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The Journal of Immunology, 2002, 168: 2332-2339.
Copyright © 2002 by The American Association of Immunologists

Evidence for the Murine IgH µ Locus Acting as a Hot Spot for Intrachromosomal Homologous Recombination1

Steven J. Raynard*, Leah R. Read{dagger} and Mark D. Baker2,*,{dagger}

* Department of Molecular Biology and Genetics, College of Biological Science, and {dagger} Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Homologous recombination accomplishes the exchange of genetic information between two similar or identical DNA duplexes. It can occur either by gene conversion, a process of unidirectional genetic exchange, or by reciprocal crossing over. Homologous recombination is well known for its role in generating genetic diversity in meiosis and, in mitosis, as a DNA repair mechanism. In the immune system, the evidence suggests a role for homologous recombination in Ig gene evolution and in the diversification of Ab function. Previously, we reported the occurrence of homologous recombination between repeated, donor and recipient alleles of the Ig H chain µ gene C (Cµ) region residing at the Ig µ locus in mouse hybridoma cells. In this study, we constructed mouse hybridoma cell lines bearing Cµ region heteroalleles to learn more about the intrachromosomal homologous recombination process. A high frequency of homologous recombination (gene conversion) was observed for markers spanning the entire recipient Cµ region, suggesting that recombination might initiate at random sites within the Cµ region. The Cµ region heteroalleles were equally proficient as either conversion donors or recipients. Remarkably, when the same Cµ heteroalleles were tested for recombination in ectopic genomic positions, the mean frequency of gene conversion was reduced by at least 65-fold. These results are consistent with the murine IgH µ locus behaving as a hot spot for intrachromosomal homologous recombination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The process of homologous recombination accomplishes the exchange of genetic information between two similar or identical DNA duplexes. Homologous recombination can occur either by gene conversion, a mechanism of unidirectional DNA sequence transfer that does not change the overall organization of the participating sequences, or by reciprocal crossing over. Both mechanisms can be associated (1). In the germline, meiotic recombination between homologous chromosomes is important for creating the genetic diversity upon which natural selection can act. In somatic cells, mitotic homologous recombination is important for correcting errors of replication and other forms of DNA damage. These beneficial features are overshadowed by the potentially deleterious consequences of homologous recombination between repeated sequences, such as deletions, inversion, translocations, and amplifications, which are implicated in several human diseases, including cancer (2, 3, 4).

Over evolutionary time, homologous recombination has most likely been important in shaping the organization of complex, multigene families (5). One such example is the family encoding the Ig H and L chain genes, whose individual members consist of a hierarchically related set of kindred sequences (6, 7, 8, 9). In somatic cells, an ever-increasing body of evidence supports an important role for homologous recombination in remodelling of the chromosomal Ig genes. Gene conversion is important in generating Ab diversity in birds and rabbits (10, 11), and may also play a role in the diversification of some mammalian Ab responses (12, 13, 14, 15, 16, 17, 18, 19, 20). Both gene conversion and crossing over occur between Ig H chain constant segments within regions of shared homology (21, 22, 23, 24). The process of mammalian somatic hypermutation is thought to involve strand invasion, homologous pairing, and copying of sequences from the sister chromatid, similar to the process of gene conversion (25). In transgenic mice, gene conversion was found to occur between an integrated Ig transgene and the endogenous Ig H chain locus (26, 27) and between tandem V(D)J segments (28). Receptor editing and receptor revision may also involve homologous pairing between the interacting V region segments (29). Thus, homologous interactions between Ig gene segments are important for proper functioning of the humoral immune compartment.

Our laboratory has been investigating homologous recombination in mammalian cells. The experimental system monitors recombinational exchanges between homologous repeats of the Ig µ gene C (Cµ)3 region positioned in the haploid, chromosomal µ locus of mouse hybridoma cells (30, 31). Interestingly, homologous recombination between the repeated Cµ segments is several orders of magnitude higher than the frequency of recombination between heteroalleles of selectable marker genes integrated in random genomic positions in different mammalian cell lines (32, 33, 34, 35, 36). It is highly unlikely that the small differences in the extent of shared homology and distance separating the repeats (<2-fold) account for the huge discrepancy in recombination frequency. Also, the high frequency of intrachromosomal recombination appears inconsistent with a general elevation of recombination proteins in the hybridoma cells because in comparison with other cell types (37), the frequency of another form of homologous recombination, namely, gene targeting (10-6 recombinants/cell), is not particularly enhanced in the hybridoma cells.

The above studies prompted further investigation of the unusually high frequency of intrachromosomal homologous recombination. Mouse hybridoma cell lines bearing Cµ region heteroalleles integrated at the chromosomal µ locus were constructed to investigate the effect of marker position on the rate and mechanism of intrachromosomal homologous recombination. The results revealed that genetic markers spanning the entire Cµ region were converted to the wild-type sequence at high frequency. The absence of directionality is consistent with the possibility that gene conversion initiates from random lesions within the Cµ region. Other experiments revealed that Cµ region heteroalleles differing by only a single genetic marker also underwent high frequency gene conversion with both Cµ regions serving equally well as either conversion donors or recipients. Remarkably, when the same Cµ region heteroalleles were placed in ectopic positions in the hybridoma genome, the mean frequency of gene conversion was reduced by at least 65-fold. Thus, the results are consistent with the chromosomal µ locus acting as a hot spot for homologous recombination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hybridoma cell lines

The hybridoma cell line, Sp6/HL, bears a single copy of the trinitrophenyl (TNP)-specific chromosomal Ig µ H chain and makes normal, cytolytic TNP-specific IgM ({kappa}-chain) (38, 39). The following Sp6/HL-derived hybridoma cell lines were used in this study. The hybridoma cell line, igm10, is a mutant that has lost the TNP-specific chromosomal µ gene (39). The hybridoma cell lines, Im/RCµ2A2, Im/RCµD7, and Im/RCµ482-3/1, were constructed by gene targeting in the Cµ region of the haploid, TNP-specific chromosomal µ gene, as described previously (40, 41). The conditions for hybridoma cell growth have been described elsewhere (38, 39).

Vector construction

Four enhancer-trap, gene-targeting vectors were constructed to investigate the effect of marker position on the frequency of Cµ region gene conversion. Each vector contains a 7.2-kb SpeI/XbaI fragment (Fig. 1GoA) from the cloned µ gene of the Sp6/HL hybridoma (42) that includes the Cµ region and a residual, 0.4-kb segment of the µ gene switch (Sµ) region inserted into a pSV2neo backbone from which the SV40 enhancer driving neo expression was removed (43, 44). The vector-borne Cµ region shares 5.8 kb of homology with the Sp6/HL chromosomal Cµ region. An endogenous restriction enzyme site present in one of the four vector-borne Cµ region exons was converted to a BspHI site by site-directed mutagenesis (45). The following oligonucleotides were synthesized at MOBIX (McMaster University, Hamilton, Ontario, Canada) and used in the mutagenesis: 5'-GGCAAAAACAGAGATCATGATGCATGTGCCCATTCC-3', to replace the BglII site in exon Cµ1 at genomic position bp 404; 5'-CGAGAACAAAGGATCATGACACACCCCAAACC-3', to replace the BamHI site in exon Cµ2 at genomic position bp 824; 5'- CATGGAAAGCCATCATGACCAATGGCACCTTCAGTGC-3', to replace the BstXI site in exon Cµ3 at genomic position bp 1329; 5'-GGAGGAATGGAACTCATGACGGAGAGACCTATACC-3', to replace the BspEI site in exon Cµ4 at genomic position bp 1921. All Cµ region genomic positions are numbered according to the system in Goldberg et al. (46). The following information applies to each oligonucleotide: the novel BspHI site is underlined; the four nucleotides that were inserted to create the BspHI site are indicated in boldface type; the original Cµ region restriction enzyme site that was destroyed by the 4-bp (ATGA) insertion is indicated in italics. The products of site-directed mutagenesis were verified by DNA sequencing.



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  		FIGURE 1. Construction of mutant hybridoma cell lines. A, Four 12.6-kb enhancer-trap insertion vectors, each bearing a BspHI marker that replaces one of the endogenous BglII, BamHI, BstXI, or BspEI restriction enzyme sites in Cµ region exon 1, 2, 3, or 4, respectively, were targeted to the recipient, haploid chromosomal µ gene in the wild-type hybridoma cell line, Sp6/HL. With the exception of the inserted marker, each vector shares 5.8 kb of uninterrupted homology to the chromosomal Cµ region. For illustrative purposes, only the vector with the mutation in Cµ region exon 1 is drawn. In all panels, the mutant exons are denoted by the open rectangles, while the wild-type exons are denoted by the filled rectangles. B, The structure of the chromosomal µ gene in the four mutant hybridoma cell lines generated by targeted vector integration. In each cell line, the mutant, recipient 5' Cµ region bears the BspHI marker in one of the four Cµ exons and is separated from the wild-type, donor 3' Cµ region by the integrated pSV2neo vector sequences. Gene conversion between the Cµ region heteroalleles will restore the sequence of the 5' mutant Cµ region exon in favor of the wild-type restriction enzyme site present in the corresponding 3' Cµ region exon. To verify the gene conversion event, the 5' Cµ region is specifically amplified to yield a 4.8-kb product using primer pair AB9703/AB9745 and the PCR product tested for the gain of the wild-type restriction enzyme site and loss of the mutant site according to PCR and gel analysis methods described previously (44 ). Sp, SpeI; X, XbaI; neo, neomycin phosphotransferase gene. This figure is not drawn to scale.

 
The vector, pCµ-repeat, bears two 4.3-kb XbaI segments of Cµ region genomic DNA inserted into a pSV2neo vector backbone in tandem orientation. The downstream (3') Cµ region is from the wild-type Sp6 hybridoma (Fig. 1GoA), while the upstream (5') Cµ region bears a 2-bp Cµ3 deletion from the Sp6-derived mutant hybridoma cell line, igm482 (38, 39). The vector bears a unique SwaI site for linearizing the vector before electroporation.

Recovery and characterization of targeted hybridoma cells

Transfer of vector DNA to the Sp6/HL hybridoma cells was performed by electroporation, as described previously (40). Identification of hybridoma cells, in which the haploid, chromosomal µ gene is modified by targeted vector insertion, was performed according to previously published procedures (44, 47). Verification of the correct µ gene structure in the targeted recombinants was performed by Southern and PCR analysis of hybridoma genomic DNA according to standard procedures, as described previously (44, 48). The procedures used to obtain both glycosylated and unglycosylated [35S]methionine biosynthetically labeled µ-H chain proteins have been described (38, 39).

Quantitative PCR (qPCR)

The primers used for qPCR were synthesized at MOBIX (McMaster University), and their sequences are as follows: primer 1, 5'-ACT TGA GAA GCC AGG ATC TAG G-3'; primer 2, 5'-CTT ACC GCT GTT GAG ATC CAG T-3'; primer 3, 5'-CCA TTG GGT TTT GAG ATG AAT T-3'; and primer 4, 5'-CCA TTG GGT TTT GAG ATG AAT C-3'. The following conditions were used for qPCR per 50-µl reaction volume: 1 µg of template DNA, 1x GeneAmp PCR buffer II (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 0.2 mM dNTPs, 0.36 µM primers, 2 mM MgCl2, 10-9 pmol vector control (see below), 3.5 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA), and 50 µl of mineral oil overlay. The cycling conditions were: 95°C for 15 min to denature the DNA and activate the Taq polymerase, followed by 40 cycles consisting of 94°C for 45 s, 59°C for 45 s, 72°C for 150 s, and finally, renaturation at 72°C for 10 min. To analyze the products of PCR, 10 µl of the reaction was loaded onto a 0.7% agarose gel and visualized by ethidium bromide (EtBr) staining.

The control vector used as an internal standard in the qPCR was constructed by insertion of two oligonucleotides containing the primer 1/2 binding sites and the primer 3/4 binding sites in reverse orientation into the multiple cloning site of pBluescript IIks. The appropriate primer pairs amplify a 1.3-kb segment from the HSV-1 thymidine kinase gene that was placed between the oligonucleotide insertions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influence of marker position on the frequency of intrachromosomal gene conversion between Cµ heteroalleles inserted in the murine IgH µ locus

To study intrachromosomal gene conversion, we have used enhancer-trap gene-targeting vectors and recipient Sp6/HL hybridoma cells (Fig. 1GoA) to construct four targeted hybridoma cell lines designated as Im/TCµEmut-1, Im/TCµEmut-2, Im/TCµEmut-3, and Im/TCµEmut-4 (Fig. 1GoB). Each hybridoma cell line bears a pair of Cµ region heteroalleles inserted in the haploid, chromosomal IgH µ locus: the 3' Cµ region is wild type and the 5' Cµ region is mutant as a consequence of replacing an endogenous restriction enzyme site within one of the four Cµ region exons with the sequence, 5'-ATGA-3', creating a BspHI restriction enzyme site. The mutant 5' Cµ region is present in its correct position to be expressed, downstream of the TNP-specific H chain V (VHTNP) region gene, and is separated from the wild-type 3' Cµ region by the pSV2neo backbone of the gene-targeting vector. Complement-dependent, TNP-specific erythrocyte lysis tests and SDS-PAGE analysis of 35S-biosynthetically labeled µ-chains (data not shown) (38, 39) verified that each hybridoma cell line synthesized a truncated µ H chain and produced noncytolytic, TNP-specific IgM.

During growth under G418-selective conditions, conversion of the recipient, 5' mutant Cµ region by the donor, 3' wild-type Cµ region can restore normal, TNP-specific IgM production in the hybridoma cells, allowing them to be detected as plaque-forming cells (PFC) in a sensitive, TNP-specific plaque assay (40). To determine the influence of marker position on the frequency of intrachromosomal Cµ region gene conversion, three subclone cultures of each parental, mutant hybridoma cell line were started from a single cell. When checked, Southern analysis confirmed that the µ gene structure in the subclones and parental cultures was the same (data not shown). Each subclone culture was grown for a defined number of generations (generation time, ~18 h) and then subjected to the TNP-specific plaque assay. As shown in Table IGo, the mean frequency of generating TNP-specific PFC in each cellline was very high. According to a {chi}2 test, the means did not differ significantly from equivalence ({chi}2 = 0.7944; 0.90 < p < 0.75). Therefore, a high, uniform frequency of gene conversion was observed across the entire 5' Cµ region.


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  		Table I. Gene conversion between Cµ region heteroalleles

 
As verification of the gene conversion event, a total of 31 TNP-specific PFC was recovered from mutants Im/TCµEmut-2, Im/TCµEmut-3, and Im/TCµEmut-4, and purified by limited dilution cloning. PFC genomic DNA was PCR amplified using primer pair AB9703/AB9745 generating the 4.8-kb product specific for the 5' Cµ region (44) (Fig. 1GoB). In separate reactions, the PCR products were digested with BamHI, BstXI, and BspEI to test for the wild-type markers in 5' Cµ exons 2, 3, and 4, respectively, and with BspHI to verify loss of the insertion mutation. The cleavage products were analyzed for the fragment sizes predicted by these enzymes by agarose gel electrophoresis. The results (data not shown) confirmed conversion of the mutant, 5' Cµ region BspHI site to the wild-type restriction enzyme site present in the corresponding position of the 3' Cµ region. Therefore, in the TNP-specific PFC, correction of the mutant 5' Cµ region exon sequence to wild type was consistent with a gene conversion event.

Use of a qPCR assay to measure the frequency of gene conversion in both the 5' and 3' Cµ regions

It was of interest to determine whether the 3' Cµ region in the hybridoma cells enjoyed the same high frequency of gene conversion as did the 5' Cµ region. As the TNP-specific plaque assay could not be used to measure gene conversion in the 3' Cµ region, a qPCR assay was developed. The finding that gene conversion was not subject to Cµ region position effects made it possible to develop the qPCR assay with hybridoma cell lines in which the Cµ region heteroallelic pair differed by only a single genetic marker.

To standardize the qPCR assay, two related hybridoma cell lines were used, both having been described previously (31, 40). In hybridoma cell line, Im/RCµ482-3/l-7 (abbreviated 3/1-7), both the 5' and 3' Cµ regions are wild type (designated, double wild type), whereas, in hybridoma cell line, Im/RCµD7-7 (abbreviated D7-7), they both contain a 2-bp deletion in the Cµ3 exon (designated double mutant) (Fig. 2GoA). Four PCR primers were designed. Primers 1 and 2 bind upstream of the 5' and 3' Cµ regions, respectively, whereas primers 3 and 4 are specific for the wild type and mutant Cµ3 exons, respectively. The sequence of primers 3 and 4 differs by a single base at their 3' terminus. As a result, the mismatch created when either primer binds to the wrong allele prevents extension of the PCR product. The wild-type and mutant sequence is specifically amplified from the 5' Cµ region using primer pairs l/3 and 1/4, respectively, generating a 1.6-kb product. Similarly, the presence of the wild-type and mutant sequence in the 3' Cµ region is determined by the production of a specific 1.9-kb product using primer pairs 2/3 and 2/4, respectively. To circumvent the problem of variability in efficiency of amplification, each PCR included 10-9 pmol of a heterologous vector control, sharing only the primer binding sites with the target sequence. Amplification of the vector generates a specific 1.3-kb product (Fig. 2GoB).



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  		FIGURE 2. qPCR assay to measure the rate of gene conversion between the Cµ region heteroalleles. A, Hybridoma 3/1-7 has the wild-type sequences in both the 5' and 3' Cµ regions, while hybridoma D7-7 bears the mutant Cµ3 exon in both the 5' and 3' Cµ regions ({rectangle}). The PCR primers and the sizes of the amplified products they produce are indicated. Primer 1 binds upstream of the 5' Cµ region and outside the region of homology in the 3' Cµ region. Primer 2 binds within pSV2neo vector sequences upstream of the 3' Cµ region. Primers 3 and 4 bind specifically to the complementary strand of the wild-type and mutant Cµ3 exon, respectively. The vector control contains the primer 1 and 2 binding sites and primer 3 and 4 binding sites on opposite strands flanking a segment of the thymidine kinase (tk) gene. B, Standard solutions of genomic DNA consisting of D7-7–3/1-7 prepared in ratios of 1:20, 1:50, 1:200, 1:500, 1:1000 along with a constant amount of vector control (10-9 pmol) were amplified using primer pair 1/4 for generation of the 1.6-kb mutant 5' Cµ region product and 1.3-kb vector control product. Quantitation of the EtBr staining in the target band divided by that in the vector control band yielded the standard curve (inset). Lanes 9–13 (inclusive), Replicate DNA samples from the hybridoma cell line, Im/RCµ2A2, that, along with the vector control, were amplified with primer pair 1/4 to detect gene conversion in the 5' Cµ region. From the ratio of product:vector in each sample, the frequency of gene conversion was determined from the standard curve. neo, Neomycin phosphotransferase gene; tk, HSV-1 thymidine kinase gene fragment. This figure is not drawn to scale.

 
The qPCR method requires that both competitor and target fragments amplify with the same efficiency, ensuring the target/vector ratio remains constant throughout the amplification process. The kinetic analysis shown in Fig. 3GoA revealed two identical curves up to 60 cycles of amplification. Thus, the ratio of these amplified products should precisely reflect the initial concentration of target vs that of the added competitor vector, irrespective of cycle number.



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  		FIGURE 3. Validation of the qPCR assay. A, Amplification kinetics of genomic target and vector control DNA. Equivalent copy numbers of the double wild-type genomic DNA (dotted line) and vector control DNA (solid line) were mixed and amplified for 32, 34, 36, 38, 40, 50, 55, and 60 cycles using primer pair 1/3. The data are plotted as a function of PCR product band intensity vs cycle number. B, A constant amount of vector control (3 x 103 copies) was added to mixtures of genomic DNA consisting of double mutant-double wild type in ratios of 1:20, 1:50, 1:200, 1:500, and 1:1000. The genomic target copy number in each mixture was calculated to be 5 x 103, 2 x 103, 5 x 102, 2 x 102, and 1 x 102, respectively. PCR amplification (40 cycles) was performed with primer pair 1/4. The ratio of target:vector band intensity was plotted vs the ratio of input target:vector copy number for five replicate determinations of each sample.

 
To determine the level of sensitivity of the qPCR assay, mixtures of genomic DNA consisting of double mutant-double wild type were prepared in ratios of 1:20, 1:50, 1:200, 1:500, and 1:1000, and amplified using primer pair l/4 for generation of the mutant, 1.6-kb 5' Cµ region product. Following gel electrophoresis, quantitation of the EtBr staining in the 1.6-kb target band divided by that in the 1.3-kb vector control band yields a specific ratio that, when plotted against the mutant Cµ region copy number, generates a standard curve (Fig. 2GoB, inset). The qPCR assay is specific and of sufficient sensitivity to detect recombinants at the frequency of ~10-3 (Fig. 2GoB, lane 6). To verify the validity of the qPCR assay over the range of input target copy numbers used in the standard curve, a series of PCR were performed using a constant amount of vector control added to the known genomic mixtures described above. Following amplification using primer pair 1/4 and gel electrophoresis, the product target/vector band ratios were plotted against the known input target/vector ratios (Fig. 3GoB). The plot showed a nearly linear relationship (R = 0.91) within this range. In a similar manner, the qPCR assay was standardized with the primer pairs l/3, 2/3, and 2/4 to detect the wild-type 5' Cµ, wild-type 3' Cµ, and mutant 3' Cµ regions, respectively.

Out of concern that PCR-mediated recombination (49) might interfere with specificity by annealing and extension of incomplete Cµ region PCR products, long extension times (150 s) were used to generate full-length products. Control experiments verified that under these conditions, there was no PCR-mediated recombination: no PCR product was detected when primers 2 and 3 were tested for amplification of an equimolar mixture of genomic DNA from the wild-type Sp6/HL hybridoma and the double mutant, and none was obtained from a DNA mixture consisting of equal copy number of a mutant Cµ region-bearing plasmid vector and the double wild type using primers l and 4 (Fig. 4Go).



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  		FIGURE 4. Control experiments for detection of PCR-mediated recombination. A, A mixture consisting of an equivalent copy number of 3/1-7 genomic DNA and the mutant Cµ region-bearing plasmid, pRCµ482 (40 ), was amplified using primer pair 1/4, a combination that amplifies the mutant 5' Cµ region to generate a 1.6-kb product. Since 3/1-7 contains the wild-type 5' Cµ region, the 1.6-kb product can only arise through PCR-mediated recombination. As a positive control, the 1:1000 mixture of double mutant-double wild type was included. Hybridoma 3/1-7 alone was included as a negative control. A constant amount of vector control was included in each PCR. No recombinant PCR product was detected. B, An equimolar mixture of genomic DNA from the wild-type Sp6/HL hybridoma and the double mutant, D7-7, was amplified using primer pair 2/3, which amplifies the wild-type 3' Cµ region. PCR-mediated recombination would generate a 1.9-kb product. Hybridoma 3/1-7 was included as a positive control. As in A above, no recombinant PCR product was detected. neo, Neomycin phosphotransferase gene. This figure is not drawn to scale.

 
We exploited the qPCR assay to measure the frequency of gene conversion in the 5' and 3' Cµ regions of the independent hybridoma cell lines, Im/RCµD7 and Im/RCµ2A2. These cell lines were constructed by gene targeting, as described previously (40), and both bear the same Cµ region heteroalleles inserted in the haploid chromosomal µ locus: the wild-type sequence is present in the 5' Cµ region, while the 3' Cµ region contains the 2-bp Cµ3 deletion. Three subclone cultures of each hybridoma were started from a single cell. When checked, Southern blot analysis revealed that the µ gene structure in each subclone was identical to that in the parent cell line (data not shown). Each subclone was grown for 25 generations in medium supplemented with 0.6 mg/ml G418. Following growth, five replicate samples of genomic DNA prepared from each Im/RCµD7 and Im/RCµ2A2 subclone were tested in the qPCR assay using primer pair l/4, which detects gene conversion in the direction of the mutant sequence in the 5' Cµ region, and in a separate reaction, primer pair 2/3, which detects gene conversion in the direction of the wild-type sequence in the 3' Cµ region. Following gel electrophoresis, the ratio of product:vector in each sample was determined by quantitation of EtBr staining, and the frequency of gene conversion was determined from the standard curve. An example of this analysis for the 5' Cµ region in Im/RCµ2A2 subclone 1 is presented in Fig. 2GoB (lanes 9–13). As indicated in Table IIGo, the mean frequency of gene conversion was very high in both the 5' and 3' Cµ regions in these hybridoma cell lines. According to a t test, the means were not significantly different (5' Cµ, p = 0.307; 3' Cµ, p = 0.205). Furthermore, the frequency of gene conversion between the Cµ regions as determined by qPCR is similar to that measured by the TNP-specific plaque assay (Table IGo).


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  		Table II. Gene conversion between Cµ region heteroalleles

 
Frequency of gene conversion between ectopic donor and recipient Cµ regions

It was of interest to determine whether the high frequency of gene conversion was also a feature of Cµ heteroalleles positioned outside the chromosomal Ig µ locus. For these studies, the plasmid vector, pCµ-repeat, was constructed. It bears 4.3-kb segments encoding the mutant and wild-type Cµ regions flanking a pSV2neo vector backbone (Fig. 5GoA). The vector was linearized at the unique SwaI site and transferred by electroporation into the Sp6-derived hybridoma cell line igm10, which lacks the endogenous chromosomal µ gene (39). Individual G418R transformants were isolated and screened by PCR analysis in conjunction with Southern blotting using both single cutters and noncutters to determine the structure and copy number of the integrated vector (results not shown). Five cell lines, designated R/CµRepeat-13, R/CµRepeat-20, R/CµRepeat-34, R/CµRepeat-35, and R/CµRepeat-49, were identified as having the structure shown in Fig. 5GoB. Hybridoma cell lines R/CµRepeat-20, R/CµRepeat-35, and R/CµRepeat-49 each contain a single fully intact integrated vector bearing both 4.3-kb Cµ regions. Cell line R/CµRepeat-13 contains a single integrated vector; however, the 3' Cµ region is truncated slightly, but retains at least 3.3 kb of homology to its heteroallelic, 5' Cµ region recombination partner. Cell line R/CµRepeat-34 contains two unlinked copies of the construct, both of which are fully intact.



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  		FIGURE 5. Insertion of Cµ region heteroalleles in ectopic positions in the hybridoma genome. A, The vector pCµ-repeat bears the 4.3-kb Cµ region heteroalleles inserted into a pSV2neo vector backbone in the tandem orientation. The upstream (5') Cµ region bears the 2-bp Cµ3 deletion ({rectangle}), but is otherwise isogenic with the downstream (3') Cµ region. The vector was linearized at the unique SwaI site and transfected into the hybridoma cell line, igm10, which lacks the endogenous chromosomal µ gene (39 ). B, Tandem repeated Cµ region structure in the independent G418R transformants, R/CµRepeat-13, 20, 34, 35, and 49, as determined by Southern analysis. S, SwaI; neo, neomycin phosphotransferase gene. This figure is not drawn to scale.

 
Each transformant was expanded from a single cell and grown for at least 25 generations under G418 selection. Genomic DNA was prepared from each culture, and qPCR utilizing primer pair l/3 was performed to measure the frequency of gene conversion in the 5' Cµ region. This analysis revealed the conversion frequency to be below the sensitivity of the assay (<0.001) in all five cell lines. To determine the conversion frequency in these cell lines, they were plated at a density of 500 cells/well in 24-well plates. Following cell growth, genomic DNA was prepared from each culture well and qPCR analysis was repeated on the separate DNA preparations. As an example, Fig. 6Go presents the relevant standards together with seven representative genomic DNA samples of the 30 tested for transformant R/CµRepeat-35. From the fraction of negative wells in the qPCR assay and the Poisson distribution, the mean gene conversion frequency between the ectopic Cµ regions in the transformants (Table IIIGo) was determined to be at least 65-fold lower than the corresponding mean frequency for the same Cµ heteroalleles positioned in the IgH chromosomal µ locus (Table IIGo). ANOVA of the logarithm-transformed data revealed a significant difference between the means at the level of {alpha} = 0.05. According to a t test, the reduced mean frequency of gene conversion between the Cµ regions removed from the chromosomal µ locus is highly significant (p = 0.00002).



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  		FIGURE 6. qPCR of R/CµRepeat-35 subcultures to determine the frequency of gene conversion in the 5' Cµ region. The transformant, R/CµRepeat-35, was distributed in 24-well plates at a density of 500 cells/well. Following growth, genomic DNA was prepared and amplified along with a constant amount of vector control (10-9 pmol) using primer pair 1/3 to detect gene conversion in the 5' Cµ region to the wild-type sequence. If a recombinant was deposited in the well during subculturing, it would be detected at a frequency of 0.002. From the fraction of negative wells and the Poisson distribution, the frequency of gene conversion was determined. This figure shows a representative gel from the qPCR analysis. Lanes 2–6, amplification products generated from standard solutions of genomic DNA consisting of 3/1-7-D7-7 prepared in ratios of 1:20, 1:50, 1:200, 1:500, and 1:1000. Amplification of the wild-type 5' Cµ region and the vector control generates 1.6-kb and 1.3-kb PCR products, respectively. Lanes 8–14, Representative samples from R/CµRepeat-35 subculture wells 1–7. Positive signals are present in subcultures 4, 5, and 7.

 

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  		Table III. Gene conversion between Cµ region heteroalleles in ectopic positions

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated an unusual feature of the chromosomal Ig µ locus, namely, the ability to support a high frequency of intrachromosomal homologous recombination. The experimental system is based on the wild-type, mouse hybridoma cell line, Sp6/HL, which bears a single copy of the chromosomal µ gene and synthesizes TNP-specific IgM({kappa}) (38, 39). The haploid µ locus in Sp6/HL was modified by gene targeting so that it contained a pair of stably integrated Cµ region heteroalleles: one member of the heteroallelic pair served as the conversion recipient, while the other member served as the conversion donor. In the first series of experiments, the effect of marker position on the frequency of gene conversion between the Cµ heteroalleles was investigated. The recipient, mutant 5' Cµ region was present in its correct position to be expressed downstream of the VHTNP region and was separated from the donor, wild-type 3' Cµ region by the integrated pSV2neo vector sequences. Gene conversion of the mutant sequence to wild type restores normal TNP-specific IgM production in the hybridoma cells, allowing them to be detected as PFC in a TNP-specific plaque assay (40). The results revealed that genetic markers spanning the entire recipient 5' Cµ region underwent gene conversion at a very high frequency (on average, ~3 x 10-3 PFC/cell). Both the high frequency of gene conversion and its uniformity across the chromosomal Cµ region are noteworthy. Previously, Cµ region heteroalleles that differed by a 2-bp Cµ3 deletion were also shown to undergo high frequency gene conversion (30, 31). As only a single marker was studied, it could be argued that the high conversion frequency reflected a peculiarity of the marker, or perhaps its position within the Cµ region. However, these concerns are ruled out by the results of the present study, since the four Cµ region markers were identical and they were all converted to wild type with a similar high efficiency.

The high frequency of gene conversion in the 5' Cµ region raised the question as to whether or not the 3' Cµ region could be converted with the same high efficiency. However, the TNP-specific plaque assay could not be used to measure gene conversion in the 3' Cµ region. Therefore, a qPCR assay was developed that, with the appropriate primer pairs, was capable of measuring gene conversion in both the 5' and 3' Cµ regions in the hybridoma cells. In the independent, Sp6/HL-derived hybridoma cell lines, Im/RCµD7 and Im/RCµ2A2, the measurements revealed that the frequency of converting the wild-type 5' Cµ region to the sequence of the mutant 3' Cµ region was equivalent to the frequency with which the mutant 3' Cµ region was converted to the sequence of the wild-type 5' Cµ region. Therefore, both the 5' and 3' Cµ regions are equally proficient as either conversion donors or recipients. In contrast, a different result was obtained in an earlier study (31) with the related hybridoma cell line, Im/RCµ482-3/1, in which the position of the wild-type and mutant Cµ alleles in the chromosomal µ locus was reversed. Using a colony-screening assay for recombinants, the 5' Cµ region was found to be at least 8-fold more efficient as a conversion recipient than the 3' Cµ region, suggesting a preferred, 5' directionality to the conversion process. Using the more sensitive qPCR assay, we have determined that the 5' Cµ region in Im/RCµ482-3/1 is favored 18-fold over the 3' Cµ region as a conversion recipient (Raynard and Baker, unpublished results). In these same studies, it was also shown that the frequency of gene conversion in the 5' Cµ region of both Im/RCµ482-3/1 and Im/RCµD7 is similar. As the hybridoma cell lines Im/RCµD7 and Im/RCµ2A2 do not display directionality, the results obtained with Im/RCµ482-3/1 might reflect a peculiarity of this particular cell line. However, in all of the hybridoma cell lines, gene conversion might involve the formation and repair of a hybrid or heteroduplex DNA intermediate, as is also the case for some gene conversion in yeast (1). Therefore, the observed conversion directionality in Im/RCµ482-3/1 might be a property of the reversed markers either through their influence on heteroduplex DNA formation, or on the direction of its repair. At present, we cannot distinguish between these alternative possibilities.

It is clear that the Cµ region heteroalleles are subject to an exceptionally high frequency of gene conversion. Therefore, it was important to determine whether this was a property of the IgH µ locus itself or, perhaps, a feature intrinsic to the Cµ regions or intervening pSV2neo vector sequences. To accomplish this, we investigated the frequency of gene conversion in five independent hybridoma cell lines in which the same Cµ heteroalleles were stably integrated in an ectopic position in the genome. In all cell lines, the endogenous chromosomal µ locus was absent. Use of the qPCR assay revealed that the mean frequency of gene conversion between the ectopic Cµ heteroalleles was at least 65-fold lower than for the same heteroalleles positioned in the chromosomal µ locus. These results suggest that the chromosomal µ locus promotes high frequency gene conversion between the repeated Cµ regions.

Double-strand breaks (DSBs) are potent stimulators of homologous recombination in both meiosis and mitosis (1, 50). In mammalian cells, DSB-induced mitotic recombination is elevated 1000-fold over the background of spontaneous recombination (51). Interestingly, this enhancement in recombination is similar to that observed for spontaneous recombination between the Cµ regions positioned in the IgH µ locus as compared with spontaneous recombination between heteroallelic selectable marker genes (32, 33, 34, 35, 36). This information is consistent with the possibility of a chromosomal µ locus-induced DSB within a member of the heteroallelic Cµ region pair. However, the observed uniformity in the frequency of gene conversion across the Cµ region is not consistent with a specific, recombination-initiating DSB site. Rather, it is more compatible with the notion that the recombination initiates from random DSBs across the entire Cµ region.

The results of this study suggest that the chromosomal IgH µ locus is a hot spot for intrachromosomal homologous recombination. The high frequency appears to be a property of intrachromosomal recombination since the frequency of gene targeting at this locus is similar to that observed at other loci in different mammalian cell types (37). Previous work has identified transcriptional activity (1, 52, 53, 54, 55) and the binding of cellular proteins to DNA elements (56, 57) as being important in promoting intrachromosomal homologous recombination in other eukaryotic systems. Proposed mechanisms include an altered, recombinationally active chromatin environment and enhanced accessibility of the DNA to lesions that promote recombination. Interestingly, the chromosomal µ locus is highly transcribed in B lineage cells, and the binding sites for several tissue-specific, developmentally regulated cellular proteins have been identified in the promoter, enhancer, and switch regions of the µ gene (58, 59). Thus, transcriptional activity and/or regulatory element binding by cellular proteins present equally plausible explanations for the enhanced homologous recombination between the repeated Cµ regions. It is tempting to speculate that the unusual capacity of the IgH µ locus to support a high frequency of intrachromosomal homologous recombination reflects the extreme genomic plasticity of this locus that is required for the generation of new Abs with diverse Ag-binding capabilities. Accordingly, our current work focuses on identifying the mechanism(s) responsible for its recombination hot spot activity.


   Acknowledgments
 
We thank the members of our laboratory, Erin Birmingham, Patricia Bell, and Richard McCulloch, for their helpful comments during the course of this work.


   Footnotes
 
1 This work was supported by the Canadian Institutes of Health Research (Operating Grant MF-13443) to M.D.B. and a Canadian Institutes of Health Research Ph.D. studentship award to S.J.R. Back

2 Address correspondence and reprint requests to Dr. Mark D. Baker, Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1. E-mail address: mdbaker{at}uoguelph.ca Back

3 Abbreviations used in this paper: Cµ region, Ig µ gene C region; DSB, double-strand break; EtBr, ethidium bromide; PFC, plaque-forming cell; qPCR, quantitative PCR; Sµ region, Ig µ gene switch region; TNP, trinitrophenyl. Back

Received for publication March 29, 2001. Accepted for publication January 4, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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