Mitotic recombination in the absence of synaptonemal complexes in Saccharomyces cerevisiae.

Summary Mitotic cells of a diploid strain of Saccharomyces cerevisiae with appropriate markers for the detection of mitotic crossing-over and mitotic gene conversion were irradiated with X-rays. Induction of these recombinational events was strong. After irradiation, cells were incubated in a rich growth medium and samples were removed for studying the possible formation of synaptonemal complexes up to a time when most cells had completed the first post-irradiation cell division. No complexes were found during the entire period of sampling, during which mitotic recombination in G₁ (mitotic gene conversion), DNA replication and G₂ (mitotic crossing-over) had occurred. These results are interpreted to mean that synaptonemal complexes are not required for mitotic recombination.     

Biochemical analysis of genetic recombination in eukaryotes.

Recent studies concerning molecular mechanisms of genetic recombination in eukaryotes are reviewed. Since many of these studies have focused on the testable predictions arising from the hybrid DNA theory of genetic recombination, this theory is summarised. Experiments to determine the time of meiotic crossing-over and the structure of the synaptonemal complex which facilitates meiotic crossing-over are described. Investigations of DNA nicking and repair events implicated in recombination are discussed. Properties of proteins which may facilitate hybrid DNA formation, and biochemical evidence for hybrid DNA formation are presented. Finally, a nuclease which has been implicated in gene conversion is described.     

Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae

The meiotic effects of several cell division cycle (cdc) mutations of Saccharomyces cerevisiae have been investigated by electron microscopy and by genetic and biochemical methods. Diploid strains homozygous for cdc mutations known to confer defects on vegetative DNA synthesis were subjected to restrictive conditions during meiosis. Electron microscopy revealed that all four mutants were conditionally arrested in meiosis after duplication of the spindle pole bodies but before spindle formation for the first meiotic division. None of these mutants became committed to recombination or contained synaptonemal complex at the meiotic arrest. — The mutants differed in their ability to undergo premeiotic DNA synthesis under restrictive conditions. Both cdc8 and cdc21, which are defective in the propagation of vegetative DNA synthesis, also failed to undergo premeiotic DNA synthesis. The arrest of these mutants at the stage before meiosis I spindle formation could be attributed to the failure of DNA synthesis because inhibition of synthesis by hydroxyurea also caused arrest at this stage. — Premeiotic DNA synthesis occurred before the arrest of cdc7, which is defective in the initiation of vegetative DNA synthesis, and of cdc2, which synthesizes vegetative DNA but does so defectively. The meiotic arrest of cdc7 homozygotes was partially reversible. Even if further semiconservative DNA replication was inhibited by the addition of hydroxyurea, released cells rapidly underwent commitment to recombination and formation of synaptonemal complexes. The cdc7 homozygote is therefore reversibly arrested in meiosis after DNA replication, whereas vegetative cultures have previously been shown to be defective only in the initiation of DNA synthesis.     

Nutritional regulation of late meiotic events in Saccharomyces cerevisiae through a pathway distinct from initiation.

The IME1 gene is essential for initiation of meiosis in the yeast Saccharomyces cerevisiae, although it is not required for growth. Here we report that in stationary-phase cultures containing low concentration of glucose, cells overexpressing IME1 undergo the early meiotic events, including DNA replication, commitment to recombination, and synaptonemal complex formation and dissolution. In contrast, later meiotic events, such as chromosome segregation, commitment to meiosis, and spore formation, do not occur. Thus, nutrients can repress the late stages of meiosis independently of their block of initiation. Cells arrested at this midpoint in meiosis are relatively stable and can resume meiotic differentiation if transferred to sporulation conditions. Resumption of meiosis does not require repression of IME1 expression, since IME1 RNA levels stay high after transfer of the arrested cells to sporulation medium. These results suggest that meiosis in S. cerevisiae is a paradigm of a differentiation pathway regulated by signal transduction at both early and late stages.     

EXO1 and MSH4 differentially affect crossing-over and segregation

The 5′-3′ exonuclease Exo1p from Saccharomyces cerevisiae is required for wild-type levels of meiotic crossing-over and normal meiotic chromosome segregation as is the meiosis-specific MutS homologue, Msh4p. Mutations in both genes reduce crossing-over by approximately two-fold, but Δmsh4 strains have significantly lower viability and a higher frequency of meiosis I non-disjunction. Epistasis analysis indicates a complex interaction between the two genes. Although crossing-over was not detectably lower in the double mutant, viability was significantly worse than either single mutant. Such a result suggests that the two genes are affecting meiotic viability by distinct mechanisms. We propose that Δexo1 affects chromosome segregation by reducing crossing-over, while Δmsh4 affects both the frequency and distribution of crossovers. Mutation in EXO1 reduces gene conversion frequencies significantly at some but not all loci, suggesting that other enzymes are also involved in DNA resection. We propose that Exo1p plays an early role in establishing some recombination intermediates by generating single-stranded tails. The role of Msh4p is suggested to be in determining whether some recombination intermediates are resolved as crossover events and in generating crossover interference. The synergistic effect of Δexo1Δmsh4 on spore viability suggests that the two genes have partially compensatory roles in a process affecting meiotic success. Received: 10 November 1999; in revised form: 14 January 2000 / Accepted: 14 January 2000     

Linear element-independent meiotic recombination in Schizosaccharomyces pombe

Most organisms form protein-rich, linear, ladder-like structures associated with chromosomes during early meiosis, the synaptonemal complex. In Schizosaccharomyces pombe, linear elements (LinEs) are thread-like, proteinacious chromosome-associated structures that form during early meiosis. LinEs are related to axial elements, the synaptonemal complex precursors of other organisms. Previous studies have led to the suggestion that axial structures are essential to mediate meiotic recombination. Rec10 protein is a major component of S. pombe LinEs and is required for their development. In this report we study recombination in a number of rec10 mutants, one of which (rec10-155) does not form LinEs, but is predicted to encode a truncated Rec10 protein. This mutant has levels of crossing over and gene conversion substantially higher than a rec10 null mutant (rec10-175) and forms cytologically detectable Rad51 foci indicative of meiotic recombination intermediates. These data demonstrate that while Rec10 is required for meiotic recombination, substantial meiotic recombination can occur in rec10 mutants that do not form LinEs, indicating that LinEs per se are not essential for all meiotic recombination.     

Recombinationless meiosis in Saccharomyces cerevisiae.

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.     

Stage-specific damage to synaptonemal complexes and metaphase chromosomes induced by X rays in male mouse germ cells

Synaptonemal complexes reveal mutagen-induced effects in germ cell meiotic chromosomes. This study was aimed at characterizing relationships between damage to synaptonemal complexes and metaphase I chromosomes following radiation exposure at various stages of spermatogenesis. Male mice were irradiated with doses of 0, 2, or 4 Gy, and spermatocytes were harvested at times consistent with earlier exposures as spermatogonial stem cells, preleptotene cells (premeiotic DNA synthesis), or meiotic prophase cells. After stem-cell exposure, twice as many rearrangements were observed in synaptonemal complexes as in metaphase I chromosomes. Irradiation during premeiotic DNA synthesis resulted in dose-related increases in synaptonemal complex breakage and rearrangements (including novel forms) and in metaphase chromosomal aberrations. Following prophase exposure, various types and levels of damage to synaptonemal complexes and metaphase chromosomes were observed. Irradiation of zygotene cells led to high frequencies of chromosome multivalents in metaphase I without a correspondingly high level of damage in preceding prophase synaptonemal complexes. Thus irradiation of premeiotic and meiotic cells results in variable relationships between damage to synaptonemal complexes and metaphase chromosomes. Interpretations of these relationships are based upon what is known about both radiation clastogenesis and the structural/temporal relationships between synaptonemal complexes at prophase and chromosomes at metaphase I of meiosis.     

Genetic control of chromosome synapsis in yeast meiosis

Both meiosis-specific and general recombination functions, recruited from the mitotic cell cycle, are required for elevated levels of recombination and for chromosome synapsis (assembly of the synaptonemal complex) during yeast meiosis. The meiosis-specific SPO11 gene (previously shown to be required for meiotic recombination) has been isolated and shown to be essential for synaptonemal complex formation but not for DNA metabolism during the vegetative cell cycle. In contrast, the RAD52 gene is required for mitotic and meiotic recombination but not for synaptonemal complex assembly. These data suggest that the synaptonemal complex may be necessary but is clearly not sufficient for meiotic recombination. Cytological analysis of spread meiotic nuclei demonstrates that chromosome behavior in yeast is comparable with that observed in larger eukaryotes. These spread preparations support the immunocytological localization of specific proteins in meiotic nuclei. This combination of genetic, molecular cloning, and cytological approaches in a single experimental system provides a means of addressing the role of specific gene products and nuclear structures in meiotic chromosome behavior.     

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Summary Gene conversion - apparently non-reciprocal transfer of sequence information between homologous DNA sequences - has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo ⁺ gene by homologous recombination. We found that all the neo ⁺ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo ⁺ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec ⁺ strain. or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec ⁺ strain, led us to propose a mechanism for this biased gene conversion. This successive half crossing-over model proposes that the elementary recombinational process is half crossing;-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.     

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.     

Saccharomyces cerevisiae cells lacking the homologous pairing protein p175 SEP1 arrest at pachytene during meiotic prophase

Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175 ^SEP1 enter pachytene with a delay. They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I. At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene. In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type. A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically. Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest. Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point. Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest. The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage.     

An expanded inventory of conserved meiotic genes provides evidence for sex in Trichomonas vaginalis

Meiosis is a defining feature of eukaryotes but its phylogenetic distribution has not been broadly determined, especially among eukaryotic microorganisms (i.e. protists)-which represent the majority of eukaryotic 'supergroups'. We surveyed genomes of animals, fungi, plants and protists for meiotic genes, focusing on the evolutionarily divergent parasitic protist Trichomonas vaginalis. We identified homologs of 29 components of the meiotic recombination machinery, as well as the synaptonemal and meiotic sister chromatid cohesion complexes. T. vaginalis has orthologs of 27 of 29 meiotic genes, including eight of nine genes that encode meiosis-specific proteins in model organisms. Although meiosis has not been observed in T. vaginalis, our findings suggest it is either currently sexual or a recent asexual, consistent with observed, albeit unusual, sexual cycles in their distant parabasalid relatives, the hypermastigotes. T. vaginalis may use meiotic gene homologs to mediate homologous recombination and genetic exchange. Overall, this expanded inventory of meiotic genes forms a useful "meiosis detection toolkit". Our analyses indicate that these meiotic genes arose, or were already present, early in eukaryotic evolution; thus, the eukaryotic cenancestor contained most or all components of this set and was likely capable of performing meiotic recombination using near-universal meiotic machinery.     

Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1''s strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51''s strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1''s chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1''s dominance in promoting strand exchange between homologs involves repression of Rad51''s strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.     

Differential effect of UV irradiation on induction of intragenic and intergenic recombination during commitment to meiosis in Saccharomyces cerevisiae

A comparison was made between the induction of intragenic and intergenic recombinations during meiosis in a wild-type diploid of Saccharomyces cerevisiae. Under non-irradiated normal conditions, production of both intragenic and intergenic recombinants greatly increased in the cells with commitment to meiosis. The susceptibility of cells to the induction of both the spontaneous intra- and intergenic recombinations in meiotic cells was similar. However, under condition of UV irradiation, there were striking differences between intra- and intergenic recombinations. Susceptibility to induction of intragenic recombination by UV irradiation was not enhanced at meiosis compared with mitosis, and was not altered through commitment to meiotic processes. In contrast, however, susceptibility to the induction of intergenic recombination by UV irradiation was enhanced markedly during commitment to meiosis compared with mitosis. Genetic analysis suggested that the enhanced susceptibility to recombination during meiosis is specifically concerned with reciprocal-type recombination (crossing-over) but not non-reciprocal-type recombination (gene conversion). Hence it is concluded that the meiotic process appears to be intimately concerned with the mechanism(s) of induction of recombination, especially reciprocal-type recombination.     

The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over

BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases. Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis. The sgs1 null mutant sporulates poorly and displays reduced spore viability. RESULTS: We have identified novel functions for Sgs1 in meiosis. Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation. In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis. Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion. The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase. A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation. CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions. The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis. Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange. We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase.     

Decreasing Gradients of Gene Conversion on Both Sides of the Initiation Site for Meiotic Recombination at the Arg4 Locus in Yeast

We have constructed eight restriction site polymorphisms in the DED81-ARG4 region and examined their behavior during meiotic recombination. Tetrad analysis reveals decreasing gradients of gene conversion on both sides of the initiation site for meiotic recombination at the ARG4 locus, extending on one side into the ARG4 gene, and on the other side into the adjacent DED81 gene. Gene conversion events can extend in both directions from the initiation site as the result of a single meiotic event. There is a second gradient of gene conversion in DED81, with high levels near the 5' end of the gene and low levels near the middle of the gene. The peaks of gene conversion activity for the DED81 and ARG4 gradients map to regions where double-strand breaks are found during meiosis. The implications of these results for models of meiotic gene conversion are discussed.     

Abnormal meiosis in bisporic strains of white button mushroom Agaricus bisporus (Lange) imbach

A formerly developed method of microspreading of mushroom basidial nuclei was applied to study meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The anomalous phenotypes in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After partial veil breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Synaptonemal complex karyotype of Eimeria tenella

In most organisms, biological variability rests on the behaviour of the chromosomes in the meiotic context. Despite the importance of meiosis, very little is known about the meiotic behaviour of the Eimeria chromosomes. The aim of the present study is to describe the standard synaptonemal complex karyotype from Eimeria tenella oocyst spreads by electron microscopy. For that purpose, complete sets of pachytene synaptonemal complexes were obtained and the morphological pachytene karyotype was determined. The authors used a previously reported method that overcomes the difficulty of the extreme resistance of protozoan oocysts to disruption and permits the release of intact meiotic chromosomes. The chromosomes were selected under a light microscope and those selected were stained with phosphotungtic acid and studied by transmission electron microscopy. The authors confirmed 14 chromosomes, which were observed as synaptonemal complexes, and the karyotype was constructed by arranging synaptonemal complexes according to their relative lengths and kinetochore position. Components of the synaptonemal complex, lateral elements, central element, recombination nodules and kinetochore were observed. Measures of the kynetochore, width of the synaptonemal complex, diameter of the recombination nodule and length of the telomeres are given. Minimal and no significant differences were found between measures of chromosomes isolated from different Eimeria tenella strains. To the best of our knowledge, the present investigation for the first time identifies and describes the morphological characteristics of the synaptonemal complex of Eimeria tenella during the meiosis that occurs within the oocysts. In addition, the authors provide evidence of the presence of recombination nodules, suggesting that the recombination process may play an important role in the molecular evolution of this parasite.     

人精母细胞重组频率和年龄相关性的分析

减数分裂遗传重组对同源染色体的正确分离和单倍体的正确形成起至关重要的作用,但人们对人精母细胞减数分裂遗传重组机制了解的还很少。通过免疫荧光染色技术标记减数分裂I联会复合体上的MLH1(DNA错配修复蛋白)位点可以检测人精母细胞的重组。文章对10例可育男性进行分析,发现每个细胞中重组位点数平均为49.4士4.4,范围为33~63,具有显著的个体差异,只有0.4%(1/220)的常染色体SC上缺少MLH1位点。进一步通过Spearman相关性分析,分析了年龄因素与个体间重组位点差异的相关性,结果提示年龄因素对常染色体及性染色体的重组均无影响。