Karyotyping of Brassica oleracea L. based on rDNA and Cot-1 DNA fluorescence in situ hybridization

To explore an effective and reliable karyotyping method in Brassica crop plants,Cot-1 DNA was isolated from Brassica oleracea genome,labeled as probe with Biotin-Nick Translation Mix kit,in situ hybridized to mitotic spreads,and where specific fluorescent bands showed on each chromosome pair.25S and 5S rDNA were labeled as probes with DIG-Nick Translation Mix kit and Biotin-Nick Translation Mix kit,respectively,in situ hybridized to mitotic preparations,where 25S rDNA could be detected on two chromosome pairs and 5S rDNA on only one.Cot-1 DNA contains rDNA and chromosome sites identity between Cot-1 DNA and 25S rDNA was determined by dual-colour fluorescence in situ hybridization.All these showed that the karyotyping technique based on a combination of rDNA and Cot-1 DNA chromosome landmarks is superior to all but one.A more exact karyotype ofB.oleracea has been analyzed based on a combination of rDNA sites,Cot-1 DNA fluorescent bands,chromosome lengths and arm ratios.     

水稻45S rDNA和5S rDNA的染色体定位研究

45SrDNA和5SrDNA是水稻中与核糖体RNA合成有关的2个功能片段,有关这2个序列在水稻染色体上的位置,不同研究者的研究结果不尽相同,在获得水稻染色体清晰制片的基础上,通过FISH确定了45SrDNA序列位于水稻的第9号和第10号染色体的短臂末端,并且第9号染色体上的拷贝数多于第10号染色体,5SrDNA序列位于第11号染色体短臂靠近着丝点处。     

Relationships among Nicotiana species revealed by the 5S rDNA spacer sequence and fluorescence in situ hybridization

To investigate phylogenetic relationships in Nicotiana, the intergenic spacer sequences of 5S rDNA were analyzed in species with 2n=18, 20 or 24, and amphidiploid species with 2n=48. The chromosomal localization of the 5S rDNA was determined by fluorescence in situ hybridization (FISH). In species with 2n=24 and their descendants, a major 5S rDNA-specific PCR fragment of 400–650 bp was obtained. The amphidiploid species contained similar length of 5S rDNA units derived from putative diploid progenitors. Among the five clones from each representative PCR fragment, some nucleotide exchanges and length heterogeneity were observed. The latter was due to variation in the spacer region, such as differences in the length of poly A and/or poly T tracts as well as insertions/deletions. Interspecific comparisons of each 5S rDNA sequence demonstrated that the spacer sequence could be divided into three regions. Excluding gaps from the aligned spacer sequences of 5S rDNA, phylogenetic trees were constructed. Each phylogenetic tree showed an almost identical topology even if different algorithms were applied. The chromosomal locations of the 5S rDNA in each species correlated with the phylogenetic topology. The phylogenetic trees were generally in agreement with the current classification. Received: 15 January 2001 / Accepted: 15 February 2001     

A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization （FISH）

The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA). Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution [Current Zoology 55(2):145-149,2009].     

Immunohistochemical localization of albumin and in situ hybridization of albumin mRNA

Hepatocytes actively involved in albumin synthesis were identified by immunohistochemical method. In sections of perioidate-lysine-2 per cent (w/v) paraformaldehyde fixed normal rat liver, albumin was detected in all hepatocytes. At the ultrastructural level, albumin was localized in the rough endoplasmic reticulum and in Golgi complexes located near the nucleus in only a small subpopulation of hepatocytes, while all other hepatocytes contained albumin only in Golgi complexes located near the bile canaliculi. Stimulation of albumin synthesis by puromycin aminonucleoside-induced nephrosis resulted in an altered intracellular distribution of albumin at the light microscopic level. When examined at the ultrastructural level, albumin was localized in the rough endoplasmic reticulum as well as in Golgi complexes located near the nucleus in nearly all these hepatocytes. Hepatocytes with the potential to synthesize albumin were identified by in situ hybridization of albumin mRNA. In sections of 0.1 per cent (v/v) glutaraldehyde perfusion fixed normal rat liver, albumin mRNA was detected in the cytoplasm of only a few hepatocytes scattered throughout the lobule. Following stimulation of albumin synthesis by the induction of nephrosis, albumin mRNA was detected in the cytoplasm of the hepatocytes. The source of albumin in those hepatocytes which lacked albumin mRNA was identified in analbuminemic rats injected with rat albumin. At 6 h post injection, the light microscopic distribution of albumin in the liver of these animals was virtually indistinguishable from that in normal rat liver. At the ultrastructural level, injected albumin was localized in lysosomes and in Golgi complexes located near the bile canaliculi.     

Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.     

Simultaneous visualization of rDNA clusters and the nucleolus by combining fluorescence in situ hybridization and silver staining

We designed two procedures to visualize simultaneously clusters of ribosomal RNA genes (rDNA) and the nucleolus in plant cells. The procedures combine fluorescence in situ hybridization (FISH) to visualize the rDNA clusters and silver staining to observe the nucleolus. When FISH is followed by silver staining, many minute FISH signals are localized in the nucleolus, and several large FISH signals are seen on the nucleolar periphery. When FISH was applied to the specimens with silver nitrate staining, large FISH signals were visualized in the nucleoplasm associated with the nucleolar periphery, but no signals were seen in the nucleoli. Thus, the two combinations of FISH and silver staining provided different details regarding the arrangement of rDNA clusters in the nucleolus of plant cells.     

Multiplication of nucleolar fibrillar centres and absence of rDNA amplification in mouse ooctye during meiotic prophase I

During meiotic prophase I the nucleolus of the mouse oocyte assumes a reticulate structure of ‘nucleolonema’ type. This change coincides with the appearance of several secondary fibrillar centres. The number of these centres at diplotene (97–113), largely exceeds that of nucleolar organizers (4c DNA = 20 NORs). The quantitatative analysis of autoradiographs after hybridization in situ with -³H-uridine labelled rRNA, enabled us to demonstrate that the multiplication of the fibrillar centres in mouse oocyte nucleolus during meiotic prophase I is not the result of an amplification of the rDNA. The number of silver grains in pachytene and diplotene nuclei was twice that counted for somatic cell and oogonium nuclei (2c DNA).     

Ultrastructural in situ hybridization: A review of technical aspects

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters: the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.     

Location of ribosomal genes in CHO cells; in situ hybridization with a non-isotopic DNA probe on G-banded chromosomes.

A novel in situ hybridization technique using sulfonated probes is described. This non-radioactive approach, which employs chemically modified DNA and immunocytochemical procedures, is compatible with pre-G-banding and allows a rapid localization of the hybridized sequences on chromosomal spreads with a high spatial resolution. Using this technique we have localised the Chinese hamster ribosomal genes in the telomeric region of ten chromosomes, and among them in the subtelomeric q region of the Z5 chromosome. These results are discussed, the genetic markers confirming and locating the origin of Z group chromosomes by rearrangements of Chinese hamster chromosomes.     

Tentative chromosomal localization of the bovine major histocompatibility complex by in situ hybridization

Summary. A genetic region, most likely the major histocompatibility complex, was assigned to bands q13–23 of cattle chromosome 23 by in situ hybridization using a cloned DNA sequence of a class I gene of the pig major histocompatibility complex.     

Chromosome evolution in ground beetles: localization of the rDNA loci in the tribe Harpalini (Coleoptera,Carabidae)

The pattern of localization of the ribosomal genes was studied by means of fluorescence in situ hybridization in 39 species of the tribe Harpalini. Most of them show one pair of autosomes carrying the ribosomal genes in a distal position of a single chromosome arm. This pattern is hypothesized to be ancestral for the whole tribe. Both, chromosome number and the number and localization of rDNA loci, show little variation and are therefore of little phylogenetic value. Only in the subtribe Ditomina is there enough variation to characterize phyletic relationships. The stability of rDNA loci is even higher than the constancy of chromosome number, as most species of Ditomina (genera Dixus, Eocarterus, Carterus, Odontocarus and Ditomus) have the usual pair of autosomes with rDNA loci, in spite of remarkable differences in the diploid number. Only Dixus sphaerocephalus and Dixus clypeatus have two autosomal pairs with a fluorescent signal. These results do not support the hypothesis that the high chromosome numbers found within Ditomina are the result of polyploid change from the ancestral 2n = 37 karyotype of the tribe Harpalini. Chromosomal translocations or the presence of mobile genetic elements are plausible sources of the few cases of intraspecific polymorphism in the rDNA loci found in species of Harpalus.     

Cytogenetic comparisons between A and G genomes in Oryza using genomic in situ hybridization

The genomic structures of Oryza sativa （A genome） and O. meyeriana （G genome） were comparatively studied using bicolor genomic in situ hybridization （GISH）. GISH was clearly able to discriminate between the chromosomes of O. sativa and O. meyeriana in the interspecific F1 hybrids without blocking DNA, and co-hybridization was hardly detected. The average mitotic chromosome length of O. meyeriana was found to be 1.69 times that of O. sativa. A comparison of 4,6-diamidino-2-phenylindole staining showed that the chromosomes of O. meyeriana were more extensively labelled, suggesting that the G genome is amplified with more repetitive sequences than the A genome. In interphase nuclei, 9-12 chromocenters were normally detected and nearly all the chromocenters constituted the G genome-specific DNA. More and larger chromocenters formed by chromatin compaction corresponding to the G genome were detected in the hybrid compared with its parents. During pachytene of the F1 hybrid, most chromosomes of A and G did not synapse each other except for 1-2 chromosomes paired at the end of their arms. At meiotic metaphase I, three types of chromosomal associations, i.e.O, sativa-O, sativa （A-A）, O. sativa-O, meyeriana （A-G） and O. meyeriana-O, meyeriana （G-G）, were observed in the F1 hybrid. The A-G chromosome pairing configurations included bivalents and trivalents. The results provided a foundation toward studying genome organization and evolution of O. meyeriana.     

3D gold in situ labelling in the EM

We have developed a novel pre-embedding in situ hybridization labelling method for electron microscopy which has given much greater sensitivity and higher labelling levels than have been achieved previously, together with good ultrastructural preservation. Vibratome sections of plant tissue were labelled throughout their thickness with 1 nm gold antibodies and then silver enhanced, embedded in resin and sectioned for electron microscopy. Because the labelling extends throughout the depth of the specimen, this method permits the study of the 3D arrangement of the labelling at the electron microscope level by either stereo-pair recording, tomographic reconstruction or 3D reconstruction from serial sections. In this paper we describe the application of this method to study the organization of rDNA in pea root tissue.     

花生45S rDNA和5S rDNA的染色体定位研究

对四粒红和蜀花四号花生材料进行了核型分析,四粒红为2B核型,核型公式为2n=4x=40=38m＋2sm（4SAT）;蜀花四号为1B核型,核型公式为2n=4x=40=40 m（2SAT）。利用双色荧光原位杂交技术,对45S rDNA和5S rDNA这两个材料有丝分裂中期染色体上的物理位置进行了定位分析。定位结果表明,四粒红有6对45S rDNA位点,位于A2L、A7S、A9L、B3L、B7S、B8L（A和B分别代表基因组A和基因组B,L和S代表长臂和短臂,数字代表染色体序号,下同）;2对5S rDNA位点,位于A3S和B3S;蜀花四号有5对45S rDNA位点,位于A2L、A9L、B3L、B7S、B9L;2对5S rDNA位点,位于A3S和B3S。花生的45S rDNA位点具有可变性,5S rDNA则相对保守。     

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome l-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using β-galactosidase/ ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. the described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.