Nejvíce citovaný článek - PubMed ID 2973607
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
- Klíčová slova
- DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
- MeSH
- buněčný cyklus MeSH
- chromozomy rostlin * genetika MeSH
- metafáze MeSH
- průtoková cytometrie MeSH
- rostliny * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The Cervidae family comprises more than fifty species divided into three subfamilies: Capreolinae, Cervinae and Hydropotinae. A characteristic attribute for the species included in this family is the great karyotype diversity, with the chromosomal numbers ranging from 2n = 6 observed in female Muntiacus muntjak vaginalis to 2n = 70 found in Mazama gouazoubira as a result of numerous Robertsonian and tandem fusions. This work reports chromosomal homologies between cattle (Bos taurus, 2n = 60) and nine cervid species using a combination of whole chromosome and region-specific paints and BAC clones derived from cattle. We show that despite the great diversity of karyotypes in the studied species, the number of conserved chromosomal segments detected by 29 cattle whole chromosome painting probes was 35 for all Cervidae samples. The detailed analysis of the X chromosomes revealed two different morphological types within Cervidae. The first one, present in the Capreolinae is a sub/metacentric X with the structure more similar to the bovine X. The second type found in Cervini and Muntiacini is an acrocentric X which shows rearrangements in the proximal part that have not yet been identified within Ruminantia. Moreover, we characterised four repetitive sequences organized in heterochromatic blocks on sex chromosomes of the reindeer (Rangifer tarandus). We show that these repeats gave no hybridization signals to the chromosomes of the closely related moose (Alces alces) and are therefore specific to the reindeer.
The nucleolus is a nuclear compartment that plays an important role in ribosome biogenesis. Some structural features and epigenetic patterns are shared between nucleolar and non-nucleolar compartments. For example, the location of transcriptionally active mRNA on extended chromatin loop species is similar to that observed for transcriptionally active ribosomal DNA (rDNA) genes on so-called Christmas tree branches. Similarly, nucleolus organizer region-bearing chromosomes located a distance from the nucleolus extend chromatin fibers into the nucleolar compartment. Specific epigenetic events, such as histone acetylation and methylation and DNA methylation, also regulate transcription of both rRNA- and mRNA-encoding loci. Here, we review the epigenetic mechanisms and structural features that regulate transcription of ribosomal and mRNA genes. We focus on similarities in epigenetic and structural regulation of chromatin in nucleoli and the surrounding non-nucleolar region and discuss the role of proteins, such as heterochromatin protein 1, fibrillarin, nucleolin, and upstream binding factor, in rRNA synthesis and processing.
- MeSH
- buněčné jadérko genetika metabolismus ultrastruktura MeSH
- chromatin genetika ultrastruktura MeSH
- epigeneze genetická * MeSH
- genetická transkripce MeSH
- geny rRNA MeSH
- histony metabolismus MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- ribozomální DNA genetika MeSH
- ribozomy genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- srovnávací studie MeSH
- Názvy látek
- chromatin MeSH
- histony MeSH
- messenger RNA MeSH
- ribozomální DNA MeSH
The application of flow cytometry and sorting (flow cytogenetics) to plant chromosomes did not begin until the mid-1980s, having been delayed by difficulties in preparation of suspensions of intact chromosomes and discrimination of individual chromosome types. These problems have been overcome during the last ten years. So far, chromosome analysis and sorting has been reported in 17 species, including major legume and cereal crops. While chromosome classification by flow cytometry (flow karyotyping) may be used for quantitative detection of structural and numerical chromosome changes, chromosomes purified by flow sorting were found to be invaluable in a broad range of applications. These included physical mapping using PCR, high-resolution cytogenetic mapping using FISH and PRINS, production of recombinant DNA libraries, targeted isolation of markers, and protein analysis. A great potential is foreseen for the use of sorted chromosomes for construction of chromosome and chromosome-arm-specific BAC libraries, targeted isolation of low-copy (genic) sequences, high-throughput physical mapping of ESTs and other DNA sequences by hybridization to DNA arrays, and global characterization of chromosomal proteins using approaches of proteomics. This paper provides a comprehensive review of the methodology and application of flow cytogenetics, and assesses its potential for plant genome analysis.
PURPOSE: Chromosomal aberrations and the nuclear topography of retinoblastoma tumour cells as well as lymphocytes of patients suffering from the familiar or sporadic form of retinoblastoma were studied. METHODS: Fluorescence in situ hybridisation (FISH) on fresh, paraffin-embedded tumour tissues and on peripheral blood leukocytes was used for cytogenetic analysis. The cell cycle profile and induction of apoptosis was studied by flow cytometry and gene expression changes were detected by RT-PCR. RESULTS: Using the repeated FISH technique, the average distances between the nuclear membrane and the fluorescence gravity centre (FGC) of seven selected chromosomes were determined in the same tumour population and three other cell types. Chromosome order in positioning from the nuclear membrane was similar in all cell populations investigated. Our experimental studies were focused on specific genetic loci relevant for retinoblastoma tumour pathogenesis. We revealed a certain heterogeneity in the copy number of the Rb1, N-myc, and TP53 gene loci in tumour cells. In addition, in lymphocytes isolated from peripheral blood of the patients, a high degree of copy number heterogeneity was also detected. In 60% of analysed retinoblastomas we observed numerical aberration involving the centromeric region of chromosome 6. In these tumours, apoptotic bodies were found irrespective of clinical therapy. Chromosome instability seems to be a typical feature of primary retinoblastomas as well as of the human pseudodiploid cell line Y79. These cells, of a hereditary form of retinoblastoma (Y79), were irradiated by gamma rays and exposed to anti-tumour drugs such as etoposide, vincristine, and cisplatin. These treatments induced apoptosis, changes in the cell cycle profile, and specific modifications in the nuclear topography of selected loci. Treatment with a non-lethal concentration of hydroxyurea was shown to induce the loss of the amplified N-myc gene involved in the homogenously staining region (HSR) that was found to be associated with the nuclear membrane of retinoblastoma Y79 cells. CONCLUSIONS: We assume that not only cytological and cytogenetic parameters but also aberrant chromatin structures and their nuclear topography can be useful tools for optimal tumour marker specification.
- MeSH
- apoptóza MeSH
- hybridizace in situ fluorescenční MeSH
- lidé MeSH
- nádory sítnice genetika patologie MeSH
- ploidie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- průtoková cytometrie MeSH
- retinoblastom genetika patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The spatial arrangement of some genetic elements relative to chromosome territories and in parallel with the cell nucleus was investigated in human lymphocytes. The structure of the chromosome territories was studied in chromosomes containing regions (clusters) of highly expressed genes (HSA 9, 17) and those without such clusters (HSA 8, 13). In chromosomes containing highly expressed regions, the elements pertaining to these regions were found close to the centre of the nucleus on the inner sides of chromosome territories; those pertaining to regions with low expression were localized close to the nuclear membrane on the opposite sides of the territories. In chromosomes with generally low expression (HSA 8, 13), the elements investigated were found symmetrically distributed over the territories. Based on the investigations of the chromosome structure, the following conclusions are suggested: (1) Chromosome territories have a non-random internal 3D structure with defined average mutual positions between elements. For example, RARalpha, TP53 and Iso-q of HSA 17 are nearer to each other than they are to the HSA 17 centromere. (2) The structure of a chromosome territory reflects the number and chromosome location of clusters of highly expressed genes. (3) Chromosome territories behave to some extent as solid bodies: if the territory is found closer to the nuclear centre, the individual genetic elements of this chromosome are also found, on average, closer the centre of the nucleus. (4) The positions of centromeres are, on average, nearer to the fluorescence weight centre of the territory (FWCT) than to genes. (5) Active genes are not found near the centromeres of their own territory. A simple model of the structure of chromosome territory is proposed.
- MeSH
- buněčné jádro genetika MeSH
- centromera genetika MeSH
- euchromatin genetika MeSH
- geny MeSH
- heterochromatin genetika MeSH
- hybridizace in situ fluorescenční MeSH
- jaderný obal genetika MeSH
- kompartmentace buňky MeSH
- lidé MeSH
- lidské chromozomy, pár 17 ultrastruktura MeSH
- lidské chromozomy ultrastruktura MeSH
- lymfocyty diagnostické zobrazování MeSH
- metoda Monte Carlo MeSH
- modely genetické MeSH
- počítačová simulace MeSH
- počítačové zpracování obrazu MeSH
- ultrasonografie MeSH
- zobrazování trojrozměrné * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- euchromatin MeSH
- heterochromatin MeSH
