Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Mutation Research, 220 (1989) 221-234 221 Elsevier

MTR02619

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

Suresh Subramani Department of Biology, B-022, University of California, San Diego, La Jolla, CA 92093 (U.S.A.)

(Accepted 27 September 1988)

Keywords: Mitotic recombination; Mammalian cells; SV40; Vectors; Reciprocal recombination; Gene conversion

Summary

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammal ian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct D N A segments to predetermined chromosomal locations by homologous recombination.

Non-homologous and homologous recombination events play important roles in determining the ability of mammalian cells to adapt to their environment. The balance between these two recombination pathways also determines whether a cellular D N A segment introduced into a cell will integrate at a homologous or non-homologous chromosomal location. The ability to control the

Correspondence: Dr. Suresh Subramani, Department of Bi- ology, B-022, University of California, San Diego, La Jolla, CA 92093 (U.S.A.). Phone: (619) 534-2327 (office), (619) 534-3092 (lab).

Abbreviations: amp, ampicillin-resistance gene; An, polyadenylation signal; G418 r, resistance to the drug G418; gpt, xanthine-guanine phosphoribosyltransferase gene; hyg, hygromycin-resistance gene; neo, neomycin-resistance gene; ori, origin of DNA replication; SV40, simian virus 40; tk, thymidine kinase gene.

choice between these alternative modes of recombination is crucial for studies involving the in- activation, regulation, correction or alteration of genes, particularly because the non-homologous pathway appears to predominate in mammalian cells. This is in contrast with organisms such as S. cerevisiae (Rothstein, 1983) and A. nidulans

(Miller et al., 1985), in which homologous events are favored over non-homologous ones, though the latter are clearly detectable. Consequently, a great deal of effort has been expended towards at tainment of an understanding of the frequencies and mechanisms of these recombination pathways in animal cells.

Much of our current knowledge regarding recombination enzymes and intermediates is derived from a study of bacteria and bacteriophages (Clark, 1973; Shulman et al., 1970; Mosig et al., 1979; Cox and Lehman, 1987). In the absence of recombination-deficient mutants it is not surpris-

0165-1110/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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ing that animal viruses, and subsequently virus- based vectors, have played a dominant role in providing insights into recombinational pathways in mammalian cells. This review will focus prim- arily on our own use of SV40-derived vectors to analyze non-homologous and homologous recombination. More comprehensive reviews of these subjects in connection with mammalian cells are available elsewhere (Subramani and Seaton, 1988; Wilson, 1988).

Viral vectors for non-homologous and homologous recombination

Early attempts to examine homologous recombination involved the use of mutant DNA viruses such as simian virus 40 (SV40) (Dubbs et al., 1974; Upcroft et al., 1980; Vogel, 1980; Vogel et al., 1977), adenovirus (Volkert and Young, 1983; Wolgemuth and Hsu, 1980; Young and Silver- stein, 1980), polyoma virus (Miller et al., 1976), or herpes simplex virus (Dasgupta and Summers, 1980; Mocarski and Roizman, 1982). The observa- tions that oligomeric SV40 molecules were infec-

tious (Rigby and Berg, 1978) and that SV40 recombinants were produced at high frequency during infection of cells with genetically mixed oligomeric DNAs (Wake and Wilson, 1979) sug- gested to us that SV40-pBR322 hybrid molecules, from which wild-type or mutant viral genomes could be excised by intramolecular recombination, could in fact be used to examine both homologous and non-homologous events. The experimental strategy involved measurements of the frequency of production of infectious virions (containing wild-type or defective genomes) after transfection of permissive CV-1 monkey kidney cells with pBR322-SV40 hybrid plasmids that contained either a single SV40 genome length (Fig. 1) or a single genome length plus increasing homologous lengths of SV40 (Fig. 2).

The substrates pBSV1 and pBSV2 underwent non-homologous recombination to generate defective SV40 molecules, a subset of which could be rescued and quantitated by using an appropriate temperature-sensitive helper virus (at the non-permissive temperature) to complement the recombinant for plaque formation. In contrast, the sub-

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Fig. 1. Substrates used to detect non-homologous recombination. Plasmids of the pBSV series have one genome length of SV40 (solid black segments) inserted into the BamHI site of pBR322 (thin lines). Two orientations (A and B) of the SV40 insert relative to the pBR322 vector are shown for pBSV1 (SV40 interrupted at BamHI site in the late region) and pBSV2 (SV40 interrupted at BclI site in the early region). The early and late regions are denoted by E and L, respectively. These SV40-containing molecules are defective and too large to be packaged into virions. Intramolecular non-homologous recombination in monkey CV1 cells could generate a variety of molecules varying in size and the portions of SV40 or pBR322 they contain. SV40 recombinants defective in either the early or late region can be scored in a plaque assay (by complementat ion with appropriate temperature-sensitive helper virus at 41° C), provided they contain an SV40 origin of D N A replication and can be packaged in virions. Thus the frequency of plaque formation provides an estimate of the min imum amount of non-homologous recombination occurring in these cells. Independent recombinants are obtained from individual plaques.

223

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Fig. 2. Substrates for homologous recombination. Molecules of the pBSVD series contain more than one genome length of SV40 cloned in pBR322. Each plasmid has a head-to-tail non-tandem duplication (hatched area) of an SV40 sequence whose length in bp is indicated by the number following pBSVD in the name of the substrate. Intramolecular homologous recombination within these duplicated regions of each molecule generates wild-type SV40 which can be quantitated by plaque assay (without helper virus). These molecules can also undergo non-homologous recombination which can be estimated using helper virus as described in the legend to Fig. 1. The early and late regions are denoted by E and L. The SV40 origin of DNA replication (ori) and some SV40 map units (0.325 and 0.14) are shown.

s t ra tes in Fig. 2 could excise wi ld - type SV40 by homologous r ecombina t i on or genera te defect ive SV40 molecules by non -homologous r ecombina - tion. F o r these substrates , the homologous and non -homologous p roduc t s could be isola ted f rom p laques fo rmed at 37 ° C wi thout he lper virus or at 4 1 ° C with he lper virus, respectively. The effi- c iency of p laque fo rma t ion (relat ive to wi ld - type SV40 D N A ) p rov ided a measure of the recombi - na t ion frequency. These subs t ra tes were ut i l ized to demons t r a t e the increase in the p r o b a b i l i t y of in t r amolecu la r homologous r ecombina t i on as the length of dup l i ca t ed sequence h o m o l o g y was in- creased. N o n - h o m o l o g o u s r e c o m b i n a t i o n was found to occur in at least 5 -10% of the cells and

at a h igher f requency than homologous events be tween 5.2-kb regions of homology. D N A se- quenc ing revealed that l i t t le or no h o m o l o g y was invo lved in n o n - h o m o l o g o u s r e c o m b i n a t i o n . Ne i the r rep l ica t ion of the D N A subs t ra tes nor any of the viral p ro te ins were essential for e i ther mode l of r e c ombina t i on (Sub ra ma n i and Berg, 1983). Other work on n o n - h o m o l o g o u s r ecombina t i on using SV40-der ived vectors is descr ibed in the ar t ic le by Wi l son (1988).

The minimum homology requirement for homologous recombination

The sys tem for s tudy ing homologous recombi - na t ion was exp lo i t ed fur ther to examine the ho-

224

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Fig. 3. Relationship between extrachromosomal recombination frequency and length of homology in monkey kidney (CV1P) cells. The recombination frequencies expressed as specific infectivities (i.e., plaque-forming units//~g DNA) relative to wild-type SV40 DNA (100%) are plotted as a function of the length of homology.

mology requirements for homologous recombination. Using a series of plasmids containing 14-5243 bp of homology, we were able to demonstrate that the recombination frequency (i.e., efficiency of plaque formation) decreased linearly as the homology was reduced from 5243 bp to 214 bp (Fig. 3). Then the recombination frequency dropped by about 10-fold between 214 and 163 bp of homology. Low recombination frequencies were also observed with as little as 14 bp of homology (Rubnitz and Subramani, 1984).

Interestingly, results obtained from intra- chromosomal recombination experiments suggest that greater than 200 bp of homology are required for efficient gene conversion between repeated chromosomal sequences in mammalian (L tk - ) cells (Liskay et al., 1987). This is quite consistent with our observation that the minimum homology requirement for extrachromosomal homologous recombination is about 200 bp.

Because of the great sensitivity of the viral plaque assay (10 6 plaques//~g SV40 DNA), these assays based on the generation of infectious viral DNA molecules from appropriate recombination substrates, proved extremely useful and straight- forward. Furthermore, large numbers of independent recombinants could be isolated with ease from individual viral plaques for further analysis.

The principal limitations, however, were that such studies were restricted by the host range and lytic nature of the virus and could not be used to investigate chromosomal recombination events. This transition necessitated a switch to SV40-based vectors carrying selectable marker genes.

Extrachromosomal recombination using substrates based on selectable marker genes

Plasmid- or virus-based vectors carrying 2 mutant copies of a selectable marker gene have served as excellent substrates for the analysis of recombination events in mammalian cells. The mutants used include linker insertions, point mutations or deletions that render the marker gene nonfunctional. Recombination regenerates the functional marker gene following transfection of the substrates into cells. Certain markers (e.g., thymidine kinase, tk) are applicable only in aux- otrophic cell lines, whereas dominant markers (e.g., the neomycin-resistance gene, neo; xanthine-guanine phosphoribosyltransferase gene, gpt) may be used in many cell types (Subramani and Southern, 1983). By appropriate placement of the recombination partners on the same or different molecules, either intramolecular (Lin et al., 1984a; Rubnitz and Subramani, 1985, 1986, 1987; Subra-

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Conversion Fig. 4. Reciprocal or non-reciprocal recombination between duplicated sequences. Homologous segments in the selectable marker (hatched areas) and in flanking DNA (solid bars) are shown. Thin lines denote vector D N A s that do not share homology. (A) Recombination between 2 mutant alleles of a marker gene. The letter x denotes a point mutation, deletion or linker-insertion mutation. (B) Recombination between truncated but homologous fragments of the marker gene. If the homology extends into vector sequences as shown, then reciprocal or non-reciprocal recombination can regenerate the functional marker gene.

mani and Rubnitz, 1985) or intermolecular (Brenner et al., 1984, 1984, 1986: Folger et al., 1982, 1985; Kucherlapati et al., 1984; Shapira et al., 1983; Smithies et al., 1985b) events have been examined. In general, either reciprocal recombination or gene conversion could generate a functional copy of the marker gene (Fig. 4).

Several strategies have permitted the determination of recombination frequencies and the isolation of products. The first, based on direct selection for the recombined selectable marker gene, provides information regarding the number of cells in the transfected cell population in which at least 1 recombination event has occurred. The substrates in Fig. 5 contain 2 truncated, nonfunctional but overlapping segments of the neo gene such that inter- or intramolecular reciprocal recombination or intermolecular gene conversion generates a neo gene which confers resistance to G418 and neomycin in mammalian cells and bacteria, respectively. However, in our experience intramolecular events are favored over intermolecular ones by about 10-fold. Typically the selection for the recombined gene is applied a few days after transfection. Consequently, intra- or inter-

molecular recombination is presumed to occur extrachromosomally followed by the integration of the DNA into the host genome in a stable manner. These experiments showed that 5-20% of the cells that were capable of expressing a marker gene stably were also competent to mediate at least 1 recombination event. Recombination frequencies were similar for direct or inverted repeat configurations of the homologous sequences and replication of the substrate was not a prerequisite for recombination (Subramani and Rubnitz, 1985).

The recombination products from the parental cells and recombinant cell lines containing the integrated extrachromosomal recombination product have been characterized either by Southern blot analysis of the genornic DNAs (Brenner et al., 1985; Folger et al., 1982; Lin et al., 1984b; Shapira et al., 1983; Subramani and Rubnitz, 1985) or by plasmid rescue (Brenner et al., 1985; Folger et al., 1985; Kucherlapati et al., 1984) of the integrated vector DNA in recombination-deficient E. coil.

In intermolecular recombination events between 2 mutant alleles of the neo gene, non-reciprocal recombination was predominant over reciprocal events (Folger et al., 1985), the frequency

226

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Fig. 5. Extrachromosomal recombination substrates based on a dominant selectable marker gene. Each molecule has 2 truncated non-tandem but overlapping fragments of the neo gene (neo-I and neo-2). The 420-bp region of homology (hatched area) is in the direct repeat configuration in DR1 and the inverted repeat arrangement in IR1. Intra- or intermolecular reciprocal recombination or intermolecular gene conversion (see Fig. 4) can regenerate the neo gene, whose expression can then confer resistance to G418 in mammalian cells and neomycin in bacteria. The neo segments in each plasmid are separated by a complete transcription unit capable of expressing the gpt gene. The plasmids also contain the ampicillin-resistance gene and origin of replication from pBR322. A~ denotes a polyadenylation signal.

of recombination was enhanced by double-strand breaks within but not outside the region of homology (Brenner et al., 1986), and most of the events occurred within an hour after delivery of the plasrnid substrates into the nucleus by micro- injection (Folger et al., 1985).

An equally important measure of extrachromosomal recombination frequency involves the determination of the ratio of recombined to unre-

combined molecules after passage of the substrates through mammalian cells. The ampicillin- resistant plasmid recombination substrates in Fig. 5 were allowed to replicate extrachromosomally in monkey COS cells for 1 -4 days. The plasmid DNAs were recovered by the Hirt procedure (Hirt, 1967) and used to transform recombination-deficient bacteria to either ampicillin or neomycin resistance. The recombination frequencies, measured by the ratio of neo r to amp r colonies, were 1-4 × 10 2 for intramolecular reciprocal recombination between directly repeated sequences shar- ing 420 bp of homology. Interestingly, for inverted repeated sequences the frequencies were 1-6 x 10 -3, but the events appeared to be intermolecular, and intramolecular inversions were not seen. These frequencies for the direct and inverted repeat substrates were 30-200-fold higher after passage through COS cells, suggesting that recombination had indeed occurred in the monkey cells. Recombination could be detected as early as 16 h after transfection and reached a plateau at about 2-3 days following transfection. Intermolecular recombination between plasmids carrying the truncated fragments of the neo gene was at a frequency of 10 -3 -10-4 (Rubnitz and Subramani, 1985).

This bacterial transformation assay for intra- or intermolecular, reciprocal or non-reciprocal recombination takes only a few days to perform and has the advantage that large numbers of truly independent recombination products can be isolated and characterized. Though replication of the DNA substrate and product are not essential for recombination, the act of replication or the COS cell environment does stimulate recombination more than can be accounted for by a simple increase in plasmid copy number (Rubnitz and Subramani, unpublished). Thus, recombination assays involving the screening of recombinants in bacteria are best suited for mammalian cells in which the recombination substrates replicate. These include COS cells for plasmids carrying the SV40 origin of DNA replication (ori) or lymphoid cells for plasmids with the Epstein-Barr virus (EBV) ori and EBNA-1 gene (Yates et al., 1985).

The high frequency of recombination deduced from the bacterial transformation assay raised concerns that the frequency might be aberrantly

high because events initiated in the mammalian cells may have been completed in bacteria. This problem has been circumvented by the use of Southern blots of DNAs which have been pas- saged through COS cells (Rubnitz and Subramani, 1985). The recombination substrate and product DNAs are recovered from the COS cells and used directly for Southern blot analysis. The use of suitable radioactive probes and restriction enzyme digests easily discriminates between the recombination substrate and the product. Densitometric scanning of the autoradiograms showed that the recombination frequency (10 -2 ) was similar to that obtained using the transformation assays and substantially higher than that obtained in control experiments involving direct transformation of bacteria with the substrates. Moreover, no recombination products were visible in a Southern blot analysis of the recombination substrates not pas- saged through COS cells. Thus, these assays do indeed measure the frequencies of recombination events that occur in the animal cells. Southern blot analysis alone is not sufficient, however, for an analysis of independent recombination products.

Double-strand breaks within or at the boundary of a region of homology can stimulate recombination events as they do in S. cerevisiae (Orr-Weaver et al., 1981). The Southern blot procedure has also provided estimates of the stimulation (10-50-fold) of the frequency of extrachromosomal intramolecular recombination by double-strand breaks. The increase in the absolute amount of recombination product can be used to determine the stimulation in experiments in which the circular and linear substrates are transfected into mammalian cells separately (Rubnitz and Subramani, 1985). In fact, the bacterial transformation assay described earlier is unreliable when linear substrates are used, because linearization within a region of homology activates recA-independent pathways of recombination in E. coli (Symington et al., 1985).

Another procedure for measuring the population of recombined molecules in mammalian cells is based on gene expression from the regenerated copy of a highly sensitive reporter gene (e.g., firefly luciferase, de Wet et al., 1987). Cells were infected transiently with a substrate containing 2 nonfunctional but overlapping segments of the

227

reporter gene. Protein extracts of these cells made 2 days later showed 10-17% enzymatic activity relative to that in cells transfected with an intact copy of the reporter gene (Seaton and Subramani, unpublished). In addition, the extrachromosomal DNA recovered from the mammalian cells can be used to transform bacteria, and colonies expressing the recombined luciferase gene can be identified by a film assay that detects glowing colonies (Wood and DeLuca, 1987).

For the extrachromosomal recombination assays in which replication of the substrates and products occurs, the inverse relationship between the ability of the plasmids to replicate and their size should be borne in mind (Calos, 1986). If the recombination product is smaller or bigger than the substrate, then the frequency of recombination may be overestimated or underestimated depend- ing on the extent to which the product replicates with a higher or lower efficiency, respectively, than the substrate.

Extrachromosomal gene conversion

In the substrates described earlier (Fig. 5), either intra- or intermolecular reciprocal recombination or intermolecular non-reciprocal recombination could have restored the marker or reporter gene. Because intermolecular events generally occurred at about a 10-fold lower frequency than intramolecular events, we presume that these substrates measured predominantly intramolecular reciprocal events. Therefore, we developed other substrates in which gene conversion alone restores the marker gene. These substrates contain a 10-bp linker-insertion (Fig. 6) or a deletion (22 or 167 bp) mutation in an otherwise complete neo gene, and also an internal homologous fragment of the gene (Rubnitz and Subramani, 1986, Rubnitz and Sub- ramani, 1987). Although non-reciprocal gene conversion or double-reciprocal recombination can restore a functional copy of the marker gene, no double-reciprocal recombination was detectable and gene conversion appeared to predominate. As measured by the bacterial transformation assay; the frequency of gene conversion in COS Cells was 1-6 x 10 -4 for these substrates, and the frequencies were 14-750-fold higher than those obtained upon introduction of the substrates directly into

228

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Fig. 6. Substrates to detect gene conversion and not reciprocal recombination. The plasmids contain a mutated neo gene (neoX) which has an XhoI linker-insertion, and an internal 526-bp fragment of the neo gene (neo-526). The 526-bp homology shown by the hatched areas is in the direct and inverted repeat configurations in pGCD1 and pGCI1, respectively. The plasrnids also contain a gpt transcription unit, the ampicillin-resistance gene and the pBR322 ori. A functional neo gene could be generated by intra- (or intrachromatid) or intermolecular (or sister-chromatid) double-reciprocal recombination or gene conversion. However, only gene conversion has been observed both extrachromosomally and within the chromosome with these substrates. Similar substrates containing deletions adjacent to the site of the XhoI linker insertion have also been described (Rubnitz and Subramani, 1987).

bacteria without prior passage through COS cells. Irrespective of the type of mutat ion, linearization of the molecules at the site of the inser t ion/deletion prior to transfection stimulated the frequency 5-12-fold, when cells expressing G418 r were selected directly after transfection of mouse 3T6 cells with the substrates (Rubni tz and Subramani, 1987).

Analysis of chromosomal recombination

An examinat ion of the frequencies and mechanisms of in t rachromosomal homologous recombinat ion requires that 2 or more copies of a selectable gene be present on the same chromosome. Because endogenous chromosomal genes do not fit these criteria, the experimental strategy required the initial placement of an appropriate recombinat ion substrate (preferably as a single copy) into the mammal ian genome. Recombina- tion between 2 nonfunct ional copies of a selectable marker gene then allows the selection of recombinants which grow out as colonies in appropriate selective medium. Because the stable integration of D N A s (in this case, the substrate) is inefficient, a second dominan t selectable marker gene (e.g., gpt, neo or hygromycin resistance, hyg) has also been placed in the substrate plasmid to facilitate the identification of clones of cells that have integrated the recombinat ion substrate into the genome. Screening of genomic D N A s from independent clonal lines confirms that the recombina t ion substrate is stably integrated in the unrecombined configurat ion and preferably as a unique copy. These cells are then switched into a suitable selective growth medium to isolate cell lines in which an in t rachromosomal recombina- t ion event (either in t rachromat id or between sister chromatids) has occurred. It is impor tant that ch romosomal recombinat ion rates be measured by fluctuation tests (Luria and Delbruck, 1943), without which the frequencies can be aberrantly high (as much as 10-100-fold) because sibling clones are counted as independent products.

The genomic D N A s from the parental and recombinant cell fines have been analyzed by com- parative Southern blot analysis. In certain cases, plasmid rescue has been used to analyze the structure of the produc t D N A s in detail. It should be noted that while the selection for recombinants facilitates the measurements of frequencies and the characterizat ion of products, it is also limiting in that only cells containing the chromatids with the recombined marker gene are analyzed. Conse- quently, other partners or products (chromatids) in the recombinat ion event are often lost, resulting in some ambigui ty as to whether the event being analyzed occurred within a chromat id or between mutan t genes or sister chromatids.

229

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© Fig. 7. Chromosomal recombination between directly repeated sequences. Sister chromatids, each containing 2 different mutant alleles (x) of the same selectable marker gene (hatched areas) are shown. Open arrows between hatched boxes indicate a second marker gene, and thin arrows denote the orientation of the repeated sequences. In intrachromatid or unequal sister-chromatid reciprocal exchange, the chromatid undergoing recombination suffers a deletion. The extrachromosomal DNA is lost by degradation or diluted out because it cannot replicate during cell division. Though the two mechanisms are distinguishable, in principle, by comparison of the other non-recombinant chromatid, this is not possible in practice because cells containing these chromatids are not recovered during the selection for the marker gene. Intra- or sister-chromatid gene conversion generate similar products. The donor of genetic information for gene conversion could be the same or the sister chromatid.

For recombinat ion substrates conta ining directly repeated homologous sequences, reciprocal recombinat ion within a chromatid, or unequal ly between sister chromatids, results in a deletion of sequences present between the 2 nonfunct ional copies of the gene. This leaves 1 intact, functional copy of the marker gene on the chromatid. In contrast, gene conversion ( intrachromatid or between sister chromatids) generally results in the correction of one or bo th copies of the gene without any deletions. Thus it is impossible to distinguish in t rachromat id versus sister-chromatid exchanges for direct repeat substrates (Fig. 7). While recombinat ion between directly repeated sequences could occur either by in t rachromat id or unequal sister-chromatid exchange (reciprocal or non-reciprocal), reciprocal recombinat ion between inverted repeats is likely to occur only in an int rachromatid fashion because reciprocal exchange between sister chromatids would generate dicentric and acentric chromatids. However, non- reciprocal exchange between inverted repeats could occur within or between sister chromatids. In- t rachromatid reciprocal recombinat ion between inverted repeats is accompanied by an inversion of sequences between the 2 nonfunct ional copies of the gene. In contrast , gene conversion (intra-

chromat id or between sister chromatids) results in the correction of one or both copies of the gene without accompanying inversions.

Substrates conta in ing truncated, nonfunct ional copies of either neo or tk genes have been used for ch romosomal recombinat ion in 3T6 and L T k - cells, respectively (Subramani and Rubnitz , 1985; Lin et al., 1984a). These studies were done with multiple copies of the substrates in the chromosomes. Similar experiments have been under- taken with 2 mutan t alleles of the neo or tk gene (each as a single copy) in 3T6 and L T k - cells, respectively (Smith and Berg, 1984; Liskay et al., 1984; Liskay and Stachelek, 1986).

The rates of recombinat ion were 1 0 - 6 - 1 0 - 8 / cell generat ion in 3T6 cells (Subramani and Rubnitz , 1985) and 10 -6 in L T k - cells (Liskay and Stachelek, 1986). Abou t 80% of the events observed in L cells arose by gene conversion, and the remaining 20% by reciprocal recombinat ion (Liskay and Stachelek, 1986; Liskay et al., 1987). Recombina t ion rates were independent of the site of integrat ion of the substrate in L T k - cells (Liskay and Stachelek, 1986; Liskay et al., 1987), in contrast with our work in 3T6 cells (Subramani and Rubnitz , 1985; Rubni tz and Subramani, 1987). Chromosomal deletion and inversion events were

230

observed as predicted for substrates with direct or inverted repeat configurations of the homologous sequences, and the rates of recombination were similar for interactions between these two types of repeats (Subramani and Rubnitz, 1985). The effect of carcinogens on chromosomal recombination has also been evaluated (Wang et al., 1988).

Chromosomal gene conversion in the absence of reciprocal exchanges

Most of the substrates described in the pre- ceding section were capable of undergoing either reciprocal or non-reciprocal exchanges. An analysis of the products, however, revealed that non-reciprocal gene conversion was about 4 times more frequent than reciprocal events (Liskay and Stachelek, 1986), We have also designed substrates to examine gene conversion in the absence of reciprocal recombination. These substrates contain a linker-insertion mutation in the neo gene and an internal homologous fragment (overlapping the site of linker insertion) of the gene on the same molecule (Fig. 6). The rates of chromosomal gene conversion were 1 0 - 6 - 1 0 - 8 / c e l l generation for these substrates. Similar molecules were used to demonstrate that the coconversion tract length for chromosomal gene conversion in L T k - cells was less than 358 bp (Liskay and Stachelek, 1986). Though double-reciprocal recombination could also have generated these recombinants, no evidence was found for it (Rubnitz and Subramani, 1986, 1987). We cannot, however, rigorously ex- clude double-reciprocal recombination between sister chromatids except on the grounds that such events are unlikely to occur at a rate of 10-8/cel l generation unless a very high degree of negative interference was also involved. Furthermore, since interactions within chromatids are probably as likely as those between chromatids, the failure to detect intrachromatid double-reciprocal events argues that such interactions are also rare between sister chromatids. Interchromosomal recombination rates have not been measured, although these events have been reported with endogenous genes on homologous chromosomes (Cavanee et al., 1983; Potter et al., 1987; Wasmuth and Hall, 1984).

The higher rate of the gene conversion relative to reciprocal recombination (Liskay and Stache-

lek, 1986; Rubnitz and Subramani, 1986) supports the proposal that gene conversion plays a dominant role in the concerted evolution of multigene families. An obvious advantage of this is that gene conversion does not alter gene dosage or gene organization and is therefore less likely to be deleterious than reciprocal recombination.

Homologous interactions between extrachromosomal molecules and the chromosome

In spite of the ubiquity of non-homologous recombination events, it is clear from several studies that homologous interactions between extrachromosomal and chromosomal D N A s are indeed detectable at low frequencies. Smith and Berg (1984) and Lin et al. (1985) generated mouse cell lines containing integrated copies of nonfunctional neo or tk genes. Transfection of the lines with plasmids carrying nonfunctional but homologous copies of the marker gene led to the restora- tion of the marker gene in about 1/105 cells that could have taken up the DNAs and expressed it stably. Linearization of the D N A was found to be essential for the recombination event to be detectable.

Subsequently, Thomas et al. (1986) and Song et al. (1987) corrected mutant neo genes placed in the chromosome of either L or human cells, respectively, via homologous recombination. The frequencies ranged from 1/76 transfected cells to 1/1000 transfected cells. In the study by Thomas et al. (1986), 2 classes of G418 r cell lines were observed. The first was one in which the wild-type neo gene was generated either by gene conversion of the chromosomal target or of the incoming plasmid which then integrated elsewhere into the genome. The second class became G418 r by a process termed 'heteroduplex-induced mutagene- sis' in which new mutations were introduced into the chromosomal neo gene (to activate the gene), perhaps by repair of a heteroduplex formed between the introduced and chromosomal sequences (Thomas and Capecchi, 1986).

These initial studies with artificially placed genes in chromosomes have now been extended to document gene-targeting of natural, endogenous loci (Smithies et al., 1985a; Thomas et al., 1987; Doetschman et al., 1987). Thus the work on re-

231

A.

R.

x x k..,h,,&._ ~ M , . M ~ M,A...A._ ~ k. . .A.,A A_A..,/ /x

Fig. 8. Mechanisms for rescue of chromosomal sequences onto plasmids. Either gene conversion (A) or double-reciprocal recombination (B) could result in a repaired extrachromosomal product. The two mechanisms differ in the structure of the chromosomal partner after the recombination event. In double-reciprocal recombination the deletion (A) in the incoming plasmid is transferred to the chromosome, whereas in gene conversion the chromosome remains unchanged. Southern blots of the chromosomal D N A of recombinant cell lines can therefore be used to ascertain the mechanism of recombination. Wavy lines refer to flanking cell DNA.

mosomal sequences onto plasmids occurs prim- arily by gene conversion and not by double-reciprocal recombination using an analogous reac- tion in which the cells are not killed as the result of recombination. We generated a cell line (COS/neo2) containing the 3' two-thirds of the neo gene integrated into the COS cell genome as a single copy. Introduction of a linearized plasmid containing the 5' two-thirds of the neo gene re- suited in G418 r colonies. The mode of recombination (double-reciprocal or non-reciprocal) could be determined by analyzing the chromosomal DNA of the recombinants (Fig. 8). Results of Southern blot analysis of the genomic DNA sug- gests that the recombinants arose by gene conversion of the plasmid from the chromosome, followed by integration of the repaired plasmid elsewhere in the genome (Seaton and Subramani, 1988).

Summary

combination that was initiated with viruses and viral-vectors has finally come of age. It should be possible now to apply this knowledge towards targeting genes to autosomal a n d / o r nonselec- table loci in mammalian cells.

Information transfer from chromosomes to extrachromosomal molecules has also been docu- mented using SV40 vectors. These studies, all per- formed in monkey COS cells (containing an integrated copy of the SV40 early promoter /enhancer and T-antigen gene), demonstrated that all (Shaul et al., 1985) or part of the T-antigen gene (Subramani, 1986) or the SV40 enhancer (Jasin et al., 1985) could be rescued from the chromosome onto extrachromosomally replicating, defective SV40 molecules to produce wild-type SV40.

In the work just described, either double-reciprocal recombination or gene conversion could have generated the final product (Fig. 8). These mechanisms differ only in the nature of the chromosomal T-antigen gene remaining after the recombination event. However, the chromosomal DNA of the COS cells was not available for analysis because the cells were killed by the wild- type SV40 produced as a consequence of recombination.

We have determined that the rescue of chro-

Animal viruses have provided important insights into many cellular processes such as replication, repair, transcription, translation, mRNA processing, cellular growth control and the sorting of proteins within mammalian cells. In view of this historical tradition, it should not be surprising that viruses and viral vectors have played a crucial role in our understanding of the recombinational pathways in mammalian cells. Specifically we have used them to study non-homologous and homologous recombination events. The latter include extrachromosomal and chromosomal, reciprocal and non-reciprocal events as well as interactions between extrachromosomal molecules and the mammalian genome. In the last 5 years, the field of recombination has advanced tremendously from a point when one doubted and despaired whether gene targeting would ever be possible in mammalian cells, to a stage where it has certainly become feasible. All of this has been accomplished with relatively little insight into the enzymology and fundamental mechanisms of homologous and non-homologous recombination. A number of systems have been described for the analysis of the enzymology of recombination in vitro, using extracts from mammalian cells (Ganea et al., 1987; Kucherlapati et al., 1985; Lopez and Coppey,

232

1987; Lopez et al., 1987; Hsieh et al., 1986). In addition, many large D N A viruses such as vac- cinia or herpes viruses probably carry some of their own recombination enzymes. Cells infected with these viruses may be a good source of such enzymes. A recent report on the joining of non- homologous ends in vitro by X. laevis egg extracts may shed light on the enzymes involved in non- homologous recombination (Pfeiffer and Viel- metter, 1988). As we learn more about these different systems in the years to come, there is no doubt that with it will come the knowledge to tilt the balance further in favor of homologous recombination.

Acknowledgements

This work was supported by grants GM31253 and CA01062. This is a publication of the Center for Molecular Genetics at UCSD.

I would like to acknowledge the work of various members of my lab, without whose diligent efforts this review would not have been possible.

References

Brenner, D.A., S. Kato, R.A, Anderson, A.C. Smigocki and R.D. Camerini-Otero (1984) The recombination and integration of DNAs introduced into mouse L cells, Cold Spring Harbor Symp. Quant. Biol., 49, 123-138.

Brenner, D.A., A.C. Smigocki and R.D. Camerini-Otero (1985) Effects of insertions, deletions and double-strand breaks on homologous recombination in mouse L cells, Mol. Cell. Biol. 5, 684-691.

Brenner, D.A., A.C. Smigocki and R.D. Camerini-Otero (1986) Double-strand gap repair results in homologous recombination in mouse L cells, Proc. Natl. Acad. Sci. (U.S.A.), 83, 1762-1766.

Calos, M.P. (1986) Mutation of autonomously replicating plasmids, in: R. Kucherlapati (Ed.), Gene Transfer, Plenum, New York, Ch. 9, pp. 243-262.

Cavenee, W.K., T.P. Dryja, R.A. Phillips, W.F. Benedict, R. Godbout, B.L. Gallie, A.L. Murphree, L.C. Strong and R.L. White (1983) Expression of recessive alleles by chromosomal mechanisms in retinoblastoma, Nature (London), 305, 779-784.

Clark, A.J. (1973) Recombination-deficient mutants of Escherichia coli and other bacteria, Annu. Rev. Genet., 7, 67-86.

Cox, M.M., and I.R. Lehman (1987) Enzymes of general recombination, Annu. Rev. Biochem., 56, 229-262.

Dasgupta, U.B., and W.C. Summers (1980) Genetic recombination of Herpes-simplex virus, the role of the host cell and UV-irradiation of the virus, Mol. Gen. Genet., 178,617-623.

de Wet, J.R., K.V. Wood, M. DeLuca, D.R. Helinski and S. Subramani (1987) Firefly luciferase gene: structure and expression in mammalian cells, Mol. Cell. Biol. 7, 725-737.

Doetschman, T., R.G. Gregg, N. Maeda, M.L. Hooper, D.W. Melton, S. Thompson and O. Smithies (1987) Targetted correction of a mutant HPRT gene in mouse embryonic stem cells, Nature (London), 330, 576-578.

Dubbs, D.R., M. Rachmeler and S. Kit (1974) Recombination between temperature-sensitive mutants of simian virus 40, Virology, 57, 161-174.

Folger, K.R., E.A. Wong, G. Wahl and M.R. Capecchi (1982) Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules, Mol. Cell. Biol., 2, 1372-1387.

Folger, K.R., K. Thomas and M.R. Capecchi (1985) Nonre- ciprocal exchanges of information between DNA duplexes coinjected into mammalian cell nuclei, Mol. Cell. Biol., 5, 59-69.

Ganea, D., P. Moore., L. Chekuri and R. Kucherlapati (1987) Characterization of an ATP-dependent DNA strand trans- ferase from human cells, Mol. Cell. Biol., 7, 3124-3130.

Hirt, B. (1967) Selective extraction of polyoma DNA from infected mouse cell cultures, J. Mol. Biol., 26, 365-369.

Hsieh, P., M.S. Meyn and R.D. Camerini-Otero (1986) Partial purification and characterization of a recombinase from human cells, Cell, 44, 885-894.

Jasin, M., J. De Villiers, F. Weber and W. Schaffner (1985) High frequency of homologous recombination in mammalian cells between endogenous and introduced SV40 genomes, Cell, 43, 695-703.

Kucherlapati, R.S, E.M. Eves, K.-Y. Song, B.S. Morse and O. Smithies (1984) Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA, Proc. Natl. Acad. Sci. (U.S.A.), 81, 3153-3157.

Kucherlapati, R.S., J. Spencer and P.D. Moore (1985) Homolo- gous recombination catalyzed by human cell extracts, Mol. Cell. Biol., 5,714-720.

Lin, F.-L., K. Sperle and N. Sternberg (1984a) Homologous recombination in mouse L cells, Cold Spring Harbor Symp. Quant. Biol., 49, 139-149.

Lin, F.-L., K. Sperle and N. Sternberg (1984b) Model for homologous recombination during transfer of DNA into mouse L cells: role for DNA ends in the recombination process, Mol. Cell. Biol., 4, 1020-1034.

Lin, F.-L., K. Sperle and N. Sternberg (1985) Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences, Proc. Natl. Acad. Sci. (U.S.A.), 82, 1391-1395.

Liskay, R.M., and J.L. Stachelek (1986) Information transfer between duplicated chromosomal sequences in mammalian cells involves contiguous regions of DNA, Proc. Natl. Acad. Sci. (U.S.A.), 83, 1802-1806.

Liskay, R.M., A. Letsou and J.L. Stachelek (1987) Homology requirement for efficient gene conversion between duplicated chromosomal sequences in mammalian cells, Genet- ics, 115, 161-167.

Liskay, R.M., J.L. Stachelek and A. Letsou (1984) Homologous recombination between repeated chromosomal sequences in mouse cells, Cold Spring Harbor Symp. Quant. Biol., 49, 183-189.

Lopez, B., and J. Coppey (1987) Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA, Nucl. Acids Res., 15, 6813-6826.

Lopez, B., S. Rousset and J. Coppey (1987) Homologous recombination intermediates between two duplex DNA catalyzed by human cell extracts, Nucl. Acids Res., 15, 5643-5655.

Luria, S.E., and M. Delbruck (1943) Mutations of bacteria from virus sensitivity to virus resistance, Genetics, 28, 491-511.

Miller, L.K., B.E. Cooke and M. Fried (1976) Fate of mis- matched base-pair regions in polyoma heteroduplex DNA during infection of mouse cells, Prec. Natl. Acad. Sci. (U.S.A.), 73, 3073-3077.

Miller, B.L., K.Y. Miller and W.E. Timberlake (1985) Direct and indirect gene replacements in Aspergillus nidulans, Mol. Cell. Biol., 5, 1714-1721.

Mocarski, E.S., and B. Roizman (1982) Herpes virus-dependent amplification and inversion of cell-asseciated viral thymidine kinase gene flanked by viral a sequences and linked to an origin of viral DNA replication, Proc. Natl. Acad. Sci. (U.S.A.), 79, 5626-5630.

Mosig, G., R. Dannenberg, D. Ghosal, A. Luder, S. Benedict and S. Beck (1979) General genetic recombination in bacteriophage T4, Stadler Genet. Symp., 11, 31-55.

Orr-Weaver, T.L., J.W. Szostak and R.J. Rothstein (1981) Yeast transformation: a model for the study of recombination, Prec. Natl. Acad. Sci. (U.S.A.), 78, 6354-6358.

Pfeiffer, P., and W. Vielmetter (1988) Joining of non-homologous DNA double strand breaks in vitro, Nucl. Acids Res., 16, 907-924.

Potter, T.A., R.A. Zeff, W. Frankel and T.V. Rajan (1987) Mitotic recombination between homologous chromosomes generates H-2 somatic cell variants in vitro, Prec. Natl. Acad. Sci. (U.S.A.), 84, 1634-1637.

Rigby, P.W.J., and P. Berg (1978) Does simian virus 40 DNA integrate into cellular DNA during productive infection? J. Virol., 28, 475-489.

Rothstein, R.J. (1983) One-step gene disruption in yeast, Meth. Enzymol., 101,202-211.

Rubnitz, J., and S. Subramani (1984) The minimum amount of homology required for homologous recombination in mammalian ceils, Mol. Cell. Biol., 4, 2253-2258.

Rubnitz, J., and S. Subramani (1985) Rapid assay for extrachromosomal homologous recombination in monkey cells, Mol. Cell. Biol., 5, 529-537.

Rubnitz, J., and S. Subramani (1986) Extrachromosomal and chromosomal gene conversion in mammalian cells, Mol. Cell. Biol., 6, 1608-1614.

Rubnitz, J., and S. Subramani (1987) Correction of deletions in mammalian ceils by gene conversion, Somat. Cell. Mol. Genet., 13, 183-190.

Seaton, B.L., and S. Subramani (1988) Transfer of chro-

233

mosomal sequences onto plasmids by gene conversion, in: Gene Transfer and Gene Therapy, ICN-UCLA Symp. Mol. Biol., in press.

Shapira, G., J.L. Stachelek, A. Letsou, L.K. Soodak and R.M. Liskay (1983) Novel use of synthetic oligonucleotide insertion mutants for the study of homologous recombination in mammalian cells, Prec. Natl. Acad. Sci. (U.S.A.), 80, 4827-4831.

Shaul, Y., O. Laub, M.D. Walker and W.J. Rutter (1985) Homologous recombination between a defective virus and a chromosomal sequence in mammalian cells, Prec. Natl. Acad. Sci. (U.S.A.), 82, 3781-3784.

Shulman, M.J., L.M. Hallick, H. Echols and E.R. Signer (1970) Properties of recombination-deficient mutants of bacteriophage lambda, J. Mol. Biol., 98, 503-517.

Smith, A.J.H., and P. Berg (1984) Homologous recombination between defective neo genes in mouse 3T6 cells, Cold Spring Harbor Symp. Quant. Biol., 49, 171-181.

Smithies, O., R.G. Gregg, S,S. Boggs, M.A. Koralewski and R.S. Kucherlapati (1985a) Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination, Nature (London), 317, 1230-1234.

Smithies, O., M.A. Koralewski, K.-Y. Song and R.S. Kucherlapati (1985b) Homologous recombination with DNA introduced into mammalian cells, Cold Spring Harbor Symp. Quant. Biol., 49, 161-170.

Song, K.-Y., F. Schwartz, N. Maeda, O. Smithies and R. Kucherlapati (1987) Accurate modifications of a chromosomal plasmid by homologous recombination in mammalian cells, Prec. Natl. Acad. Sci. (U.S.A.), 84, 6820-6824.

Subramani, S. (1986) Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating defective simian virus 40 DNA by homologous recombination, Mol. Cell. Biol., 6, 1320-1325.

Subramani, S., and P. Berg (1983) Homologous and nonho- mologous recombination in mammalian cells, Mol. Cell. Biol., 3, 1040-1052.

Subramani, S., and J. Rubnitz (1985) Recombination events after transient infection and stable integration of DNA into mouse cells, Mol. Cell. Biol., 5, 659-666.

Subramani, S., and B.L. Seaton (1988) Homologous recombination in mitotically dividing mammalian cells, in: R. Kucherlapati and G. Smith (Eds.), Genetic Recombination, ASM, in press.

Subramani, S., and P.J. Southern (1983) Analysis of gene expression using simian virus 40 vectors, Anal. Biechem., 135, 1-15.

Symington, L.S., P. Morrison and R. Kolodner (1985) In- tramolecular recombination of linear DNA catalyzed by the Escherichia coli recombination system, J. Mol. Biol., 186, 515-525.

Thomas, K.R., and M.R. Capecchi (1986) Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene, Nature (London), 324, 34-38.

Thomas, K.R., and M.R. Capecchi (1987) Site-directed muta- genesis by gene targeting in mouse embryo-derived stem cells, Cell, 51, 503-512.

234

Thomas, K.R., K.R. Folger and M.R. Capecchi (1986) High frequency targeting of genes to specific sites in the mammalian genome, Cell, 44, 419-428.

Upcroft, P., B. Carter and C. Kidson (1980) Analysis of recombination in mammalian cells using SV40 genome segments having homologous overlapping termini, Nucl. Acids Res., 8, 2725-2736.

Vogel, T. (1980) Recombination between endogenous and exogenous simian virus 40 genes, Virology, 104, 73-83.

Vogel, T., Y. Gluzman and E. Winocour (1977) Recombination between endogenous and exogenous simian virus 40 genes: biochemical evidence for genetic exchange, J. Virol., 24, 541-550.

Volkert, F.C., and C.S.H. Young (1983) The genetic analysis of recombination using adenovirus overlapping terminal DNA fragments, Virology, 125, 175-193.

Wake, C.T., and J.H. Wilson (1979) Simian virus 40 recombinants are produced at high frequency during infection with genetically mixed oligomeric DNA, Proc. Natl. Acad. Sci. (U.S.A.), 76, 2876-2880.

Wang, Y., V.M. Maher, R.M. Liskay and J.J. McCormick (1988) Carcinogens can induce homologous recombination

between duplicated chromosomal sequences in mouse L cells, Mol. Cell. Biol., 8, 196-202.

Wasmuth, J.J., and L.V. Hall (1984) Genetic demonstration of mitotic recombination in cultured Chineses hamster cell hybrids, Cell, 36, 697-707.

Wilson, J. (1988) Illegitimate recombination in mammalian cells, in: R. Kucherlapati and G. Smith (Eds.), Genetic Recombination, ASM, in press.

Wolgemuth, D.J., and M.-T. Hsu (1980) Visualization of genetic recombination intermediates of human adenovirus type 2 DNA from infected HeLa cells, Nature (London), 287, 168-171.

Wood, K., and M. Dehica (1987) Photographic detection of luminescence in E. coli containing the gene for firefly luciferase, Anal. Biochem., 161, 501-507.

Yates, J.L., N. Warren and B. Sugden (1985) Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells, Nature (London), 313, 812-815.

Young, C.S.H., and S.J. Silverstein (1980) The kinetics of adenovirus recombination in homotypic and heterotypic genetic crosses, Virology, 101, 503-515.

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors

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