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
- Algorithms MeSH
- Cell Cycle genetics MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Neoplasms genetics metabolism pathology MeSH
- Cell Cycle Proteins genetics metabolism MeSH
- Transcription Factors metabolism MeSH
- Transcriptome * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
- Validation Study 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
- Cell Cycle physiology MeSH
- Time Factors MeSH
- Chromosomes, Plant * metabolism MeSH
- Genome, Plant genetics MeSH
- Genomics methods MeSH
- Hydroxyurea MeSH
- Metaphase MeSH
- Mitotic Index MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
- Flow Cytometry methods MeSH
- Buffers MeSH
- Saccharum cytology genetics MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- Staining and Labeling methods MeSH
- Cell Division MeSH
- Cell Culture Techniques methods MeSH
- Cell Cycle MeSH
- Chlamydomonas reinhardtii cytology genetics growth & development MeSH
- Chlorophyta cytology genetics growth & development MeSH
- Chloroplasts genetics MeSH
- DNA, Plant genetics MeSH
- Photoperiod * MeSH
- Cell Fractionation methods MeSH
- DNA Replication MeSH
- Light MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- MeSH
- Cell Cycle * MeSH
- Chlorophyta cytology genetics MeSH
- DNA Replication MeSH
- Light MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
This study is a thorough examination of the effects of the DNA polymerase inhibitor aphidicolin on the nuclear cycle and cell cycle progression characteristics, as well as their reversibility, in Giardia intestinalis. Giardia trophozoites are arrested in the G1/S-junction after aphidicolin treatment according to their DNA content. However, cell growth continues and trophozoites arrested with aphidicolin resemble cells in the G2 phase and trophozoites in ageing cultures. Extensive treatment with aphidicolin causes side effects and we detected positive signals for phosphorylated histone H2A, which, in mammalian cells, is involved in a signalling pathway triggered as a reaction to double stranded DNA breaks. These results suggest that aphidicolin causes dissociation of the nuclear and cytoplasmic cycles, a phenomenon that has also been described for other inhibitors in mammalian cell lines. Thus, if aphidicolin is used for synchronization of Giardia trophozoites, this fact must be accounted for, and treatment with aphidicolin must be minimal. Copyright 2009 Elsevier Inc. All rights reserved.
- MeSH
- Aphidicolin pharmacology MeSH
- Bromodeoxyuridine metabolism MeSH
- Cell Cycle drug effects MeSH
- Time Factors MeSH
- Cyclin B analysis MeSH
- DNA-Directed DNA Polymerase MeSH
- Fluorescent Antibody Technique MeSH
- Phosphorylation drug effects MeSH
- Giardia lamblia cytology genetics drug effects MeSH
- Histones metabolism MeSH
- Enzyme Inhibitors pharmacology MeSH
- Nucleic Acid Synthesis Inhibitors MeSH
- Mitotic Index MeSH
- DNA Damage drug effects MeSH
- DNA, Protozoan biosynthesis drug effects MeSH
- Flow Cytometry MeSH
- DNA Replication drug effects MeSH
- Trophozoites cytology drug effects MeSH
- Publication type
- Research Support, Non-U.S. Gov't 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.
- MeSH
- Cell Division * MeSH
- Cell Cycle * MeSH
- DNA antagonists & inhibitors biosynthesis MeSH
- Humans MeSH
- DNA Replication * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
