Table 1, the order of the Hind111 DNA fragments along the viral genome was determined to be I J E D A C G F H and B from their overlappings in each partial-digestion product.
We still do not know which fragment of either HindIII-I or HindIII-B would be located at right- or left-hand end. However, we have tentatively placed the EcoRI-A fragment of Ad 7 DNA at the left-hand end which contains HindIII-I fragments (Fig. 2). Rodent cell lines transformed by Ad 5 or Ad 2 contain some part of the GC-rich
512
SHORT
COMMUNICATIONS 866 &
A
ItJt E t 43 81 175
D
t
292
A
FIG. 2. The Hind111 and EcoRI cleavage (distance from left end/length of Ad 7 DNA)
t
514
C
t tFtt 644G705
791H840
mans of the Ad 7 DNA x 160.
left half of the viral genome (4-, is), and the transforming segment of Ad 2 and Ad 5 DNA is located from 1 to 7% at the lefthand end of the viral genome (2). We reported previously that 200-400 copies of about lo-20% of Ad 7 genome were present in a diploid quantity of Ad ‘I-transformed hamster cell DNA (5). Recent preliminary experiments showed the presence of the HindIII-I fragment and the HindIII-J fragment in Ad 7 transformed cell DNA (Sekikawa and Fujinaga, in preparation). The quantification of the DNA fragments present in Ad 7-transformed cells, and the identification of the DNA fragment with the transforming ability is now in progress with the positioning of early and late genes. ACKNOWLEDGMENTS We thank Dr. Hiroto Shimojo, Institute of Medical Science, Tokyo University, and Dr. Mitsuru Takanami, Institute for Chemical Research, Kyoto University, for their critical readings of the manuscript. We also thank Dr. Yukio Sugino, Biological Research Laboratory, Takeda Chemical Industries Ltd. for generous gift of EndoR.EcoRI. 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. SHARP, P. A., GALLIMOR, P. H., and FLINT, S. J., Cold Spring Harbor Symp. Quant. Biol. 39, 457-474 (1974).
B
B
molecule.
EC& I Hind II
Map units
are given
as
2. GRAHAM, F. L., ABRAHAMS, P. J., MULDER, C., HEIJNEKER, H. L., WARNMR, 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). 3. GRODZICKER, T., WILLIAMS, J., SHARP, P., and SAMBROOK, J., Cold Spring Harbor Symp. Quant. Biol. 39, 439-446 (1974). 4. SAMBROOK, J., BOTCHAN, M., GALLIMOR, P., OZANNE, B., PETTERSSON, U., WILLIAMS, J., and SHARP, P. A., Cold Spring Harbor Symp. Quant. Biol. 39, 615-632 (1974). 5. FUJINAGA, K., SEKIKAWA, K., YAMAZAKI, H., and GREEN, M., Cold Spring Harbor Symp. Quant. Biol. 39, 633-636 (1974). 6. GREEN, M., and PINA, M., Virology 20, 199-207 (1963). 7. GREEN, M., and PINA, M., Proc. Natl. Acad. Sci. USA 51, 1251-1259 (1964). 8. TAKANAMI, M., In “Methods in Molecular Biology” (R. Wecker, ed.), Vol. 7. Marcel Dekker Inc., New York, 1974. 9. LAI, C.-J., and NATHANS, D., J. Mol. Biol. 89, 179-193 (1974). 10. MULDER, C., SHARP, P. A., DELIUS, H., and PETTERSSON, U., J. Virol. 14, 68-77 (1974). 11. SHARP, P. A., SUGDEN, W., and SAMBROOK, J., Biochemistry 12, 3055-3063 (1973). 12. THURING, R. W. J., SANDERS, J. P. M., and BORST, P., Anal. Biochem. 66, 213-220 (1975). 13. MAIZEL, J. V., JR., In “Methods in Virology” (K. Maramorosh, and H. Koprowski, eds.), Vol. 5. Academic Press, New York and London, 1971. 14. GREEN, M., PINA, M., KIMES, R., WENSINK, P. D., MACHATTIE, L. A., and THOMAS, C. A., JR., Proc. Nat. Acad. Sci. USA 57, 1302-1309, (1967). 15. GALLIMORE, P. H., SHARP, P. A., and SAMBROOK, J., J. Mol. Biol. 89, 49-72 (1974).
82, 509-512 (1977)
SHORT Cleavage
Maps of Human Adenovirus Endonuclease HindIll KENJI
Department
COMMUNICATIONS
of Molecular
Biology,
SEKIKAWA
AND
Type 7 DNA by Restriction and EcoRI KEI FUJINAGA’
Cancer Research Institute,
Sapporo Medical
College, Sapporo,
Japan
Accepted July 5,1977 Adenovirus type 7 DNA (Grider strain) was cleaved into ten and two specific fragments by the restriction endonuclease Hind111 and EcoRI, respectively. These specific fragments were mapped on the adenovirus type 7 genome by analyzing partial digestion products and by the double-digestion experiment.
Specific cleavages and physical mappings of DNA tumor virus genomes by restriction endonucleases provide powerful tools for analyzing viral genomes. Locations of early, late, and transforming genes (I, 2) along the viral genome, locations of ts mutations (31, and the portion of the viral genome present in viral-transformed cells (4, 5) were studied by these techniques. We previously reported that restriction endonuclease (Endo R) Hind cleaved the adenovirus type 7 (Ad 7) DNA into 23 unique fragments and these fragments were used to characterize viral DNA sequences in Ad 7 tumor cells (5). In this communication we analyzed partial-digestion and double-digestion products of EndoReHindIII and EndoR*EcoRI and constructed physical maps for further investigations of the structure and functions of the Ad 7 viral genome. Grider strain of Ad 7 was grown in KB cells, and virions were purified as described (6). Viral DNA was extracted from purified virions by the method of Green and Pina (6,7). 32P-Labeled Ad 7 was prepared from infected cells labeled with 50 &i/ml of carrier-free [32P]orthophosphate in phosphate-free Eagle’s minimum essential medium (MEM) supplemented with 5% dialyzed calf serum for 72 hr at 37”. The specific activity of Ad 7 [32P]DNA was l-2 x lo6 cpmlpg. EndoReHind was prepared 1 Author dressed.
to whom reprint
requests
should be ad-
from Haemophilus influenzae Rd strain by the procedure of Takanami (8) and EndoRsHindIII, one of the components of EndoR *Hind, was purified as described by Lai and Nathans (9). EndoRaEcoRI was generously provided by Dr. Yukio Sugino (Biological Research Laboratory, Takeda Chemical Industries Ltd. Osaka, Japan). One unit of the enzyme was defined as the minimum amount which could digest 1.0 pg of Ad 2 DNA in 60 min at 37”. EndoR digestion was carried out by incubating viral DNA in 20 to 50 ~1 of the reaction mixture at 37” for 2 hr with 1 unit/p1 of EcoRI, or for 4 hr with 1.7 unit/5 ~1 of HindIII. Buffer used for EcoRI digestion contained 100 mM Tris-HCl, pH 7.6, 10 n-&f MgC12, 50 mM NaCl, and that for Hind111 digestion contained 10 mM TrisHCl pH 7.6, 7 mM MgC12, 7 n&f KCl, 7 mM 2-mercaptoethanol. Ad 7 [32PlDNA was cleaved by Hind111 and resolved by 1.4% agarose slab gel electrophoresis. As shown in Fig 1B (left slot), ten distinct bands were observed. Molecular sizes of the ten fragments were estimated from the distribution of 32P radioactivity in each fragment and from electrophoretic mobility of each fragment using intact Ad 2 DNA and Ad 2 and Ad 12 DNA fragments produced by EcoRI digestion as size references (data not shown). Averages of the two determinations are listed in footnote b for Table 1. The plot of the relative 32Pradioactivity in each fragment versus each migration distance on a semiloga509
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
510
SHORT
COMMUNICATIONS TABLE
I
COMPLETE CLEAVAGE OF PARTIAL-DIGESTION PRODUCTS WITH Hind111 Partialdigestion product
Molecular weight” ‘“D”f#
b” FI ; f i j kc 1 m n 0
7.6 13.4 14.5 17.0 19.0 22.0 24.0 26.0 28.0 30.0 33.0 38.0 40.0 52.0 59.0
Complete
cleavage product
7
I, J F, H F, G E, I, J C, G B, H D, E D, E, J C, F, G B, F, H 2A, A, A, A A,
C, C, Q C, C,
D G E D, E D, F, G
Overlapping
Sum of product molecular weightb (% of Ad 7 DNA) 8.1 13.5 14.7 17.5 19.1 20.9 21.1 24.9 27.7 29.5 69.1 41.3 43.1 56.3 61.6
I
orders
J FH GF
I
JE CG HB ED JED CGF FHB DAC ACG EDA EDAC DACGF
IJEDACGFHB a Molecular weight was estimated from relative mobility of each fragment in agarose gel electrophoresis and percentage of viral genome was calculated from molecular weight of each fragment using 2.3 x 10’ for the molecular weight of whole Ad 7 DNA (14). b Molecular weight of each complete-digestion product by Hind111 was estimated as described in the text, and percentage of viral genome was calculated from molecular weight as described in footnote a. Values are as follows: A(22.2%), B(16.0%), C(13.081, D(I1.7%), E(9.4%), F(8.6%), G(6.1%), H(4.9%), I(4.381, J(3.8%). c Parital digestion product k is composed of two different DNA fragments, A-C and A-D.
rithmic scale showed that all fragments were present in equimolar amounts (data not shown). EcoRI digestion of Ad 7 yields two fragments, and their molecular weights are 19.9 x lo6 and 3.1 x 106, respectively. This result agrees with that of Ad 7 Cl 9 strain reported by Mulder et al. (10). Ad 7 DNA (Grider) has only one cleavage site and differs from Ad 7 DNA (Cl E46-LL) which has two cleavage sites for EcoRI (10). It was also shown that EcoRI cleaved the HindIII-B fragment (16.0%) into two fragments with molecular lengths of 13.4 and 2.6% of the whole viral genome. The order of Ad 7 Hind111 fragments along the viral genome was determined by the analysis of partial-digestion products. Partial digestion of Ad 7 [32P]DNA was carried out by incubating viral DNA with Hind111 for a short period. Products were separated by 0.9% agarose slab gel electrophoresis. As shown in Fig IA, several discrete bands other than those of the com-
plete-digestion product are present in partial-digestion products. The molecular size of each partial-digestion product was estimated from its electrophoretic mobility (Table 1). Fifteen partial-digestion products were eluted, purified by phenol extraction, and redigested with an excess amount of HindIII, followed by the separation on a 1.4% agarose gel electrophoresis. Typical results are presented in Fig 1B. The partial-digestion product rca,” for instance, yielded I and J; the partial “b” yielded F and H, the partial “c” yielded F and G, and the partial “d” yielded E, I, and J fragments, respectively. The film was scanned after autoradiography of the gel using a Gel Scanner (Varian M635 Gel Scanner). The area of each peak was measured, and the relative area of each peak was plotted on a logarithmic scale against its relative mobility. Thus, it was shown, for instance, that the partial ‘3” fragment yielded B, F, and H in an equimolar amount and the partial “k” fragment
SHORT
COMMUNICATIONS
511
FIG. 1. Partial digestion and redigestion of Ad 7 DNA by EndoR:HindIII. Ad 7 [32P]DNA (22.5 pg, 3.31 x 10’ cpm) in a 50-~1 reaction mixture was partially digested with 8.5 unit (25 ~1) ofHind at 37” for 15 min. The digestion products were electrophoresed in a 0.9% agarose slab gel (16 x 22 x 0.5 cm) in the buffer containing 36 mM Tris, 32 mM KH,PO,, 1 r&f EDTA for 24 hr at 50 V. Gels were stained with 0.5 pg/ml of ethidium bromide and visualized under uv light (II ) (A). Elution of DNA fragments from agarose gels was carried out by the freeze and squeeze method of Thuring et al. (12). Ethidium bromide was removed by passing through the Dowex 5OW-X8 resin (Bio-Rad, analytical grade) column (0.8 x 1 cm), and DNA fragments were purified by phenol extraction followed by dialysis against 0.1 x SSC (SSC: 0.15 M NaCl, 0.015 M Na citrate). Complete digestion of DNA fragments with Hind111 was carried out as described in the text, and digestion products were resolved by electrophoresis in a 1.4% agarose slab gel (16 x 22 x 0.2 cm) for 20 hr at 50 V. Gels were dried over steam heat as described by Maize1 (13). and then autoradiographed on Kodak blue X-ray films (B).
yielded 2A, C, and D. The result of the analysis of partial products are summarized in Table 1. As shown in the last column of Table 1, the order of the Hind111 DNA fragments along the viral genome was determined to be I J E D A C G F H and B from their overlappings in each partial-digestion product.
We still do not know which fragment of either HindIII-I or HindIII-B would be located at right- or left-hand end. However, we have tentatively placed the EcoRI-A fragment of Ad 7 DNA at the left-hand end which contains HindIII-I fragments (Fig. 2). Rodent cell lines transformed by Ad 5 or Ad 2 contain some part of the GC-rich
512
SHORT
COMMUNICATIONS 866 &
A
ItJt E t 43 81 175
D
t
292
A
FIG. 2. The Hind111 and EcoRI cleavage (distance from left end/length of Ad 7 DNA)
t
514
C
t tFtt 644G705
791H840
mans of the Ad 7 DNA x 160.
left half of the viral genome (4-, is), and the transforming segment of Ad 2 and Ad 5 DNA is located from 1 to 7% at the lefthand end of the viral genome (2). We reported previously that 200-400 copies of about lo-20% of Ad 7 genome were present in a diploid quantity of Ad ‘I-transformed hamster cell DNA (5). Recent preliminary experiments showed the presence of the HindIII-I fragment and the HindIII-J fragment in Ad 7 transformed cell DNA (Sekikawa and Fujinaga, in preparation). The quantification of the DNA fragments present in Ad 7-transformed cells, and the identification of the DNA fragment with the transforming ability is now in progress with the positioning of early and late genes. ACKNOWLEDGMENTS We thank Dr. Hiroto Shimojo, Institute of Medical Science, Tokyo University, and Dr. Mitsuru Takanami, Institute for Chemical Research, Kyoto University, for their critical readings of the manuscript. We also thank Dr. Yukio Sugino, Biological Research Laboratory, Takeda Chemical Industries Ltd. for generous gift of EndoR.EcoRI. 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. SHARP, P. A., GALLIMOR, P. H., and FLINT, S. J., Cold Spring Harbor Symp. Quant. Biol. 39, 457-474 (1974).
B
B
molecule.
EC& I Hind II
Map units
are given
as
2. GRAHAM, F. L., ABRAHAMS, P. J., MULDER, C., HEIJNEKER, H. L., WARNMR, 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). 3. GRODZICKER, T., WILLIAMS, J., SHARP, P., and SAMBROOK, J., Cold Spring Harbor Symp. Quant. Biol. 39, 439-446 (1974). 4. SAMBROOK, J., BOTCHAN, M., GALLIMOR, P., OZANNE, B., PETTERSSON, U., WILLIAMS, J., and SHARP, P. A., Cold Spring Harbor Symp. Quant. Biol. 39, 615-632 (1974). 5. FUJINAGA, K., SEKIKAWA, K., YAMAZAKI, H., and GREEN, M., Cold Spring Harbor Symp. Quant. Biol. 39, 633-636 (1974). 6. GREEN, M., and PINA, M., Virology 20, 199-207 (1963). 7. GREEN, M., and PINA, M., Proc. Natl. Acad. Sci. USA 51, 1251-1259 (1964). 8. TAKANAMI, M., In “Methods in Molecular Biology” (R. Wecker, ed.), Vol. 7. Marcel Dekker Inc., New York, 1974. 9. LAI, C.-J., and NATHANS, D., J. Mol. Biol. 89, 179-193 (1974). 10. MULDER, C., SHARP, P. A., DELIUS, H., and PETTERSSON, U., J. Virol. 14, 68-77 (1974). 11. SHARP, P. A., SUGDEN, W., and SAMBROOK, J., Biochemistry 12, 3055-3063 (1973). 12. THURING, R. W. J., SANDERS, J. P. M., and BORST, P., Anal. Biochem. 66, 213-220 (1975). 13. MAIZEL, J. V., JR., In “Methods in Virology” (K. Maramorosh, and H. Koprowski, eds.), Vol. 5. Academic Press, New York and London, 1971. 14. GREEN, M., PINA, M., KIMES, R., WENSINK, P. D., MACHATTIE, L. A., and THOMAS, C. A., JR., Proc. Nat. Acad. Sci. USA 57, 1302-1309, (1967). 15. GALLIMORE, P. H., SHARP, P. A., and SAMBROOK, J., J. Mol. Biol. 89, 49-72 (1974).