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Cloning of the terminal loop of vaccinia virus DNA
VIROLOGY
124, 215-217
(1983)
Cloning of the Terminal DAVID
Loop of Vaccinia
J. PICKUP, DEEPAK BASTIA, AND WOLFGANG K. JOKLIK’ Lk-partment of Microbidogy Medical Received
Center,
and Immunhgy,
Lb-ham,
Septembe-r24,
North
1982;auxpted
An EcoRI fragment of vaccinia virus strain flop” sequence has been cloned in pBR322.
Terminal fragments of the genomes of both vaccinia virus and cowpox virus DNA have been cloned into plasmid vectors. In both cases a nuclease (either Sl nuclease or the vaccinia virus ss DNA-specific nuclease) was used to remove the terminal crosslink prior to cloning, and as a result 40-50 bp comprising all or most of the terminal flip-flop loop sequences were lost (1-3). In this communication we describe the cloning and characterization of an EcoRIgenerated terminal fragment of the DNA of vaccinia virus strain WR. This cloned fragment contains the terminal flip-flop loop sequence. Vaccinia virus strain WR was grown in mouse L fibroblasts which were themselves growing in suspension culture in Eagle’s minimal essential medium (Joklik’s modification, Grand Island Biological Co.) containing 3% fetal calf serum. The virus was purified as described by Joklik (4) and DNA was isolated from it as described by Nevins and Joklik (5) except that after proteinase K digestion the DNA was extracted gently with phenol and chloroform, and then dialyzed overnight against 1 mM EDTA in 10 mM Tris * HCl (pH 8) at 4’. In order to clone the EcoRI-generated terminal fragments of vaccinia DNA, its terminal loops were cleared by digestion for 5 min at 55” with Sl nuclease (50 pg DNA in 250 ~1 of reaction mixture containing 30 mMNaOAc (pH 4.5), 0.3 MNaCl, i To whom
reprint
Virus DNA
requests
should
Duke University
Carolina
27710
&@ember28,
WR DNA
that
1982
contains
its terminal
“flip-
1 mM ZnSO1, 5% glycerol, and 750 units of Sl nuclease). The DNA was then repaired with the Klenow fragment of E. wli DNA polymerase I (Collaborative Research), blunt-end ligated to EcoRI linker, and digested with EcoRI as described by Goodman and MacDonald (6). The DNA fragments were separated by agarose gel electrophoresis, and the terminal fragments extracted from the gel matrix according to the procedure of Vogelstein and Gillespie (7). They were ligated into pBR322 DNA which had been digested with EcoRI and treated with alkaline phosphatase, and the ligated DNA was then used to transform E. coli HBlOl (8). Transformants containing recombinant plasmids were screened by standard methods (9, lo), using as a probe a nick-translated cloned terminal SaZI fragment of vaccinia virus
I 0
1000 moo
3000 4000 SW0 6000 Base pBir*
FIG. 1. Physical map of the cloned terminal fragment of vaccinia virus DNA in pPJ20. The EcoRI site at the left-hand end corresponds to the EcoRI linker ligated to the U-treated end of the viral genome. Mapping was done by standard methods (11,12). Restriction endonuclease fragments of h DNA and pBR322 DNA (13, 14) were used as size standards. The orientation of the cloned EcoRI fragment was determined by comparing its SaWWel/Alul restriction map with that of a cloned end-fragment of vaccinia DNA described by Wittek and Moss (1.5).
be addressed. 215
0042~6822/83/010215-03$03.00/O Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
216
SHORT
COMMUNICATIONS
strain WR DNA kindly provided by Dr. Bernard Moss. A clone containing a 6.4-kbp-long terminal EcoRI fragment of vaccinia virus was isolated and characterized (pPJ20). The SuZI map of this fragment is shown in Fig. 1. Restriction fragments of pPJ20 were subcloned into phage Ml3 vectors mp8 and mp9 (16). Nucleotide sequence analysis was done according to the method of Sanger et
and it then continues on into the blocks of repeated sequences as described by Baroudy et al. (2). In other words, the end of the viral genome corresponds to nucleotides 48-49, and nucleotides 8-48 and 4996 are the two stems of the terminal flipflop region. The manner in which this cloned insert appears to have been generated is illustrated in Fig. 3. The upper part of the figure shows the flip-flop loop (S form) (2). It appears that in the construction of pPJ20 the Sl nuclease cleaved this loop at about nucleotide 90, removing nucleotides 90 and 91. Subsequent repair synthesis by the Klenow fragment of DNA polymerase 1 appears to have been primed from nucleotide 92, generating a strand complementary to the loop and forming a linear duplex intermediate (Fig. 3, lower part). It should be noted that of the two slightly mismatched loop stems near the junction between the flip-flop loop sequence and the nonrepeated constant region (residues l13 and 92-104), pPJ20 contains the strand
al. (17).
The sequence of the cloned region corresponding to the terminus of the viral genome is shown in Fig. 2. As expected, the sequence starts with the EcoRI linker sequence, then follows a symmetrical region (residues 8-48 and 49-83) which represents the linearized 35 terminal bp of the S form of the terminal flip-flop loop (and six inserted unpaired residues in one strand) (2). This is followed by residues 84-96 which represent the internal 13 bp of one strand of the S form of the flip-flop region. After this the sequence is identical to the first nonrepeated constant region,
A-*-C-T.T.A-G-T4~-A-A-~-r-A-~-A.~-A~~-A-~-A-A-~-~-~-r-A~~-A-A-~-~-A-A-~-~-~!~-A-~-~-~-=-Ac~AA-r-~-~-=-A-,. A-A
T-A-A-A-A-T
A-I-A-A-A-A-T-5,
‘T-A’ >
II A-T-C-A-A-T-CTA-T-T-T-A-A-A~~-Ar~-A-~-A-~-A-~-~~A-A-A-A-~~A-~~~-A-A-~-~ 50
60
70
so
:-:
90
100
FIG. 3. Putative mode of generation of the repaired loop sequence in pPJ20. The upper part of the figure shows the sequence of the flip-flop terminal loop (S form) as determined by Baroudy et al. (2). Nucleotides of the flip-flop loop are numbered. The six base pairs at the right are part of the nonrepeated constant region. Beneath this is shown the putative structure of the intermediate formed after treatment of vaccinia DNA with Sl nuclease and repair with the Klenow fragment of DNA polymerase I. The direction and extent of repair synthesis is indicated by the arrow.
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COMMUNICATIONS
complementary to the repair-synthesized strand of the intermediate (see Fig. 2). The cloned terminal flip-flop loop of vaccinia virus DNA is likely to prove a useful reagent. In the course of vaccinia virus DNA replication, various enzymes must act upon both the terminal loop structure and the dimer replicative intermediate structure (2, 18, 19). Moreover, the existence of the flip-flop variants and the precise maintenance of the terminal loop structure indicate that at least one sitespecific nuclease acts upon the loop region. The cloned loop region will be useful both as a substrate for the isolation and characterization of enzymes that interact specifically with the termini of the vaccinia virus genome, and as a substrate for in. vitro studies of the mechanism of vaccinia virus DNA replication. Such studies are currently underway. ACKNOWLEDGMENTS We would like to thank Lily Lou, Dhaval Patel, and Rodger Pryzant for expert technical assistance. This work was supported by Research Grant lRO1 AI 08909. REFERENCES 1. WI~EK, R., BARBOSA, E., COOPER, J. A., GARON, C. F., CHAN, H., and Moss, B., Nature (London) 285, 21-25 (1980).
217
2. BAROUDY, B. M., VENKATESAN, S., and Moss, B., CeU 28,315-324 (1982). S. PICKUP, D. J., BASTIA, D., STONE, H. O., and JOKLIK, W. K., Proc. Nat. Acad Sci USA, in press (1982). 4. JOKLIK, W. K., Virdogy 18,9-18 (1962). 5. NEVINS, J. R., and JOKLIK, W. K., J. Bid Chem. 252, 6930-6938 (1977). 6. GOODMAN, H. M., and MCDONALD, R. J., In “Methods in Enzymology” (R. Wu, ed.), Vol. 68, pp. 75-99. Academic Press, New York (1979). 7. VOGELSTEIN, B., and GIUESPIE, D., Proc. Nat. Acud. Sci USA 76, 615-619 (1979). 8. COHEN, S. N., CHANG, A. C. Y., and Hsu, L., Proc Nat. Ad Sti USA 69,2110-2114 (1972). 9. GRUNSTEIN, M., and HOGNESS, D. S., Proc. Nat. Acad Sci USA 72,3961-3965 (1975). 10. BIRNBOIM, H. C., and DOLY, J., Nuc!.eic Acids Res, 7, 1513-1523 (1979). II. SOUTHERN, E. M., J. MoL Biol. 93,503-517 (1975). 1.6. SMITH, H. O., and BIRNSTIEL, M. L., Nucleic Acids Res. 3.2387-2398 (1976). 1.9. DANIELS, D. L., DEWEIT, J. R., and BLAZER, F. R., J. V+ol 33,390-400 (1980). 14.SUTCLIFFE, J. G., Cdd Spring Harbor Sgmp Quunt. Biol. 43,77-90 (1979). 15. WINK, R., and MOSS, B., CeU 21,277-284 (1980). 16. MESSING, J., CREA, R., and SEEBURG, P. H., Nucleic Acids Res. 9,309-321 (1981). 17. SANGER, F., CO~JLSON, A. R., BARELL, B. G., SMITH, A. J. H., and ROE, B. A., J. MOL BioL 143,161178 (1980). 18. MOYER, R. W., and GRAVES, R. L., Cell 27, 391401 (1981). 19. BAROUDY, B. M., VENKATESAN, S., and Moss, B., Cold Sprint Harbor Symp. C&ant. Bid, in press (1982).
124, 215-217
(1983)
Cloning of the Terminal DAVID
Loop of Vaccinia
J. PICKUP, DEEPAK BASTIA, AND WOLFGANG K. JOKLIK’ Lk-partment of Microbidogy Medical Received
Center,
and Immunhgy,
Lb-ham,
Septembe-r24,
North
1982;auxpted
An EcoRI fragment of vaccinia virus strain flop” sequence has been cloned in pBR322.
Terminal fragments of the genomes of both vaccinia virus and cowpox virus DNA have been cloned into plasmid vectors. In both cases a nuclease (either Sl nuclease or the vaccinia virus ss DNA-specific nuclease) was used to remove the terminal crosslink prior to cloning, and as a result 40-50 bp comprising all or most of the terminal flip-flop loop sequences were lost (1-3). In this communication we describe the cloning and characterization of an EcoRIgenerated terminal fragment of the DNA of vaccinia virus strain WR. This cloned fragment contains the terminal flip-flop loop sequence. Vaccinia virus strain WR was grown in mouse L fibroblasts which were themselves growing in suspension culture in Eagle’s minimal essential medium (Joklik’s modification, Grand Island Biological Co.) containing 3% fetal calf serum. The virus was purified as described by Joklik (4) and DNA was isolated from it as described by Nevins and Joklik (5) except that after proteinase K digestion the DNA was extracted gently with phenol and chloroform, and then dialyzed overnight against 1 mM EDTA in 10 mM Tris * HCl (pH 8) at 4’. In order to clone the EcoRI-generated terminal fragments of vaccinia DNA, its terminal loops were cleared by digestion for 5 min at 55” with Sl nuclease (50 pg DNA in 250 ~1 of reaction mixture containing 30 mMNaOAc (pH 4.5), 0.3 MNaCl, i To whom
reprint
Virus DNA
requests
should
Duke University
Carolina
27710
&@ember28,
WR DNA
that
1982
contains
its terminal
“flip-
1 mM ZnSO1, 5% glycerol, and 750 units of Sl nuclease). The DNA was then repaired with the Klenow fragment of E. wli DNA polymerase I (Collaborative Research), blunt-end ligated to EcoRI linker, and digested with EcoRI as described by Goodman and MacDonald (6). The DNA fragments were separated by agarose gel electrophoresis, and the terminal fragments extracted from the gel matrix according to the procedure of Vogelstein and Gillespie (7). They were ligated into pBR322 DNA which had been digested with EcoRI and treated with alkaline phosphatase, and the ligated DNA was then used to transform E. coli HBlOl (8). Transformants containing recombinant plasmids were screened by standard methods (9, lo), using as a probe a nick-translated cloned terminal SaZI fragment of vaccinia virus
I 0
1000 moo
3000 4000 SW0 6000 Base pBir*
FIG. 1. Physical map of the cloned terminal fragment of vaccinia virus DNA in pPJ20. The EcoRI site at the left-hand end corresponds to the EcoRI linker ligated to the U-treated end of the viral genome. Mapping was done by standard methods (11,12). Restriction endonuclease fragments of h DNA and pBR322 DNA (13, 14) were used as size standards. The orientation of the cloned EcoRI fragment was determined by comparing its SaWWel/Alul restriction map with that of a cloned end-fragment of vaccinia DNA described by Wittek and Moss (1.5).
be addressed. 215
0042~6822/83/010215-03$03.00/O Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
216
SHORT
COMMUNICATIONS
strain WR DNA kindly provided by Dr. Bernard Moss. A clone containing a 6.4-kbp-long terminal EcoRI fragment of vaccinia virus was isolated and characterized (pPJ20). The SuZI map of this fragment is shown in Fig. 1. Restriction fragments of pPJ20 were subcloned into phage Ml3 vectors mp8 and mp9 (16). Nucleotide sequence analysis was done according to the method of Sanger et
and it then continues on into the blocks of repeated sequences as described by Baroudy et al. (2). In other words, the end of the viral genome corresponds to nucleotides 48-49, and nucleotides 8-48 and 4996 are the two stems of the terminal flipflop region. The manner in which this cloned insert appears to have been generated is illustrated in Fig. 3. The upper part of the figure shows the flip-flop loop (S form) (2). It appears that in the construction of pPJ20 the Sl nuclease cleaved this loop at about nucleotide 90, removing nucleotides 90 and 91. Subsequent repair synthesis by the Klenow fragment of DNA polymerase 1 appears to have been primed from nucleotide 92, generating a strand complementary to the loop and forming a linear duplex intermediate (Fig. 3, lower part). It should be noted that of the two slightly mismatched loop stems near the junction between the flip-flop loop sequence and the nonrepeated constant region (residues l13 and 92-104), pPJ20 contains the strand
al. (17).
The sequence of the cloned region corresponding to the terminus of the viral genome is shown in Fig. 2. As expected, the sequence starts with the EcoRI linker sequence, then follows a symmetrical region (residues 8-48 and 49-83) which represents the linearized 35 terminal bp of the S form of the terminal flip-flop loop (and six inserted unpaired residues in one strand) (2). This is followed by residues 84-96 which represent the internal 13 bp of one strand of the S form of the flip-flop region. After this the sequence is identical to the first nonrepeated constant region,
A-*-C-T.T.A-G-T4~-A-A-~-r-A-~-A.~-A~~-A-~-A-A-~-~-~-r-A~~-A-A-~-~-A-A-~-~-~!~-A-~-~-~-=-Ac~AA-r-~-~-=-A-,. A-A
T-A-A-A-A-T
A-I-A-A-A-A-T-5,
‘T-A’ >
II A-T-C-A-A-T-CTA-T-T-T-A-A-A~~-Ar~-A-~-A-~-A-~-~~A-A-A-A-~~A-~~~-A-A-~-~ 50
60
70
so
:-:
90
100
FIG. 3. Putative mode of generation of the repaired loop sequence in pPJ20. The upper part of the figure shows the sequence of the flip-flop terminal loop (S form) as determined by Baroudy et al. (2). Nucleotides of the flip-flop loop are numbered. The six base pairs at the right are part of the nonrepeated constant region. Beneath this is shown the putative structure of the intermediate formed after treatment of vaccinia DNA with Sl nuclease and repair with the Klenow fragment of DNA polymerase I. The direction and extent of repair synthesis is indicated by the arrow.
SHORT
COMMUNICATIONS
complementary to the repair-synthesized strand of the intermediate (see Fig. 2). The cloned terminal flip-flop loop of vaccinia virus DNA is likely to prove a useful reagent. In the course of vaccinia virus DNA replication, various enzymes must act upon both the terminal loop structure and the dimer replicative intermediate structure (2, 18, 19). Moreover, the existence of the flip-flop variants and the precise maintenance of the terminal loop structure indicate that at least one sitespecific nuclease acts upon the loop region. The cloned loop region will be useful both as a substrate for the isolation and characterization of enzymes that interact specifically with the termini of the vaccinia virus genome, and as a substrate for in. vitro studies of the mechanism of vaccinia virus DNA replication. Such studies are currently underway. ACKNOWLEDGMENTS We would like to thank Lily Lou, Dhaval Patel, and Rodger Pryzant for expert technical assistance. This work was supported by Research Grant lRO1 AI 08909. REFERENCES 1. WI~EK, R., BARBOSA, E., COOPER, J. A., GARON, C. F., CHAN, H., and Moss, B., Nature (London) 285, 21-25 (1980).
217
2. BAROUDY, B. M., VENKATESAN, S., and Moss, B., CeU 28,315-324 (1982). S. PICKUP, D. J., BASTIA, D., STONE, H. O., and JOKLIK, W. K., Proc. Nat. Acad Sci USA, in press (1982). 4. JOKLIK, W. K., Virdogy 18,9-18 (1962). 5. NEVINS, J. R., and JOKLIK, W. K., J. Bid Chem. 252, 6930-6938 (1977). 6. GOODMAN, H. M., and MCDONALD, R. J., In “Methods in Enzymology” (R. Wu, ed.), Vol. 68, pp. 75-99. Academic Press, New York (1979). 7. VOGELSTEIN, B., and GIUESPIE, D., Proc. Nat. Acud. Sci USA 76, 615-619 (1979). 8. COHEN, S. N., CHANG, A. C. Y., and Hsu, L., Proc Nat. Ad Sti USA 69,2110-2114 (1972). 9. GRUNSTEIN, M., and HOGNESS, D. S., Proc. Nat. Acad Sci USA 72,3961-3965 (1975). 10. BIRNBOIM, H. C., and DOLY, J., Nuc!.eic Acids Res, 7, 1513-1523 (1979). II. SOUTHERN, E. M., J. MoL Biol. 93,503-517 (1975). 1.6. SMITH, H. O., and BIRNSTIEL, M. L., Nucleic Acids Res. 3.2387-2398 (1976). 1.9. DANIELS, D. L., DEWEIT, J. R., and BLAZER, F. R., J. V+ol 33,390-400 (1980). 14.SUTCLIFFE, J. G., Cdd Spring Harbor Sgmp Quunt. Biol. 43,77-90 (1979). 15. WINK, R., and MOSS, B., CeU 21,277-284 (1980). 16. MESSING, J., CREA, R., and SEEBURG, P. H., Nucleic Acids Res. 9,309-321 (1981). 17. SANGER, F., CO~JLSON, A. R., BARELL, B. G., SMITH, A. J. H., and ROE, B. A., J. MOL BioL 143,161178 (1980). 18. MOYER, R. W., and GRAVES, R. L., Cell 27, 391401 (1981). 19. BAROUDY, B. M., VENKATESAN, S., and Moss, B., Cold Sprint Harbor Symp. C&ant. Bid, in press (1982).