Deletion and linkage mapping of eight markers from the proximal short arm of chromosome 6

Eight chromosome 6p markers (MUT, D6S4, D6S5, D6S19, D6S29, PIM, HLA, and F13A) were regionally mapped using somatic cell hybrid deletion cell lines that retained different regions of chromosome 6p. New restriction fragment length polymorphisms were identified at the D6S5 and PIM loci using newly isolated genomic clones at these loci. Genetic linkage among the eight loci was determined using the 40 CEPH reference families. Linkage analyses showed that these loci are in one linkage group spanning 48 cM in males and 128 cM in females. Using both the deletion mapping data and multipoint linkage analyses, chromosomal order for these loci was determined as centromere-(MUT, D6S4)-(D6S5, D6S19)-(D6S29, PIM)-HLA-F13-A-telomere. Analyses of sex-specific recombination frequencies revealed a higher rate of recombination in females in the region between D6S4 and D6S29, while the recombination rate in males was higher for the interval between D6S29 and the HLA loci.     

Intrachromosomal recombination in plants.

Molecular evidence for intrachromosomal recombination between closely linked DNA repeats within the plant genome is presented. The non-overlapping complementary deletion derivatives of the selectable neomycin phosphotransferase gene (nptII), when intact conferring kanamycin resistance, were inserted into the genome of Nicotiana tabacum. The functional marker gene was restored with frequencies between 10(-4) and 10(-6) per proliferating cell clone. Prolonged tissue culture prior to kanamycin selection did not increase the number of recombinant kanamycin-resistant (KanR) cell clones. DNA analysis of KanR clones derived from cells carrying multiple tandem recombination units suggested that these units have a tendency to undergo concerted recombination. Recovery and analysis of kanamycin-sensitive seedlings with patches of KanR cells provided direct evidence for mitotic recombination in plant tissue.     

The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.—Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.—Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6^cs, ca^nd, mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6^cs causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by ca^nd, mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability.     

Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology.

We have designed and constructed integration-defective retroviral vectors to explore their potential for gene targeting in mammalian cells. Two nonoverlapping deletion mutants of the bacterial neomycin resistance (neo) gene were used to detect homologous recombination events between viral and chromosomal sequences. Stable neo gene correction events were selected at a frequency of approximately 1 G418r cell per 3 x 10(6) infected cells. Analysis of the functional neo gene in independent targeted cell clones indicated that unintegrated retroviral linear DNA recombined with the target by gene conversion for variable distances into regions of nonhomology. In addition, transient neo gene correction events which were associated with the complete loss of the chromosomal target sequences were observed. These results demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.     

Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome.

Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated. Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome. Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element. Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination. Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.     

The spectra of large second-step mutations are similar for two different mouse autosomes

Loss of tumor suppressor gene expression via mutations plays a critical role in cancer development, particularly when occurring in heterozygous cells. These so-called "second-step" mutational events are often large in size and arise most often from chromosome loss, mitotic recombination, or interstitial deletion. An open question in cancer research is whether different chromosomes are equally susceptible to formation of large mutations, or alternatively if the unique sequence of each chromosome will lead to chromosome-specific mutational spectra. To address this question, the spectra of second-step mutations were determined for chromosomes 8 and 11 in Aprt and Tk mutants, respectively, isolated from primary kidney clones heterozygous for both loci. The results showed that the spectra of large mutational events were essentially the same. This observation suggests that internal and external cellular environments provide the driving force for large autosomal mutational events, and that chromosome structure per se is the substrate upon which these forces act.     

Analysis of mutations in the Tk gene of Tk(+/-) mice treated as neonates with 3'-azido-3'-deoxythymidine (AZT)

Mother-to-child transmission of the human immunodeficiency virus (HIV) is reduced by perinatal treatment with the antiretroviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT, zidovudine). AZT, however, is genotoxic and carcinogenic in mice when administered either transplacentally or neonatally, suggesting a possible cancer risk for children later in life. In a previous study we found that treating B6C3F1/Tk(+/-) mice on postnatal days 1 through 8 with intraperitoneal injections of 200 mg AZT per kg body weight per day significantly increased spleen lymphocyte mutant frequencies in the autosomal Tk gene. Allele-specific PCR of Tk mutants from treated mice indicated that 61% had lost the Tk(+) allele (loss of heterozygosity; LOH), compared with 35% of Tk mutants from control mice, a difference that was significant. In the present study, Tk mutant lymphocyte clones were analyzed further using polymorphic microsatellite markers that flank the Tk gene along the length of mouse chromosome 11. The analysis indicated that allele-loss mutations in control mice were due to either total chromosome loss, mitotic recombination, or both. The pattern of marker loss in mutants from AZT-treated mice differed significantly from the control mice and was consistent with chromosome loss, mitotic recombination, interstitial deletion, gene conversion, and an unusual discontinuous LOH. The results indicate that AZT induced a unique pattern of mutations in the Tk gene of mice and that the major mechanisms of mutation by AZT involved deletion and recombination.     

Isolation of paraquat-tolerant mutants from tomato cell cultures

Summary Tomato callus clones selected for the ability to grow at paraquat concentrations lethal to wild-type cells were found at an approximate frequency of 5×10^–8 per viable cell. Diploid plants were regenerated from nine of the nineteen paraquat-tolerant callus clones isolated. Although some of these plants appeared normal, others had altered morphology and reduced vigor and fertility. New callus cultures initiated from these regenerated plants typically had at least a 30-fold increase over the wild type in tolerance to paraquat. Tests on callus from sexual progeny showed that the paraquat-tolerant phenotypes of clones PQT⁴, PQT⁶, and probably also PQT¹³ resulted from dominant nuclear mutations, but the number of loci involved is not yet known. Paraquat spray experiments indicated that slight paraquat-tolerance was expressed at the plant level in PQT¹³, but not in any of the other clones tested.     

RFLP Analysis of Chromosomal Segregation in Progeny from an Interspecific Hexaploid Somatic Hybrid between Solanum Brevidens and Solanum Tuberosum

Segregation of restriction fragment length polymorphism (RFLP) loci was monitored to determine the degree of homeologous pairing and recombination in a hexaploid somatic hybrid, A206, the result of protoplast fusion between Solanum tuberosum (PI 203900, a tetraploid cultivated potato) and Solanum brevidens (PI 218228), a diploid, sexually incompatible, distant relative harboring several traits for disease resistance. Somatic hybrid A206 was crossed to Katahdin, a tetraploid potato cultivar, to generate a segregating population of pentaploid progeny. Although the clones of the tetraploid S. tuberosum lines PI 203900 and Katahdin were highly polymorphic, the diploid S. brevidens clone was homozygous at all but two of the tested RFLP loci. Thus, homeologous recombination could be detected only when S. tuberosum and S. brevidens chromosomes paired and the S. brevidens homologs then segregated into separate gametes. A bias toward homologous pairing was observed for all 12 chromosomes. At least four and perhaps six chromosomes participated in homologous pairing only; each of 24 progeny contained all S. brevidens-derived RFLP markers for chromosomes 4, 8, 9 and 10. The remaining six chromosomes paired with their homolog(s) about twice as often as expected if hexaploid pairings were completely random. Where detectable with RFLPs, homeologous recombinations (both single and double) occurred at a frequency of 1.31 per chromosome. Cytological observations of meiosis I in the somatic hybrid indicated that homeologous pairing had occurred. Enhanced recombinational activity was observed for chromosome 2. A specific small deletion from chromosome 4 was detected in A206 and 11 other somatic hybrids out of 14 screened. These hybrids represent 13 independent fusion events between the same clones of S. brevidens and S. tuberosum. In one instance, this deletion occurred in one of two plants resulting from the same callus, indicating that the loss occurred in culture after fusion had taken place. It is possible that this deletion contributes to somaclonal variation.     

一种利用Cre/loxP系统进行标记基因删除与靶基因置换的细胞模型研究

Cre/lox系统可以介导DNA的定点插入和定点删除,可利用其实现转基因动物中"友好位点"的重复利用及标记基因的有效删除.为直观地评估该系统介导的以上两种重组反应的效果,通过标记基因并利用大鼠乳腺癌细胞系SHZ-88进行了模型研究.首先构建了两个载体:a.整合载体pTE-lox2272-DsRed-loxP-GFP-loxP,含有红色荧光标记基因DsRed和绿色荧光标记基因GFP;b.置换载体pT-lox2272-neo-loxP,含有筛选标记基因neo,用以置换DsRed基因.然后,用整合载体转染SHZ-88细胞,并随机挑取了3个同时表达DsRed和GFP的稳定整合细胞克隆.随后用置换载体和Cre表达载体PBS185对以上每个克隆分别进行了3次共转染,通过G418筛选并扩增培养后,总共获得1 070个克隆.通过分析标记基因DsRed和GFP在这些克隆中的表达情况:Cre介导的删除效率为91.1%,定点置换效率为29.3%.最后对部分克隆进行了PCR和DNA印迹鉴定,分子鉴定结果与发光的表型状况一致.这一方法为Cre/lox系统在转基因家畜生产中的进一步应用提供了实验依据.     

Control of recombination ability of vegetative cells in Saccharomyces cerevisiae

Summary In a diploid strain heteroallelic at the ade3 locus, the mitotic intragenic recombination frequency is enhanced ten fold when the cell population is starved for histidine (Hénaut et Luzzati, 1971). By studying simultaneous recombinational events at two independant loci, it is shown that the effect of histidine starvation is most simply explained in term of an increase in the frequency of cells capable of recombination. In these competent cells, intragenic recombination frequencies during mitosis are equal to those found during meiosis. However, the frequency of recombination between the gene and the centromere appears to be lower during mitosis than during meiosis.We believe that histidine starvation in ade3 strains stimulates chromosome pairing, and that there is no fundamental difference between mitotic and meiotic recombination.     

Soybean pedigree analysis using map-based molecular markers: recombination during cultivar development

An analysis of the genome structure of soybean cultivars was conducted to determine if cultivars are composed of large regions of chromosomes inherited intact from one parent (indicative of minimal recombination) or if the chromosomes are a mixture of one parent's DNA interspersed with the DNA from the other parent (indicative of maximal recombination). Twenty-one single-cross-derived and 5 single-backcross-derived soybean cultivars and their immediate parents (47 genotypes) were analyzed at 89 RFLP loci to determine the minimal number and distribution of recombination events detected. Cultivars derived from single-cross and single-backcross breeding programs showed an average of 5.2 and 8.0 recombination events per cultivar, respectively. A homogeneity Chi-square test based upon a Poisson distribution of recombination events across 13 linkage groups indicated that the number of recombinations observed among linkage groups was random for the single-cross cultivars, but not for the single-backcross-derived cultivars. A twotailed t-test demonstrated that for some linkage groups, the number of recombinations per map unit exceeded the confidence interval developed from a t-distribution of recombinations standardized for map unit distance. Paired t-tests of the number of recombinations observed between linkage-group ends and the mid-portion of the linkage groups indicated that during the development of the cultivars analyzed in this study more recombinations were associated with the ends of linkage groups than with the middle region. Detailed analysis of each linkage group revealed that large portions of linkage groups D, F, and G were inherited intact from one parent in several cultivars. A portion of linkage group G, in contrast, showed more recombination events than expected, based on genetic distance. These analyses suggest that breeders may have selected against recombination events where agronomically favorable combinations of alleles are present in one parent, and for recombination in areas where agronomically favorable combinations of alleles are not present in either parent.Names are necessary to report factually on the available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be available. Contribution of the Midwest Area, USDA-ARS, Project No. 3236 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011. Journal Paper No. J-16533     

Comparison of homoeologous group-6 short arm physical maps of wheat and barley reveals a similar distribution of recombinogenic and gene-rich regions

Eighty two new loci, mapped with 51 DNA clones, were added to the earlier deletion maps of the homoeologous group-6 short arms of hexaploid wheat ( Triticum aestivum L. em Thell., 2n = 6 x = 42, AABBDD). There are now 41, 56 and 52 loci mapped on deletion maps of 6AS, 6BS and 6DS, respectively. The linear order of orthologous loci in all three arms appears to be identical. The majority of the loci are located in the distal one-half of the three arms. There seems to be an increased marker/gene density from the centromeric to the telomeric regions in each arm, and the marker density in comparable physical regions is similar on all three maps. Recombination is not uniformly distributed along the chromosome arms; 60% of recombination occurs in the distal one-third of each arm. Recombination increases from the proximal region to the distal end in a nonlinear pattern. The distribution of loci and recombination along each of the three chromosome arms is highly correlated. Comparison of the 6BS deletion map from this study and a 6HS physical map of barley ( Hordeum vulgare L., 2n = 2 x = 14, HH) reveals a remarkably similar distribution of recombinogenic and gene-rich regions between the two chromosome arms, suggesting that the distribution patterns of genes may be conserved in the homoeologous group-6 chromosome short arms of wheat and barley. A consensus map of wheat group-6 short arms containing 46 orthologous loci was constructed. Comparison of the consensus map with published linkage maps of Triticeae group-6 chromosome short arms indicates that the linear order of the loci on the maps has been largely conserved. Evidence from this study does not support the existence of a 2BS-6BS reciprocal terminal translocation.     

Recombinational error and deletion formation in Neisseria gonorrhoeae: a role for RecJ in the production of pilE L deletions

Genetic linkage within Neisseria gonorrhoeae populations is in equilibrium, yet the physical linkage map indicates a relatively stable chromosome structure, despite an apparently vast potential for mispairing between repeated sequences (e.g. between the multiple pil or opa alleles, or through mispairing of any of the numerous small repeated sequences that are liberally scattered throughout the chromosome). Therefore, the stability of the physical linkage map suggests that aberrant recombination between repeated sequences is a rare event. This study was undertaken to explore some of the parameters that may govern deletion events between short direct oligonucleotide repeats, using a chromosomal locus that appears to be especially prone to deletions (the pilin expression locus; pilE). In this report, we demonstrate that deletion formation at pilE occurs primarily through recombinational error following a pilE/pilS interaction; illegitimate (i.e. RecA-independent) events can occur, but they are infrequent. In contrast, when genetically engineered opa deletion substrates were constructed and placed in the chromosome, deletions at the opa loci were infrequent even under rec(+) conditions. A model is presented in which the gonococcal RecA and RecJ proteins promote pilE deletions through a recombination event that is templated or stabilised by a pilE/pilS interaction.     

Parameters of Mitotic Recombination in Minute Mutants of DROSOPHILA MELANOGASTER

A sample of 16 Minutes, representing 12 loci distributed over all the chromosome arms and including 3 pairs of alleles and 4 deficiencies, has been studied with respect to several developmental and recombinational parameters. Cell marker mutants located in most of the chromosome arms were used to assess (1) spontaneous and X-ray-induced mitotic recombination frequencies of each Minute, and (2) clone sizes of the different cell marker clones. These parameters were analyzed both in the wing disc and in the abdominal histoblasts.—Whereas spontaneous frequencies are not affected by the presence of the Minutes studied, the different Minutes characteristically increase the frequency of recombination clones arising after X-irradiation. The recombinant clones which are M⁺/M⁺ are significantly larger than clones in the same fly which retain the M⁺/M condition. This is particularly striking in clones in the wing disc, slightly so in clones in the tergites. The occurrence of mitotic recombination in the fourth chromosome is reported for the first time.—Chaeta length and developmental delay correlates with the recombinational parameters in different ways. Possible causal interrelationships of the different traits of the Minute syndrome are discussed.     

Fragile Site Instability in Saccharomyces cerevisiae Causes Loss of Heterozygosity by Mitotic Crossovers and Break-Induced Replication

Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.     

DNA copy-number instability in low-dose gamma-irradiated TK6 lymphoblastoid clones

Genomic instability that might occur early during low-dose, fractionated radiation exposures may be traceable in radiogenic compared to spontaneous cancers. Using a human 18K cDNA microarray-based comparative genome hybridization protocol, we measured changes in DNA copy number at over 14,000 loci in nine low-dose (137)Cs gamma-irradiated (acute exposure to 10 cGy/day x 21 days) and nine unirradiated TK6 clones and estimated locus-specific copy-number differences between them. Radiation induced copy-number hypervariability at thousands of loci across all chromosomes, with a sevenfold increase in low-level, randomly positioned DNA gains. Recurrent gains at 40 loci occurred among irradiated clones and were distributed nonrandomly across the genome, with the highest densities in 3q, 13q and 20q at sites that were hypodiploid without irradiation. Another nonrandomly distributed set of 94 loci exhibited relative recurrent gains from a hypodiploid state to a diploid state, suggesting hemizygous-to-homozygous transitions. Frequently recurring losses at 57 loci were concentrated on the single X-chromosome but were sparsely distributed at 0-2 loci per autosome. These results suggest induced mitotic homologous recombination as a possible mechanism of low-dose radiation-induced genomic instability. Genomic instability induced in TK6 cells resembled that seen in radiogenic tumors and suggests a way that radiation could induce genomic instability in preneoplastic cells.     

Segregation of Recombinant Chromatids following Mitotic Crossing over in Yeast

It has long been assumed that chromatid segregation following mitotic crossing over in yeast is random, with the recombinant chromatids segregating to opposite poles of the cell (x-segregation) or to the same pole of the cell (z-segregation) with equal frequency. X-segregation events can be readily identified because heterozygous markers distal to the point of the exchange are reduced to homozygosity. Z-segregation events yield daughter cells which are identical phenotypically to nonrecombinant cells and thus can only be identified by the altered linkage relationships of genetic markers on opposite sides of the exchange. We have systematically examined the segregation patterns of chromatids with a spontaneous mitotic exchange in the CEN5-CAN1 interval on chromosome V. We find that the number of x-segregation events is equal to the number of z-segregations, thus demonstrating that chromatid segregation is indeed random. In addition, we have found that at least 5% of the cells selected for a recombination event on chromosome V are trisomic for this chromosome, indicating a strong association between mitotic recombination and chromosome nondisjunction.     

Spontaneous Mitotic Recombination and Evidence for an X-Ray-Inducible System for the Repair of DNA Damage in DROSOPHILA MELANOGASTER

Spontaneous mitotic recombination in the left arm of chromosome 3 was examined in both unirradiated control flies and sibs irradiated early in development by determining the sizes and frequencies of multiple-wing-hair (mwh) clones in the wing blade of heterozygous mwh/+ flies. Approximately 16% of the spontaneous mwh clones arise from events generating cells with normal division rates. The remaining 84% result from events generating cells with an average cell division rate one-third that of the surrounding cells; these are thought to result from events that generate aneuploid cells. Such clones probably arise from a failure correctly to repair spontaneous DNA damage. The frequency of spontaneous events late in development decreases significantly after irradiation as much as 150 hours earlier in development. The suppression of spontaneous events decreases with a longer period of time between irradiation and the final cell divisions in the wing blade. These results suggest the existence of a repair system for DNA damage in Drosophila that is induced by irradiation. The decrease in effect with time following irradiation could result from slow degradation or dilution by subsequent cell growth and division.     

Extensive loss of heterozygosity is suppressed during homologous repair of chromosomal breaks

Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 x 10(-5); however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.