杨山牡丹×紫牡丹杂交后代“梦幻”的细胞遗传学

进行了杨山牡丹×紫牡丹杂交后代"梦幻"体细胞的细胞遗传学研究,结果表明该品种为混倍体.对其不同染色体数目的细胞进行核型分析,核型类别分别是3B,2B,2C,与父母本有很大的差异.用植物染色体去壁低渗制片法研究了"梦幻"大孢子母细胞减数分裂双线期-后期Ⅰ的分裂过程,结果显示:所有的细胞减数分裂行为均异常,在终变期-中期Ⅰ,分别出现了多价体、单价体、二价体提前分离、染色体胶连、赤道外染色体、染色体断裂等异常现象,这些异常导致了后期Ⅰ的不等数分离,二价体或多价体不分离,染色体胶连及出现大量的染色体断片,最终不能形成正常的雌配子体.此外,在体细胞中观察到的混倍现象在生殖细胞的减数分裂过程中也得到了进一步的证实,在终变期分别出现了5_Ⅱ+断片、4_Ⅱ+3_Ⅰ,5_Ⅱ+2_Ⅰ+断片、6_Ⅱ+3_Ⅰ+断片等染色体配对构型.     

雌核发育高背鲫成熟分裂前期的细胞学研究

雌核发育的高背鲫，成熟分裂前期共经历了细线期、偶线期、凝线期、粗线期和双线期５个发育阶段，显示出第一次减数分裂的基本特征．但也存在一些差异，表现在偶线期染色体可能没有正常配对，进入粗线期在等长上全面发生了分离，以致双线期没有交叉和二价体灯刷染色体形成．这些差异是前期的一种改变，是雌核发育高背鲫由第一减数分裂前期演变成没有同源染色体分离，而只有一次姐妹染色体分开的一种己有的成熟分裂机制     

一个G/G易位型Down氏综合征患者家系的染色体分析和随体联合的研究

本文报道了一个G/G易位型Down氏综合征患者的家系染色体分析和随体联合研究的情况.结果表明患者的双亲核型正常,患者异常染色体的来源可能是双亲之一的生殖细胞在减数分裂过程中,由于某些因素的影响,21号染色体发生了不分离,并且产生了21号同源染色体之间新的罗伯逊易位.统计患者双亲及其兄弟的随体联合频率,发现显著高于对照组正常人.为国外某些学者认为Down氏综合征患者双亲的随体联合频率高于一般人.提供了一个例证.     

黄牡丹花粉母细胞减数分裂过程的细胞遗传学观察

对黄牡丹花粉母细胞的减数分裂过程进行了全面的观察，发现在其减数分裂中存在诸如单价体、四价体、多价体、同源染色体联会不分离，后期Ⅰ～Ⅱ染色体桥及染色体断片、落后染色体、微核等一系列异常现象．统计了异常现象出现的频率；从细胞遗传学水平上分析了黄牡丹在野外结实率低与花粉母细胞减数分裂异常的关系．     

葎草单染色体的显微分离及DOP-PCR技术体系建立

葎草是具有XX/XY1Y2性染色体系统的雌雄异株植物,是研究植物性染色体演化的模式材料之一.利用染色体显微分离技术从葎草根尖有丝分裂中期分裂相中将单条染色体进行了显微分离及DOP-PCR(Degenerate oligonucleotide primer-PCR)扩增,并构建了单染色体DOP-PCR扩增产物的荧光探针,对葎草根尖有丝分裂中期分裂相染色体进行了荧光原位杂交,其结果表明荧光信号分布在所有的染色体上,表明所建立的技术体系能够成功分离葎草单染色体并进行DNA扩增.本研究结果为进一步进行葎草X,Y染色体的细胞及分子生物学研究提供了技术支持.     

电子显微镜分离小鼠染色体的研究

本实验把小白鼠的骨髓细胞，制成电镜染色体样品，探索了利用电子显微镜所产生的高度聚焦的电子束，将目标染色体或染色体片段与其他染色体或染色体片段分离、切割开的过程，检验了电子束分离、切割染色体的可行性的有效性，开发出了一套较为成熟的电子束分离、切割染色体的新技术或方法，使之成为染色体及基因研究的有效手段。     

加拿大披碱草×老芒麦杂种F_1加倍一代株系Ⅱ花粉母细胞减数分裂行为的观察

[目的]探讨植物远缘杂交不育的原因,以期为后期作物远缘杂交育种提供理论依据。[方法]利用常规的压片方法对加拿大披碱草×老芒麦杂种F1加倍一代株系Ⅱ花粉母细胞减数分裂过程中的染色体行为进行研究。[结果]结果发现加拿大披碱草×老芒麦杂种F1加倍一代株系Ⅱ花粉母细胞减数分裂过程中出现了一些异常分裂现象,即发现在加拿大披碱草×老芒麦杂种F1加倍一代株系Ⅱ花粉母细胞减数分裂过程中一些染色体行为不规则的现象,出现如:单价体、四价体、多价体、染色体桥及染色体断片、落后染色体、微核等一系列异常现象。[结论]加拿大披碱草×老芒麦杂种F1加倍一代株系Ⅱ花粉母细胞减数分裂过程高频率异常现象的发生直接导致其大部分花粉发育异常,表现出远缘杂种的不育。     

大葱(Allium fistulosum L.)染色体Giemsa C—带型的研究

本文用铁矾——苏木精染色法分析了大葱染色体组型。结果表明,大葱(2n=16)染色体是中部或近端看丝点,第6号染色休带有随体。根据染色体大小、着丝点位置、有无随体等特点排列了染色体组型图。用修改的Vosa(1972)Giemsa C—带方法研究了大葱的C—带带型。观察到全部染色体都显清楚的瑞带,个别染色体显中间带和着丝点带。第6号染色体显付缢痕带,整个随体被浓染, 大葱(A. fistulosum L.)是石蒜科的一年生或二年生蔬菜作物,原产于东方。目前它作为我国北方的重要蔬菜而大面积栽培。近几年国内外一些作者曾研究过葱属作物的染色体,但他们的工作都没有提到葱。本工作的目的是对大葱的染色体形态和Giemsa C—带带型作一次比较深入系统的研究,为育种工作和探讨亲缘关系提供必要的依据。现将我们的工作报告如下:     

减数分裂型黏连蛋白亚基SMC1β和STAG3在染色体运动中的功能启动时期研究

维持姐妹染色单体的黏着是保证染色体正确附着在纺锤体上,从而实现正确分离的关键。黏连蛋白在有丝分裂和减数分裂过程中对染色体结构和染色体分离具有重要作用。在迄今为止发现的四种减数分裂型黏连蛋白亚基RAD 21L、REC8、SMC1β和STAG3中,RAD 21L是具有配对同源染色体的主导蛋白质,在减数分裂前细线期同源染色体配对的启动过程中起着中心作用。为揭示SMC1β和STAG3在减数分裂期染色体运动中的作用起始点,观察了异位表达蛋白在体细胞中对染色体运动的影响。结果显示:与对照(未转染)细胞相比,异位表达的GFP标记SMC1β或STAG3对两个FISH荧光信号之间的距离没有显著影响,这两个FISH荧光信号代表核内一对X染色体或一对11染色体。研究结果表明:至少在减数分裂期同源染色体配对过程开始之前,SMC1β或STAG3都不履行各自的功能。     

中国西南特有植物云南金合欢的染色体数目报道

采用常规根尖压片法对云南金合欢（Acacia yunnanensis）进行了染色体研究。研究结果表明,云南金合欢为八倍体植物,染色体数目为2n=8x=112,属小型染色体。首次报道了云南金合欢的染色体数。     

细胞中心体的复制与调控

中心体复制异常与基因组不稳定存在相关性,并能引起某些肿瘤疾病发生。中心体复制与细胞周期事件相耦合,许多细胞周期调节蛋白和中心体蛋白调节着中心体的复制,中心体复制还受癌基因和抑癌基因及其表达产物的调节。研究中心体复制调控机制,对肿瘤的早期诊断和治疗有重要的作用。     

Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos

Polarity establishment requires a symmetry-breaking event, resulting in an axis along which determinants are segregated. In Caenorhabditis elegans, oocytes are apolar and are triggered to polarize rapidly along one axis after fertilization. The establishment of this first polarity axis is revealed by the asymmetric distribution of PAR proteins and cortical activity in the one-celled embryo. Current evidence suggests that the centrosome-pronucleus complex contributed by the sperm is involved in defining the polarization axis. Here we directly assess the contribution of the centrosome to polarity establishment by laser ablating the centrosome before and during polarization. We find that the centrosome is required to initiate polarity but not to maintain it. Initiation of polarity coincides with the proximity of the centrosome to the cortex and the assembly of pericentriolar material on the immature sperm centrosome. Depletion of microtubules or the microtubule nucleator gamma-tubulin did not affect polarity establishment. These results demonstrate that the centrosome provides an initiating signal that polarizes C. elegans embryos and indicate that this signalling event might be independent of the role of the centrosome as a microtubule nucleator.     

Asymmetric inheritance of centrosomally localized mRNAs during embryonic cleavages

During development, different cell fates are generated by cell-cell interactions or by the asymmetric distribution of patterning molecules. Asymmetric inheritance is known to occur either through directed transport along actin microfilaments into one daughter cell or through capture of determinants by a region of the cortex inherited by one daughter. Here we report a third mechanism of asymmetric inheritance in a mollusc embryo. Different messenger RNAs associate with centrosomes in different cells and are subsequently distributed asymmetrically during division. The segregated mRNAs are diffusely distributed in the cytoplasm and then localize, in a microtubule-dependent manner, to the pericentriolar matrix. During division, they dissociate from the core mitotic centrosome and move by means of actin filaments to the presumptive animal daughter cell cortex. In experimental cells with two interphase centrosomes, mRNAs accumulate on the correct centrosome, indicating that differences between centrosomes control mRNA targeting. Blocking the accumulation of mRNAs on the centrosome shows that this event is required for subsequent cortical localization. These events produce a complex pattern of mRNA localization, in which different messages distinguish groups of cells with the same birth order rank and similar developmental potentials.     

Abnormal expression of centrosome protein (centrin) in spermatozoa of male human infertility

To study the relations between male infertility and centrosome protein (centrin) and the functions of centrin in spermatogenesis, the matured spermatozoa of 10 normal male people and 18 male infertility patients were stained by immunofluorescence labeling antibody against centrin. The results showed that two fluorescence signal dots appeared in the normal male spermatozoa and were located at the base of flagellum. They are proximal centriole and distal centriole. However, in some spermatozoa of the male infertility, centrin protein was located abnormally at the base of flagellum and its staining signals were spread, the normal proximal and distal centrioles were confused and could not be recognized separately. These results suggest that abnormality of centrosome protein may be related to male infertility. This discovery may be used as a marker of abnormal sperm and male infertility.     

Identification of a novel resident centrosomal protein

One human autoimmune serum was identified to react with centrosomes by immunofluorescence. We applied the affinity purification of membrane-bound antibody technique and demonstrated that the antibodies present in this antiserum reacted with a 31/29 ku centrosomal antigen. Immunofluorescence showed that this antigen is located at centrosome in a cell-cycle independent manner, and thereby it belongs to the family of centrosomal residents. We then uti- lized this autoimmune serum and antibodies against centrin and gamma-tubulin to investigate changes of centrosome cycle kinetics during premature chromosome condensation (PCC) artificially induced in V79-8 cells. We show here that centrosomal proteins continue to express when cells are synchronized at G1/S boundary and S phase by Hydroxyurea (HU). During this time, the addition of caffeine causes cells with unreplicated genome to go into mitosis, and induces the separation of the replicated centrosomes. These results suggest that the coordination of DNA synthesis and centrosome replication in the normal cell cycle can be uncoupled. Cells ensure that centrosome duplicates once, and only once during each DNA synthesis cycle through the tight and subtle coordination of cell cycle engine molecules, and thereby the assembly of bipolar spindle and the accurate transmission of genetic information.     

Release of mature starfish oocytes from interphase arrest by microinjection of human centrosomes

Mature oocytes (unfertilized eggs) are arrested at definite cell-cycle stages which vary from species to species. In frogs and mammals, the oocytes are arrested at the second metaphase of meiosis whereas in echinoderms they are blocked later, at the pronucleus stage. What causes the maturing oocytes to stop at some point in the cell cycle is not entirely clear. In frogs, the metaphase arrest seems to be maintained by a cytostatic factor. In echinoderms, which stop at interphase, no such a factor has so far been found. The fertilization process, beyond the introduction of paternal chromosomes, releases the oocyte from cell-cycle arrest and provides a functional centrosome to replace the endogenous centrosome which is apparently lost during oogenesis in most species. Several lines of evidence suggest that release from cell-cycle arrest is mediated by a Ca2+ burst which is associated with fertilization, and it is known that the functional centrosome provided by the sperm is necessary for mitotic spindle formation and cleavages. We report here that microinjection of purified human centrosomes into mature starfish oocytes is sufficient to release them from arrest at interphase and to support many cleavages leading to the occasional formation of normal embryos. In this species centrosome induced re-entry into the cell cycle does not require a transient calcium burst nor does it require intact microtubules.     

Mechanism limiting centrosome duplication to once per cell cycle

The centrosome organizes the microtubule cytoskeleton and consists of a pair of centrioles surrounded by pericentriolar material. Cells begin the cell cycle with a single centrosome, which duplicates once before mitosis. During duplication, new centrioles grow orthogonally to existing ones and remain engaged (tightly opposed) with those centrioles until late mitosis or early G1 phase, when they become disengaged. The relationship between centriole engagement/disengagement and centriole duplication potential is not understood, and the mechanisms that control these processes are not known. Here we show that centriole disengagement requires the protease separase at anaphase, and that this disengagement licences centriole duplication in the next cell cycle. We describe an in vitro system using Xenopus egg extract and purified centrioles in which both centriole disengagement and centriole growth occur. Centriole disengagement at anaphase is independent of mitotic exit and Cdk2/cyclin E activity, but requires the anaphase-promoting complex and separase. In contrast to disengagement, new centriole growth occurs in interphase, is dependent on Cdk2/cyclin E, and requires previously disengaged centrioles. This suggests that re-duplication of centrioles within a cell cycle is prevented by centriole engagement itself. We propose that the 'once-only' control of centrosome duplication is achieved by temporally separating licensing in anaphase from growth of new centrioles during S phase. The involvement of separase in both centriole disengagement and sister chromatid separation would prevent premature centriole disengagement before anaphase onset, which can lead to multipolar spindles and genomic instability.     

Centrosome polarization delivers secretory granules to the immunological synapse

Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes--termed 'lytic granules'--at the immunological synapse formed between the CTL and the target. On contact with the target cell, the microtubule organizing centre of the CTL polarizes towards the target and granules move along microtubules in a minus-end direction towards the polarized microtubule organizing centre. However, the final steps of secretion have remained unclear. Here we show that CTLs do not require actin or plus-end microtubule motors for secretion, but instead the centrosome moves to and contacts the plasma membrane at the central supramolecular activation cluster of the immunological synapse. Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane. These data show that CTLs use a previously unreported mechanism for delivering secretory granules to the immunological synapse, with granule secretion controlled by centrosome delivery to the plasma membrane.     

Potential existence of two independent centrosometargeting domains in PP4

Protein phosphatase 4 (PP4) is an important member in the PPP family of protein Ser/Thr phosphatases. It has been proven to regulate a variety of cellular processes such as centrosome maturation, micro- tubule nucleation, splicesome assembly, and JNK pathway activation. Compared to the crystallized and structurally well defined phosphatase PP1 and PP2B, little is known about the structure of PP4. Besides the conserved motifs characteristic of the PPP family, no information is available on the other domains of PP4. PP4 is reported to localize to the centrosome in many species such as Drosophila, Caenor- habditis elegans and mammalian cells, which suggests a conserved role of PP4 in the regulation of centrosome function. Unlike several other centrosomal proteins, no sequence has been identified for PP4 that can target it to specific centrosomal localization. In this study, we used a combination of PCR mutagenesis and transient expression of GFP-tagged proteins in mammalian cells, and identified two PP4 centrosome-targeting domains of 68―136 and 134―220 aa. These two domains may be associated for appropriate localization to the centrosome. The findings are useful for further elucidating the func- tion of the domains and other structural characteristics of PP4.     

Cdc42 and mDia3 regulate microtubule attachment to kinetochores

During mitosis, the mitotic spindle, a bipolar structure composed of microtubules (MTs) and associated motor proteins, segregates sister chromatids to daughter cells. Initially some MTs emanating from one centrosome attach to the kinetochore at the centromere of one of the duplicated chromosomes. This attachment allows rapid poleward movement of the bound chromosome. Subsequent attachment of the sister kinetochore to MTs growing from the other centrosome results in the bi-orientation of the chromosome, in which interactions between kinetochores and the plus ends of MTs are formed and stabilized. These processes ensure alignment of chromosomes during metaphase and their correct segregation during anaphase. Although many proteins constituting the kinetochore have been identified and extensively studied, the signalling responsible for MT capture and stabilization is unclear. Small GTPases of the Rho family regulate cell morphogenesis by organizing the actin cytoskeleton and regulating MT alignment and stabilization. We now show that one member of this family, Cdc42, and its effector, mDia3, regulate MT attachment to kinetochores.