着丝点的结构与机能

本文根据近年来免疫化学,生物化学,细胞化学及超微结构技术的发展,对着丝点的结构与机能的研究进展进行了扼要的综述。特别是硬皮病(CREST)综合症病人血清中抗着丝点抗体的发现,以免疫化学的方法,检测出着丝点中含有不同分子量的蛋白,其机能也各有不同,随细胞周期的进展合成的着丝点蛋白也有不同。     

黑麦染色体银染的初步研究

本文对黑麦染色体的银染条件进行了探索,并对黑麦染色体上银染正反应分布区进行了研究。首次发现黑麦染色体的核仁组成中心区、着丝点和端粒均能用硝酸银染色。对此现象的原因进行了讨论。     

一例硬皮病人自发抗核仁抗体的免疫荧光及其与银染的比较研究

从硬皮病人血清中筛选出一例含有自发抗核仁抗体的血清,利用这个血清对核仁抗原的性质及其在细胞中随分裂周期不同产生的分布变化做了初步研究,并把结果与核仁嗜银蛋白做了比较。间接免疫荧光染色及细胞化学分析表明,这种核仁抗原的性质是蛋白质,其分布与嗜银蛋白相似,在间期,抗原呈颗粒状簇集在核仁中,而在分裂中期,抗原颗粒与染色体NORs部位接合,但有证据指出,这种抗原蛋白与核仁嗜银蛋白有所不同,同时还发现,经长时间秋水仙素处理诱导产生微核化的多核细胞中尽管微核的数目远多于细胞中NORs的数目,免疫荧光染色和银染都显示出每个微核中类核仁小体的存在。这说明(1)类核仁小体也是由核仁物质构成;(2)某些类核仁小体的产生可能与NORs无关。对这个现象的意义进行了讨论。     

辽宁产粗皮蛙的染色体组型研究(图版Ⅳ,下)

用骨髓细胞制片法分析了粗皮蛙的染色体组型 ,结果表明其二倍体染色体数为 2 6 ,可配成 13对 ,有5对大型染色体 (相对长度 >9)和 8对小型染色体 (相对长度 <6 5 ) ,其中 ,第 1、 5、 6、 7、 8对为中部着丝点染色体 ,第 12对为端部着丝点染色体 ,第 2、 3、 4、 9、 10、 11、 13对为亚中部着丝点染色体 ,第 4对为性染色体 ,属XY型 ,X染色体为亚中部着丝点 (相对长度为 10 70 ,臂比指数为 1 72 ) ,Y染色体为亚中部着丝点(相对长度为 12 83,臂比指数为 2 0 2 )。     

用免疫荧光技术研究细胞周期中核基质的分布变化

本文利用含有抗核基质自发抗体的硬皮人血清,以小鼠艾氏腹水癌细胞为材料,用间接免疫荧光染色的方法,追踪了对应核基质抗原在细胞周期中分布的变化。结果显示,在末期和间期之间存在一个核基质抗原从细胞质向细胞核内转移的过程。由于这一过程是通过核膜进行的,从而提示核基质结构可能有解聚和再聚合的行为。用酶化学结合间接免疫荧光染色的方法,初步研究了抗原的化学性质。染色形态的比较研究显示所用血清中可能含有不同于以前发现的、抗新的核基质抗原的自发抗体。     

染色体的着丝点命名系统

本文简要介绍了有关着丝点位置的四个命名系统,即二点二区、二点四区、四点四区和四点六区的命名系统,对生物教学和有关科研有一定参考价值。     

热休克诱导虹鳟四倍体

虹鳟卵受精后5—9小时期间,用热休克处理,12月龄时检查,四倍体出现频率为5％。较高的温度处理可导致卵的高死亡率。2n=60的虹鳟核型中,有中部和亚中部着丝点染色体22对,近端着丝点染色体1对,端部着丝点染色体7对,总臂数NF=104。4n=120的虹鳟四倍体核型中,有22套中部和亚中部着丝点染色体,1套近端着丝点染色体,和7套端部着丝点染色体,总臂数NF=208。未发现有染色体倍性镶嵌的个体。分析比较了二倍体和四倍体两类鱼的红细胞及其核的9个度量值(DNA相对含量,细胞及核的长轴、短轴,面积和体积),为应用红细胞鉴定四倍体虹鳟提供了倍性标准。在形态、解剖和生长速度方面未发现两类鱼有什么差别。     

黑麦染色体的银染研究

在对黑麦染色体银染过程的盐酸解离条件进行探索的同时,对黑麦染色体的银染正反应区进行了研究,首次发现经短时间空气干燥(4-24h)的黑麦染色体制片,随着盐酸解离强度的递增,分别出现了核仁组织区(NOR)、NOR和端粒以及NOR和着丝点的银染正反应,就此现象讨论了端粒和着丝点的银染机理。     

草鱼、中华鳖T淋巴细胞表面抗原的研究

以草鱼脑组织、中华鳖脑组织和胸腺细胞为抗原制备兔抗草鱼脑血清（RACBS）、兔抗顺华鳖脑血清（RATBS）和兔抗中华鳖胸腺细胞血清（RATTS）。补体依赖性细胞毒试验和不同组织对RATBS、RATTS的吸收试验结果表明：中华鳖胸腺细胞和脑组织均存在Thy1抗原（亦称脑组织抗原或胸腺-脑组织T细胞抗原）;草鱼脑组织缺乏Thy1抗原。应用间接酶标免疫组化染色技术显示：Thy1抗原阳性反应物沉淀于中华鳖胸腺细胞和外周一部分淋巴细胞表面,进一步用抗人白细胞分化抗原CD4、CD8单克隆抗体进行免疫组化交叉反应提示中华鳖淋巴细胞膜上含有与人类T淋巴细胞表面抗原CD4、CD8类似结构的物质。本文讨论了Thy1抗原、CD抗原的出现及其意义。探讨了淋巴细胞异质性及RATBS、RATTS的特异性和作用。     

制备肺炎衣原体抗原片检测血清抗体

目的:探索肺炎衣原体抗原片检测血清抗体法在诊断Cpn感染中的实际应用前景。方法:应用进口肺炎衣原体(Cpn)毒株感染Hep-2细胞,分别以瑞氏-姬母萨染色、吖啶橙染色和直接免疫荧光染色等3种方法鉴定Cpn感染细胞。纯化获取大量Cpn抗原,用于制备斑点抗原片。建立微量免疫荧光染色法(MIF)检测血清抗体,诊断Cpn感染。结果:Cpn感染Hep-2细胞的最适条件是用含1μg/mL放线菌酮的维持液,在35℃、5%CO2孵箱中培养7d,并在培养的第0、3、4、5天以2600r/min离心1h,感染成功率极高。染色反应显示,瑞氏-姬母萨染色可将Cpn包涵体染成蓝紫色或红紫色;吖啶橙染色则使Cpn感染的Hep-2细胞呈现鲜明的橘红色;免疫荧光抗体染色后,在Cpn感染细胞内可见亮苹果绿色包涵体。通过斑点抗原荧光抗体染色的方法抽样检测了100份病人血清中的Cpn抗体,其中抗Cpn-IgG抗体的阳性血清共61份,阳性率为61%。与Cpn-外周血单核细胞(Cpn-PBMC)抗原片比较,阳性检出率无明显差别。结论:用Cpn感染细胞制作的Cpn斑点抗原片可用于临床检测血清Cpn-IgG抗体,且具有特异性、敏感性高的特点,但要求检测人员有一定的经验。     

Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients

The specificity of the staining of CREST scleroderma patient serum was investigated by immunofluorescence and immunoelectron microscopy. The serum was found to stain the centromere region of mitotic chromosomes in many mammalian cell types by immunofluorescence. It also localized discrete spots in interphase nuclei which we have termed "presumptive kinetochores." The number of presumptive kinetochores per cell corresponds to the chromosome number in the cell lines observed. Use of the immunoperoxidase technique to localize the antisera on PtK2 cells at the electron microscopic level revealed the specificity of the sera for the trilaminar kinetochore disks on metaphase and anaphase chromosomes. Presumptive kinetochores in the interphase nuclei were also visible in the electron microscope as randomly arranged, darkly stained spheres averaging 0.22 micrometers in diameter. Preabsorption of the antisera was attended using microtubule protein, purified tubulin, actin, and microtubule-associated proteins. None of these proteins diminished the immunofluorescence staining of the sera, indicating that the antibody-specific antigen(s) is a previously unrecognized component of the kinetochore region. In some interphase cells observed by both immunofluorescence and immunoelectron microscopy, the presumptive kinetochores appeared as double rather than single spots. Analysis of results obtained using a microspectrophotometer to quantify DNA in individual cells double stained with scleroderma serum and the DNA fluorescent dye, propidium iodide, led to the conclusion that the presumptive kinetochores duplicate in G2 of the cell cycle.     

CENP—B的基因表达与细胞周期关系的研究

本文以HeLa细胞为材料研究一种着丝粒蛋白CENP-B的基因表达与细胞周期及细胞核骨架的关系。将HeLa细胞同步在不同周期时相,以流式细胞光度术、同位素掺入和ACA着丝粒染色等方法检测细胞同步化效果。我们分别提取了各周期时相细胞的总RNA和Poly(A)~ RNA,用Dot blot和Northern blot杂交方法研究CENP-B在细胞周期中的表达。结果表明,CENP-B基因在细胞周期中的各个时相均有表达,但表达的强度差别很大:G2期表达最强,S期最弱,G1期中的表达介于二者之间;有意义的是CENP-B基因在M期仍然有较强的表达,表现出其在细胞周期中表达的持续性;这种表达的持续性反映了一种可能性:着丝粒、动粒蛋白不断合成,但直到S期后进入G2期时着丝粒、动粒蛋白到一定临界浓度时才开始组装新的动粒。另外,着丝粒、动粒蛋白的持续合成对着丝粒、动粒功能的发挥可能是必需的。用Bam H I限制性内切酶消化处于不同细胞周期时相的HeLa细胞核骨架,提取与核骨架紧密结合的DNA,用~(32)P标记的cDNA为探针研究CENP-B基因与细胞核骨架的结合与其表达的关系。结果证明,在G2期细胞中CENP-B基因表达最强,与细胞核骨架结合最为紧密,G1期细胞中次之,S期中CENP-B基因与核骨架结合最弱,说明CENP-B基因与细胞核骨架结合的紧密度影响其表达强度。     

激光照射对中华大蟾蜍早期胚胎染色体的影响

应用YAG倍频激光器对中华大蟾蜍四细胞期胚胎进行照射,观察胚胎的生长、发育,并对激光照射致畸的胚胎在尾芽期用压片法进行染色体分析。实验结果表明：激光照射后正常的胚胎其染色体无变化,激光照射致畸的胚胎其染色体有丢失和加倍等变化。     

Serosal and mucosal facilitated transport of urea in urinary bladder of of Bufo bufo: evidence for an alleged water uptake

In Bufo bufo urinary bladder an urea facilitated transport has been localised on the luminal membrane. The transport fulfils the criteria for such a mechanism, i.e. is saturable and is inhibited by phloretin, a specific inhibitor for urea transport. Similarly to that of Bufo marinus and Rana esculenta the luminal membrane of Bufo bufo urinary bladder shows an ADH stimulated facilitated transport. Experiments wtih Amphotericin B, serosal phloretin (with and without ADH), have demonstrated the presence of a facilitated urea transport localised on basolateral membrane. Urea uptake on the isolated epithelial cells of Bufo bufo urinary bladder shows a characteristic feature, different from molecules passively transported such as glycerol yet inhibited by phloretin. Allegedly with urea, water flows in to the cells by a dragging or osmotic effect.     

中华大蟾蜍染色体“单位线”结构的研究

适量BrdU处理中华大蟾蜍外周血淋巴细胞,常规制片后可观察到直径恒定为0.4μm的染色质纤维——单位线结构。本实验表明两栖类染色体同样具有单位线结构,并发现组成染色体两单体的姐妹单位线相伴排列,且与中期染色体有明显的形态联系。通过分析显示染色体和单位线过渡变化的分裂相,认为染色体高级结构由单位线进一步螺旋化完成,从而支持Bak模型。     

Comparison of hyperosmotic shock-induced changes in kinetochore structure with chromosome motion in PtK1 cells

Summary Treatment of metaphase PtK₁ cells with 0.2 M to 0.5 M sucrose and anaphase cells with 0.5 M sucrose has previously been shown to stop chromosome motion probably due to a significant alteration in the functional attachment of kinetochore microtubules (kMTs) with the kinetochore lamina. The work presented here examines the effects of 0.15 M to 0.25 M sucrose on PtK₁ metaphase and anaphase cells with a focus on the ultrastructural changes in the kinetochore and rates of chromosome motion. Metaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose from 5 to 15 min showed spindle elongation with sister chromatids remaining at the metaphase plate; these cells failed to enter anaphase. Ultrastructural analysis revealed MTs did not insert directly into the kinetochore lamina but rather associated tangentially with an amorphous material proximal to the kinetochore region much like that described previously with higher concentrations of osmotica. Treatment of metaphase cells with 0.25 M sucrose arrested the cell in metaphase and ultrastructural analysis revealed novel osmiophilic spherical structures approximately 0.50 m in diameter located proximal to kinetochores. MTs appeared to stop just short of. or associate laterally with, these spherical structures. Anaphase PtK₁ cells treated with 0.15 M and 0.20 M sucrose showed reduced rates of chromosome segregation during 5 min treatments, suggesting they retained functional kinetochore/kMT interactions. However, treatment of anaphase cells with 0.25 M sucrose blocked anaphase A chromosome motion and produced electron dense spherical structures approximately 0.50 m in diameter, identical to those observed in similarly treated metaphase cells. Removal of 0.25 M sucrose in treated anaphase cells resulted in normal chromosome segregation within 1 min. Cells released from sucrose treatment showed the absence of spherical structures and reformation of normal kinetochore/MT interactions which was temporally correlated with the resumption of chromosome motion.Abbreviations DIC differential interference contrast - kMT(s) kinetochore microtubule(s) - MT(s) microtubule(s) - nkMT(s) non-kinetochore microtubule(s)     

Structure of kinetochore fibres in crane-fly spermatocytes after irradiation with an ultraviolet microbeam: neither microtubules nor actin filaments remain in the irradiated region

We studied chromosome movement after kinetochore microtubules were severed. Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region. After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving. We examined actin and/or microtubules in irradiated cells by means of confocal fluorescence microscopy or serial-section reconstructions from electron microscopy. For each cell thus examined, chromosome movement had been recorded continuously until the moment of fixation. Kinetochore microtubules were completely severed by the ultraviolet microbeam in cells in which chromosomes continued to move poleward after the irradiation: none were seen in the irradiated regions. Similarly, actin filaments normally present in kinetochore fibres were severed by the ultraviolet microbeam irradiations: the irradiated regions contained no actin filaments and only local spots of non-filamentous actin. There was no difference in irradiated regions when the associated chromosomes continued to move versus when they stopped moving. Thus, one cannot explain motion with severed kinetochore microtubules in terms of either microtubules or actin-filaments bridging the irradiated region. The data seem to negate current models for anaphase chromosome movement and support a model in which poleward chromosome movement results from forces generated within the spindle matrix that propel kinetochore fibres or kinetochore stubs poleward.     

Changes in prometaphase chromosome motion are dependent on experimentally induced changes in kinetochore structure.

Prometaphase PtK₁ cells are treated with low concentrations of sucrose in order to analyze its effects on kinetochore structure, microtubule (MT) associations with the developing kinetochore and chromosome congression. Prometaphase cells treated with 0.15M sucrose slows chromosome congression, yet chromosomes form a metaphase configuration. However, 0.2M sucrose treatment prevents chromosome congression and affects some of the kinetochore MT linkages with the kinetochore, resulting in loss of chromosome congression. We use time lapse video microscopy and ultrastructural analysis to correlate changes in the linkages in the kinetochore MTs and the kinetochore to explain these findings. It appears hyperosmotic shock treatment can produce non-functional linkages between kinetochore MTs and kinetochores such that chromosome congression is affected. When non-functional linkages are formed, the presence of both a corona and matrix-like material is also present, proximal to the kinetochore. The role of this material and its organization at the klnetochore is discussed in its relation to generating mitotic forces.     

Mechanisms of kinetochore‐microtubule attachment errors in mammalian oocytes

Proper kinetochore‐microtubule attachment is essential for correct chromosome segregation. Therefore, cells normally possess multiple mechanisms for the prevention of errors in kinetochore‐microtubule attachments and for selective stabilization of correct attachments. However, the oocyte, a cell that produces an egg through meiosis, exhibits a high frequency of errors in kinetochore‐microtubule attachments. These attachment errors predispose oocytes to chromosome segregation errors, resulting in aneuploidy in eggs. This review aims to provide possible explanations for the error‐prone nature of oocytes by examining key differences among other cell types in the mechanisms for the establishment of kinetochore‐microtubule attachments.     

CaMtw1, a member of the evolutionarily conserved Mis12 kinetochore protein family, is required for efficient inner kinetochore assembly in the pathogenic yeast Candida albicans

Proper assembly of the kinetochore, a multi-protein complex that mediates attachment of centromere DNA to spindle microtubules on each chromosome, is required for faithful chromosome segregation. Each previously characterized member of the Mis12/Mtw1 protein family is part of an essential subcomplex in the kinetochore. In this work, we identify and characterize CaMTW1, which encodes the homologue of the human Mis12 protein in the pathogenic budding yeast Candida albicans. Subcellular localization and chromatin immunoprecipitation assays confirmed CaMtw1 is a kinetochore protein. CaMtw1 is essential for viability. CaMtw1-depleted cells and cells in which CaMtw1 was inactivated with a temperature-sensitive mutation had reduced viability, accumulated at the G2/M stage of the cell cycle, and exhibited increased chromosome missegregation. CaMtw1 depletion also affected spindle length and alignment. Interestingly, in C. albicans, CaMtw1 and the centromeric histone, CaCse4, influence each other for kinetochore localization. In addition, CaMtw1 is required for efficient kinetochore recruitment of another inner kinetochore protein, the CENP-C homologue, CaMif2. Mis12/Mtw1 proteins have well-established roles in the recruitment and maintenance of outer kinetochore proteins. We propose that Mis12/Mtw1 proteins also have important co-dependent interactions with inner kinetochore proteins and that these interactions may increase the fidelity of kinetochore formation.