Sorting
Dotaz
Zobrazit nápovědu
- MeSH
- buněčné linie MeSH
- hyaluronoglukosaminidasa MeSH
- myši MeSH
- transplantační imunologie MeSH
- Check Tag
- myši MeSH
Nuclear genomes of many important plant species are tremendously complicated to map and sequence. The ability to isolate single chromosomes, which represent small units of nuclear genome, is priceless in many areas of plant research including cytogenetics, genomics, and proteomics. Flow cytometry is the only technique which can provide large quantities of pure chromosome fractions suitable for downstream applications including physical mapping, preparation of chromosome-specific BAC libraries, sequencing, and optical mapping. Here, we describe step-by-step procedure of preparation of liquid suspensions of intact mitotic metaphase chromosomes and their flow cytometric analysis and sorting.
Analysis and sorting of plant chromosomes (plant flow cytogenetics) is a special application of flow cytometry in plant genomics and its success depends critically on sample quality. This unit describes the methodology in a stepwise manner, starting with the induction of cell cycle synchrony and accumulation of dividing cells in mitotic metaphase, and continues with the preparation of suspensions of intact mitotic chromosomes, flow analysis and sorting of chromosomes, and finally processing of the sorted chromosomes. Each step of the protocol is described in detail as some procedures have not been used widely. Supporting histograms are presented as well as hints on dealing with plant material; the utility of sorted chromosomes for plant genomics is also discussed. © 2016 by John Wiley & Sons, Inc.
- MeSH
- chromozomy rostlin metabolismus MeSH
- DNA rostlinná genetika MeSH
- hybridizace in situ fluorescenční MeSH
- karyotypizace MeSH
- meristém cytologie účinky léků MeSH
- metafáze účinky léků MeSH
- molekulová hmotnost MeSH
- oxid dusný farmakologie MeSH
- proteomika MeSH
- průtoková cytometrie metody MeSH
- rostliny genetika MeSH
- semena rostlinná účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
191 s. : il.
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.
In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.
- MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- jaderné proteiny analýza MeSH
- ječmen (rod) cytologie metabolismus MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- proteomika metody MeSH
- průtoková cytometrie MeSH
- rostlinné proteiny analýza MeSH
- Publikační typ
- časopisecké články MeSH
Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.
- MeSH
- buněčný cyklus MeSH
- chromozomy rostlin MeSH
- cytogenetika MeSH
- DNA rostlinná genetika MeSH
- hybridizace in situ fluorescenční metody MeSH
- karyotypizace MeSH
- meristém cytologie MeSH
- průtoková cytometrie metody MeSH
- rostlinné buňky MeSH
- rostliny genetika MeSH
- semena rostlinná růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.
BACKGROUND: Methamphetamine is a neurotoxic agent. Its chronic abuse may result in cognitive impairment with negative consequences for patients' treatment and rehabilitation. The aim of the study was to compare Wisconsin Card Sorting Test profiles of Czech subjects dependent on methamphetamine with healthy individuals. SUBJECTS AND METHODS: Forty-three hospitalized Czech Caucasian patients including twenty-seven men at the average age of 25.3±5.2 years dependent on methamphetamine for 6.2±3.3 years were assessed by the Wisconsin Card Sorting Test. We used the same neurocognitive test for the comparison group of healthy controls with the same ethnicity (N=52, men N=28, average age of 38.7±12.1 years). We applied the Chi-Square Test, Two-Sample T Test, Mann-Whitney U Test and Kolmogorov-Smirnov Test to compare methamphetamine dependent patients with healthy volunteers. RESULTS: All recorded Wisconsin Card Sorting Test parameters were significantly different in the group of methamphetamine dependent patients versus healthy volunteers (P=0.04-0.006; Mann-Whitney U Test, Two-Sample T Test). The results showed a higher error rate and a smaller achievement quality in the patients as against healthy subjects. We ascertained a significant cognitive deterioration in the patients as compared to healthy volunteers even if the average patients' achievements were in the normal range according to the test norms. CONCLUSIONS: A cognitive impairment was present in the group of patients as compared to healthy controls. Better understanding of neurocognitive symptoms in methamphetamine dependent subjects should help to generate modern therapeutic approaches, both pharmacological and psychosocial, to prevent or attenuate the long-term negative consequences of methamphetamine use disorders.
- MeSH
- dospělí MeSH
- kognitivní poruchy chemicky indukované komplikace MeSH
- lidé středního věku MeSH
- lidé MeSH
- methamfetamin škodlivé účinky MeSH
- mladiství MeSH
- neuropsychologické testy MeSH
- poruchy spojené s užíváním amfetaminu komplikace psychologie MeSH
- stimulanty centrálního nervového systému škodlivé účinky MeSH
- studie případů a kontrol MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
