Vol. 29, No. 1
JOURNAL OF VIROLOGY, Jan. 1979, p. 153-160
0022-538X/79/01-0153/08$02.00/0
Altered Restriction Endonuclease Cleavage Pattern of Simian Virus 40 DNA TIKVA VOGEL,* YAAKOV GLUZMAN, AND NAAMA KOHN Department of Virology, Weizmann Institute of Science, Rehovot, Israel Received for publication 8 August 1978
Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101-1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaeIII site at position 0.21 and a defective HinIlI site at 0.655 map units; and (iii) tsC219 carries a new HinlIl site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.
The simian virus 40 (SV40) genome has been well mapped with restriction endonucleases which specifically fragment the DNA (6, 12). By this technique, the mapping of the origin of DNA replication (5), of temperature-sensitive (ts) mutations (15, 22), of transformation properties (1, 10, 13), and of transcription products (14) has been achieved. Recently, restriction fragments have been widely used in DNA sequencing studies (19, 23, 24). The unique property of restriction enzymes, recognition of a specific deoxynucleotide sequence in DNA (17), has been used as a tool to study genetic crosses in various systems (3, 20) and to demonstrate recombination between endogenous and exogenous SV40 genes (25). In those studies, in which the fragments obtained by cleavage of DNAs of individual recombinants were compared with those obtained from the parental viruses, it was possible to distinguish the regions of the recombinant genomes which consist of one parental sequence from those consisting of the other. However, to estimate precisely the borders of the recombination and its frequency, the genomes of the two viruses must have sufficiently different sequences that restriction endonucleases can discriminate between
modifications which affect the cleavage pattern obtained by several restriction enzymes. tsD202, which carries a ts lesion on fragment E of Hin(II + III) at 0.86 to 0.945 map units (15, 22), has a base modification at position 0.9 map units. tsB204, which has a ts lesion on fragment E of Hin(II + III) at 0.985 to 0.06 map units (15), has been shown also to carry base modifications at positions 0.21 and 0.655 map units. tsC219, with a ts lesion within fragment J of Hin(II + III) at 0.06 to 0.105 map units (15), has a base substitution at position 0.15 map units. From each of these ts mutants, we have isolated several revertants which grow as wild type at the nonpermissive temperature. The cleavage pattern of each revertant was identical to the parental ts mutant. MATERIALS AND METHODS Viruses and viral DNA. The SV40 ts mutants
tsD202, tsB204, and tsC219 and their wild-type progenitor strain (designated WT-M) were kindly provided by R. Martin (2). The origin of our SV40 strain 777 was described previously (18). The wild-type revertants D202, B204, and C219 were obtained from plaques isolated from monkey CV1 cells infected and cultivated at 40.5°C (8, 9). To prepare viral DNA, monkey BSC-1 cells were
infected at a multiplicity of approximately 2 to 5 them. at either 33.5°C (for the ts In the present study we describe restriction PFU/cell and incubated viruses). Infected culall or (for 37°C mutants) endonuclease analysis of three ts mutants of tures were labeled at 24 other h postinfection with [3H]SV40 isolated by Chou and Martin (2). The thymidine (5 ,uCi/ml, 24 Ci/mmol) or ["4C]thymidine mutants fall into three different complementa- (0.5 !tCi/ml, 58 mCi/mmol). At 48 to 72 h postinfection, tion groups and are all mapped in the late re- viral DNA was extracted by the Hirt procedure (11), gions of the SV40 genome (2, 15, 16). They carry, and the supercoiled SV40 DNA I was purified by in addition to the well-characterized and ethidium bromide-cesium chloride density gradient mapped ts lesions (15, 16), additional sequence centrifugation as described elsewhere (4). The SV40 153
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DNA I was further purified by sedimentation on 5 to 20% neutral sucrose gradients (4). Restriction endonucleases. The endonucleases Hin(II + III), HinIII, HpaII, HaeII, and HaeIII (17) were purchased from New England Biolabs, Beverly, Mass. The endonuclease Bgl was prepared by an unpublished method of Zain and Roberts of Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (personal communication). The endonuclease BamHI was purified essentially by the technique of Wilson and Young (26). The DNAs (0.4 to 0.7 t.g) were digested with excess restriction endonucleases at 37°C for 4 to 18 h in a buffer solution containing 10 mM Tris-hydrochloride (pH 7.8), 6 mM MgCl2, 6 mM f8-mercaptoethanol, and 50 mM NaCl. Reactions were stopped by addition of EDTA and sodium dodecyl sulfate to 10 mM and 0.5%, respectively. The reaction mixture was then incubated at 37°C for an additional 15 min. Gel electrophoresis. The restriction endonuclease cleavage products were analyzed on 4 or 6% polyacrylamide or 1.4% agarose vertical slab gels (22 by 14 by 0.4 cm) in a running buffer solution containing 40 mM Tris-hydrochloride, 20 mM sodium acetate, and 2 mM EDTA (pH 7.8). Samples of cleaved DNA in 15% sucrose and 0.1% bromophenol blue were applied to each gel slot, and electrophoresis was carried out at 4°C (for the polyacrylamide gels) or at room temperature (for the agarose gels) at the voltages and for the times indicated in the figure legends. Visualization of the unlabeled cleavage products and quantitation of labeled fragments were performed as described in earlier studies (21, 25).
RESULTS Defective HaeM cleavage site on tsD202 and revertant D202. The mutant tsD202 belongs to the D complementation group of the ts mutants of SV40 (2). It is a late mutant with an early phenotype, exhibiting a defective uncoating property at the nonpermissive temperature (12). The tsD lesion is located within HinIlI fragment E, at approximately 0.9 map units from the EcoRI site (Fig. 1) (15, 22). Using endonuclease cleavage techniques, we have been able to show that the HaeIII restriction cleavage pattern of tsD202 differs extensively from that of the progenitor strain WT-M. The DNAs of tsD202 and two standard SV40 strains, WT-M and 777, were digested to completion with HaeIII. The cleavage products were then separated by gel electrophoresis as shown in Fig. 2. HaeIII cleaves SV40 DNA into 10 larger classes of fragments (A through J) and a number of smaller ones. The gel electropherogram of WT-M and 777 DNAs (Fig. 2, slots 1 and 2) shows the 10 major classes, A though J (the minor classes run off the gel under the applied conditions). HaeIII cleavage of SV40 tsD202 (Fig. 2, slot 3) generated nine major classes of fragments; two of the expected classes, H and I, are missing, whereas a new class of
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FIG. 1. Hin, Hae, BamHI, Bgl, HpaII, and HaeII cleavage sites on S V40 DNA. Hin refers to the mixture Hin(II + III); HinIII cleavage sites are designated by arrows. Hae refers to HaeIII. Fractional map lengths indicate relative distances in arbitrary map units from the EcoRI site. Only the major classes of fragments in the Hin and Hae maps are lettered. Adapted from Nathans and Smith (1 7) and Kelly and Nathans (12).
A a
D
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FIG. 2. Hae digestion of SV40 WT-M, 777, tsD202, and revertant D202 DNAs. The conditions of digestion and separation of the products on 4% polyacrylamide gels (10 h, 4°C, 115 V) are described in the text. The cleavage patterns of WT-M, 777, tsD202, and revertant D202 are shown in slots 1, 2, 3, and 4, respectively. The Hae fragment classes are designated A though J. Arrows indicate the location of the new Hae fragment.
fragment (Fig. 2, arrow) is present. These results can be explained by assuming that the tsD202 genome lacks the cleavage site at position 0.9 map units which generated the H and I frag-
SV40 CLEAVAGE PATTERN
VOL. 29, 1979
ments; consequently, H and I migrate as a single fragment marked by the arrow (for additional evidence, see reference 25). The HaeIII cleavage pattern of three independently isolated D202 revertants all displayed a pattern identical to that of the parental tsD202. The HaeIII cleavage pattern of one of the revertants is shown in Fig. 2, slot 4. Defective HaeM site on tsB204 and B204 revertants. The mutant tsB204 belongs to the SV40 B complementation group defective at the nonpermissive temperature in normal formation of the major SV40 capsid protein VP-1 (12). By using the marker rescue technique, the tsB lesion has been mapped within Hin(II + III) fragment F at 0.96 to 0.06 map units (15, 16). The DNAs of tsB204, WT-M, and two independently isolated B204 revertants (designated
1
2
3
4
5
155
333 and 334) were digested to completion with HaeIII. The gel electrophoresis profile of WT-M DNA cleaved by HaeIII displayed the expected 10 major classes of fragments (Fig. 3, slot 3). However, HaeIII cleavage of tsB204 DNA generated only eight of the expected fragments (Fig. 3, slot 1); fragments C and D are missing, whereas a new fragment is present (Fig. 3, third band of slot 1). The assumption that the HaeIII cleavage site between fragments C and D is missing in tsB204 DNA (Fig. 1) would explain these results. This assumption is based on the following considerations. (i) The estimated size of the new HaeIII fragment (940 bp) fits well with the sum of the estimated sizes of the two missing fragments, HaeIII-C and -D (565 and 380 bp, respectively). (ii) When the restriction endonuclease BamHI, which normally cleaves
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J FIG. 3. Hae digestion of SV40 tsB204, WT-M, and two B204 revertants (333 and 334). The DNA preparations of tsB204, WT-M, 333, and 334 were digested with either Hae alone (slots 1, 3, 5, and 7, respectively) or with Hae and BamHI (slots 2, 4, 6, and 8, respectively). The cleavage products were separated on a 4% polyacrylamide slab gel (11 h, 4°C, 200 V). The Hae fragment classes of standard WT-M DNA are designated A through J.
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SV40 DNA at position 0.15 map units (Fig. 1), is added to the HaeIII cleavage products of WTM DNA, HaeIII fragment C (0.105 to 0.21 map units) no longer appears, whereas two fragments of 320 and 240 bp are generated (the two new bands between fragments H and I in slot 4 of Fig. 3). The addition of BamHI to HaeIII cleavage products of tsB2O4 results, as would be expected, in the disappearance of the new HaeIII fragment and its conversion into a small fragment of 240 bp, and a second larger fragment of 700 bp (Fig. 3, slot 2). We have also analyzed the DNAs of two independently isolated B204 revertants; both displayed a HaeIII cleavage pattern similar to their tsB204 parental strain (Fig. 2, slots 5 through 8). Defective HinlIl site on tsB204 and B204 revertants. HinIII cleaved SV40 DNA into six classes of fragments, designated A through F (Fig. 1 and 4). The cleavages of WT-M and tsB204 DNAs by this enzyme show significant differences; whereas the HinIII cleavage of WTM DNA generated the six expected classes of fragments (Fig. 4, slot 8), cleavage of tsB204 with HinIII resulted in only five fragments. Four of
FIG. 4. HinIII digestion of SV40 WT-M and tsB204. The DNA preparations of WT-M and tsB204 were digested either with HinIII alone (slots 8 and 4, respectively), with HinIII and added HpaII (slots 6 and 2), with HinIII and added HaeII (slots 7 and 3), or with HinIII and added Bgl (slots 5 and 1). The cleavage products were separated on a 1.4% agarose slab gel (16 h, 240C, 50 V). The HinIII fragment classes are designated A through F. The arrow indicates the location of the new HinIII fragment.
J. VIROL.
the fragments were identical to HinIII-A, -D, -E, and -F, whereas two of the normal fragments, HinIII-B and -C, are missing and a new large fragment is present (Fig. 4, slot 4, arrow). HinIII-B and -C are included within the new HinIII fragment, presumably due to the lack of a normal HinIII cleavage site at the HinIII-B -C junction (0.655 map units). This conclusion is based on the following observations. (i) The estimated size of the new HinIII fragment (2,300 bp) corresponds well to the sum of the estimated sizes of the two missing fragments, B and C (1,215 and 1,000 bp, respectively). (ii) The new HinIII fragment of tsB204 contains the sites for the restriction enzymes HaeII, HpaII, and Bgl. These enzymes, which cleave DNA of the standard strain WT-M at positions 0.84, 0.735, and 0.67 map units, respectively (Fig. 1 and Fig. 4, slots 7, 6 and 5), also cleave the new HinIII fragment and release cleavage products of the expected mobility (Fig. 2, slots 3, 2, and 1). (iii) Hin(II + III) endonuclease normally cleaves SV40 DNA at multiple sites, generating 11 major classes of fragments, designated Hin(II + III)-A through -K. The cleavage products of a mixture of 3H-labeled tsB204 and "4C-labeled WT-M DNA with Hin(II + III) endonuclease are shown in Fig. 5. Evidently, the fragments Hin(II + III)A and -C are lacking in the tsB204 cleavage products, and a new class of fragment with a higher mobility (Fig. 5, arrow) is present. Its size (approximately 1,900 bp) is equal to the sum of the two missing fragments, Hin(II + III)-A and -C (1,215 and 567 bp, respectively). The HinIll and Hin(II + III) cleavage profiles of three revertants isolated from tsB204 were found to be identical to that of the parental tsB204 (data not shown), exhibiting a defective HinIll site at position 0.655 map units. New HinlI site on tsC219 and C219 revertants. The late mutant tsC219 belongs to the C complementation group of SV40 ts mutants (2). The mutation was mapped within fragment J of Hin(II + III) at 0.06 to 0.105 map units (15, 16). As was already shown in Fig. 4, cleavage of WT-M DNA with the endonuclease HinIII generates the six expected classes of fragments, HinIII-A through -F (Fig. 6, slot 6). However, when tsC219 is cleaved with HinIII, seven classes of fragments are present (Fig. 6, slot 3), of which five are the expected HinIII-B through -F, but the largest class of fragment, HinIII-A, is missing and two new fragments emerge (Fig. 6, third and fourth fragments in slot 3; see arrows). Because the sum of the estimated sizes of the two new fragments (1,080 and 760 bp, respectively) fits well with the size of the missing HinIII fragment A (approximately 1,845 bp), we assume the presence of a new HinIII site on
SV40 CLEAVAGE PATTERN
VOL. 29, 1979 35
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40 60 80 Mobility FIG. 5. Electrophoretic mobilities of Hin(II + III) fragments from WT-M and tsB204. '4C-labeled SV40 WT-M DNA (0) was mixed with 3H-labeled tsB204 DNA (x) and digested with Hin(II + III). The cleavage products were separated by electrophoresis on a 4% polyacrylamide slab gel (18 h, 4°C, 100 V). The gel was sliced, and the distribution of 3H- and '4C-labeled material was determined as described in the text. The Hin(II + III) fragment classes are designated A through K. The arrow indicates the location of the new Hin(II + III) fragment.
HinIII fragment A of tsC219, based on the following data. (i) On cleavage of the standard WTM DNA with HinIII and then with BamHI, HinIII A did not emerge, but two fragments appeared (Fig. 6, third and fourth fragments of slot 5) having respective mobilities similar to those of the two new HinIII fragments of tsC219. (ii) Cleavage of tsC219 DNA with HinIII endonuclease and then with BamHI gave a pattern essentially similar to that obtained originally by cleavage of tsC219 with HinIII, namely, seven fragments. However, a slight decrease in mobility of the fourth fragment was observed (Fig. 6, slot 4), indicating that the BamHI site is present on the fourth fragment and is close to the new HinIII site. (iii) In the Hin(II + III) cleavage pattern obtained by cleavage of a DNA mixture containing 3H-labeled tsC219 and 14C-labeled WT-M (Fig. 7), the Hin(II + III) fragment G of tsC219 (0.105 to 0.175 map units; approximately 375 bp) is missing, and two new fragments are present. One comigrates with Hin(II + III) fragment K (approximately 212 bp), and a second fast-moving fragment (approximately 160 bp; Fig. 7, arrow) is observed. Analysis of four independently isolated tsC219 revertants shows that all carry the new HinlIl site on Hin(II + III) fragment G, similar to the parental tsC219. The cleavage pattern of one of these tsC219 revertants is shown in Fig. 6, slots 1 and 2. From Fig. 6 and 7, it is apparent that the new
HinIII site of tsC219 is approximately 10 bp, distant from the known BamHI site (at approximately 0.15 map units). Other experiments (data not shown) indicated that the third HinIII fragment of tsC219 contains the site for EcoRI. Thus, the relative location of the new HinIII site of tsC219 is on the left of the BamHI cleavage site.
DISCUSSION We have described alterations in restriction endonuclease cleavage patterns of various SV40 ts mutants and their wild-type revertants. We have mapped the altered cleavage sites by cleaving the DNAs of the mutants with several restriction endonucleases and comparing the cleavage products with those of the parental wild-type DNA. The physical location of the ts lesions of the various SV40 ts mutants having already been mapped (15, 16, 22), we have attempted to correlate the known sites of the ts lesions with the altered restriction cleavage sites on the same DNA molecule. Our findings indicate that there is such a correlation in the case of the mutant tsD202, but not for the mutants tsB204 and tsC219. In tsD202, we have shown a defective HaeIII cleavage site at approximately position 0.9 map units, that is, in the same region as the D202 ts lesion (15, 16, 22). However, we have also shown
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J. VIROL.
that the defective HaeIII s,ite at position 0.9 map units was not repaired iri each of three independently isolated D202 irevertants, suggesting that the HaeIII site may be located a few base pairs distant from the cornresponding position of the tsD lesion. Alternativedy, the tsD lesion and u
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FIG. 6. HinIII cleavage of tsC219, revertant C219, and WT-M DNAs. The DNA preparations of WT-M, tsC219, and revertant C219 wwere digested with either HinIII alone (slots 6, 3, and 1, respectively) or with HinIII and BamHI (slots 5, 4, and 2, respectively), The cleavage products were. acrylamide slab gel (16 h, 4 00, 120 V). The fragment classes are designa ted A through F. Arrows indicate the location of the tw o new HinIII fragments.
Hinly
the HaeIII site may overlap, but the repair of the tsD lesion may have been carried out by a base modification at a distant location from the 0.9-map unit position. At the present time, we have no way of distinguishing between these two possibilities. In earlier studies, we were able to demonstrate a repair of the HaeIII cleavage site concomitantly with the rescue of the tsD202 mutation, due to a recombination event between integrated SV40 genome of SV40-transformed monkey cells and the genome of a superinfecting tsD202 (25). In the two other mutants, tsB204 and tsC219, the locations of the ts lesion (15, 16) are remarkably distant from the altered restriction cleavage sites observed in our studies. In tsB204, the ts lesion was mapped on fragment F of Hin(II + III) (0.985 to 0.06 map units), whereas we have shown an altered HaeIII site at the HaeIII-C -* -D junction (0.21 map units) and also an altered HinIII site at the HinIII-B -C -C junction (0.655 map units). In tsC219, the ts lesion was mapped on fragment J of Hin(II + III) (0.06 to 0.105 map units), whereas we have shown a new HinIII site on fragment G of Hin(II + III) (at about 0.15 map units). Several revertants isolated from tsB204 and tsC219 displayed identical restriction patterns to those of their parental ts mutants, suggesting that the altered nucleotide sequences on the DNA molecule, reflected by the altered restriction cleavage sites, might be related to a class of silent mutations which has no substantial effect on viral growth and infectivity. This is interesting, because the altered nucleotide sequences happen to reside in some crucial locations on the SV40 genome, i.e. 0.15,
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FIG. 7. Electrophoretic mobilities of Hin(II + III) fragments trom ts0219 and WT-M. 'H-labeled tsC219 DNA (x) was mixed with '4C-labeled WT-M DNA (@) and digested with Hin(II + III). The cleavage products were separated and quantitated as described in the legend to Fig. 5. The Hin(II + III) fragments are designated A through K. The arrow indicates the location of the smaller fragment of the two new HinIII
fragments.
SV40 CLEAVAGE PATTERN
VOL. 29, 1979
0.21, and 0.655 map units, which correspond respectively to the coding regions for the 3' termini of VP-1 and U-antigen and the 5' termini of the early RNA (12). The possibility should also be kept in mind that more sites on the DNA mutant molecules might have been modified, but were not detected because of the limitation to those sequences which are recognized by the specific restriction enzymes used in this study. The finding of altered nucleotide sequences is not surprising per se, because these SV40 ts mutants have been isolated by mutagenizing the infected virus with hydroxylamine (2), which is known to react predominantly with cytosine residues, inducing the mutagenic transition G (7.An c T(7). Hence, one could envisage the generation of the new HinIII site on position 0.15 of the tsC219 genome. It has been shown already that the nucleotide sequence on the parental wild-type DNA at about position 0.15 map units is GI AGCTT (19); thus, by the mutagenic transition of G -s A caused by hydroxylamine, the known cleavage site for HinIII, Al AGCTT (12), could be generated. DNA sequence analyses also confirm our finding that the normal BamHI recognition sequence is present 10 to 11 bp to the right of the new HinIII site. The availability of various SV40 strains with modified restriction endonuclease cleavage patterns covering all regions of the genome would be highly useful for extending the genetic and physiological analysis of SV40. The altered restriction sites would constitute powerful biochemical markers for detailed studies of genetic crosses and recombination frequencies. New types of restriction fragments would also facilitate sequencing studies of mutants of SV40. We are currently using these SV40 mutants in recombination studies.
5. 6.
7. 8. 9.
10.
11. 12. 13.
14.
15.
16. 17. 18. 19.
ACKNOWLEDGMENTS We thank E. Winocour for his support and encouragement and B. Danovitch, T. Koch, and B. Yakobson for their expert technical assistance. This work was supported in part by Public Health Service contract NOI CP33220 from the National Cancer Institute and by grants from the United States-Israel Binational Science Foundation and the German Science Fund.
1.
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Clements, J. B., R. Cortini, and N. M. Wilkie. 1976. Analysis of Herpes virus DNA substructure by means
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Simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. U.S.A. 68:2913-2917. Danna, K., and D. Nathans. 1972. Bidirectional replication of Simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 69:3097-3100. Danna, K., G. H. Sack, and D. Nathans. 1973. Studies of Simian virus 40 DNA. VII. A cleavage map of the SV40 genome. J. Mol. Biol. 78:363-376. Freese, E., E. Bautz, and E. Bautz-Fruse. 1961. The chemical and mutagenic specificity of hydroxylamine. Proc. Natl. Acad. Sci. U.S.A. 47:845-855. Gluzman, Y., J. Davison, M. Oren, and E. Winocour. 1977. Properties of permissive monkey cells transformed by UV-irradiated simian virus 40. J. Virol. 22:256-266. Gluzman, Y., E. L. Kuff, and E. Winocour. 1977. Recombination between endogenous and exogenous simian virus 40 genes. I. Rescue of a simian virus 40 temperature-sensitive mutant by passage in permissive transformed monkey lines. J. Virol. 24:534-540. Graham, F. L., P. J. Abrahams, C. Mulder, H. L. Heijneker, S. 0. Warnaar, F. A. J. de Vries, W. Fiers, and A. J. van der Eb. 1974. Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and Simian virus 40. Cold Spring Harbor Symp. Quant. Biol. 39:637-650. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. Kelly, T. J., Jr., and D. Nathans. 1977. The genome of Simian virus 40, p. 85-123. In Advances in virus research, vol. 21. Academic Press Inc., New York. Ketner, G., and T. J. Kelly, Jr. 1976. Integrated Simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases. Proc. Natl. Acad. Sci. U.S.A. 73:1102-1106. Khoury, G., M. A. Martin, T. N. H. Lee, K. J. Danna, and D. Nathans. 1973. A map of Simian virus 40 transcription sites expressed in productively infected cells. J. Mol. Biol. 78:377-389. Lai, C. J., and D. Nathans. 1974. Mapping temperaturesensitive mutants of Simian virus 40: rescue of mutants by fragments of DNA. Virology 60:466-475. Lai, C. J., and D. Nathans 1975. A map of temperaturesensitive mutants of Simian virus 40. Virology 66:70-81. Nathans, D., and H. 0. Smith. 1975. Restriction endonucleases in the analysis and restructuring of DNA molecules. Annu. Rev. Biochem. 44:273-293. Oren, M., E. L. Kuff, and E. Winocour. 1976. The presence of common host sequences in different populations of substituted SV40 DNA. Virology 73:419-430. Reddy, V. B., B. Thimmappaya, R. Dhar, K. N. Subramanian, B. S. Zain, J. Pan, P. K. Ghosh, M. L. Celma, and S. M. Weissman. 1978. The genome of Simian virus 40. Science 200:494-502. Sambrook, J., J. Williams, P. A. Sharp, and T. Grodzicker. 1975. Physical mapping of temperature-sensitive mutations of adenoviruses. J. Mol. Biol. 97: 369-390. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agaroseethidium bromide electrophoresis. Biochemistry 12:
3055-3063. 22. Shenk, T. E., C. Rhodes, W. J. Rigby, and P. Berg. 1974. Mapping of mutational alterations in DNA with S, nuclease: the location of deletions, insertions and
temperature-sensitive mutations in SV40. Cold Spring Harbor Symp. Quant. Biol. 39:61-67. 23. Subramanian, N. K., R. Dahr, and S. M. Weissman. 1977. Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G frag-
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ment of SV40 DNA. J. Biol. Chem. 252:355-367. 24. Van Heuverswyn, H., A. Van de Voorde, and W. Fiers. 1977. Nucleotide sequence of SV40 DNA restriction fragment Hin C-Hap 2. Nucleic Acids Res. 4: 1015-1024. 25. Vogel, T., Y. Gluzman, and E. Winocour. 1977. Recom-
J. VIROL. bination between endogenous and exogenous simian virus 40 genes. II. Biochemical evidence for genetic exchange. J. Virol. 24:541-550. 26. Wilson, G. A., and F. F. Young. 1975. Isolation of a sequence-specific endonuclease (Baml) from Bacillus amyloliquefaciens H. J. Mol. Biol. 97:123-125.
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0022-538X/79/01-0153/08$02.00/0
Altered Restriction Endonuclease Cleavage Pattern of Simian Virus 40 DNA TIKVA VOGEL,* YAAKOV GLUZMAN, AND NAAMA KOHN Department of Virology, Weizmann Institute of Science, Rehovot, Israel Received for publication 8 August 1978
Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101-1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaeIII site at position 0.21 and a defective HinIlI site at 0.655 map units; and (iii) tsC219 carries a new HinlIl site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.
The simian virus 40 (SV40) genome has been well mapped with restriction endonucleases which specifically fragment the DNA (6, 12). By this technique, the mapping of the origin of DNA replication (5), of temperature-sensitive (ts) mutations (15, 22), of transformation properties (1, 10, 13), and of transcription products (14) has been achieved. Recently, restriction fragments have been widely used in DNA sequencing studies (19, 23, 24). The unique property of restriction enzymes, recognition of a specific deoxynucleotide sequence in DNA (17), has been used as a tool to study genetic crosses in various systems (3, 20) and to demonstrate recombination between endogenous and exogenous SV40 genes (25). In those studies, in which the fragments obtained by cleavage of DNAs of individual recombinants were compared with those obtained from the parental viruses, it was possible to distinguish the regions of the recombinant genomes which consist of one parental sequence from those consisting of the other. However, to estimate precisely the borders of the recombination and its frequency, the genomes of the two viruses must have sufficiently different sequences that restriction endonucleases can discriminate between
modifications which affect the cleavage pattern obtained by several restriction enzymes. tsD202, which carries a ts lesion on fragment E of Hin(II + III) at 0.86 to 0.945 map units (15, 22), has a base modification at position 0.9 map units. tsB204, which has a ts lesion on fragment E of Hin(II + III) at 0.985 to 0.06 map units (15), has been shown also to carry base modifications at positions 0.21 and 0.655 map units. tsC219, with a ts lesion within fragment J of Hin(II + III) at 0.06 to 0.105 map units (15), has a base substitution at position 0.15 map units. From each of these ts mutants, we have isolated several revertants which grow as wild type at the nonpermissive temperature. The cleavage pattern of each revertant was identical to the parental ts mutant. MATERIALS AND METHODS Viruses and viral DNA. The SV40 ts mutants
tsD202, tsB204, and tsC219 and their wild-type progenitor strain (designated WT-M) were kindly provided by R. Martin (2). The origin of our SV40 strain 777 was described previously (18). The wild-type revertants D202, B204, and C219 were obtained from plaques isolated from monkey CV1 cells infected and cultivated at 40.5°C (8, 9). To prepare viral DNA, monkey BSC-1 cells were
infected at a multiplicity of approximately 2 to 5 them. at either 33.5°C (for the ts In the present study we describe restriction PFU/cell and incubated viruses). Infected culall or (for 37°C mutants) endonuclease analysis of three ts mutants of tures were labeled at 24 other h postinfection with [3H]SV40 isolated by Chou and Martin (2). The thymidine (5 ,uCi/ml, 24 Ci/mmol) or ["4C]thymidine mutants fall into three different complementa- (0.5 !tCi/ml, 58 mCi/mmol). At 48 to 72 h postinfection, tion groups and are all mapped in the late re- viral DNA was extracted by the Hirt procedure (11), gions of the SV40 genome (2, 15, 16). They carry, and the supercoiled SV40 DNA I was purified by in addition to the well-characterized and ethidium bromide-cesium chloride density gradient mapped ts lesions (15, 16), additional sequence centrifugation as described elsewhere (4). The SV40 153
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DNA I was further purified by sedimentation on 5 to 20% neutral sucrose gradients (4). Restriction endonucleases. The endonucleases Hin(II + III), HinIII, HpaII, HaeII, and HaeIII (17) were purchased from New England Biolabs, Beverly, Mass. The endonuclease Bgl was prepared by an unpublished method of Zain and Roberts of Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (personal communication). The endonuclease BamHI was purified essentially by the technique of Wilson and Young (26). The DNAs (0.4 to 0.7 t.g) were digested with excess restriction endonucleases at 37°C for 4 to 18 h in a buffer solution containing 10 mM Tris-hydrochloride (pH 7.8), 6 mM MgCl2, 6 mM f8-mercaptoethanol, and 50 mM NaCl. Reactions were stopped by addition of EDTA and sodium dodecyl sulfate to 10 mM and 0.5%, respectively. The reaction mixture was then incubated at 37°C for an additional 15 min. Gel electrophoresis. The restriction endonuclease cleavage products were analyzed on 4 or 6% polyacrylamide or 1.4% agarose vertical slab gels (22 by 14 by 0.4 cm) in a running buffer solution containing 40 mM Tris-hydrochloride, 20 mM sodium acetate, and 2 mM EDTA (pH 7.8). Samples of cleaved DNA in 15% sucrose and 0.1% bromophenol blue were applied to each gel slot, and electrophoresis was carried out at 4°C (for the polyacrylamide gels) or at room temperature (for the agarose gels) at the voltages and for the times indicated in the figure legends. Visualization of the unlabeled cleavage products and quantitation of labeled fragments were performed as described in earlier studies (21, 25).
RESULTS Defective HaeM cleavage site on tsD202 and revertant D202. The mutant tsD202 belongs to the D complementation group of the ts mutants of SV40 (2). It is a late mutant with an early phenotype, exhibiting a defective uncoating property at the nonpermissive temperature (12). The tsD lesion is located within HinIlI fragment E, at approximately 0.9 map units from the EcoRI site (Fig. 1) (15, 22). Using endonuclease cleavage techniques, we have been able to show that the HaeIII restriction cleavage pattern of tsD202 differs extensively from that of the progenitor strain WT-M. The DNAs of tsD202 and two standard SV40 strains, WT-M and 777, were digested to completion with HaeIII. The cleavage products were then separated by gel electrophoresis as shown in Fig. 2. HaeIII cleaves SV40 DNA into 10 larger classes of fragments (A through J) and a number of smaller ones. The gel electropherogram of WT-M and 777 DNAs (Fig. 2, slots 1 and 2) shows the 10 major classes, A though J (the minor classes run off the gel under the applied conditions). HaeIII cleavage of SV40 tsD202 (Fig. 2, slot 3) generated nine major classes of fragments; two of the expected classes, H and I, are missing, whereas a new class of
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FIG. 1. Hin, Hae, BamHI, Bgl, HpaII, and HaeII cleavage sites on S V40 DNA. Hin refers to the mixture Hin(II + III); HinIII cleavage sites are designated by arrows. Hae refers to HaeIII. Fractional map lengths indicate relative distances in arbitrary map units from the EcoRI site. Only the major classes of fragments in the Hin and Hae maps are lettered. Adapted from Nathans and Smith (1 7) and Kelly and Nathans (12).
A a
D
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FIG. 2. Hae digestion of SV40 WT-M, 777, tsD202, and revertant D202 DNAs. The conditions of digestion and separation of the products on 4% polyacrylamide gels (10 h, 4°C, 115 V) are described in the text. The cleavage patterns of WT-M, 777, tsD202, and revertant D202 are shown in slots 1, 2, 3, and 4, respectively. The Hae fragment classes are designated A though J. Arrows indicate the location of the new Hae fragment.
fragment (Fig. 2, arrow) is present. These results can be explained by assuming that the tsD202 genome lacks the cleavage site at position 0.9 map units which generated the H and I frag-
SV40 CLEAVAGE PATTERN
VOL. 29, 1979
ments; consequently, H and I migrate as a single fragment marked by the arrow (for additional evidence, see reference 25). The HaeIII cleavage pattern of three independently isolated D202 revertants all displayed a pattern identical to that of the parental tsD202. The HaeIII cleavage pattern of one of the revertants is shown in Fig. 2, slot 4. Defective HaeM site on tsB204 and B204 revertants. The mutant tsB204 belongs to the SV40 B complementation group defective at the nonpermissive temperature in normal formation of the major SV40 capsid protein VP-1 (12). By using the marker rescue technique, the tsB lesion has been mapped within Hin(II + III) fragment F at 0.96 to 0.06 map units (15, 16). The DNAs of tsB204, WT-M, and two independently isolated B204 revertants (designated
1
2
3
4
5
155
333 and 334) were digested to completion with HaeIII. The gel electrophoresis profile of WT-M DNA cleaved by HaeIII displayed the expected 10 major classes of fragments (Fig. 3, slot 3). However, HaeIII cleavage of tsB204 DNA generated only eight of the expected fragments (Fig. 3, slot 1); fragments C and D are missing, whereas a new fragment is present (Fig. 3, third band of slot 1). The assumption that the HaeIII cleavage site between fragments C and D is missing in tsB204 DNA (Fig. 1) would explain these results. This assumption is based on the following considerations. (i) The estimated size of the new HaeIII fragment (940 bp) fits well with the sum of the estimated sizes of the two missing fragments, HaeIII-C and -D (565 and 380 bp, respectively). (ii) When the restriction endonuclease BamHI, which normally cleaves
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J FIG. 3. Hae digestion of SV40 tsB204, WT-M, and two B204 revertants (333 and 334). The DNA preparations of tsB204, WT-M, 333, and 334 were digested with either Hae alone (slots 1, 3, 5, and 7, respectively) or with Hae and BamHI (slots 2, 4, 6, and 8, respectively). The cleavage products were separated on a 4% polyacrylamide slab gel (11 h, 4°C, 200 V). The Hae fragment classes of standard WT-M DNA are designated A through J.
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SV40 DNA at position 0.15 map units (Fig. 1), is added to the HaeIII cleavage products of WTM DNA, HaeIII fragment C (0.105 to 0.21 map units) no longer appears, whereas two fragments of 320 and 240 bp are generated (the two new bands between fragments H and I in slot 4 of Fig. 3). The addition of BamHI to HaeIII cleavage products of tsB2O4 results, as would be expected, in the disappearance of the new HaeIII fragment and its conversion into a small fragment of 240 bp, and a second larger fragment of 700 bp (Fig. 3, slot 2). We have also analyzed the DNAs of two independently isolated B204 revertants; both displayed a HaeIII cleavage pattern similar to their tsB204 parental strain (Fig. 2, slots 5 through 8). Defective HinlIl site on tsB204 and B204 revertants. HinIII cleaved SV40 DNA into six classes of fragments, designated A through F (Fig. 1 and 4). The cleavages of WT-M and tsB204 DNAs by this enzyme show significant differences; whereas the HinIII cleavage of WTM DNA generated the six expected classes of fragments (Fig. 4, slot 8), cleavage of tsB204 with HinIII resulted in only five fragments. Four of
FIG. 4. HinIII digestion of SV40 WT-M and tsB204. The DNA preparations of WT-M and tsB204 were digested either with HinIII alone (slots 8 and 4, respectively), with HinIII and added HpaII (slots 6 and 2), with HinIII and added HaeII (slots 7 and 3), or with HinIII and added Bgl (slots 5 and 1). The cleavage products were separated on a 1.4% agarose slab gel (16 h, 240C, 50 V). The HinIII fragment classes are designated A through F. The arrow indicates the location of the new HinIII fragment.
J. VIROL.
the fragments were identical to HinIII-A, -D, -E, and -F, whereas two of the normal fragments, HinIII-B and -C, are missing and a new large fragment is present (Fig. 4, slot 4, arrow). HinIII-B and -C are included within the new HinIII fragment, presumably due to the lack of a normal HinIII cleavage site at the HinIII-B -C junction (0.655 map units). This conclusion is based on the following observations. (i) The estimated size of the new HinIII fragment (2,300 bp) corresponds well to the sum of the estimated sizes of the two missing fragments, B and C (1,215 and 1,000 bp, respectively). (ii) The new HinIII fragment of tsB204 contains the sites for the restriction enzymes HaeII, HpaII, and Bgl. These enzymes, which cleave DNA of the standard strain WT-M at positions 0.84, 0.735, and 0.67 map units, respectively (Fig. 1 and Fig. 4, slots 7, 6 and 5), also cleave the new HinIII fragment and release cleavage products of the expected mobility (Fig. 2, slots 3, 2, and 1). (iii) Hin(II + III) endonuclease normally cleaves SV40 DNA at multiple sites, generating 11 major classes of fragments, designated Hin(II + III)-A through -K. The cleavage products of a mixture of 3H-labeled tsB204 and "4C-labeled WT-M DNA with Hin(II + III) endonuclease are shown in Fig. 5. Evidently, the fragments Hin(II + III)A and -C are lacking in the tsB204 cleavage products, and a new class of fragment with a higher mobility (Fig. 5, arrow) is present. Its size (approximately 1,900 bp) is equal to the sum of the two missing fragments, Hin(II + III)-A and -C (1,215 and 567 bp, respectively). The HinIll and Hin(II + III) cleavage profiles of three revertants isolated from tsB204 were found to be identical to that of the parental tsB204 (data not shown), exhibiting a defective HinIll site at position 0.655 map units. New HinlI site on tsC219 and C219 revertants. The late mutant tsC219 belongs to the C complementation group of SV40 ts mutants (2). The mutation was mapped within fragment J of Hin(II + III) at 0.06 to 0.105 map units (15, 16). As was already shown in Fig. 4, cleavage of WT-M DNA with the endonuclease HinIII generates the six expected classes of fragments, HinIII-A through -F (Fig. 6, slot 6). However, when tsC219 is cleaved with HinIII, seven classes of fragments are present (Fig. 6, slot 3), of which five are the expected HinIII-B through -F, but the largest class of fragment, HinIII-A, is missing and two new fragments emerge (Fig. 6, third and fourth fragments in slot 3; see arrows). Because the sum of the estimated sizes of the two new fragments (1,080 and 760 bp, respectively) fits well with the size of the missing HinIII fragment A (approximately 1,845 bp), we assume the presence of a new HinIII site on
SV40 CLEAVAGE PATTERN
VOL. 29, 1979 35
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40 60 80 Mobility FIG. 5. Electrophoretic mobilities of Hin(II + III) fragments from WT-M and tsB204. '4C-labeled SV40 WT-M DNA (0) was mixed with 3H-labeled tsB204 DNA (x) and digested with Hin(II + III). The cleavage products were separated by electrophoresis on a 4% polyacrylamide slab gel (18 h, 4°C, 100 V). The gel was sliced, and the distribution of 3H- and '4C-labeled material was determined as described in the text. The Hin(II + III) fragment classes are designated A through K. The arrow indicates the location of the new Hin(II + III) fragment.
HinIII fragment A of tsC219, based on the following data. (i) On cleavage of the standard WTM DNA with HinIII and then with BamHI, HinIII A did not emerge, but two fragments appeared (Fig. 6, third and fourth fragments of slot 5) having respective mobilities similar to those of the two new HinIII fragments of tsC219. (ii) Cleavage of tsC219 DNA with HinIII endonuclease and then with BamHI gave a pattern essentially similar to that obtained originally by cleavage of tsC219 with HinIII, namely, seven fragments. However, a slight decrease in mobility of the fourth fragment was observed (Fig. 6, slot 4), indicating that the BamHI site is present on the fourth fragment and is close to the new HinIII site. (iii) In the Hin(II + III) cleavage pattern obtained by cleavage of a DNA mixture containing 3H-labeled tsC219 and 14C-labeled WT-M (Fig. 7), the Hin(II + III) fragment G of tsC219 (0.105 to 0.175 map units; approximately 375 bp) is missing, and two new fragments are present. One comigrates with Hin(II + III) fragment K (approximately 212 bp), and a second fast-moving fragment (approximately 160 bp; Fig. 7, arrow) is observed. Analysis of four independently isolated tsC219 revertants shows that all carry the new HinlIl site on Hin(II + III) fragment G, similar to the parental tsC219. The cleavage pattern of one of these tsC219 revertants is shown in Fig. 6, slots 1 and 2. From Fig. 6 and 7, it is apparent that the new
HinIII site of tsC219 is approximately 10 bp, distant from the known BamHI site (at approximately 0.15 map units). Other experiments (data not shown) indicated that the third HinIII fragment of tsC219 contains the site for EcoRI. Thus, the relative location of the new HinIII site of tsC219 is on the left of the BamHI cleavage site.
DISCUSSION We have described alterations in restriction endonuclease cleavage patterns of various SV40 ts mutants and their wild-type revertants. We have mapped the altered cleavage sites by cleaving the DNAs of the mutants with several restriction endonucleases and comparing the cleavage products with those of the parental wild-type DNA. The physical location of the ts lesions of the various SV40 ts mutants having already been mapped (15, 16, 22), we have attempted to correlate the known sites of the ts lesions with the altered restriction cleavage sites on the same DNA molecule. Our findings indicate that there is such a correlation in the case of the mutant tsD202, but not for the mutants tsB204 and tsC219. In tsD202, we have shown a defective HaeIII cleavage site at approximately position 0.9 map units, that is, in the same region as the D202 ts lesion (15, 16, 22). However, we have also shown
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that the defective HaeIII s,ite at position 0.9 map units was not repaired iri each of three independently isolated D202 irevertants, suggesting that the HaeIII site may be located a few base pairs distant from the cornresponding position of the tsD lesion. Alternativedy, the tsD lesion and u
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FIG. 6. HinIII cleavage of tsC219, revertant C219, and WT-M DNAs. The DNA preparations of WT-M, tsC219, and revertant C219 wwere digested with either HinIII alone (slots 6, 3, and 1, respectively) or with HinIII and BamHI (slots 5, 4, and 2, respectively), The cleavage products were. acrylamide slab gel (16 h, 4 00, 120 V). The fragment classes are designa ted A through F. Arrows indicate the location of the tw o new HinIII fragments.
Hinly
the HaeIII site may overlap, but the repair of the tsD lesion may have been carried out by a base modification at a distant location from the 0.9-map unit position. At the present time, we have no way of distinguishing between these two possibilities. In earlier studies, we were able to demonstrate a repair of the HaeIII cleavage site concomitantly with the rescue of the tsD202 mutation, due to a recombination event between integrated SV40 genome of SV40-transformed monkey cells and the genome of a superinfecting tsD202 (25). In the two other mutants, tsB204 and tsC219, the locations of the ts lesion (15, 16) are remarkably distant from the altered restriction cleavage sites observed in our studies. In tsB204, the ts lesion was mapped on fragment F of Hin(II + III) (0.985 to 0.06 map units), whereas we have shown an altered HaeIII site at the HaeIII-C -* -D junction (0.21 map units) and also an altered HinIII site at the HinIII-B -C -C junction (0.655 map units). In tsC219, the ts lesion was mapped on fragment J of Hin(II + III) (0.06 to 0.105 map units), whereas we have shown a new HinIII site on fragment G of Hin(II + III) (at about 0.15 map units). Several revertants isolated from tsB204 and tsC219 displayed identical restriction patterns to those of their parental ts mutants, suggesting that the altered nucleotide sequences on the DNA molecule, reflected by the altered restriction cleavage sites, might be related to a class of silent mutations which has no substantial effect on viral growth and infectivity. This is interesting, because the altered nucleotide sequences happen to reside in some crucial locations on the SV40 genome, i.e. 0.15,
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FIG. 7. Electrophoretic mobilities of Hin(II + III) fragments trom ts0219 and WT-M. 'H-labeled tsC219 DNA (x) was mixed with '4C-labeled WT-M DNA (@) and digested with Hin(II + III). The cleavage products were separated and quantitated as described in the legend to Fig. 5. The Hin(II + III) fragments are designated A through K. The arrow indicates the location of the smaller fragment of the two new HinIII
fragments.
SV40 CLEAVAGE PATTERN
VOL. 29, 1979
0.21, and 0.655 map units, which correspond respectively to the coding regions for the 3' termini of VP-1 and U-antigen and the 5' termini of the early RNA (12). The possibility should also be kept in mind that more sites on the DNA mutant molecules might have been modified, but were not detected because of the limitation to those sequences which are recognized by the specific restriction enzymes used in this study. The finding of altered nucleotide sequences is not surprising per se, because these SV40 ts mutants have been isolated by mutagenizing the infected virus with hydroxylamine (2), which is known to react predominantly with cytosine residues, inducing the mutagenic transition G (7.An c T(7). Hence, one could envisage the generation of the new HinIII site on position 0.15 of the tsC219 genome. It has been shown already that the nucleotide sequence on the parental wild-type DNA at about position 0.15 map units is GI AGCTT (19); thus, by the mutagenic transition of G -s A caused by hydroxylamine, the known cleavage site for HinIII, Al AGCTT (12), could be generated. DNA sequence analyses also confirm our finding that the normal BamHI recognition sequence is present 10 to 11 bp to the right of the new HinIII site. The availability of various SV40 strains with modified restriction endonuclease cleavage patterns covering all regions of the genome would be highly useful for extending the genetic and physiological analysis of SV40. The altered restriction sites would constitute powerful biochemical markers for detailed studies of genetic crosses and recombination frequencies. New types of restriction fragments would also facilitate sequencing studies of mutants of SV40. We are currently using these SV40 mutants in recombination studies.
5. 6.
7. 8. 9.
10.
11. 12. 13.
14.
15.
16. 17. 18. 19.
ACKNOWLEDGMENTS We thank E. Winocour for his support and encouragement and B. Danovitch, T. Koch, and B. Yakobson for their expert technical assistance. This work was supported in part by Public Health Service contract NOI CP33220 from the National Cancer Institute and by grants from the United States-Israel Binational Science Foundation and the German Science Fund.
1.
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Clements, J. B., R. Cortini, and N. M. Wilkie. 1976. Analysis of Herpes virus DNA substructure by means
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Simian virus 40 DNA by restriction endonuclease of Hemophilus influenzae. Proc. Natl. Acad. Sci. U.S.A. 68:2913-2917. Danna, K., and D. Nathans. 1972. Bidirectional replication of Simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 69:3097-3100. Danna, K., G. H. Sack, and D. Nathans. 1973. Studies of Simian virus 40 DNA. VII. A cleavage map of the SV40 genome. J. Mol. Biol. 78:363-376. Freese, E., E. Bautz, and E. Bautz-Fruse. 1961. The chemical and mutagenic specificity of hydroxylamine. Proc. Natl. Acad. Sci. U.S.A. 47:845-855. Gluzman, Y., J. Davison, M. Oren, and E. Winocour. 1977. Properties of permissive monkey cells transformed by UV-irradiated simian virus 40. J. Virol. 22:256-266. Gluzman, Y., E. L. Kuff, and E. Winocour. 1977. Recombination between endogenous and exogenous simian virus 40 genes. I. Rescue of a simian virus 40 temperature-sensitive mutant by passage in permissive transformed monkey lines. J. Virol. 24:534-540. Graham, F. L., P. J. Abrahams, C. Mulder, H. L. Heijneker, S. 0. Warnaar, F. A. J. de Vries, W. Fiers, and A. J. van der Eb. 1974. Studies on in vitro transformation by DNA and DNA fragments of human adenoviruses and Simian virus 40. Cold Spring Harbor Symp. Quant. Biol. 39:637-650. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. Kelly, T. J., Jr., and D. Nathans. 1977. The genome of Simian virus 40, p. 85-123. In Advances in virus research, vol. 21. Academic Press Inc., New York. Ketner, G., and T. J. Kelly, Jr. 1976. Integrated Simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases. Proc. Natl. Acad. Sci. U.S.A. 73:1102-1106. Khoury, G., M. A. Martin, T. N. H. Lee, K. J. Danna, and D. Nathans. 1973. A map of Simian virus 40 transcription sites expressed in productively infected cells. J. Mol. Biol. 78:377-389. Lai, C. J., and D. Nathans. 1974. Mapping temperaturesensitive mutants of Simian virus 40: rescue of mutants by fragments of DNA. Virology 60:466-475. Lai, C. J., and D. Nathans 1975. A map of temperaturesensitive mutants of Simian virus 40. Virology 66:70-81. Nathans, D., and H. 0. Smith. 1975. Restriction endonucleases in the analysis and restructuring of DNA molecules. Annu. Rev. Biochem. 44:273-293. Oren, M., E. L. Kuff, and E. Winocour. 1976. The presence of common host sequences in different populations of substituted SV40 DNA. Virology 73:419-430. Reddy, V. B., B. Thimmappaya, R. Dhar, K. N. Subramanian, B. S. Zain, J. Pan, P. K. Ghosh, M. L. Celma, and S. M. Weissman. 1978. The genome of Simian virus 40. Science 200:494-502. Sambrook, J., J. Williams, P. A. Sharp, and T. Grodzicker. 1975. Physical mapping of temperature-sensitive mutations of adenoviruses. J. Mol. Biol. 97: 369-390. Sharp, P. A., B. Sugden, and J. Sambrook. 1973. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agaroseethidium bromide electrophoresis. Biochemistry 12:
3055-3063. 22. Shenk, T. E., C. Rhodes, W. J. Rigby, and P. Berg. 1974. Mapping of mutational alterations in DNA with S, nuclease: the location of deletions, insertions and
temperature-sensitive mutations in SV40. Cold Spring Harbor Symp. Quant. Biol. 39:61-67. 23. Subramanian, N. K., R. Dahr, and S. M. Weissman. 1977. Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G frag-
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ment of SV40 DNA. J. Biol. Chem. 252:355-367. 24. Van Heuverswyn, H., A. Van de Voorde, and W. Fiers. 1977. Nucleotide sequence of SV40 DNA restriction fragment Hin C-Hap 2. Nucleic Acids Res. 4: 1015-1024. 25. Vogel, T., Y. Gluzman, and E. Winocour. 1977. Recom-
J. VIROL. bination between endogenous and exogenous simian virus 40 genes. II. Biochemical evidence for genetic exchange. J. Virol. 24:541-550. 26. Wilson, G. A., and F. F. Young. 1975. Isolation of a sequence-specific endonuclease (Baml) from Bacillus amyloliquefaciens H. J. Mol. Biol. 97:123-125.
