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A general approach to construct double deletion mutants of SV40
VIROLOGY
96, 27'7-280(1979)
A General
Approach
to Construct
Double
Deletion
MONIKA K(ZNIG AND CHING-JUH Laboratory National
of Molecular Institutes
Mutants
of SV40
LAI’
Virology, National Cancer Institute, of Health, Bethesda, Maryland 20014
Accepted March 2, 1979
A procedure is described for the construction of double deletion mutants of SV40 from two parental viruses, each carrying a deletion sequence at a specificregion.The construction steps involve heteroduplex formation between two parental DNA molecules, nuclease Sl treatment, and subsequent cloning of the duplexes after infection in the permissive cell. Two such mutants were obtained: One contains both deletions in the late gene region; and the other contains one deletion in the early region and another in the late region. These mutants were derived from their respective deletion mutants as shown by restriction enzyme analysis of their DNA genomes.
We attempted to further delineate viral sequences that are involved in gene expression and to obtain viral mutants that contain desirable sequences of suitable genomic size. In this communication we present a general approach to construct such mutants from two parental mutants, each carrying a deletion at a specific location. This approach can be further extended to obtain mutants containing other physical as well as biological markers from two isogenic parental viruses. For this purpose we have chosen viable deletion mutants to demonstrate the construction of two sets of mutants. The first set of mutant originated from two mutants, both containing a deletion in the late region, one in the leader sequences and the other in the body sequences of the previously mapped 16 S mRNA structure (16). The second set of mutants was constructed from a viable mutant which lacks sequences in the early region, and another viable mutant which lacks sequences in the late leader region. Figure 1 outlines the procedures for construction of such mutants. The circular DNA from each mutant of the parental pair is singly cleaved at a separate site in the genome with different restriction enzymes to obtain their respective linear DNA molecules. (15,lS). Specifically, mutant dl-2303 DNA which 1 Present address: Laboratory of Infectious Diseases, lacks sequences in the late leader region (deletion mapping between 0.725-0.760 unit) NIAID, NIH, Bethesda, Maryland 20014. The circular DNA genome of SV40, consisting of more than 5200 base pairs, can be dissected into approximately two equal halves: one half responsible for the early gene transcripts and the other half for the late gene transcripts (1, 2). Results from marker rescue mapping of temperaturesensitive mutants belonging to different complementation groups suggest the genomic locations of these complementing viral functions (3-6). Further localization of the viral genes was obtained from experiments using defective deletion mutants isolated from higher multiplicity passage infection and from in vitro construction by restriction enzyme excision (Y-9). In addition, viable deletion mutants have been obtained and the deletions mapped in several specific genomic regions (10-B; Lai et al., manuscript in preparation). Experiments from mapping the viral RNA in the early region demonstrated that there is a related region which exhibits a discontinuity (intervening region) in the cytoplasmic transcripts from the lytically infected cells (13,14). In the late segment of the genome, the deletion region of viable mutants includes the templates for the leader RNA sequences of the late cytoplasmic transcripts that are not known to be translated
277
0042-6822/79/090277-04$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
278
from the circular duplexes by electrophoresis on 1.4% agarose gels. The circular DNA molecules were eluted from the gels and used for infection of BSC-1 cells. Several dl-1 dl-2 virus plaques were isolated and their genomes analyzed by restriction enzyme digestion. Figure 2A shows the results of endo R Isolate Form II DNA Hind11 + III digestion of DNAs from both parental mutant viruses and the constructed mutant. As compared to the wildtype DNA digest, mutant dl-2303 is missing the Hin-C and Hin-D fragments and shows Nuclease Sl the appearance of a 17% unit length fragment derived from a fusion of the remaining Hin-C and Hin-D. The other parental virus dl-1017 gave a digest in which Hin-E and Hin-K fragments are absent, and a new 12% unit length fragment from fused Hin-E-K Infect BSC~l Cells is present. The Hind11 + III digest of the constructed mutant dl-2320, shows the absence of all four fragments, Hin-C, -D, -E, and -K, and the presence of their respective fusion fragments (17 and 12%). Thus the Double Deletion Mutants specific absence and the reappearance of the FIG. 1. Scheme of procedures to construct double Hind fragments of dl-2320 are characterisdeletion mutants of SV40. For heteroduplex formation tics of both parental viral genomes. This see Nathans and Lai (197’7). analysis indicates that both deletions present separately in the parents are now combined and reside in the progeny viral ge(Lai et al., manuscript in preparation) was nome. The Hind11 + III analysis shown in cleaved with HaeII, and dl-1017 DNA Fig. 2B presents the results of construction (kindly supplied by Dr. Daniel Nathans; Lai of the second type of mutants derived from et al., manuscript in preparation) which one parent dl-2303 which lacks template selacks sequences in the late body RNA re- quences for the late leader RNA and angion was cleaved with HpaII. The DNA other parent, dl-884, which lacks sequences of a third mutant, dl-884 (kindly supplied in the early region. In comparison to wildby Dr. Thomas Shenk), containing a dele- type DNA digest, the Hind digest of dl-884 tion, in the early region was similarly shows an altered Hin-A fragment (18.5% cleaved with H@I. An equal quantity (5 unit length in contrast to normal 22.5% pug) of the separately cleaved DNA from Hin-A fragment), whereas other fragments each mutant pair was mixed, denatured in of dl-884 remain normal. The Hind cleavage 1 ml of 0.1 M NaOH, and neutralized with pattern of dl-2303 was described earlier. 0.1 ml of 1 M Tris-HCl, pH 7.0. The DNA The progeny virus dl-2330 gave a digestion mixture was briefly reassociated at 68” and pattern characteristic of its parental getreated subsequently with nuclease Sl. The nomes. These characteristic features innuclease Sl treatment removes any unhy- clude missing Hin-A, Hin-C, and Hin-D bridized single-stranded DNA and the sin- fragments and the presence of two new gle-stranded loops present in the circular fragments of sizes equivalent to 18.5 and heteroduplex molecules between two dele- 17% unit length which derived from the tion mutants. Reassociated linear homo- shortened Hin-A and fused Hin-C-D fragduplex DNA molecules are resistant to the ments, respectively. It should be noted that nuclease Sl digestion and were separated the new viable mutant dl-2330 obtained by
0
I
0t
D
SHORT
COMMUNICATIONS
A M
A
--
dl-2303 d1.2320
279
B
dl-1017
dl-2330
dl-884
,;” mb II II* m
G-M
C
D
IiI-J-
g* -
K-
EF
G
J K
FIG. 2. Endo R Hind analysis of parental and progeny viral DNA’s. 32P-labeled DNAs (Form I) from both parents and the constructed progeny viruses were prepared by CsCl-dye centrifugation and digested to completion with Hind11 + III. The digests were separated by electrophoresis on 4% polyacrylamide gels (Danna and Nathans, 1971) and an autoradiogram was made from the dried gels. (A) Two parental viable deletion mutants, dl-2303 and dl-1017, and the progeny mutant dl-2320. (B) Mutants dl-2303 and dl-334 that are used to generate dl-2330. Hind B’ and other smaller minor fragments present in dl-334 were probably derived from a population of contaminating mutants. This population was not selected for mutant construction by this procedure. Wild-type SV40 DNA, (M), is reference standard.
this approach contains only 92.5% of the wild-type genomic information. Taken together, these results indicate that mutants constructed by this procedure contain deletions identical to the ones present in the parental genomes as determined by analysis with restriction enzymes. Furthermore, these progeny viruses also retain the viability that was used for mutant selection. It is conceivable that this approach can be applied to obtain mutants with combinations of other physical and functional markers as well. ACKNOWLEDGMENTS The authors wish to thank Dr. George Khoury for his continuing encouragement, Drs. Daniel Nathans and Thomas Shenk for generously supplying their deletion SV40 mutants.
REFERENCES 1. KHOURY, G., MARTIN, M. A., LEE, T. N. H., DANNA, K. J., and NATHANS, D., J. Mol. Biol. 78,3’77-389 (1973). 2. SAMBROOK,J., SUGDEN, B., KELLER, W., and SHARP, P. A., Proc. Nat. Acad. Sci. USA 70, 3711-3715 (1973).
8. TEGTMEYER, P., and OZER, H. L., J. Viral. 8, 516-524 (1971). 4. CHOU, J. Y., and MARTIN, R. G., J. Viral. 13, 1101-1109 (1974).
5. LAI, C.-J. and NATHANS, D., Virology
60,466-475
(1974).
6. LAI, C.-J. and NATHANS, D., Virology
66, 70-81
(1975).
7. LAI, C. J. and NATHANS, D., Virology 75,335-345 (1976). 8. BROCKMAN,W. W., and NATHANS, D., Proc. Nat. Acad. Sci. USA 71, 942-946 (1974).
280
SHORT
COMMUNICATIONS
9. SCOTT, W. A., BROCKMAN, W. W., and NATHANS, D., Virology 75, 319-334 (19’76). 10. MERTZ, J. E., and BERG, P., Proc. Nat. Acad. Ski. USA 71, 4879-4883 (1974). II. SHENK, T. E., CARBON, J., and BERG, P., J. Viral. 18, 664-671 (1976). 12. FEUNTEUN, J., KRESS, M., GARDES, M., and MONIER, R., Proc. Nat. Acad. Sci. USA 75, 4455-4459 (1978). IS. BERK, A., and SHARP, P., Proc. Nat. Acad. Sci. USA 75, 1274-1278 (1978). I$. CRAWFORD, L. V., COLE, C. N., SMITH, A. E., PAUCHA, E., TEGTMEYER, P., RUNDELL, K.,
15.
16. 17.
18.
and BERG, P., Proc. Nat. Acad. Sci. USA 75, 117-121 (1978). DHAR, R., SUBRAMANIAN, K. N., PAN, J., and WEISSMAN, S. M., Proc. Natl. Acad. Sci. USA 74,827-831 (1977). LAI, C.-J., DHAR, R., and KHOURY, G., Cell 14, 971-982 (1978). NATHANS, D., and LAI, C.-J., In “The Molecular Biology of the Mammalian Genetic Apparatus” (P. 0. Tso, ed.), pp. 269-275. Elsevieri North-Holland, Amsterdam, 1977. DANNA, K. J., and NATHANS, D., Proc. Nat. Acad. Sci. USA 68, 2913-2917 (1971).
96, 27'7-280(1979)
A General
Approach
to Construct
Double
Deletion
MONIKA K(ZNIG AND CHING-JUH Laboratory National
of Molecular Institutes
Mutants
of SV40
LAI’
Virology, National Cancer Institute, of Health, Bethesda, Maryland 20014
Accepted March 2, 1979
A procedure is described for the construction of double deletion mutants of SV40 from two parental viruses, each carrying a deletion sequence at a specificregion.The construction steps involve heteroduplex formation between two parental DNA molecules, nuclease Sl treatment, and subsequent cloning of the duplexes after infection in the permissive cell. Two such mutants were obtained: One contains both deletions in the late gene region; and the other contains one deletion in the early region and another in the late region. These mutants were derived from their respective deletion mutants as shown by restriction enzyme analysis of their DNA genomes.
We attempted to further delineate viral sequences that are involved in gene expression and to obtain viral mutants that contain desirable sequences of suitable genomic size. In this communication we present a general approach to construct such mutants from two parental mutants, each carrying a deletion at a specific location. This approach can be further extended to obtain mutants containing other physical as well as biological markers from two isogenic parental viruses. For this purpose we have chosen viable deletion mutants to demonstrate the construction of two sets of mutants. The first set of mutant originated from two mutants, both containing a deletion in the late region, one in the leader sequences and the other in the body sequences of the previously mapped 16 S mRNA structure (16). The second set of mutants was constructed from a viable mutant which lacks sequences in the early region, and another viable mutant which lacks sequences in the late leader region. Figure 1 outlines the procedures for construction of such mutants. The circular DNA from each mutant of the parental pair is singly cleaved at a separate site in the genome with different restriction enzymes to obtain their respective linear DNA molecules. (15,lS). Specifically, mutant dl-2303 DNA which 1 Present address: Laboratory of Infectious Diseases, lacks sequences in the late leader region (deletion mapping between 0.725-0.760 unit) NIAID, NIH, Bethesda, Maryland 20014. The circular DNA genome of SV40, consisting of more than 5200 base pairs, can be dissected into approximately two equal halves: one half responsible for the early gene transcripts and the other half for the late gene transcripts (1, 2). Results from marker rescue mapping of temperaturesensitive mutants belonging to different complementation groups suggest the genomic locations of these complementing viral functions (3-6). Further localization of the viral genes was obtained from experiments using defective deletion mutants isolated from higher multiplicity passage infection and from in vitro construction by restriction enzyme excision (Y-9). In addition, viable deletion mutants have been obtained and the deletions mapped in several specific genomic regions (10-B; Lai et al., manuscript in preparation). Experiments from mapping the viral RNA in the early region demonstrated that there is a related region which exhibits a discontinuity (intervening region) in the cytoplasmic transcripts from the lytically infected cells (13,14). In the late segment of the genome, the deletion region of viable mutants includes the templates for the leader RNA sequences of the late cytoplasmic transcripts that are not known to be translated
277
0042-6822/79/090277-04$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT COMMUNICATIONS
278
from the circular duplexes by electrophoresis on 1.4% agarose gels. The circular DNA molecules were eluted from the gels and used for infection of BSC-1 cells. Several dl-1 dl-2 virus plaques were isolated and their genomes analyzed by restriction enzyme digestion. Figure 2A shows the results of endo R Isolate Form II DNA Hind11 + III digestion of DNAs from both parental mutant viruses and the constructed mutant. As compared to the wildtype DNA digest, mutant dl-2303 is missing the Hin-C and Hin-D fragments and shows Nuclease Sl the appearance of a 17% unit length fragment derived from a fusion of the remaining Hin-C and Hin-D. The other parental virus dl-1017 gave a digest in which Hin-E and Hin-K fragments are absent, and a new 12% unit length fragment from fused Hin-E-K Infect BSC~l Cells is present. The Hind11 + III digest of the constructed mutant dl-2320, shows the absence of all four fragments, Hin-C, -D, -E, and -K, and the presence of their respective fusion fragments (17 and 12%). Thus the Double Deletion Mutants specific absence and the reappearance of the FIG. 1. Scheme of procedures to construct double Hind fragments of dl-2320 are characterisdeletion mutants of SV40. For heteroduplex formation tics of both parental viral genomes. This see Nathans and Lai (197’7). analysis indicates that both deletions present separately in the parents are now combined and reside in the progeny viral ge(Lai et al., manuscript in preparation) was nome. The Hind11 + III analysis shown in cleaved with HaeII, and dl-1017 DNA Fig. 2B presents the results of construction (kindly supplied by Dr. Daniel Nathans; Lai of the second type of mutants derived from et al., manuscript in preparation) which one parent dl-2303 which lacks template selacks sequences in the late body RNA re- quences for the late leader RNA and angion was cleaved with HpaII. The DNA other parent, dl-884, which lacks sequences of a third mutant, dl-884 (kindly supplied in the early region. In comparison to wildby Dr. Thomas Shenk), containing a dele- type DNA digest, the Hind digest of dl-884 tion, in the early region was similarly shows an altered Hin-A fragment (18.5% cleaved with H@I. An equal quantity (5 unit length in contrast to normal 22.5% pug) of the separately cleaved DNA from Hin-A fragment), whereas other fragments each mutant pair was mixed, denatured in of dl-884 remain normal. The Hind cleavage 1 ml of 0.1 M NaOH, and neutralized with pattern of dl-2303 was described earlier. 0.1 ml of 1 M Tris-HCl, pH 7.0. The DNA The progeny virus dl-2330 gave a digestion mixture was briefly reassociated at 68” and pattern characteristic of its parental getreated subsequently with nuclease Sl. The nomes. These characteristic features innuclease Sl treatment removes any unhy- clude missing Hin-A, Hin-C, and Hin-D bridized single-stranded DNA and the sin- fragments and the presence of two new gle-stranded loops present in the circular fragments of sizes equivalent to 18.5 and heteroduplex molecules between two dele- 17% unit length which derived from the tion mutants. Reassociated linear homo- shortened Hin-A and fused Hin-C-D fragduplex DNA molecules are resistant to the ments, respectively. It should be noted that nuclease Sl digestion and were separated the new viable mutant dl-2330 obtained by
0
I
0t
D
SHORT
COMMUNICATIONS
A M
A
--
dl-2303 d1.2320
279
B
dl-1017
dl-2330
dl-884
,;” mb II II* m
G-M
C
D
IiI-J-
g* -
K-
EF
G
J K
FIG. 2. Endo R Hind analysis of parental and progeny viral DNA’s. 32P-labeled DNAs (Form I) from both parents and the constructed progeny viruses were prepared by CsCl-dye centrifugation and digested to completion with Hind11 + III. The digests were separated by electrophoresis on 4% polyacrylamide gels (Danna and Nathans, 1971) and an autoradiogram was made from the dried gels. (A) Two parental viable deletion mutants, dl-2303 and dl-1017, and the progeny mutant dl-2320. (B) Mutants dl-2303 and dl-334 that are used to generate dl-2330. Hind B’ and other smaller minor fragments present in dl-334 were probably derived from a population of contaminating mutants. This population was not selected for mutant construction by this procedure. Wild-type SV40 DNA, (M), is reference standard.
this approach contains only 92.5% of the wild-type genomic information. Taken together, these results indicate that mutants constructed by this procedure contain deletions identical to the ones present in the parental genomes as determined by analysis with restriction enzymes. Furthermore, these progeny viruses also retain the viability that was used for mutant selection. It is conceivable that this approach can be applied to obtain mutants with combinations of other physical and functional markers as well. ACKNOWLEDGMENTS The authors wish to thank Dr. George Khoury for his continuing encouragement, Drs. Daniel Nathans and Thomas Shenk for generously supplying their deletion SV40 mutants.
REFERENCES 1. KHOURY, G., MARTIN, M. A., LEE, T. N. H., DANNA, K. J., and NATHANS, D., J. Mol. Biol. 78,3’77-389 (1973). 2. SAMBROOK,J., SUGDEN, B., KELLER, W., and SHARP, P. A., Proc. Nat. Acad. Sci. USA 70, 3711-3715 (1973).
8. TEGTMEYER, P., and OZER, H. L., J. Viral. 8, 516-524 (1971). 4. CHOU, J. Y., and MARTIN, R. G., J. Viral. 13, 1101-1109 (1974).
5. LAI, C.-J. and NATHANS, D., Virology
60,466-475
(1974).
6. LAI, C.-J. and NATHANS, D., Virology
66, 70-81
(1975).
7. LAI, C. J. and NATHANS, D., Virology 75,335-345 (1976). 8. BROCKMAN,W. W., and NATHANS, D., Proc. Nat. Acad. Sci. USA 71, 942-946 (1974).
280
SHORT
COMMUNICATIONS
9. SCOTT, W. A., BROCKMAN, W. W., and NATHANS, D., Virology 75, 319-334 (19’76). 10. MERTZ, J. E., and BERG, P., Proc. Nat. Acad. Ski. USA 71, 4879-4883 (1974). II. SHENK, T. E., CARBON, J., and BERG, P., J. Viral. 18, 664-671 (1976). 12. FEUNTEUN, J., KRESS, M., GARDES, M., and MONIER, R., Proc. Nat. Acad. Sci. USA 75, 4455-4459 (1978). IS. BERK, A., and SHARP, P., Proc. Nat. Acad. Sci. USA 75, 1274-1278 (1978). I$. CRAWFORD, L. V., COLE, C. N., SMITH, A. E., PAUCHA, E., TEGTMEYER, P., RUNDELL, K.,
15.
16. 17.
18.
and BERG, P., Proc. Nat. Acad. Sci. USA 75, 117-121 (1978). DHAR, R., SUBRAMANIAN, K. N., PAN, J., and WEISSMAN, S. M., Proc. Natl. Acad. Sci. USA 74,827-831 (1977). LAI, C.-J., DHAR, R., and KHOURY, G., Cell 14, 971-982 (1978). NATHANS, D., and LAI, C.-J., In “The Molecular Biology of the Mammalian Genetic Apparatus” (P. 0. Tso, ed.), pp. 269-275. Elsevieri North-Holland, Amsterdam, 1977. DANNA, K. J., and NATHANS, D., Proc. Nat. Acad. Sci. USA 68, 2913-2917 (1971).