Cell, Vol. 11, 845-857, August 1977, Copyright 0 1977 by MIT The Nucleotide Sequence of Repetitive Monkey DNA Found in Defective Simian Virus 40 Martin Rosenberg Laboratory of Molecular Biology Shoshana Segal, Edward L. Kuff and Maxine F. Singer Laboratory of Biochemistry National Cancer Institute Bethesda, Maryland 20014 Summary DNA fragments Containing monkey DNA se- quences have been isolated from defective SV40 genomes that carry host sequences in place of portions of the SV40 genome. The fragments were isolated by restriction endonuclease cleavage and contain segments homologous to sequences in both the highly repetitive and unique (or less repetitive) classes of monkey DNA. The complete nucleotide sequence of one such fragment [151 base pairs (bp)] predominantly homologous to the highly reiterated class of monkey DNA was deter- mined using both RNA and DNA sequencing methods. The nucleotide sequence of this homo- geneous DNA segment does not contain dlscernl- ble multiple internal repeating units but only a few short oligonucleotide repeats. The reiteration fre- quency of the sequence in the monkey genome is >lo6. Digestion of total monkey DNA (from unin- fected cells) with endonuclease R-Hind Ill pro- duces relatively large amounts of discrete DNA fragments that contain extensive regions homolo- gous to the fragment isolated from the defective SV40 DNA. A second fragment, also containing monkey se- quences, was isolated from the same defective substituted SV40 genome. The nucleotide se- quence of the 33 bp of this second fragment that are contiguous to the 151 bp fragment has also been determined. The sequences in both fragments are also pres- ent in other, independently derived, defective substituted SV40 genomes. Introduction Several defective variants of simian virus 40 (SV40) with genomes that contain DNA sequences de- rived from the host (monkey) genome have been reported in recent years (Lavi and Winocour, 1972; Martin et al.. 1973; Lavi et al., 1973; Rozenblatt et al., 1973; Brockman and Nathans, 1974; Frenkel, Lavi and Winocour, 1974; Lee, Brockman and Na- thans, 1975; Oren, Kuff and Winocour, 1976; Davoli et al., 1977; Rao and Singer, 1977b). The genomes of the defective variants are closed circular duplex molecules (DNA I), and typically consist of several tandem repeats of a DNA segment containing the monkey sequences and a portion of the wild-type SV40 DNA sequences including the origin of repli- cation. The monkey sequences in a given defective may be derived from either the highly repetitive portion of the monkey genome or from the less repetitive or unique portion or from both. Further- more, there is evidence indicating that the monkey sequences present in independently derived defec- tive variants are not a random selection from the monkey genome. Rather, certain sequences, both of the repetitive and unique classes, are more likely to occur than are others (Frenkel et al., 1974; Oren et al., 1976). It is presumed that recombinational events between monkey DNA and infecting or repli- cating viral DNA give rise to these "substituted" variants, but the mechanism of the recombination is unknown. It may involve chromosomal DNA or extrachromosomal DNA known to exist in monkey kidney cells in tissue culture (Smith and Vinograd, 1972). The recombination may reflect site-specific integration into the cellular DNA and subsequent excision, but it is also possible that nonspecific recombination is involved. Segments containing the monkey sequences and few, if any, SV40 sequences can be isolated from purified, substituted defective genomes by cleav- age with restriction endonucleases (Rozenblatt et al., 1973; Lee et al., 1975; Segal et al., 1976; Rao and Singer, 1977b). The substituted defective DNA called CVP8/11P2 (Eco RI res) DNA I (Figure 1) yields two fragments of interest (Rao and Singer, 1977b). [Restriction endonucleases and fragments of DNA derived from cleavage with restriction en- donucleases are abbreviated according to the rec- ommendations of Smith and Nathans (1973).] The (Hind II/Hind Ill)-E fragment is approximately 3.1% of a wild-type SV40 genome in length and contains monkey DNA of high reiteration frequency: the (Hind II/Hind Ill)-C fragment is approximately 4.3% of a wild-type genome in length and does not hy- bridize to filters containing either SV40 or monkey DNA, and may therefore consist predominantly of monkey DNA from the low repetitive or single-copy class (Segal et al., 1976; Rao and Singer, 1977b). Since the CVPB/l/P2 (Eco RI res) DNA I consists of a 4 fold tandeom repeat, each fragment occurs 4 times within the defective genome. Fragments (Hind II/Hind Ill)-C and (Hind II/Hind Ill)-E are con- tiguous within each tandem repeat, and each has one end generated by endonuclease ReHind II and one generated by endonuclease R.Hind 111; the Hind 111 cleavage site separates fragments C and E. No SV40 sequences are detectable in either frag- ment by filter hybridization. We have undertaken experiments designed to characterize the se- Cell 846 rplpl monkey 'unique' sequences w monkey 'repetitive' sequences SV40 sequences Figure 1. A Schematic Drawing of the Structure of the Substi- tuted Defective Genome CVP8/1/P2 (Eco RI res) DNA I The figures shows the sites sensitive to endonucleases R. Hind II. R.Hind Ill, R.Bam I and R.Hap 11, as well as (inner circle) the fragments generated by combined cleavage with endonuclease R.Hind II and R'Hind 111 (Rao and Singer, 1977b). Fragments (Hind IllHind ill)-C (WOO) and (Hind IVHind Ill)-€ (NW) are indi- cated. - quences in (Hind II/Hind Ill)-C and -E in a variety of ways. Radioactively labeled cRNA copies of (Hind II/ Hind Ill)-E have been prepared and used to study the location of the sequences in the fragment in African green monkey chromosomes by in situ hy- bridization (Segal et al., 1976). The results indi- cated many possible chromosomal origins for the (Hind IVHind Ill)-E sequences, both centromeric and noncentromeric. The sequence was detected in the arms of between 9 and 11 chromosomes; this location is consistent with an interspersion of re- petitive and unique sequences in the monkey ge- nome as has been observed in a variety of other eucaryotic DNAs (Davidson and Britten, 1973). In the present paper, we report the complete nucleotide sequence of (Hind II/Hind Ill)-E frag- ment (151 residues), as well as the sequence of the first 33 nucleotide residues of (Hind II/Hind Ill)-C from the endonuclease R.Hind 111 site that sepa- rates it from (Hind II/Hind Ill)-E. The monkey se- quences in fragment E are shown to have a reitera- tion frequency of approximately 1.6 x lo6 in the monkey genome. It was recently reported that cleavage of total monkey DNA with endonuclease R.Hind 111 results in a series of discrete DNA frag- ments resolvable on polyacrylamide or agarose gels (Gruss and Sauer, 1975). Our results indicate that the monkey sequence in the (Hind WHind Ill)-E fragment hybridizes to each of the fragments ob- tained directly from monkey DNA. Finally, we pres- ent hybridization data indicating that sequences in both (Hind II/Hind Ill)-E and (Hind IVHind Ill)-C are found within the genomes of other independently derived, substituted SV40 defectives. Results The Complete Nucleotide Sequence of (Hind Ill Hind Ill)-E Fragment Both RNA and DNA sequencing techniques were used to determine an unambiguous sequence for the 151 residues of the (Hind II/Hind Ill)-E fragment (Figure 2). The RNA sequencing methods involved preparing and characterizing a cRNA transcript of the DNA fragment, and analyzing the oligonucleo- tide products generated from both complete and partial digests of the cRNA with T1 and pancreatic RNAase. As previously described (Segal et al., 1976). the major transcription product was an es- sentially full-length copy of one strand of (Hind II/ Hind Ill)-E fragment. A second (minor) transcript, readily separable from the major product on poly- acrylamide gels, yielded oligonucleotides consist- ent with its being a covalently linked RNA copy of both strands of (Hind IVHind III)-E. Analysis and comparison of the T1 and pancreatic oligonucleo- tide products obtained from these two transcripts designated two separate sets of oligonucleotides that derived from each of the two complementary strands of the DNA fragment (Figure 3). As shown in Figure 2, the complementary products were used to help specify the relative order of certain of the oligonucleotides from the major transcript. These data, in conjunction with the data obtained from the partial T1 ribonuclease digestions (see Figure 2, RNA), allowed deduction of a unique sequence for most of the fragment, although some ambigui- ties remained. Direct DNA sequence analysis was also carried out on (Hind IVHind Ill)-E, using both the tech- niques of partial snake venom analysis (Sanger et al., 1973) and the new chemical procedures intro- duced by Maxam and Gilbert (1977). For both pro- cedures, (Hind II/Hind Ill)-E was labeled with 32P at one or the other of its 5'-hydroxy termini, as de- scribed in Experimental Procedures. The dimethyl sulfate-hydrazine (DMS-Hz) (Maxam and Gilbert, 1977) technique for direct DNA sequencing was applied to (Hind II/Hind Ill)-E labeled separately at either its Hind II or Hind 111 end, and the nucleotide sequences were deduced from a comparison of the various polyacrylamide gels obtained. Representative examples of these Monkey DNA in Defective SV40 847 t Fragment C ""I 'I' Fragment E - -- Fragment E mm I" Hlnd 111 Fragment C with -30 -20 -10 ->*I( DNA pah Hind 111 end ... GTT"C.CCTEICTTCW 5' SVD OMS- Hind 111 end ... ~TCE~~~~O~.O~FYTI~~.~~,~~,,. 5' HZ Figure 2. The Derivation of the Nucleotide Sequence of (Hind il/Hind Ill)-€ Fragment and Part of (Hind IllHind Ill)-C Fragment Ail nucleotide positions are numbered from the R. Hind 111 site separating the C and E fragments (-1/+1). RNA (€fragment only): numerical designation of TI (RNAase (1) and pancreatic RNAase (P) oligonucleotides associated with either the major strand or minor strand transcript are those indicated in Figure 3. Only oligonucleotides which were actually used to deduce the relative order of the T1 RNAase products from the major strand transcript are depicted. Partial T1 RNAase digestion products (see Experimental Procedures) obtained from the major strand transcript are designated by brackets (m). Nearest-neighbor nucleotides to each oligonucleotide product are underlined (for example, T23, AAACUGC). The nucleotide sequence of each oligonucleotide product was determined by standard RNA sequencing techniques (see Experimental Procedures). These sequences and their relative order within the overall sequence were confirmed by direct DNA sequencing methods. DNA: the sequence of nucleotides depicted for each of the DNA sequencing methods (that is, DMS-Hz; partial SVD) denote only those residues which could be unambiguously determined from that technique at the level of single-nucleotide resolution. gel patterns are shown in Figure 4, and the derived sequences are indicated in Figure 2 (DNA:DMS- Hz). Partial snake venom analysis of the same end- labeled fragments allowed correlation of the first identifiable nucleotide residue in the DMS-Hz gel patterns with a particular nucleotide position within the fragment (Figure 2, DNA:SVD). Analysis of the products of the partial snake venom diges- tions by both two-dimensional "homochromatog- raphy" (Brownlee and Sanger, 1969) (Figure 5) and one- and two-dimensional paper electrophoresis (Rf values given in Table 1) gave nucleotide se- quences for 20-25 nucleotide residues at each end of the fragment. These sequences overlapped the sequences obtained by both the RNA and DNA sequencing techniques, and also confirmed the se- quences predicted from the known specificity of the restriction endonuclease cleavage sites at the termini of the fragment (Old, Murray and Roizes, 1975; Smith, 1974). The results summarized in Figure 2 indicate that the RNA and DNA sequencing methods gave com- pletely consistent data. Although each approach generated certain sequence ambiguities, in con- junction these methods allowed deduction of an unambiguous sequence of 151 nucleotide residues for (Hind II/Hind lll)-E. It is important to point out, for assessing the accuracy of the sequencing data, that in addition to using the RNA and DNA se- quence information separately, as confirmatory of one another, the techniques were also used to complement one another. Given a relatively easily obtainable catalogue of all the T1 and pancreatic RNAase products obtained from the RNA analysis, they may be ordered within the sequence by refer- ence to the gels obtained from the DNA sequencing method. Knowing the sequence and the nearest- neighbor nucleotide of a particular T1 or pan- creatic RNAase product predicts a very specific pattern on the DNA sequencing gels. These pat- terns can readily be picked out and the oligonucle- otides ordered far beyond the single residue reso- lution of the gel. Thus when used in conjunction, the RNA and DNA sequencing methods allow for greater flexibility and accuracy than does either one alone. Cel I e48 B :-I 102 \, pH 3.5 - 3 00'01 D ,-, ! ., l111 pH 3.5 - Figure 3. T1 RNAase Fingerprint (A, Autoradiograph; 6, Schematic Sketch) and Pancreatic RNAase Fingerprint (C, Autoradiograph; 0, Schematic Sketch) of Total cRNA Transcription Product Labeled with &*P-GTP First dimension separation: electrophoresis on Cellogel in 8.0 M urea at pH 3.5 (Sanger et al., 1965). Second dimension: ascending thin- layer homochromatography on DEAE-cellulose (9/1, celluloselDEAE-cellulose, 40 x 20 cm) (Brownlee and Sanger, 1969). Closed circles (0) depict oligonucleotides derived from the major strand transcript; stippled circles (.:.) depict oligonucleotides derived from the minor strand transcript (see Figure 2). Monkey DNA in Defective SV40 849 I GATC iGCY T. - ,T - c, - .c - A- 'G - A- A- A- A- A- A- G- T- T- T- T- T- A- G- T- G- G- 2 GATC AGC? v II 3 AGTC GX 4 AGTC Kii - T, - - Gs: - T: - TIG -c -T - - - c_T - T/G - - -c --G -T -c -A -A -A -G -A -G -T -c -T Figure 4. Representative Autoradiographs of DNA Sequencing Gels Obtained from the DMS-Hz Chemical Sequencing Methods (Maxam and Gilbert, 1977) as Applied to (Hind IllHind Ill)-E Fragment (1) a2P-labeled at the Hind II end; first discernible residue (at the bottom) isthe G 6 nucleotides from the end at position 146 in the sequence shown in Figure 2: (2) identical to (1). except for longer electrophoresis such that first discernible residue is 22 nucleotides from the end, at sequence posltion 130; (3) "P-labeled at the Hind 111 end; first discernible residue (at the bottom) is at position 6 (Figure 2); (4) identical to (3), except that first discernible residue is at position 17. Partial Nucleotide Sequence of (Hind IVHind Ill)-C Fragment A sequence of 33 nucleotide residues at the Hind Ill end of (Hind WHind Ill)-C fragment [contiguous with (Hind IVHind Ill)-E) was determined using only the direct DNA sequencing techniques (Figures 2 and 58). The fragment was labeled with 32P at the Hind 111 terminus (see Experimental Procedures) Cell 850 Figure 5. Autoradiographs of Two-Dimensional Fractionation (as Described In Experimental Procedures and Identical to That Used in Figure 3) of the Products Resulting from Partial Digestion with Snake Venom Exonuclease of (Hind IllHind Ill)-E Fragment (A) and (Hind 111 Hind Ill)-C Fragment (6) Labeled with V-at Their Hind 111 5' Termini and analyzed by partial snake venom digestion (Figure 2, SVD, and Figure 56) and the DMS-Hz sequencing technique (Figure 2, DMS-Hz). The Occurrence of Sequences in (Hind II/Hind 111)- E in the Monkey Genome-Cot Analysis 3H-labeled (Hind IVHind Ill)-E fragment was allowed to reanneal in the presence of heterologous DNA (E. coli) or DNA prepared from the BSC-1 line of monkey kidney cells, and the percentage of (Hind II/Hind)-E in double-stranded form was determined by chromatography on hydroxylapatite (Figure 6). (Hind IVHind Ill)-E alone reannealed with a CotllZ of 1.7 x mole-sec/l; the reaction was accelerated about 7 times in the presence of a 126 fold weight excess about 7 times in the presence of a 126 fold weight excess of BSC-1 DNA. Reactants harvested in the plateau regions of the two reannealing curves were almost equally resistant to digestion with single-strand-specific nuclease S1 (82% in the case of fragment E alone, and 72% for the mixture with BSC-1 DNA). In addition, the melting of rean- nealed (Hind II/Hind Ill)-E itself was compared with the melting of duplexes between (Hind IVHind Ill)-E and BSC-1 DNA (Figure 6, insert). Melting was sharp in both cases. The Tm value for the duplex between (Hind II/Hind Ill)-E and BSC-1 DNA was about 1°C lower than was that of the reanneled fragment itself. Both the S1 nuclease data and the melting curves indicate extensive matching be- tween (Hind IVHind Ill)-E and the homologous se- quences in the cellular DNA, but suggest the possibility of a small percentage of mismatching. The observed Tm values, 83-84"C, are consistent with the fact that (Hind IVHind Ill)-E contains 59.6% A.T bp (Figure 2). From the acceleration in reannealing of (Hind II/ Hind Ill)-E by a known quantity of BSC-1 DNA (see above), it can be calculated that fragment se- quences may represent as much as 4.8% of the cell genome. Since monkey cells contain about 5 x lo8 bp of DNA, (Hind II/Hind Ill)-E sequences may Monkey DNA in Defective SV40 851 Table 1. Rf Values of Oligonucleotide Products of Partial Snake Venom Phosphodiesterase Digestion of (Hind II/Hind Ill)-E Rf Values Oligonucleotide Product pH 3.5 pH 1.7 'pdA 1.90 2.25 'pdApdG 0.78 2.00 'pd ApdGpdC 0.42 1 .85 'pdApdGpdCpTpTpT 0 0.08 'pdApdApdC 0.72 2.00 'pdApdApdCpdC 0.47 1.95 'pdApdApdCpdCpdA 0.15 1 .80 ' pdApdApdCpdCpdApdG 0.05 1.22 'pdApdApdCpdCpdApdG pdGpT 0 0.21 'pdApdG pdCpT 0.20 0.72 'pdApdGpdCpTpT 0.09 0.25 'pdApdA 1.06 2.05 'pdApdApdCpdCpdApdG pdG 0 0.56 e Denotes Rf values = mobility relative to blue marker dye, xylene cyano1 FF, determined by electrophoresis on DEAE paper at either pH 3.5 (2.5 hr at 250 ma) or pH 1.7 (4.5 hr at 250 ma) (Brownlee. 1972); appropriate markers were obtained from other DNA frag- ments of known sequence or purchased from Collaborative Re- search. The first six oligonucleotides listed were obtained from the diges- tion of (Hind WHind Ill)-E labeled with 31P at the 5'-hydroxyl of the Hind 111 terminus. The last seven oligonucleotides listed were obtained from fragment labeled at the 5'-hydroxyl of the Hind II terminus. * Denotes a radioactive phosphate; dA, dC, dG and T are the four deoxyribonucleosides. comprise as much as 2.4 x IO8 bp per cell. This value corresponds to a reiteration frequency of about 1.6 x lo6 in the monkey genome. The Occurrence of Sequences in (Hind II/Hind 111)- E in the Monkey Genome-Hybridization to Fragments of Monkey DNA on Filters As previously observed (Gruss and Sauer, 1975; F. L. Brown, P. R. Musich and J. J. Maio, personal communication; M. Israel and M. Martin, personal communication), cleavage of total DNA from Afri- can green monkey kidney cells with endonuclease I?. Hind 111 and analysis of the products on agarose or polyacrylamide gels yielded several prominent fragments of defined size, as well as a mass of unseparable fragments of relatively high molecular weight (Figure 7). Additional cleavage of the mon- key DNA fragments with Hind II [which is necessary to generate (Hind II/Hind Ill)-E from the defective SV401 did not alter the Hind 111 digestion pattern seen in Figure 7 in a detectable manner (not shown). At least five discrete bands were usually visible. The smallest discrete class of fragments [called AGMr (Hind 111)-1] is also the predominant class and is slightly larger than the 151 bp (Hind IIIHind Ill)-E marker (Figure 7). Other experiments using the endonuclease R. Hind 111 fragments of wild-type SV40 as markers indicated that AGMr (Hind 111)-1 is Cot [mole x recllilerl Figure 6. Reassociation of (Hind II/Hind Ill)-E JH-labeled (Hind II/Hind Ill)-E fragment (349,600 cpmlpg, 4.8 ngl ml) was allowed to reanneal at 68% in 0.12 M sodium phosphate (pH 6.8) in the presence of 605 nglml of denatured sonicated BSC-1 DNA (0) or E. coli DNA (0). The extent of hybridization at each time was analyzed by hydroxyapatite chromatography. The abscissa is the molar concentration of nucleotide residues of (Hind IllHind ill)-E fragment times seconds. The insert shows a thermal denaturation of samples first reannealed under the same conditions as above to a Cot value of lo-*. approximately 170 bp in length (preliminary se- quence analysis indicates a chain length of 172 bp; unpublished experiments). The next largest class AGMr (Hind 111)-2 is about 350 bp in length, and the remaining fragments appear to be consecutively larger multiples of the size of AGMr (Hind 111)-1. The products of the digestion of BSC-1 DNA with endonuclease R. Hind 111 were transferred from the agarose slab gel (see Figure 7) directly to nitrocel- lulose strips and hybridized against 32P-labeled cRNA from (Hind II/Hind Ill)-E fragment as de- scribed in Experimental Procedures. As shown in Figure 7, each of the five discrete bands detected by staining with ethidium bromide hybridized with the cRNA; no other hybrid was detected. Thus all or part of the sequences contained in the (Hind Ill Hind Ill)-E fragment of the defective SV40 genome also occur in each size class of discrete monkey Hind 111 fragments. Although all the classes of dis- crete monkey fragments contain some sequences homologous to at least a portion of (Hind IVHind III)-E, the nature of the additional sequences in the fragments longer than AGMr (Hind 111)-1 is not known. Sequences Present in (Hind IVHind Ill)-E and -C Fragments Are Present in Other Substituted Defective SV40 Variants Previous work (Rozenblatt et al., 1973; Frenkel et al., 1974; Oren et al., 1976) indicated that the DNA associated with the defective SV40 variant CVBIII P4 contains segments homologous to both highly reiterated and infrequently reiterated or unique monkey sequences. Furthermore, using cRNA Cell 852 AB C -Frg.E Figure 7. DNA Fragments Obtained from the Digestion of BSC-1 DNA with Endonuclease R.Hind 111 and Hybridization of the Frag- ments with (Hind ll/Hind Ill)-E BSC-1 DNA (3 pg, total volume 50 rl) was digested with endonu- clease R.Hind 111, and the material was electrophoresed on a 1.4% agarose gel (slot 6). A mixture of defective SV40 DNAs (CVP8/1/ P2) was digested with endonuclease R.(Hind II/Hind Ill) to provide a marker of (hind II/Hind Ill)-E fragment which is the shortest fragment in the digest (slot C) (Rao and Singer, 1977b). The gel was stained with ethidium bromide and photographed (slots B and C). The BSG1 digest was transferred to nitrocellulose sheets and hybridized with a2P-labeled cRNA to fragment E (spec. ra- dioact. about 1 X 10' dpmlpg), and an autoradiogram of the nitrocellulose strip was made (slot A). probes copied from the monkey sequences present in CVB/l/P4 and other independently derived de- fective viral DNAs, Oren and his co-workers (1976) demonstrated that the same sequences occur in several of the independently isolated populations of substituted SV40 variants. It seemed probable that the same sequences might be present in CVP8/1/P2 (Eco RI res) DNA 1, since it shares a common ancestry with CVB/l/P4 (Rao and Singer, 1977a). The data in Table 2 indicate that sequences in (Hind IVHind Ill)-E fragment hybridize signifi- cantly to several of the independent SV40 variants previously described. We consider that hybridiza- tion of >5% of the radioactive probe represents a clear indication of positive homology. It is espe- cially noteworthy that the two defectives 776-P11 and 1103 contain sequences homologous to those of (Hind II/Hind Ill)-E, since these were derived from passage of wild-type strain 776 and not wild- type strain 777, as was CVPB/I/P2 (Eco RI res). No hybridization of the cRNA to (Hind II/Hind Ill)-E was detected with the defective variants 1101 or DAR- d5, which only contain monkey sequences that are infrequently reiterated (Lee et al., 1975; Davoli et Table 2. Hybridization of (Hind II/Hind Ill)-E Fragment Sequences to Independently Derived, Substituted Variants of SV40 DNA on Filter % Input Experiment Type Pg Hybridized 1 CVP8/1/P2 0.5 16 CVGIllP10 1.25 29 777-T0 1.25 13 CVB14lP8 0.5 12 CVB/2/P5 1.25 4 RH91 1 0.5 3 CV112/P7 1.25 1 CVl/I/P5 1.25 0.4 2 BSC-I 18 65 777(CVB) 1.8 0.8 776-P11 1.2 25 3 777(CVB) 6 2 CVP8/11P2 1.25 15 DAR-d5 1.5 0.8 4 BSC-1 12.5 24 1101 1.25 23 1103 1.25 10 The filter hybridization procedure is described in Experimental Procedures. In experiments 1, 2 and 3, the input was 12P-cRNA (7.5 x lo*, 4.5 x lo4 and 3.0 x 10` cpm. respectively). In experi- ment 4, the input was (Hind IllHind Ill)-E fragment (DNA) labeled with snP at the Hind II 5'-hydroxyl terminus (9.5 x IO'cpm). When first prepared, the cRNA had a specific radioactivity of about 1 .I X 10ncpm/pg. The inputs of cRNA correspond, in each instance, to approximately 0.7 ng of RNA. The defective CVP8lllP2 is the source of (Hind II/Hind Ill)-E fragment. The other defectives have been described previously by Oren et al. (1976), except for DAR- d5, whichwasdescribed byDavolietal. (1977), and1011 and1103 which were described by Lee et al. (1975). The viral stocks 777-T8, RH 911 and 776-PI1 were derived from the indicated strains of SV40, and have been carried at high multiplicity of infection over a period of years in the laboratory of E. Winocour (Oren et al., 1976). 777(CVB), a plaque-purified strain 777 SV40, and DNA isolated from BSC-1 cells SeNed, respectively, as negative and positive controls for hybridization. The percentage of the input radioactivity bound to blank filters (no DNA) was subtracted from the values shown. In experiments 1,2 and 3, the blank values were <0.4% of the input; in experiment 4, the blank values were <1 5%. It should be noted that the cRNA-DNA hybridization procedure (experiments 1, 2 and 3) affords lower blank values and greater efficiency of hybridization than the DNA-DNA procedure (experi- ment 4). al., 1977). We conclude that the (Hind II/Hind 111)-E sequences are frequently incorporated into substi- tuted SV40 defectives. Discussion The mechanisms responsible for the formation of defective SV40 genomes that are substituted with host (monkey) DNA are not known. Furthermore, those properties of the substituted genomes that Monkey DNA in Defective SV40 853 account for their perpetuation during multiple se- rial passages of defective viral stocks are also not known. It is clear from the available data (reviewed in the Introduction) that such molecules are gener- ated during permissive infection of monkey kidney cells in tissue culture by SV40 and, after high multi- plicity passaging, may come to represent a sub- stantial portion of the viral genomes synthesized during infection. The formation of these genomes and their subsequent evolution during serial high multiplicity passage (Brockman, Lee and Nathans, 1973; Martin et al., 1973; Rozenblatt et al., 1973) must involve recombinational events within the SV40 genome, as well as between SV40 and mon- key genome DNA. These events may include inte- gration of infecting wild-type SV40 genomes into the chromosomal DNA of the cells and subsequent faulty excision, or exchanges of DNA segments between the DNA of the two genomes in the ab- sence of true integration. It is also possible that they arise by recombination between the wild-type SV40 genomes and nonchromosomal host DNA molecules such as the small polydisperse circular DNA molecules known to occur in uninfected cells of the BSC-1 monkey kidney line (Smith and Vino- grad, 1972). Both low and high reiteration frequency classes of monkey DNA occur in the substituted SV40 defective genomes. Winocour and his co-workers (Frenkel et al., 1974; Oren et al., 1976) have demonstrated that in a variety of independently isolated defectives, a nonrandom selection from the total monkey genome is present. A set of highly reiterated monkey sequences and a set of sequences of low reiteration frequency were de- tected in several independently isolated defective variants. Other substituted variants did not appear to contain the same monkey sequences. With re- gard to the reiterated sequences, the data in this paper confirm the earlier observation. Sequences contained in fragment (Hind IVHind Ill)-E of the defective SV40 genome CVP8/1/P2 (Eco RI res) DNA I are present in several of those defectives previously characterized as sharing common mon- key reiterated sequences. In addition, a completely separate isolate, variant 1103 (Leeet al., 1975), also has (Hind II/Hind Ill)-E sequences. While it might appear that even random recombination would fre- quently result in the incorporation of a sequence reiterated 1.6 x lo6 times in the monkey genome, in fact, the (Hind II/Hind Ill)-E sequences represent 10% of the genome and yet are found in about half the substituted variants tested. Thus particular monkey sequences containing at least portions of (Hind II/Hind Ill)-E fragment are commonly found in substituted variants. Selection of these sequences may occur during the initial recombination events or during the subsequent replication of DNA. The availability of well characterized, substituted SV40 variants offers an opportunity to elucidate the structures involved in recombination and, perhaps, thereby to increase understanding of the corre- sponding mechanisms. The incorporation of the monkey sequences present in (Hind II/Hind Ill)-E fragment into the defective SV40 variant has also provided a convenient tool for the isolation and characterization of a portion of the monkey ge- nome. We describe here the entire nucleotide se- quence of the (Hind II/Hind Ill)-E fragment isolated from the defective CVP8/1/P2 (Eco RI res) DNA I, as well as that of 33 residues in (Hind II/Hind Ill)-C that are contiguous with (Hind IVHind Ill)-E in the defec- tive genome. The direct DNA sequencing data indi- cate that (Hind IVHind Ill)-E and (Hind IVHind Ill)-C are each single homogeneous sequences, and not mixtures of sequences as might have been ex- pected if the molecules of CVP8/P2 (Eco RI res) DNA I contained a variety of host sequences. (Hind II/Hind Ill)-E fragment is 151 residues long, has an A-T content of 59.6%, and is bounded by an endonuclease R. Hind 111 restriction site at one end and an endonuclease R.Hind II restriction site at the other. Although the (Hind II/Hind Ill)-E se- quence has a high reiteration frequency in the monkey genome, the nucleotide sequence is mark- edly different from the sequences reported previ- ously for reiterated satellite and spacer regions of eucaryote genomes (Brownlee, Cartwright and Brown, 1974; Biro et al., 1975; Endow, Polan and Gall, 1975; Saker et al., 1976). The latter are char- acterized by extensive internal repetitions of rela- tively short segments (6-15 residues). On the other hand, inspection of the sequence of (Hind II/Hind Ill)-E (Figure 2) does not indicate extensive internal repetition, although there are a few short repeats. Recent data (Roizes, 1976) suggest that the highly repetitive bovine satellite DNA I (Botchan, 1974) may also contain rather long basic internal repeat units. There are several short regions of 2 fold rota- tional symmetry within the fragment (Hind II/Hind Ill)-E sequence. The longest contain a total of 10 and 12 symmetrically positioned residues, and oc- cur at positions 4-14 and 68-84, respectively (Fig- ure 2). The longer region of symmetry (positions 68-84) contains an unsymmetrical core sequence of five residues (positions 74-78), and potentially gives rise to a stem and loop structure in the single- stranded cRNA copy of this region. In fact, under the conditions used for partial endonuclease diges- tion of the cRNA transcript, this region was found to be most resistant to ribonuclease attack (Figure 2). Most strikingly, the sequence contains relatively long stretches of asymmetrically distributed, alter- nating arrays of purine- and pyrimidine-rich seg- Cell 854 ments. Examination of either strand indicates that most of these stretches vary from between six and twelve consecutive purine or pyrimidine residues, and have little, if any, exact sequence homology among them. There is also an interesting array of sequences at the R.Hind II end of (Hind IVHind Ill)-E. The G at position 149 is in the recognition site GTTPuAC for endonuclease R.Hind II (Smith, 1974). The same G residue is contained within a recognition site for endonuclease R. Eco RII-that is, CCTGG, residues 145-149 (Bigger, Murray and Murray, 1973; Boyer et al., 1973). Similarly, the two C residues at the 5' end of the endonuclease R.Eco RII recognition site are the last two residues of a recognition site for the endonuclease R.Hph, TCACC (residues 142- 146) (Kleid et al., 1976). There is no apparent rea- son to expect that sequences for three different bacterial restriction endonucleases should occur in a row in this fragment. (Hind IVHind Ill)-E also contains two sites susceptible to cleavage by endo- nuclease R.Eco RI under the relatively nonspecific conditions termed RI" (Polisky et al., 1975). These sites are the sequences AATT at positions 32-35 and 95-98. Digestion of total monkey DNA with endonucle- ase R.Hind 111 yields a series of well defined DNA fragments of various molecular weights (Gruss and Sauer, 1975). As shown here, all size classes of these fragments contain sequences that hybridize with (Hind II/Hind Ill)-E. The monkey fragments are of various molecular weights, the smallest and most abundant [AGMr (Hind 111)-1] being about 172 bp in length. The other fragments are of increasing size and appearto be multimers of AGMr (Hind 111)-1 in length. It is not known whether these longer fragments contain multiple copies of all or of only a part of the (Hind II/Hind Ill)-E sequence. However, studies on the nucleotide sequence of AGMr (Hind 111)-1 (M. Rosenberg, H. Rosenberg and M. F. Singer, manuscript in preparation) indicate that a sequence of 124 residues from one end of AGMr (Hind 111)-1 is identical to the 124 residue sequence from the endonuclease Re Hind 111 terminus of (Hind II/Hind Ill)-E. Similarly, the 33 determined residues of (Hind II/Hind Ill)-C fragment are identical, in 32 positions, to the 33 residues at the end of AGMr (Hind 111)-1 distal to the (Hind II/Hind Ill)-E se- quences. These data provide direct sequence con- firmation of the origin of portions of both (Hind II/ Hind Ill)-C and -E in the monkey genome, and allow us to define the exact positions at which the highly reiterated monkey DNA contained in the SV40-de- fective genome diverges from the AGMr (Hind 111)-1 sequence as it is found in the monkey genome. Subsequent to residue 124, (Hind IVHind Ill)-E is different from AGMr (Hind 111)-1. This sequence might be an SV40 sequence or, alternatively, a monkey sequence not present in AGMr (Hind 111)-1. As reported earlier by Gruss and Sauer (1975), no Hind II sites are detectable in the series of frag- ments generated from the BSC-1 DNA by cleavage with endonuclease R. Hind 111. Thus if residues 125- 151 of (Hind IVHind Ill)-E were derived from the monkey genome, they must occur in an infrequent segment containing a portion of the highly reiter- ated AGMr (Hind 111)-1 sequence. Furthermore, while it is improbable that the sequence is derived from wild-type SV40 DNA, since nowhere within the wild-type SV40 genome are three contiguous sites specific for endonucleases ReEco RII and R.Hind II, as there are in (Hind IVHind Ill)-E, it could repre- sent an altered wild-type sequence. Sequence de- termination at the Hind ll end of the (Hind WHind III)-B fragment known to contain SV40 sequences and to be contiguous to (Hind II/Hind Ill)-E in the substituted defective CVP8/1/P2 (Eco RI res) DNA I (Rao and Singer, 1977b) should allow a clearer identification of the origin of the sequences at the Hind II end of (Hind WHind Ill)-E. It is possible, of course, that recombinational events themselves generated the nucleotide sequence of residues 125 through 151, and that the observed sequence can- not be characterized either as monkey or SV40 in origin. About 20-25% of the chromosomal DNA of Afri- can green monkey cells is contained in the highly repetitive fraction designated as component CY (Maio, 1971; Kurnit, Shafit and Maio, 1973). Gruss and Sauer (1975) reported that component CY DNA gives the same series of well defined low molecular weight DNA fragments as does total monkey DNA upon digestion with endonuclease R.Hind 111. The reiteration frequency of component CY has been estimated to be about 7 x lo6, but it has not yet been shown that a is a homogeneous sequence element. It appears probable that the sequence described in this report is a sequence that is in- cluded in component CY. Experimental Procedures Materlals The following materials were prepared as previously described: defective substituted SV40 variant CVP8/1/P2 (Eco RI res) DNA I (Rao and Singer, 1977a); (Hind ll/Hind Ill)-€ fragment from CVP8/ 1/P2 (Eco RI res) DNA I (both unlabeled and lH-labeled), purified by electrophoresis on polyacrylamide gels after digestion with the endonuclease R.(Hind Ii/Hind 111) (Segal et al., 1976); Rao and Singer. 1977b; "P-labeled cRNA to fragment E (Segal et al., 1976). Monkey DNA was isolated from BSC-1. an established line of monkey kidney cells, by the procedure of Aloni et al. (1969). E. coli DNA (type VIII) was obtained from Sigma. Endonucleases R.Hind 111 (Smith, 1974) and R.Hind 11, an isochizomerof R.Hind II (Landy et al., 1974), were obtained from New England Bioiabs. NE-260 scintillatlon fluid was obtained from Nuclear Enterprlses, Inc. (San Carlos, California). Monkey DNA in Defective SV40 855 RNA Sequencing Methods "P-labeled cRNA to fragment E was prepared by labeling the RNA separately with each of the four a-"P-ribonucleoside triphos- phates (New England Nuclear). The transcription products were analyzed either directly or subsequent to electrophoresis on 5% polyacrylamide gels in 8 M urea (Segal et al., 1976). The RNA was digested with either T1 or pancreatic RNAase, and the resulting oligonucleotides were fractionated by two-dimensional homo- chromatography (Figure 3) (Brownlee and Sanger, 1969). These products were further characterized by a variety of standard se- quencing techniques (Erownlee, 1972) identical to those used previously (Rosenberg, Weissman and de Crombrugghe, 1975; Kramer and Rosenberg. 1976). These methods include subse- quent digestion of oligonucleotides with the appropriate enzymes (Tl, pancreatic and U2 RNAases) and fractionation of the prod- ucts in one dimension on DEAE paper (Whatman DE 81) at pH 1.7 and 3.5 (Brownlee, 1972); determination of base composition and nearest-neighbor analyses by alkaline hydrolysis; and analysis of certain oligonucleotide products subsequent to their modification with a carbodiimide reagent (Barrell, 1971). In addition, partial digestion of the cRNA with T1 RNAase was carried out as previously described (Rosenberg and Kramer, 1977). The products were fractionated two-dimensionally as de- scribed above using homochromatography solutions which effec- tively separated oligomers up to 45 residues in chain length. All partial products were further characterized by complete,digestion with the appropriate enzymes and fractionation in either one or two dimensions by standard electrophoretic and/or chromato- graphic techniques (Sanger. Brownlee and Barrell, 1965; Brown- lee and Sanger, 1969; Brownlee, 1972). The results of these partial enzymatic digestions are summarized in Figure 2. Preparatlon of Fragments Labeled wlth 32P at a Slngle S'-Hy- droxy Terminus The 5' ends of the DNA fragments were labeled, after dephospho- rylation with bacterial alkaline phosphatase (Sigma), by phospho- rylation with y-12P-ATP (ICN; spec. act. >1 Ci/pmoie) using T4 polynucleotide kinase as described by Maniatis, Jeffrey and Kleid (1975). The strategy for obtaining the appropriate fragment [(Hind IllHind Ill)-C or -E] labeled selectively at only one end will be understood by reference to Figure 1 and as follows: CVP8/1/P2 (Eco RI res) DNA I was cleaved with restriction endonuclease R.Hind 111, and the 5' ends of the mixed DNA fragments were labeled with 12P as described above. These fragments were then redigested with endonuclease R. Hind 11, and the products were resolved by electrophoresis on polyacrylamide gels and eluted from the gels as previously described (Rosenberg et al., 1975). Hind IVHind Ill-C and -E fragments, labeled uniquely at their Hind 111 termini, were well resolved (Rao and Singer, 1977a) and can be obtained in pure form directly from these gels. (Hind IVHind Ill)-€ fragment, labeled selectively at its Hlnd II end, was obtained by simply reversing the order of the restriction endonuclease digestions. CVP8/1/P2 (Eco AI res) DNA I was first cleaved with endonuclease R.Hinc II, end-labeled and then cleaved with endonuclease R.HInd 111. Again, gel electrophoresis yielded the purified fragments. DNA Sequenclng by Partial Venom Phosphodletterase Digeo- tlon DNA fragments labeled selectively at one end were partially di- gested with snake venom exonuclease (Sanger et al., 1973; Man- iatis et al., 1975). The resulting products were fractionated by electrophoresis on Cellogel (Kalex) at pH 3.5 followed by either homochromatography on DEAE-cellulose thin-layer plates (see Figure 5) or electrophoresis on DEAE paper at pH 1.7 and/or 3.5 (Sanger et al.. 1965; Brownlee. 1972). DNA Sequencing by Dimethyl Sulfate and Hydrazlne Analysis DNA fragments labeled at one 5'-hydroxy terminus were sub- jected to direct DNA sequencing according to the methods de- scribed by Maxam and Gilbert (1977). Only representative autoradiograms of the many gel electro- pherograms obtained in the course of this work are presented here. The autoradiograms of all additional gels used to determine the reported sequences are available to interested investigators upon request to the authors. Determination of Reatroclation Kinetics BSC-1 and E. coli DNA were sonicated for 30 min at full strength in a 10 kc Raytheon sonifier: the average length of the resulting fragments was about 150 bp as determined on alkaline sucrose gradients. The unlabeled BSC-1 or E. coli DNA was mixed with %HH- labeled (Hind II/Hind Ill)-€ fragment in 8 mM NaOH and denatured by heating at 100°C for 5 min. Thereafter thesamples were cooled to 68°C and neutralized with 8 mM HCI. The salt concentration was adjusted to 0.12 M sodium phosphate (pH 6.8). and incuba- tion was carried out at 68'C. At various time intervals, samples were removed and diluted with cold distilled HzO to give a final concentration of 0.04 M sodium phosphate. The extent of hybridi- zation was determined by hydroxyapatite chromatography at 60°C as described (Britten, Graham and Neufeld. 1974). In brief, unhy- bridized (Hind II/Hind Ill)-€ was eluted from the columns at 0.12 M sodium phosphate, and hybridized molecules were removed at 0.4 M sodium phosphate. Radioactivity was determined in NE-260 scintillation fluid. Thermal Denaturation of Reannealed "-Labeled (Hlnd Ii/Hlnd 3H-labeled (Hind IllHind Ill)-€ fragment was allowed to reassociate as described above in the presence of sonicated denatured BSC-1 or E. coli DNA to a Cot value of (based on the concentration of the fragment as in Figure 6). The samples were diluted to a final concentration 0.04 M sodium phosphate and applied to hydroxy- apatite columns at 60°C. The columns were washed at 60°C with 0.12 M sodium phosphate until no additional radioactivity emerged. The temperature was then increased at 5'C intervals (up to 98"C), and the total radioactivity eluted by 0.12 M sodium phosphate at each temperature was determined in NE-260 scintil- lation fluid. Digestion of BSC-1 DNA wlth Endonuclease R.Hlnd 111 (and Hybridization of Resulting Fragments wlth cRNA lo (Hlnd Ii/Hlnd ill)-E BSC-1 DNA was digested with 2 units of endonuclease R.Hind 111 per pg for 44 hr at 37°C in 0.06 M NaCI, 7 mM MgClr, 7 mM Tris- HCI (pH 7.4). The reaction mixture was analyzed by electrophore- sis through 1.4% agarose slab gels for 5 hrat 40 V: the electropho- resis buffer was 0.04 M Tris-HCI, 0.02 M sodium acetate, 2 mM EDTA (pH 7.8). The gel was then stained with a 0.5 pglml solution of ethidium bromide (Sharp, Sugden and Sambrook. 1973). and the DNA was visualized with ultraviolet light. Photographs of gels were taken with a Polaroid camera and high speed type 57 Kodak film. The fragments were then transferred to nitrocellulose sheets by the method of Southern (1975) as modified by Botchan. Topp and Sambrook (1976). and hybrldlzatlon was carried out with s2P- labeled cRNA to (Hind IllHind III)-E. Hybridization was in the presence of 50% formamide (Fluka) and 0.75 M NaCI, 0.5% 50- dium dodecylsulfate, 0.05 M Tris-HCI (pH 7.8) for 24 hr at 37°C. After appropriate washing, the nitrocellulose sheet was placed in contact with Kodak No-Screen film for 48 hr. Filter Hybrldlzatlon Hybridization of aaP-labeled cRNA transcribed from (Hind IIIHind Ill)-E fragment or of lPP-labeled (Hind WHInd Ill)-E itself to DNA immobilized on nitrocellulose filters was carried out by the proce- dures described previously (Lavi and Winocour. 1972: Segal et el., 1976), except that the samples were treated at 68°C for 30 min in 99% formamide prior to dilution to 50% formamide and addition to Ill)-E Cell 856 the filter suspended in hybridization buffer. DNA from a variety of independently derived, substituted SV40 variants (Oren et al., 1976) was prepared in the laboratory of Dr. Ernest Winocour. The DNA of the substituted variant DAR-d5 (Davoli et al.. 1977) was supplied by Dr. George Fareed. 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