玉米rRNA基因的组织和表达模式

玉米(Zea mays)只有1对45S rDNA位点并在分裂期染色体形成次缢痕, 是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交(fluorescence in situ hybridization, FISH)、CPD(PI与DAPI组合)染色和银染技术, 分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号: 荧光强烈地位于核仁周边的纽, 而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出; 点的数目越多, 纽变得越小; 点的数目多少与细胞的活性呈正相关。研究结果表明, 纽代表了处于凝缩状态的非活性的rDNA染色质, 纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现; 不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示, 大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示, 同一间期细胞的2个同源rDNA位点的表达水平存在差异, 同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示, 早中期细胞的rDNA染色质相对解凝缩, 银染在所有早中期细胞和部分中期细胞显示了明显的核仁, 表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录, 其转录在晚中期才停止。     

玉米rRNA基因的组织和表达模式

玉米（Zea mays）只有1对45S rDNA位点并在分裂期染色体形成次缢痕,是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交（fluorescence in situ hybridization,FISH）、CPD（PI与DAPI组合）染色和银染技术,分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号：荧光强烈地位于核仁周边的纽,而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出;点的数目越多,纽变得越小;点的数目多少与细胞的活性呈正相关。研究结果表明,纽代表了处于凝缩状态的非活性的rDNA染色质,纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现;不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示,大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示,同一间期细胞的2个同源rDNA位点的表达水平存在差异,同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示,早中期细胞的rDNA染色质相对解凝缩,银染在所有早中期细胞和部分中期细胞显示了明显的核仁,表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录,其转录在晚中期才停止。     

遗传学实验

（十六）哺乳动物染色体的银染法实验原理 银染法（Ag-As技术）专染哺乳动物染色体上的核 仁组成区（nucleolar organizer region, NOR)。已知哺 乳动物中编码18S rRNA, 28S rRNA的基因（rDNA）是 位于特定染色体的核仁组成区。当用银染法染色时, rDNA能选择性地还原银,呈深黑色,染色深黑的可染 区即为核仁组成区（Ag-NORs )。可染区的数目反映了 有转录活性的核糖体RNA基因的数目。     

多囊卵巢病人淋巴细胞的rRNA基因活性

人类的rRNA基因位于5对近端着丝点染色体短臂的次缢痕上。间期细胞中,该区域转录45S rRNA并聚集形成核仁,因此把该区域称为核仁形成区(NOR)。在中期染色体制片上,核仁形成区常常靠近,这种现象叫做随体联合。一般认为随体联合是间期细胞核仁的残余。     

图像反卷积复原技术在玉米45S rDNA转录图式研究中的运用

由于成像焦平面外杂光的干扰,宽场荧光显微镜所得的图像往往较模糊,使其不适合用来观察植物细胞核内的精细结构。该实验讨论了反卷积软件中对图像复原结果有较大影响的几个重要参数的设置。经过合适的图像反卷积复原,由宽场荧光显微镜获取的玉米45S rRNA原位杂交信号图像得以清楚显示。对所得杂交图像的分析表明:玉米45S rDNA转录往往先发生在核仁外围,且随着核仁转录活性的提高逐渐向核仁内部扩散,并最终与5S rRNA一起,在核仁内部的空洞结构中形成成熟rRNA。研究结果显示,反卷积复原可有效提高宽场荧光显微镜所得二维图像的分辨率,从而使宽场荧光显微镜在植物细胞核内精细结构研究中发挥更多的作用。     

小鼠肝核仁体外转录的研究

真核细胞基因的表达及其调控是当前分子 生物学领域中极为重要的研究课题之一,尤其 是特定基因的表达与调控更加引人注意。真核 细胞的核仁是核搪体装配的场所,它含有多拷 贝的特定基因— 核糖体RNA基因（rDNA) 和专门负责转录rDNA的RNA聚合酶I,唯 一的基因产物是rRNA。因此,核仁及其染色 质是研究特定基因转录及其调节控制的一个理 想系统。Busch 等〔141于1964年第一次证明离 体核仁能在体外系统中合成rRNA。其后,不少 工作者分别用鼠肝细胞^[7,15]、蛙卵母细胞^[8] 、四 膜虫细胞^[5],以及多种人工培养细胞(Novikoff 肝癌细胞、Hela细胞、Ehrlich 腹水癌细胞 等^[1][2]的核仁及其染色质对rRNA的体外合 成进行了研究。我们以小鼠肝核仁为材料,研 究核仁染色质的结构与功能,试图寻找与rRNA 体外合成有关的某种调控蛋白,以便阐明真核 细胞核糖体基因表达的调控机理。     

肝癌患者外周血淋巴细胞的rDNA活性及其四维时空遗传意义初探

人类D组和G组染色体的随体茎部为18S和28S rRNA基因(即rDNA)的座位,该区亦称为核仁形成区。应用细胞化学上的银染法可以判断rDNA的活性。至今已经证明,银染物质是同rRNA转录有密切联系的蛋白质,因而这是一个判别该区有何功能而不是有没有形态结构的简便有效方法。周宪庭等报道,在中国人中,随着年龄增长,该基因活性有丢失的趋势。银染法也很快用在肿瘤遗传学的研究中。H.Hubbell和徐道觉发现,一些肿瘤的细胞株,其每细胞的Ag-NORs(表示银染后出现阳性的rRNA基因数目)与正常对照组相     

核仁是怎样行使其生理功能的

真核细胞间期核中最显著的细胞结构是核仁。在光学显微镜下,核仁通常呈均质而致密的球体;在电子显微镜下,核仁主要分为三个区,即.1)颗粒区,含有核糖体前体颗粒;2)致密的丝状区即纤维区,包含有正在转录的RNA分子;3)浅染色区,或称为核仁内染色质,该区包含有从染色体核仁组织者区来的DNA,其中含有rRNA基因。因而,从成份上说,核仁是由DNA、RNA和蛋白质组成的,它的主要功能是转录rRNA和装配核糖体亚单位。     

肝癌患者外周血淋巴细胞的rDNA活性及其四维时空遗传意义初探

人类D组和G组染色体的随体茎部为18S 和28S rRNA基因（即rDNA）的座位,该区 亦称为核仁形成区。应用细胞化学上的银染法 可以判断rDNA的活性。至今已经证明,银 染物质是同rRNA 转录有密切联系的蛋白质, 因而这是一个判别该区有何功能而不是有没有 形态结构的简便有效方法。周宪庭等田报道, 在中国人中,随着年龄增长,该基因活性有丢失 的趋势。银染法也很快用在肿瘤遗传学的研究 中。H. Hubbell和徐道觉［[41发现,一些肿瘤的 细胞株,其每细胞的纯-NORs（表示银染后出 现阳性的rRNA基因数目）与正常对照组相 接近,然而由于肿瘤细胞株的大多数细胞的染 色体数目大于46,故其D和G组的染色体数目 也超过正常者的10条,因此实际上有更多的 rDNA处于失活状态。D. A. Miller等【31运用 分子杂交、细胞杂交等方法得出,肿瘤细胞株 中不但含有更多的D和G组染色体,而且由于 纯-NOR基因并不比对照组有更多的失活,所以 肿瘤细胞中活性rDNA总数比正常细胞要多。 鉴于不同工作者对同一问题得出相互矛盾的结 果,因此有必要在病人的细胞中观察有活性的 rDNA数目究竟有没有变化,若有变化,则其可 能的意义又是什么？这便是本文探讨的目的。     

波兰小麦和矮兰麦45S rDNA和5S rDNA基因位点FISH分析

采用双色荧光原位杂交技术, 以45S rDNA和5S rDNA基因为探针, 对波兰小麦(Triticum polonicum L.)和矮兰麦(T. turgidum L. cv. Ailanmai)进行了分析。结果表明, 高秆波兰小麦(T. polonicum L. High)和矮兰麦的45S rDNA和5S rDNA基因位点高度一致, 都显示4个45S rDNA和6个5S rDNA基因位点; 矮秆波兰小麦(T. polonicum L. Dwarf)的45S rDNA基因位点与高秆波兰小麦和矮兰麦也一致表现出4个位点, 而其5S rDNA基因位点有8个。同时讨论了rDNA基因位点的数目和分布位置在种间和种内存在差异的原因。     

Abiotic stress and induced DNA hypomethylation cause interphase chromatin structural changes in rice rDNA loci

Global climate change, i.e. higher and more variable temperatures, and a gain in soil salinity are increasing plant stress with direct consequences on crop yield and quality levels. Rice productivity is strongly affected by abiotic stress conditions. The regulation of chromatin structure in response to environmental stress is poorly understood. We investigated the interphase chromatin organization from rice plants in non-stress versus stress conditions. We have used a cytogenetic approach, based on fluorescence in situ hybridization (FISH) with 45S, 5S rDNA and centromeric probes on rice tissue sections. The abiotic stress conditions included cold, heat and mild salinity and were applied during seed germination. In contrast to cold, saline and heat stresses caused extensive decondensation of 45S rDNA chromatin and also an increase in the distance between the 2 homologous 5S rDNA loci. 5-Azacytidine (5-AC), a DNA hypomethylating drug, greatly increased 45S rDNA chromatin decondensation and interestingly was able to induce polarization of centromeres in rice interphase nuclei. The abiotic stresses tested did not perturb the spatial position of centromeres, typically with circular arrangement around the nucleolus. The results suggest a role for chromatin plasticity in a world of climate changes.     

3D organization of interphase fibroblast nuclei in two closely related shrew species, Sorex granarius and S. araneus, differed by the structure of chromosome termini

To study 3D organization of fibroblast interphase nuclei in two sibling shrew species, Sorex araneus from Cordon race and S. granarius, FISH with probe to telomeric and rDNA repeats, and immunofluorescence with ANA CREST and antibodies to nucleolus protein B23 were used. Karyotypes of studied species are composed of near identical chromosomal arms and differ by the number of metacentrics and the structure of terminal chromosome regions. The large telomeres containing on the average 218 kbp of telomere repeats characterize the short arms in all of 32 S. granarius acrocentrics. Telomere repeats in them alternate with nbosomal repeats. These regions also contain active NORs. In contrast, active NORs in S. araneus are localized at the terminal regions of 8 chromosomal arms (Zhdanova et al., 2005, 2007b). We have shown that telomere associations of chromosomes and contacts of a part of telomere clusters with inner nuclear membrane and nucleolus characterize interphase nuclei of both S. granarius and S. araneus. Moreover, the partial colocalization of telomere and ribosomal clusters, and spatial nearness of centomeric and telomeric regions were revealed in the interphase nuclei of S. granarius. Evidently, only those ribosomal clusters that contain a number of active ribosomal genes display connection with nucleolus. The stripping of nucleolus materials during transition of fibroblasts to mitosis and the role of B23 protein in this process has been studied.     

Condensation of chromatin into chromosomes preserves an open configuration but alters the DNase I hypersensitive cleavage sites of the transcribed gene

DNase I was used to probe the molecular organization of the chicken ovalbumin (OV) gene and glyceraldehyde 3-phosphate dehydrogenase (GPD) gene in interphase nuclei and in metaphase chromosomes of cultured chicken lymphoblastoid cells (MSB-1 line). The OV gene was not transcribed in this cell line, whereas the GPD gene was constitutively expressed. The GPD gene was more sensitive to DNase I digestion than the OV gene in both interphase nuclei and metaphase chromosomes, as determined by Southern blotting and liquid hybridization techniques. In addition, we observed DNase I hypersensitive sites around the 5' region of the GPD gene. These hypersensitive sites were not always at the same locations between the interphase nuclei and metaphase chromosomes. Our results suggest that chromatin condensation and decondensation during cell cycle alters nuclease hypersensitive cleavage sites.     

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55–65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.