A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light-dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light-dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in such cultures is presented, including analyses of nuclear, cellular, and chloroplast divisions. Modifications of basic protocols that enable changes in cell cycle progression are also suggested so that nuclear or chloroplast divisions can be followed separately.

• Klíčová slova
• Cell division, Chloroplast division, DNA replication, Green algae, Growth, Illumination regime, Multiple fission cell cycle, Nuclear division, Synchronization,
• MeSH
• barvení a značení metody   MeSH
• buněčné dělení MeSH
• buněčné kultury metody   MeSH
• buněčný cyklus MeSH
• Chlamydomonas reinhardtii cytologie   genetika   růst a vývoj   MeSH
• Chlorophyta cytologie   genetika   růst a vývoj   MeSH
• chloroplasty genetika   MeSH
• DNA rostlinná genetika   MeSH
• fotoperioda * MeSH
• frakcionace buněk metody   MeSH
• replikace DNA MeSH
• světlo MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH

The analysis of structure and metabolism of a cell at a defined phase of cell cycle is often difficult because cell cycle progression in somatic tissues is asynchronous and only a fraction of cells are cycling. An elegant solution to obtain populations of cells enriched for single stage of the cell cycle is to impose the synchrony artificially. Different systems have been used to obtain synchronized populations of plant cells, including suspension-cultured cells, leaf mesophyll protoplasts and root tip meristems. Root tips have been frequently used in a variety of studies ranging from chromosome analysis to cell cycle and its regulation. Seedlings with actively growing roots may be obtained in most plant species, they are easy to handle, the experimental system is well defined, reproducible and can be easily modified for different species. This paper describes a protocol for cell cycle synchronization in root tips of Vicia faba, which is based on the use of DNA synthesis inhibitor hydroxyurea [18]. Modifications of the protocol for Pisum sativum, Medicago sativa, Hordeum vulgare, Secale cereale, Triticum aestivum, and Zea mays are also given. Flow cytometric data indicate that about 90% of root tip cells are synchronized. On average, mitotic indices exceeding 50% are obtained with the method. Synchronized cells may be accumulated at metaphase using a mitotic spindle inhibitor to achieve metaphase indices exceeding 50%.

• MeSH
• časové faktory MeSH
• CDC geny fyziologie   MeSH
• fyziologie rostlin * MeSH
• kořenová čepička rostlin fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development.

• MeSH
• algoritmy MeSH
• buněčný cyklus genetika   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory genetika   metabolismus   patologie   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• transkriptom * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• validační studie MeSH
• Názvy látek
• proteiny buněčného cyklu MeSH
• transkripční faktory MeSH

The success of somatic cell nuclear transfer depends critically on the cell cycle stage of the donor nucleus and the recipient cytoplast. Karyoplasts in the G0 or G1 stages are considered to be the most suitable for nuclear transfer. In the present study, we used a reversible cell cycle inhibitor, mimosine, to synchronize porcine granulosa cells (GCs) in G1 phase of the cell cycle. Porcine GCs were obtained from 3 to 5mm ovarian follicles of slaughtered gilts. The effect of mimosine on the proliferation, DNA synthesis and cell cycle stage of cultured cells was examined by incorporation of radiochemical 3H-thymidine, immunocytochemical detection of incorporated thymidine analogue 5-bromo-2-deoxyuridine (BrdU) and flow cytometry analyses. Mimosine treatment of pig GCs for 24h resulted in proliferation arrest in vitro. Treatment with 0.5mM mimosine significantly (P<0.05) inhibited 3H-thymidine incorporation after 24h of culture (4.6% +/- 0.1) and after 24h of culture in serum deprived medium (41.3% +/- 3.8), in comparison to controls (100%). Inhibition of DNA synthesis was further confirmed by immunocytochemical and flow cytometry analyses. Compared with controls (78.2%), mimosine treatment for 24h increased the proportion of G0/G1 cells in the culture (85.7%) more effectively than serum starvation (SS; 81.2%). Mimosine-caused G1 arrest of porcine GCs was fully reversible and cells continued to proliferate after removing the drug, especially when they were stimulated by EGF.

• MeSH
• buněčné dělení účinky léků   MeSH
• buněčný cyklus účinky léků   MeSH
• časové faktory MeSH
• DNA biosyntéza   MeSH
• epidermální růstový faktor farmakologie   MeSH
• folikulární buňky cytologie   účinky léků   MeSH
• G1 fáze * MeSH
• imunohistochemie MeSH
• kultivované buňky MeSH
• mimosin aplikace a dávkování   farmakologie   MeSH
• prasata * MeSH
• průtoková cytometrie MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• epidermální růstový faktor MeSH
• mimosin MeSH

The extremophilic unicellular red microalga Galdieria sulphuraria (Cyanidiophyceae) is able to grow autotrophically, or mixo- and heterotrophically with 1% glycerol as a carbon source. The alga divides by multiple fission into more than two cells within one cell cycle. The optimal conditions of light, temperature and pH (500 µmol photons m-2 s-1, 40 °C, and pH 3; respectively) for the strain Galdieria sulphuraria (Galdieri) Merola 002 were determined as a basis for synchronization experiments. For synchronization, the specific light/dark cycle, 16/8 h was identified as the precondition for investigating the cell cycle. The alga was successfully synchronized and the cell cycle was evaluated. G. sulphuraria attained two commitment points with midpoints at 10 and 13 h of the cell cycle, leading to two nuclear divisions, followed subsequently by division into four daughter cells. The daughter cells stayed in the mother cell wall until the beginning of the next light phase, when they were released. Accumulation of glycogen throughout the cell cycle was also described. The findings presented here bring a new contribution to our general understanding of the cell cycle in cyanidialean red algae, and specifically of the biotechnologically important species G. sulphuraria.

• Klíčová slova
• Galdieria, cell cycle, cell division, growth, light intensity, red algae, synchronization, temperature, trophic regimes,
• MeSH
• buněčný cyklus fyziologie   MeSH
• heterotrofní procesy fyziologie   MeSH
• kultivované buňky MeSH
• mikrořasy cytologie   růst a vývoj   MeSH
• Rhodophyta cytologie   růst a vývoj   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks-deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations.

• Klíčová slova
• DNA replication, S phase, cell cycle, deoxyribonucleotide triphosphates synthesis, ribonucleotide reductase, thymidine, thymidylate synthase,
• MeSH
• buněčné dělení * MeSH
• buněčný cyklus * MeSH
• DNA antagonisté a inhibitory   biosyntéza   MeSH
• lidé MeSH
• replikace DNA * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH

Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.

• MeSH
• buněčný cyklus fyziologie   MeSH
• časové faktory MeSH
• chromozomy rostlin * metabolismus   MeSH
• genom rostlinný genetika   MeSH
• genomika metody   MeSH
• hydroxymočovina MeSH
• metafáze MeSH
• mitotický index MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny MeSH
• průtoková cytometrie metody   MeSH
• pufry MeSH
• Saccharum cytologie   genetika   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amiprophos methyl MeSH Prohlížeč
• hydroxymočovina MeSH
• nitrobenzeny MeSH
• organothiofosforové sloučeniny MeSH
• pufry MeSH

During environmental stress, the vegetative cells of the facultative pathogenic amoeba Acanthamoeba castellanii reversibly differentiate into resistant dormant stages, namely, cysts or pseudocysts. The type of resistant stage depends on the nature and duration of the stressor. Cell differentiation is accompanied by changes in morphology and cellular metabolism. Moreover, cell differentiation is also expected to be closely linked to the regulation of the cell cycle and, thus, to cellular DNA content. While the existence of the resistant stages in A. castellanii is well known, there is no consensus regarding the relationship between differentiation and cell cycle progression. In the present work, we used flow cytometry analysis to explore the changes in the DNA content during Acanthamoeba encystation and pseudocyst formation. Our results strongly indicate that A. castellanii enters encystation from the G2 phase of the cell cycle. In contrast, differentiation into pseudocysts can begin in the G1 and G2 phases. In addition, we present a phylogenetic analysis and classification of the main cell cycle regulators, namely, cyclin-dependent kinases and cyclins that are found in the genome of A. castellanii.

• Klíčová slova
• Acanthamoeba, Cell cycle, Cyclin-dependent kinase, Cysts, Pseudocysts, Synchronization,
• MeSH
• Acanthamoeba castellanii klasifikace   genetika   MeSH
• buněčná diferenciace genetika   MeSH
• fylogeneze MeSH
• fyziologický stres genetika   MeSH
• proteiny buněčného cyklu genetika   MeSH
• protozoální DNA analýza   MeSH
• průtoková cytometrie MeSH
• stadia vývoje genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny buněčného cyklu MeSH
• protozoální DNA MeSH

Multiple fission is a cell cycle variation leading to the production of more than two daughter cells. Here, we used synchronized cultures of the chlorococcal green alga Parachlorella kessleri to study its growth and pattern of cell division under varying light intensities. The time courses of DNA replication, nuclear and cellular division, cell size, total RNA, protein content, dry matter and accumulation of starch were observed at incident light intensities of 110, 250 and 500 µmol photons m-2s-1. Furthermore, we studied the effect of deuterated water on Parachlorella kessleri growth and division, to mimic the effect of stress. We describe a novel multiple fission cell cycle pattern characterized by multiple rounds of DNA replication leading to cell polyploidization. Once completed, multiple nuclear divisions were performed with each of them, immediately followed by protoplast fission, terminated by the formation of daughter cells. The multiple fission cell cycle was represented by several consecutive doublings of growth parameters, each leading to the start of a reproductive sequence. The number of growth doublings increased with increasing light intensity and led to division into more daughter cells. This study establishes the baseline for cell cycle research at the molecular level as well as for potential biotechnological applications, particularly directed synthesis of (deuterated) starch and/or neutral lipids as carbon and energy reserves.

• Klíčová slova
• Parachlorella kessleri, cell cycle pattern, deuterated lipid, deuterated starch, deuterium, energy reserves, growth processes, light intensity, reproduction events,
• MeSH
• buněčné kultury * MeSH
• buněčný cyklus * MeSH
• Chlorophyta růst a vývoj   MeSH
• světlo * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Green algae dividing by multiple fission comprise unrelated genera but are connected by one common feature: under optimal growth conditions, they can divide into more than two daughter cells. The number of daughter cells, also known as the division number, is relatively stable for most species and usually ranges from 4 to 16. The number of daughter cells is dictated by growth rate and is modulated by light and temperature. Green algae dividing by multiple fission can thus be used to study coordination of growth and progression of the cell cycle. Algal cultures can be synchronized naturally by alternating light/dark periods so that growth occurs in the light and DNA replication(s) and nuclear and cellular division(s) occur in the dark; synchrony in such cultures is almost 100% and can be maintained indefinitely. Moreover, the pattern of cell-cycle progression can be easily altered by differing growth conditions, allowing for detailed studies of coordination between individual cell-cycle events. Since the 1950s, green algae dividing by multiple fission have been studied as a unique model for cell-cycle regulation. Future sequencing of algal genomes will provide additional, high precision tools for physiological, taxonomic, structural, and molecular studies in these organisms.

• Klíčová slova
• Cell division, DNA replication, cell size, green algae, growth, light, multiple fission cell cycle, nuclear division, temperature.,
• MeSH
• buněčný cyklus * MeSH
• Chlorophyta cytologie   genetika   MeSH
• replikace DNA MeSH
• světlo MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH