VIROLOGY 233, 423–429 (1997) ARTICLE NO. VY978597

Cloning and Sequencing of the Cellular–Viral Junctions from the Human Adenovirus

View metadata, citation and similar papers at core.ac.ukType 5 Transformed 293 Cell Line brought to you by CORE provided by Elsevier - Publisher Connector Nathalie Louis,*,1 Carole Evelegh,* and Frank L. Graham*,†,2

*Department of Biology and †Department of Pathology, McMaster University, Hamilton, Ontario L8S 4K1, Canada Received February 11, 1997; returned to author for revision April 14, 1997; accepted May 5, 1997

The human–viral junctions of integrated adenovirus type 5 (Ad5) DNA in 293 cells have been cloned and sequenced. The Ad5 sequences extend from nucleotides (nt) 1 to 4344 and are located in the pregnancy-specific b-1-glycoprotein 4 (PSG 4) . This maps the insertion of Ad5 DNA to human 19 (19q13.2). The Ad5 sequences are represented as a single collinear insertion of viral DNA with no rearrangements. There were 19 bp of PSG4 DNA deleted at the site of insertion and an extra 3 nt (GTC) at the left viral/cellular junction. A short patch of homology between viral DNA and PSG4 DNA (6 nt with 1 mismatch) was present at the right junction. ᭧ 1997 Academic Press

INTRODUCTION restricted 293 cell DNA has indicated that the integration of Ad sequences into the host genome probably occurred The 293 cell line was derived from human embryonic at a single site although the cells contain several copies kidney cells transfected with fragments of mechanically of the left end of Ad5 DNA (Ruben de Campione, 1983; sheared adenovirus 5 DNA (Ad5) by selection for cells Spector, 1983). The limits of the integrated Ad5 DNA were that exhibited many of the characteristics of Ad transfor- narrowed to sequences between viral nucleotides (nt) 1 mation (Graham et al., 1977). The transforming region of and 270 with respect to the left-end of the viral genome the human adenovirus is within the left 11% of the viral and between nt 4123 and 5372 at the right end of the genome and contains early region 1 (E1), consisting of inserted fragment and the Ad5 sequences in 293 cells two transcription units, E1a and E1b, whose products appeared to be collinear with viral genomic DNA be- are necessary and sufficient for mammalian cell transfor- tween nt 270 and nt 4123 (Spector, 1983). In the present mation by Ads. Because 293 cells express E1a and E1b study we have determined the precise sequences at both viral gene products they are permissive for growth of junctions of viral and cellular DNA and mapped the prob- Ad2 and Ad5 viruses that are defective in E1 functions able integration site of the viral DNA to chromosome and, as a consequence, the cell line is used extensively region 19q13.2. for the production and propagation of E1-defective mu- tants and especially recombinant E10 adenoviral vectors (see Hitt et al., 1994, 1995, for review). MATERIALS AND METHODS Considerable attention has been given to the possibil- PCR and cloning into pUC19 ity of using replication-defective E1 replacement vectors for gene therapy because of many advantageous fea- For inverse PCR to amplify the left junction, DNA iso- tures of the adenovirus system (Hitt et al., in press). How- lated from 293 cells was digested at 37Њ overnight with ever, it has become apparent in recent years that homolo- XbaI. The digested genomic DNA was then resolved by gous recombination between E10 viral genomes and the low-melting-point agarose gel electrophoresis, and DNA integrated E1 sequences in 293 cells frequently results fragments in the range 2 to 2.5 kb were gel-purified using in replication-competent Ads (RCA) that contaminate the Wizard PCR DNA Purification System (Promega, Mad- stocks of E10 vectors (Hehir et al., 1996; Lochmuller et ison, WI). Next, the purified DNA fragments were self- al., 1994). To understand how this recombination occurs, ligated by T4 ligase treatment. An aliquot of the unpuri- and perhaps facilitate the design of vectors that prevent fied ligation reaction (10 ml) was amplified by PCR using it, knowledge of the precise viral DNA sequences present primers designed with the Primerselect program from in 293 cells would be helpful. Southern blot analysis of DNASTAR (DNASTAR Inc., Madison, WI). Following a 3- min preincubation at 94Њ, PCR was carried out in an Perkin–Elmer thermocycler with 25 cycles of a 45-s 94Њ 1 Present address: Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche Cedex, France. denaturing step, a 30-s 55Њ annealing step, and a 90-s 2 To whom correspondence and reprint requests should be ad- 72Њ elongation step. The product was treated with the dressed. Fax: (905) 521-2955. Klenow fragment of Escherichia coli polymerase I, ligated

0042-6822/97 $25.00 423 Copyright ᭧ 1997 by Academic Press All rights of reproduction in any form reserved.

AID VY 8597 / 6a3b$$$$61 06-06-97 14:58:26 vira AP: Virology 424 LOUIS, EVELEGH, AND GRAHAM to HincII-digested pUC19, and used to transform E. coli mately 1.3 kb was detected and this fragment was gel- DH5a by electroporation. The correct clones were identi- purified and cloned into pUC19. Recombinant clones fied by restriction enzyme digestion. DNA sequencing were screened by HindIII, HindIII/BamHI, and XbaI diges- was performed using an automatic sequencer [Dye-La- tion to identify inserts of the expected size and structure, beled Dideoxyterminators Cycle Sequencing protocol and a clone with an insert of 1.3 kb was isolated and with Taq-FS enzyme using ABS373 A Stretch automatic sequenced using pUC19 universal primers (New En- sequencer (PE/Applied Biosystems, Rexdale, Ontario, gland Biolabs, Mississauga, Ontario, Canada). The se- Canada)]. Oligonucleotides (except pUC19 universal quence obtained for Ad5 was identical to the left terminus primers) were purchased from and sequencing was per- of the viral genome up to and including the first nucleo- formed by The Central Facility of the Institute for Molecu- tide of the left inverted terminal repeat (ITR). Thus, all lar Biology and Biotechnology (MOBIX), McMaster Uni- nucleotides from the left end of the viral DNA were re- versity (Hamilton, Ontario, Canada). tained during integration of the transforming region into the human embryonic kidney cell genome. Southern blot In order to identify with precision the cellular integra- tion site, 300 nucleotides of the adjacent cellular se- Genomic DNA from 293 cells was digested with appro- quence were compared (Altschul et al., 1990) with DNA priate restriction enzymes, and digestion products were sequences from the GenBank nucleotide database and separated on 1% agarose gels and transferred to posi- 97% homology was found with the C-terminal exon of the tively charged nylon membranes (Boehringer-Mannheim pregnancy-specific b-1-glycoprotein 11 (PSG11) gene GmbH) by capillary transfer. The probe (a gel-purified (Joe et al., 1994). The human pregnancy-specific glyco- Eco47III fragment from pXC1 (McKinnon et al., 1982) rep- (PSG) family consists of 11 highly related resenting the left 4.3 kb of Ad5 DNA) was labeled with that are expressed mainly in the placenta throughout digoxigen by random priming using a Boehringer-Mann- pregnancy and the functions of which are unknown. They heim GmbH DIG high prime labeling and detection kit, form a subgroup of the carcinoembryonic antigen family Catalog No. 1585614. Hybridization was carried out ac- and are localized on the long arm of cording to instructions provided with the detection kit or (19q13.1–q13.3) (Khan et al., 1992; Teglund et al., 1995; as previously described (Haj-Ahmad and Graham, 1986). Thompson et al., 1990). Detection was by chemiluminescence with a 5-min expo- sure. Sequence of the right junction and exact structure of the integrated viral DNA fragment RESULTS AND DISCUSSION Sequence of the left-end cellular–viral junction Based on the results described above it was possible in 293 cells to design a second PCR strategy to amplify the right-end junction of Ad5–human DNA. We made the assumption To isolate the left-end junction of viral and 293 cellular that Ad5 DNA had inserted into the PSG11 gene without DNA, we used inverse PCR (Ochman et al., 1988), a pro- introducing large deletions or rearrangements of the tar- cedure that allows amplification of sequences that lie get site and designed two new primers that would anneal outside the boundaries of known sequences. Ruben to either side of the junction (Table 1). The forward primer (1983) had shown that hybridization of 32P-labeled Ad5 (AB8658) hybridized to Ad5 E1b DNA and the reverse DNA to Southern blots of XbaI-digested 293 cell DNA primer (AB8659) to PSG11 sequences approximately 900 detected two bands of approximately 5.5 and 2.3 kb and bp 3؅ of the presumed integration site. A PCR was carried the extreme left-end Ad5 sequences in 293 cells were out with DNA extracted from 293 cells, or human A549 contained in the 2.3-kb XbaI fragment. This information cells as a negative control, essentially as described by was used in the design of our PCR amplification strategy Innis et al. (1990). When the PCR products were analyzed as outlined below and in Fig. 1. Briefly, 293 cellular DNA on a 1% agarose gel stained with ethidium bromide (Fig. was digested to completion with XbaI, DNA fragments 2), a distinct band of approximately 2.3 kb was obtained of around 2–2.5 kb were gel-purified and ligated, and with genomic DNA extracted from 293 cells but not from the resulting circular fragments were amplified by PCR A549 DNA. This fragment, after treatment with the Klenow using primers which hybridized to viral sequences. The fragment of DNA polymerase, was cloned into the HincII reverse primer (AB8288) corresponded to nt 123–94 of site of pUC19 and a plasmid containing a 2.3-kb insert Ad5 and the forward primer (AB8287) corresponded to with the predicted structure was sequenced using a nt 1127–1156 (Table 1). The PCR primers were directed sense primer corresponding to nucleotides 3889–4009 away from each other in the native genome, but after of the viral genome and pUC19 universal primers (New digestion and circularization, the oligonucleotides anneal England Biolabs). The sequence obtained allowed us to in the correct orientation for PCR amplification of the determine the right-end junction of the viral DNA se- cell–viral DNA junction. A single PCR product of approxi- quence and together with the left end junction provides

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FIG. 1. Amplification of the Ad5 left-end DNA sequence in 293 cells by inverse PCR. DNA isolated from 293 cells and digested with XbaI was fractionated by agarose gel electrophoresis and DNA fragments in the range 2 to 2.5 kb were purified, ligated, and amplified by PCR using primers AB8287 and AB8288 as described under Materials and Methods. The PCR products were treated with the Klenow fragment of E. coli DNA polymerase I and cloned into the HincII site of pUC19. a complete structure for the integrated Ad5 sequences cellular DNA flanking the viral DNA insert and DNA se- in 293 cells (Fig. 3). We conclude that a DNA fragment quences from the PSG family suggested that the insert of 4344 bp, including the complete transforming E1 re- was in the PSG4 gene (19q13.2) rather than in PSG11. gion and pIX gene, had been integrated. Viral sequences In a stretch of 237 nt immediately adjacent to the left end at each end of the insert (nts 1–120 and nts 3910–4344) of Ad5 only a single nucleotide differed from sequences were identical to the viral genomic sequence and there- derived from the 3؅ end of PSG4 mRNA (Zimmermann et fore had not undergone rearrangement during the inte- al., 1989) versus 14 mismatches over the same segment gration process. for PSG11. Of 147 nt at the right flank of Ad5 there was Further examination of sequence similarities between 1 mismatch relative to the PSG4 sequence versus 6 for

TABLE 1 Oligonucleotides Used for PCR

Gene Name Sequence 5؅ to 3؅ Locationa Orientationb

E1a AB8287 TGGGCAGTGGGTGATAGAGTGGTGGGTTTG nt 1127 Sense E1a AB8288 ATCACACTTCCGCCACACTACTACGTCACC nt 123 Antisense Eb1 AB8658 AATGTGGCCTCCGACTGTGGTTG nt 2991 Sense Pregnancy-specific b1-glycoprotein-11 gene AB8659 TCTGGGGTCTGGGTCTTGGCTTAC nt 2617c Antisense

.a Position of the 5؅ nt b Relative to the direction of Ad5 E1 transcription. c Nucleotide 2617 of the C-terminal exon.

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no rearrangements in the immediate vicinity of the Ad5 DNA insert occurred in 293 cells.

Southern blot hybridization Hehir et al. (1996) recently used Southern blot hybrid- ization analysis to estimate the rightward extent of Ad5 DNA in 293 cells and concluded that the integrated se- quences ended between nt 4137 and nt 4280, a conclu- FIG. 2. PCR amplification of junction between Ad5 right-end and human genomic DNA from 293 cells. Following extraction of 293 cell sion which differs from our results. These workers also genomic DNA, PCR amplification was conducted with the primers found that additional bands were illuminated in addition AB8658 and AB8659 as described under Materials and Methods, ex- to those expected from a single insert of a collinear frag- cept the annealing temperature was 60Њ and the extension time was ment of Ad5 DNA and suggested that some of the Ad5 2 min. PCR products were analyzed by agarose gel electrophoresis sequences in 293 cells might be rearranged. In the clon- and staining with ethidium bromide. Lane 1, PCR products from a reaction with A549 cell DNA. Lane 2, PCR products from a reaction ing and sequence analyses described above we se- with 293 cell DNA. Lane 3, 1-kb ladder. The locations of molecular size quenced only a single clone for each junction and it markers (in kb) are shown to the right. remained formally possible that we had cloned out a minor species of Ad5 DNA that was not representative of the ‘‘true’’ structure. The sequence of the left and right PSG11. There was no significant homology between Ad5 junctions of Ad5 DNA can be used to make certain pre- DNA and cellular DNA at the target site of integration. dictions with respect to the size of DNA restriction frag- Between the left end of Ad5 and adjacent PSG4 DNA ments that can be illuminated on blots hybridized with there was an insertion of GTC and at the right junction Ad5 DNA. In particular, there should be an Eco47III site the sequence CCA was common to both Ad5 DNA and at nt 4297 in Ad5, just 47 nt 5؅ of the right viral–cell DNA PSG4 DNA. A short stretch of homology between Ad5 nt junction, and a BspHI site in PSG4 DNA, just 25 nt to the 4339–4344 and PSG4 DNA (6 nt with one mismatch) may right of the junction. At the left-end junction our sequence have played a role in the recombination event that led results predicted that there should be XbaI, BspHI, and to insertion of the Ad5 sequences (Fig. 3). The presence EcoRI sites in PSG4 DNA located 872, 739, and 223 nt of such ‘‘patchy’’ homologies at the junctions of integrated from the insert of Ad5 DNA. To test these predictions we viral DNA in transformed cells has been well docu- digested 293 cell DNA with HindIII, or with HindIII plus mented by Doerfler and his colleagues (cf. Knoblauch et one Eco47III, XbaI, BspHI, or EcoRI endonuclease, and al., 1996). A segment of 19 bp was deleted from PSG4 probed Southern blots with a labeled Ad5 DNA fragment DNA during or subsequent to insertion of Ad5 se- spanning nts 1 to 4297. The results of these analyses quences. Integration of foreign DNA by illegitimate re- are shown in Table 2 and Fig. 4. In Table 2, predicted combination is a poorly understood phenomenon but the sizes of fragments that should hybridize with the probe structure of the Ad5 DNA insertion in 293 cells is consis- are presented in the second column and observed frag- tent with that of other integration sites that have been ment sizes (determined from electrophoretic mobility by characterized: a short ‘‘filler’’ sequence (GTC) at one junc- comparison with a molecular weight standard prepared tion and a short stretch of homology at the other (Roth by digestion of pXC1 DNA) from three independent gels and Wilson, 1988). Such integration events are frequently are presented in the adjacent three columns. The aver- associated with chromosomal rearrangements but the age values for fragment sizes are shown in the last col- near-identity of flanking cellular sequences to PSG4 DNA umn. Figure 4 represents the Southern blot hybridization suggests that, other than deletion of 19 bp of PSG4 DNA, of gel 3. The observed fragment sizes are in good agree-

FIG. 3. Junctions between Ad5 and human genomic DNA in 293 cells. (PSG4) PSG4 mRNA, 3؅ end, from Zimmermann et al. (1989). The 19 nt deleted from PSG4 and substituted by Ad5 sequences are indicated in boldface. (293) Left and right junctions between PSG4 DNA and Ad5 DNA (capitalized) in 293 cells. An insert of GTC at the left junction is underlined and a short region of partial homology between Ad5 and PSG4 DNA at the right end is indicated by shaded parallelograms. (Ad5) Leftmost 18 nt of Ad5 DNA and sequence from nt 4327 to 4355.

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TABLE 2 tion reactions to measure the amounts of Ad5 DNA in Predicted and Observed Fragments Hybridizing 293 cells. Contamination of the HindIII F fragment, de- to Ad5 Left-End DNA rived from the right end of the genome (map units (mu) 89–97), with HindIII G (mu 0–8) and hybridization of the Predicted Average HindIII I fragment (mu 97–100) to ITR sequences might a b b b 293 DNA digest size (bp) Gel 1 Gel 2 Gel 3 (kb) explain the accelerated hybridization rates seen with HindIII — 7.2 7.2 8.5 7.7 these probes in the presence of 293 cell DNA. — 5.6 5.7 6.2 5.8 HindIII / EcoRI — 4.4 4.8 4.8 4.7 Implications for vector design 3028 3.2 3.2 3.0 3.2 HindIII / XbaI — 3.8 4.2 3.9 4.0 The structure determined for integrated Ad5 se- 2212 2.2 2.4 2.2 2.2 quences in 293 cells has implications for possible mod- 1465 1.4 1.6 1.4 1.5 els to explain formation of E1/ RCA during propagation HindIII / BspHI 3544 3.4 3.7 3.6 3.6 of E1 replacement vectors in these cells. Almost all E10 1565 1.7 1.6 1.5 1.6 vectors have deletions of sequences starting from nt HindIII / Eco47III — 5.6 5.8 6.3 5.9 1492 1.4 1.6 1.5 1.5 350–450 and extending to nt 3330–3530. Thus on both sides of the foreign DNA insertions there is significant a Fragment sizes predicted from the sequences of flanking PSG-4 overlap homology between integrated Ad5 sequences in DNA and the Ad5 DNA sequence. 293 cells and vector DNA. The presence of a perfect Ad b Observed fragment sizes (kb) calculated from electrophoretic mobil- ITR in 293 cells would in theory permit the rescue of a ity relative to marker DNA fragments. complete E1 region into the virus by a single recombina- tion event followed by ‘‘panhandle’’ formation (annealing of the 293-derived ITR with the ITR at the right end of ment with predicted values and no evidence was seen the Ad genome) and exonucleolytic trimming to generate of minor species of Ad5 DNA hybridizing with the left- an ITR duplex that could serve as an origin of viral DNA end probe. Hehir et al. (1996) failed to detect a 1115-nt replication (see Haj-Ahmad and Graham, 1986; Lippe and BsrI fragment in Southern blots of 293 cell DNA hybrid- Graham, 1989, for examples). This process occurs quite ized with an Ad2 probe containing nt 3664–5143 and efficiently when left-end viral sequences linked to plas- concluded that the integrated sequences did not extend past the BsrI site in Ad5 DNA at nt 4280. This site is present in our sequence and the plasmid containing the right junction was cleaved at the expected position by BsrI. Thus we have no explanation for the observations reported by Hehir et al. From the calculated fragment sizes determined from the Southern blot and from the sequence analysis we could prepare a restriction map for the Ad5 DNA and flanking PSG4 DNA in 293 cells between two HindIII sites on either side of the Ad5 DNA insert. The results are shown in Fig. 5. From the combined cloning, sequencing, and Southern blot analyses presented above it is clear that there is a single insertion of Ad5 DNA in 293 cells. We have seen no evidence of additional bands other than those predicted by the map shown in Fig. 5 although, of course, the possibility that small segments of viral DNA might have been integrated cannot be ruled out. In addi- tion, we have never seen evidence for the presence of Ad5 DNA sequences from other regions of the viral DNA (Ruben, 1983, unpublished results), in contrast to an early report that 293 cells contained sequences from the right end of the genome (Aiello et al., 1979) and in agreement FIG. 4. Southern blot hybridization analysis of restricted 293 cell with results reported by Spector (1983). Our analyses DNA (gel 3). DNA was digested as indicated and 10 mg DNA was were done with 293 cells at the earliest available pas- electrophoresed through a 1% agarose gel. After blotting by capillary sages and it is unlikely that the cells acquired right-end transfer to a nylon membrane, the membrane was hybridized against digoxigen labeled left-end Ad5 DNA as described in Materials and DNA at later passages. Aiello et al. (1979) used labeled Methods. The marker was prepared by digestion of pXC1 DNA with DNA probes prepared from gel-purified HindIII fragments HindIII and with HindIII / SphI to produce fragments of 6734, 3171, from the left and right ends of Ad DNA in liquid hybridiza- and 1476 bp that hybridized with the probe.

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FIG. 5. Restriction map of a 13.5-kb segment of 293 cell DNA spanning the insert of Ad5 DNA. Locations of restriction enzyme sites determined by sequencing are indicated in boldface. Sites are numbered relative to the leftmost nucleotide of the Ad5 DNA insertion. The Ad5 DNA segment representing nt 1–4344 is indicated by the solid bar. mid DNA are ligated to viral DNA fragments containing resulting in the initial formation of E1/ virus and the the right ITR followed by transfection into 293 cells (Stow, relative growth rates of the vector and RCA derived from 1981). This mechanism would not require any homology it. If RCA did not replicate more efficiently than E10 vec- between vector DNA and 293 viral sequences at the left tors (even in 293 cells) they would probably always be end of the genome. Alternatively, a double-recombination present at low, perhaps undetectable, levels. Vectors between homologous DNA on either side of the E1 sub- such as the one studied by Hehir et al. (Ad2/CFTR-1 stitution in Ad vectors and viral sequences in 293 cells with a deletion extending into pIX coding sequences and could result in reconstitution of a complete E1 region unable to synthesize pIX) that replicate poorly can gener- and generation of RCA. Recent results reported by Hehir ate detectable RCA rapidly even though the efficiency of et al. (1996) suggest that it is this latter mechanism that recombination might be reduced. may predominate in the formation of RCA. These authors Our finding that Ad5 DNA sequences are integrated analyzed several RCA generated following propagation into chromosome region 19q13.2 is interesting in view of Ad2-based E1 replacement vectors in 293 cells and, of the fact that a preferred region of integration for adeno- making use of polymorphisms between Ad5 and Ad2 virus-associated virus (AAV) DNA is located in 19q13– DNA at the left end of the genome, were able to show qter (Kotin et al., 1990, 1991). Whether the Ad5 integration that most if not all RCA generated in their system retained site in 293 cells is close to the site of AAV integration Ad2 sequences on the left (as well as right) flank of the mapped in latently infected human cell lines and whether E1 region substituted with foreign DNA in the original this is of some fundamental significance or is purely vector. coincidental will require further study. The complete sequence of Ad5 DNA in 293 cells avail- able as a result of our study and the results reported by ACKNOWLEDGMENTS Hehir et al. (1996) may aid in the design of vector-helper We thank D. Gooden and B. Allore for oligonucleotide synthesis and cell systems that reduce or prevent the generation of DNA sequencing, and S. Bacchetti and R. Parks for critically reading the RCA during propagation of Ad E1 replacement vectors manuscript. This work was supported by grants from the National Insti- in 293 cells. The finding that RCA appear to result from tutes of Health, Natural Sciences and Engineering Research Council, two recombination events suggests that an intact ITR in Medical Research Council (NSERC), and the National Cancer Institute of Canada (NCIC). F.L.G is a Terry Fox Research Scientist of the NCIC. the complementing cell line is not necessary for RCA formation. However, a certain degree of overlap is likely REFERENCES to be essential. Hehir et al. (1996) showed that extending the E1 deletion in their vector rightward from Ad2 nt 3328 Aiello, L., Guilfoyle, R., Huebner, K., and Weinmann, R. (1979). Adenovi- to 4020, decreasing homology on the right flank from rus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology approximately 1010 to 310 bp, reduced the frequency of 94, 460–469. RCA generation to nondetectable levels. Extension of the Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. E1 deletion leftward can be achieved by moving the pack- (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. aging signal (nt 194 to 358) to the right end of the genome Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977). Character- (Hearing and Shenk, 1983) and might be expected to istics of a human cell line transformed by DNA from human adenovi- rus type 5. J. Gen. Virol. 36, 59–72. have a similar effect on RCA formation. The rate of ap- Haj-Ahmad, Y., and Graham, F. L. (1986). Characterization of an adeno- pearance of RCA in vector preparations is determined by virus type 5 mutant carrying embedded inverted terminal repeats. at least two parameters: the efficiency of recombination Virology 153, 22–34.

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