Intrachromosomal recombination between well-separated, homologous sequences in mammalian cells.

In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin mu-gene bearing a mutation in the constant (Cmu) region, was separated from the integrated single copy wild-type donor Cmu region by approximately 1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cmu region occurred with high frequency, correcting the mutant chromosomal mu-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cmu region, as they contained the three Cmu-bearing fragments expected from this recombination, the original donor Cmu region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cmu region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cmu regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.     

Requirements for ectopic homologous recombination in mammalian somatic cells.

Ectopic recombination occurs between DNA sequences that are not in equivalent positions on homologous chromosomes and has beneficial as well as potentially deleterious consequences for the eukaryotic genome. In the present study, we have examined ectopic recombination in mammalian somatic (murine hybridoma) cells in which a deletion in the mu gene constant (Cmu) region of the endogenous chromosomal immunoglobulin mu gene is corrected by using as a donor an ectopic wild-type Cmu region. Ectopic recombination restores normal immunoglobulin M production in hybridomas. We show that (i) chromosomal mu gene deletions of 600 bp and 4 kb are corrected less efficiently than a deletion of only 2 bp, (ii) the minimum amount of homology required to mediate ectopic recombination is between 1.9 and 4.3 kb, (iii) the frequency of ectopic recombination does not depend on donor copy number, and (iv) the frequency of ectopic recombination in hybridoma lines in which the donor and recipient Cmu regions are physically connected to each other on the same chromosome can be as much as 4 orders of magnitude higher than it is for the same sequences located on homologous or nonhomologous chromosomes. The results are discussed in terms of a model for ectopic recombination in mammalian somatic cells in which the scanning mechanism that is used to locate a homologous partner operates preferentially in cis.     

The molecular basis of multiple vector insertion by gene targeting in mammalian cells

Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.     

Ectopic recombination within homologous immunoglobulin mu gene constant regions in a mouse hybridoma cell line.

We have transferred a pSV2neo vector containing the wild-type constant region of the immunoglobulin mu gene (C mu) into the mutant hybridoma igm482, which bears a 2-bp deletion in the third constant-region exon of its haploid chromosomal mu gene (C mu 3). Independent igm482 transformants contain the wild-type immunoglobulin C mu region stably integrated in ectopic chromosomal positions. We report here that the wild-type immunoglobulin C mu region can function as the donor sequence in a gene conversion event which corrects the 2-bp deletion in the mutant igm482 chromosomal C mu 3 exon. The homologous recombination event restores normal immunoglobulin M production in the mutant cell.     

Evidence for the murine IgH mu locus acting as a hot spot for intrachromosomal homologous recombination

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 mu gene C (Cmu) region residing at the Ig mu locus in mouse hybridoma cells. In this study, we constructed mouse hybridoma cell lines bearing Cmu 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 Cmu region, suggesting that recombination might initiate at random sites within the Cmu region. The Cmu region heteroalleles were equally proficient as either conversion donors or recipients. Remarkably, when the same Cmu 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 mu locus behaving as a hot spot for intrachromosomal homologous recombination.     

Homologous recombination between transferred and chromosomal immunoglobulin kappa genes.

Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP). As recipient cells, we used the Sp6-derived mutant hybridoma igk14, which has a deletion of the kappa TNP gene and consequently does not synthesize TNP-specific immunoglobulin M. igk14 retains the mu TNP gene and two additional rearranged kappa genes, denoted kappa M21B1 and kappa M21G. As a transfer vector, we used pSV2neo bearing the functionally rearranged TNP-specific V kappa segment. Following DNA transfer by electroporation, we isolated rare transformants which produced normal amounts of the functional kappa TNP chain. Analysis of the DNA of these transformants indicated that in all cases, a functional kappa TNP gene had been formed as the result of a homologous integrative recombination event with the igk14 kappa M21B1 gene. These results suggest that homologous recombination might be used for mapping and introducing immunoglobulin gene mutations and for more conveniently engineering specifically altered immunoglobulins.     

The mechanism of mammalian gene replacement is consistent with the formation of long regions of heteroduplex DNA associated with two crossing-over events

In this study, the mechanism of mammalian gene replacement was investigated. The system is based on detecting homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin mu-delta region in a murine hybridoma cell line. The backbone of the gene replacement vector (pCmuCdeltapal) consists of pSV2neo sequences bounded on one side by homology to the mu gene constant (Cmu) region and on the other side by homology to the delta gene constant (Cdelta) region. The Cmu and Cdelta flanking arms of homology were marked by insertions of an identical 30-bp palindrome which frequently escapes mismatch repair when in heteroduplex DNA (hDNA). As a result, intermediates bearing unrepaired hDNA generate mixed (sectored) recombinants following DNA replication and cell division. To monitor the presence and position of sectored sites and, hence, hDNA formation during the recombination process, the palindrome contained a unique NotI site that replaced an endogenous restriction enzyme site at each marker position in the vector-borne Cmu and Cdelta regions. Gene replacement was studied under conditions which permitted the efficient recovery of the product(s) of individual recombination events. Analysis of marker segregation patterns in independent recombinants revealed that extensive hDNA was formed within the Cmu and Cdelta regions. In several recombinants, palindrome markers in the Cmu and Cdelta regions resided on opposite DNA strands (trans configuration). These results are consistent with the mammalian gene replacement reaction involving two crossing-over events in homologous flanking DNA.     

High-frequency gene conversion between repeated C mu sequences integrated at the chromosomal immunoglobulin mu locus in mouse hybridoma cells.

The occurrence of mitotic recombination between repeated immunoglobulin mu gene constant (C mu) region sequences stably integrated at the haploid chromosomal immunoglobulin mu locus in murine hybridoma cells was investigated. Recombination events are detected as changes in hapten-specific immunoglobulin M production. Recombination occurs with high frequency (0.5 to 0.8%) by a mechanism consistent with gene conversion. A double-strand break repair-like mechanism is suggested by the finding that repair of a 2-bp deletion mutation and a 2-bp insertion mutation occurs with parity in a donor-directed manner. The results also suggest that the gene conversion process is directional in that the 5' C mu region sequence is preferentially converted.     

Homologous recombination in hybridoma cells: dependence on time and fragment length.

Mutant hybridoma-myeloma cell lines that are defective in immunoglobulin production are expected to be useful for defining the molecular requirements of immunoglobulin gene expression. The analysis of such mutants would be greatly facilitated if they could be mapped by marker rescue, i.e., by identifying the segments of wild-type DNA that can restore the normal phenotype by homologous recombination with the mutant chromosomal immunoglobulin gene. To assess the feasibility of this type of mapping, we have measured the efficiency with which fragments of wild-type DNA recombine with a mutant hybridoma immunoglobulin gene and restore normal immunoglobulin production. We found that most if not all recombinants were detectable 2 days after DNA transfer and that the frequency of gene restoration increased with increasing length of the transferred mu gene fragments, between 1.2 and 9.5 kilobases. These results indicate that the available technology should be adequate to map mutations in the mu gene to within approximately 1 kilobase.     

An element in the endogenous IgH locus stimulates gene targeting in hybridoma cells.

Gene targeting of the immunoglobulin (Ig) heavy chain locus is the basis of improved methods of investigating gene expression and of antibody engineering. The VH-Cmu intron is a convenient region for mediating homologous recombination events which result in production of Ig bearing an altered heavy chain. Also, this segment includes several elements which are important for gene expression, replication and isotype switching: in some cases it will be advantageous to alter these processes by modifying this intron. Considering that multiple targeting steps might be needed to accomplish all the requisite changes, it is important to know whether any of the anticipated modifications also alter the recombinogenicity of the IgH locus. To test this possibility we have measured the frequency at which a mutation in the Cmu3 exon of the endogenous mu gene is corrected by homologous recombination with a transfected segment of Cmu DNA. Comparison of recombination frequencies in several engineered hybridomas indicates that deletion of a 7.1 kb segment from the VH-Cmu intron depresses recombination by approximately 10-fold.     

Immunoglobulin molecular genetics: the prospects for mutational analysis of the chromosomal immunoglobulin genes

Homologous recombination between transferred and chromosomal DNA can be used to effect precise, predetermined modifications of the chromosomal genes. Ultimately this phenomenon should allow the assessment of genetic regulatory elements as they function in the normal chromosomal environment. We have previously described a system for isolating mutant hybridoma cells that are defective in immunoglobulin (Ig) production, with a view toward using these mutants to define cis-acting elements that influence Ig gene expression. Here we describe results that indicate that homologous recombination between transferred and chromosomal Ig genes can be used to map Ig mutations by marker rescue.     

Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells.

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.     

End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells.

We have studied the mechanism of targeted recombination in mammalian cells using a hemizygous adenine phosphoribosyltransferase-deficient (APRT-) Chinese hamster ovary (CHO) cell mutant as a recipient. Three structurally different targeting vectors with a 5' or a 3', or both, end-deleted aprt sequence, in either a closed-circular or linear form, were transfected to the cells with a mutated aprt gene by electroporation. APRT-positive (APRT+) recombinant clones were selected and analyzed to study the gene correction events of the deletion mutation. Some half of 58 recombinant clones obtained resulted from corrections of the deleted chromosomal aprt gene by either gene replacement or gene insertion, a mechanism which is currently accepted for homologous recombination in mammalian cells. However, the chromosomal sequence in the remaining half of the recombinants remained uncorrected but their truncated end of the aprt gene in the incoming vectors was corrected by extending the end beyond the region of homology to the target locus; the corrected vector was then randomly integrated into the genome. This extension, termed end extension repair, was observed with all three vectors used and was as far as 4.6-kilobase (kb) or more long. It is evident that the novel repair reaction mediated by homologous recombination, in addition to gene replacement and gene insertion, is also involved in gene correction events in mammalian cells. We discuss the model which may account for this phenomenon.     

Somatic and Meiotic Chromosomal Recombination between Inverted Duplications in Transgenic Tobacco Plants

Homologous recombination has been extensively studied in bacteria, yeast, and more recently in animal cells, but little is known about this process in plants. We present here an analysis of meiotic and somatic chromosomal recombination between closely linked inverted duplications located on a single chromosomal region in tobacco. Transgenic tobacco lines were constructed by Agrobacterium transformation with plasmid vectors containing a functional hygromycin phosphotransferase (hyg) selectable marker flanked by a pair of defective neomycin phosphotransferase (neo) genes positioned as inverted repeats. As each neo gene is mutated in a different site, recombination between the two defective genes can be detected following selection for kanamycin-resistant plant cells. The recombination substrates were designed to allow investigation into the nature of molecular events underlying homologous recombination by restriction endonuclease analysis. Chromosomal recombination was studied in mitotically dividing cells (cultured leaf mesophyll cells) and after meiosis (germinated seedlings). Spontaneous somatic recombinants were recovered at frequencies between ~3 x 10-5 to 10-6 events per cell. Low dose [gamma] irradiation of somatic cells resulted in a threefold maximum increase in the recovery of recombinants. Recombinants were also detected at low frequency when transgenic T3 seeds were germinated under kanamycin selection. DNA gel blot analyses demonstrated that homologous recombination occurred mainly as gene conversion unassociated with reciprocal exchange, although a variety of other events including gene coconversion were also observed.     

Nonhomologous recombination at sites within the mouse JH-C delta locus accompanies C mu deletion and switch to immunoglobulin D secretion.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.     

Inverted duplication of JH associated with chromosome 14 translocation and T-cell leukemia in ataxia-telangiectasia.

A specific 14q32 breakpoint is observed in a homologous chromosome 14 translocation [t(14;14)q12q32] occurring in the T-cells of about 10% of patients with ataxia-telangiectasia (AT). To investigate whether the 14q32 breakpoint in AT occurs within the immunoglobulin gene cluster as is frequently detected in B-cell lymphoma, immunoglobulin clones were hybridized to Southern blots of DNA isolated from the T-cells of two AT patients with this chromosome 14 translocation. The 14q32 translocation breakpoints in these patients are apparently not within JH, S mu, C mu, S alpha-1 or -2, or C alpha-1 or -2, but one of the patients has an inverted duplication of at least 26 kilobases (kb) of the C mu region, with an associated 5' flanking deletion. The point of origin of the inverted duplication is within JH near the recombination signal for the J4 gene. This suggests that normal JH recombination mechanisms may have played a role in the development of this 14q32 chromosomal aberration. The presence of AT chromosomal breakpoints near other rearranging genes suggests a role for exaggerated recombination in the pathogenesis of chromosomal instability in AT.     

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.     

Extrachromosomal and chromosomal gene conversion in mammalian cells.

We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.     

Mechanisms of double-strand-break repair during gene targeting in mammalian cells

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.