Nucleolar dominance does not occur in root tip cells of allotetraploid Brassica species.

Using in situ hybridization and silver staining methods, the numbers of active and inactive rDNA loci have been established for three allotetraploid species of Brassica (B. napus, B. carinata, and B. juncea) and their diploid ancestors (B. campestris, B. nigra, and B. oleracea). The allotetraploid species have chromosome numbers equal to the sum of the numbers in their diploid relatives, but have fewer rDNA loci. All species investigated have lower numbers of active NORs (AgNORs, nucleolar organizer regions) compared with the numbers of rDNA sites revealed by in situ hybridization. The number of active rDNA loci of the allotetraploid species is equal to the number of AgNORs in their diploid ancestors, indicating the absence of nucleolar dominance in amphidiploid Brassica species, at least in root meristematic cells.     

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。     

rDNA site number polymorphism and NOR inactivation in natural populations ofAllium schoenoprasum

Nucleolar-organiser activity has been studied by silver staining and byin situ hybridization with an rDNA probe in two populations ofAllium schoenoprasum. One population is monomorphic with NORs and rDNA sites terminal on the short arm of pair 8 in all individuals. The other populations is monomorphic for pair 8 NORs but is also polymorphic for NORs on the long arm of pair 7. All plants in this population carry ribosomal cistrons on both chromosomes of pair 7 but 0, 1 or 2 of these sites can be active in rRNA synthesis. Cis-acting nucleolar-suppression affects the pair 7 locus. We suggest that there has been progressive reduction in the number of NORs during the evolution ofA. schoenoprasum.     

植物rRNA基因组织模式的荧光原位杂交比较分析

采用荧光原位杂交技术,对分属5个科的10种植物的分生细胞的18S-25S rRNA基因(45S rDNA)的组织模式进行了比较分析.45S rDNA探针在所有供试植物的间期核都产生了两种杂交信号:荧光强、位于核仁周边的纽和荧光较弱分布于核仁内的点,表明不同植物间期核的rDNA染色质的组织模式相似.在每种植物的部分间期细胞都观察到点与纽相连或从纽发出的情况,而且点的数目越多纽就变得越小,点的有无和数目的多少与细胞的活性呈正相关.这些事实表明,纽代表了处于凝缩状态的非活性的rDNA染色质,点是由纽解凝缩而来,rDNA异染色质解凝缩形成点是植物rRNA基因活跃转录的细胞学表现,在同一物种中点的多少代表了间期核rDNA转录活性的强弱.我们的结果支持点是核仁内活性rRNA基因组织的结构单位及rRNA合成发生地点的推论.我们的结果还显示,不同植物间期核的rDNA染色质的组织也存在一些差异,其中核仁内点的最大数目有较大的不同.在所有供试植物的有丝分裂前中期细胞,45S rDNA探针在rDNA位点都产生了松散的信号块和许多点,表明植物的rDNA位点在有丝分裂前中期还有较活跃的转录.     

Evolution of 5S rDNA units and their chromosomal localization in Allium cepa and Allium schoenoprasum revealed by microdissection and FISH

Allium cepa and Allium schoenoprasum each possess 5S rDNA units of two different sizes. The evolution of the two repeat units and their chromosomal localization were investigated. A. cepa has 5S rDNA loci in the proximal and distal regions of the short arm of chromosome 7. When the proximal and distal segments of the short arm of chromosome 7 were microdissected separately, and used as templates for PCR, the short and long 5S rDNA fragments were amplified predominantly from the proximal and distal segments, respectively. The nucleotide sequence of the long 5S rDNA unit resulted from partial duplication of a non-transcribed spacer (NTS) and the insertion of a unique sequence. FISH using a probe consisting of the unique sequence demonstrated that the long unit was distally localized. In A. cepa, the long 5S rDNA unit is only present distally and the short unit is predominantly located proximally on the short arm of chromosome 7. In A. schoenoprasum, the NTSs of the two different-sized 5S rDNAs had quite different sequences. The two 5S rDNA loci were localized very close together in the interstitial region of chromosome 6. FISH, using long and short 5S rDNA unit probes with a competitor of a 120-bp sequence of the 5S rRNA gene, indicated that the long 5S rDNA unit was localized proximally and the short unit distally. Although the NTSs of the 5S rDNA of A. cepa and A. schoenoprasum had quite different nucleotide sequences, the long 5S rDNA units of A. cepa and A. schoenoprasum share a common 75-bp sequence. This sequence might act in the formation of the long 5S rDNA unit in Allium species.     

Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae)

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region (NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic populations of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied, even those of distant islands. Diploid karyotypes (2 n = 16) always possessed two NOR-bearing chromosome pairs with pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluorochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands was noticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was correlated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as number and size of silver stained NORs, existed between cells and between individuals of the same population. The different size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only 5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich constitutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed between homologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 99–108.     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝（Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝（宽翅菘蓝,Isatis violascens Bunge）的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

Cyanamide mode of action during inhibition of onion (Allium cepa L.) root growth involves disturbances in cell division and cytoskeleton formation

Cyanamide is an allelochemical produced by hairy vetch (Vicia villosa Roth.). Its phyotoxic effect on plant growth was examined on roots of onion (Allium cepa L.) bulbs. Water solution of cyanamide (2-10 mM) restricted growth of onion roots in a dose-dependent manner. Treatment of onion roots with cyanamide resulted in a decrease in root growth rate accompanied by a decrease in accumulation of fresh and dry weight. The inhibitory effect of cyanamide was reversed by its removal from the environment, but full recovery was observed only for tissue treated with this chemical at low concentration (2-6 mM). Cytological observations of root tip cells suggest that disturbances in cell division may explain the strong cyanamide allelopathic activity. Moreover, in cyanamide-treated onion the following changes were detected: reduction of mitotic cells, inhibition of proliferation of meristematic cells and cell cycle, and modifications of cytoskeleton arrangement.     

Correlation between the size of the intergenic regulatory region, the status of cytosine methylation of rRNA genes and nucleolar expression in wheat

Summary A large number of wheat rRNA genes are methylated at all the CCGG sites that are present in the intergenic regions. A smaller number of rRNA genes are not methylated at one or more CCGG sites. A subset of genes was found unmethylated at a specific CCGG site just downstream of the array of 135 by A repeats in the intergenic region. In all the genotypes studied, the rDNA loci with larger intergenic regions between their genes also possess a greater number of rRNA genes that are unmethylated at one or more CCGG sites in the intergenic regions than do the loci with shorter intergenic regions. In four genotypes (for which data were available), rDNA loci with longer intergenic regions had larger secondary constrictions on metaphase chromosomes, a measure of relative locus activity, than the loci with shorter intergenic regions. The results have been integrated into a model for the control of rDNA expression based on correlations between cytosine methylation patterns and the number of upstream 135 by repeats in intergenic regions. According to this model the 135 by repeats play a part in the control of gene activity by binding a protein(s) that is in limiting supply, thereby predisposing the neighbouring gene to become active preferentially.     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝 （Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝(宽翅菘蓝,Isatis violascens Bunge)的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae

BACKGROUND AND AIMS: The Brassicaceae family encompasses numerous species of great agronomic importance, belonging to such genera, as Brassica, Raphanus, Sinapis and Armoracia. Many of them are characterized by extensive intraspecific diversity of phenotypes. The present study focuses on the polymorphism of number, appearance and chromosomal localization of ribosomal DNA (rDNA) sites and, when possible, in relation to polyploidy, in 42 accessions of Brassica species and ten accessions of Diplotaxis, Eruca, Raphanus and Sinapis species. METHODS: Chromosomal localization of ribosomal DNA was carried out using dual colour fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as probes on enzymatically digested root-tip meristematic cells. KEY RESULTS: Loci for 5S and 18S-5.8S-25S rDNA were determined for the first time in six taxa, and previously unreported rDNA constellations were described in an additional 12 accessions. FISH revealed frequent polymorphism in number, appearance and chromosomal localization of both 5S and 25S rDNA sites. This phenomenon was most commonly observed in the A genome of Brassica, where it involves exclusively pericentromeric sites of 5S and 25S rRNA genes. The intraspecific polymorphism was between subspecies/varieties or within a variety or cultivar (i.e. interindividual). CONCLUSIONS: The number of rDNA sites can differ up to 5-fold in species with the same chromosome number. In addition to the eight previously reported chromosomal types with ribosomal genes, three new variant types are described. The extent of polymorphism is genome dependent. Comparing the A, B and C genomes revealed the highest rDNA polymorphism in the A genome. The loci carrying presumably inactive ribosomal RNA genes are particularly prone to polymorphism. It can also be concluded that there is no obvious polyploidization-related tendency to reduce the number of ribosomal DNA loci in the allotetraploid species, when compared with their putative diploid progenitors. The observed differences are rather caused by the prevailing polymorphism within the diploids and allotetraploids. This would make it difficult to predict expected numbers of rDNA loci in natural polyploids.