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Comparative sequence analysis of the inverted terminal repetition in the genomes of animal and avian adenoviruses
VIROLOGY
B25,491-495
(1983)
imperative Sequence Analysis of the Inverted ~errn~~~~ in the Genomes of Animal and Avian Aden~v~r~ses ORIKAZ~ SHINAGAWA, *J TOSHIRO ISHIYAMA,* R. ~A~MA~A~XAN,~ KEI FUJINAGA,# MASANOBU KAMADA,§ ANDGIMEI SATO* Public Health, School of Veterinary Medicine, Obihiro University oj Veterinary Meckne, Obihiro 080, Japan; tDepartrnent of Biochemistry, The University of Kansas Medical Center, College of Health Science and Hospital, Karxsas City, Kansas 66103; $Cancer Research Institute, Sapporo Medical College, Sapporo 060, Japan, and $Equine Research Institute, Japan Racing Association, Tochigi 329-04, Japan
*Department Agriculture
of Veterinary
and
Received September 23, 1982; accepted December 8, 1982 The nucieotide sequences at the inverted terminal repetitions from two animal adenoviruses, infectious canine hepatitis virus and equine adenovirus, and from one avian adenovirus, CELO, were analyzed. DNAs from infectious canine hepatitis virus and equine adenovirus contain a homologous region which is 23 nucleotides long from the terminus. The first 17 nucleotides of this region are identical to the ones in human adenovirus type 2 DNA. The striking homologous sequence of 14 nucleotides, conserved in the inverted terminal repetitions of several human adenovirus strains and in simian adenovirus type 7, is only partially conserved in the two animal and one avian adenoviruses reported here.
Ade~oviruses (Ad) have been isolated from humans, animals, and fowl, and more than 10 Ad types have been characterized to date (8, 26, 28). They are classified into two main groups, the genus Mastadencvirus and the genus Aviadenovirus (16). The genome of Ad is a liner duplex DNA molecule with a molecular weight of 2030 X 106 (10,16,27). The 5’ end of each DNA strand is covalently bound to a terminal protein with an M, of 55,000 (17, 18). In addition, Ad DNA possesses a unique inverted terminal repetition (ITR) of the type abc . . . &‘a (6, 29). Although the exact function of ITR is unknown, it is thought to have an important role in DNA replication. There is evidence that initiation of DNA replication occurs within the terminal 15 base pairs (bp) or near the ends ($ 23). ITRs of human Ad types 2, 3, 4, 5, ?, 12, and 18, simian Ad7, and mouse Ad FL were shown to be perfect repetitions (1, 5, 19, 20, 2.2, 2.4-26). Comparative sequence analysis of ITRs from three groups * To whom
reprint
requests
should
be addressed.
of human Ads, A, B, and 6, revealed an interesting correlation between of the ITR and the oneogenir pro the viruses; the length of this region increases with the Q types (20). The huma B, C, and E contain 14 nucleotides long, 9-22 from each termi mologous region is also present in the I of simian Ad7 (2@, but it is only ~~~ti~~~~~ represented in mouse Ad FL (24). We undertook a detailed analysis of the nucleotide sequences at from different species to function and the extent of homologous regions in DN ent Ads. In this communi the nucleotide sequences at the ITRs of two animal Ads, infectious ea titis virus (ICI-IV) an and one avian Ad quences are compare man, simian, and mouse Ads. ICHV strain Woe-4 (21) an T-l (22) were propagated in 491
0042”6822/83 Copyright All rights
$3.00
0 1983 by Academic Press, I~c. of reproduction in any form reserved.
SHORT
COMMUNICATIONS
bP 233009500640043624200~ '56' '6722001800-
159513631106-
625FIG. 1. Restriction enzyme cleavage patterns of ICHV, EAd, and CELO DNAs. ICHV, EAd, and CELO DNAs were cleaved with BamHI or EcoRI and electrophoresed in an 0.8% agarose gel in the presence of ethidium bromide. In the right lane of CELO/ EcoRI, the DNA fragments were electrophoresed in a 1% agarose gel for a shorter time to show the smallest fragment H. Terminal fragments are underlined. ICHV/BomHI-D fragment and CELO/Bon2HI-J fragment are not shown in this figure. The size markers used were a mixture of restriction fragments of DNA from bacteriophage X and plasmid pBR322.
CELO strain Ote (13) was inoculated into allantoic cavities of lo-day-old chick embryos and infected allantoic fluid was harvested 4 days later. Infected culture fluids including cells and cell debris or infected allantoic fluids were concentrated by precipitation with polyethylene glycol No. 6000 (10%) and 0.5 M NaCI. The precipitates were suspended in 20 to 50 ml of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0). Virions were purified and DNA was extracted as described (9). DNA sequence analysis was
carried out according to the method of Maxam and Gilbert (1.5). The 3’ termini of intact Ad DNA were labeled by using [a32P]dGTP (Amersham, sp act 2000-3000 Ci/ mmol) and T4 DNA polymerase (20). The 3’-labeled DNA was cleaved with BarnHI for CELO, and EcoRI for ICHV and EAd. The generated fragments were fractionated by agarose gel electrophoresis and the labeled terminal fragments were isolated. The sequence data derived from the 3’ end were confirmed by the data derived from the DNA labeled at the 5’ termini. Before the labeling of the 5’ termini of intact Ad DNA by using [Y-~‘P]ATP and polynucleotide kinase, the Ad DNA was treated with 0.1 N NaOH, 1 mM EDTA at 3’7” for 3 hr, and with bacterial alkaline phosphatase (20, 25).
Figure 1 shows the cleavage patterns after digestion of ICHV, EAd, and CELO DNA with BarnHI and EcoRI. Denisova et al. (4) have reported that CELO DNA was cleaved by EcoRI into seven fragments and the terminal fragments were B and F. In this study, however, EcoRI cleaved the DNA into eight fragments (Fig. 1), and the terminal fragments were F and the smallest fragment H which were found by using with the end-labeled DNA (data not shown). The lengths of the fragments and molecular weight of the three Ad DNAs estimated from the sum of the fragment lengths are shown in Table 1. The molecular weight of ICHV (22.0 X 106) and EAd (23.8-24.5 X 106) are within the range of the molecular weights of human Ad DNAs (10,27), and that of CELO (29.3-30.4 X 106) agrees with the values reported elsewhere (14. 16). Figure 2 shows the nucleotide sequence at the ITR from ICHV, EAd, and CELO. The ITRs of ICHV, EAd, and CELO are 160, 103, and 54 bp in length, respectively. GC contents of ICHV-, EAd-, and CELOITR were 41,67, and 39%, respectively. To compare the nucleotide sequence of ITRs from ICHV, EAd, and CELO with those of ITRs from other Ads, available sequences are compiled in Fig. 2. The first 23 nucleotides are identical from the ends of ICHV and EAd. The highly conserved homologous sequence of
SHORT
4&
COMMUNICATIONS TABLE
1
GALCULRTED~OLE~ULARWEIGHTSOFRESTRICTIONFRAGME~TTSFROMI@WV,EA~,P,ND
-
ICHV Fragment A B c D F F G H 1 J Sum
Ns, (Da)”
EAd
BamHI
EcoRI
15,85oaa* 12,050 5,500 463
21,200 3,610 2,575 2,245 1,750 1,605 930
19,800 7,650 6,600 1,790 765
15,000 13,750 3,940 2,640 2,335
11,500 8,600 5,940 5,290 4,900 3,550 2,065 1,660 1,170 385
16,108 9:lOO 5,220 4$x? 4,880 3,360 3,LOO $5
33,863
33,915
36,605
37,665
45,060
46,725
22.0 x lo6
23.8 X lo6
24.5 X lo6
29.3 x 106
22.0 x 10”
a Base pairs. *Numbers of base ‘Six hundred fifty
pairs represent the mean of duplicate Da per base pair is assumed.
14 bp (nucleotides 9-22) present in several human Ad DNAs (20, 25) is only partially conserved in ICHV and EAd (nucleotides 9-17) and in CELO (nucleotides 9-14). Thus, the hexanucleotide sequence ATAATA (nucleotides 9-14) is present in every ITR. Comparison of ITR sequences of ICHV or EAd with human Ad type 2 shows that the first 17 nucleotides are common among these Ads, whereas ICHV, EAd, and mouse Ad FL commonly share the first 19 nueleotides. This is in contrast to the simian and human Ads (except human Ad4) which share very similar sequences up to about 50 nucleotides. The sequence GCCAATAT between nueleotides 34-41 (or 35-42 in the case of Ad7) is conserved within the ITR of all human Ads except Ad4 (26). Interestingly, this octanucleotide sequence includes “CCAAT,” which has been found upstream from the capping site for several mammalian P-like globin genes and from the TATA box for the sea urchin H3 genes (2, 11). Although “‘CCAAT” is conserved in simian Ad7, it is absent from ICHV, EAd, CELO, and mouse Ad FL as well as from human Ad4. Another interesting structural feature
-
GEL0
EcoRI
BamHI
GEL0 DNAS -
BamHI
ECOIEI
30.4 x IO6
experiments.
of the ITRs of several human Ads except Ad4 is the hexanucleotide seq.uence TGACGT, which is present at a site where the ITR terminates in Ad3,7, or 12, or two nucleotides penultimate to the t~~rn~~~~ tion site of the ITRs of Ad2 and Ad18 (7). Flowever, this bexanucleotide is a Ad4 at that site (26). nucleotides beyond the end of Ad4 DNA, but n This hexanucleotide sequence is ~o~se~v~d within the ITR of simian Ad7 at position 66-71 and in the ITR of I 125-130, several nucleotides the termination site of the If Ads have been evolving origin, avian Ad (CELO) verged from human and animal Ads at an early stage of evolution. ~ubseq~ent~y~ imal Ads diverged from a group consist, of human and simian Ads. Among the animal Ads, mouse Ad might earlier than ICHV and EAd. does not have a homologous other human Ads and simian Ad7 b nueleotide number 22 from the ends. and animal Ads, ICIIV, EAd, and mouse Ad FL do not have any marked ~~rn~~~~r beyond the well-conserved sequence (after
SHORT
COMMUNICATIONS
the 14th nucleotide in CELO, the 23rd in ICHV and EAd, and the 19th in mouse Ad FL). The hexanucleotide sequence ATAATA at position 9-14 might have been conserved in all Ads from an early stage of evolution. One of the possible functions of this sequence may be to serve as a recognition signal for the terminal proteinprecursor (80 k protein), which is involved in the initiation of DNA replication (3). ACKNOWLEDGMENTS This work was partly supported by a grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan, and by a Grant 56480067, from the same Ministry. REFERENCES 1. ARRAND, J. R., and ROBERTS, R. J., J. Mel Biol. 128,577-594 (1979). 2. BENOIST, C., O’HARE, K. O., BREATHNACH, R., and CHAMBON, P., Nucl. Acids Res. 4, 4371-4389 (1980). 3. CHALLBERG, M. D., OSTROVE, J. M., and KELLY, T. J., J. viral. 41, 265-270 (1982). 4. DENISOVA, T. S., SITNIKOV, B. S., and GHIBADULIN, R. A., Mol. BioZ. (USSR) 13, 1021-1034 (1979). 5. DIJKEMA, R., and DEKKER, B. M. M., Gene 8,7-15 (1979). 6. GARON, C. F., BERRY, K. W., and ROSE, J. A., Proc. Nat. Acad Sci. USA 69, 2391-2395 (1972). 7. GARON, C. F., PARR, R. P., PADMANABHAN, R., ROBINSON, I., GARRISON, J. W., and ROSE, J. A., viro.!Qgy 121,230-239 (1982). 8. GREEN, M., MACKEY, J. K., WOLD, W. S. M., and RIGDEN, P., viro&y 93, 481-492 (1979). 9. GREEN, M., and PIRA, M., Proc. Nat. Acad. Sci. USA 51, 1251-1255 (1964). 10. GREEN, M., PIQA, M., KIMES, R., WENSINK, P. C., MACHATTIE, L. A., and THOMAS, C. A., Proc Nat. Acad. Sci USA 57, 1302-1304 (1967). 11. HENTSCHEL, C. C., and BIRNSTELL, M. L., Cell 25, 301-313 (1981). 12. KAMADA, M., AKIYAMA, Y., SATO, K., and KODERA, S., Japan J. Vet. Sci 39, 661-664 (1977). 1.3. KAWAMURA, H., SATO, T., TSUBAHARA, H., and IsOGAI, S., Nat. Inst. An&. Health C&at. (Tokyo) 3, l-10 (1963). 14. LAVER, W. G., YOUNGHUSBAND, H. B., and WRIGLEY, N. G., virology 45, 598-614 (1971). 15. MAXAM, A. M., and GILBERT, W., Proc. Nat. Acad. Sci. USA 74,560-564 (1977). 16. MOHANTY, S. B., and DLITTA, S. K., “Veterinary Virology,” pp. 3-38. Lea & Febiger, Philadelphia (1981). 17’. REKOSH, D. M. K., RUSSELL, W. C., BELLETT,
SHORT A. J. D., and (1977).
ROBINSON,
COMMUNICATIONS
A. J., CelE 11, 283-295
18. ROBINSON, A. J., YOUNGFIUSBAND, H. B., and BELLETT, A. J. D., firology 56,54-69 (1973). 19. SHINAGAWA, Biophys.
M., and PADMANABHAN, R., B&hem Res. Cmmun. 87, 671-678 (1979).
20. SHINAGAWA, M., and PADMANABHAN, R., Proc. Aead. 5X. USA 77,3831-3835 (1980). 21. SHINAGAWA, M., ETOH, S., and YANAGAWA, rology 66, 161-171 (1975). 22. STEENBERGW, 6, 307-318
P. II., (1979).
and SUSSENBACH,
Nat
R., Vi-
J. S., Gene
d&s
23. SUSSENB~CH, J. S., and JUIJK, M. Z., %rolnyy 84, 509-517 (1978). 24. TEMPLE, M., ANTOINE, G., DELIUS, II., STAML, S., and WINNACKER, E. L., virology 199,1-12 (1982). 25. TOLUN, A., ALESTROM, P., and PETTERSSON, U., Cell 17,705-713 (1979). 26. TOKUNAGA, O., SHINAGAWA, M., and T?ADMAKAEHAN, R., Gene I&321-327 (1982). 27. VAN DER EB, A. J.; and VAN KERSTERN, L. M Biochim Biophys. Acta 129,441-444 (1966). 28. WADELL, G., Intervirology 11, 47-57 (1979). 29. WOLFSON, J., and DRESSLER, D., Proc. Nat. Acad Ski. USA 69,3054-3057 (1972).
B25,491-495
(1983)
imperative Sequence Analysis of the Inverted ~errn~~~~ in the Genomes of Animal and Avian Aden~v~r~ses ORIKAZ~ SHINAGAWA, *J TOSHIRO ISHIYAMA,* R. ~A~MA~A~XAN,~ KEI FUJINAGA,# MASANOBU KAMADA,§ ANDGIMEI SATO* Public Health, School of Veterinary Medicine, Obihiro University oj Veterinary Meckne, Obihiro 080, Japan; tDepartrnent of Biochemistry, The University of Kansas Medical Center, College of Health Science and Hospital, Karxsas City, Kansas 66103; $Cancer Research Institute, Sapporo Medical College, Sapporo 060, Japan, and $Equine Research Institute, Japan Racing Association, Tochigi 329-04, Japan
*Department Agriculture
of Veterinary
and
Received September 23, 1982; accepted December 8, 1982 The nucieotide sequences at the inverted terminal repetitions from two animal adenoviruses, infectious canine hepatitis virus and equine adenovirus, and from one avian adenovirus, CELO, were analyzed. DNAs from infectious canine hepatitis virus and equine adenovirus contain a homologous region which is 23 nucleotides long from the terminus. The first 17 nucleotides of this region are identical to the ones in human adenovirus type 2 DNA. The striking homologous sequence of 14 nucleotides, conserved in the inverted terminal repetitions of several human adenovirus strains and in simian adenovirus type 7, is only partially conserved in the two animal and one avian adenoviruses reported here.
Ade~oviruses (Ad) have been isolated from humans, animals, and fowl, and more than 10 Ad types have been characterized to date (8, 26, 28). They are classified into two main groups, the genus Mastadencvirus and the genus Aviadenovirus (16). The genome of Ad is a liner duplex DNA molecule with a molecular weight of 2030 X 106 (10,16,27). The 5’ end of each DNA strand is covalently bound to a terminal protein with an M, of 55,000 (17, 18). In addition, Ad DNA possesses a unique inverted terminal repetition (ITR) of the type abc . . . &‘a (6, 29). Although the exact function of ITR is unknown, it is thought to have an important role in DNA replication. There is evidence that initiation of DNA replication occurs within the terminal 15 base pairs (bp) or near the ends ($ 23). ITRs of human Ad types 2, 3, 4, 5, ?, 12, and 18, simian Ad7, and mouse Ad FL were shown to be perfect repetitions (1, 5, 19, 20, 2.2, 2.4-26). Comparative sequence analysis of ITRs from three groups * To whom
reprint
requests
should
be addressed.
of human Ads, A, B, and 6, revealed an interesting correlation between of the ITR and the oneogenir pro the viruses; the length of this region increases with the Q types (20). The huma B, C, and E contain 14 nucleotides long, 9-22 from each termi mologous region is also present in the I of simian Ad7 (2@, but it is only ~~~ti~~~~~ represented in mouse Ad FL (24). We undertook a detailed analysis of the nucleotide sequences at from different species to function and the extent of homologous regions in DN ent Ads. In this communi the nucleotide sequences at the ITRs of two animal Ads, infectious ea titis virus (ICI-IV) an and one avian Ad quences are compare man, simian, and mouse Ads. ICHV strain Woe-4 (21) an T-l (22) were propagated in 491
0042”6822/83 Copyright All rights
$3.00
0 1983 by Academic Press, I~c. of reproduction in any form reserved.
SHORT
COMMUNICATIONS
bP 233009500640043624200~ '56' '6722001800-
159513631106-
625FIG. 1. Restriction enzyme cleavage patterns of ICHV, EAd, and CELO DNAs. ICHV, EAd, and CELO DNAs were cleaved with BamHI or EcoRI and electrophoresed in an 0.8% agarose gel in the presence of ethidium bromide. In the right lane of CELO/ EcoRI, the DNA fragments were electrophoresed in a 1% agarose gel for a shorter time to show the smallest fragment H. Terminal fragments are underlined. ICHV/BomHI-D fragment and CELO/Bon2HI-J fragment are not shown in this figure. The size markers used were a mixture of restriction fragments of DNA from bacteriophage X and plasmid pBR322.
CELO strain Ote (13) was inoculated into allantoic cavities of lo-day-old chick embryos and infected allantoic fluid was harvested 4 days later. Infected culture fluids including cells and cell debris or infected allantoic fluids were concentrated by precipitation with polyethylene glycol No. 6000 (10%) and 0.5 M NaCI. The precipitates were suspended in 20 to 50 ml of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0). Virions were purified and DNA was extracted as described (9). DNA sequence analysis was
carried out according to the method of Maxam and Gilbert (1.5). The 3’ termini of intact Ad DNA were labeled by using [a32P]dGTP (Amersham, sp act 2000-3000 Ci/ mmol) and T4 DNA polymerase (20). The 3’-labeled DNA was cleaved with BarnHI for CELO, and EcoRI for ICHV and EAd. The generated fragments were fractionated by agarose gel electrophoresis and the labeled terminal fragments were isolated. The sequence data derived from the 3’ end were confirmed by the data derived from the DNA labeled at the 5’ termini. Before the labeling of the 5’ termini of intact Ad DNA by using [Y-~‘P]ATP and polynucleotide kinase, the Ad DNA was treated with 0.1 N NaOH, 1 mM EDTA at 3’7” for 3 hr, and with bacterial alkaline phosphatase (20, 25).
Figure 1 shows the cleavage patterns after digestion of ICHV, EAd, and CELO DNA with BarnHI and EcoRI. Denisova et al. (4) have reported that CELO DNA was cleaved by EcoRI into seven fragments and the terminal fragments were B and F. In this study, however, EcoRI cleaved the DNA into eight fragments (Fig. 1), and the terminal fragments were F and the smallest fragment H which were found by using with the end-labeled DNA (data not shown). The lengths of the fragments and molecular weight of the three Ad DNAs estimated from the sum of the fragment lengths are shown in Table 1. The molecular weight of ICHV (22.0 X 106) and EAd (23.8-24.5 X 106) are within the range of the molecular weights of human Ad DNAs (10,27), and that of CELO (29.3-30.4 X 106) agrees with the values reported elsewhere (14. 16). Figure 2 shows the nucleotide sequence at the ITR from ICHV, EAd, and CELO. The ITRs of ICHV, EAd, and CELO are 160, 103, and 54 bp in length, respectively. GC contents of ICHV-, EAd-, and CELOITR were 41,67, and 39%, respectively. To compare the nucleotide sequence of ITRs from ICHV, EAd, and CELO with those of ITRs from other Ads, available sequences are compiled in Fig. 2. The first 23 nucleotides are identical from the ends of ICHV and EAd. The highly conserved homologous sequence of
SHORT
4&
COMMUNICATIONS TABLE
1
GALCULRTED~OLE~ULARWEIGHTSOFRESTRICTIONFRAGME~TTSFROMI@WV,EA~,P,ND
-
ICHV Fragment A B c D F F G H 1 J Sum
Ns, (Da)”
EAd
BamHI
EcoRI
15,85oaa* 12,050 5,500 463
21,200 3,610 2,575 2,245 1,750 1,605 930
19,800 7,650 6,600 1,790 765
15,000 13,750 3,940 2,640 2,335
11,500 8,600 5,940 5,290 4,900 3,550 2,065 1,660 1,170 385
16,108 9:lOO 5,220 4$x? 4,880 3,360 3,LOO $5
33,863
33,915
36,605
37,665
45,060
46,725
22.0 x lo6
23.8 X lo6
24.5 X lo6
29.3 x 106
22.0 x 10”
a Base pairs. *Numbers of base ‘Six hundred fifty
pairs represent the mean of duplicate Da per base pair is assumed.
14 bp (nucleotides 9-22) present in several human Ad DNAs (20, 25) is only partially conserved in ICHV and EAd (nucleotides 9-17) and in CELO (nucleotides 9-14). Thus, the hexanucleotide sequence ATAATA (nucleotides 9-14) is present in every ITR. Comparison of ITR sequences of ICHV or EAd with human Ad type 2 shows that the first 17 nucleotides are common among these Ads, whereas ICHV, EAd, and mouse Ad FL commonly share the first 19 nueleotides. This is in contrast to the simian and human Ads (except human Ad4) which share very similar sequences up to about 50 nucleotides. The sequence GCCAATAT between nueleotides 34-41 (or 35-42 in the case of Ad7) is conserved within the ITR of all human Ads except Ad4 (26). Interestingly, this octanucleotide sequence includes “CCAAT,” which has been found upstream from the capping site for several mammalian P-like globin genes and from the TATA box for the sea urchin H3 genes (2, 11). Although “‘CCAAT” is conserved in simian Ad7, it is absent from ICHV, EAd, CELO, and mouse Ad FL as well as from human Ad4. Another interesting structural feature
-
GEL0
EcoRI
BamHI
GEL0 DNAS -
BamHI
ECOIEI
30.4 x IO6
experiments.
of the ITRs of several human Ads except Ad4 is the hexanucleotide seq.uence TGACGT, which is present at a site where the ITR terminates in Ad3,7, or 12, or two nucleotides penultimate to the t~~rn~~~~ tion site of the ITRs of Ad2 and Ad18 (7). Flowever, this bexanucleotide is a Ad4 at that site (26). nucleotides beyond the end of Ad4 DNA, but n This hexanucleotide sequence is ~o~se~v~d within the ITR of simian Ad7 at position 66-71 and in the ITR of I 125-130, several nucleotides the termination site of the If Ads have been evolving origin, avian Ad (CELO) verged from human and animal Ads at an early stage of evolution. ~ubseq~ent~y~ imal Ads diverged from a group consist, of human and simian Ads. Among the animal Ads, mouse Ad might earlier than ICHV and EAd. does not have a homologous other human Ads and simian Ad7 b nueleotide number 22 from the ends. and animal Ads, ICIIV, EAd, and mouse Ad FL do not have any marked ~~rn~~~~r beyond the well-conserved sequence (after
SHORT
COMMUNICATIONS
the 14th nucleotide in CELO, the 23rd in ICHV and EAd, and the 19th in mouse Ad FL). The hexanucleotide sequence ATAATA at position 9-14 might have been conserved in all Ads from an early stage of evolution. One of the possible functions of this sequence may be to serve as a recognition signal for the terminal proteinprecursor (80 k protein), which is involved in the initiation of DNA replication (3). ACKNOWLEDGMENTS This work was partly supported by a grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture, Japan, and by a Grant 56480067, from the same Ministry. REFERENCES 1. ARRAND, J. R., and ROBERTS, R. J., J. Mel Biol. 128,577-594 (1979). 2. BENOIST, C., O’HARE, K. O., BREATHNACH, R., and CHAMBON, P., Nucl. Acids Res. 4, 4371-4389 (1980). 3. CHALLBERG, M. D., OSTROVE, J. M., and KELLY, T. J., J. viral. 41, 265-270 (1982). 4. DENISOVA, T. S., SITNIKOV, B. S., and GHIBADULIN, R. A., Mol. BioZ. (USSR) 13, 1021-1034 (1979). 5. DIJKEMA, R., and DEKKER, B. M. M., Gene 8,7-15 (1979). 6. GARON, C. F., BERRY, K. W., and ROSE, J. A., Proc. Nat. Acad Sci. USA 69, 2391-2395 (1972). 7. GARON, C. F., PARR, R. P., PADMANABHAN, R., ROBINSON, I., GARRISON, J. W., and ROSE, J. A., viro.!Qgy 121,230-239 (1982). 8. GREEN, M., MACKEY, J. K., WOLD, W. S. M., and RIGDEN, P., viro&y 93, 481-492 (1979). 9. GREEN, M., and PIRA, M., Proc. Nat. Acad. Sci. USA 51, 1251-1255 (1964). 10. GREEN, M., PIQA, M., KIMES, R., WENSINK, P. C., MACHATTIE, L. A., and THOMAS, C. A., Proc Nat. Acad. Sci USA 57, 1302-1304 (1967). 11. HENTSCHEL, C. C., and BIRNSTELL, M. L., Cell 25, 301-313 (1981). 12. KAMADA, M., AKIYAMA, Y., SATO, K., and KODERA, S., Japan J. Vet. Sci 39, 661-664 (1977). 1.3. KAWAMURA, H., SATO, T., TSUBAHARA, H., and IsOGAI, S., Nat. Inst. An&. Health C&at. (Tokyo) 3, l-10 (1963). 14. LAVER, W. G., YOUNGHUSBAND, H. B., and WRIGLEY, N. G., virology 45, 598-614 (1971). 15. MAXAM, A. M., and GILBERT, W., Proc. Nat. Acad. Sci. USA 74,560-564 (1977). 16. MOHANTY, S. B., and DLITTA, S. K., “Veterinary Virology,” pp. 3-38. Lea & Febiger, Philadelphia (1981). 17’. REKOSH, D. M. K., RUSSELL, W. C., BELLETT,
SHORT A. J. D., and (1977).
ROBINSON,
COMMUNICATIONS
A. J., CelE 11, 283-295
18. ROBINSON, A. J., YOUNGFIUSBAND, H. B., and BELLETT, A. J. D., firology 56,54-69 (1973). 19. SHINAGAWA, Biophys.
M., and PADMANABHAN, R., B&hem Res. Cmmun. 87, 671-678 (1979).
20. SHINAGAWA, M., and PADMANABHAN, R., Proc. Aead. 5X. USA 77,3831-3835 (1980). 21. SHINAGAWA, M., ETOH, S., and YANAGAWA, rology 66, 161-171 (1975). 22. STEENBERGW, 6, 307-318
P. II., (1979).
and SUSSENBACH,
Nat
R., Vi-
J. S., Gene
d&s
23. SUSSENB~CH, J. S., and JUIJK, M. Z., %rolnyy 84, 509-517 (1978). 24. TEMPLE, M., ANTOINE, G., DELIUS, II., STAML, S., and WINNACKER, E. L., virology 199,1-12 (1982). 25. TOLUN, A., ALESTROM, P., and PETTERSSON, U., Cell 17,705-713 (1979). 26. TOKUNAGA, O., SHINAGAWA, M., and T?ADMAKAEHAN, R., Gene I&321-327 (1982). 27. VAN DER EB, A. J.; and VAN KERSTERN, L. M Biochim Biophys. Acta 129,441-444 (1966). 28. WADELL, G., Intervirology 11, 47-57 (1979). 29. WOLFSON, J., and DRESSLER, D., Proc. Nat. Acad Ski. USA 69,3054-3057 (1972).