During mitotic prophase, chromosomes of the pathogenic unicellular eukaryote Giardia intestinalis condense in each of the cell's two nuclei. In this study, Giardia chromosomes were investigated using light microscopy, high-resolution field emission scanning electron microscopy, and in situ hybridization. For the first time, we describe the overall morphology, condensation stages, and mitotic segregation of these chromosomes. Despite the absence of several genes involved in the cohesion and condensation pathways in the Giardia genome, we observed chromatin organization similar to those found in eukaryotes, i.e., 10-nm nucleosomal fibrils, 30-nm fibrils coiled to chromomeres or in parallel arrangements, and closely aligned sister chromatids. DNA molecules of Giardia terminate with telomeric repeats that we visualized on each of the four chromatid endings of metaphase chromosomes. Giardia chromosomes lack primary and secondary constrictions, thus preventing their classification based on the position of the centromere. The anaphase poleward segregation of sister chromatids is atypical in orientation and tends to generate lagging chromatids between daughter nuclei. In the Giardia genome database, we identified two putative members of the kleisin family thought to be responsible for condensin ring establishment. Thus far, Giardia chromosomes (300 nm to 1.5 μm) are the smallest chromosomes that were analyzed at the ultrastructural level. This study complements the existing molecular and sequencing data on Giardia chromosomes with cytological and ultrastructural information.
- MeSH
- adenosintrifosfatasy analýza MeSH
- buněčné jádro ultrastruktura MeSH
- chromozomy fyziologie ultrastruktura MeSH
- DNA vazebné proteiny analýza MeSH
- Giardia lamblia genetika ultrastruktura MeSH
- mitóza MeSH
- multiproteinové komplexy analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The genus Xenopus represents important model organisms in the field of developmental biology and chromosomal evolution. Developmental processes are tightly coupled with the analysis of gene function via genetic linkage and mapping. Cytogenetic techniques such as chromosome banding or FISH are essential tools for the determination of gene position and subsequently for the construction of linkage and physical maps. Here, we present a summary of key achievements in X. tropicalis and X. laevis cytogenetics with emphasis on the gene localization to chromosomes. The second part of this review is focused on the chromosomal evolution regarding both above-mentioned species. With respect to methodology, hybridization techniques such as FISH and chromosome-specific painting FISH are highlighted.
- MeSH
- chromozomy genetika ultrastruktura MeSH
- diploidie MeSH
- druhová specificita MeSH
- genetické markery MeSH
- genom MeSH
- malování chromozomů MeSH
- mapování chromozomů MeSH
- molekulární evoluce * MeSH
- oocyty ultrastruktura MeSH
- polymorfismus genetický MeSH
- syntenie genetika MeSH
- tandemové repetitivní sekvence MeSH
- tetraploidie MeSH
- vznik druhů (genetika) * MeSH
- Xenopus laevis genetika MeSH
- Xenopus klasifikace genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations. 2011 Macmillan Publishers Limited. All rights reserved.
- MeSH
- buněčné jádro * metabolismus MeSH
- buněčný cyklus MeSH
- časové faktory MeSH
- chromatin metabolismus MeSH
- chromozomy * ultrastruktura MeSH
- DNA-helikasy metabolismus MeSH
- DNA * genetika metabolismus MeSH
- intracelulární signální peptidy a proteiny * metabolismus MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- mitóza * MeSH
- nádorové buněčné linie MeSH
- poškození DNA MeSH
- regulace genové exprese u nádorů MeSH
- replikace DNA * MeSH
- Check Tag
- lidé MeSH
The highly repetitive nature of the Trichomonas vaginalis genome and massive expansion of various gene families has caused difficulties in genome assembly and has hampered genome mapping. Here, we adapted fluorescence in situ hybridization (FISH) for T. vaginalis, which is sensitive enough to detect single copy genes on metaphase chromosomes. Sensitivity of conventional FISH, which did not allow single copy gene detection in T. vaginalis, was increased by means of tyramide signal amplification. Two selected single copy genes, coding for serine palmitoyltransferase and tryptophanase, were mapped to chromosome I and II, respectively, and thus could be used as chromosome markers. This established protocol provides an amenable tool for the physical mapping of the T. vaginalis genome and other essential applications, such as development of genetic markers for T. vaginalis genotyping.
- MeSH
- antigeny protozoální genetika MeSH
- chromozomy chemie genetika ultrastruktura MeSH
- DNA fingerprinting metody MeSH
- fluorescence MeSH
- genetické markery MeSH
- genom MeSH
- genotyp MeSH
- genová dávka MeSH
- hybridizace in situ fluorescenční metody MeSH
- lidé MeSH
- mapování chromozomů metody MeSH
- senzitivita a specificita MeSH
- serin-C-palmitoyltransferasa genetika MeSH
- Trichomonas vaginalis chemie cytologie genetika MeSH
- trichomoniáza genetika parazitologie MeSH
- tryptofanasa genetika MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The organisation of dinoflagellate chromosomes is exceptional among eukaryotes. Their genomes are the largest in the Eukarya domain, chromosomes lack histones and may exist in liquid crystalline state. Therefore, the study of the structural and functional properties of dinoflagellate chromosomes is of high interest. In this work, we have analysed the telomeres and telomerase in two Dinoflagellata species, Karenia papilionacea and Crypthecodinium cohnii. Active telomerase, synthesising exclusively Arabidopsis-type telomere sequences, was detected in cell extracts. The terminal position of TTTAGGG repeats was determined by in situ hybridisation and BAL31 digestion methods and provides evidence for the linear characteristic of dinoflagellate chromosomes. The length of telomeric tracts, 25-80 kb, is the largest among unicellular eukaryotic organisms to date. Both the presence of long arrays of perfect telomeric repeats at the ends of dinoflagellate chromosomes and the existence of active telomerase as the primary tool for their high-fidelity maintenance demonstrate the general importance of these structures throughout eukaryotes. We conclude that whilst chromosomes of dinoflagellates are unique in many aspects of their structure and composition, their telomere maintenance follows the most common scenario.
- MeSH
- chromatin genetika metabolismus MeSH
- chromozomy genetika metabolismus ultrastruktura MeSH
- Dinoflagellata genetika růst a vývoj metabolismus ultrastruktura MeSH
- kapalné krystaly MeSH
- protozoální DNA genetika MeSH
- repetitivní sekvence nukleových kyselin MeSH
- replikace DNA genetika MeSH
- telomerasa metabolismus MeSH
- telomery genetika metabolismus ultrastruktura MeSH
- zlomy DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mammalian chromosomes occupy chromosome territories within nuclear space the positions of which are generally accepted as non-random. However, it is still controversial whether position of chromosome territories/chromatin is maintained in daughter cells. We addressed this issue and investigated maintenance of various chromatin regions of unknown composition as well as nucleolus-associated chromatin, a significant part of which is composed of nucleolus organizer region-bearing chromosomes. The photoconvertible histone H4-Dendra2 was used to label such regions in transfected HepG2 cells, and its position was followed up to next interphase. The distribution of labeled chromatin in daughter cells exhibited a non-random character. However, its distribution in a vast majority of daughter cells extensively differed from the original ones and the labeled nucleolus-associated chromatin differently located into the vicinity of different nucleoli. Therefore, our results were not consistent with a concept of preservation chromatin position. This conclusion was supported by the finding that the numbers of nucleoli significantly differed between the two daughter cells. Our results support a view that while the transfected daughter HepG2 cells maintain some features of the parental cell chromosome organization, there is also a significant stochastic component associated with reassortment of chromosome territories/chromatin that results in their positional rearrangements.
- MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- časové faktory MeSH
- chromatin chemie metabolismus MeSH
- chromozomy ultrastruktura MeSH
- fibroblasty metabolismus MeSH
- financování organizované MeSH
- fluorescenční mikroskopie metody MeSH
- histony metabolismus MeSH
- lidé MeSH
- počítačové zpracování obrazu MeSH
- Check Tag
- lidé MeSH
The terminal chromatin structures at the ends of eukaryotic chromosomes, the telomeres, are a focus of intensive research due to their importance for the maintenance of chromosome integrity. Their shortening due to incomplete replication functions as a molecular clock counting the number of cell divisions, and ultimately results in cell-cycle arrest and cellular senescence. Telomere shortening can be compensated by the nucleoprotein enzyme complex called telomerase, which is able to extend shortened telomeres. In humans, only embryonic and germ cells show telomerase activity that is sufficient for telomere length stability and cellular immortality. Unfortunately, telomerase is activated in cancer cells, which, thus, achieve unlimited growth and a malignant phenotype. Even if there were no any other links of telomere biology to other essential processes in the cell nucleus such as DNA repair, chromosome positioning, and nuclear architecture in mitosis and meiosis, the close connection of telomere biology to aging and cancer makes telomeres and techniques for their analysis important enough from the point of view of us, mortal and disease-prone people. In this chapter, we describe the most common types of analyses used in telomere biology: screening for typical and variant telomeric sequences, determination of telomere lengths, and measurement of telomerase activity.
- MeSH
- buněčný cyklus MeSH
- chromozomy ultrastruktura MeSH
- endodeoxyribonukleasy metabolismus MeSH
- fenotyp MeSH
- genetické techniky MeSH
- hybridizace in situ fluorescenční MeSH
- lidé MeSH
- molekulová hmotnost MeSH
- oprava DNA MeSH
- polymerázová řetězová reakce MeSH
- restrikční enzymy metabolismus MeSH
- sefarosa chemie MeSH
- stárnutí buněk MeSH
- telomerasa metabolismus MeSH
- telomery ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
