Meiotic Recombination Analyses in Pigs Carrying Different Balanced Structural Chromosomal Rearrangements

Correct pairing, synapsis and recombination between homologous chromosomes are essential for normal meiosis. All these events are strongly regulated, and our knowledge of the mechanisms involved in this regulation is increasing rapidly. Chromosomal rearrangements are known to disturb these processes. In the present paper, synapsis and recombination (number and distribution of MLH1 foci) were studied in three boars (Sus scrofa domestica) carrying different chromosomal rearrangements. One (T34he) was heterozygote for the t(3;4)(p1.3;q1.5) reciprocal translocation, one (T34ho) was homozygote for that translocation, while the third (T34Inv) was heterozygote for both the translocation and a pericentric inversion inv(4)(p1.4;q2.3). All three boars were normal for synapsis and sperm production. This particular situation allowed us to rigorously study the impact of rearrangements on recombination. Overall, the rearrangements induced only minor modifications of the number of MLH1 foci (per spermatocyte or per chromosome) and of the length of synaptonemal complexes for chromosomes 3 and 4. The distribution of MLH1 foci in T34he was comparable to that of the controls. Conversely, the distributions of MLH1 foci on chromosome 4 were strongly modified in boar T34Inv (lack of crossover in the heterosynaptic region of the quadrivalent, and crossover displaced to the chromosome extremities), and also in boar T34ho (two recombination peaks on the q-arms compared with one of higher magnitude in the controls). Analyses of boars T34he and T34Inv showed that the interference was propagated through the breakpoints. A different result was obtained for boar T34ho, in which the breakpoints (transition between SSC3 and SSC4 chromatin on the bivalents) seemed to alter the transmission of the interference signal. Our results suggest that the number of crossovers and crossover interference could be regulated by partially different mechanisms.     

Study of Homologous Recombination and Chromosomal Rearrangements inEscherichia coliStrains Carrying a Heterozygous Tandem Duplication

Heterozygous tandem duplications formed in conjugational matings in Escherichia coliprovides a convenient model system for studying the evolution of bacterial chromosome. Heterozygous duplications segregate various classes of haploid and diploid recombinants that appear as a result of unequal crossing over between sister chromosomes. In this work, an extended tandem duplication in the deooperon of E. colicarrying deoA deoB::Tn5/deoC deoD thr::Tn9alleles was examined. Recombination between homologous DNA repeats in the duplication was studied in strains carrying different combinations of recBC, sbcBC, recB::Tn10, recQ::Tn3and recF::Tn3mutations. The frequency of recombination between homologous DNA repeats was very high in all strains and did not decrease when the RecBCD and RecF recombinational pathways were simultaneously damaged in strains with the recB sbcBC recQ(or recF) genotype. It is assumed that unequal crossing over between direct DNA repeats in duplications may proceed through a particular pathway of adaptive recombination.     

Stimulation of Chromosomal Rearrangements by Ribonucleotides

We show by whole genome sequence analysis that loss of RNase H2 activity increases loss of heterozygosity (LOH) in Saccharomyces cerevisiae diploid strains harboring the pol2-M644G allele encoding a mutant version of DNA polymerase ε that increases ribonucleotide incorporation. This led us to analyze the effects of loss of RNase H2 on LOH and on nonallelic homologous recombination (NAHR) in mutant diploid strains with deletions of genes encoding RNase H2 subunits (rnh201Δ, rnh202Δ, and rnh203Δ), topoisomerase 1 (TOP1Δ), and/or carrying mutant alleles of DNA polymerases ε, α, and δ. We observed an ∼7-fold elevation of the LOH rate in RNase H2 mutants encoding wild-type DNA polymerases. Strains carrying the pol2-M644G allele displayed a 7-fold elevation in the LOH rate, and synergistic 23-fold elevation in combination with rnh201Δ. In comparison, strains carrying the pol2-M644L mutation that decreases ribonucleotide incorporation displayed lower LOH rates. The LOH rate was not elevated in strains carrying the pol1-L868M or pol3-L612M alleles that result in increased incorporation of ribonucleotides during DNA synthesis by polymerases α and δ, respectively. A similar trend was observed in an NAHR assay, albeit with smaller phenotypic differentials. The ribonucleotide-mediated increases in the LOH and NAHR rates were strongly dependent on TOP1. These data add to recent reports on the asymmetric mutagenicity of ribonucleotides caused by topoisomerase 1 processing of ribonucleotides incorporated during DNA replication.     

Cre-loxP Recombination System for Large Genome Rearrangements in Lactococcus lactis

We have used a new genetic strategy based on the Cre-loxP recombination system to generate large chromosomal rearrangements in Lactococcus lactis. Two loxP sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal loxP sites was highly efficient (approximately 1 × 10⁻¹/cell) when the Cre recombinase was provided in trans, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-loxP recombination system described can potentially be used for other gram-positive bacteria without further modification.     

Recombination between Chromosomal Is200 Elements Supports Frequent Duplication Formation in Salmonella Typhimurium

Spontaneous tandem chromosomal duplications are common in populations of Escherichia coli and Salmonella typhimurium. They range in frequency for a given locus from 10(-2) to 10(-4) and probably form by RecA-dependent unequal sister strand exchanges between repetitive sequences in direct order. Certain duplications have been observed previously to confer a growth advantage under specific selective conditions. Tandem chromosomal duplications are unstable and are lost at high frequencies, representing a readily reversible source of genomic variation. Six copies of a small mobile genetic element IS200 are evenly distributed around the chromosome of S. typhimurium strain LT2. A survey of 120 independent chromosomal duplications (20 for each of six loci) revealed that recombination between IS200 elements accounted for the majority of the duplications isolated for three of the loci tested. Duplications of the his operon were almost exclusively due to recombination between repeated IS200 elements. These data add further support to the idea that mobile genetic elements provide sequence repeats that play an important role in recombinational chromosome rearrangements, which may contribute to adaptation of bacteria to stressful conditions.     

Interpreting Chromosomal Rearrangements in the Context of 3-Dimentional Genome Organization: A Practical Guide for Medical Genetics

In this exciting era of “next-gen cytogenetics”, the use of novel molecular methods such as comparative genome hybridization and whole genome and whole exome sequencing becomes more and more common in clinics. This results in generation of large amounts of high-resolution patient-specific data and challenges the development of new approaches for interpretation of obtained information. Usually, interpretation of chromosomal rearrangements is focused on alterations of linear genome sequence, underestimating the role of spatial chromatin organization. In this article, we describe the main features of 3-dimentional genome organization, emphasizing their role in normal and pathological development. We highlight some tips to help physicians estimating the impact of chromosomal rearrangements on the patient phenotype. A separate section describes available tools that can be used to visualize and analyze human genome architecture.     

Pairing for Recombination in Lgv of Caenorhabditis Elegans: A Model Based on Recombination in Deficiency Heterozygotes

The effect of deficiencies on recombination was studied in Caenorhabditis elegans. Heterozygous deficiencies in the left half of linkage group V [LGV(left)] were shown to inhibit recombination to their right. Fourteen deficiencies, all to the left of unc-46, were analyzed for their effect on recombination along LGV. The deficiencies fell into two groups: 10 "major inhibitors" which reduce recombination to less than 11% of the expected rate between themselves and unc-46; and four "minor inhibitors" which reduce recombination, but to a much lesser extent. All four minor inhibitors delete the left-most known gene on the chromosome, while six of the ten major inhibitors do not (i.e., these are "internal" deficiencies). Where recombination could be measured on both sides of a deficiency, recombination was inhibited to the right but not to the left. In order to explain these results we have erected a model for the manner in which pairing for recombination takes place. In doing so, we identify a new region of LGV, near the left terminus, that is important for the pairing process.     

Chromosomal Rearrangements between Serotype A and D Strains in Cryptococcus neoformans

Cryptococcus neoformans is a major human pathogenic fungus that can cause meningoencephalitis in immunocompromised hosts. It contains two divergent varieties, var. grubii (serotype A) and var. neoformans (serotype D), as well as hybrids (serotype AD) between these two varieties. In this study, we investigated the extent of chromosomal rearrangements between the two varieties, estimated the effects of chromosomal rearrangements on recombination frequencies, and surveyed the potential polymorphisms of the rearrangements among natural strains of the three serotypes. Through the analyses of two sequenced genomes from strains H99 (representing var. grubii) and JEC21 (representing var. neoformans), we revealed a total of 32 unambiguous chromosome rearrangements, including five translocations, nine simple inversions, and 18 complex rearrangements. Our analyses identified that overall, rearranged regions had recombination frequencies about half of those around syntenic regions. Using a direct PCR screening strategy, we examined the potential polymorphisms of 11 rearrangements among 64 natural C. neoformans strains from five countries. We found no polymorphism within var. neoformans and very limited polymorphism within var. grubii. However, strains of serotype AD showed significant polymorphism, consistent with their hybrid origins coupled with differential loss of heterozygosity. We discuss the implications of these results on the genome structure, ecology, and evolution of C. neoformans.     

Chromosomal Distribution of Transposable Elements in Drosophila Melanogaster: Test of the Ectopic Recombination Model for Maintenance of Insertion Site Number

Data of insertion site localization and site occupancy frequency of P, hobo, I, copia, mdg1, mdg3, 412, 297, and roo transposable elements (TEs) on the polytene chromosomes of Drosophila melanogaster were extracted from the literature. We show that TE insertion site number per chromosomal division was significantly correlated with the amount of DNA. The insertion site number weighted by DNA content was not correlated with recombination rate for all TEs except hobo, for which a positive correlation was detected. No global tendency emerged in the relationship between TE site occupancy frequency, weighted by DNA content, and recombination rate; a strong negative correlation was, however, found for the 3L arm. A possible dominant deleterious effect of chromosomal rearrangements due to recombination between TE insertions is thus not the main factor explaining the dynamics of TEs, since this hypothesis implies a negative relationship between recombination rate and both TE insertion site number and site occupancy frequency. The alternative hypothesis of selection against deleterious effects of insertional mutations is discussed.     

Pathways for Homologous Recombination between Chromosomal Direct Repeats in Salmonella Typhimurium

Homologous recombination pathways probably evolved primarily to accomplish chromosomal repair and the formation and resolution of duplications by sister-chromosome exchanges. Various DNA lesions initiate these events. Classical recombination assays, involving bacterial sex, focus attention on double-strand ends of DNA. Sexual exchanges, initiated at these ends, depend on the RecBCD pathway. In the absence of RecBCD function, mutation of the sbcB and sbcC genes activates the apparently cryptic RecF pathway. To provide a more general view of recombination, we describe an assay in which endogenous DNA damage initiates recombination between chromosomal direct repeats. The repeats flank markers conferring lactose utilization (Lac(+)) and ampicillin resistance (Ap(R)); recombination generates Lac(-) Ap(S) segregants. In this assay, the RecF pathway is not cryptic; it plays a major role without sbcBC mutations. Others have proposed that single-strand gaps are the natural substrate for RecF-dependent recombination. Supporting this view, recombination stimulated by a double-strand break (DSB) in a chromosomal repeat depended on RecB function, not RecF function. Without RecBCD function, sbcBC mutations modified the RecF pathway and allowed it to catalyze DSB-stimulated recombination. Sexual recombination assays overestimate the importance of RecBCD and DSBs, and underestimate the importance of the RecF pathway.     

一种两步基因同源重组敲除酵母靶标蛋白基因的方法

目的:建立一种在代谢工程改造的毕赤酵母菌中高效敲除靶标蛋白基因的方法。方法:构建敲除载体,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因为靶基因,以尿嘧啶合成关键酶URA3基因为营养缺陷型筛选标记,第一步重组利用ura3正筛选获得敲除载体在酵母染色体中的定点整合,第二步通过5-氟乳清酸(5-FOA)筛选到表型为ura3-的克隆,ura3基因被剔除的同时,目的基因GM-CSF也随之丢失,实现第二次重组;利用基因组PCR和蛋白电泳进行鉴定。结果:通过两步基因同源重组,敲除了毕赤酵母GJK01的报告蛋白GM-CSF的编码基因388 bp,PCR结果显示该基因已完全丢失,SDS-PAGE分析无GM-CSF表达。结论:仅通过2周时间的筛选、鉴定,在毕赤酵母GJK01中敲除了报告蛋白GM-CSF的编码基因,突变菌株的阳性率达到50%,最终建立了以URA3为筛选标记的两步基因同源重组敲除目的基因的方法。     

Enhancement of Escherichia Coli Plasmid and Chromosomal Recombination by the Ref Function of Bacteriophage P1

The Ref activity of phage P1 enhances recombination between two defective lacZ genes in the Escherichia coli chromosome (lac- x lac- recombination). Plasmid recombination, both lac- x lac- and tet- x tet-, was measured by transformation of recA strains, and was also assayed by measurement of beta-galactosidase. The intracellular presence of recombinant plasmids was verified directly by Southern blotting. Ref stimulated recombination of plasmids in rec+ and rec(BCD) cells by 3-6-fold, and also the low level plasmid recombination in recF cells. RecA-independent plasmid recombination, either very low level (recA cells) or high level (recB recC sbcA recA cells), was not stimulated. Ref stimulated both intramolecular and intermolecular plasmid recombination. Both normal and Ref-stimulated lac- x lac- chromosomal recombination, expected to be mostly RecBC-dependent in wild-type bacteria, were affected very little by a recF mutation. We have previously reported Ref stimulation of lac- x lac- recombination in recBC sbcB bacteria, a process known to be RecF-dependent. Chromosomal recombination processes thought to involve activated recombination substrates, e.g., Hfr conjugation, P1 transduction, were not elevated by Ref activity. We hypothesize that Ref acts by unknown mechanisms to activate plasmid and chromosomal DNA for RecA-mediated recombination, and that the structures formed are substrates for both RecF-dependent (plasmid, chromosomal) and Rec(BCD)-dependent (chromosomal) recombination pathways.     

Recombination of Escherichia coli Chromosomal Segments in Salmonella typhosa

Approximately half of Salmonella typhosa hybrids resulting from mating with Escherichia coli Hfr donors inherit the selected donor marker by recombination, and the length of the E. coli chromosomal segment most frequently incorporated in these recombinants is between 1 and 2 min.