The Experts below are selected from a list of 108 Experts worldwide ranked by ideXlab platform
Jaroslav Doležel - One of the best experts on this subject based on the ideXlab platform.
-
Sorting of plant Chromosomes.
Methods in Cell Biology, 2020Co-Authors: Jaroslav Doležel, Hana Šimková, Martin A. Lysak, Marie Kubaláková, Jiří Macas, Sergio LucrettiAbstract:Publisher Summary This chapter provides an overview of plant flow cytogenetics. The high-yield procedure for preparation of Chromosome suspensions based on the use of root meristems is relatively simple and can be modified to different plant species. The use of hairy root cultures represents an elegant solution for those genotypes that are difficult to propagate via seeds. A common procedure for Chromosome Isolation and flow sorting consists of three basic steps: (1) cell cycle synchronization and metaphase accumulation; (2) preparation of Chromosome suspensions; and (3) flow cytometric analysis and sorting. The chapter presents high-yield procedures for preparation of Chromosome suspensions from synchronized root tips in two legume and two cereal species. Procedures for univariate and bivariate flow cytometric analysis of isolated Chromosomes are presented, including those for Chromosome sorting. Two different methods are reviewed for testing the identity and purity of sorted Chromosome fractions, namely, fluorescence microscopy and polymerase chain reaction (PCR).
-
Current Protocols in Cytometry - Flow analysis and sorting of plant Chromosomes.
Current protocols in immunology, 2016Co-Authors: Jan Vrána, Petr Cápal, Hana Šimková, Miroslava Karafiátová, Jana Čížková, Jaroslav DoleželAbstract:The use of flow cytometry for evaluation of plant Chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of Chromosome suspensions, flow analysis and sorting of Chromosomes, and processing of the sorted Chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted Chromosomes for plant genome mapping.. Keywords: flow cytometry; plant Chromosomes; cell cycle synchronization; Chromosome Isolation; Chromosome sorting; physical gene mapping; Chromosome-specific DNA libraries
-
Chromosome Isolation by flow sorting in aegilops umbellulata and ae comosa and their allotetraploid hybrids ae biuncialis and ae geniculata
PLOS ONE, 2011Co-Authors: Istvan Molnar, Hana Šimková, Marie Kubaláková, Andras Cseh, Marta Molnarlang, Jaroslav DoleželAbstract:This study evaluates the potential of flow cytometry for Chromosome sorting in two wild diploid wheats Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Flow karyotypes obtained after the analysis of DAPI-stained Chromosomes were characterized and content of Chromosome peaks was determined. Peaks of Chromosome 1U could be discriminated in flow karyotypes of Ae. umbellulata and Ae. biuncialis and the Chromosome could be sorted with purities exceeding 95%. The remaining Chromosomes formed composite peaks and could be sorted in groups of two to four. Twenty four wheat SSR markers were tested for their position on Chromosomes of Ae. umbellulata and Ae. comosa using PCR on DNA amplified from flow-sorted Chromosomes and genomic DNA of wheat-Ae. geniculata addition lines, respectively. Six SSR markers were located on particular Aegilops Chromosomes using sorted Chromosomes, thus confirming the usefulness of this approach for physical mapping. The SSR markers are suitable for marker assisted selection of wheat-Aegilops introgression lines. The results obtained in this work provide new opportunities for dissecting genomes of wild relatives of wheat with the aim to assist in alien gene transfer and discovery of novel genes for wheat improvement.
-
Isolation of Chromosomes from Pisum sativum L. hairy root cultures and their analysis by flow cytometry
Plant Science, 1998Co-Authors: Pavel Neumann, Jaroslav Doležel, Martin A. Lysak, Jiří MacasAbstract:Abstract Continuously growing hairy-root cultures of pea ( Pisum sativum L.) line L-84 have been induced by transformation of seedlings by Agrobacterium rhizogenes A4-24. The cultures were then used for cell cycle synchronisation in vitro and for preparation of Chromosome suspensions. Various concentrations of hydroxyurea (0.5–2.0 mM) and timing of amiprophos-methyl (APM, 2–4 h) treatment have been tested to achieve the highest mitotic synchrony, and to accumulate the highest number of synchronised cells in metaphase, respectively. Optimal conditions (2 mM hydroxyurea for 18 h, 10 μM APM for 2 h applied 6 h after a release from hydroxyurea block) resulted in a high frequency of cells in metaphase (43%). The synchronised root tips were fixed in formaldehyde and Chromosomes were released into a lysis buffer by mechanical homogenisation. The Chromosomes in suspension showed a well preserved morphology, and could be used for flow cytometric analysis. While only two Chromosomes (5 and 7) were discriminated in a standard karyotype, four Chromosomes (3, 5, 6 and 7) could be clearly discriminated in the line L-84 which contains a stable reciprocal translocation between Chromosomes 3 and 6. The establishment of hairy root cultures and the development of an efficient Chromosome Isolation procedure from a translocation line L-84 represent a basic prerequisite for subsequent flow-sorting of selected Chromosomes of pea.
Toshio Tsukiyama - One of the best experts on this subject based on the ideXlab platform.
-
Dynamic changes in histone acetylation regulate origins of DNA replication
Nature Structural & Molecular Biology, 2010Co-Authors: Ashwin Unnikrishnan, Philip R Gafken, Toshio TsukiyamaAbstract:Although histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here we describe an efficient single-step method to specifically purify histones located around an origin of replication from Saccharomyces cerevisiae . Using high-resolution MS, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that acetylation of histone H3 and H4 is dynamically regulated around an origin of replication, at the level of multiply acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S phase. Histone modifications have been implicated in many DNA transactions. Using a mini-Chromosome Isolation and mass spectroscopy technique applicable to specifically probing many DNA binding factors, the acetylation of histone residues surrounding an S. cerevisiae origin are now defined during the cell cycle, with further experiments implicating residues in efficient origin activation.
Ashwin Unnikrishnan - One of the best experts on this subject based on the ideXlab platform.
-
Dynamic changes in histone acetylation regulate origins of DNA replication
Nature Structural & Molecular Biology, 2010Co-Authors: Ashwin Unnikrishnan, Philip R Gafken, Toshio TsukiyamaAbstract:Although histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here we describe an efficient single-step method to specifically purify histones located around an origin of replication from Saccharomyces cerevisiae . Using high-resolution MS, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that acetylation of histone H3 and H4 is dynamically regulated around an origin of replication, at the level of multiply acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S phase. Histone modifications have been implicated in many DNA transactions. Using a mini-Chromosome Isolation and mass spectroscopy technique applicable to specifically probing many DNA binding factors, the acetylation of histone residues surrounding an S. cerevisiae origin are now defined during the cell cycle, with further experiments implicating residues in efficient origin activation.
Magnus Nordenskjöld - One of the best experts on this subject based on the ideXlab platform.
-
Complete characterization of a large marker Chromosome by reverse and forward Chromosome painting
Human Genetics, 1992Co-Authors: Elisabeth Blennow, Håkan Telenius, Catharina Larsson, Svetlana Bajalica, Bruce A. J. Ponder, Magnus NordenskjöldAbstract:Marker Chromosome are small supernumerary Chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker Chromosomes need complete characterization. By combining Chromosome Isolation by flow sorting and the “degenerate oligonucleotide primed — polymerase chain reaction”, we have constructed a DNA library specific for a marker Chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads (“reverse Chromosome painting“) and were thus able to determine that the marker consists of the centromeric part of Chromosome 7, the telomeric region of the long arm of Chromosome 5 and the telomeric region of the short arm of the X-Chromosome. Subsequently, we hybridized normal Chromosome-specific libraries of the relevant Chromosomes onto metaphases containing the marker Chromosome (“forward Chromosome painting”) and could in this manner establish the precise location of the different Chromosome regions on the marker Chromosome itself. This is a general approach suitable for outlining marker Chromosomes in detail, and will aid the identification of the genes involved.
Hana Šimková - One of the best experts on this subject based on the ideXlab platform.
-
Sorting of plant Chromosomes.
Methods in Cell Biology, 2020Co-Authors: Jaroslav Doležel, Hana Šimková, Martin A. Lysak, Marie Kubaláková, Jiří Macas, Sergio LucrettiAbstract:Publisher Summary This chapter provides an overview of plant flow cytogenetics. The high-yield procedure for preparation of Chromosome suspensions based on the use of root meristems is relatively simple and can be modified to different plant species. The use of hairy root cultures represents an elegant solution for those genotypes that are difficult to propagate via seeds. A common procedure for Chromosome Isolation and flow sorting consists of three basic steps: (1) cell cycle synchronization and metaphase accumulation; (2) preparation of Chromosome suspensions; and (3) flow cytometric analysis and sorting. The chapter presents high-yield procedures for preparation of Chromosome suspensions from synchronized root tips in two legume and two cereal species. Procedures for univariate and bivariate flow cytometric analysis of isolated Chromosomes are presented, including those for Chromosome sorting. Two different methods are reviewed for testing the identity and purity of sorted Chromosome fractions, namely, fluorescence microscopy and polymerase chain reaction (PCR).
-
Current Protocols in Cytometry - Flow analysis and sorting of plant Chromosomes.
Current protocols in immunology, 2016Co-Authors: Jan Vrána, Petr Cápal, Hana Šimková, Miroslava Karafiátová, Jana Čížková, Jaroslav DoleželAbstract:The use of flow cytometry for evaluation of plant Chromosomes requires some specialized attention to preparation and instrumentation. This unit deals exclusively with plant cytogenetics and presents an outline of this area as well as methods for accumulation of cells in metaphase, preparation of Chromosome suspensions, flow analysis and sorting of Chromosomes, and processing of the sorted Chromosomes. Each method is described in tremendous detail because in many aspects dealing with plant cells is quite different from dealing with mammalian cells. Supporting histograms are presented as well as a range of special hints on dealing with plant material and a discussion of the utility of sorted Chromosomes for plant genome mapping.. Keywords: flow cytometry; plant Chromosomes; cell cycle synchronization; Chromosome Isolation; Chromosome sorting; physical gene mapping; Chromosome-specific DNA libraries
-
Chromosome Isolation by flow sorting in aegilops umbellulata and ae comosa and their allotetraploid hybrids ae biuncialis and ae geniculata
PLOS ONE, 2011Co-Authors: Istvan Molnar, Hana Šimková, Marie Kubaláková, Andras Cseh, Marta Molnarlang, Jaroslav DoleželAbstract:This study evaluates the potential of flow cytometry for Chromosome sorting in two wild diploid wheats Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Flow karyotypes obtained after the analysis of DAPI-stained Chromosomes were characterized and content of Chromosome peaks was determined. Peaks of Chromosome 1U could be discriminated in flow karyotypes of Ae. umbellulata and Ae. biuncialis and the Chromosome could be sorted with purities exceeding 95%. The remaining Chromosomes formed composite peaks and could be sorted in groups of two to four. Twenty four wheat SSR markers were tested for their position on Chromosomes of Ae. umbellulata and Ae. comosa using PCR on DNA amplified from flow-sorted Chromosomes and genomic DNA of wheat-Ae. geniculata addition lines, respectively. Six SSR markers were located on particular Aegilops Chromosomes using sorted Chromosomes, thus confirming the usefulness of this approach for physical mapping. The SSR markers are suitable for marker assisted selection of wheat-Aegilops introgression lines. The results obtained in this work provide new opportunities for dissecting genomes of wild relatives of wheat with the aim to assist in alien gene transfer and discovery of novel genes for wheat improvement.