Genetic control of intrachromosomal recombination

Intrachromosomal recombination between direct repeats can occur either as gene conversion events, which maintain exactly the number of repeat units, or as deletions, which reduce the number of repeat units. Gene conversions are classical recombination events that utilize the standard chromosome recombination machinery. Spontaneous deletions between direct repeats are generally recA-independent in E. coli and RAD52-independent in S. cerevisiae. This independence from the major recombination genes does not mean that deletions form through a nonrecombinational process. Deletions have been suggested to result from sister chromatid exchange at the replication fork in a recA-independent process. The same type of exchange is proposed to be RAD52-independent in Saccharomyces cerevisiae. RAD52-dependent events encompass all events that involve the initial steps of a recombination reaction, which include strand invasion to form a heteroduplex intermediate.     

The mechanism of extrachromosomal homologous DNA recombination in plant cells

Summary By cotransfecting plasmids carrying particular mutations in the -glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.     

Topological aspects of site-specific DNA-inversion

Site-specific recombination events are of fundamental importance in many biological systems. In vitro experiments using purified proteins and DNA sub-strates are yielding insights into strand exchange mechanisms and synapsis of recombination sites. By examining results across a range of systems - prokaryotic and eukaryotic - two distinct classes of site-specific recombinase enzymes can be defined.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

Mutations at the human minisatellite MS32 integrated in yeast occur with high frequency in meiosis and involve complex recombination events

Minisatellites are composed of tandem repetitive DNA sequences and are present at many positions in the human genome. They frequently mutate to new length alleles in the germline, by complex and incompletely understood recombination mechanisms which may operate during meiosis. In several minisatellites the mutation events are restricted to one end of the repeat array, indicating a possible association with elements that act in cis. Mutant alleles do not show exchange of flanking regions. To construct a model system suitable for further investigations of the mutation process, we have integrated the human minisatellite MS32, flanked by synthetic markers, in the vicinity of a meiotic recombination hot spot upstream of the LEU2 locus in the yeast Saccharomyces cerevisiae. Here we provide direct evidence for a meiotic origin of MS32 mutations. Mutation events were polarised towards both ends of the minisatellite and varied from simple duplications and deletions to complex intra- and interallelic events. Interallelic events were frequently accompanied by exchange of regions flanking the minisatellite. The results also support the notion that cis-acting elements are involved in the mutational process. The fact that MS32 mutant structures are similar in yeast and human shows that meiotic recombination plays a crucial role in both organisms and emphasises the usefulness of yeast strains harbouring minisatellites as a model system for the study of minisatellite mutation. Received: 1 March 1997 / Accepted: 16 May 1997     

The interchromosomal effect of inversions in maize

Summary The interchromosomal effect of inversions in maize along the short arm of chromosome 9 yields results which are distinctly different from those which are reported with Drosophila melanogaster. Recombination was increased in the c ₁-sh₁ region of chromosome 9 while the sh ₁-wx region was unaffected. This increased frequency of recombination appears to be due to an increase in single exchange events as multiple events were unchanged. Increases in recombination were accompanied by either an increase in chromosome interference or normal interference levels. The magnitude of increase in recombination was much smaller than that seen in interchromosomal effects in Drosophila and is consistent with other observations made in maize. When two inversions were present in the same nucleus simultaneously, the effect on recombination was of the same magnitude as the effect of a single inversion. All inversions tested, regardless of size or position with respect to the centromere showed the same magnitude of increase.     

Recombination events are concentrated in the spike protein region of Betacoronaviruses

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.     

Action of RecBCD enzyme on Holliday structures made by RecA

In vitro, Escherichia coli RecA protein acts upon gapped and partially homologous linear duplex DNA to generate recombination products linked by Holliday junctions. When strand exchange reactions are supplemented with purified RecBCD enzyme, we observe the formation of products that resemble "patch" recombinants. The formation of "splice" recombinant products was not observed. The individual subunits, RecB, RecC, or RecD, had no effect on RecA protein-mediated strand exchange nor on the Holliday junctions formed in the reaction. Analysis of the way in which patch products arise indicates exonucleolytic digestion of the linear arms of the recombination intermediates (alpha-structures) by RecBCD enzyme. We find no evidence for specific resolution events at the site of the Holliday junction by RecBCD enzyme using these DNA substrates.     

Recombination nodules and synaptonemal complex in recombination-defective females of Drosophila melanogaster

The cytological effects of mutant alleles of the mei-9, mei-218, and mei-41 loci during prophase I have been examined by electron microscopy. None of these mutants affect synaptonemal complex structure, continuity, or temporal behavior. Both the precondition-defective mutants mei-218 and mei-41 affect both number and morphology of spherical recombination nodules and apparently affect at least the numbers of ellipsoidal recombination nodules, whereas in the exchange-defective mutant mei-9 the numbers and morphologies of both ellipsoidal and spherical recombination nodules are normal. The parallel effects of mei-218 and mei-41 on meiotic recombination and on recombination nodules indicate that spherical recombination nodules at least mark the site of exchange events; the effects of these mutants on nodule morphology suggest that the nodule performs an active role in the recombination process. The nodule phenotype of mei-9 indicates that spherical nodules are present, and presumably functioning, well before the concluding stages of the recombination event. The parallel effects of all 3 mutants on ellipsoidal and spherical nodules indicate that these are indeed related structures but does not ellucidate the nature of the relationship. It is suggested that all aspects of meiotic recombination are under the aegis of recombination nodules.     

Role of Recbc Function in Formation of Chromosomal Rearrangements: A Two-Step Model for Recombination

The role of recBC functions has been tested for three types of chromosomal recombination events: (1) recombination between direct repeats to generate a deletion, (2) recombination between a small circular fragment and the chromosome, and (3) recombination between inversely oriented repeats to form an inversion. Deletion formation by recombination between direct repeats, which does not require a fully reciprocal exchange, is independent of recBC function. Circle integration and inversion formation are both stimulated by the recBC function; these events require full reciprocality. The results suggest that half-reciprocal exchanges can occur without recBC, but recBC functions greatly stimulate completion of a fully reciprocal exchange. We propose that chromosomal recombination is a two-step process, and recBC functions are primarily required for the second step.     

Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system

The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.     

Hybrid dysgenesis in Drosophila: The mechanism of T-007-induced male recombination

Summary The term hybrid dysgenesis describes a syndrome of genetic effects which sometimes results when Drosophila melanogaster from wild populations are outcrossed; this syndrome often includes male recombination as well as enhanced rates of genic and chromosomal mutation, sterility, and transmission ratio distortion. In this study, we have examined the mechanism of T-007-induced male recombination by genetically characterizing third chromosomes generated by an exchange in a well-marked euchromatic region. Most recombinant chromosomes were sequentially normal, and no recessive lethal events at the point of exchange were recovered. The results demonstrate that although some recombinants may be generated by nonhomologous chromosome (or chromatid) breakage and reunion, the predominant effect of T-007 is through an enhanced rate of normal mitotic exchange. The rate of mitotic exchange is also increased by ionizing radiation and chemical mutagens; we suggest that the common factor in all three cases is the induction of single strand breaks.     

Factors affecting ectopic gene conversion in mice

Duplicated genes and repetitive sequences are distributed throughout the genomes of complex organisms. The homology between related sequences can promote nonallelic (ectopic) recombination, including gene conversion and reciprocal exchange. Resolution of these events can result in translocations, deletions, or other harmful rearrangements. In yeast, ectopic recombination between sequences on nonhomologous chromosomes occurs at high frequency. Because the mammalian genome is replete with duplicated sequences and repetitive elements, high levels of ectopic exchange would cause aneuploidy and genome instability. To understand the factors regulating ectopic recombination in mice, we evaluated the effects of homology length on gene conversion between unlinked sequences in the male germline. Previously, we found high levels of gene conversion between lacZ transgenes containing 2557 bp of homology. We report here that genetic background can play a major role in ectopic recombination; frequency of gene conversion was reduced by more than an order of magnitude by transferring the transgenes from a CF1 strain background to C57BL/6J. Additionally, conversion rates decreased as the homology length decreased. Sequences sharing 1214 bp of sequence identity underwent ectopic conversion less frequently than a pair sharing 2557 bp of identity, while 624 bp was insufficient to catalyze gene conversion at significant levels. These results suggest that the germline recombination machinery in mammals has evolved in a way that prevents high levels of ectopic recombination between smaller classes of repetitive sequences, such as the Alu family. Additionally, genomic location appeared to influence the availability of sequences for ectopic recombination. Received: 12 September 1997 / Accepted: 29 December 1997     

On the Origin of Plasmid-borne, Extended-spectrum, Antibiotic Resistance Mutations in Bacteria

Many antibiotic resistance mutations arise in pathogenic bacteria that harbor plasmids (R-plasmids). Resistance to third generation cephalosporins, for instance, largely occurs by one or more point mutations in plasmidblagenes that expand the resistance spectrum of β-lactamases. Here I review relevant evidence underlying the worldwide emergence of extended spectrum β-lactamases (ESBLs). The conclusion reached is that the origin of these resistance-conferring mutations cannot be explained by a series of single point mutation and selection events. Instead, highly advantageous stochastic processes might exist that generate alterations in the sequence or the conformation of particular regions in chromosomal or plasmid genomes such asbla, i.e., recombination or mutation. Several explanations for the origin of ESBLs are reviewed but direct experimental evidence to support or to invalidate them is still lacking. The cellular conditions under which ESBLs arise are unknown; however, involvement of nutritional stresses inside natural animal hosts and of plasmid conjugal functions appear likely.     

A comparative study of the recombination pattern in three species of Platyrrhini monkeys (primates)

Homologous chromosomes exchange genetic information through recombination during meiotic synapsis, a process that increases genetic diversity and is fundamental to sexual reproduction. Meiotic studies in mammalian species are scarce and mainly focused on human and mouse. Here, the meiotic recombination events were determined in three species of Platyrrhini monkeys (Cebus libidinosus, Cebus nigritus and Alouatta caraya) by analysing the distribution of MLH1 foci at the stage of pachytene. Moreover, the combination of immunofluorescence and fluorescent in situ hybridisation has enabled us to construct recombination maps of primate chromosomes that are homologous to human chromosomes 13 and 21. Our results show that (a) the overall number of MLH1 foci varies among all three species, (b) the presence of heterochromatin blocks does not have a major influence on the distribution of MLH1 foci and (c) the distribution of crossovers in the homologous chromosomes to human chromosomes 13 and 21 are conserved between species of the same genus (C. libidinosus and C. nigritus) but are significantly different between Cebus and Alouatta. This heterogeneity in recombination behaviour among Ceboidea species may reflect differences in genetic diversity and genome composition.     

The Red Queen theory of recombination hotspots

Recombination hotspots are small chromosomal regions, where meiotic crossover events happen with high frequency. Recombination is initiated by a double‐strand break (DSB) that requires the intervention of the molecular repair mechanism. The DSB repair mechanism may result in the exchange of homologous chromosomes (crossover) and the conversion of the allelic sequence that breaks into the one that does not break (biased gene conversion). Biased gene conversion results in a transmission advantage for the allele that does not break, thus preventing recombination and rendering recombination hotspots transient. How is it possible that recombination hotspots persist over evolutionary time (maintaining the average chromosomal crossover rate) when they are self‐destructive? This fundamental question is known as the recombination hotspot paradox and has attracted much attention in recent years. Yet, that attention has not translated into a fully satisfactory answer. No existing model adequately explains all aspects of the recombination hotspot paradox. Here, we formulate an intragenomic conflict model resulting in Red Queen dynamics that fully accounts for all empirical observations regarding the molecular mechanisms of recombination hotspots, the nonrandom targeting of the recombination machinery to hotspots and the evolutionary dynamics of hotspot turnover.     

A plasmid model to study genetic recombination in yeast

Chimeric plasmids have been constructed containing two heteroallelic mutant copies of the yeast HIS3 gene as an inverted repetition. Intramolecular exchange events between these two allelic mutant copies are capable of generating a wild-type allele. Plasmids containing two mutant heteroalleles have been transformed into appropriate his3^? yeast strains, and the frequency of exchange events generating His⁺ prototrophs has been measured during mitotic division. After 20 generations of growth under nonselective conditions, between 0.1 and 1 % of the transformed yeast cells become His⁺ prototrophs. This percentage decreases at least ten-fold in a strain with a rad52 mutation. Plasmid molecules having undergone exchange events have been isolated from yeast cells and have been examined after transfer to Escherichia coli. Physical examination shows that less than 10 % of the plasmids having undergone genetic exchange have also undergone an internal reciprocal recombination event as evidenced by reorientation of linked restriction sites. The remainder of the plasmids having undergone genetic exchange do not exhibit reciprocal recombination. Characterization of the individual allelic copies within a plasmid having undergone exchange reveals that in 24 of 25 examples only one of the two HIS3 copies has become wild type, and that either copy is equally likely to become wild type. We conclude that the model plasmid we have constructed undergoes intramolecular genetic exchange events and will be useful for studying genetic recombination.     

The Effect of mei-41 on Rdna Redundancy in DROSOPHILA MELANOGASTER

The recombination and repair defective mutant, mei-41, exhibits three rather striking effects on the genetic properties and chromosomal stability of rDNA in Drosophila. First, mei-41 inhibits rDNA magnification. However, mei-9, another recombination and repair defective mutation has no similar effect. This indicates that magnification requires some, but not all, of the gene products necessary for meiotic exchange. Second, under magnifying conditions, mei-41 induces interchanges between the X rDNA and either arm of the Ybb- chromosome. These interchanges occur at high frequency and are independent of rDNA orientation. Third, in mei-41 bb+/Ybb+ males, bobbed mutants in the X, but not the Y, also arise at high frequency. Evidence suggests that these events involve the rDNA type I insertion. The recombination and repair defective properties of mei-41 together with our results regarding its unusual and specific effects involving rDNA are explained in a simple model that has general implications for chromosome structure.     

Pulsed-field gel analysis of the pattern of DNA double-strand breaks in the Saccharomyces genome during meiosis

Pulsed-field gel electrophoresis (PFGE) has been used to study the timing, frequency, and distribution of double-strand breaks (DSBs) in chromosomal-sized DNA during meiosis in yeast. It has previously been shown that DSBs are associated with some genetic hotspots during recombination, and it is important to know whether meiotic recombination events routinely initiate via DSBs. Two strains have been studied here—a highsporulating homothallic wild type and a congenic mutant strain carrying a rad50S mutation. This mutant has previously been reported to accumulate broken molecules in meiosis to much higher frequencies than wild type and to abolish the characteristic wild-type processing of DNA that has been observed at the break sites. When whole chromosomes are resolved by PFGE, both strains show some broken molecules starting at the time that cells commit to genetic recombination. Breakage has been assessed primarily on Chromosome III and Chr. XV, using Southern hybridization to identify these chromosomes and their fragments. At any one time, break frequency in wild type is much lower than the cumulative frequency of recombination events that occur during meiosis. However, there is suggestive evidence that each break is short-lived, and it is therefore difficult to estimate the total number of breaks that may occur. In rad50S, chromosome breaks accumulate to much higher levels, which are probably broadly consistent with the estimated number of recombination events in wild type. However, since rad50S is substantially defective in completing recombination, it is not known for certain if it initiates events at wild-type frequencies. A surprising feature of the data is that a strong banding pattern is observed in the fragment distribution from broken chromosomes in both strains, implying that at least much of the breakage occurs at specific sites or within short regions. However, with the exception of the rDNA region on Chr. XII, assessment of the genetic map indicates that recombination can occur almost anywhere in the genome, although some regions are much hotter than others. Possible reasons for this apparent paradox are discussed. It may in part result from breakage levels too low for adequate detection in cold regions but may also imply that recombination events are localized more than previously realized. Alternatively, there may be a more indirect relationship between break sites and the associated recombination events. © 1993 Wiley-Liss, Inc.     

Dengue virus serotype infection specifies the activation of the unfolded protein response

Background

Tomato-infecting begomoviruses are widely distributed across the world and cause diseases of high economic impact on wide range of agriculturally important crops. Though recombination plays a pivotal role in diversification and evolution of these viruses, it is currently unknown whether there are differences in the number and quality of recombination events amongst different tomato-infecting begomovirus species. To examine this we sought to characterize the recombination events, estimate the frequency of recombination, and map recombination hotspots in tomato-infecting begomoviruses of South and Southeast Asia.

Results

Different methods used for recombination breakpoint analysis provided strong evidence for presence of recombination events in majority of the sequences analyzed. However, there was a clear evidence for absence or low Recombination events in viruses reported from North India. In addition, we provide evidence for non-random distribution of recombination events with the highest frequency of recombination being mapped in the portion of the N-terminal portion of Rep.

Conclusion

The variable recombination observed in these viruses signified that all begomoviruses are not equally prone to recombination. Distribution of recombination hotspots was found to be reliant on the relatedness of the genomic region involved in the exchange. Overall the frequency of phylogenetic violations and number of recombination events decreased with increasing parental sequence diversity. These findings provide valuable new information for understanding the diversity and evolution of tomato-infecting begomoviruses in Asia.