Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3356-3360, May 1986 Genetics Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA (mutagenesis/retroviral vector/base substitution/deletion) CHARLES R. ASHMAN, PUDUR JAGADEESWARAN, AND RICHARD L. DAVIDSON Center for Genetics, University of Illinois College of Medicine at Chicago, 808 South Wood Street, Chicago, IL 60612 Communicated by Theodore T. Puck, December 26, 1985

ABSTRACT A retroviral shuttle vector was constructed by was mutagen-induced. In addition, the selectable gene is introducing the Escherichia coli xanthine (guanine) phospho- integrated into chromosomal DNA in the mammalian cells ribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector rather than being maintained in an autonomously replicating [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) CeU molecule. 37, 1053-1062]. This vector was packaged into infectious virus The selectable gene used in this study is the E. coli gpt which then was used to infect a hypoxanthine (guanine) gene, which codes for xanthine (guanine) phosphoribosyl- phosphoribosyltransferase-deficient mouse cell line. Cell lines transferase (XPRTase). This gene was chosen because there that expressed the gpt gene were isolated, and it was found that are selective systems for and against XPRTase activity in these cells contained a single integrated copy of the vector in a both bacteria and mammalian cells. The gpt gene was proviral form. Treatment of these cell lines with either ethyl inserted into the retroviral shuttle vector pZip-NeoSV(X)1 methanesulfonate or BrdUrd produced a >10-fold increase in (8), which can be packaged into a virus that integrates into the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact chromosomal DNA of infected cells. Also, the vector se- gpt genes have been recovered from a number of Sgur cell lines quences can be efficiently recovered from infected cells and after COS cell fusion and introduced into E. coli as part of a introduced into E. coli as plasmids. In this paper we describe plasmid. The complete DNA sequences of three mutant genes a system that uses this retroviral shuttle vector to rescue have been determined. Two of the mutant genes have a single mutant gpt genes from mammalian cells, and we have base substitution, whereas the third has a 34-base-pair dele- sequenced three mutant genes recovered after mutagenesis. tion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical MATERIALS AND METHODS mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is Cell Culture, DNA Transfection, and Virus Infection. Line that the mutations are induced in genes integrated into mam- A9 is a hypoxanthine (guanine) phosphoribosyltransferase malian chromosomes rather than in genes existing as part of (HPRTase)-deficient derivative of mouse L cells (9). COS-1 autonomously replicating plasmids. cells (10) were provided by K. Subramanian and psi-2 cells (11) by R. Mulligan. The basic cell culture medium was "Shuttle vectors" capable of replication in both animal cells Dulbecco's modified Eagle's medium supplemented with and bacteria have been used to study mutagenesis in mam- 10% fetal bovine serum; psi-2 cells were grown in medium malian cells (1-7). The basic strategy has been to introduce supplemented with 10% calf serum. HAT medium (12) is a marker bacterial gene into mammalian cells as part of a supplemented with 0.1 mM hypoxanthine, 0.4 mM amino- shuttle vector, recover populations of shuttle-vector mole- pterin, and 16 AM thymidine. Selections for G418 (13), cules from the cells, transform Escherichia coli, and score 6-thioguanine (Sgu), and ouabain resistance were carried out mutants in the marker gene in the E. coli transformants. The in medium supplemented with 1 mM G418, 36 pzM Sgu, or 1 major advantages of this approach are that large numbers of mM ouabain, respectively. mutant genes are made available for analysis while the A retroviral shuttle vector containing the E. coli gpt gene amount of mammalian cell culture is minimized. However, (hereafter called pZip-GptNeo) was constructed by inserting shuttle-vector molecules spontaneously acquire unusually the gpt gene into the pZip-NeoSV(X)1 vector, which was high frequencies ofbase substitution, deletion, and insertion provided by C. Cepko. Plasmid construction was performed mutations when passaged through mammalian cells. according to standard procedures (14). Here we describe a somewhat different approach that has For DNA transfections, 60-mm culture dishes were inoc- enabled us to efficiently recover mutant genes from mam- ulated with 4 x 105 psi-2 cells. After 24 hr, the cells were malian cells while avoiding the problem of high spontaneous treated with vector DNA by the calcium phosphate method mutation frequencies. We have introduced a selectable gene (15). Four hours later, the DNA was removed and the cells into mammalian cells as part of a retroviral shuttle vector and were given a 20% (vol/vol) glycerol shock for 4 min. After 2 isolated infected cells that express the gene. After mutagen- days of growth under nonselective conditions, the cells from esis, cell lines with mutations in the selectable gene were each dish were harvested and plated in four 100-mm dishes isolated. Shuttle-vector sequences containing the mutant containing G418 medium. gene were then recovered from the mutant cell lines and For virus infections, G418r psi-2 colonies that had been introduced into E. coli. In contrast to earlier studies, the transfected with the pZip-GptNeo vector were pooled and population of shuttle-vector molecules used to transform E. coli is recovered from a clonal population of mutant cells. Abbreviations: bp, base pair(s); EtMes, ethyl methanesulfonate; This increases the likelihood that the recovered mutant gene HAT, hypoxanthine/aminopterin/thymidine; HPRTase, hypoxan- thine (guanine) phosphoribosyltransferase; kb, kilobase(s); LTR, long terminal repeat; MK, minimal agar medium with kanamycin; The publication costs of this article were defrayed in part by page charge MKSgu, minimal agar medium with kanamycin and thioguaniine; payment. This article must therefore be hereby marked "advertisement" SV40, simian virus 40; Sgu, 6-thioguanine; XPRTase, xanthine in accordance with 18 U.S.C. §1734 solely to indicate this fact. (guanine) phosphoribosyltransferase. 3356 Downloaded by guest on September 28, 2021 Genetics: Ashman et al. Proc. Natl. Acad. Sci. USA 83 (1986) 3357

grown to near confluence in a 100-mm dish. The G418 from a Moloney murine leukemia virus (Mo-MuLV) provirus medium was removed and replaced with nonselective medi- cloned in pBR322. Most of the viral sequences were deleted um (6 ml per dish). Twenty-four hours later, this medium was and unique BamHI and Xho I sites were created. The viral collected and filtered through 0.45-,um filters, and Polybrene sequences remaining include the long terminal repeats (8 ug/ml) was added to the filtrate. The filtrate was added to (LTRs), which contain sequences necessary for initiation and 60-mm dishes containing 5 x 105 A9 cells for 3 hr. Two days polyadenylylation of viral transcripts as well as integration of later, the cells were harvested and plated in either G418 or viral sequences into chromosomal DNA. Also, the vector HAT medium. contains the sequences necessary for reverse transcription of Recovery and Analysis of Vector DNA. Equal numbers (1.5 the viral genome and encapsidation ofviral RNA. To give this x 106) of virus-infected, HATr A9 cells and COS cells were vector the properties of a "shuttle" vector, a fragment plated together in 100-mm dishes and were fused 2 days later containing the simian virus 40 (SV40) origin of replication, by addition of 50% PEG plus 10% dimethyl sulfoxide for 1 the pBR322 origin of replication, and the neo gene from TnS min (16). Two days later, low molecular weight DNA was was introduced into the Xho I site. The presence of the SV40 extracted (17) and used to transform E. coli (18). Bacterial and pBR322 origins of replication enable the vector to strains DH-1 (18) and DT-2 (5) were provided by R. replicate in mammalian cells and bacteria, while the neo gene Kucherlapati (University of Illinois at Chicago). Kanamycin- provides a selectable marker in bacteria (Kanr) and mamma- resistant (Kan9 transformants were selected on plates con- lian cells (G418D. taining that drug at 50 ,g/ml. Sgur transformants were High titers of virus are produced when the pZip- selected as described (5). Preparation of plasmid DNA, NeoSV(X)l vector is introduced into psi-2 cells (11). This cell restriction enzyme digestion, agarose gel electrophoresis, line, which contains a defective Mo-MuLV provirus, supplies and Southern blotting were carried out as described (5). all the functions in trans necessary for the packaging ofRNA Mutagenesis. Virus-infected A9 cells maintained in HAT into virus but is incapable of packaging its own RNA. To medium were inoculated at a density of 106 cells per dish into determine whether the pZip-GptNeo vector could likewise be 150-mm tissue culture dishes containing medium supplement- packaged into a transmissible virus, it was transfected into ed with 0.1 mM hypoxanthine, 16 AM thymidine, and ethyl the psi-2 cell line and transformants were selected in G418 methanesulfonate (EtMes). After 18 hr, the medium was medium. In one experiment, the transfection of 2 x 106 psi-2 replaced with fresh G418 medium. After 10 days of growth in cells with 10 ,ug of vector DNA gave about 100 G418r G418 medium, the cells were harvested and mutant frequen- colonies. cies were determined. For Sgu resistance, three 100-mm Medium from cultures of G418r psi-2 cells was collected dishes were inoculated with 1-2 x 105 cells per dish; for and used as a source of virus to infect A9 cells. Virus titers ouabain resistance, two 100-mm dishes were inoculated with were assayed by selection in either G418 or HAT medium. In 5 x 105 cells per dish. To determine the plating efficiency in one experiment the virus titers were about 20 G418r colony- the absence of selection, 100-mm dishes were inoculated in forming units (cfu)/ml and 0 HATr cfu/ml. Five G418r parallel with 100 cells per dish. After 10 days, the dishes were colonies were isolated and all five were also HATr, even fixed and stained, and colonies were counted. Results are though no HATr colonies were obtained by direct selection. expressed as the number of resistant colonies per 105 cells, Thus, the pZip-GptNeo vector can be packaged into infec- corrected for plating efficiency in the absence of selection. tious virus, although only extremely low virus titers are BrdUrd mutagenesis was carried out as described (19). produced. Also, both the gpt and neo genes are expressed in DNA Sequencing. Oligonucleotide primers were synthe- infected cells. sized using an Applied Biosystems DNA Synthesizer. Des- The structure ofthe vector sequences in an infected A9 cell ignating the first base of the gpt coding sequence as base no. line was examined by Southern blotting (Fig. 2). High 1, the primers extend from -75 to -56, 55 to 74, 183 to 202, molecular weight DNA was extracted from uninfected A9 and 332 to 351. Plasmid DNA was extracted from 15-ml cells and from a HATr, G418r infected line (A9I-2) and overnight bacterial cultures by the alkaline lysis method (20), digested with either Xba I or Apa I. Xba I cuts the pZip- and dideoxy extensions were carried out using 1 ,ug of GptNeo vector in the viral LTRs, producing a 5.8-kb frag- plasmid DNA (21). Wedge-shaped (22) 7% acrylamide se- ment as well as a 5.1-kb fragment which contains the gpt and quencing gels containing urea were used to reliably resolve 150-200 bases. 1 2 3 4 5 kb RESULTS Vector Structure and Virus Production. The structure ofthe 23.1 pZip-GptNeo retroviral shuttle vector is shown in Fig. 1. This vector was constructed by inserting the E. coli gpt gene into the BamHI site of pZip-NeoSV(X)1 (8), which was derived 9.4 FIG. 2. Southern blot analy- K Gpt 6.6 sis ofDNA from a virus-infected cell line. High molecular weight DNA (10 ,ug) isolated from X K B K Xh Xh X K 4.4 uninfected A9 cells was digested Neo 40 jpBR with Apa I (lane 1) or Xba I (lane ,F LjJLL 3). DNA from the virus-infected A9I-2 line was digested with Apa I (lane 2) or Xba I (lane 4). As a FIG. 1. Structure of the pZip-GptNeo vector. A 930-base-pair 2 3 control, 8 pg of pZip-GptNeo (bp) Bgl II-Apa I DNA fragment containing the E. coli gpt gene was and 10 ,g of A9 DNA were introduced into the BamHI site of pZip-NeoSV(X)1 by use of a mixed and digested with Xba I BamHI linker. The orientation of the gpt fragment was confirmed by (lane 5). Sizes [in kilobases (kb)] digestion with Kpn I. Arrows indicate the direction of transcription. of HindIII fragments of X phage The structure of the pZip-NeoSV(X)l vector has been described in DNA, run in parallel, are at detail (8). B, BamHI; K, Kpn I; X, Xba I; Xh, Xho I. right. Downloaded by guest on September 28, 2021 3358 Genetics: Ashman et al. Proc. Natl. Acad. Sci. USA 83 (1986)

neo genes. Apa I cuts the vector once in the 5.8-kb Xba I 0.7 kb long, with the gpt sequence contained in the two fragment. The blot was probed with a nick-translated 5.1-kb smallest fragments. When plasmid DNA isolated from 86 Xba I fragment. Kanr colonies was digested with Kpn I, 41 ofthe preparations A single 5.1-kb band was present in A9I-2 DNA digested gave the expected pattern. A representative gel is presented with Xba I (Fig. 2, lane 4). This band was not present in A9 in Fig. 3. Single-LTR plasmids (lanes 2, 3, 4, 5, 7, 8, 9, and DNA (lane 3). The intensity of this band is similar to the 11) were recovered from all five fusions. A variety of intensity of the band in lane 5, in which 8 pg of pZip-GptNeo restriction patterns that did not exhibit the single-LTR DNA was added to 10 Ag of A9 DNA prior to digestion with pattern (lanes 1, 6, 10, and 12) also were observed. Xba I, to simulate the presence of one copy of the vector per To confirm that the plasmids that appeared to have an cell. These results demonstrate that in A9I-2 DNA the Xba I intact gpt gene contained a functional gpt gene, a number of fragment containing the gpt gene is intact and present in a plasmids recovered from the virus-infected A9 cells were single copy, consistent with integration of the virus in a introduced into E. coli strain DT-2, a Gpt- derivative of single-copy proviral form. Also, the presence of a single, high HB101. Transformed bacteria were plated on minimal agar molecular weight Apa I band in A9I-2 DNA (lane 2) is plates supplemented with either kanamycin (MK plates) or consistent with the integration of viral sequences at a single kanamycin and Sgu (MKSgu plates). Transformants that site. contain a functional gpt gene will not survive on MKSgu Recovery of pZip-GptNeo Sequences. Integrated shuttle plates, whereas transformants that lack a functional gpt gene vectors containing the SV40 origin of replication can be will grow on both MK and MKSgu plates. A total of 28 rescued by fusion with monkey COS cells [which make SV40 plasmids having both the 0.9- and 0.7-kb Kpn Ifragments and large tumor (T) antigen constitutively (10)], apparently by 25 plasmids lacking both of these fragments were used to activation of the SV40 origin by T antigen followed by transform DT-2. Every plasmid that had both Kpn I frag- excision of vector sequences (23). In cells with repeated ments gave Sgu-sensitive (SguS) transformants, whereas integrated sequences, excision often occurs as a result of every plasmid with a different restriction pattern gave Sgur homologous recombination between the repeated sequences transformants. Thus the presence of an intact gpt gene (24). Thus, fusion of COS cells and cells containing the (indicated by the presence of both Kpn I fragments) corre- pZip-NeoSV(X)1 virus as a single-copy provirus results lated perfectly with the Gpt+ phenotype in bacteria. These predominantly in recovery of plasmid containing a single data show that intact, functional gpt genes can be rescued LTR and the sequence between the LTRs (8). This is the from the infected cells and that plasmids containing intact gpt structure expected if excision occurred as a result of recom- genes can be easily and reliably identified by restriction- bination between the proviral LTRs. Therefore, if pZip- digest analysis. GptNeo vector sequences are present in infected A9 cells in Mutagenesis of the Virus-Infected Cells. The results of one a proviral form, it should be possible to rescue the vector representative experiment in which A9I-1, -2, -3, -4, and -5, sequences between the LTRs (including the gpt gene) as a were mutagenized with EtMes at 500 ,ug/ml are presented in single LTR plasmid following COS cell fusion. Table 1. Two- to five-fold increases in mutant frequency were Five G418r, HATr A9 clones (A9I-1 to -5) were fused to observed in three ofthe lines (A9I-1, A9I-3, and A9I-5), while COS cells and low molecular weight DNA was extracted. A a substantially larger increase was observed in A9I-2. In one portion ofthis DNA was used to transform E. coli strain DH-1 line, A9I-4, a relatively high spontaneous mutant frequency and Kanr transformants were selected. Transformants were was observed, and this frequency was not increased by obtained from DNA extracted from all five fusions. DNA mutagen treatment. recovered from the fusion of 5 x 106 mouse cells with an In a second experiment (Table 1) both Sgu resistance and equal number of COS cells yielded between 10 and 1000 Kanr ouabain resistance were measured following exposure of transformants. A9I-2 to either EtMes (450 ,ug/ml) or BrdUrd (60 ,M). In this Plasmid DNA was prepared from a number of Kanr experiment, concentrations of EtMes and BrdUrd that in- colonies and examined by restriction-digest analysis. If a duced Sgur mutants also induced ouabain resistance. These pZip-GptNeo provirus were to undergo excision and form a single LTR plasmid, digestion of this plasmid with Kpn I Table 1. Mutagenesis of virus-infected A9 cells should produce three fragments, approximately 3.5, and 0.9, No. of mutants EtMes, BrdUrd, per 101 cells* Cell line ,mg/ml gm Sgr Ouabainr Experiment 1 A9M-1 0 2 500 4 A9I-2 0 1 500 16 A9I-3 0 2 500 9 A91-4 0 24 500 8 A9I-5 0 4 500 16 FIG. 3. Restriction digests of plasmids recovered from virus- Experiment 2 infected cells. Low molecular weight DNA was extracted from an A91-2-COS cell fusion. This DNA was used to transform E. coli A9I-2 0 2 0 strain DH-1, and Kanr transformants were isolated. Plasmid DNA 450 29 7 was extracted from these transformants, digested with Kpn I, and 0 17 0 electrophoresed (-0.5 ug per lane) in a 1% agarose gel (lanes 1-12). 60 105 12 Lanes M: X phage DNA digested with BstEII. Bands were visualized under UV light after staining with ethidium bromide. *Corrected for plating efficiency. Downloaded by guest on September 28, 2021 Genetics: Ashman et al. Proc. Natl. Acad. Sci. USA 83 (1986) 3359

results suggest that the sensitivity of the gpt gene to DISCUSSION mutagenic treatment is similar to that of a cellular gene. Two EtMes-induced A91-2 Sgur colonies were isolated and We have described a system for studying mutagen specificity studied in more detail. To test for reversion of the Sgur in mammalian cells that avoids the problem of high sponta- marker, the cells were grown in nonselective medium for 4 neous mutation frequencies observed in earlier studies (1-7). days (approximately four cell doublings) and then plated in In our system, a gene is introduced into mammalian chro- HAT medium. No revertant HATr colonies were observed mosomal DNA and cell lines with mutations in the foreign from 3 x 106 cells of each mutant. However, both Sgur cell gene are selected. The mutant gene is then recovered from lines grew as well as the parental A91-2 cells in G418 medium, the mammalian cells and introduced into bacteria as part of indicating that viral sequences were still present in the mutant a plasmid. Large amounts of the mutant gene can then be cells. Both the low reversion frequency and the retention of isolated and used for DNA sequencing studies. G418r in the Sgur cells are consistent with the mutant For a system of this type to be useful it is essential that (i) phenotype resulting from a mutation in the gpt gene. there be selective systems for the introduced gene in both Rescue of Mutant gpt Genes. To date, a total of 6 EtMes- mammalian cells and bacteria; (ii) the gene be relatively induced and 11 BrdUrd-induced Sgur mutants have been small, to facilitate identification of changes in base se- isolated from A9I-2 cells and fused to COS cells. In every quences; (iii) the introduced gene has a low spontaneous case, Kanr DH-1 transformants were obtained after transfor- mutation frequency that can be increased by mutagen treat- mation with low molecular weight DNA extracted from the ment; and (iv) the mutant genes can be easily rescued and fused cells. Plasmid DNA was prepared from a number ofthe introduced into bacteria. The system utilizing the pZip- Kanr transformants and analyzed by digestion with either GptNeo retroviral shuttle vector that we have described Kpn I or Hae III (the gpt gene is contained within a single satisfies all these requirements. 0.9-kb Hae III fragment). The presence of two Kpn I The E. coli gpt gene was chosen for use in this system fragments (0.9 and 0.7 kb) or a single 0.9-kb Hae III fragment because of the availability of selective systems and its small was considered to indicate the presence ofan intact gpt gene. size. HPRTase-deficient mammalian cells that express the A total of 140 plasmids recovered from the 17 Sgur mutants gpt gene can be selected in HAT medium, and both bacteria were analyzed in this manner, and 61 were found to have an and mammalian cells that do not express the gene can be intact gpt gene by these criteria. Plasmids having an intact gpt selected in medium containing Sgu. The coding sequence of gene were recovered from all but one of the 17 mutant cell the gpt gene is 456-bp long (25-27). We have found that a gene lines. Many of these plasmids (43 of61) were then introduced of this size can be easily sequenced by the Sanger method into the Gpt- E. coli strain DT-2 and the transformants were using 4 (and possibly only 3) oligonucleotide primers. plated on MK and MKSgu plates. In every instance, equal A low spontaneous frequency of a recessive mutation numbers of transformants were obtained on MK and MKSgu requires that the gene be stably expressed. Previous studies plates, indicating that the plasmids lacked a functional gpt have shown that genes introduced into mammalian cells often gene. Thus, all 43 intact gpt genes rescued from 16 Sgur lose activity at extremely high rates (28, 29). There is mutants were nonfunctional. In contrast, as described above, evidence suggesting that this loss is due to neither loss of the all 28 intact gpt genes rescued from the HATr infected cell gene nor structural gene mutations but instead may be due to lines were functional. These results strongly indicate that a change in expression of the gene (30, 31). In preliminary intact mutant gpt genes are being rescued from the mutant experiments, we observed that many spontaneous Sgur cell lines and that these mutations were generated in the variants isolated from HATr virus-infected A9 cells also lost mammalian cells prior to the rescue process. expression of the neo gene. Since the gpt and neo genes are DNA Sequencing of Mutant gpt Genes. The complete DNA part of the same transcriptional unit in the pZip-GptNeo sequences of three mutant gpt genes have been determined. vector, this suggests that many of the spontaneous Sgur One mutant gene, El, was recovered from a Sgur cell line variants arise as a result of either unstable expression of this induced by treatment of A91-2 with EtMes at 640 ,ug/ml. The transcriptional unit or loss of these genes rather than muta- DNA sequence determination of the entire mutant gene tion of the gpt gene. By maintaining the cells in G418 medium indicated that it contains only a single base change. The rather than nonselective medium prior to Sgu selection, the alteration is a C-)T transition at position 332 of the gpt frequency of spontaneous Sgur variants was greatly reduced. sequence. This change converts an ACC codon to ATC, The ability to select for the presence of a second gene in the resulting in the substitution of an isoleucine for a threonine same transcriptional unit largely overcomes the problem of residue in the XPRTase polypeptide. The second mutant unstable expression or loss of the transcriptional unit leading gene, B5, was recovered from a Sgur mutant cell line induced to high spontaneous variant frequencies. by 60 ,uM BrdUrd. DNA sequencing revealed that this gene We have shown that high frequencies of Sgur mutants can also contains only a single base change, a G-+A transition at be obtained in a single-step selection in A91-2 cells after position 92 of the gpt sequence. This change converts a GGC mutagenesis with EtMes or BrdUrd. The selection of reces- codon to GAC, resulting in a substitution of aspartic acid for sive mutants in a single step is possible because the gpt gene glycine in the polypeptide. is present in a single copy in these cells. That a cell line A third mutant gene, E8, was recovered from a Sgur cell containing a single copy of the gpt gene was so readily line induced by EtMes at 750 ,ug/ml, a concentration that is obtained is a consequence ofthe use of a retroviral vector for extremely toxic. This mutant gene was found to contain a the introduction of the gene, since retroviral vectors most 34-bp deletion extending from -14 to 20 in the gpt sequence. often integrate into chromosomal DNA as a single copy. The observation of a deletion in this mutant gene raised the Although it is possible to introduce genes in a single-copy possibility that the mutation was generated during the recov- form by other means, these methods often result in the ery process, as it has been observed that shuttle-vector integration of genes at multiple sites or as tandem repeats at molecules often suffer deletions when passaged through a single site (5, 24, 32). mammalian cells. However, the deletion in E8 produces a The use of a retroviral shuttle vector to introduce the gpt small but recognizable reduction in the size ofthe 0.7-kb Kpn gene into mammalian cells also ensures that the gene can be I fragment. Several other plasmid molecules recovered from easily recovered. The proviral structure of the integrated this same mutant cell line also have this smaller Kpn I vector ensures that the SV40 origin of replication will be fragment, suggesting that the deletion is present in the closely flanked by repeated sequences, the viral LTRs. This mammalian cell mutant. allows the vector sequences to be excised by homologous Downloaded by guest on September 28, 2021 3360 Genetics: Ashman et al. Proc. Natl. Acad. Sci. USA 83 (1986)

recombination between repeated sequences following COS 1. Calos, M. P., Lebkowski, J. S. & Botchan, M. R. (1983) Proc. cell fusion. Approximately 50% ofthe plasmid molecules that Nati. Acad. Sci. USA 80, 3015-3019. were recovered from virus-infected A9 cells and introduced 2. Razzaque, A., Mizusawa, H. & Seidman, M. M. (1983) Proc. into E. coli had the structure expected if they were excised Natl. Acad. Sci. USA 80, 3010-3014. from chromosomal 3. Razzaque, A., Chakrabarti, S., Joffee, S. & Seidman, M. DNA as a result of recombination be- (1984) Mol. Cell. Biol. 4, 435-441. tween the LTRs. It should be noted that the same percentage 4. Lebkowski, J. S., DuBridge, R. B., Antell, E. A., Greisen, of recovered molecules had intact gpt genes whether they K. S. & Calos, M. P. (1984) Mol. Cell. Biol. 4, 1951-1960. were recovered from HAT' or Sgur mammalian cells. Fur- 5. Ashman, C. R. & Davidson, R. L. (1984) Mol. Cell. Biol. 4, ther, the identification ofplasmids containing intact gpt genes 2266-2272. is easily and reliably accomplished by restriction-digest 6. Sarkar, S., Dasgupta, U. B. & Summers, W. C. (1984) Mol. analysis. Cell. Biol. 4, 2227-2230. We have identified the changes in base sequence in three 7. Ashman, C. R. & Davidson, R. L. (1985) Somatic Cell Mol. mutant genes. All of the mutant genes were recovered from Genet. 11, 499-504. 8. Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, mutant cell lines isolated following exposures to mutagens 1053-1062. that resulted in a >10-fold increase in Sgur mutant frequency. 9. Littlefield, J. W. (1964) Nature (London) 203, 1142-1144. That such large increases in mutant frequency were observed 10. Gluzman, Y. (1981) Cell 23, 175-182. is evidence that the mutations were induced by the mutagens. 11. Mann, R., Mulligan, R. C. & Baltimore, D. B. (1983) Cell 33, This system should be useful for identifying base substitu- 153-159. tions, small deletions, and insertions induced by any 12. Littlefield, J. W. (1964) Science 145, 709-710. mutagen, provided the mutagen is capable of inducing a 13. Davies, J. & Jiminez, A. (1980) Am. J. Trop. Med. Hyg. 29, significant increase in the frequency of Sgur mutants in the 1089-1092. treated cells. 14. Maniatis, T., Fritsch, E. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Labora- Another feature ofthis system that may be important is that tory, Cold Spring Harbor, NY). the mutations are produced in genes integrated into chromo- 15. Graham, F. L. & van der Eb, A. J. (1973) Virology 52, somal DNA rather than in genes existing as a part of 456-467. autonomously replicating plasmids. In another study (33) 16. Davidson, R. L. & Gerald, P. S. (1976) Somatic Cell Genet. 2, utilizing an autonomously replicating shuttle vector, sponta- 165-176. neous mutations were observed at unusually high frequencies 17. Hirt, B. (1967) J. Mol. Biol. 26, 365-369. and the spontaneous base-substitution mutations occurred 18. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. exclusively at G-C base pairs. It is possible that chromosomal 19. Ashman, C. R. & Davidson, R. L. (1981) Mol. Cell. Biol. 1, and nonchromosomal genes may differ either in their sus- 254-260. 20. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, ceptibility to certain types of damage or in their ability to 1513-1523. repair this damage. If such were the case, integrated shuttle- 21. Zagursky, R. J., Baumeister, K., Lomax, N. & Berman, M. L. vector genes may be more representative of endogenous (1985) Gene Anal. Tech. 2, 89-94. cellular genes than are genes in autonomously replicating 22. Chen, E. Y. & Seeburg, P. H. (1985) DNA 4, 165-170. plasmids. 23. Botchan, M., Topp, W. & Sambrook, J. (1979) Cold Spring It should be possible to develop systems for studying Harbor Symp. Quant. Biol. 44, 709-719. mutagen specificity utilizing the pZip-GptNeo vector in a 24. Conrad, S., Liu, C.-P. & Botchan, M. R. (1982) Science 218, variety of mammalian cell lines. This will require that the 1223-1225. vector integrate into chromosomal DNA in a single copy and 25. Richardson, K. K., Fostel, J. & Skopek, T. R. (1983) Nucleic that the viral transcript be expressed at high levels. A Acids Res. 11, 8809-8816. 26. Pratt, D. & Subramani, S. (1983) Nucleic Acids Res. 11, packaging cell line has been developed that should be capable 8817-8823. ofproducing pZip-GptNeo virus with amphotropic host range 27. Jagadeeswaran, P., Ashman, C. R., Roberts, S. & (34), so that it may be possible to introduce this vector as a Langenberg, J. (1984) Gene 31, 309-313. virus into human cell lines. Alternatively, it should be 28. Davidson, R. L., Adelstein, S. J. & Oxman, M. N. (1973) possible to introduce the vector directly into human cells by Proc. Natl. Acad. Sci. USA 70, 1912-1916. other means (e.g., microinjection or calcium phosphate- 29. Pellicer, A., Robins, D., Wold, B., Sweet, R., Jackson, J., mediated DNA uptake) and identify lines that contain a single Lowry, I., Roberts, J. M., Sim, G. K., Silverstein, S. & Axel, copy of the vector. If the vector can be stably expressed in R. (1980) Science 209, 1414-1422. these cells, this system should be useful for studying 30. Clough, D. W., Kunkel, L. M. & Davidson, R. L. (1982) mutagenesis in human cells. Overall, this system should be Science 216, 70-73. valuable for analyzing mutagenic specificity and the molec- 31. Davies, R. L., Fuhrer-Krusi, S. & Kucherlapati, R. S. (1982) ular mechanisms of chemical mutagenesis in mammalian Cell 31, 521-529. cells. 32. Breitman, M. L., Tsui, L.-C., Buchwald, M. & Siminovitch, L. (1982) Mol. Cell. Biol. 2, 966-976. We thank Pamela Broeker and Peter DeVries for expert technical 33. Miller, J. H., Lebkowski, J. S., Greisen, K. S. & Calos, M. P. assistance and Rajinder Kaul for his help in the synthesis of the (1984) EMBO J. 3, 3117-3121. oligonucleotide primers. This work was supported by Public Health 34. Cone, R. D. & Mulligan, R. C. (1984) Proc. Natl. Acad. Sci. Service Grant CA31781 from the National Institutes of Health. USA 81, 6349-6353. Downloaded by guest on September 28, 2021