short dna fragments induce site specific recombination in mammalian cells

Molecular and Cellular Biochemistry 92: 107-116, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Original Article

Short DNA fragments induce site specific recombination in mammalian cells

Katharina Hunger-Bertling, Petra Harrer and Wolf Bertling Klinischer Arbeitskreis fgtr Rheumatologie der Max-Planck Gesellschaft an der Universitiit Erlangen-Niirnberg, Schwabachanlage 10, 8520 Erlangen, FRG

Received 13 December 1988; accepted 8 May 1989

Key words: homologous recombination, polyoma capsids, electroporation, lymphocytes

Summary

A defective hprt gene was corrected by homologous recombination in a lymphocyte cell line deficient in Hypoxanthine-phosphoribosyl-transferase activity (hprt). In a novel approach, only a fragment of a cDNA clone of the functional hprt gene was used to induce homologous recombination. The mutation that was corrected corresponds to a single base change in exon III of the hprt gene.

Two transfection methods, electroporation and the previously unreported use of polyoma capsids containing only short DNA fragments, were able to induce the recombinational event. After transfection cells with a functional hprt gene were selected and homologous recombination events were identified using polymerase chain reaction.

Double stranded fragments and both coding and non-coding single stranded fragments resulted in conversion to a functional gene.

Analysis of the resulting hprt positive cells revealed that most cells had undergone a simple replacement reaction. Interestingly, however, some cells had lost an intron adjacent to the site of mutation. Potential mechanisms for this phenomenon, including the possible involvement of RNA in DNA repair, are discussed.

Introduction

Homologous recombination has been studied in bacteria [1, 2], yeast [3] and mammalian cell systems [4-6]. The fragment lengths necessary for successful site-specific recombination have been determined in bacteria [2, 6, 7] and the homology requirements and other parameters have been described for mammalian gene conversion systems [8-14]. In eukaryotic systems mostly nicked or linearized double stranded plasmids have been used to induce recombination except for single reports concerning circular single stranded DNA in mammalian cells [15] and in yeast [16]. Various mechanisms have been postulated for the integration of such constructs into the genome [3, 17] most of

which are based on the Meselson-Radding model [18]. It is thought that the ends of linearized mole- cules are involved in starting the process [19, 20], while specific enzymes catalyze the following pairing and strand exchange reactions [21-23].

Homologous recombination studies in mammals show that many DNA transfection strategies [24- 26] are relatively error prone. We decided to cir- cumvent this problem by transfecting only a fragment of a gene that meets the recombination length requirements established by Watt et al. [2] and others [5, 10]. This approach also avoids the general problems in the use of recombinant DNA for homologous recombination, e.g. the co-introduction of sequences other than the homologous fragment. We used a well defined mutation in the read-

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ily selectable hprt gene as our recombination target and the corresponding cloned cDNA sequence for this gene as our recombinant source. Defects in the hprt gene cause diseases such as gout and Lesch- Nyhan syndrome in men [27, 28]. We therefore used a lymphocyte cell line, hprtmunich, from a gout patient where the HPRT defect has been mapped by protein sequencing to a single amino acid [29]. We will show here that the defect corresponds to a single nucleotide change in exon III. The human hprt gene has been cloned as a cDNA [30] and the corresponding genomic organization is well defined [31]. Since the hprt gene is located on the X-chromosome, there is only one copy per diploid cell. This allows the selection of hprt positive cells in HAT medium (Hypoxanthine-Aminopterine- Thymidine). Aminopterine blocks a major pathway of purine metabolism, and only cells containing a functional HPRT gene can use this alternative pathway to survive.

Here we report the use of a short isolated DNA fragment spanning the part of exon III containing the point mutation and the 3' end of exon III to induce a site-specific gene alteration. We transfected the fragments into the mutant cell line by electroporation or by encapsulation into viral capsids as described before [32]. The experiments were conducted with three types of fragment: double stranded DNA, single stranded DNA in both orientations and single stranded DNA complexed with the protein rec A which is known to participate in homologous recombination in bacteria [33, 34] and may generally promote recombination. We found that both electroporation and the gentle transfection method using empty polyoma capsids [35] gave rise to HPRT positive cells independent of the type of DNA fragment used. The polymerase chain reaction [36, 37] was used to amplify recombined sequences in genomic DNA and ana- lyze the products of successful recombination products.

Interestingly, for some positive cells the recombinational event results in a loss of the adjacent intron. The reasons for the precise excision of the intron sequence are discussed. Thus small cDNA fragments can potentially be used to specify homologous insertion sites in the eukaryotic genome and

can be introduced into cells by mild transfection methods.

Experimental procedures

Cells

The lymphoid hprt negative cell line hprtmunich, was a gift from Dr. T. Palella (University of Michigan). These cells are EBV transformed [38] and have a non-functional hprt gene due to an amino acid exchange in exon III [29]. Maintenance and selection using HAT (100 ~M Hypoxanthine, 0.4/.~M Aminopterine, 16 t~M Thymidine [Flow Laborato- ries, VA]) were performed as described [39].

Plasmids

A plasmid containing a functional cDNA of the human hprt gene (p4aA8) was a gift from Dr. T. Friedmann (University of California, San Diego) [30]. We recloned a Hinc II/Bam H I-fragment into M13mpl8. The resulting clone mKW31, which no longer coded for a functional full length cDNA, was used to isolate the 165 bp Xho I/Mbo II-fragment of the exon III of the human HPRT-gene. The fragment, purified by gel electrophoresis, was used either directly for transfection assays or was separated into the 169bp and 164 bp complementary single strands [40]. A similar fragment (Xho I/Hinc II, comprising parts of the exon III and exon IV) also served as a hybridization probe after label- ing by random primer extension [41]. Commercial- ly available recA protein (United States Biochem- icals) was tested for activity [42] and coupled to single strand DNA [32]. Oligonucleotides were made on a DNA-Synthesizer (Applied Biosys- terns) and purified over a polyacrylamide gel.

In control experiments we used oligonucleotides complementary to the site of the mutation. Oli- gomers of 14 nucleotides were expected to be too short to efficiently trigger homologous recombination [2, 10]. We also used sheared salmon sperm DNA and assays without DNA as controls.

Transformation methods

10 6 cells and 2/xg of DNA without carrier were used per electroporation assay. Electroporation was performed using a Gene-Pulser apparatus (Bio- Rad) [39] as previously described.

For transfection using viral capsids the DNA was packaged into empty capsids of a small-plaque- forming polyoma virus strain [43]. Empty capsids were a gift from Dr. Aposhian (University of Ari- zona) and were prepared as described by Crawford [44]. Empty viral capsids (3.0tzg protein) were complexed with DNA (0.5 tzg) or DNA/recA complexes in PLP buffer (Tris/HC1 pH7.5 10mM, NaC1 10mM, EDTA l mM, BSA 100tzg/ml) in a 500 Ixl assay as described [32, 35]. Hprtmunich ceils (10 6) were 'infected' with vitally encapsidated DNA (0.21xg) as described [32]. After an initial incubation ( lh at 37°C) the assays were diluted with HAT medium to a final volume of 10 ml.

Analysis

Genomic DNAs derived from human lymphoid cells, hprt positive and hprt negative populations, were prepared from isolated nuclei and purified over CsC1 [45]. DNA was digested with appropriate restriction endonucleases according to the manufacturer's (Boehringer) instructions and fraction- ated on 0.8% agarose gels. For hybridization DNA was transferred to nitrocellulose by the method of Southern 846] and was blot-hybridized with appro- priately labeled probes (see above).

Polymerase chain reaction was performed according to Saiki et al. [36, 37] using Taq polymerase (New England Biolabs), 1/xg of Hind III-linearized genomic DNA and appropriate primers. We performed 30 cycles at 94 ° C, 22°C and 54°C for 1, 1 and 3 min respectively. For set-ups with the shorter primer # 2 (Fig. 1) cycles were at 94 ° C, 22 ° C, 37 ° C and 54 ° C for 1, 5, 3, and 3 minutes. After 30 cycles the DNA was precipitated and 1/3 of each assay mixture was loaded on a 4% NuSieve gel (FMC Bioproducts Corp., ME).

DNA from transformed HAT-resistant cells and from hprtmunich lymphocytes was amplified by po-

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lymerase chain reaction in two different ways. In both cases we digested the genomic DNAs with Hind III before PCR reaction. The upstream primers were identical (primer number 1 in Fig. 1). In one set of experiments we used an intron specific primer (primer number 2 in Fig. 1) as the downstream primer of the PCR reaction. In the other set a primer homologous to a part of exon IV (primer number 3 in Fig. 1) was used. With an intron specific downstream primer, a fragment of appropriate length was synthesized by Taq Polymerase with either hprt positive and hprt negative genomic DNA as template (Fig. 4). The exon IV specific primer, however, only generated a 120 bp fragment from hprt positive DNA (Fig. 4). The polymerase chain reaction is known to be not suitable for sequences longer than two to three kb. The intron between exon III and IV is 10.9 kb and since it also contains a Hind III restriction site [31] the DNA used for PCR was digested with Hind III to prevent the amplification of the intron. Amplified DNA fragments were separated on agarose gels, excised and recovered with GeneClean (Bio 101 Inc., CA). The purified DNA fragments were either heat de- natured and used directly for dideoxy sequencing [47, 48] or were phosphorylated with T4 polynucle- otide kinase (Boehringer) following the directions of the manufacturer. Phosphorylated fragments were recloned into pUC 18 and the plasmid DNA was prepared by an alkaline lysis method [45] for double strand sequencing [48].

Results

A Xba I/Mbo II fragment of a cDNA of the human hprt gene or each of its single strands (Fig. 1) was tested for its ability to participate in homologous recombination events. The DNA was neither au- tonomously replicating nor associated with foreign DNA such as plasmid or viral sequence.

Electroporation

After transfection of human lymphocytes with the double stranded cDNA fragment or with either

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S'GCTGACCTGGATTAC

primer 1

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) . . . . . . . . . . . . . ~ r ~ g l , n ~ U ~ . d f o r l u [ u t l n n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

N r v D ~ i . L,.LV~Vre~s ~.Asp~l~s,,rhr~ D Z ~ V I ~ a D P L ~ r L r ~

wild type

Ly~yr Ly~Tyr

AA AG~_~AT AA AG~TAT hprt. u.. i e h

3, ATACTAAGAAAA ~ ' ~ ' ACTGGTCAGTTGTCCC s '

primer 2 primer 3

Fig. 1. Part of the sequence of the mutant and wildtype genomic hprt gene. Parts of exon III, the adjacent intron and exon IV are shown as double stranded DNA and as amino acid sequence [capital letters: exon sequence, small letters: intron sequence, the site of mutation is enlarged]. The substitution of the C/G basepair by an A/T basepair leads to the replacement of a Serine by an Arginine in the protein sequence. Important parts of the sequence of the fragment are enlarged below and above the overview. All primers used for polymerase chain reaction and sequencing are indicated: primer 1 is the upstream primer and sequencing primer, primer 2 is the intron specific primer, and primer 3 is the downstream primer, specific for exon IV. The sequence of control oligomers (14 mer) was: 5'TGAAGAGCTATATI~GT and 5 'ACAATAGCTCTI'CA. The intron sequence information provided by Jane C. Moores [25] is: 5 ' G T G A G T A T A T ' I T A A X T A T G A T F C T Y I T X C A GT . . . . . " I T I ' I T l q T F A A CTA G 3'.

orientation of the single stranded fragments, stably transformed HAT resistant clones were detectable after two to three weeks. No HAT insensitive cells were found after transfection with control DNA.

Polyoma capsid mediated transfection

Empty polyoma capsids loaded with double stranded or single stranded fragments of either orientation were used to transfect DNA into the mutant lymphocyte cell line hprtm,nich- This system also allows the transfection of DNA-protein complexes, e.g. single stranded fragments of either orientation complexed with rec A [32]. When we transfected with homologous fragments of sufficient length [2] [> 160 bp or bases] stably transformed HAT-resistant hprt positive cells were detectable within 2-3 weeks. None of the control DNAs (neither short specific oligomers nor sheared salmon sperm DNA) nor empty capsids alone induced the growth of cells in HAT medium.

Recombination rates

Although the growth characteristics of hprtm,nich prevented us from isolating individual resistant col- onies (see discussion), reasonable estimates of the recombination rates (assuming similar growth stimulus following rescue of the mutant phenotype by the recombination event) could be obtained by comparing cell numbers at a fixed time (21 days) after transfection. According to this assumption we estimate the number of recombinants as being between 10 and 100 per transfection assay (106 cells). In 16 independent experiments, where we transfected a hprt gene fragment we observed a steep onset of growth in selection medium after 2-3 weeks. No HAT resistant cells grew in the course of 20 control experiments (Fig. 2).

In our system it was not possible to precisely determine the number and frequencies of recombination events. The variations between individual experiments using double stranded vs. single stranded vs. recA complexed DNA were also not

cells/ml

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Fig. 2_ Cell growth under selective conditions. The upper- dashed line represents the growth of HAT sensitive cells in normal growth medium. The lower dashed curve shows the reaction of these cells as well as of cells of control experiments after adding HAT medium at day 0. These two curves are averages of 25 experiments• The dotted curve shows the growth of the cell line hprtmunJch in HAT medium after transfection with DNA fragments of the exon III of the intact hprt gene at day 0. Dye exclusion assays were used to determine the number of live cells. Values of dye exclusion assays of two typical experiments with double stranded DNA fragments encapsidated in empty capsids of polyoma virus are indicated by squares.

large enough to allow a quantitative assessment of the relative recombination efficiency.

Analysis

We wanted to determine the nature of the event which had caused rescue of the resistant phenotype in hprt~...ich. Therefore we grew transfected HAT- resistant populations derived from hprtmunich cell and after several passages extracted and examined the hprt gene. Transfected hprt positive cells from two experiments using viral capsids packed with double stranded fragments were passaged several times and used to prepare genomic DNA.

Genomic DNA of untreated HAT sensitive cells (hprt negative cells) and of transformed HAT resistant cells (hprt positive cells) was subjected to

111

hybridization analysis to determine the nature of the recombinational event.

The hprt fragment had primarily integrated at one locus as evidenced by blot hybridization of Eco RI digested genomic DNA with exon III fragment. In genomic blots of both hprtmunich cells and hprt positive cells, one band corresponding to an Eco R I fragment of the correct length was detected. When using a cDNA fragment comprising 169 bp of exon III and only 14 bp of exon IV (Xho I/Hinc II) [30] to probe hprt negative and hprt positive cells only one fragment of 8.2 kb was expected in both cases [31]. Surprisingly, DNA of hprt positive cells showed a second, weaker band at 12.0 kb, which is due to the loss of an intron (Fig. 3).

We examined both species in more detail using the polymerase chain reaction (PCR). For both PCR and for hybridization analyses we used genomic DNA of cells with virally encapsidated double stranded fragments (Fig. 4).

Fragments of hprt positive DNA amplified with the downstream primer specific for exon IV [number 3 (Fig. 1)] were sequenced directly using the dideoxy sequencing reaction (Fig. 5). The amplified fragments of hprt positive and hprt negative cells generated with the intron specific primer [number 2 (Fig. 1)] were phosphorylated and recloned. Using some of these plasmid clones, we confirmed the protein sequence analysis of the original defect in hprtmunich at the DNA level. The nature of the amino acid exchange, Serine for Argi- nine [29], leaves an ambiguity in the nature of the mutated base - the wild type Serine codon AGC could have mutated to any of 5 possible Arginine codons. Our PCR-based sequence analysis indicates the most probable sequence at the mutation as being AAGAGATATTG compared to the functional hprt sequence AAGAGCTATTG.

The sequence analysis of clones derived from intron-containing hprt positive cells indicated that preparations of genomic hprt positive DNA were contaminated with genomic hprt negative DNA. Since we could not single out individual clones but rather had to isolate the DNA from liquid cultures of genomic hprt positive cells, we had probably also isolated DNA from quiescent or dead hprt negative cells which were still among the viable cells. Dye

112

, , /.\/'\ , , , / \

e × ~ II ~xon Ill ~xc~n IV

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e~oc~n II e x o n Ill ÷ I V

Fig. 3. Schematic drawing of recombination products. Marked sections (iiil) correspond to fragments reacting with the hybridization probe. Upper section: Hprt genes containing the intron between exon III and exon IV. Three Eco RI sites are in the intron between exon III and exon IV, the hybridizing fragment therefore does not comprise exon IV. Lower section: Hprt positive DNA of clones missing the intron between exon III and exon IV. The hybridizing fragment is extended to the next Eco RI site downstream of exon IV. Blocked areas (11) indicate coding regions, arrows (A) indicate Eco RI restriction sites.

exclusion assays suggested that some 1-2% dead cells were present when the cells were harvested.

We can not formally exclude the possibility that some quiescent negative cells were present or that we had occasionally induced a second site mutation compensating the original defect.

Discussion

We have used short DNA fragments derived from recombinant sources to induce homologous recom-

bination to human genomic sequences at a prede- termined site. The specific modification rescues the hprt negative phenotype of the cell line hprtm,nich allowing them to survive in HAT medium. In al- most all previous cases when homologous recombination has been observed, recombinant DNA was used, i.e. mammalian genes or gene fragments integrated into plasmids or phage genomes. Our experiments indicate that by careful design of the homologue the specific recombination can be initi- ated in mammalian cells without affecting other parts of the genome.

Fig. 4. Polymerase chain reaction products. A schematic drawing of recombination products indicating the location of primers is added. A: Hprt positive (lane 2) and hprt negative (lane 3) DNA prepared from cells containing the intron between exon III and exon IV. Arrows (--~ and ~--) indicate the polymerase chain reaction products using primer 1 and primer 2 (Fig. 1). Products were separated on a 0.8% Agarose gel. B: Only hprt positive DNA (lane 4) of cells missing the intron between exon III and exon IV yields also polymerase chain reaction products when using primer 1 and primer 3 (arrow). Hprt negative DNA (lane 6) which contains the intron does not yield a product, since the intron is 10.9 kb long and contains a Hind III site (Kim et al. 1986). All genomic DNA had been Hind III digested prior to polymerase chain reacUon. Products were separated on a 0.8% (A) and a 4,0% (B) agarose gel. Phi X DNA digested with Hae III was used as a size marker in both cases.

A B

3"

T A

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C & A A T C T

G A A Y

C T A G

A T A T

A G T T

T - "2 T T / A A t T T

A A J

F= G

A A

A A

T T

C

A A

A A

C C

T ~ T

A - ~R

T

T - 7

T T

7 T

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Fig. 5_ Sequencing reactions using primer 1. A: Sequence of the directly sequenced amplified polymerase chain reaction fragment resulting from primer 1 and 3. Note that there is no intron between the end of exon III (TATTG) and the beginning of exon IV. B: Sequence of a recloned polymerase chain reaction product of hprt negative cells using primer 1 and 2. The previously unknown nature of the base exchange of the hprt deficient cell line hprtmu,i,h is most likely AAGAGATATFG as it appeared in all sequences of hprt negative clones.

We have shown that the encapsidation of transfecting DNA in viral coats leads to similar recombination efficiencies as electroporation (as assessed by number of viable cells). Chemical or physical DNA transfection systems trigger many undesir- able side effects in the mammalian nucleus [49, 50]. Polyoma virus capsid mediated transfection is a more gentle method but also has side effects such as deregulation of expression of certain genes [51]. The viral capsid system can potentially also be used

113

for gene transfer studies in vivo, and may allow genetic engineering in adult animals.

Other investigators have shown that the modification of a specific gene in the genome is possible whether or not the target gene is actively tran- scribed [5, 6, 9, 11]. In bacteria the mechanisms, frequencies and the enzymes involved in homologous recombination have been studied extensively [l, 52]. In mammalian systems there is also evi- dence for the existence of specific recombination systems, e.g. human cell extracts catalyzing the homologous recombination between two duplex DNAs [21, 53]. The frequency and kinetics of such recombinational events have been determined for mammalian nuclei with two co-injected plasmids [54].

We can extrapolate from the growth curves of rescued cells to estimate the efficiency of homologous recombination in our system, although such an extrapolation gives only a vague idea of the initial number of recombinant cells. Given these limitations our crude estimates of recombination rates (10 -4 to 10 -5) fall in the range between 10 -1 and 10 -5 described by other workers [4, 9, 11-14, 55]. The wide spread that has been observed seems to result not only from the different lengths of the exogenous DNA, but also from the degree of homology, the length of homologous versus non-homologous flanking sequences and maybe from the primary sequence of the target gene itself. The existence and the number of pseudogenes with re- duced homology is also a likely reason for the observed wide variation. Of course the cell type and the transfection system are also very important.

Hprt~unich are non-adherent cells with a very low viability in agar after electroporation. In liquid culture, however, they recover well with good viability (80 to 90%) [32]. When we used polyoma capsid-mediated transfection we had to suspend the cells in liquid culture due to the nature of the 'in- fection' process, but there was no decrease in viability (data not shown).

Polyclonal analysis data strongly support a unique and specific integration event. Hybridiza- tion analyses showed only homologous integration events even after long exposure times. The loss of the intron between exon III and IV indicates that

114

we are not dealing with a simple reversion of a point mutation, it also hints at the existence of a system involved in repair or recombination in mammalian cells. We cannot determine the exact location of the recombination between resident and exogenous DNA at the 5' end of the fragment. However, we have obtained information about the exact location of recombination at the 3' end. Since the exon III fragment ends exactly at the exon- intron transition (Fig. 1), the results imply that a double strand break at or close to such a transition site may have caused the precise excision of the adjacent intron. Specialized repair enzymes, e.g. RNA processing enzymes, or - more likely - RNA itself might be involved in homologous recombination processes. The induction of mutations by new- ly introduced sequences in the cognate genes had been observed before [9]. We looked at some nat- urally occurring deletions or insertions to see whether available sequence data support the idea that such a repair system was involved. The sequence of at least one of the two sites of such a deletion or insertion resembles the consensus sequence for exon-intron transitions [56, 57]. The small number of cases and the high probability of randomly finding such sequences does not constitu- te a proof for the involvement of a repair system using RNA or of RNA-processing enzymes, but it cannot be disregarded.

The presence of reactive ends such as double strand breaks on the transfected and the genomic DNA [19, 20, 58, 59] is highly recombinogenic. Approaching the point of linearization to the homologous part increases the efficiency of recombination [60]. Capecchi and coworkers recently [5] observed after transfection of a linearized plasmid with only one homologous end that the non-homologous part was not cointegrated. This observation does not rule out that free DNA ends particpate in the initiation of recombination events, but suggests that homology of free ends is not the dominant factor. Recombinational events could be caused by a simple cross-over within the homologous parts [60] leading to a duplication of those sequences [11]. The insertion of an additional homologous fragment is described by Thomas and Capecchi [55] using long fragments with a relatively low grade of

homology (number of homologous vs. number of non-homologous nucleotides) compared to the single point mutation located on the 165 base pair fragment. Recombination might also be triggered by gene conversion whereby parts of the resident gene are replaced by the equivalent exogenous DNA sequence [54, 55].

The use of a non-replicating DNA fragment min- imizes the chances that unwanted foreign DNA is introduced as it is possible when retroviral integration vectors are used. The viral capsid system offers the potential application of gene transfer studies to in vivo systems and might allow genetic engineering in adult animals. Such an approach (employing encapsidation) may allow planned, specific alter- ations of gene loci in bone marrow, liver, spleen or in the lymphatic system, although many problems remain to be solved before techniques, as the one described here, can be applied to animal systems. In conclusion, the ability to utilize short, defined DNA sequences to induce homologous recombination may be the ideal vehicle to study mammalian gene regulation. It allows the change of a very short sequence without any alteration in the environ- ment of the target sequence.

Acknowledgements

I would like to thank Ron Shehee (UNC, Chapel Hill) for his support and helpful discussions and Susan Stamper at UNC at Chapel Hill who provided us with some of the oligomers. Aposhian (Univ. Arizona, Tucson) kindly provided us with empty Polyoma capsids and the corresponding virus strain. Last but not least we thank S. Goodman, who helped us prepare this manuscript.

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Address for offprints: K. Hunger-Bertling, Klinischer Arbeitskreis Rheumatologie, Universit/it Erlangen-Niirnberg, Schwabanlage 10, 8520 Erlangen, FRG

short dna fragments induce site specific recombination in mammalian cells

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