Genome maps of simian virus 40 defectives propagated in human glioblastoma cells

VIROLOGY

87, 120-129 (1978)

Genome Maps of Simian Virus 40 Defectives Propagated in Human Glioblastoma Cells DANA CARROLL* AND FRANK J . O'NEILL* t • f 'Departments of Microbiology and tPathology, University of Utah Medical Center and tResearch Service, Veterans Administration Hospital, Salt Lake City, Utah 84132 Accepted February 2, 1978 The DNA sequences present in four defective SV40 genomes propagated on human glioblastoma cells (A172) have been characterized by analysis of restriction enzyme digests and by hybridization to the wild-type genome . Like defectives grown on monkey cells, these molecules are 10 to 20% shorter than wild-type DNA and contain reiterations of specific segments of the nondefective genome, including multiple copies of the viral replication origin . However, unlike the monkey cell defectives, those grown on A172 cells have also retained multiple copies of the region of viral DNA around the BamHI site at map position 0.15 . These defectives consist only of sequences derived from SV40 and contain no detectable host cell sequencesINTRODUCTION

Defective SV40 genomes accumulated during high multiplicity passage on monkey cells have been characterized previously . At early passages, variants appear which contain duplications of segments including the viral replication origin and compensating deletions of other sequences (Mertz et al., 1975 ; Brockman et al., 1975b) . Upon repeated passage, simpler genomes arise which contain several copies of the replication origin and often some sequences derived from the host cell genome (Lee et al., 1975) . The replication origin appears to be the only cis-acting requirement for propagation of the defectives, and its presence in multiple copies is very likely responsible for the competitive advantage over standard virus which they display in high multiplicity infections . In the preceding paper (O'Neill and Carroll, 1978), we described the rapid accumulation of defective genomes during low multiplicity growth of SV40 on human glioblastoma (Al 72) cells . Left in abeyance was the molecular nature of these defective genomes . In this report, we demonstrate that the defectives consist entirely of sequences derived from SV40. We have deduced physical maps of these defectives and shown 120 0042-6822/78/0871-0120$02.00/0 Copyright o 1978 by Academic Press, Inc . All rights of reproduction in any form reserved.

which regions of the nondefective genome are present . The defectives resemble those generated on high multiplicity passage in monkey cells in being composed of reiterations of a small portion of the parent genome, including the normal replication origin. In addition, these A172-grown variants all contain a unique SV40 sequence from the opposite side of the genome . MATERIALS AND METHODS

Materials. Escherichia colt DNA polym-

erase I was purchased from BoehringerMannheim. [a- 32 P]TTP and [3H]TTP were from New England Nuclear Corporation . The restriction enzymes HindIII, EcoRI, HaeII, and HaeIII were prepared according to published procedures . (Smith, 1974; Greene et al., 1974 ; Roberts et al., 1975) . BamHI and HpaII were purchased from New England BioLabs, (Beverley, Massachusetts) . Bacteriophage and bacterial plasmid DNAs used as molecular weight markers were isolated by standard procedures . Seakem agarose (ME) was from Marine Colloids, Inc . (Rockland, Maine) ; acrylamide and bisacrylamide were from Eastman. Other chemicals were reagent grade and were purchased from various suppliers . OX174 DNA was kindly provided by Dr . Igor Dawid .

121

CENOME MAPS OF SV40 DEFECTIVES

Viral DNAs. The cell lines used and the propagation of viruses are described in the preceding paper (O'Neill and Carroll, 1978) . The defectives used in this study were grown exclusively on A172 cells for passage and for DNA isolation. They were derived from a nonplaque-purified large-plaque stock of SV40. The original virus stock, grown on TC-7 cells, was used as the source on nondefective viral DNA . Viral DNAs were prepared from Hirt supernates (Hirt, 1967) of infected cells . Electrophoresis. The electrophoresis of viral DNAs or their restriction fragments in agarose or composite agarose-polyacrylamide gels was as described previously (Carroll and Brown, 1976 ; O'Neill and Carroll, 1978) . DNA was recovered from specific bands in 1% agarose gels by homogenization of gel slices in 0 .15 M NaCl, 0 .05 M Tris, and 6 mM EDTA (pH 7.9), leaving them at room temperature overnight, and spinning out the agarose at 20,000 g for 30 min in a Beckman SW 50 .1 rotor. The supernate was extracted twice with water-saturated phenol, and the DNA was precipitated twice from 63% ethanol . Labeling of viral DNAs . Radioactive isotopes were incorporated into purified viral DNAs by nick translation with E. coli DNA polymerise I (Maniatis et al. 1975, 1976) . Specific activities of approximately 5 x 10 6 cpm/µg were achieved with [32P]TTP and 1 .5 x 106 cpm/µg with [3 H]TTP . Blot hybridizations. Restriction fragments of SV40 DNA were transferred from 2% agarose or composite 0 .5% agarose, 2 .0% polyacrylamide slab gels to Millipore filter strips as described by Southern (1975) . Although the composite gels gave sharper bands after hybridization, the efficiency of transfer of DNA from these gels was much lower than from 2% agarose and quite variable . Hybridizations were carried out overnight at 68° in 6x SSC, 1 mM EDTA, and 0.5% SDS (Botchan et al., 1976) . After washing and drying, the filters were used to expose X-ray film, either directly (`2 P) or after impregnation with PPO for fluorography (Southern, 1975) . Electron microscopy. The procedures were similar to those described by Davis et al. (1971) . For heteroduplex formation,

nondefective and defective DNAs were mixed at a slight weight excess of defective DNA, cleaved with the appropriate restriction enzyme(s), and then ethanol precipitated . The precipitate was redissolved in 95% formamide and 0.01 M EDTA (pH 8 .4) and heated briefly at 40 to 50° to ensure denaturation of the DNAs . The sample was diluted with an equal volume of 0 .2 M Tris and 0 .01 M EDTA, (pH 8.25), bringing the final total DNA concentration to approximately 2 .5 µg/ml, and annealing was allowed to continue for 1 hr at room temperature. Spreadings of native DNAs and heteroduplexes were from 50% formamide, 0 .1 M Tris, 0.01 M EDTA (pH 8.5) and 75 µg of cytochrome c/ml onto a hypophase of 20% formamide, 0 .01 M Tris, and 0 .001 M EDTA (pH 8.5) . Length standards were double-stranded pMB9 DNA (Rodriguez et al., 1975) (5480 bp,' calibrated against T7 DNA : Freifelder, 1970) and single-stranded X 174 DNA (5375 bases ; Sanger et al., 1977) . Photographs were taken on a Siemens Elmiskop 1A, Tracings were made from projected EM negatives, and DNA lengths were measured with a Numonics graphics calculator . RESULTS

Isolation of Unique Defective Genomes Passage of SV40 on A172 cells leads to the accumulation of a wide variety of defective genomes (O'Neill and Carroll, 1978) . To purify particular genomes for study, we isolated DNA from prominent bands cut from preparative 1% agarose slab gels . The sources of the DNAs isolated are illustrated in Fig. 1 . Bands D2 and D3 are from the sixth passage of nonplaque-purified SV40 on A172 cells; band D4 was the predominant species from an attempted plaque purification of a defective. Their origins are described in greater detail in the preceding paper (O'Neill and Carroll, 1978) . D2 is, in fact, a mixture of two DNAs, as will be demonstrated below. D3 and D4 are unique species . By comparison with electrophoretic mobilities of nondefective SV40 and bacterial plasmid DNAs, the sizes of these 'Abbreviation used : bp, base pairs.

1 22

CARROLL AND O'NEILL

-wt D2 04 D3

Flu . 1 . DNA bands isolated from preparative agarose gels for the preparation of pure defective species . Lane 1 contains the NPP SV40/A172 P6 Hirt supernate DNA and Lane 3 contains a plaque-purified variant DNA (Plaque 3, O'Neill and Carroll, 1978) . Electrophoresis was from top to bottom in . 10% agarose .

defectives are D2, 4260 bp ; D3, 3550 bp ; D4, 3970 bp. Restriction Enzyme Digestions of the Defectives The standard SV40 we have used is a large-plaque isolate (Estes et al., 1971) . Sites for a number of restriction enzymes appear to have the same locations in this genome as in that of the better-characterized small-plaque isolate (see, e .g ., Botchan et al ., 1976; Subramanian et al ., 1977) . However, our large-plaque wild type has two sites for Haell, one at map position 0 .835, as in the small-plaque species, the other of which we have mapped at position 0 .71

(data not shown) . This is accompanied by an increase of 15 bp, compared to our triply plaque-purified SV40 (O'Neill and Carroll, 1978), in the size of restriction fragments containing this region, e .g ., HindIIIC and HaeIIIG. Variations in this region are common among different strains of the virus (Nathans and Danna, 1972) . The locations of sites for the restriction enzymes used in this study are pictured in Figs . 3, 5, and 7. Digests of the isolated defective DNAs with several restriction enzymes are presented in Fig. 2 . The observation that the same multiband patterns are obtained with more than one enzyme is an immediate indication that the defectives are composed of reiterated sequences . EcoRI digestion of D2 yielded two sharp bands at 2340 by sand 1960 by and two bands at lower mobilities . We believe the low mobility bands represent the supercoiled and nicked circular forms of subband D2A, which is not cleaved by EcoRI . The two major bands arise from subband 23, which is cleaved twice by EcoRI. Digestion with BamHI or HaeII produced the same pattern of fragments from subband 13213 while generating a single fragment of 1470 by from D2A . A band at 1470 by was also generated by HindIII, and this and the one generated by BamHI are impervious to EcoRl treatment. Thus, we conclude that D2A is composed of a triplication of a segment 1470 by long, each copy of which contains a single site for BamHI, HaeII, and HindIII . Codigestion experiments have established the map shown in Fig . 3 . A map similarly derived for D2B is also shown in Fig . 3 . This species is essentially a duplication in which one copy has a deletion of roughly 380 by compared to the other . D3 is more complex than the other defectives, and because it was less abundant in the original DNA preparation, less material was available to complete determination of its structure . It is sufficient to say that D3 is clearly a reiteration mutant like the others, it contains one site per reiterated segment for BamHI, HaeII, and HindIII, and it has no EcoRI sites . D4 also has no site for EcoRI . Identical band patterns were generated from this DNA by digestion with BamHI, HaeII, or



123

GENOME MAPS OF SV40 DEFECTIVES

5000-

17001120 . 1010

520420-

D2

D3

D4

FIG . 2. Restriction enzyme digests of SV40 and isolated defective UNAs . Electrophoresis was from top to

bottom in a 2 .0% polyacrylamide-0 .5% agarose composite gel .

HindIII . The upper band in these digests (1470 bp) has twice the intensity expected by comparison with the lower band (1100 bp), and it has occasionally been resolved into two components. A map deduced from results of codigestion experiments is presented in Fig. 3 . D4 is a triplication, very similar to D2A, in which one copy is deleted by about 370 by compared to the others . None of the defectives was cleaved by

B E A H

H H

FIG . 3 . Sites of restriction enzyme cleavage in wildtype SV40 and defective DNAs . The circular lengths are drawn to scale and aligned with a BamHI site at position 0.15. Enzyme sites depicted are A, HeeII; B, BamHI; E, EcoRI ; H, HindII! ; and P, HpaII.



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CARROLL AND O'NEILL

Hpall under conditions in which standard SV40 DNA was completely digested . Hybridization to SV40 Restriction Fragments To determine which regions of the SV40 gemome are represented in the defectives, nick-translated defective DNAs were hybridized to restriction fragments of standard SV40 by the method of Southern

(1975) . For each defective, digests of nondefective SV40 DNA with HindIII, HindIII + HpaII, and HaeIII were used to provide fragments in the appropriate size range from all regions of the genome . The results of one such experiment are shown in Fig . 4 . In the case of each defective, some bands show hybridization while others do not . The information derived from several such experiments has been mapped onto the

Ftc . 4 . Blot hybridizations to SV40 DNA . On the left is a photograph of a portion of the ethidium bromidestained gel showing digests of wild-type SV40 DNA with HindIII, HindIIl + HpaII, and HaeIII (left to right) . Following transfer to a nitrocellulose filter, identical trios of digests were hybridized with 'P-labeled SV40, D2, and D4 DNA. The resulting autoradiographs are shown on the right . Electrophoresis was in a 2.0% polyacrylamide-0 .5% agarose composite gel.



125

GENOME MAPS OF SV40 DEFECTIVES

nondefective SV40 genome (Fig . 5) . In each defective, the region containing the origin of replication (map position 0 .67) is represented, as is a region from the opposite side of the genome . Clear gaps exist between these regions on both sides .

Heteroduplex Analysis

FIG, 5 . Schematic representation of blot hybridization data . The locations of HaeIH (outer ring) and Hind III + HpaII (inner ring) restriction fragments of SV40 DNA are pictured in the upper left . In the other three diagrams, the degree of hybridization to each of these fragments by the defective DNAs is illustrated . Shaded fragments give heavy hybridization, stippled ones are only lightly hybridized, and white ones are not hybridized at all. HaeIII fragments E and F and G and H are not separated in the gels used, so the hybridization which occurs to both of these bands with all defectives cannot be unambiguously assigned, hence the question marks on these fragments .

2Ba

BamH I

2Bb

We prepared heteroduplexes between the defective and nondefective genomes in order to map more precisely the regions of homology between them and to look for non-SV40 sequences in the defectives . The restriction enzyme digests revealed the presence of one BamHI site per reiteration in each of the defectives, and the blot hybridizations had suggested that this might well be the SV40 BamHI site, since that region of the standard genome was present in each defective . Therefore, we BamHIdigested mixtures of defective (D2 or D4) and standard SV40 DNAs and annealed these to form heteroduplexes, expecting to find structures with both ends perfectly matched . This was indeed the case, as shown in Fig. 6 . Heteroduplexes containing each of the 2Aa

4a

fthwft ∎

FIG . 6 . Heteroduplexes between defective fragments and wild-type SV40 . For each type of defective fragment, heteroduplexes formed from BamHI and BamHI + EcoRl-cut mixtures are shown along with interpretive drawings. Molecules are oriented so that the EcoRI site is nearest to the right-hand end . Magnification is approximately x42,000 .





1 26

CARROLL AND O'NEILL

expected defective BamHI fragments could be identified in each case from the total length of duplex in the structures . The total length of duplex plus single-stranded DNA was always equal to the length of nondefective SV40 DNA. The locations and sizes of the single-stranded loops were used to determine which regions of the standard genome were deleted from the defectives . In no case was there evidence for the presence of non-SV40 sequences in the defectives. In addition to the structures shown in Fig . 6, all of the expected heteroduplexes between pairs of defective fragments were found . The orientation of the heteroduplex structures with respect to the wild-type genome was determined by examination of heteroduplexes formed from BamHI + EcoRI codigests. EcoRI removes about 750 by from one end of BamHI-cleaved wildtype DNA and from segments D2Ba and .824_728 ,61 .62

0

194

.73

.97

.72

55 .90 96

.28

96

.28

.62 .70

.183

a

52

.72

30

.62

33

.30

.s1

32

2Ba 285 2An 4a

.24

45

T 0 .7

05

0.3

oz

a 'S x xi xx x 'Be w x x FIG. 7 . Schematic map of the regions of SV40 represented in the defective genomes . The nondefective genome has been broken at 0 .5 and is displayed as the horizontal axis. X is the replication origin at 0 .67, and sites for restriction enzymes used in this study are indicated below. The regions present in each BamHI fragment of the defective genomes are represented by horizontal lines with the end points determined in the heteroduplex studies. Intact defective 2B is made up of one copy each of 2Ba and 2Bb, defective 2A has three copies of segment 2Aa, and defective 4 has two copies of 4a and one of 4b . Included for comparison are maps of the two other SV40 defectives which have been shown to contain the region including the viral BamHI site (data from Davoli et at., 1977) . The defective a :, is an exact triplication of the segment illustrated and was derived From DAR virus, an SV40-like virus of human origin . a' was isolated after high multiplicity passage of SV40 on monkey cells and has been propagated as aa' or a4 ' .

D2Bb . The location of a single-stranded tail or a reduction in duplex length indicated which end of each heteroduplex structure contained the EcoRI site (Fig. 6) . A schematic illustration of the regions of the nondefective genome represented in each defective BamHI fragment is given in Fig . 7 . D2A is a triplication in which each copy is identical to the others and contains the segments from 0 .62 to 0 .72 and from 0.11 to 0 .30 of SV40 . D2B is a duplication containing essentially the same sequences around the replication origin and a larger region surrounding the BamHI site and in which only one copy has the sequences from 0.85 to 0.90. D4 is made up of three nearly identical segments, all of which have similar representations of the origin, two of which are very similar to the D2A subunit, and the third of which is additionally deleted for the region 0 .24 to 0 .30. Sites for the restriction enzymes used to characterize the defectives are indicated in Fig. 7. It can be seen that there is excellent correspondence between the heteroduplex and restriction maps. There is a BamHI site (position 0 .15) in every defective segment, and the HindIII and Haell sites at 0 .65 and 0 .71 are preserved . Only D2B has additional HindIII sites at 0 .86 (in D2Ba) and 0.98 (in D2Ba and b) and the EcoRI site at position 0. Also included in this figure are the two other defective SV40 genomes which have been shown to contain the region around the BamHI site (Khoury et al., 1974; Davoli et al ., 1977) . The overall lengths of the defectives are reported in Table 1 . Values derived as sumTABLE 1 LENGTHS AND SEQUENCE COMPpE.XITIFS OF SV4O DEFECPIVE9 AS FRACTIONS OF THE NONDEFECTIVE GENOME

Length Electron micrographs of BamHI fragments Gel of BamHI fragments Gel of intact circles Complexity"

D2A

13213

D4

0 .89

0 .87

0 .81

0 .88

0 .87

0 .79

0 .86 0 .29

0 .86 0 .47

0 .80 0 .32

"The complexities are the fraction of wild-type SV40 sequences which are represented in at least one copy in each of the defectives .



GENOME MAPS OF SV40 DEFECTIVES

mations of BamHI fragments measured from electron micrographs agree very well with those based on electrophoretic mobilities of BamHI fragments or of intact covalently closed circular DNAs . Also included in Table 1 are the fractions of the total nondefective SV40 sequence complexity retained in each defective, which range from 0.29 for D2A to 0.47 for D2B . DISCUSSION

Several laboratories have characterized defective SV40 genomes recovered following repeated high multiplicity passage of the virus on monkey cells (Brockman et al., 1975a; Martin et al., 1975 ; Mertz et al ., 1975 ; Winocour et al ., 1975; Yoshiike et al ., 1975 ; Davoli et al., 1977 ; and references therein) . These are commonly deletion-reiteration mutants which are made up of several tandem copies of a segment containing the viral replication origin and sequences contiguous to it and often a fragment of host cell DNA . The retention of the replication origin is easily understood as a necessity for propagation of the defective genome, and the degree of reiteration appears to be determined by the requirement of a minimum genome length for efficient packaging into virus particles (Ganem et al., 1976 ; Shenk and Berg, 1976) . Both the shorter size of the defectives and their multiplicity of replication origins very likely put them at an advantage in replication compared to the standard genome and thereby account for their interference activity and amplification on high multiplicity passage . The SV40 defectives amplified in human glioblastoma cells resemble the monkey cell defectives in containing multiple copies of the replication origin . A rather constant segment is retained in each defective, which includes the sequences between positions 0 .62 and 0 .72 of the nondefective genome. However, they also contain multiple copies of a region of the standard genome opposite the replication origin, including the BamHI site at 0.15 . The retention sequences from this region is more variable (Fig . 7), but the minimum in any defective segment is from map positions 0 .11 to 0 .24. This section contains the normal termini for replication (Danna and Nathans, 1972 ; Fareed et al.,

12 7

1972) and for early transcription (Acheson, 1976), so we shall refer to it as the termination region, without implying that it serves such a function in the variants . The origin and termination regions have the same relative orientation in the defectives as in the standard genome . The genomes described here are not independent in origin, but we have preliminary evidence that retention and reiteration of the termination region are characteristic of A172-grown defectives (see below) . Two previously characterized papovavirus defectives have also retained multiple copies of the termination region : one derived from the SV40-like DAR virus (Fareed et at, 1974 ; Khoury et al ., 1974), and one from SV40 (Ganem et al ., 1976 ; Davoli et al ., 1977) . The latter has the initiation and termination segments in the normal orientation, as do our A172-grown defectives. The DAR-derived defective has these regions in inverted orientation . Why has the termination region been retained in these defectives? Does it confer a selective advantage on the variants? Or is it joined by recombination to the origin segment and then passively reiterated along with the origin, which confers an obvious replicative advantage? It has been suggested that there might be preferred sites for intragenomic recombination in SV40 (Martin et al., 1975) . The ends of the origin segments illustrated in Fig . 7 might all be the same within the limits of measurement, and position 0 .11, which is prominent in defectives D2A and D4, is the common terminus of the SV40 substitutions in the Ad2'ND series of hybrids (Morrow et al ., 1973) . However, taken together, the ends of the termination segments are quite variable . Also, the recombination events leading to formation of early passage deletions occur at a multiplicity of sites (Brockman et al., 1975b; Mertz et al., 1975) . Thus, it seems unlikely that recombination at preferred sites results in the passive amplification of the termination region . The region of the viral genome including the BamHl site is not required for termination of replication. Brockman et al . (1975b) have demonstrated directly that in monkey cells termination occurs halfway



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CARROLL AND O'NEILL

around the genome from the origin of bidirectional replication, independent of the DNA sequence at that point. In addition, late passage variants of SV40 clearly have not retained the viral termination region (Lee et at., 1975 ; Davoli et al., 1977) . Is there a cis-acting function in addition to the replication origin that is required for viability of the defectives? The early passage variants which have been characterized have not lost the termination region (Mertz et al., 1975 ; Brockman et al., 1975b), and late passage variants have picked up host DNA sequences (Lee et al., 1975; Davoli et al., 1977) which may substitute in function for the termination region . The inversion of the termination region in the DAR variant (Khoury et al., 1974) can be rationalized if the sequence specifically retained has dyad symmetry . The only variants apparently containing no sequences except those around the replication origin are those constructed by Shenk and Berg (1976) . We can propose several possible roles for the termination region retained in the defectives. (1) It may provide an entry site rather than a site of action for a replication termination function. (2) It may ensure termination of early transcription, which, if unlimited, might interfere with normal replication . (3) It may act in some step of genome encapsidation . It seems unlikely that any defective-coded function is selected, since no known SV40 gene maps wholly in the retained sequences, and the region common to all these defectives stretches only from map positions 0 .11 to 0.164 (Mg, 7) . The requirement for retention of the termination region may be peculiar to or at least more stringent in human cells . The defective genomes we have characterized were selected during growth in human cells and that from the DAR virus may have been (Sack et al ., 1973 ; Fareed et al., 1974) . We now have preliminary evidence (D . Carroll, J . L . Hansen, and F. J . O'Neill, unpublished data) that defectives arising independently from passage of triply plaquepurified SV40 on A172 cells also contain multiple copies of the termination region . They are cleaved at multiple sites with

BamHI and the patterns of BamHI fragments are very nearly identical to those for cleavage with BgII, which has a single site very near the replication origin . This indicates equal reiterations of origin and termination regions . This behavior contrasts with that of defectives arising on high multiplicity passage of the same virus on monkey cells . These begin to show multiple Bgl I sites but have lost or retained only a single BamHI site. Current efforts are directed at determining whether the presence of the viral termination region is absolutely required for propagation of defectives in A172 cells. ACKNOWLEDGMENTS We are grateful to Dr. John Swanson for use of his electron microscope, to Dr. David Wolstenholme for use of his Numonics graphics calculator, and to Jean Wilcken and Laurence Renzetti for technical assistance. Critical comments from Drs . Dan Kolakofsky and Mike Botchan helped guide this work, which was supported by NIH Grants GM22232 and CA15141 and research funds of the Veterans Administration Hospital . REFERENCES AcRESON, N . H . (1976) . Transcription during productive infection with polyoma virus and simian virus 40. Cell 8, 1-12. BOTCHAN, M ., Ton', W ., and SAMBROOK, J. (1976) . The arrangement of simian virus 40 sequences in the DNA of transformed cells. Cell 9, 269-287 . BROCKMAN, W. W ., GUTAL, M . W., and NATHANS, D . (1975b) . Evolutionary variants of simian virus 40 : Characterization of cloned complementing variants, Virology 66, 36-52. BROCKMAN, W. W ., LEE, T. N. H ., and NATHANS, D . (1975a) . Characterization of cloned evolutionary variants of simian virus 40. Cold Spring Harbor Symp . Qaant. Biol. 39,119-127. CARROL , D ., and BROWN, D . D . (1976) . Repeating units of Xenopus laevis oocyte-type 5S DNA are heterogeneous in length. Cell 7, 467-475 . DANNA, K . J ., and NATHANS, D. (1972) . Bidirectional replication of simian virus 40 DNA . Proc. Nat. Acad. Sci . USA 69, 3097-3100. DAVIS, H . W ., SIMON, M ., and DAVIDSON, N. (1971) . Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids . Methods Enzymol. 21, 413-428 . DAVOLI, D ., GANEM, D ., NUSSBAUM, A. L., FARKED, G . C., HowLrv, P . M ., KHOI,RV, G ., and MARTIN, M . A. (1977). Genome structures of reiteration mutants of simian virus 40 . Virology 77,110-124.



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