Detection Of Recombination Events Research Articles

Complete genome characterization of chilli veinal mottle virus associated with mosaic and mottling disease of tomato and development of LAMP assay for quick detection.

Chilli veinal mottle virus (ChiVMV) is a potyvirus known to cause havoc in many solanaceous crops. Samples from tomato plants exhibiting typical mosaic and mottling symptoms in two locations from farmers' fields were collected and tested using DAC ELISA for the presence of ChiVMV and other viruses known to infect tomato. ChiVMV Gauribidanur isolate from infected tomato was mechanically inoculated to Datura metel, Nicotiana tabacum, Nicotiana benthamiana, Nicotiana glutinosa, chilli, and tomato plants which exhibited systemic mosaic and mottling symptoms 10days post-inoculation. This results were further confirmed by RT-PCR and DAC ELISA using CP gene-specific primers and ChiVMV antisera, respectively. Transmission electron microscopy revealed the presence of long filamentous particles (800×11nm) resembling viruses in the Potyviridae family. The complete genome of ChiVMV comprised 9716 nucleotides except for poly A tail, with a predicted open reading frame spanning 9270 nucleotides encoding polyproteins of 3089 amino acids. Comparative analysis revealed that ChiVMV-tomato isolates reported across the world shared maximum nucleotide identity (93-96.7%) with chilli isolates from India and Pakistan. These results were well supported by sequence demarcation analysis. Further, the Neibhor-Net network analysis of the complete genome of ChiVMV-tomato, along with other host isolates, formed a reticular network phylogenetic tree suggesting recombination events. Subsequently, RDP5 detected intra-specific recombination breakpoints at the positions 1656-5666 nucleotides with major parent ChiVMV (MN508960) Uravakonda and minor parent ChiVMV (MN508956) with a significant average p value of 1.905×10-22. The LAMP assay using ChiVMV-specific primers resulted in ladder-like amplified products on electrophoresed gel and a distinct red colour pattern with hydroxy naphthalene blue, indicating a positive reaction for the presence of ChiVMV in infected tomato samples. To validate LAMP-designed primers, RNA extracted from ChiVMV-infected tomato, chilli, datura, and tobacco samples were subjected to LAMP assay and it accurately detected the presence of ChiVMV in infected plant samples. Overall, this study provides holistic information of ChiVMV infecting tomato, spanning diagnosis, transmission, genetic characterization, and detection of recombination events, which collectively contribute to effective disease management, crop protection, and informed decision-making in agricultural practices.

Interbacterial Transfer of Carbapenem Resistance and Large Antibiotic Resistance Islands by Natural Transformation in Pathogenic Acinetobacter.

ABSTRACTAcinetobacter baumannii infection poses a major health threat, with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intrastrain but also interstrain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4 and AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genomes of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii.

Using Genomics to Shape the Definition of the Agglutinin-Like Sequence (ALS) Family in the Saccharomycetales.

The Candida albicans agglutinin-like sequence (ALS) family is studied because of its contribution to cell adhesion, fungal colonization, and polymicrobial biofilm formation. The goal of this work was to derive an accurate census and sequence for ALS genes in pathogenic yeasts and other closely related species, while probing the boundaries of the ALS family within the Order Saccharomycetales. Bioinformatic methods were combined with laboratory experimentation to characterize 47 novel ALS loci from 8 fungal species. AlphaFold predictions suggested the presence of a conserved N-terminal adhesive domain (NT-Als) structure in all Als proteins reported to date, as well as in S. cerevisiae alpha-agglutinin (Sag1). Lodderomyces elongisporus, Meyerozyma guilliermondii, and Scheffersomyces stipitis were notable because each species had genes with C. albicans ALS features, as well as at least one that encoded a Sag1-like protein. Detection of recombination events between the ALS family and gene families encoding other cell-surface proteins such as Iff/Hyr and Flo suggest widespread domain swapping with the potential to create cell-surface diversity among yeast species. Results from the analysis also revealed subtelomeric ALS genes, ALS pseudogenes, and the potential for yeast species to secrete their own soluble adhesion inhibitors. Information presented here supports the inclusion of SAG1 in the ALS family and yields many experimental hypotheses to pursue to further reveal the nature of the ALS family.

Recombinant CV-A6 strains related to hand-foot-mouth disease and herpangina at primary care centers (Barcelona, Spain).

Aim: To describe the genetic diversity of enteroviruses (EV) causing hand, foot and mouth disease (HFMD) and herpangina, especially of coxsackievirus (CV)-A6, from patients attended at pediatric primary care centers during the 2017-2018 season. Methods: Phylogenetic analysis of partial VP1 region was performed for genetic characterization. The complete VP1 and 3Dpol proteins were sequenced for lineage determination and detection of recombination events. Results: An 80% of samples were EV laboratory-confirmed. CV-A6 was the most detected (70%) and associated with atypical HFMD (78%). The comparison of VP1 and 3Dpol phylogenies showed evidence of recombination in three strains, in which two shifted to CV-A16 3Dpol. Conclusion: The study provides recent information regarding the nonrecombinant and recombinant EVs related to HFMD at primary care centers.

T-RECs: rapid and large-scale detection of recombination events among different evolutionary lineages of viral genomes

BackgroundMany computational tools that detect recombination in viruses are not adapted for the ongoing genomic revolution. A computational tool is needed, that will rapidly scan hundreds/thousands of genomes or sequence fragments and detect candidate recombination events that may later be further analyzed with more sensitive and specialized methods.ResultsT-RECs, a Windows based graphical tool, employs pairwise alignment of sliding windows and can perform (i) genotyping, (ii) clustering of new genomes, (iii) detect recent recombination events among different evolutionary lineages, (iv) manual inspection of detected recombination events by similarity plots and (v) annotation of genomic regions.ConclusionsT-RECs is very effective, as demonstrated by an analysis of 555 Norovirus complete genomes and 2500 sequence fragments, where a recombination hotspot was identified at the ORF1-ORF2 junction.

Detection of homologous recombination in closely related strains.

Detection of recombination events in a bacterial genome is both important from the evolutionary point of view, and of practical interest. Indeed, homologous recombination (HR) plays a major role in the exchange of antigenic determinants between strains. There exist statistical methods to detect recently recombined segments in whole-genome sequences that use a high local density of substitutions as a signal of HR events with a source outside considered strains. However, it is difficult to detect the HR events within a set of strains, which represent whole species diversity, due to a low number of substitutions in recombined segments and high level of diversity of strains. Here, we analyzed HR in 20 Escherichia coli (E. coli) strains to define what fraction of segments with a high substitution rate were introduced in a genome by HR. For detection of HR, we used the segmentation, performed by the adaptive weights smoothing (AWS) algorithm. It detects sharp changes in the structure of observed data analyzing only qualitative structural information. We validated the approach on simulated data, applied it to the analysis of E. coli strains, and determined the recombination rates between phylogroups.

Recombination detection in Aspergillus fumigatus through single nucleotide polymorphisms typing.

The first evidence of sexual reproduction in Aspergillus fumigatus was reported in 2009. Nevertheless, it remains difficult to understand how A. fumigatus is able to reproduce through this mode in its natural environment and how frequently this occurs. The aim of this study was to analyse single nucleotide polymorphisms (SNPs) in a set of environmental and clinical isolates of A. fumigatus to detect signatures of recombination. A group of closely related Portuguese A. fumigatus isolates was characterized by mating type and the genetic diversity of the intergenic regions, microsatellites and multilocus sequence typing (MLST) genes. A group of 19 SNPs, organized in nine association groups and inherited in blocks, was identified and compared. Several variations on the association panel were detected on reference isolates of A. fumigatus AF293 and A1163, the sequence types available at MLST database and six clinical and environmental Portuguese isolates. About one to four haplotype disruptions were observed per isolate. Considering clinical and environmental isolates, sexual reproduction seems to occur more frequently than previously admitted in A. fumigatus. In this study, a practical SNP approach is proposed for detection of recombination events in larger collections of A. fumigatus.

Hsphase: an R package for pedigree reconstruction, detection of recombination events, phasing and imputation of half-sib family groups.

BackgroundIdentification of recombination events and which chromosomal segments contributed to an individual is useful for a number of applications in genomic analyses including haplotyping, imputation, signatures of selection, and improved estimates of relationship and probability of identity by descent. Genotypic data on half-sib family groups are widely available in livestock genomics. This structure makes it possible to identify recombination events accurately even with only a few individuals and it lends itself well to a range of applications such as parentage assignment and pedigree verification.ResultsHere we present hsphase, an R package that exploits the genetic structure found in half-sib livestock data to identify and count recombination events, impute and phase un-genotyped sires and phase its offspring. The package also allows reconstruction of family groups (pedigree inference), identification of pedigree errors and parentage assignment. Additional functions in the package allow identification of genomic mapping errors, imputation of paternal high density genotypes from low density genotypes, evaluation of phasing results either from hsphase or from other phasing programs. Various diagnostic plotting functions permit rapid visual inspection of results and evaluation of datasets.ConclusionThe hsphase package provides a suite of functions for analysis and visualization of genomic structures in half-sib family groups implemented in the widely used R programming environment. Low level functions were implemented in C++ and parallelized to improve performance. hsphase was primarily designed for use with high density SNP array data but it is fast enough to run directly on sequence data once they become more widely available. The package is available (GPL 3) from the Comprehensive R Archive Network (CRAN) or from http://www-personal.une.edu.au/~cgondro2/hsphase.htm.

A general approach for haplotype phasing across the full spectrum of relatedness.

Many existing cohorts contain a range of relatedness between genotyped individuals, either by design or by chance. Haplotype estimation in such cohorts is a central step in many downstream analyses. Using genotypes from six cohorts from isolated populations and two cohorts from non-isolated populations, we have investigated the performance of different phasing methods designed for nominally ‘unrelated’ individuals. We find that SHAPEIT2 produces much lower switch error rates in all cohorts compared to other methods, including those designed specifically for isolated populations. In particular, when large amounts of IBD sharing is present, SHAPEIT2 infers close to perfect haplotypes. Based on these results we have developed a general strategy for phasing cohorts with any level of implicit or explicit relatedness between individuals. First SHAPEIT2 is run ignoring all explicit family information. We then apply a novel HMM method (duoHMM) to combine the SHAPEIT2 haplotypes with any family information to infer the inheritance pattern of each meiosis at all sites across each chromosome. This allows the correction of switch errors, detection of recombination events and genotyping errors. We show that the method detects numbers of recombination events that align very well with expectations based on genetic maps, and that it infers far fewer spurious recombination events than Merlin. The method can also detect genotyping errors and infer recombination events in otherwise uninformative families, such as trios and duos. The detected recombination events can be used in association scans for recombination phenotypes. The method provides a simple and unified approach to haplotype estimation, that will be of interest to researchers in the fields of human, animal and plant genetics.

Detection of recombination events, haplotype reconstruction and imputation of sires using half-sib SNP genotypes

BackgroundIdentifying recombination events and the chromosomal segments that constitute a gamete is useful for a number of applications in genomic analyses. In livestock, genotypic data are commonly available for half-sib families. We propose a straightforward but computationally efficient method to use single nucleotide polymorphism marker genotypes on half-sibs to reconstruct the recombination and segregation events that occurred during meiosis in a sire to form the haplotypes observed in its offspring. These meiosis events determine a block structure in paternal haplotypes of the progeny and this can be used to phase the genotypes of individuals in single half-sib families, to impute haplotypes of the sire if they are not genotyped or to impute the paternal strand of the offspring’s sequence based on sequence data of the sire.MethodsThe hsphase algorithm exploits information from opposing homozygotes among half-sibs to identify recombination events, and the chromosomal regions from the paternal and maternal strands of the sire (blocks) that were inherited by its progeny. This information is then used to impute the sire’s genotype, which, in turn, is used to phase the half-sib family. Accuracy (defined as R2) and performance of this approach were evaluated by using simulated and real datasets. Phasing results for the half-sibs were benchmarked to other commonly used phasing programs – AlphaPhase, BEAGLE and PedPhase 3.ResultsUsing a simulated dataset with 20 markers per cM, and for a half-sib family size of 4 and 40, the accuracy of block detection, was 0.58 and 0.96, respectively. The accuracy of inferring sire genotypes was 0.75 and 1.00 and the accuracy of phasing was around 0.97, respectively. hsphase was more robust to genotyping errors than PedPhase 3, AlphaPhase and BEAGLE. Computationally, hsphase was much faster than AlphaPhase and BEAGLE.ConclusionsIn half-sib families of size 8 and above, hsphase can accurately detect block structure of paternal haplotypes, impute genotypes of ungenotyped sires and reconstruct haplotypes in progeny. The method is much faster and more accurate than other widely used population-based phasing programs. A program implementing the method is freely available as an R package (hsphase).

Diversity of Citrus tristeza virus in Jamaica.

Citrus tristeza virus (CTV), one of the most serious pathogens affecting Rutaceae species, causes considerable economic losses to citrus production worldwide. While mild pathotypes have been known for many years in Jamaica, decline outbreaks were only recently recognized after the introduction of the vector Toxoptera citricidus in 1993. In this study, coat protein (CP) gene sequences of isolates from four major citrus-growing regions in Jamaica were obtained and their molecular diversity compared with reference CTV genotypes from other regions. CP sequences from Jamaican isolates showed identities between 90 to 100% at the nucleotide level and shared similar identities with comparable sequences reported for other Jamaican isolates in 2009 and 2010. Phylogenetic analysis revealed segregation into five of seven lineages. One third of the Jamaican isolates clustered with Florida-T30 and Spain-T385 genotypes, with 99 to 100% identity within the lineage. The remaining isolates grouped closely with Florida T36-like genotypes or with the grapefruit and orange stem pitting B249 from Venezuela and the New Zealand CTV-resistance breaking genotypes. Identities among these groups ranged between 91 and 99%. The detection of genetic variants and potential recombination events involving parents from different lineages provide further evidence of the diversity among Jamaican CTV isolates.

SSE: a nucleotide and amino acid sequence analysis platform

BackgroundThere is an increasing need to develop bioinformatic tools to organise and analyse the rapidly growing amount of nucleotide and amino acid sequence data in organisms ranging from viruses to eukaryotes.FindingA simple sequence editor (SSE) was developed to create an integrated environment where sequences can be aligned, annotated, classified and directly analysed by a number of built-in bioinformatic programs. SSE incorporates a sequence editor for the creation of sequence alignments, a process assisted by integrated CLUSTAL/MUSCLE alignment programs and automated removal of indels. Sequences can be fully annotated and classified into groups and annotated of sequences and sequence groups and access to analytical programs that analyse diversity, recombination and RNA secondary structure. Methods for analysing sequence diversity include measures of divergence and evolutionary distances, identity plots to detect regions of nucleotide or amino acid homology, reconstruction of sequence changes, mono-, di- and higher order nucleotide compositional biases and codon usage.Association Index calculations, GroupScans, Bootscanning and TreeOrder scans perform phylogenetic analyses that reconcile group membership with tree branching orders and provide powerful methods for examining segregation of alleles and detection of recombination events. Phylogeny changes across alignments and scoring of branching order differences between trees using the Robinson-Fould algorithm allow effective visualisation of the sites of recombination events.RNA secondary and tertiary structures play important roles in gene expression and RNA virus replication. For the latter, persistence of infection is additionally associated with pervasive RNA secondary structure throughout viral genomic RNA that modulates interactions with innate cell defences. SSE provides several programs to scan alignments for RNA secondary structure through folding energy thermodynamic calculations and phylogenetic methods (detection of co-variant changes, and structure conservation between divergent sequences). These analyses complement methods based on detection of sequence constraints, such as suppression of synonymous site variability.For each program, results can be plotted in real time during analysis through an integrated graphics package, providing publication quality graphs. Results can be also directed to tabulated datafiles for import into spreadsheet or database programs for further analysis.ConclusionsSSE combines sequence editor functions with analytical tools in a comprehensive and user-friendly package that assists considerably in bioinformatic and evolution research.

Genome analysis of two type 6 echovirus (E6) strains recovered from sewage specimens in Greece in 2006

Echovirus 6 (E6) is one of the main enteroviral serotypes that was isolated from cases of aseptic meningitis and encephalitis during the last years in Greece. Two E6 (LR51A5 and LR61G3) were isolated from the sewage treatment plant unit in Larissa, Greece, in May 2006, 1 year before their characterization from aseptic meningitis cases. The two isolates were initially found to be intra-serotypic recombinants in the genomic region VP1, a finding that initiated a full genome sequence analysis. In the present study, nucleotide, amino acid, and phylogenetic analyses for all genomic regions were conducted. For the detection of recombination events, Simplot and bootscan analyses were carried out. The continuous phylogenetic relationship in 2C-3D genomic region of strains LR51A5 and LR61G3 with E30 isolated in France in 2002-2005 indicated that the two strains were recombinants. SimPlot and Bootscan analyses confirmed that LR51A5 and LR61G3 carry an inter-serotypic recombination in the 2C genomic region. The present study provide evidence that recombination events occurred in the regions VP1 (intraserotypic) and non-capsid (interserotypic) during the evolution of LR51A5 and LR61G3, supporting the statement that the genomes of circulating enteroviruses are a mosaic of genomic regions of viral strains of the same or different serotypes. In conclusion, full genome sequence analysis of circulating enteroviral strains is a prerequisite to understand the complexity of enterovirus evolution.

Detection of recombination events in bacterial genomes from large population samples

Analysis of important human pathogen populations is currently under transition toward whole-genome sequencing of growing numbers of samples collected on a global scale. Since recombination in bacteria is often an important factor shaping their evolution by enabling resistance elements and virulence traits to rapidly transfer from one evolutionary lineage to another, it is highly beneficial to have access to tools that can detect recombination events. Multiple advanced statistical methods exist for such purposes; however, they are typically limited either to only a few samples or to data from relatively short regions of a total genome. By harnessing the power of recent advances in Bayesian modeling techniques, we introduce here a method for detecting homologous recombination events from whole-genome sequence data for bacterial population samples on a large scale. Our statistical approach can efficiently handle hundreds of whole genome sequenced population samples and identify separate origins of the recombinant sequence, offering an enhanced insight into the diversification of bacterial clones at the level of the whole genome. A data set of 241 whole genome sequences from an important pandemic lineage of Streptococcus pneumoniae is used together with multiple simulated data sets to demonstrate the potential of our approach.

Epigenetic QTL Mapping inBrassica napus

There is increasing evidence that epigenetic marks such as DNA methylation contribute to phenotypic variation by regulating gene transcription, developmental plasticity, and interactions with the environment. However, relatively little is known about the relationship between the stability and distribution of DNA methylation within chromosomes and the ability to detect trait loci. Plant genomes have a distinct range of target sites and more extensive DNA methylation than animals. We analyzed the stability and distribution of epialleles within the complex genome of the oilseed crop plant Brassica napus. For methylation sensitive AFLP (MSAP) and retrotransposon (RT) epimarkers, we found a high degree of stability, with 90% of mapped markers retaining their allelic pattern in contrasting environments and developmental stages. Moreover, for two distinct parental lines 97% of epialleles were transmitted through five meioses and segregated in a mapping population. For the first time we have established the genetic position for 17 of the 19 centromeres within this amphidiploid species. Epiloci and genetic loci were distributed within distinct clusters, indicating differential detection of recombination events. This enabled us to identify additional significant QTL associated with seven important agronomic traits in the centromeric regions of five linkage groups.

A mitotic recombination system for mouse chromosome 17

Mitotic recombination between homologous chromosomes is a genetic technique for mosaic analysis in model organisms. The general application of this technique in the mouse depends on establishment of effective recombination systems for individual chromosomes and reliable and sensitive methods for detection of recombination events. Here, we established a Cre/LoxP-mediated recombination system in mice for mosaic analysis of full-length chromosome 17. Cre-mediated germ-line recombination between the homologous chromosomes was observed with approximately 9% frequency in a progeny test. Mitotic recombination in somatic tissues was evaluated and scored in B and T lymphocytes with the aid of surface markers and fluorescent-activated cell sorting. We show that a lineage-specific Cre can induce mitotic recombination with a highly reproducible frequency of 0.5-1.0% in lymphoid progenitors. The recombination system established here allows for a simple and accurate detection and isolation of recombination events in live cells, making this system particularly attractive for mosaic analysis or mutagenesis studies in the immune system.

Listeria monocytogenes internalins are highly diverse and evolved by recombination and positive selection

To probe the evolution of internalins with confirmed or suspected roles in Listeria monocytogenes virulence we sequenced the full inlB, inlC2, inlC, inlD, inlE, inlF, inlG, and inlH ORFs from 40 L. monocytogenes isolated from human ( n = 10) and animal ( n = 10) clinical cases, foods ( n = 10), and the natural environment ( n = 10). inlB and inlE were present in all isolates, representing 26 and 20 alleles, respectively. inlC was found in all lineage I and II isolates and represented 21 alleles. inlC2 and inlD represented 22 and 24 alleles, respectively, and were found in all L. monocytogenes isolates, with the exception of three lineage II isolates, which carried inlH, an apparent fusion of the 5′ end of inlC2 with the 3′ end of inlD. inlF and inlG were absent from lineage I isolates and represented 16 and 11 alleles, respectively. Average pairwise nucleotide differences per site ( π) ranged from 0.00849 ( inlF) to 0.07020 ( inlE). Phylogenetic trees generally showed clustering of internalin genes into two major evolutionary lineages consistent with lineages I and II previously assigned by ribotyping. In addition to detection of recombination events within each internalin gene, inlB, inlC, inlC2, and inlF showed significant evidence for positive selection (i.e., selection for an advantageous mutant allele). Overall, our data indicated that (i) internalin genes are highly diverse, (ii) internalin gene sequences cluster consistent with the phylogenetic lineages of L. monocytogenes, (iii) both intragenic recombination and positive selection have contributed to the evolution of L. monocytogenes internalins, and (iv) L. monocytogenes internalins show distinct evolutionary histories.

The rate of extrachromosomal homologous recombination within a novel reporter plasmid is elevated in cells lacking functional ATM protein

Homologous recombination between identical stretches of DNA depends on the coordinated action of many tightly regulated proteins. Cellular defects in homologous recombination are strongly associated with increased genomic instability and tumorigenesis. In cells of the cancer-prone syndrome ataxia telangiectasia (A-T), increased intrachromosomal recombination has been demonstrated, while extrachromosomal recombination has been discussed controversially. We constructed a novel, episomally replicating pGrec recombination vector containing two mutated alleles of the enhanced green fluorescent protein ( eGFP) gene. Homologous recombination can reconstitute functional wildtype eGFP, thus allowing detection of recombination events based on cellular eGFP fluorescence. Using an isogenic cell pair of A-T fibroblasts and derivatives complemented by an ATM expression vector, we were able to demonstrate in A-T cells high extrachromosomal recombination rates, which are suppressed upon ectopic ATM expression. We thus found that ATM deficiency increases spontaneous recombination not only in intrachromosomal but also in extrachromosomal substrates, suggesting that lack of ATM increases homologous recombination independent of the chromatin structure.

Redefining bacterial populations: a post-genomic reformation.

Sexual reproduction and recombination are essential for the survival of most eukaryotic populations. Until recently, the impact of these processes on the structure of bacterial populations has been largely overlooked. The advent of large-scale whole-genome sequencing and the concomitant development of molecular tools, such as microarray technology, facilitate the sensitive detection of recombination events in bacteria. These techniques are revealing that bacterial populations are comprised of isolates that show a surprisingly wide spectrum of genetic diversity at the DNA level. Our new awareness of this genetic diversity is increasing our understanding of population structures and of how these affect host pathogen relationships.

Sensitive analysis of recombination activity using integrated cell surface reporter substrates

Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result in various malignancies. So far the study of recombination activity has frequently relied on the use of reporter substrates that are limited by low sensitivity as well as tedious and distorting readout procedures. Immunoglobulin class switch recombination substrates were generated which, upon recombination, resulted in the surface expression of human CD4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation. Recombining cells harboring integrated reporter substrates were analyzed by immunofluorescence and flow cytometry and could easily be isolated by high-gradient magnetic cell sorting (MACS). The analysis was not influenced by cloning efficiencies, as would be the case after drug selection, or prokaryotic recombination that might occur after analysis of recovered substrates in bacteria. In addition, cytometric readout is much faster, as it can be performed immediately after recombination. The substrate exhibited properties compatible with the detection of immunoglobulin class switch recombination and permitted the detection of recombination events down to 10(-5) per cell and generation. The high sensitivity of this system allows precise detection of very rare recombination events and thus permits the study of cell types with extremely low recombination activities.