During Copying of Two Distinct RNA Templates

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During Copying of Two Distinct RNA Templates Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6971-6975, July 1995 Biochemistry Homologous recombination promoted by reverse transcriptase during copying of two distinct RNA templates MATrEO NEGRONI*, MIRIA RICCHETTI*, PASCAL NOUVELt, AND HENRI Buc* *Unite de Physicochimie des Macromolecules Biologiques (Unite de Recherche Associee, 1149 du Centre National de la Recherche Scientifique) and tUnit6 de Genetique des Mammiferes, Institut Pasteur, 75724 Paris Cedex 15, France Communicated by Martin Gellert, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, March 7, 1995 (received for review October 14, 1994) ABSTRACT Retroviruses are known to mutate at high MATERIALS AND METHODS rates. An important source of genetic variability is recombi- nation taking place during reverse of internal Plasmid Construction. Plasmid pSP6 was obtained by in- transcription sertion at the Dra III site in regions of the two genomic RNAs. We have designed an in vitro pBluescript- (Stratagene) of a single SnaBI site followed by the sequence of SP6 RNA model system, involving genetic markers carried on two RNA polymerase promoter. Deletion of the 106-bp Sac I/Kpn I templates, to allow a search for individual recombination fragment resulted in pSP6M. This plasmid, pSP6M, was then events and to score their frequency of occurrence. We show modified to give rise to the required DNA sequences. Plasmid that Moloney murine leukemia virus reverse transcriptase pALA was obtained by excision of the 445-bp fragment defined alone promotes recombination homologous efficiently. While by the two Pvu II sites present in pSP6M. pLK was constructed RNA concentration has little effect on recombination fre- by replacing the f3-lactamase gene present in pSP6M with the quency, there is a clear correlation between the amount of kanamycin-resistance (Knr) gene carried in pRSV-neo (20). reverse transcriptase used in the assay and the extent of pALA and pLK thus bear an 899-nt common region spanning recombination observed. Under conditions mimicking the in from the end of the antibiotic-resistance cassette to the first vivo situation, a rate compatible with ex vivo estimates has 163 nt of the lacZ' gene and containing the origin of repli- been obtained. cation. AP DNA (used for AP RNA synthesis) was prepared by linearizing pALA at its SnaBI site and digesting it further with Recombination is one of the most frequent genomic alter- Pvu II. AP DNA is the fragment containing the SP6 promoter ations introduced during reverse transcription (1-3). It can and going from the unique SnaBI site to the unique Pvu II site allow repair of lethal mutations and is one of the major sources present in pALA. All the cloning protocols were performed of capture of oncogenes, of rescue of defective retroviruses, according to standard procedures (21). Details of the con- and of generation of wider host range viruses (4-7). In mice, structions are available on request. recombination between different endogenous retroviruses can RNA Synthesis. ALA (or AP) and LK RNAs were indepen- dently prepared from pALA (or AP DNA) and pLK, respec- lead to the generation of pathogenic retroviruses (8). Recom- tively, by incubation of linearized bination seems to occur SnaBI plasmid with 40 units mainly during RNA-directed synthesis of SP6 RNA polymerase for 1 h at 37°C followed by addition of minus-strand DNA (9-11). of 1 unit of DNase I and further incubation for 10 min at 370C. Recombination events taking place during minus-strand RNAs were purified by phenol/chloroform extraction and by synthesis can be divided into two classes, depending on the two precipitations with 2.5 M ammonium acetate. Resulting region of the genome involved. (i) Strong-stop recombination RNAs were analyzed by agarose gel electrophoresis and results from strand transfer occurring at the ends of the viral quantified by spectrophotometry. genome. The reaction involves the terminal repeated sequence Primer Sequences. The following sequences were used: of genomic RNA and is essential for proviral DNA synthesis STDW, 5'-GACTAGGCTAGCCTATTGGTTAAAAAAT- and generation of the proviral promoter (12, 13). This type of GAGCTG-3'; STUP, 5'-CCGCATGCTAGCTAGTACGT- recombination has been studied in vitro using model templates GAACCATCACCC-3'; D, 5' -AGATCCTTTTTGATA- and purified reverse transcriptases (RTs) from various sources; ATCTC-3'; M13, 5'-GTAAAACGACGGCCAGT-3'. a detailed model for this mechanism has been proposed Reverse Transcription. RT concentration was deduced from (14-16). (ii) The second type of recombination is responsible titration experiments performed with a constant amount of for reassortment of sequences lying within internal regions of primer/template and increasing amounts of enzyme under the genome (3, 11, 17, 18). In this case, a copy-choice model, initial velocity conditions as described (22). RNA templates postulating an extensive sequence homology and a sequence- were annealed to primer STDW. Subsequently, MoMLV RT dependent template switch during minus-strand synthesis, has (either RNase HI or RNase H-; both from GIBCO/BRL) was been proposed (9). In vitro studies have previously shown a added. The reaction buffer contained 50 mM Tris HCl (pH correlation between the presence of a pause site on a given 8.3), 75 mM KCl, 3 mM MgCl2, 1 mM each dNTP, 1 mM RNA template and the ability for RT to switch synthesis onto dithiothreitol, 100 units of RNasin (Promega). Incubation was a homologous region of a recipient DNA oligonucleotide (19). carried out for 1 h at 37°C in a final vol of 10 ,ul. The reaction We have developed an in vitro model system to screen and was stopped by phenol/chloroform extraction followed by study homologous recombination events occurring during incubation for 30 min at 37°C with 1 ,ug of DNase-free RNase reverse transcription of internal regions of two RNA tem- (Boehringer Mannheim) and 0.5 unit ofEscherichia coli RNase H. DNA was then purified on plates. We report here the results obtained using Moloney by chromatography G-50 murine columns. The presence of full-length DNAs was verified by leukemia virus (MoMLV) RT. PCR amplification performed in 10 mM Tris HCl (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 200 ,uM each dNTP, 1 AM each The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: RT, reverse transcriptase; MoMLV, Moloney murine accordance with 18 U.S.C. §1734 solely to indicate this fact. leukemia virus; Kn, kanamycin; Ap, ampicillin. 6971 Downloaded by guest on October 3, 2021 6972 Biochemistry: Negroni et al. Proc. Natl. Acad. Sci. USA 92 (1995) primer (STDW and STUP), and 2.5 units of Taq DNA the main experiment (recombination sample R). In these polymerase (Roche-Cetus, Alameda, CA). PCR products controls, reverse transcription was performed separately for were analyzed by agarose gel electrophoresis. the two types of RNA and the samples were pooled immedi- Cloning of Reverse Transcription Products. Second-strand ately afterward and processed as described above.t§ synthesis was achieved by using the STUP primer and the same Evidence for a Copy-Choice Model. The present system conditions as in the PCR but heating the samples at 94°C for allows analysis of any individual DNA molecule produced 90 sec in the absence of Taq polymerase, which was then added during reverse transcription. It is then possible to distinguish at 55°C for 1 min. Incubation was subsequently carried out at molecules having the molecular structure expected for recom- 72°C for 10 min and the reaction was stopped by phenol/ binants from any artefact. The primers used to initiate DNA chloroform extraction. Full-length double-stranded DNAs synthesis were specifically designed to facilitate such an anal- were gel purified. The purification was carried out by removing ysis. The primer used for first-strand synthesis hybridizes the gel slice spanning from the LK to the ALA DNA bands, internally to both RNA sequences, yielding final products with allowing the recovery of both parental and recombinant a characteristic size (Figs. 1 and 3). Moreover, DNA synthesis DNAs. Eluted DNA was incubated with 0.2 unit of the Klenow from these primers suppresses a unique SnaBI site present on fragment of E. coli DNA polymerase I in the absence of the plasmids used for RNA production (pLK and pALA) and nucleotides at 25°C for 10 min and, after the addition of 50 ,uM creates a single Nhe I site (Fig. 3). For each experiment 2 x each dNTP, for a further 60 min. The reaction was stopped by 104-106 total clones can be obtained, allowing detection of rare phenol/chloroform extraction followed by chromatography on events. We systematically analyzed the minimal amount of G-50 columns. DNA was then digested with Nhe I, circularized clones required for a significant statistical analysis. Indeed, by 0.32 unit of T4 DNA ligase, and used for transformation of numerous Apr/Kns/lac+ colonies (Kns, Kn sensitive) were XL1-Blue MRF' Epicurian Coli cells (Stratagene). found among Apr/lac- clones, and all the corresponding Statistical Analysis. Statistical analysis was carried out by plasmids were analyzed by Nhe I restriction. computing the value of p as follows: All plasmids reisolated from Apr/Kns/lac+ clones as well as plasmids from several Apr/lac- clones were analyzed by Nhe fl - f2 I restriction. All the plasmids arising from Apr/lac- clones q(-q)(Wtl + Wt2) contained a single Nhe I restriction site and showed the size expected for plasmids derived from reverse transcription. The same was true for 81% (456/561) and 68% (38/56) of Apr/ Kns/lac± clones in recombination and control samples, re- where, if r1 and r2 are the number of recombinant clones in the spectively.
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