大蒜中期染色体内的核糖核蛋白物质

研究了大蒜（Ａｌｌｉｕｍ ｓａｔｉｖｕｍ Ｌ．）中期染色体的超微结构和ＲＮＰ物质。常规染色表明,大部分染色体内部有低电子密度区,有的染色体中低电子密度区域较大而似孔洞。银染结果也证明了有大小不等的孔洞存在。Ｂｅｒｎｈａｒｄ 染色显示,在染色体周边和染色体内部都有ＲＮＰ分布。用ＮａＯＨ 处理证明了Ｂｅｒｎｈａｒｄ 染色法所显示的深染区确实含有ＲＮＡ。ＲＮＰ量的多少与ＥＤＴＡ 的分化时间呈负相关     

小麦细胞核仁中DNA原位位置与rRNA基因转录位点的研究

以小麦细胞为研究材料, 应用常规电子显微镜技术和DNA细胞化学特异染色NAMA-Ur技术, 在原位水平对核仁中DNA的分布和特征进行了直观的观察。结果表明, 小麦细胞核仁中DNA位于纤维中心(Fibrillar Centers, FC)、致密纤维组分(Dense Fibrillar Component, DFC)以及两者的过渡区域, 并呈现出环绕FC排布的构型; 应用RNP优先染色(Benhard staining)技术分析了核仁中RNP的分布及其原位位置, 直观的显示了小麦细胞核仁中RNP颗粒主要集中在 FC与DFC的过渡区域及DFC和颗粒组分(Granular Component, GC)中; 并且在FC与DFC的过渡区域, 它们不太均匀也不太连续地半围绕着FC而排布; 进一步借助于RNA/DNA杂合体抗体在原位水平标记和分析了细胞核仁中活跃基因转录的精细位点, 结果表明小麦细胞核仁rRNA基因的转录位点位于FC与DFC的过渡区域及DFC中。     

小麦核仁在细胞周期中的变化

运用Bernhard染色方法研究了小麦根端分生组织细胞核仁在细胞周期中的变化。结果显示,间期核仁染色很深,能够区分出纤维中心（FC）、致密纤维组分（DFC）和颗粒组分（G）,而染色质被漂白,在染色质间可以观察到细小的RNP颗粒。进入前期,在染色质的边缘有小的RNP颗粒分布。中期,染色体周边分布着类似于间期核仁的深染的大RNP颗粒,形成一个不完全连续的“鞘”状结构;在染色体内部看不到类似核仁的深染颗粒。到了后期时,仍可见RNP“鞘”状结构的存在。进入末期,这些RNP植物逐渐由“鞘”脱离,最后参与新核仁的形成。这些结果表明,核仁解体后的物质直接转移到了中期染色的表面,并形成不连续的表层,没有进入染色体的内部。     

A feulgen-positive nucleolus

The Feulgen and Rossenbeck staining procedure reveals in all embryonic and adult cell types of the quail (Coturnix coturnix japonica) one or several chromatin condensations in the interphase nucleus. The Unna-Pappenheim technique, combined with RNAase treatment according to Brachet, shows that these chromatin masses are associated with the nucleolar RNA. Electron microscopic studies confirm this observation and the EDTA preferential staining procedure for RNP according to Bernhard makes it possible to distinguish three main types of nucleoli in the various tissues of the quail showing different patterns of RNA and DNA relationships. The functional significance of the large amount of nucleolus-associated chromatin in the quail, and of the more or less intimate relationships between RNA and DNA in the various types of nucleoli are discussed.     

Development of the synaptonemal complex and polycomplex formation in three species of grasshoppers

This paper describes the development of the synaptonemal complex in three species of grasshopper: Chorthippus bicolor, Oedipoda coerulescens and Paracinema bicolor. In all three cases the development seems similar. A typical synaptonemal complex is observed during pachytene. Diplotene bivalents show a low density material associated with the chromatin and during first metaphase the beginnings of polycomplex formations are seen. Well organized polycomplexes can be recognized from first anaphase to early spermatids. The elements of the polycomplexes, as well as elements of the synaptonemal complex, show themselves to be positive after preferential staining for ribonucleoproteins. Polycomplexes observed after spreading and positive staining present similar characteristics to those observed after sectioning.This paper is dedicated to the memory of W. Bernhard for his contribution to the knowledge of the cell nucleus     

An EM Study on the Nucleolar Residual in Meristematic Cells of Vicia faba

A special structure which originates from the nucleolus and persists during mitosis is observed in meristematic cells of Vicia faba, and is called nucleolar residual in this paper. The result of conventional staining showed that the nucleolar residual (NR) was a medium electron-dense structure that could be distinguished from chromosomes. At the end of prophase, the NR was observed in the dispersing nucleolar material, which consisted principally of granules and fibrils about 15 to 20 nm in diameter. In prometaphase, the NR, composed mainly of the similar granules and fibrils, was found attached to chromosomes. Results of Bernhard's technique for RNP (ribonucleoprotein) preferential staining indicated that the main chemical component of the NR was RNP, and sometimes bleached regions showing the same reaction as chromatin in Bernhard's staining appeared in the structure. In metaphase and anaphase some of the NRs were attached to chromosomes while others existed randomly in the cytoplasm. The NR is supposed, according to its cytochemical features and its behavi0ur during the nucleolar disintegration, to be a structure composed mainly of nucleolar matrix material or nucleolar skeleton.     

Correlation between isolated lampbrush chromosomes and nuclear structures of Pleurodeles waltl oocytes: An electron microscopic study

The organization of the lampbrush chromosomes of Pleurodeles waltl was studied by fixation and embedding of oocytes in toto and correlated with that observed in end-embedded preparations of isolated chromosomes. Particular attention was focused on marker loops, like the granular and globular loops, and atypical structures known as spheres (S) and M. In both types of preparations, the majority of the loops, the so-called normal loops, and the granular loops appeared to share a common basic organization, with ribonucleoprotein (RNP) fibrils appearing as strings of 30 nm particles, as described by earlier authors. Some new types of loop organization were observed: (i) P loops with 45 nm RNP particles; (ii) dense granular loops; (iii) loops with a cylindrical organization. RNP fibrils formed by 60 nm particles were found to occur in association with the globular loops. EDTA staining suggested the presence of large amounts of RNP in the sphere but very little in M. Three morphologically different types of RNP granules could be observed free in the nucleoplasm.     

蚕豆核仁残体的电镜研究

本文报道了有丝分裂过程中蚕豆(Vicia faba)根端分生组织细胞内一种由核仁物质组成的特殊结构,我们将其称作核仁残体。经常规染色后,可在前期末正在分散的核仁物质中看到由直径15—20nm 的颗粒和纤维组成的核仁残体;前中期时,核仁残体附着在染色体上,其电子密度低于染色体。Bernhard 染色结果表明,核仁残体的主要成分是核糖核蛋白(RNP)颗粒和纤维,一些核仁残体中存在着与染色质染色反应相似的被漂白区。有的核仁残体附着在中、后期染色体上,有的游离存在于细胞质中。本文讨论了核仁残体的成分及其本质等问题。     

The ultrastructure and possible role of the vacuolar component of hepatocyte nucleoli

A study was made of the nucleolar vacuoles in guinea pig hepatocytes that are poorly investigated for animal cells. A comparison of ultrathin sections, contrasted by heavy metals, with those treated according to Bernhard allowed to reveal the following intravacuolar structures: 1) fibrils 10-15 nm and 20-30 nm thick similar to perinucleolar chromatin fibrils; 2) RNP-granules 15-20 nm in diameter resembling the granular component of the nucleolus; 3) RNP-fibrils 15-20 nm thick with high electron density. The latter were visualized for the first time, their function still remains obscure. Upon stimulation of hepatocytes with partial hepatectomy, the vacuolar component changed. In 2.5 and 5 hours after the operation the vacuoles became smaller, the number of RNP structures of two types increased. Further, in 9 hours, the enlarging of vacuoles was accompanied by a sharp decrease in the number of these RNP-structures. The results obtained allow to suppose that the vacuoles of nucleolonemic nucleoli may be functioning elements, linking intra- and perinucleolar chromatin fibrils. They are depots for the RNP synthesized in the nucleolus; the rRNP is transported through the vacuolar system.     

Cytochemical aspects of the nucleolar organizer in Allium cepa microspores

In Allium cepa mierospores, the nucleolar organizing region appears as an area of low density situated between two nucleolar masses. It consists of a series of zones with a density similar to that of the chromatin surrounded by areas of lower density. The dense zones are sometimes arranged in an orderly pattern of 2–4 rows. The organizing region consists of filaments, about 100 Å in diameter, which are seen to be concentrated in the dense areas, and more scattered in the rest of the region. — The alcoholic PTA staining technique reveals the presence of an appreciable quantity of arginine-Iysine rich histones in the organizing region, as well as a similarity between the dense areas of this region and the rest of the chromatin: a similarity which is also brought out by the thallium alcoholate technique, used for DNA staining. By means of uranyl-BDTA-lead RNP material can be shown to be predominantly located in the low-density areas of the organizing region in the form of fibres of about 80 Å in diameter, presumably representing the newly synthetized r-RNA (45 S RNA). A pattern is suggested for the organizing region, in which areas of functional chromatin (euchromatin) would appear alternating with areas of non-functional chromatin (heterochromatin).     

Cytochemical studies on RNP complexes produced by puff 2-48BC in Drosophila hydei

Differential staining of the core and RNP particles of RNP complexes in puff 2–48 BC in salivary gland chromosomes of Drosophila hydei was achieved with aqueous uranyl acetate (UA) at low pH, with UA in acetone, with phosphotungstic acid (PTA) in organic solvents, and with aqueous PTA at pH 5 and 6. A comparison of the results of UA and PTA staining under various conditions indicate that the proteins in the core region and in the RNP particles connected to it differ with respect to their amino-acid composition (arginine and lysine residues). — The staining mechanism of PTA and UA is discussed.     

牛肾细胞染色体中染色质纤维的包装及RNP的分布

应用培养的牛肾（ＢＫ）细胞及分离的ＢＫ细胞染色体做常规透射电镜样品,经表面舒展技术和临界点干燥制备ＢＫ细胞染色体的扫描电镜样品,并结合电镜细胞化学研究了ＢＫ染色质纤维的包装及核糖核蛋白（ＲＮＰ）的分布。观察到ＢＫ染色体具有多级双股螺旋结构。在染色体横切面中,可见染色体中央有一低电子密度的无染色质区,该区内有大量ＲＮＰ物质。在染色质区ＲＮＰ较少,分布在染色质纤维间,与中央轴区的ＲＮＰ相连续,表明ＲＮＰ在染色体中呈不均匀分布。     

Characterization of proteins associated with nuclear ribonucleoprotein particles by two-dimensional polyacrylamide gel electrophoresis.

Rat liver nuclear ribonucleoprotein particles were prepared by two different methods and defined as 40S ribonucleoprotein (40S RNP) and heterogeneous nuclear ribonucleoprotein (HnRNP) particles. The RNP particles were either solubilized in 8 M urea--6 mM 2-mercaptoethanol--20 mM glycine--20 mM Tris--HCl (pH 8.4) or subjected to removal of RNA by phenol extraction prior to solubilizing the proteins in the urea buffer. The proteins associated with 40S RNP and HnRNP were heterogeneous and very similar in their electrophoretic patterns when analyzed by two-dimensional PAGE, except a protein with molecular weight of 62 000 and an isoelectric point (pI) of 6.2 was present only in HnRNP particles. At least 12 major and 22 minor components could be identified in both preparations. The major proteins were found at pI values varying from 6.0 to 8.5 and with molecular weights from 32 000 to 42 000, and a group of proteins with molecular weight approximately 65 000 were more prominent in HnRNP than in 40S RNP. The other components were found mainly at pI ranges from 5.0 to 6.5 with molecular weights from 43 000 to 65 000. The phenol method extracted essentially all proteins associated with either 40S RNP and HnRNP, but was less effective in extracting a group of proteins with pI values from 5.0 to 5.5 and more efficient for proteins with pI values from 7.5 to 8.5. When chromatin proteins isolated by phenol extraction were compared with HnRNP particle proteins isolated by the same method, the electrophoretic mobilities of the HnRNP particle proteins were found to be identical with a fraction nonhistone chromatin proteins. The 40S RNP particles were further purified by metrizamide isopycnic density gradient centrifugation. The electrophoretic patterns of these proteins were very similar to those prepared by sucrose density gradient centrifugation. Therefore, we concluded that the proteins of RNP particles constituted part of the chromatin proteins.     

Isolation and Properties of an RNA Polymerase from Influenza Virus-Infected Cells

Structures with RNA polymerase activity were isolated from influenza virus-infected cells, and consisted of ribonucleoprotein (RNP) complexes, similar in morphology to the viral internal component or nucleocapsid. The isolation procedure involved fractionation of infected cells in a discontinuous sucrose gradient, in which enzyme activity was concentrated in a fraction of intermediate density which contains both smooth and rough cytoplasmic membranes. The RNPs with polymerase activity were further purified in a velocity gradient, after which the peak fractions showed a 35-fold purification of the polymerase activity when compared with cytoplasmic extracts. The NP polypeptide, which is the subunit of the virion RNP, was the only virus-specific polypeptide detected in these RNP structures.     

Ribonucleoproteins containing mRNA in the cytoplasm of mouse plasmacytoma cells]

The rapidly labelled postribosomal ribonucleoprotein (RNP) found in the cytoplasm of mouse plasmacytoma cells were investigated. It has been shown that 45S and 80S particles contain relatively high molecular weight (approximately 12-17S) pulse-labelled RNA similar to the polyribosomal mRNA. No other postribosomal RNP was found which would contain an RNA with similar sedimentation characteristics. In CsC1 density gradients, the postribosomal RNP gives two peaks. One of them, the rapidly labelled component (rho 1.52 g/cm3) is found only in 45S RNP. The other rapidly labelled component (rho 1.36-1.41 g/cm3) is revealed in all investigated regions of sucrose gradients. The latter contains relatively low molecular weight RNA (approximately7-9S). These RNP are supposed to be informosome-like particles. The components with a buoyant density of 1.52 g/cm3 may represent an mRNP-45S subparticles complex. The rapidly labelled mRNA of 80S particles is released after EDTA treatment in the form of mRNP with a buoyant density of 1.45-1.47 g/cm3.     

Cytochemistry of nucleoproteids and some cathionic proteins in the peripheral blood leukocytes of patients with lung cancer.

In 40 patients with untreated lung cancer cytochemical studies of the peripheral blood leukocytes were conducted by means of a cytological method for the simultaneous staining of nucleoproteids (RNP and DNP) and some cathionic proteins (after Zvetkova and Zvetkov [60]). Changes were detected in the RNP cytoplasmic contents of lymphocytes, of which the most outstanding were the reduction and uneven distribution of RNP granules, their frequent extracellular expulsion by means of microclasmatoses, as well as changes in the staining of cathionic proteins of RNP accompanied by an increased nuclear chromatin condensation in the small and medium-sized lymphocytes. Parellel to reducing of the percentage of these cells in the peripheral blood of patients with advanced neoplastic disease an increased number of lymphoblastoid and monoblastoid cells is established with RNP diffusely stained, but reduced in quantity and localized in the cytoplasmic periphery and projections (compared to Downey type II atypical cells). By means of one of the variants of the method (modified type of Feulgen's reaction) a characteristic distribution and structuring of the nuclear chromatin is established in mono- and polymorphonuclear cells, most clearly expressed in the nuclei of monocytes and monoblastoid cells, as well as in nuclei of neutrophil granulocytes. In these cellular types a more specific nuclear modelling (microhypersegmentation) is observed resulting in multiple irregular nuclear projections on the nuclear surface, probably caused by subkaryolemal distribution of uneven chromatin thickenings. The changes are also recorded in the cathionic protein containing secondary cytoplasmic granules in granulocytes-neutrophils and eosinophils, probably associated with changes in the lysosomal and phagocytic functions of these cells in neoplastic diseases. The authors discuss the importance of the obtained results in connection with data on the participation of lymphocytes and neutrophils in the immune response to tumour antigenic stimuli during the course of the neoplastic process, as well as with data on the suppressive effect of antigenic (serum, viral) factors, possibly affecting the synthesis and the transport of cellular nucleoproteids (RNP and DNP) in leukocytes of cancer patients.     

关于蚕豆根端分生组织细胞核中类胀泡结构的研究

本文对蚕豆根端分生组织细胞核中类胀泡结构的超微结构变化和细胞化学特点进行了研究。我们观察到,类胀泡结构是与集缩染色质紧密相连的核内结构。该结构是由直径30nm左右的纤维组成的较为疏松的网络,其中的30nm纤维可以进一步解集缩并释放出直径约10nm的颗粒,这些颗粒可能是核小体。Bernhard染色结果表明,类胀泡结构含有RNP。放射自显影结果表明,类胀泡结构具有转录活性。我们推测,蚕豆细胞核中的类胀泡结构可能是非核仁基因表达的一种形态。     

Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine antibody

A commercially available bromodeoxyuridine (BrdUrd) antibody was used to demonstrate sister chromatid differentiation (SCD) and to evaluate sister chromatid exchanges (SCEs) in V79 Chinese hamster cells. V79 cells were cultivated for one cell cycle in the presence of BrdUrd, followed by a second cell cycle in the absence of BrdUrd. Chromosome preparations were stained by a common immunologic staining technique. The staining pattern observed is similar to that after FPG (fluorescent plus Giemsa) staining, though with reverse staining specificity. The sensitivity of BrdUrd detection is enhanced by a factor of 20 compared to the FPG technique and thus allows the evaluation of SCEs at very low BrdUrd concentrations. The application of the antibody technique gives information about the origin and localization of SCEs and produces further evidence for the spontaneous occurrence of SCEs.