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.     

Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells

Homologous recombination (HR) is important in repairing errors of replication and other forms of DNA damage. In mammalian cells, potential templates include the homologous chromosome, and after DNA replication, the sister chromatid. Previous work has shown that the mammalian recombination machinery is organized to suppress interchromosomal recombination while preserving intrachromosomal HR. In the present study, we investigated spontaneous intrachromosomal HR in mouse hybridoma cell lines in which variously numbered tandem repeats of the µ heavy chain constant (Cµ) region reside at the haploid, chromosomal immunoglobulin µ heavy chain locus. This organization provides the opportunity to investigate recombination between homologous gene repeats in a well-defined chromosomal locus under conditions in which recombinants are conveniently recovered. This system revealed several features about the mammalian intrachromosomal HR process: (i) the frequency of HR was high (recombinants represented as much as several percent of the total of recombinants and non-recombinants); (ii) the recombination process appeared to be predominantly non-reciprocal, consistent with the possibility of gene conversion; (iii) putative gene conversion tracts were long (up to 13.4 kb); (iv) the recombination process occurred with precision, initiating and terminating within regions of shared homology. The results are discussed with respect to mammalian intrachromosomal HR involving interactions both within and between sister chromatids.     

Increase in incidence of chromosome instability and non-conservative recombination between repeats in Saccharomyces cerevisiae hpr1Δ strains

Null hprl Δ strains show a large increase (up to 2000-fold) over wild type in the frequency of occurrence of deletions between direct repeats on three different chromosomes. However, we show that hprl Δ mutations have little or no effect on reciprocal exchange, gene conversion or unequal sister chromatid exchange, as determined using intrachromosomal, interchromosomal and plasmid-chromosome assay systems. A novel intrachromosomal recombination system has allowed us to determine that over 95% of deletions in hpr1 Δ strains do not occur by reciprocal exchange. On the other hand, hpr1 Δ strains show chromosome loss frequencies of up to 100 times the wild-type level. Our results suggest that yeast cells have a very efficient non-conservative recombination mechanism, dependent on RADI and RAD52, that causes deletions between direct DNA repeats, and this mechanism is strongly stimulated in hpr1 Δ strains. The results indicate that the Hpr1 protein is required for stability of DNA repeats and chromosomes. We propose that in the absence of the Hprl protein the cell destabilizes the genome by allowing the initiation of events that lead to deletions of sequences between repeats, and to chromosome instability. We discuss the roles that proteins such as Hprl have in maintaining direct repeats and in preventing non-conservative recombination and consider the importance of these functions for chromosome stability.     

Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy,interchromosomal rearrangement,and the simultaneous incidence of both events during mitotic growth

The SGS1 gene of Saccharomyces cerevisiae is a member of the RecQ helicase family, which includes the human BLM, WRN and RECQL4 genes responsible for Bloom and Werner's syndrome and Rothmund-Thomson syndrome, respectively. Cells defective in any of these genes exhibit a higher incidence of genome instability. We previously demonstrated that various genetic alterations were detectable as events leading to loss of heterozygosity (LOH) in S. cerevisiae diploid cells, utilizing a hemizygous URA3 marker placed at the center of the right arm of chromosome III. Analyses of chromosome structure in LOH clones by pulse field gel electrophoresis (PFGE) and PCR, coupled with a genetic method, allow identification of genetic alterations leading to the LOH. Such alterations include chromosome loss, chromosomal rearrangements at various locations and intragenic mutation. In this work, we have investigated the LOH events occurring in cells lacking the SGS1 gene. The frequencies of all types of LOH events, excluding intragenic mutation, were increased in sgs1 null mutants as compared to the wild-type cells. Loss of chromosome III and chromosomal rearrangements were increased 13- and 17-fold, respectively. Further classification of the chromosomal rearrangements confirmed that two kinds of events were especially increased in the sgs1 mutants: (1) ectopic recombination between chromosomes, that is, unequal crossing over and translocation (46-fold); and (2) allelic crossing over associated with chromosome loss (40-fold). These findings raise the possibility that the Sgs1 protein is involved in the processing of recombination intermediates as well as in the prevention of recombination repair during chromosome DNA replication. On the other hand, intrachromosomal deletions between MAT and HMR were increased only slightly (2.9-fold) in the sgs1 mutants. These results clearly indicate that defects in the SGS1 gene function lead to an elevated incidence of LOH in multiple ways, including chromosome loss and interchromosomal rearrangements, but not intrachromosomal deletion.     

Spontaneous loss of heterozygosity in diploid Saccharomyces cerevisiae cells

To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells. Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberration were the major classes of genetic alterations leading to LOH. The frequencies of chromosome loss and chromosome aberration were significantly affected when the marker was located in different chromosomes, suggesting that chromosome-specific elements may affect the processes that led to these alterations. Aberrant-sized chromosomes were detected readily in approximately 8% of LOH events when the URA3 marker was placed in chromosome III. Molecular mechanisms underlying the chromosome aberrations were further investigated by studying the fate of two other genetic markers on chromosome III. Chromosome aberration caused by intrachromosomal rearrangements was predominantly due to a deletion between the MAT and HMR loci that occurred at a frequency of 3.1 x 10(-6). Another type of chromosome aberration, which occurred at a frequency slightly higher than that of the intrachromosomal deletion, appeared to be caused by interchromosomal rearrangement, including unequal crossing over between homologous chromatids and translocation with another chromosome.     

Control of Large Chromosomal Duplications in Escherichia Coli by the Mismatch Repair System

Excessive recombination between repeated, interspersed, and diverged DNA sequences is a potential source of genomic instability. We have investigated the possibility that a mechanism exists to suppress genetic exchange between these quasi-homologous (homeologous) sequences. We examined the role of the general mismatch repair system of Escherichia coli because previous work has shown that the mismatch repair pathway functions as a barrier to interspecies recombination between E. coli and Salmonella typhimurium. The formation of large duplications by homeologous recombination in E. coli was increased some tenfold by mutations in the mutL and mutS genes that encode the mismatch recognition proteins. These findings indicate that the mismatch recognition proteins act to prevent excessive intrachromosomal exchanges. We conclude that mismatch repair proteins serve as general controllers of the fidelity of genetic inheritance, acting to suppress chromosomal rearrangements as well as point mutations.     

Ectopic mitotic recombination in Drosophila probed with bacterial beta-galactosidase gene-based reporter transgenes.

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.     

Association of reciprocal exchange with gene conversion between the repeated segments of 2-micron circle

The occurrence of reciprocal exchange of flanking DNA during gene conversion between the repeated segments of the yeast plasmid, 2-micron circle has been examined. The conversion event is induced by making a double-stranded gap within one of the repeats in vitro and allowing the gap to be repaired in vivo. The repair takes place with frequent recombination of flanking markers. Neither the topology of the plasmid substrates (linear or circular) nor the relative orientation of the repeats affects the association rule significantly. These events are reminiscent of meiotic gene conversion between homologous chromosomes but contrast sharply with mitotic or meiotic intrachromosomal gene conversion. It would appear that the difference between the outcomes of intramolecular gene conversion on a chromosome and on a plasmid gapped in vitro does not result from the different physical states of intracellular versus transformed DNA. A gene conversion event in a 2-micron circle : : Tn5 plasmid mediated by the 2-micron circle recombinase (FLP) in vivo, which is formally analogous to the yeast mating type interconversion, often results in recombination of flanking markers. The reaction can be mimicked, in the absence of FLP, by gapping the plasmid within one of the 2-micron circle repeats in vitro and carrying out gap repair in vivo.     

Rapid induction of large chromosomal deletions by a Cre/inverted loxP system in mouse ES Cell hybrids

Loss of heterozygosity by whole or partial loss of chromosomal regions is crucial to genetic disorders, cancers and diseases. It is difficult to analyze the mechanisms of pathogenesis caused by large-scale chromosomal abnormalities due to the extreme rarity of this mutagenesis. Using a Cre/inverted loxP system, we have generated a chromosome elimination cassette (CEC) that induces a selective loss of embryonic-stem-cell-derived chromosomes in undifferentiated embryonic stem cell-somatic cell hybrids. Here, due to the increased expression of Cre, rapid formation of Cre recombination products and immediate loss of CEC-tagged chromosomes were detected by fluorescence in situ hybridization. Cre also initiated intrachromosomal recombination between identical short sequences outside loxP, leading to large chromosomal deletions of CEC-tagged regions. The Cre-mediated antiparallel synapses likely act as a scaffold to bring the identical short sequences into close proximity for recombination. This CEC technology might allow better understanding of the modulator sequences responsible for the tangled structure formation and its solution mechanism, inducing mitotic recombination leading to chromosomal deletions.     

Chromosome evolution of MMU16 and RNO11: conserved synteny associated with gene order rearrangements explicable by intrachromosomal recombinations and neocentromere emergence

Comparative mapping between the rat and mouse genomes has shown that some chromosomes are entirely or almost entirely conserved with respect to gene content. Such is the case of rat chromosome 11 (RNO11) and mouse chromosome 16 (MMU16). We determined to what extent such an extensive conservation of synteny is associated with a conserved gene order. Therefore, we regionally localized several genes on RNO11. The comparison of the gene map of RNO11 and MMU16 unambiguously shows that the gene order has not been conserved in the Murinae lineage, thereby implying the occurrence of intrachromosomal evolutionary rearrangements. The transition from one chromosome configuration to the other one can be explained either by two intrachromosomal recombinations or by a single intrachromosomal recombination accompanied by neocentromere emergence.     

Unveiling novel RecO distant orthologues involved in homologous recombination

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.     

Generation of a Restriction Fragment Length Polymorphism Linkage Map for Toxoplasma Gondii

We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.     

Evolutionary Relationships and Sequence Variation of α-Amylase Variants Encoded by Duplicated Genes in the Amy Locus of Drosophila Melanogaster

To infer the genealogical relationships of α-amylase electromorphs of Drosophila melanogaster, we determined the nucleotide sequences of a collection of electromorphs sampled throughout the world. On average there were 1.0 amino acid substitutions between identical electromorphs and 3.9 between different electromorphs, respectively. We found that the evolution of AMY(1) through AMY(6) electromorphs occurred by sequential accumulation of single amino acid substitutions each causing one charge difference. The nucleotide diversities at synonymous sites within Amy(1),Amy(2),Amy(3),Amy(4) and Amy(6) were 0.0321, 0.0000, 0.0355, 0.0059 and 0.0030, respectively. We also obtained evidence of genetic exchanges, such as intrachromosomal recombination, interchromosomal recombination or gene conversion, between the two duplicated Amy genes as well as among the alleles.     

A plant-transformation-competent BIBAC/BAC-based map of rice for functional analysis and genetic engineering of its genomic sequence.

Sequencing of the rice genome has provided a platform for functional genomics research of rice and other cereal species. However, multiple approaches are needed to determine the functions of its genes and sequences and to use the genome sequencing results for genetic improvement of cereal crops. Here, we report a plant-transformation-competent, binary bacterial artificial chromosome (BIBAC) and bacterial artificial chromosome (BAC) based map of rice to facilitate these studies. The map was constructed from 20 835 BIBAC and BAC clones, and consisted of 579 overlapping BIBAC/BAC contigs. To facilitate functional analysis of chromosome 8 genomic sequence and cloning of the genes and QTLs mapped to the chromosome, we anchored the chromosomal contigs to the existing rice genetic maps. The chromosomal map consists of 11 contigs, 59 genetic markers, and 36 sequence tagged sites, spanning a total of ca. 38 Mb in physical length. Comparative analysis between the genetic and physical maps of chromosome 8 showed that there are 3 "hot" and 2 "cold" spots of genetic recombination along the chromosomal arms in addition to the "cold spot" in the centromeric region, suggesting that the sequence component contents of a chromosome may affect its local genetic recombination frequencies. Because of its plant transformability, the BIBAC/BAC map could provide a platform for functional analysis of the rice genome sequence and effective use of the sequencing results for gene and QTL cloning and molecular breeding.     

Interplay between recombination, cell division and chromosome structure during chromosome dimer resolution in Escherichia coli

Chromosome dimers form in bacteria by recombination between circular chromosomes. Resolution of dimers is a highly integrated process involving recombination between dif sites catalysed by the XerCD recombinase, cell division and the integrity of the division septum-associated FtsK protein and the presence of dif inside a restricted region of the chromosome terminus, the dif activity zone (DAZ). We analyse here how these phenomena collaborate. We show that (i) both inter- and intrachromosomal recombination between dif sites are activated by their presence inside the DAZ; (ii) the DAZ-specific activation only occurs in conditions supporting the formation of chromosome dimers; (iii) overexpression of FtsK leads to a general increase in dif recombination irrespective of dif location; (iv) overexpression of FtsK does not improve the ability of dif sites inserted outside the DAZ to resolve chromosome dimers. Our results suggest that the formation of an active XerCD-FtsK-dif complex is restricted to when a dimer is present, the features of chromosome organization that determine the DAZ playing a central role in this control.     

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.     

Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae.

An ectopic recombination system using ura3 heteroalleles varying in size from 80 to 960 bp has been used to examine the effect of substrate length on spontaneous mitotic recombination. The ura3 heteroalleles were positioned either on nonhomologous chromosomes (heterochromosomal repeats) or as direct or inverted repeats on the same chromosome (intrachromosomal repeats). While the intrachromosomal events occur at rates at least 2 orders of magnitude greater than the corresponding heterochromosomal events, the recombination rate for each type of repeat considered separately exhibits a linear dependence on substrate length. The linear relationships allow estimation of the corresponding minimal efficient processing segments, which are approximately 250 bp regardless of the relative positions of the repeats in the yeast genome. An examination of the distribution of recombination events into simple gene conversion versus crossover events indicates that reciprocal exchange is more sensitive to substrate size than is gene conversion.     

Some genetic properties of intrachromosomal recombination

Summary Intrachromosomal recombination was estimated by the occurrence of unequal crossing over without marker gene exchange in three different, independent tandem duplicaltions in Drosophila melanogaster. Each tandem duplication gave rise to intrachromosomal recombinants. The frequency of intrachromosomal recombination is independent of the genetic length of the tandem duplication. Further, intrachromosomal recombinants were recovered as frequently in ring as in rod X chromosomes implying that the recombination event is not equatable to a single interchromosomal crossover.Communicated by Ch. Auerbach