Molecular characterization of Ph1 as a major chromosome pairing locus in polyploid wheat

The foundation of western civilization owes much to the high fertility of bread wheat, which results from the stability of its polyploid genome. Despite possessing multiple sets of related chromosomes, hexaploid (bread) and tetraploid (pasta) wheat both behave as diploids at meiosis. Correct pairing of homologous chromosomes is controlled by the Ph1 locus. In wheat hybrids, Ph1 prevents pairing between related chromosomes. Lack of Ph1 activity in diploid relatives of wheat suggests that Ph1 arose on polyploidization. Absence of phenotypic variation, apart from dosage effects, and the failure of ethylmethane sulphonate treatment to yield mutants, indicates that Ph1 has a complex structure. Here we have localized Ph1 to a 2.5-megabase interstitial region of wheat chromosome 5B containing a structure consisting of a segment of subtelomeric heterochromatin that inserted into a cluster of cdc2-related genes after polyploidization. The correlation of the presence of this structure with Ph1 activity in related species, and the involvement of heterochromatin with Ph1 (ref. 6) and cdc2 genes with meiosis, makes the structure a good candidate for the Ph1 locus.     

小麦染色体配对基因Ph1结构剖析及调控机制研究进展

近年来,关于小麦抑制部分同源染色体配对基因Ph1的研究有了突破性进展.本文对该基因的结构和调控机理的最新研究进行综述.通过创造和分子标记鉴定Ph1缺失突变体,利用分子生物学及比较基因组学技术,该基因位点被界定于5BL上一个2.5 Mb的区域内,含有一个类cdk基因簇,且在该类cdk基因簇中插入一个亚端粒异染色质片段.细胞学研究显示,Ph1基因通过控制亚端粒的互作启始染色体识别和配对伙伴选择.与此同时,生物信息学揭示,这些类cdk基因与人类和老鼠的cdk2基因高度同源,它们与细胞周期中DNA复制、染色质凝集、碱基错配修复等事件相关.减数分裂时,该基因位点通过"感知"染色体的同源性程度而触发染色质的构象变化,从而控制染色体的配对和重组.此外,小麦中可能存在一种与Ph1相关的类似于酵母中的粗线期检查点机制.预测未来的研究将可能集中在Ph1对染色体同源性的"感知"机制、Ph1的开启与关闭、植物减数分裂重组的忠实性及减数分裂过程的检查点机制等方面.     

Pre-meiotic S phase is linked to reductional chromosome segregation and recombination

Meiosis is initiated from G1 of the cell cycle and is characterized by a pre-meiotic S phase followed by two successive nuclear divisions. The first of these, meiosis I, differs from mitosis in having a reductional pattern of chromosome segregation. Here we show that meiosis can be initiated from G2 in fission yeast cells by ectopically activating the meiosis-inducing network. The subsequent meiosis I occurs without a pre-meiotic S phase and with decreased recombination, and exhibits a mitotic pattern of equational chromosome segregation. The subsequent meiosis II results in random chromosome segregation. This behaviour is similar to that observed in cells lacking the meiotic cohesin Rec8 (refs 3, 4), which becomes associated with chromosomes at G1/S phase, including the inner centromere, a region that is probably critical for sister-centromere orientation. If the expression of Rec8 is delayed to S phase/G2, then the centromeres behave equationally. We propose that the presence of Rec8 in chromatin is required at the pre-meiotic S phase to construct centromeres that behave reductionally and chromosome arms capable of a high level of recombination, and that this explains why meiosis is initiated from G1 of the cell cycle.     

着丝粒生物学

着丝粒的关键作用是保证细胞减数分裂和有丝分裂的顺利进行，保证生物的遗传．近年来随着对多个物种的着丝粒测序之后对着丝粒的功能提出了很多相互矛盾的假说．本文阐述了低等真核生物的着丝粒没有重复序列而高等真核生物的着丝粒具有大量的重复序列，并且简述了各物种着丝粒的组成和各类与组蛋白H3、核仁、着丝粒DNA序列及DNA的高级结构相关的着丝粒功能模型．     

Hypervariable telomeric sequences from the human sex chromosomes are pseudoautosomal

Pairing of human X and Y chromosomes during meiosis initiates within the so-called pairing region at the telomeres or the chromosome short arms. Using DNA from the Y chromosome we found sequence homology in the pairing region of the human X and Y chromosomes. This DNA is telomeric, contains repetitive sequences and is highly polymorphic in the population. The polymorphism has allowed family studies which show the sequences are not inherited as though linked to the sex chromosomes. This 'pseudoautosomal' pattern of inheritance points to an obligate recombination in the pairing region of the sex chromosomes during male meiosis.     

Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.     

The conserved kinetochore protein shugoshin protects centromeric cohesion during meiosis

Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast. We also identify a homologue of Sgo1 in budding yeast. We provide evidence that shugoshin is widely conserved among eukaryotes. Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.     

Movement and segregation of kinetochores experimentally detached from mammalian chromosomes

The kinetochore is a specialized structure at the centromere of eukaryotic chromosomes that attaches chromosomes to the mitotic spindle. Recently, several lines of evidence have suggested that kinetochores may have more than a passive role in the movement of chromosomes during mitosis and meiosis. Kinetochores seem to attract and 'capture' microtubules that grow from the spindle poles and microtubules may lengthen or shorten by the addition or subtraction of tubulin subunits at their kinetochore-associated ends. An attractive hypothesis is that kinetochores function as 'self-contained engines running on a microtubule track'. Here, we show that kinetochores can be experimentally detached from chromosomes when caffeine is applied to Chinese hamster ovary cells that are arrested in the G1/S phase of the cell cycle. The detached kinetochore fragments can still interact with spindle microtubules and complete all the mitotic movements in the absence of other chromosomal components. As these cells enter mitosis before DNA synthesis is completed, chromosome replication need not be a prerequisite for the pairing, alignment and segregation of kinetochores.     

Genetic evidence that a Y-linked gene in man is homologous to a gene on the X chromosome

The mammalian sex chromosomes are thought to be related to each other by sharing a common origin. That is, the X and Y chromosomes originally evolved from a pair of chromosomes that only differed at the locus determining sexual differentiation. For example, this evolutionary relationship is reflected during meiosis in chromosomal pairing between the tip of the human X chromosome short arm and the Y chromosome which presumably implies sequence homology. However, compelling genetic evidence for functional homology between the mammalian X and Y chromosome is lacking. We describe here the localization of a gene to the tip of the short arm of the human X chromosome and evidence for a related gene on the Y chromosome.     

Rec8 phosphorylation and recombination promote the step-wise loss of cohesins in meiosis

During meiosis, cohesins--protein complexes that hold sister chromatids together--are lost from chromosomes in a step-wise manner. Loss of cohesins from chromosome arms is necessary for homologous chromosomes to segregate during meiosis I. Retention of cohesins around centromeres until meiosis II is required for the accurate segregation of sister chromatids. Here we show that phosphorylation of the cohesin subunit Rec8 contributes to step-wise cohesin removal. Our data further implicate two other key regulators of meiotic chromosome segregation, the cohesin protector Sgo1 and meiotic recombination in bringing about the step-wise loss of cohesins and thus the establishment of the meiotic chromosome segregation pattern. Understanding the interplay between these processes should provide insight into the events underlying meiotic chromosome mis-segregation, the leading cause of miscarriages and mental retardation in humans.     

Centromere inactivation in a dicentric rice chromosome during sexual reproduction

T0135 is a variant selected from the progeny of a rice line telotrisomic for the short arm of chromosome 11 （2n＋IIS＇）. Fluores- cent in situ hybridization （FISH） results indicated that T0135 contained two telocentric chromosomes, which have two centro- mere-specific molecular markers （5S rDNA） for chromosome 11; thus T0135 is a newly-described rice chromosome variant with two dicentric chromosomes, named 22＋11L-＋11L＇＋I IS.11S-＋I 1S-11S. （22 represents the 22 chromosomes excluding chromo- some 11 in the rice genome, ＂-＂ represents the centromere）. To investigate the genetic stability of the rice dicentric chromosomes during sexual reproduction, we observed the chromosome types in the progeny. Ninety-four percent of the progeny had the same chromosome type as the parental line. This result indicates that the dicentric chromosomes are mostly stable during mitosis and meiosis. Immunofluorescence analysis for centromere specific histone H3 （CENH3） revealed that only one centromere is active and the other centromere is inactivated in the rice dicentric chromosomes.     

The inhibiting effect of 1·4 recombinant P chromosome of wheat-<Emphasis Type="Italic">Agropyron cristatum</Emphasis> addition line on the <Emphasis Type="Italic">Ph</Emphasis> gene

P chromosomes may carry a genetic system that inhibits the Ph gene in wheat. Abnormal chromosome synapsis in wheat-Agropyron cristatum addition line II-21-2 (additional 1·4 recombinant P chromosome) was observed in this study. The results of cytogenetics and Ph1 gene amplification showed that the Ph1 gene was normal and the average number of quadrivalents or hexavalents was determined to be 0.41 and 0.13, respectively, in pollen-mother cells of wheat-Agropyron cristatum addition line II-21-2. The analysis o...     

Molecular cytogenetic analysis of Triticum aestivum-Leymus racemosus reciprocal chromosomal translocation T7DS·5LrL/T5LrS·7DL

In order to induce chromosome translocation between wheat chromosomes and chromosome 5Lr of Leymus racemosus, the mi- crosporocytes during meiosis of T. aestivum-L. racemosus disomic addition line DA5Lr were irradiated by 60Co γ-rays 800 R (100 R/min). Before flowering, the treated spikes were emasculated and bagged. After 2-3 d, the emasculated flowerets were pollinated using pollens from T. aestivum cv. Chinese Spring. One plant with two translocation chromosomes involved in both the long and short arm of...     

Shugoshin collaborates with protein phosphatase 2A to protect cohesin

Sister chromatid cohesion, mediated by a complex called cohesin, is crucial--particularly at centromeres--for proper chromosome segregation in mitosis and meiosis. In animal mitotic cells, phosphorylation of cohesin promotes its dissociation from chromosomes, but centromeric cohesin is protected by shugoshin until kinetochores are properly captured by the spindle microtubules. However, the mechanism of shugoshin-dependent protection of cohesin is unknown. Here we find a specific subtype of serine/threonine protein phosphatase 2A (PP2A) associating with human shugoshin. PP2A colocalizes with shugoshin at centromeres and is required for centromeric protection. Purified shugoshin complex has an ability to reverse the phosphorylation of cohesin in vitro, suggesting that dephosphorylation of cohesin is the mechanism of protection at centromeres. Meiotic shugoshin of fission yeast also associates with PP2A, with both proteins collaboratively protecting Rec8-containing cohesin at centromeres. Thus, we have revealed a conserved mechanism of centromeric protection of eukaryotic chromosomes in mitosis and meiosis.     

Heterochromatin links to centromeric protection by recruiting shugoshin

The centromere of a chromosome is composed mainly of two domains, a kinetochore assembling core centromere and peri-centromeric heterochromatin regions. The crucial role of centromeric heterochromatin is still unknown, because even in simpler unicellular organisms such as the fission yeast Schizosaccharomyces pombe, the heterochromatin protein Swi6 (HP1 homologue) has several functions at centromeres, including silencing gene expression and recombination, enriching cohesin, promoting kinetochore assembly, and, ultimately, preventing erroneous microtubule attachment to the kinetochores. Here we show that the requirement of heterochromatin for mitotic chromosome segregation is largely replaced by forcibly enriching cohesin at centromeres in fission yeast. However, this enrichment of cohesin is not sufficient to replace the meiotic requirement for heterochromatin. We find that the heterochromatin protein Swi6 associates directly with meiosis-specific shugoshin Sgo1, a protector of cohesin at centromeres. A point mutation of Sgo1 (V242E), which abolishes the interaction with Swi6, impairs the centromeric localization and function of Sgo1. The forced centromeric localization of Sgo1 restores proper meiotic chromosome segregation in swi6 cells. We also show that the direct link between HP1 and shugoshin is conserved in human cells. Taken together, our findings suggest that the recruitment of shugoshin is the important primary role for centromeric heterochromatin in ensuring eukaryotic chromosome segregation.     

Cohesin Rec8 is required for reductional chromosome segregation at meiosis.

When cells exit from mitotic cell division, their sister chromatids lose cohesion and separate to opposite poles of the dividing cell, resulting in equational chromosome segregation. In contrast, the reductional segregation of the first stage of meiotic cell division (meiosis I) requires that sister chromatids remain associated through their centromeres and move together to the same pole. Centromeric cohesion is lost as cells exit from meiosis II and sister chromatids can then separate. The fission yeast cohesin protein Rec8 is specific to and required for meiosis. Here we show that Rec8 appears in the centromeres and adjacent chromosome arms during the pre-meiotic S phase. Centromeric Rec8 persists throughout meiosis I and disappears at anaphase of meiosis II. When the rec8 gene is deleted, sister chromatids separate at meiosis I, resulting in equational rather than reductional chromosome segregation. We propose that the persistence of Rec8 at centromeres during meiosis I maintains sister-chromatid cohesion, and that its presence in the centromere-adjacent regions orients the kinetochores so that sister chromatids move to the same pole. This results in the reductional pattern of chromosome segregation necessary to reduce a diploid zygote to haploid gametes.     

三种国外冷杉的核型研究

首次报道了３种国外冷杉的核型。ＡｂｉｅｓｃｅｐｈａｌｏｎｉｃａＬｏｕｄ。有９对中部着丝粒和３对近中着丝粒染色体,Ａ．ｎｕｍｉｄｉｃａＤｅＬａｎｎｏｎｅｘＣａｒｒ．文中还讨论了３种冷杉及其氯不组的进化地位。     

Microtubule-motor activity of a yeast centromere-binding protein complex.

During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.     

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A (ref. 2). A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH3 (refs 3, 4). The structural basis of this specification is of particular interest. Yeast Scm3 and human HJURP are conserved non-histone proteins that interact physically with the (CenH3-H4)(2) heterotetramer and are required for the deposition of CenH3 at centromeres in vivo. Here we have elucidated the structural basis for recognition of budding yeast (Saccharomyces cerevisiae) CenH3 (called Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 in complex with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved amino terminus and a shorter α-helix at the carboxy terminus of Scm3(CBD) wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3(CBD) induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.