Extensive coloured identification of dog chromosomes to support karyotype studies: the colour code

The identification of individual dog chromosomes is problematic because the 38 pairs of autosomes are small and acrocentric. Here we describe the design and application of a FISH tool that enables definitive identification of each dog autosome in a normal karyotype, without relying on subjective interpretation of DAPI banding patterns. From a high-resolution physical map of the canine genome, we have chosen a panel of 80 canine chromosome-specific BAC clones. DNA from each clone is labeled with one of five different fluorochrome-conjugated nucleotides. By selecting one to three spatially separated BACs per chromosome, and labelling them with a distinctive combination of colours, each autosome can be identified objectively and orientated accurately, irrespective of the quality of DAPI chromosome banding. This tool, or part of it, can be used for any purpose where accurate identification of canine autosomes in a normal karyotype is essential. In this study, we demonstrate use of the 'colour code' for chromosome identification following CGH analysis of unbalanced genomic aberrations in a canine brain tumour. Our method is an improvement of an earlier procedure, featuring chromosome-specific BACs and sequential FISH hybridisations, as it enables simultaneous identification of all chromosomes in a single hybridisation.     

In situ chromosome preparation technique for simultaneous cytogenetic and immunocytochemical studies on cell cultures of solid tumors

Summary Immunophenotyping of cultured cancer cells requires intact antigenic structures; these are mostly destroyed by conventional chromosome preparation techniques. Thus, the simultaneous cytogenetic and immunocytochemical characterization of solid tumor cells appears unfeasible. Here, we describe a novel method that allows in situ chromosome preparation from monolayer cultures of solid tumor cells without affecting their immunological features. Using this technique, it is possible to achieve detailed cytogenetic data including chromosome banding together with the demonstration of cytoplasmic and nuclear antigens within the same tumor cell.     

In situ hybridization and chromosome banding in mammalian species

Chromosome banding is often required in conjunction with fluorescent in situ hybridization of labelled probes for chromosome painting, satellite DNA and low-copy sequences to allow identification of chromosomes and simultaneous probe localization. Here, we present a method that reveals both patterns with only one observation step. The band pattern is produced by restriction-enzyme digestion of chromosomes, followed by fixation with paraformaldehyde in PBS, a short chromosome denaturation step in hybridization solution, and then standard in situ hybridization, washing and detection protocols. Using a range of different mammalian species, chromosome-banding patterns were immediately recognizable, although synchronisation procedures normally required for high- resolution G-banding were not needed. Unlike other methods available, only one round of observation is required using a conventional fluorescence microscope, the method works without modification in many species, and in situ hybridization is not used for chromosome identification (allowing multiple targets and minimizing background). The banding pattern is probably generated by a combination of DNA dissolution and heterochromatin reorganisation after enzyme digestion, followed by paraformaldehyde fixation of the new chromatin structure and incomplete denaturation. The method is of widespread utility in comparative genomics and genome organization programmes.     

Dimeric bisbenzimidazole Hoechst 33258-related dyes as novel AT-specific DNA-binding fluorochromes for human and plant cytogenetics

Ten new fluorescent dyes have been tested as possible tools for human and plant chromosome banding; eight of the tested dyes were dimer derivatives of bisbenzimidazole (Hoechst 33248). The dimer compounds differed from each other by the length of the linker connecting benzimidazole moieties and by the structure of the moieties themselves. Four compounds selected for the study, namely, DB(7), DB(8), DB(17), and DB(18), upon UV excitation (365 nm) exhibited a bright blue fluorescence in nuclear heterochromatic granules, but at longer excitation wavelengths (blue or green) did not show any fluorescence in nucleus or cytoplasm. All these compounds were shown to produce a high-quality distinct banding in human and plant chromosomes, comparable to that obtained after standard DAPI staining. Further study was carried out so that to evaluate contrast of the band borders, fluorescence intensity, as well as a photobleaching rate for different dyes. As a result, we selected DB(17) as a fluorochrome of choice for chromosome banding. Spectral characteristics of this compound allow employing it in combination with FISH analysis. Investigation of linum karyotypes using DB(17) has demonstrated applicability of this dye for banding of very short plant chromosomes. The known higher stability of dimer DNA-binding fluorophors, as compared to that of monomer ones, suggests the possibility of using DB(17) for banding and identification of flow-sorted chromosomes.     

Banded karyotypes from bone marrow: A clinical useful approach

Summary We have developed a new protocol for the preparation of banded chromosomes from human bone marrow. This protocol incorporates new procedures with improvements in conventional ones to rapidly produce high quality banded karyotypes from bone marrow aspirates. Tissue culture is completely climinated and replaced with a truly direct method of chromosome preparation in which a small amount of marrow is treated with a solution containing trypsin, hypotonic salts and colcemid (THC). The THC protocol, when compared with standard short term culture methods for marrow chromosome preparation, produces more extended and more readily banded chromosomes. Rapid banding is further facilitated by replacement of standard G-banding techniques with Wright's staining. These technical developments allow karyotypic analysis within 2–4 h after receipt of the specimen. The high quality and rapidity of the THC protocol have important implications for the clinical usefulness of cytogenetic analysis of bone marrow in studying congenital defects as well as leukemias and lymphomas.     

In situ hybridization banding of human chromosomes with Alu-PCR products: A simultaneous karyotype for gene mapping studies

Polymerase chain reaction products generated from a single Alu primer and human genomic DNA produce a distinct and highly reproducible R-banding pattern when hybridized to metaphase chromosome spreads. Individual chromosomes can be readily identified and karyotyped. Compared to conventional fluorescence banding on heat-denatured chromosomes, the in situ hybridization banding (ISHB) shows high contrast and definition. We demonstrate that this banding method can be employed effectively in double-labeling experiments for the rapid and simultaneous assignment of probes to specific chromosomal bands. Since virtually any fluorochrome can be used to delineate chromosomal bands, ISHB should provide added flexibility for multicolor mapping strategies.     

Multicolor banding technique, spectral color banding (SCAN): new development and applications

Conventional banding techniques can characterize chromosomal aberrations associated with tumors and congenital diseases with considerable precision. However, chromosomal aberrations that have been overlooked or are difficult to analyze even by skilled cytogeneticists were also often noted. Following the introduction of multicolor karyotyping such as spectral karyotyping (SKY) and multiplex-fluorescence in situ hybridization (M-FISH), it is possible to identify this kind of cryptic or complex aberration comprehensively by a single analysis. To date, multicolor karyotyping techniques have been established as useful tools for cytogenetic analysis. However, since this technique depends on whole chromosome painting probes, it involves limitations in that the origin of aberrant segments can be identified only in units of chromosomes. To overcome these limitations, we have recently developed spectral color banding (SCAN) as a new multicolor banding technique based on the SKY methodology. This new technique may be deemed as an ideal chromosome banding technique since it allows representation of a multicolor banding pattern matching the corresponding G-banding pattern. We applied this technique to the analysis of chromosomal aberrations in tumors that had not been fully characterized by G-banding or SKY and found it capable of (1) detecting intrachromosomal aberrations; (2) identifying the origin of aberrant segments in units of bands; and (3) precisely determining the breakpoints of complex rearrangements. We also demonstrated that SCAN is expected to allow cytogenetic analysis with a constant adequate resolution close to the 400-band level regardless of the degree of chromosome condensation. As compared to the conventional SKY analysis, SCAN has remarkably higher accuracy for a particular chromosome, allowing analysis in units of bands instead of in units of chromosomes and is hence promising as a means of cytogenetic analysis.     

Banding patterns on lampbrush chromosomes of Triturus marmoratus (Amphibia Urodela) by the Giemsa stain

Lampbrush chromosome preparations from the newt species Triturus marmoratus have been submitted to a banding procedure by using a Giemsa stain technique (C-banding) as well as variants of the method. Centromeres, most of telomeres, the nucleolus organizing region and some segments along the chromosome axes appear to be differently stained. The centromere positions have been indicated on the maps of the lampbrush complement of the species. The possible relationships between banding and chromosome structure and organization are briefly discussed.     

应用染色体涂染技术分析两例染色体结构异常 Analysis of the Structure of Abnormal Karyotypes by Using Chromosome Painting

为了确定两例细胞遗传学提示染色体结构异常的核型,应用通过显微切割技 术构建的人类18号和7号染色体探针池,分别对这两例病例的中期分裂相进行染色体涂染,结合显带染色体,确定两者核型分别为46,XY,t(3;18) (q12;q21)和46,XX,dir ins(1;7)(p3104;q34q36)。染色体涂染技术是染色体显带技术的重要补充和发展,为染色体结构异常提供了一种直观、准确的检测手段,在遗传咨询和产前诊断方面有重要作用。 Abstract:In this study,chromosome painting technique was performed to analyse the abnormal karyotypes of two carriers.Chromosome 18 and 7 specific libraries,which were generated by chromosome microdissection technique,were used as probe pools to hybridize the carriers metaphase chromosomes respectively.Unlabled human genomic DNA was used to inhibit the hybridization of sequences in the library that bind to mutiple chromosomes.Structure abnormality was detected clearly in metaphase.Combined with the banding chromosomes,we concluded that their karytypes were 46,XY,t(3;18)(q12;q21)and 46,XX,dir ins(1;7)(p3104;q34q36).Chromosome painting,as a direct and concise method in analysing chromosome structure abnormality,is an important complement and development of chromosome banding technique,and has important application in genetic counselling and prenatal diagnosis.     

An improved method for chromosome banding after fluorescence in situ hybridization: Use of a tetramethylrodhamine-conjugated BrdU antibody

A method for high quality chromosome banding after in situ hybridization with biotinylated probes has been developed. Fluoresceine-conjugated avidin is used for probe detection, while chromosome banding is performed with a tetramethylrodhamine-conjugated anti-BrdU antibody. In this way probe localization and chromosome identification can be performed simultaneously simply by changing the incidental light wavelength.Abbreviations BAT BrdU antibody technique - DABCO 1,4 diazobicyclo-(2.2.2)octane - FITC fluorescein isothiocyanate - FPG fluorochrome plus giemsa - PHA phytohemagglutinin - RBA R-banding BrdU acridine - TRITC tetramethylrhodamine isothiocyanate     

Application of Giemsa banding to orchid karyotype analysis

A method for obtaining orchid chromosome squash preparations from ovular tissues and a Giemsa C-band technique are described. Jointly applied, they result in well-defined chromosome banding patterns. Preliminary tests with two species of the genusCephalanthera show that Giemsa banding is also well suited for orchids. Besides aiding in chromosome identification and karyotype analysis, it should prove valuable in studies of chromosomal variation and karyotype evolution of this large family.     

Homologous chromosomes characteristics by sequential banding procedures in rainbow trout Oncorhynchus mykiss

The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. Possibility of detection both FISH and DAPI signals can help in procedures of gene mapping on chromosomes.     

Karyotype and chromosomal banding pattern in Heteropeza pygmaea

The chromosomes of somatic and germ line cells of female embryos produced by paedogenesis were studied. The haploid set in somatic cells consists of one long submetacentric chromosome, one large acrocentric, one medium metacentric and two small acrocentrics. The length vs arm index karyogram makes it possible to distinguish all but the two pairs of small acrocentric chromosomes. — Attempts were made to develope a method for banding pattern visualization. The best result was obtained using trypsin which induced banding in the chromosomes of the somatic cells and occasionally also of the germ line cells. The resulting banding patterns were frequently not identical in members of a chromosome pair. There was also a variation between metaphases within an embryo as well as from different embryos. Some tentative explanations for these results are discussed.     

FISH analysis comparing genome organization in the domestic horse (Equus caballus) to that of the Mongolian wild horse (E. przewalskii)

Przewalski's wild horse (E. przewalskii, EPR) has a diploid chromosome number of 2n = 66 while the domestic horse (E. caballus, ECA) has a diploid chromosome number of 2n = 64. Discussions about their phylogenetic relationship and taxonomic classification have hinged on comparisons of their skeletal morphology, protein and mitochondrial DNA similarities, their ability to produce fertile hybrid offspring, and on comparison of their chromosome morphology and banding patterns. Previous studies of GTG-banded karyotypes suggested that the chromosomes of both equids were homologous and the difference in chromosome number was due to a Robertsonian event involving two pairs of acrocentric chromosomes in EPR and one pair of metacentric chromosomes in ECA (ECA5). To determine which EPR chromosomes were homologous to ECA5 and to confirm the predicted chromosome homologies based on GTG banding, we constructed a comparative gene map between ECA and EPR by FISH mapping 46 domestic horse-derived BAC clones containing genes previously mapped to ECA chromosomes. The results indicated that all ECA and EPR chromosomes were homologous as predicted by GTG banding, but provide new information in that the EPR acrocentric chromosomes EPR23 and EPR24 were shown to be homologues of the ECA metacentric chromosome ECA5.     

Heterochromatin and silver banding of rye (Secale cereale,Gramineae) chromosomes

Air-dried chromosomes of rye when stained with aqueous silver nitrate show differential banding patterns. In addition to staining the NOR sites, the silver nitrate stains all regions of constitutive heterochromatin, as identified by Giemsa C-banding, as well as a number of small interstitial regions. However, the heterochromatin on the B chromosome is not stained by the silver method. This is proposed as a rapid and reliable banding method.     

Chromosomal localization of the ovine beta-casein gene by non-isotopic in situ hybridization and R-banding.

The ovine beta-casein gene (CNS2) has been mapped to a specific chromosome band using nonradioactive in situ hybridization and simultaneous fluorescent R-banding. The probe pTZ-E4 was a fragment of the ovine beta-casein gene inserted in the plasmid pTZ18R and labeled with biotin-11-dUTP. It hybridized to band q32 of ovine chromosome 4. The discrepancy between this result and the previous localization of this gene on cattle chromosome 6 may be explained by the very great similarity of the banding patterns of ovine and bovine chromosomes 4 and 6.     

马尾松染色体荧光带型的研究

对马尾松有丝分裂中期染色体荧光带纹的分析结果表明,其色霉素Ａ的染色体的荧光带赤;１对为着丝粒区和臂间我均有带纹的中间着丝染色体。６对为臂间区有带纺的中间着丝粒染色体;２对为着丝粒区有带纹的中间着丝粒染色体;３对无带纹的中间或近中着丝粒染色体;１对为着丝粒区有带纹的近中着丝粒染色体。     

The 5S ribosomal RNA gene clusters in Tetrahymena thermophila: strain differences, chromosomal localization, and loss during micronuclear ageing

Summary The organization of the 5S genes in the genome of Tetrahymena thermophila was examined in various strains, with germinal ageing, and the 5S gene clusters were mapped to the MIC chromosomes. When MIC or MAC DNA is cut with the restriction enzyme EcoRI, electrophoresed, blotted, and probed with a 5S rDNA probe, the banding patterns represent the clusters of the 5S rRNA genes as well as flanking regions. The use of long gels and 60 h of electrophoresis at 10 mA permitted resolution of some 30–35 5S gene clusters on fragments ranging in size from 30-2 kb (bottom of gel). The majority of the 5S gene clusters were found in both MIC and MAC genomes, a few being MIC limited and a few MAC limited. The relative copy number of 5S genes in each cluster was determined by integrating densitometric tracings made from autoradiograms. The total number of copies in the MAC was found to be 33% greater than in the MIC. When different inbred strains were examined, the majority of the 5S gene clusters were found to be conserved, with a few strain-specific clusters observed. Nine nullisomic strains missing both copies of one or more MIC chromosomes were used to map the 5S gene clusters. The clusters were distributed non-randomly to four of the five MIC chromosomes, with 17 of them localized to chromosome 1. A deletion map of chromosome 1 was constructed using various deletion strains. Some of these deletion strains included B strain clones which had been in continuous culture for 15 years. Losses of 5S gene clusters in these ageing MIC could be attributed to deletions of particular chromosomes. The chromosomal distribution of the 5S gene clusters in Tetrahymena is unlike that found for the well-studied eukaryotes, Drosophila and Xenopus.     

Rapid identification of chromosomes carrying silver-stained nucleolus-organizing regions

Summary The use of a combination of transmitted light and epiluminescence after silver and fluorescent staining of chromosome preparations makes it possible to achieve simultaneous visualization of silver-stained NORs and fluorescent chromosomes. This technique permits exact localization of silver precipitates on normal and BrdU-substituted chromosomes. After previous silver impregnation, fluorescent staining by actinomycin-daunomycin-DAPI was used to induce a banding pattern that enables identification of specific chromosomes while observing silver-stained NORs at the same time. Application of this method to Down's syndrome patient revealed a 21/21 Robertsonian translocation with NORs eliminated.