Proc. Natl. Acad. Sci. USA Vol. 74, No. 4, pp. 1631-1634, April 1977
Cell Biology
Mapping of inverted repeated DNA sequences within the genome of simian virus 40 (viral DNA secondary structure/electron microscopy)
MING-TA HSU AND WARREN R. JELINEK The Rockefeller University, New York, New York 10021
Communicated by James E. Darnell, February 2, 1977
ABSTRACT Single-stranded, linear DNA of simian virus 40 (SV40) created by denaturing the endonuclease EcoRI- or Hpa II-generated, linear, double-stranded products from form I DNA of SV40 was analyzed for regions of inverted repeated sequences by visualization with the electron microscope. Six hairpin loops were found at positions 0.11-0.30 (two loops forming a "rabbit ears" structure), 0.47-0.52, 0.63-0.68, 0.700.76, and 0.90-0.96. The nucleotide sequences within all of these inverted repeats may be related since the looped regions can crosshybridize with one another and, thus, the SV40 genome may contain regions of interspersed repeated and unique sequences. The map positions of the 3' and 5' ends of the early and late messenger RNAs, as determined by others, lie within regions of inverted repeated sequences. Previously recorded recombination events that occurred either within the SV40 genome or between SV40 DNA and other genomes have apparently occurred frequently at positions of inverted repeated sequences within the SV40 DNA. The presence of inverted repeated DNA sequences in the genomes of a variety organisms has been described (1-5). Such sequences have also been shown to be present in the RNA of various organisms (6-10), and at least in prokaryotes regions of secondary structure are believed to be involved in posttranscriptional processing of RNA molecules (9, 10). We have determined whether regions of inverted repeated DNA sequences might be located within the genome of a simple eukaryotic virus coincident with the positions at which the ends of the viral messenger RNAs have been mapped. To do this we have used the electron microscope to visualize regions of secondary structure within the denatured DNA of simian virus 40 (SV40) and mapped their positions in the viral genome. MATERIALS AND METHODS SV40 DNA was purified from CV-1 cells infected with a plaque-purified isolate of strain SVS (28) grown at low multiplicities of infection (0.1 plaque-forming unit per cell). The form I viral DNA was isolated by the procedure of Hirt (11), followed by extraction with phenol and chloroform, equilibrium centrifugation in CsCI/ethidium bromide, passage of the DNA over Dowex 50X resin (12), and extensive dialysis. The DNA was cleaved by either endonuclease EcoRI (in 0.1 M Tris, pH 7.4/0.01 M MgCl2) or endonuclease Hpa II (in 0.1 M Tris, pH 7.4/0.01 M MgCl2/1 mM 2-mercaptoethanol) restriction enzyme. After cleavage was complete (i.e., not more than 5% of the intact circular DNA remained), the DNA was heated in a boiling water bath for 2 min in 1 mM Tris, pH 7.4/0.01 M NaCl/0.1 mM EDTA, diluted to 0.1 ,ug/ml in 50% formamide/1 M ammonium acetate/0.1 M Tris, pH 8.5/0.01 M EDTA, spread on a hypophase of 20% formamide/0.01 M ammonium acetate/0.01 M Tris, pH 8.5/1 mM EDTA, and picked up on Parlodian-coated electron microscope grids. The
RE
A
a)
7n0
E
a)
z
Fractional length
RE
B
a) 0
E
50
E z
Fractional length
FIG. 1. Histograms showing the number of molecules containing a region of secondary structure at each 2% interval along the SV40 DNA. Form I SV40 DNA was cleaved with either the EcoRI or the Hpa II restriction endonuclease. The resulting linear double-stranded DNA molecules were then denatured by boiling and spread for visualization by electron microscopy (see Materials and Methods) and regions of secondary structure were scored with respect to their positions on the linear single-stranded DNA. RE, "rabbit ears." (A) Histogram for molecules cleaved by EcoRI; (B) histogram for mole-
Abbreviation: SV40, simian virus 40.
cules cleaved by Hpa II.
1631
1632
Cell Biology: Hsu and Jelinek
Proc. Natl. Acad. Sci. USA 74 (1977)
FIG. 2. Electron micrographs of SV40 single-stranded DNA resulting from cleavage of form I SV40 DNA by either EcoRI or Hpa II. Linear single-stranded SV40 DNA was obtained by treating form I SV40 DNA with either the EcoRI or the Hpa II restriction nuclease and boiling the resulting products, which were then spread for visualization by electron microscopy (see Materials and Methods). (a-e) Molecules resulting from EcoRI cleavage showing hairpin loops; (f-I) molecules resulting from Hpa II cleavage showing hairpin loops; (m-s) molecules resulting from either EcoRI or Hpa II cleavage showing crosshybridization between regions of hairpin loops as well as some hairpin loops. The bars of the micrographs represent 0.2 t1m.
grids were examined in a Philips 201 electron microscope after staining with uranyl acetate and rotary shadowing with platinum-palladium. RESULTS SV40 form I DNA was cleaved once at 0.0/1.0 by the Escherichua coli restriction endonuclease RI (EcoRI) or at 0.73 by the Haemophilis parainfluenza restriction endonuclease II (Hpa II) (13-16), denatured by boiling and spread for observation in the electron microscope as described in Materials and Methods. Ninety-three percent of the molecules contained at least one region of secondary structure. At lower concentrations of ammonium acetate the regions of secondary structure began to be lost, and without the addition of any ammonium acetate no regions of secondary structure could be seen; at higher concentrations of ammonium acetate the molecules were too compacted to be accurately measured. The most easily distin-
guishable structure was a "rabbit ears" that was seen in 10-20% of all molecules at position 0.11-0.30 on the map of the SV40 genome. This genomic position was deduced by determining the distances from the "rabbit ears" structure to the ends of the EcoRI- or the Hpa II-cleaved molecules. The use of two restriction enzymes, each of which cleaves the circular molecule once at a site different from the other, allows the determination not only of the position from the ends of the molecules of a particular structure, but also of the polarity of the resulting linear molecules. The "rabbit ears" in the EcoRI-generated linear molecules began either 0.11 or 0.72 from one end and comprised 0.19 of the entire length of the EcoRI-cleaved DNA. One loop of the "rabbit ears" contained 10.8% of the genome and the other 7.8%. In the linear molecules generated by the Hpa II enzyme, the "rabbit ears" began either at 0.37 or at 0.45 from one end and covered 0.19 of the entire length of the molecule. By convention, the EcoRI enzyme cleaves the SV40 circular DNA at 0.0/1.0; the Hpa II enzyme cleaves at 0.73
Cell Biology: Hsu and jelinek Table 1. Size of inverted
Proc. Natl. Acad. Sci. USA 74 (1977)
relOeats
Loop no.
Genome position
No. of molecules containing loop
RE I II III IV
(0.11-0.30) (0.47-0.52) (0.63-0.69) (0.70-0.76) (0.90-0.96)
150 42 .39 34 62
'0*
'" '
--I-- HP a II
1633
0 AAA.
--_C
Mean size, % of genome 19.01 + 5.50 + 6.66 + 6.40 + 6.23 +
3.22 2.23 2.512.29 2.35 .-di-816
The sizes of the inverted repeated sequences were measured and are expressed here as percentage of the total SV40 genome ±SD.
(13-16). The data presented in Fig. 1 for the position of the "rabbit ears" are consistent with only one interpretation, the "rabbit ears" extend from the map position 0.11 to 0.30. Thus with this position fixed the positions of other regions of secondary structure could then be uniquely established. Additional regions of secondary structure were located in molecules in which the "rabbit ears" could be seen and mapped with respect to the "rabbit ears" and the ends of the molecules created by either the EcoRI or the Hpa II enzyme. Fig. 1A shows a histogram in which the number of EcoRI-cleaved molecules containing a region of secondary structure at each 2% interval on the viral genome is plotted as a function of the fractional length along the linear, single-stranded DNA. Fig. 2a-e contains photographs of such molecules. Likewise, Fig. 1B shows similar data for molecules generated with the Hpa II endonuclease and Fig. 2f-l shows photographs of some of these molecules. We find regions of secondary structures at positions 0.11-0.30 (RE), 0.47-0.53 (I), 0.63-0.68 (II), 0.70-0.76 (III), and 0.90-0.96 (IV). There is a 5% difference between the positions of loops I and II in the EcoRI-cleaved and the Hpa II-cleaved molecules (see Fig. 1) which we attribute to measuring errors in the Hpa ITcleaved molecules. The Hpa IT endonuclease cleaves in the middle of hairpin loop III, creating a short loop at the end of the molecules that is difficult to measure accurately. The regions of secondary structure can take either of two forms; they can form hairpin loops (as shown in Fig. 2a-l) in which the two complementary regions are immediately adjacent to one another with a small "turn around" region between them, or they can crosshybridize with one another to form "stem" regions. Several examples of these crosshybridized configurations are shown in Fig. 2m-s. Table 1 shows the number of molecules containing each of the secondary structures, its position within the SV40 genome, its mean size, expressed as percent of the total genome size, and the standard deviation of the mean size, also expressed as percent of the total genome size. DISCUSSION
Single-stranded SV40 DNA can exist in multiple configurations when spread for visualization by electron microscopy as described here. These configurations appear to be determined by inverted repeated sequences that can either form hairpin loops or can crosshybridize with one another to form large, looped out, single-stranded regions. Apparently all of the regions of hairpin loops described here can crosshybridize with one another. Seven of the 10 possible crosshybridizations between the hairpin loops are shown in Fig. 2m-s, i.e., RE/I, RE/TI, RE/Ill, I/II, I/III, II/IV, and III/IV; we have also observed molecules with the RE/IV, I/IV, and IT/III hybrids (photographs not shown).
..,E46*
o WL~~~~~~~~~~6
En
UO 1: ,£-
FIG. 3. Map of the SV40 genome. The darker continuous circular line represents the SV40 DNA. The darkest arcs represent regions of inverted repeated DNA sequences and are designated RE ("rabbit ears"), I, II, III, and IV. The outermost, discontinuous circular line marks fractional positions on the genome. Immediately internal to this discontinuous line are wavy lines representing the early and late SV40 mRNAs (18, 19); the arrowheads denote the 3' ends of these mRNAs. The regions coding for the known SV40 proteins are designated by the boldface lettering (ref. 4; and P. Berg, personal communication, abstract in the Cold Spring Harbor abstracts of papers presented at the 1976 Tumor Virus Meeting). Inside the circular line representing the SV40 DNA are two concentric circular arrays showing the positions on the SV40 genome that have been mapped as recombination sites within the SV40 DNA (20, 21) (inner of the two circles) and between SV40 DNA and other genomes (22-27) (outermost of the two circles).
The hairpin loops must have only short regions of perfect complementarity since they are readily lost at ammonium acetate concentrations below 1 M when the spreading solution also contains 50% formamide. Probably loop I and the second loop of the "rabbit ears" (clockwise from the EcoRI cut site) are not caused simply by collapse of G-C rich sequentes since these are the regions that are the first to melt in the double-stranded molecules (15, 17) and thus are probably A-T rich. When RNA transcribed from SV40 form I DNA with E. coli RNA polymerase was spread for visualization by electron microscopy, loops were seen even in 50% formamide without the addition of any ammonium acetate (Hsu and Jelinek, unpublished observations), conditions in which no DNA secondary structures could be seen. Perhaps the regions of secondary structure function in RNA where they would form hairpin loops of greater stability than in the DNA. Fig. 3 shows a map of the SV40 genome on which are indicated the positions of the inverted repeated sequences. Also indicated are the positions of the early and late messenger RNA coding regions as determined by May et al. (18) and Khoury et al. (19), as well as the positions of recombination events previously reported to have taken place within the SV40 genome (20, 21) and between SV40 DNA and other DNAs (22-27). We note that the 3' and 5' ends of the SV40 early and late messenger RNAs are located (18, 19) within regions of inverted repeated DNA sequences (Fig. 3). We note also that recombination events have occurred frequently within regions of in-
1634
Cell Biology: Hsu and Jelinek
verted repeated DNA (20, 21), and, finally, that in vkrious defective and nondefective adenovirus-SV40 hybrids (22-27) the inserted SV40 DNA frequently has one or both of its ends'either within or near an inverted repeated sequence on the SV40 genome (Fig. 3). The presence of regions of homology within SV4O DNA has already been predicted by Khoury et al. (20), who proposed tharthe locations of such regions would determine patterns of recombination within the genome of this virus. We thank Joseph Cozzitorto for excellent technical assistance. This work was supported by grants from the National Institutes of Health (CA 19073 tq M-T.H. and Al 12564 to W.R.J.) and the National Science Foundation (BMS 74-18317 to W.R.J.). 1. Wilson, D. A. & Thomas, C. A. (1974) J. Mol. Biol. 84, 115144. 2. Schmid, C. W. & Deininger, P. L. (1975) Cell 6,345-358. 3. Davidson, E., Hough, D., Amenson, C. & Britten, R. (1973) J. Mol. Biol. 77, 1-23. 4. Graham, D., Newfeld, D., Davidson, E. & Britten, R. (1974) Cell 1, 127-137. 5. Schmid, C., Manning, J. & Davidson, N. (1975) Cell 5, 159172. 6. Ryskov, A., Saunders, G., Farshyan, V. & Georgiev, G. (1973) Biochim. Biophys. Acta 312, 152-164. 7. Jelinek, W. & Darnell, J. E. (1972) Proc. Natl. Acad. Sci. USA 69,2537-2541. 8. Jelinek, W., Molloy, G., Fernandez-Munoz,. R., Salditt, M. & Darnell, J. E. (1974) J. Mol. Biol. 82,361-370. 9. Dunn, J. J. & Studier, F. W. (1973) Proc. Natl. Acad. Sci. USA
70,3296-3300. 10. Rosenberg, M., Kramer, R. A. & Steitz, J. (1974) J. Mol. Biol. 89, 777-782.
Proc. Natl. Acad. Sci. USA 74 (1977) 11. Hirt, B. (1967) J. Mol. Biol. 26,365-369. 12. Radloff, R., Bauer, W. & Vinograd, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1514-1521. 13. Morrow, J. F. & Berg, P. (1972) Proc. Natl. Acad. Sci. USA 69, 3365-3369. 14. Sharp, P. A., Sugden, B. & Sambrook, J. (1972) Biochemistry 2, 3055-3063. 15. Mulder, C. & Delius, H. (1972) Proc. Natl. Acad. Sci. USA 69, 3215-3219. 16. Dana, K. J., Sack, G. H. & Nathans, D. (1973) J. Mol. Biol. 78, 363-376. 17. Beard, P., Morrow, J. F. & Berg, P. (1973) J. Virol. 12, 13031313. 18. May, E., Maizel, J. V. & Salzman, N. P. (1977) Proc. Natl. Acad. Sci. USA 74,496-500. 19. Khoury, G., Martin, M. A., Lee, T. N. H., Danna, K. J. & Nathans, D. (1973) J. Mol. Biol. 78,377-389. 20. Khoury, G., Fareed, G. C., Berry, K., Martin, M. A., Lee, T. N. H. & Nathans, D. (1974) J. Mol. Biol. 87,289-301. 21. Mertz, J. E., Carbon, J., Hertzberg, M., Davis, R. W. & Berg, P. (1975) Cold Spring Harbor Symp. Quent. Biol. 39,69-84. 22. Kelley, T. J., Lewis, A. M., Levine, A. S. & Siegel, S. (1974) J. Mol. Biol. 89, 113-126. 23. Kelley, T. J., Lewis, A. M., Levine, A. S. & Siegel, S. (1974) Cold Spring Harbor Symp. Quant. Biol. 39,409-417. 24. Kelley, T. J. (1975) J. Virol. 15, 1267-1272. 25. Lebowitz, P., Kllley, T. J., Nathans, D., Lee, T. N. H. & Lewis, A. M. (1974) Proc. Nat. Acad. Sci. USA 71, 441-445. 26. Brockman, W. W., Lee, T. N. H. & Nathans, D. (1974) Cold Spring Harbor Symp. Quent. Biol. 39, 119-127. 27. Chow, L. T., Bayer, HF W., Tischer, E. G. & Goodman, H. (1974) Cold Spring Harbor Symp. Quent. Biol. 39,109-117. 28. Takemoto, K. K., Kirschstein, R. L. & Habel, K. (1966) J. Bacteriol. 92, 990-994.
Cell Biology
Mapping of inverted repeated DNA sequences within the genome of simian virus 40 (viral DNA secondary structure/electron microscopy)
MING-TA HSU AND WARREN R. JELINEK The Rockefeller University, New York, New York 10021
Communicated by James E. Darnell, February 2, 1977
ABSTRACT Single-stranded, linear DNA of simian virus 40 (SV40) created by denaturing the endonuclease EcoRI- or Hpa II-generated, linear, double-stranded products from form I DNA of SV40 was analyzed for regions of inverted repeated sequences by visualization with the electron microscope. Six hairpin loops were found at positions 0.11-0.30 (two loops forming a "rabbit ears" structure), 0.47-0.52, 0.63-0.68, 0.700.76, and 0.90-0.96. The nucleotide sequences within all of these inverted repeats may be related since the looped regions can crosshybridize with one another and, thus, the SV40 genome may contain regions of interspersed repeated and unique sequences. The map positions of the 3' and 5' ends of the early and late messenger RNAs, as determined by others, lie within regions of inverted repeated sequences. Previously recorded recombination events that occurred either within the SV40 genome or between SV40 DNA and other genomes have apparently occurred frequently at positions of inverted repeated sequences within the SV40 DNA. The presence of inverted repeated DNA sequences in the genomes of a variety organisms has been described (1-5). Such sequences have also been shown to be present in the RNA of various organisms (6-10), and at least in prokaryotes regions of secondary structure are believed to be involved in posttranscriptional processing of RNA molecules (9, 10). We have determined whether regions of inverted repeated DNA sequences might be located within the genome of a simple eukaryotic virus coincident with the positions at which the ends of the viral messenger RNAs have been mapped. To do this we have used the electron microscope to visualize regions of secondary structure within the denatured DNA of simian virus 40 (SV40) and mapped their positions in the viral genome. MATERIALS AND METHODS SV40 DNA was purified from CV-1 cells infected with a plaque-purified isolate of strain SVS (28) grown at low multiplicities of infection (0.1 plaque-forming unit per cell). The form I viral DNA was isolated by the procedure of Hirt (11), followed by extraction with phenol and chloroform, equilibrium centrifugation in CsCI/ethidium bromide, passage of the DNA over Dowex 50X resin (12), and extensive dialysis. The DNA was cleaved by either endonuclease EcoRI (in 0.1 M Tris, pH 7.4/0.01 M MgCl2) or endonuclease Hpa II (in 0.1 M Tris, pH 7.4/0.01 M MgCl2/1 mM 2-mercaptoethanol) restriction enzyme. After cleavage was complete (i.e., not more than 5% of the intact circular DNA remained), the DNA was heated in a boiling water bath for 2 min in 1 mM Tris, pH 7.4/0.01 M NaCl/0.1 mM EDTA, diluted to 0.1 ,ug/ml in 50% formamide/1 M ammonium acetate/0.1 M Tris, pH 8.5/0.01 M EDTA, spread on a hypophase of 20% formamide/0.01 M ammonium acetate/0.01 M Tris, pH 8.5/1 mM EDTA, and picked up on Parlodian-coated electron microscope grids. The
RE
A
a)
7n0
E
a)
z
Fractional length
RE
B
a) 0
E
50
E z
Fractional length
FIG. 1. Histograms showing the number of molecules containing a region of secondary structure at each 2% interval along the SV40 DNA. Form I SV40 DNA was cleaved with either the EcoRI or the Hpa II restriction endonuclease. The resulting linear double-stranded DNA molecules were then denatured by boiling and spread for visualization by electron microscopy (see Materials and Methods) and regions of secondary structure were scored with respect to their positions on the linear single-stranded DNA. RE, "rabbit ears." (A) Histogram for molecules cleaved by EcoRI; (B) histogram for mole-
Abbreviation: SV40, simian virus 40.
cules cleaved by Hpa II.
1631
1632
Cell Biology: Hsu and Jelinek
Proc. Natl. Acad. Sci. USA 74 (1977)
FIG. 2. Electron micrographs of SV40 single-stranded DNA resulting from cleavage of form I SV40 DNA by either EcoRI or Hpa II. Linear single-stranded SV40 DNA was obtained by treating form I SV40 DNA with either the EcoRI or the Hpa II restriction nuclease and boiling the resulting products, which were then spread for visualization by electron microscopy (see Materials and Methods). (a-e) Molecules resulting from EcoRI cleavage showing hairpin loops; (f-I) molecules resulting from Hpa II cleavage showing hairpin loops; (m-s) molecules resulting from either EcoRI or Hpa II cleavage showing crosshybridization between regions of hairpin loops as well as some hairpin loops. The bars of the micrographs represent 0.2 t1m.
grids were examined in a Philips 201 electron microscope after staining with uranyl acetate and rotary shadowing with platinum-palladium. RESULTS SV40 form I DNA was cleaved once at 0.0/1.0 by the Escherichua coli restriction endonuclease RI (EcoRI) or at 0.73 by the Haemophilis parainfluenza restriction endonuclease II (Hpa II) (13-16), denatured by boiling and spread for observation in the electron microscope as described in Materials and Methods. Ninety-three percent of the molecules contained at least one region of secondary structure. At lower concentrations of ammonium acetate the regions of secondary structure began to be lost, and without the addition of any ammonium acetate no regions of secondary structure could be seen; at higher concentrations of ammonium acetate the molecules were too compacted to be accurately measured. The most easily distin-
guishable structure was a "rabbit ears" that was seen in 10-20% of all molecules at position 0.11-0.30 on the map of the SV40 genome. This genomic position was deduced by determining the distances from the "rabbit ears" structure to the ends of the EcoRI- or the Hpa II-cleaved molecules. The use of two restriction enzymes, each of which cleaves the circular molecule once at a site different from the other, allows the determination not only of the position from the ends of the molecules of a particular structure, but also of the polarity of the resulting linear molecules. The "rabbit ears" in the EcoRI-generated linear molecules began either 0.11 or 0.72 from one end and comprised 0.19 of the entire length of the EcoRI-cleaved DNA. One loop of the "rabbit ears" contained 10.8% of the genome and the other 7.8%. In the linear molecules generated by the Hpa II enzyme, the "rabbit ears" began either at 0.37 or at 0.45 from one end and covered 0.19 of the entire length of the molecule. By convention, the EcoRI enzyme cleaves the SV40 circular DNA at 0.0/1.0; the Hpa II enzyme cleaves at 0.73
Cell Biology: Hsu and jelinek Table 1. Size of inverted
Proc. Natl. Acad. Sci. USA 74 (1977)
relOeats
Loop no.
Genome position
No. of molecules containing loop
RE I II III IV
(0.11-0.30) (0.47-0.52) (0.63-0.69) (0.70-0.76) (0.90-0.96)
150 42 .39 34 62
'0*
'" '
--I-- HP a II
1633
0 AAA.
--_C
Mean size, % of genome 19.01 + 5.50 + 6.66 + 6.40 + 6.23 +
3.22 2.23 2.512.29 2.35 .-di-816
The sizes of the inverted repeated sequences were measured and are expressed here as percentage of the total SV40 genome ±SD.
(13-16). The data presented in Fig. 1 for the position of the "rabbit ears" are consistent with only one interpretation, the "rabbit ears" extend from the map position 0.11 to 0.30. Thus with this position fixed the positions of other regions of secondary structure could then be uniquely established. Additional regions of secondary structure were located in molecules in which the "rabbit ears" could be seen and mapped with respect to the "rabbit ears" and the ends of the molecules created by either the EcoRI or the Hpa II enzyme. Fig. 1A shows a histogram in which the number of EcoRI-cleaved molecules containing a region of secondary structure at each 2% interval on the viral genome is plotted as a function of the fractional length along the linear, single-stranded DNA. Fig. 2a-e contains photographs of such molecules. Likewise, Fig. 1B shows similar data for molecules generated with the Hpa II endonuclease and Fig. 2f-l shows photographs of some of these molecules. We find regions of secondary structures at positions 0.11-0.30 (RE), 0.47-0.53 (I), 0.63-0.68 (II), 0.70-0.76 (III), and 0.90-0.96 (IV). There is a 5% difference between the positions of loops I and II in the EcoRI-cleaved and the Hpa II-cleaved molecules (see Fig. 1) which we attribute to measuring errors in the Hpa ITcleaved molecules. The Hpa IT endonuclease cleaves in the middle of hairpin loop III, creating a short loop at the end of the molecules that is difficult to measure accurately. The regions of secondary structure can take either of two forms; they can form hairpin loops (as shown in Fig. 2a-l) in which the two complementary regions are immediately adjacent to one another with a small "turn around" region between them, or they can crosshybridize with one another to form "stem" regions. Several examples of these crosshybridized configurations are shown in Fig. 2m-s. Table 1 shows the number of molecules containing each of the secondary structures, its position within the SV40 genome, its mean size, expressed as percent of the total genome size, and the standard deviation of the mean size, also expressed as percent of the total genome size. DISCUSSION
Single-stranded SV40 DNA can exist in multiple configurations when spread for visualization by electron microscopy as described here. These configurations appear to be determined by inverted repeated sequences that can either form hairpin loops or can crosshybridize with one another to form large, looped out, single-stranded regions. Apparently all of the regions of hairpin loops described here can crosshybridize with one another. Seven of the 10 possible crosshybridizations between the hairpin loops are shown in Fig. 2m-s, i.e., RE/I, RE/TI, RE/Ill, I/II, I/III, II/IV, and III/IV; we have also observed molecules with the RE/IV, I/IV, and IT/III hybrids (photographs not shown).
..,E46*
o WL~~~~~~~~~~6
En
UO 1: ,£-
FIG. 3. Map of the SV40 genome. The darker continuous circular line represents the SV40 DNA. The darkest arcs represent regions of inverted repeated DNA sequences and are designated RE ("rabbit ears"), I, II, III, and IV. The outermost, discontinuous circular line marks fractional positions on the genome. Immediately internal to this discontinuous line are wavy lines representing the early and late SV40 mRNAs (18, 19); the arrowheads denote the 3' ends of these mRNAs. The regions coding for the known SV40 proteins are designated by the boldface lettering (ref. 4; and P. Berg, personal communication, abstract in the Cold Spring Harbor abstracts of papers presented at the 1976 Tumor Virus Meeting). Inside the circular line representing the SV40 DNA are two concentric circular arrays showing the positions on the SV40 genome that have been mapped as recombination sites within the SV40 DNA (20, 21) (inner of the two circles) and between SV40 DNA and other genomes (22-27) (outermost of the two circles).
The hairpin loops must have only short regions of perfect complementarity since they are readily lost at ammonium acetate concentrations below 1 M when the spreading solution also contains 50% formamide. Probably loop I and the second loop of the "rabbit ears" (clockwise from the EcoRI cut site) are not caused simply by collapse of G-C rich sequentes since these are the regions that are the first to melt in the double-stranded molecules (15, 17) and thus are probably A-T rich. When RNA transcribed from SV40 form I DNA with E. coli RNA polymerase was spread for visualization by electron microscopy, loops were seen even in 50% formamide without the addition of any ammonium acetate (Hsu and Jelinek, unpublished observations), conditions in which no DNA secondary structures could be seen. Perhaps the regions of secondary structure function in RNA where they would form hairpin loops of greater stability than in the DNA. Fig. 3 shows a map of the SV40 genome on which are indicated the positions of the inverted repeated sequences. Also indicated are the positions of the early and late messenger RNA coding regions as determined by May et al. (18) and Khoury et al. (19), as well as the positions of recombination events previously reported to have taken place within the SV40 genome (20, 21) and between SV40 DNA and other DNAs (22-27). We note that the 3' and 5' ends of the SV40 early and late messenger RNAs are located (18, 19) within regions of inverted repeated DNA sequences (Fig. 3). We note also that recombination events have occurred frequently within regions of in-
1634
Cell Biology: Hsu and Jelinek
verted repeated DNA (20, 21), and, finally, that in vkrious defective and nondefective adenovirus-SV40 hybrids (22-27) the inserted SV40 DNA frequently has one or both of its ends'either within or near an inverted repeated sequence on the SV40 genome (Fig. 3). The presence of regions of homology within SV4O DNA has already been predicted by Khoury et al. (20), who proposed tharthe locations of such regions would determine patterns of recombination within the genome of this virus. We thank Joseph Cozzitorto for excellent technical assistance. This work was supported by grants from the National Institutes of Health (CA 19073 tq M-T.H. and Al 12564 to W.R.J.) and the National Science Foundation (BMS 74-18317 to W.R.J.). 1. Wilson, D. A. & Thomas, C. A. (1974) J. Mol. Biol. 84, 115144. 2. Schmid, C. W. & Deininger, P. L. (1975) Cell 6,345-358. 3. Davidson, E., Hough, D., Amenson, C. & Britten, R. (1973) J. Mol. Biol. 77, 1-23. 4. Graham, D., Newfeld, D., Davidson, E. & Britten, R. (1974) Cell 1, 127-137. 5. Schmid, C., Manning, J. & Davidson, N. (1975) Cell 5, 159172. 6. Ryskov, A., Saunders, G., Farshyan, V. & Georgiev, G. (1973) Biochim. Biophys. Acta 312, 152-164. 7. Jelinek, W. & Darnell, J. E. (1972) Proc. Natl. Acad. Sci. USA 69,2537-2541. 8. Jelinek, W., Molloy, G., Fernandez-Munoz,. R., Salditt, M. & Darnell, J. E. (1974) J. Mol. Biol. 82,361-370. 9. Dunn, J. J. & Studier, F. W. (1973) Proc. Natl. Acad. Sci. USA
70,3296-3300. 10. Rosenberg, M., Kramer, R. A. & Steitz, J. (1974) J. Mol. Biol. 89, 777-782.
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