Methylation-sensitive restriction endonuclease digestion patterns revealed in Vicia faba L. chromosomes by in situ nick-translation

Restriction endonucleases sensitive to cytosine methylation (HpaII, MspI and HhaI) and 5-azacitidine were used to study the localization of target sequences in Vicia faba metaphase chromosomes by in situ digestion and radioactive or non-radioactive nick-translation. In control experiments, neither isolated DNA nor chromosomes in situ were digested by HpaII and MspI. Pretreatment with demethylating agent, 5-azacitidine resulted both in increased effectiveness of in situ digestion and nick-translation. In 5-azacitidine-treated material, negative bands in M chromosomes appeared. HhaI cleaved isolated DNA, digested it in situ and gave positive signals as a result of nick-translation procedure in metaphase chromosomes. In S chromosomes containing heterochromatin without target sequences for HpaII and MspI, negative bands were shown after nick-translation. Such heterochromatin contains FokI sequences and in situ nick-translation driven by that restriction enzyme resulted in positive bands.     

Localization of the ribosomal RNA genes in Drosophila simulans

In situ hybridization of cloned rRNA genes from Drosophila melanogaster to D. simulans metaphase chromosomes shows that in the tested wild type strains both sex chromosomes contain a nucleolus organizer region. Silver grain counts support the published data that the X chromosomal rRNA gene number is significantly higher than the Y chromosomal.     

In situ hybridization with fluoresceinated DNA.

We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.     

Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.     

电镜原位杂交观察小鼠中期染色体内RNA分布特点及其来源

本文用RNase-gold标记法处理小鼠中期染色体切片,发现染色单体切面内部及周边有大量较均匀分布的RNA。以玉米18 s rRNA的cDNA及水稻5srRNA的DNA为探针,经电镜原位杂交观察到了小鼠18srRNA和5srRNA均在中期染色单体切面内部及边缘有分布,从而首次证明小鼠中期染色体RNA除来源于核仁外,还可以来源于核基质中的5srRNA,但这并非仅有的两种来源。     

The methylation peculiarities of pericentromeric heterochromatin of chromosomes 1,9 and 16 in human embryo

By means of in situ nick-translation technique, methylation patterns of pericentric heterochromatin of chromosomes 1, 9 and 16 in extraembryonic (chorion) and embryonic cells of 5-8 week old human fetuses with normal karyotype (5), and in one specimen with trisomy for chromosome 16 were studied. Fixed metaphase chromosomes from direct chromosome preparations were digested with either endonuclease Msp I or its isoshizomer Hpa II recognizing and restricting the same sDNA sequence C decreases CGC with Hpa II, but not Msp I sensitive to methylation state of internal cytosin. According to our results, heterochromatin of extraembryonic, but not embryonic cells is hypomethylated. An obvious difference was registered in signal strength between homologous regions in iq12 of both parental chromosomes 1 in early (5-6 week old), but not in more advanced fetuses. Methylation pattern difference was detected in pericentric chromatin of triple copies of chromosome 16 in extraembryonic tissues of the 47,XY, + 16 fetus. These results are in line with a hypothesis of intraheterochromatin location of "early" genes governing initial stages of embryonic development in humans.     

Locations of 18S and 28S ribosomal genes on the chromosomes of the Indian muntjac

The locations of genes coding for 18S and 28S ribosomal RNA have been mapped on metaphase chromosomes of the Indian muntjac M. muntjak by in situ hybridization with (3H)rRNA from the toad X. laevis. The results show that, in the muntjac, rDNA clusters are associated with the prominent secondary constrictions on the X and the Y1 chromos. In addition a cluster of rDNA is found near the tip of one arm on the longest pair of autosomes. The autosomal cluster of rDNAs usually does not express as a secondary constriction at metaphase.     

In situ hybridization of DNA sequences in human metaphase chromosomes visualized by an indirect fluorescent immunocytochemical procedure

In situ hybridization and immunocytochemical procedures are described which allow identification and localization of specific DNA sequences in human chromosomes by fluorescence microscopy. With this method the genes coding for 18S and 28S ribosomal RNA (rRNA) were localized on human metaphase chromosomes by in situ hybridization of 18S or 28S rRNA followed by an immunocytochemical incubation with specific anti-RNA-DNA hybrid antiserum. Visualization of the immunocytochemically localized RNA-DNA hybrids was achieved by indirect immuno-fluorescence. The antiserum against RNA-DNA hybrid molecules was raised in a rabbit injected with poly(rA)-poly(dT). The specificity of the sera was determined using a model system of Sephadex beads to which various nucleic acids had been coupled. To obtain optimal specific fluorescence and very low aspecific background staining, several modifications of the in situ hybridization and the immunocytochemical procedures were investigated. The use of aminoalkylsilane-treated glass slides, removal of unbound fluorochrome molecules from the fluorochromelabelled antibody solutions and application of a proteinase K treatment during the hybridization procedure and the immunocytochemical procedure proved to be essential for optimal results.     

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。     

Genetic and chromosomal localization of the 5S rDNA locus in sugar beet (Beta vulgaris L.).

A digoxigenin-labelled 5S rDNA probe containing the 5S rRNA gene and the adjacent intergenic spacer was used for in situ hybridization to metaphase and interphase chromosomes of a trisomic stock from sugar beet (Beta vulgaris L.). Three chromosomes of primary trisomic line IV (T. Butterfass. Z. Bot. 52: 46-77. 1964) revealed signals close to the centromeres. Polymorphisms of 5S rDNA repeats in a segregating population were used to map genetically the 5S rRNA genes within a cluster of markers in linkage group II of sugar beet. The concentration of genetic markers around the centromere presumably reflects the suppressed recombination frequency in centromeric regions. The correlation of physical and genetic data allowed the assignment of a linkage group to sugar beet chromosome IV according to line IV of the primary trisomics.     

Chromosome banding in Amphibia

The karyotypes of 14 species of Anura from 9 genera of the suborders Amphicoela, Aglossa, Opisthocoela and Anomocoela were analysed with various banding techniques and conventional cytogenetic methods. The 18S + 28S and 5S ribosomal RNA genes were localized by means of in situ hybridization. No Q-, R- and G-banding patterns in the euchromatic segments of the metaphase chromosomes could be demonstrated in any of the species; this does not seem to be caused by a higher degree of spiralization of the amphibian chromosomes, but by the special DNA organization in these organisms. In most karyotypes, constitutive heterochromatin is present at centromeres, telomeres and nucleolus organizer regions (NORs), but rarely in interstitial positions. The heterochromatic regions are either quinacrine positive and mithramycin negative or vice versa. All species examined possess only one homologous pair of NORs; these display the brightest mithramycin fluorescence in the karyotypes. Many specimens exhibited unequal labelling of the two NORs both after silver and mithramycin staining as well as after in situ hybridization with ³H-18S + 28S rRNA. In four species, between one and six chromosome pairs with homologous 5S rRNA sites could be identified. The 5S rRNA genes and the 18S + 28S rRNA genes are closely linked in two species. In the male meiosis of the Amphicoela and Opisthocoela, there are intersitial, subterminal and terminal chiasmata in the bivalents, whereas only terminal chiasmata are observed in the bivalents of the Aglossa and Anomocoela. No heteromorphic sex-specific chromosomes could be demonstrated in any of the species. The differential staining techniques revealed that the chromosomal structure in these four suborders is largely the same as in the highly evolved anuran suborders Procoela and Diplasiocoela.     

薄片牡蛎的核型及18S-28S核糖体rRNA基因的染色体定位

以薄片牡蛎(Dendostrea folium)成体鳃组织为材料制备有丝分裂中期染色体标本,对其染色体核型进行了分析,并运用荧光原位杂交技术(FISH)将18S-28S核糖体RNA基因定位于中期染色体上。FISH探针是通过PCR扩增介于18S-28S rRNA基因之间的ITS和5.8S rRNA基因序列,并在PCR扩增过程中掺入了Biotin-11-dUTP进行生物素标记。结果显示,薄片牡蛎的单倍染色体数目为n=10,全部为中部着丝粒染色体。与大多数已知巨蛎属牡蛎的染色体核型相似。ITS探针在薄片牡蛎中期分裂体相上产生两簇FISH信号,分别杂交于2号染色体短臂的近端粒区域。本研究首次报道了薄片牡蛎的中期染色体核型以及18S-28S核糖体RNA基因在染色体上的定位。     

Studies on RNA in the Metaphase Chromosome of Zea mays by Electron Microscopy in Situ Hybridization

In situ hybridization using biotinylated cDNA probes of 18S rRNA, 5S rRNA, tRNAfMet, tRNAcys, tRNAAsn was performed on ultra-thin sections of K4M-embedded maize root tip. After hybridization, the biotinylated hybrids were detected with avidin coupled to 10 nm gold particles and then examined under the electron microscopy. The results showed that 18S rRNA, 5S rRNA and tRNA all existed in the metaphase chromosomes at random. They were distributed not only in the interior of the chromosomes, but also in the periphery of the chromosomes. Three tRNAs and 5S rRNA in the chromosomes were equal in amount to that in the cytoplasm, but the amount of 18S rRNA in the chromosomes was much higher than that in the cytoplasm. These results indicated that a part of the RNAs in the chromosomes came from the nucleolus, while others came from the nucleoplasm.     

Radiolabelling of DNA/polypeptide complexes in isolated bulk DNA and in residual nuclear matrix DNA by nick-translation

Conditions are described that allow 32P-radiolabelling and detection of tight complexes between DNA and polypeptides by nick-translation. Prolonged nick-translation of purified bulk DNA results in radiolabelled complexes migrating on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 54 kd respectively. Residual nuclear matrix DNA which is not accessible to DNase I on the nuclear level becomes accessible to radiolabelling by nick-translation on the nuclear matrix level. In this case the in situ radiolabelled complexes migrate on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 100 kd. The DNA/polypeptide complexes are stable during treatments with SDS, beta-mercapto ethanol and alkali which points to covalent bonds between the polypeptides and DNA strands.     

Ag-NOR staining in the Bennett wallaby, Macropus rufogriseus: evidence for dosage compensation

Silver staining of cells in metaphase and interphase nuclei of both sexes of the Bennett wallaby, Macropus rufogriseus, has shown that (1) the nucleolus organizer region (NOR) is located only on the X chromosome (single Ag-NOR); (2) both X chromosomes in the female cells stain with silver; (3) the amounts of silver staining of metaphase chromosomes and interphase nuclei of both sexes are very similar; (4) the single X chromosome is hyperactive in male cells to equalize the expression of rRNA genes in the female cells with two X chromosomes; and (5) the mechanism of dosage compensation for rRNA genes in this species is similar to that reported for Drosophila salivary gland cells.     

Syrian hamster 5S rRNA genes are assigned to 6q2 with PNA-FISH by an R-banded karyotype

A karyotype for the Syrian hamster is proposed based on an R-banding pattern. R-bands were obtained by BrdU incorporation into the cells followed by a combined DAPI and propidium iodide staining of the fixed metaphase spreads. In situ hybridisation was performed with two biotinylated 18-mer PNA (peptide nucleic acid) probes complementary to sequences within the 5S rRNA gene. The 5S rRNA gene repeats map to chromosome 6q2. The present PNA-FISH procedure is an abbreviated and simpler version of that previously published.     

Guinea pig (Cavio cambayo) 5S rRNA genes map to 7q2, 20q2 and 30q2 shown by an R-banded karyotype with PNA-FISH

A karyotype for the guinea pig (Cavio cambayo) is proposed based on an R-banding pattern. R-bands were obtained by BrdU incorporation into the cells followed by a combined DAPI and propidium iodide staining of the fixed metaphase spreads. In situ hybridization was performed with two biotinylated 18-mer PNA (peptide nucleic acid) probes complementary to sequences within the 5S rRNA gene. The 5S rRNA gene repeats map to chromosomes 7q2. 20q2 and 30q2, respectively.     

Amplification of ribosomal genes and formation of extrachromosomal nucleoli in oocytes of starfish Henricia hayashi (Asteroidea: Echinasteridae)

DNA and RNA specific dyes, Ag-NOR staining and in situ hybridization were used for studying the nucleolar apparatus in the growing oocytes of Henricia hayashi (Asteroidea: Echinasteridae). A plasmid containing ribosomal genes of Drosophila melanogaster (Kolchinsky et al., 1980) labelled with 3H by nick-translation served as an rDNA probe. Multiple extrachromosomal nucleoli are formed by the cascade type as a result of growth and subsequent fragmentation of the chromosomal (primary) rDNA body and its derivative extrachromosomal (secondary) rDNA bodies. Ribosomal genes were shown in all nucleolar structures. Argentophilia of the primary and secondary DNA bodies appears to be due to the dense packing of the rDNA-containing material. Ag(+) NORs were detected in the extrachromosomal multiple nucleoli and NOR complexes. Amplification of rDNA is a highly probable conclusion from the existing data.