一种改进的植物染色体Ag—NOR染色方法及其应用

自1975年,Howell等和Goodpasture等报道人类染色体的Ag-NOR(银染核仁组成区)染色技术以来,银染色技术已广泛用于人类和哺乳动物染色体的核仁、核仁组成区、中心粒、着丝点、染色体轴心以及联会复合体等结构和行为的研究。但银染技术在植物     

维生素A酸诱导HL-60细胞分化后银染核仁形成区的研究

用Ag-NOR、Brachet反应和作者改进的悬浮培养细胞Ag-aNOR的TEM技术等,综合地对RA诱导HL-60细胞分化后银染核仁形成区的变化进行了研究。实验结果表明,银染NOR的均值在给药组与对照组细胞中差异不显著。对间期细胞进行Ag-aNOR反应,结果??表明其数量在给药组比对照组明显减少,同时发现Brachet反应显示富含rRNA的核仁数与Ag-aNOR数改变极为一致,提示虽然活性的rRNA基因的数量在RA诱导分化的HL-60细胞中不变,但其转录明显受抑,使rRNA合成减少,进而在细胞水平出现了恶性表达的核仁变得少而小、甚至消失的分化表型。HL-60细胞的Ag-aNOR电镜观察更进一步证明了这一结论。     

人类双生子的银染核仁形成区的研究

近二十年来,人们为了探讨遗传与环境在 产生表型中的相互作用,已在解剖、生理、生化、 心理、行为及病理状态等各方面对人类双生子 进行了详尽的研究。1975年Goodpasture等⁵⁾ 应用银染技术使近端着丝粒染色体的副缴痕, 即人类核仁形成区（NOR）特异染色,并证卖 银染的位置是：RNA基因的位置。为了进一 步了解银染核仁形成区（Ag-NOR）和银染近 端着丝粒染色体联合（Ag-AA）的遗传特征, 我们用银染技术进行了双生子的Ag-NOR和 Ag-AA的研究。     

癌症患者淋巴细胞核仁形成区活性变异的研究

应用银染-G带复合显示方法研究了肺癌、胃癌、肠癌、乳腺癌患者的外周淋巴细胞核仁形成区(NOR)活性。与正常对照相比,肺癌第15号染色体的Ag-NOR频率及Ag-NOR总频率增加,乳腺癌第14号染色体的Ag-NOR频率减少,胃癌第14号染色体Ag-NOR频率减少而第22号染色体的Ag-NOR频率增加。肠癌未见明显变异。结果提示不同部位的肿瘤具有不同的优势银染型,rRNA基因的表达可能存在肿瘤部位的特异性。     

正常造血细胞的核仁组成区计数定量研究

本文应用银染技术对正常造血细胞的核仁组成区进行了计数定量研究。结果显示,在粒系和红系中,随细胞的成熟,细胞中簇状ＡｇＮＯＲ的数量逐渐减少．而点状ＡｇＮＯＲ数量逐渐增多,无分裂能力的成熟细胞仅有少数点状ＡｇＮＯＲ。淋巴细胞中为一完全集结的银染颗粒,而巨核细胞内为各自分离的点状银染颗粒。本结果为正常造血细胞的核仁组成区提供了基础数值。     

大麦染色体银带的研究

本文以六棱、四棱和二棱大麦为材料,用去壁低渗火焰干燥法制备染色体标本,标本在60℃恒温下经70—80％AgNO_3水溶液处理12—14小时,可在核仁组成区、着丝粒和端粒出现黑色银染区。细胞化学反应说明这些不同区域的银染物质具有相同的性质。银染带型与C带型和N带型迥然不同,3种大麦的银染带型也有差别。试验还证明染色体标本制备技术对银染效果影响极大,只有合适的酶解和火焰干燥处理才能促使着丝粒和端粒显示出银染正反应。     

不良孕产史和自发性流产夫妇Dp~ 、Gp~ 染色体研究

应用细胞遗传学的方法对15例不良孕产史及自发性流产患者Dp~ 、Gp~ 染色体,和10例年龄相近的正常成年人染色体进行了研究,两组间随体联合(SAS),银染核仁形成区(Ag-NOR)及银染随体联合(Ag-AA)的结果表明,各组间频率差异无显著性意义。     

小麦根端分生组织细胞核中核仁通道结构的电镜观察

用常规电镜和整体银染电镜观察技术对小麦根端分生组织细胞核进行了研究。发展核仁与其周边染色质之间存在通道结构。初步分析认为,染色体NORs中的rDNA是通过该通道进入到核仁的纤维中心的。     

一种新的核仁组成区胶银染色技术

核仁组成区(Nucleolar OrganizerRegions,NORs)是位于细胞核仁内的DNA 环,具有 rRNA 基因,与 rRNA 的转录活性有关、在蛋白质合成中起重要的作用。遗传学家曾利用银染技术显示染色体核仁组成区来研究各种遗传缺陷性疾病。1986年 Ploton 建立丁一科改良的一步法胶银染色技术显示核仁组成区相关蛋白(Nucleoar Organizer Region Associa-ted Protein,AgNORs),虽然现在对这些蛋白染色的性质还不清楚,但该技术提供了一种快速检测核仁结构和活性改变的有效手段,已受到病理学界的高度重视,     

不良孕产史和自发性流产夫妇Dp、Gp 染色体研究

应用细胞遗传学的方法对巧例不良孕产史及自发性流产患者Dp⁺ 、Gp⁺ 染色体,和10例年龄相近的正常成年人染色体进行了研究,两组间随体联合（SAs),银染核仁形成区(Ag-NOR)助及银染随体联合（Ag-AA）的结果表明,各组间频率差异无显著性意义。     

The effect of pH on silver staining of nucleolus organizer regions

The effect of pH on silver staining of the nucleolus organizer regions (NORs) of human chromosomes has been investigated between pH 6.5 and 12.0. Nonvolatile mixtures of ethanolamine and ethanolammonium nitrate replaced the ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5 by the silver staining procedure of Howell and Black served as a standard; this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR staining was incomplete, and between pH 8.5 and 6.5 there was no NOR staining.     

The Effect of Ph on Silver Staining of Nucleolus Organizer Regions

The effect of pH on silver staining of the nucleolus organizer regions (NORs) of human chromosomes has been investigated between pH 6.5 and 12.0. Nonvolatile mixtures of ethanolamine and ethanolammonium nitrate replaced the ammonia of standard procedures. The optimal NOR staining obtained at pH 3.5 by the silver staining procedure of Howell and Black served as a standard; this procedure stained all ten NORs in 90% of mitoses. Similar NOR staining was found in 75% of mitoses stained at pH 11.7 or 11.8, but only in 10-15% of mitoses stained between pH 11.6 and 10.0. Between pH 10.0 and 9.0 NOR staining was incomplete, and between pH 8.5 and 6.5 there was no NOR staining.     

Ag staining of the nucleolus organizer (NO) and its relationship to satellite association

Summary The frequency of involvement in satellite association and the frequency of selective staining of the secondary constrictions with silver solutions have been studied in five phenotypically normal individuals, all carriers of morphological variants of the nucleolus organizing region (NOR). The results show the preferential involvement of some morphological markers in satellite association, and also their preferential staining with Ag-I. It has also been shown that acrocentric chromosomes involved in satellite association are always stained by silver.     

Detection of fibrillarin in nucleolar remnants and the nucleolar matrix

In order to gain further insights into the fundamental structure of the nucleolus, nucleolar remnants of Xenopus and chickens were examined for the presence of fibrillarin and nucleolus organizer region (NOR) silver staining. Nucleolar remnants of Xenopus nucleated red blood cells were found to contain easily detectable amounts of fibrillarin and NOR silver staining. Upon examination of various tissues, fibrillarin and NOR silver staining were detected in nucleoli of Xenopus liver hepatocytes and within nucleoli of oocytes and follicle cells from ovaries of mature female toads. By comparison, nucleolar remnants of adult chicken nucleated red blood cells contained only trace amounts of fibrillarin and NOR silver staining, whereas red blood cell nucleolar remnants of immature chicks had easily detectable amounts of fibrillarin and NOR silver staining. Nucleoli from hepatocytes of both adult and immature chickens demonstrated comparable levels of fibrillarin and NOR silver staining. Since fibrillarin was found in nucleolar remnant structures, we tested for (and detected) its presence in residual nucleoli of in situ nuclear matrix derived from HeLa cells. These findings are discussed in terms of the basic structural and functional organization of the nucleolus.     

Polymorphism of nucleolar organizer regions of chromosomes in human embryos

NOR activity in metaphase chromosomes from extraembryonic and embryonic tissues of 9-12 week human fetuses was studied after standard silver staining. Significant interidividual variations in the average cummulative NOR activity was assessed by means of one-factor dispersion analysis. No significant intertissue fluctuation of NOR activity was found. Total number of NOR+ chromosomes demonstrated no correlation with the embryonic age. Steady growth of an average cummulative NOR activity respective of progressive embryonic age was proven by correlation analysis method. Unequal participation of NOR-bearing chromosomes of D- and G-groups during early embryonic development in human was shown.     

Osteopontin is histochemically detected by the AgNOR acid-silver staining

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.     

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus)

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag-NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.     

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（4～24 h）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。     

Cytogenetic examination of the NOR activity in a proband with 13/13 translocation and in her relatives

Summary NOR activity in a proband with 13/13 translocation and in her relatives was examined by NOR silver impregnation and by determination of the association frequencies. In the proband, besides the fused chromosomes 13, also a chromosome 14 and a 15 showed no NOR staining. Therefore the possiblity could be ruled out that the loss of NORs was compensated by the activation of inactive NORs. However, in the proband, one chromosome 22 seemed to be more intensively stained by silver nitrate than in her parents. As in the proband, the association frequency remained constant because of an increased association tendency of chromosomes 22. The possibility is discussed that the loss of NORs was compensated by a higher NOR activity of one chromosome 22.Parts of this work are included in the doctoral thesis (M. D.) of S. H.