Nejvíce citovaný článek - PubMed ID 16408852
Visualization of DNA-containing structures in various species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR Green I dye
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
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
Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.
- Klíčová slova
- Desmodesmus quadricauda, cell cycle, confocal Raman microscopy, guanine, lipids, microalgae, polyphosphate, starch,
- MeSH
- buněčná stěna chemie MeSH
- buněčný cyklus * MeSH
- časové faktory MeSH
- Chlorophyta cytologie růst a vývoj MeSH
- guanin analýza MeSH
- lipidová tělíska metabolismus MeSH
- lipidy analýza MeSH
- mikroskopie * MeSH
- polyfosfáty analýza MeSH
- Ramanova spektroskopie * MeSH
- škrob analýza MeSH
- velikost buňky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- guanin MeSH
- lipidy MeSH
- polyfosfáty MeSH
- škrob MeSH
Phosphorus is an essential element for life on earth and is also important for modern agriculture, which is dependent on inorganic fertilizers from phosphate rock. Polyphosphate is a biological polymer of phosphate residues, which is accumulated in organisms during the biological wastewater treatment process to enhance biological phosphorus removal. Here, we investigated the relationship between polyphosphate accumulation and electron-dense bodies in the green alga Parachlorella kessleri. Under sulfur-depleted conditions, in which some symporter genes were upregulated, while others were downregulated, total phosphate accumulation increased in the early stage of culture compared to that under sulfur-replete conditions. The P signal was detected only in dense bodies by energy dispersive X-ray analysis. Transmission electron microscopy revealed marked ultrastructural variations in dense bodies with and without polyphosphate. Our findings suggest that the dense body is a site of polyphosphate accumulation, and P. kessleri has potential as a phosphate-accumulating organism.
- MeSH
- barvení a značení MeSH
- biologické modely MeSH
- Chlorophyta cytologie růst a vývoj metabolismus ultrastruktura MeSH
- elektrony * MeSH
- fosfáty metabolismus MeSH
- lipidy chemie MeSH
- polyfosfáty metabolismus MeSH
- sekvenční analýza RNA MeSH
- síra metabolismus MeSH
- transkriptom genetika MeSH
- zobrazování trojrozměrné MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfáty MeSH
- lipidy MeSH
- polyfosfáty MeSH
- síra MeSH
DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.
- MeSH
- bleomycin farmakologie MeSH
- buněčný cyklus účinky léků genetika MeSH
- Chlorophyta MeSH
- floxuridin farmakologie MeSH
- G2 fáze genetika MeSH
- kofein farmakologie MeSH
- oprava DNA účinky léků MeSH
- poškození DNA fyziologie MeSH
- proteiny buněčného cyklu MeSH
- Scenedesmus cytologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bleomycin MeSH
- floxuridin MeSH
- kofein MeSH
- proteiny buněčného cyklu MeSH
- Zeocin MeSH Prohlížeč
Suitable morphological characteristics for identification of zygnematalean algae were determined using a combination of classical light microscopy (LM) techniques, fluorescence microscopy (with blue and green excitation), scanning electron microscopy (SEM) and specialized culture methods. Characteristics of spore germination, growth and reproduction under culture conditions identified Zygnema chalybeo-spermum in a mixture of zygnematalean spores collected from a small fishpond in Czechia. Reproduction in general, particularly aplanospore formation and lateral conjugation was more frequent and occurred earlier in a nutrient poor medium than in a nutrient rich medium, where vegetative growth was more vigorous. Variability in spore size was wide and influenced by the origin (sexual and/or asexual) of the spores. Asexual spores, particularly partenospores were rounded and significantly smaller than sexual ones. Thus spore morphology alone (size and shape) is not a particularly helpful characteristic for species identification. The mesospore of mature spores contained lipids and a sporopolenin-like material (algaenan), which showed green autofluorescence with blue excitation. The mesospore ornamentation, the only characteristic found that is suitable for identification purposes, can be observed easily in LM and SEM after exospore removal by KOH treatment. Detailed LM and SEM observations of the zygospores of all Zygnema species thus could provide basic data necessary for the preparation of an atlas and key for species identification which, after completion with molecular methods, brings clarification into the genetic relationships between morphospecies.
- MeSH
- druhová specificita MeSH
- Eukaryota klasifikace izolace a purifikace fyziologie MeSH
- kultivační média MeSH
- mikrobiologie vody MeSH
- mikroskopie MeSH
- sladká voda mikrobiologie MeSH
- spory hub izolace a purifikace ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- kultivační média MeSH
A technique was developed to evaluate alternative support systems to test tubes used in the standard most probable number technique, for simultaneous isolation and enumeration of cyanobacteria. Five different support systems were tested for their suitability in terms of accuracy, sensitivity, economics and ease of handling. PCR plates with 96 wells and carrying capacity of 300 microL per well were found to be most sensitive, besides being cost- and time-effective. This technique can also be useful for isolation of cyanobacteria, due to immobilization of colonies in the gel matrix and storage of samples at room temperature, without loss of viability for 5-6 weeks. This technique can help to process large sample size with ease--both for enumeration and isolation and can be extended for enumeration of other microorganisms from diverse sources.
