Cyclin-dependent kinases (CDK) play a key role in coordinating cell division in all eukaryotes. We investigated the capability of cyclin-dependent kinases CDKA and CDKB from the green alga Chlamydomonas reinhardtii to complement a Saccharomyces cerevisiae cdc28 temperature-sensitive mutant. The full-length coding regions of algal CDKA and CDKB cDNA were amplified by RT-PCR and cloned into the yeast expression vector pYES-DEST52, yielding pYD52-CDKA and pYD52-CDKB. The S. cerevisiae cdc28-1N strain transformed with these constructs exhibited growth at 36 degrees C in inducing (galactose) medium, but not in repressing (glucose) medium. Microscopic observation showed that the complemented cells had the irregular cylindrical shape typical for G2 phase-arrested cells when grown on glucose at 36 degrees C, but appeared as normal budded cells when grown on galactose at 36 degrees C. Sequence analysis and complementation tests proved that both CDKA and CDKB are functional CDC28/cdc2 homologs in C. reinhardtii. The complementation of the mitotic phenotype of the S. cerevisiae cdc28-1N mutant suggests a mitotic role for both of the kinases.
- MeSH
- Chlamydomonas reinhardtii enzymologie MeSH
- cyklin-dependentní kinasy chemie metabolismus MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- proteinkinasa CDC28, S cerevisiae metabolismus MeSH
- Saccharomyces cerevisiae cytologie enzymologie MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- teplota * MeSH
- testy genetické komplementace * MeSH
- transformace genetická MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cyklin-dependentní kinasy MeSH
- proteinkinasa CDC28, S cerevisiae MeSH
We developed an alternative method of staining cell nuclei and chloroplast nucleoids of algal cells using SYBR Green I (the fluorescent dye used commonly for detecting dsDNA in agarose and polyacrylamide gels as an alternative to highly mutagenic ethidium bromide and for DNA staining of viruses and bacteria followed by flow cytometry, digital image analysis or real-time PCR), which enabled routine staining in vivo. Cells do not need to be fixed or treated chemically or physically before staining, thus the shape, size and position of DNA-containing structures are not affected. The fluorescence signal is sharp and reproducible. Examples of application of the method are shown in color microphotographs for representatives of eukaryotic algae from the taxa Chlorophyta, Rhodophyta and the prokaryotic Cyanophyta. The method is also useful for studying progress of the cell cycle in algal cells dividing by multiple fission, as shown by observation of changes in nuclear number during the cell cycle of the green alga Chlamydomonas reinhardtii and Scenedesmus quadricauda. Staining with SYBR Green I can be recommended as a fast, safe and efficient method for the detection of DNA-containing structures in vivo.
- MeSH
- barvení a značení metody MeSH
- benzothiazoly MeSH
- buněčný cyklus MeSH
- chinoliny MeSH
- Chlorophyta genetika fyziologie ultrastruktura MeSH
- diaminy MeSH
- DNA řas analýza MeSH
- fluorescenční barviva metabolismus MeSH
- organické látky metabolismus MeSH
- permeabilita buněčné membrány MeSH
- počítačové zpracování obrazu MeSH
- polymerázová řetězová reakce MeSH
- průtoková cytometrie MeSH
- reprodukovatelnost výsledků MeSH
- Rhodophyta genetika fyziologie ultrastruktura MeSH
- sinice genetika fyziologie ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- benzothiazoly MeSH
- chinoliny MeSH
- diaminy MeSH
- DNA řas MeSH
- fluorescenční barviva MeSH
- organické látky MeSH
- SYBR Green I MeSH Prohlížeč
The effect of cadmium on growth processes (accumulation of RNA, proteins and cell volume), cell cycle reproductive events (DNA replication, mitosis, protoplast fission and daughter-cell formation) and the regulatory activity of histone H1 kinases were monitored in synchronized cultures of the chlorococcal alga Scenedesmus quadricauda. Distinct dosage-dependent inhibitory effects of cadmium ions were found in individual growth and reproductive processes. At concentration of about 60 mumol/L CdCl2, the growth processes were slowed down after about half of the cell cycle but the cells grew to the same or larger size than did untreated cells. At higher concentration, the growth became progressively inhibited, being completely blocked above 240 mumol/L. Total RNA accumulation was the most sensitive growth process. Each of the reproductive events was a target for cadmium ions with increasing sensitivity in the following order: DNA replication, mitosis, protoplast fission and daughter cell formation. Throughout the entire experiment, the activity of "mitosis-specific" histone H1 kinases was negligible in the cadmium (60 mumol/L CdCl2) treated cultures, whilst that of the control culture varied, peaking just prior to nuclear divisions. The activity of "growth-associated" histone H1 kinases was not affected by cadmium ions. No effect was found if cadmium was present during the precommitment period. The longer the period in the presence of cadmium, the stronger inhibition of reproductive events.
- MeSH
- buněčné dělení účinky léků MeSH
- buněčné jádro účinky léků MeSH
- chlorid kademnatý toxicita MeSH
- kultivační média farmakologie MeSH
- proteinkinasy metabolismus MeSH
- Scenedesmus cytologie účinky léků růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorid kademnatý MeSH
- histone H1 kinase MeSH Prohlížeč
- kultivační média MeSH
- proteinkinasy MeSH
Routinely prepared gametes (by flooding 3 week-old agar cultures) showed about 80% mating competence if the opposite sexual partners were mixed together. The mating competence exhibited a strict dependence on the composition of the solution in which the cells were suspended before mixing; it decreased progressively with increasing concentration of nitrates. In contrast, no inhibiting effect was found if urea was used as the source of nitrogen. Other ions present in nutrient media did not show any effect. Mating activity varied according to the spectral composition of light, being higher with a blue light than with a red one. Blue light caused accumulation of vis-à-vis pairs, which were blocked to form zygotes. Freshly released daughter cells in vegetatively grown synchronous cultures had a dual nature--vegetative and sexual one. In these daughter cells, similar rules were found for governing of mating competence to those valid for standard gametes obtained from flooded agar cultures. High mating competence was found in daughter cells released the during dark period in distilled water, nitrate-free media, in the presence of Mg2+ or Ca2+ ions, or in media containing urea. The conditions during which daughter cells are released and the conditions under which they mate can be considered crucial for expression of gametic nature as a mating competence.
- MeSH
- Chlamydomonas růst a vývoj fyziologie MeSH
- dusičnany metabolismus MeSH
- kultivační média chemie MeSH
- močovina metabolismus MeSH
- rozmnožování fyziologie MeSH
- světlo * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dusičnany MeSH
- kultivační média MeSH
- močovina MeSH
Using both monoclonal and polyclonal antibodies against mammalian plectin (multifunctional protein cross-linking cytoskeletal structures, mainly intermediate filaments, in mammalian cells), several putative isoforms of plectin-like proteins were found in protein extracts from the green alga Chlamydomonas eugametos (Volvocales). Immunofluorescence and immunoblotting revealed that some of the plectin-like proteins were present in perinuclear region or localized near the cell wall, probably being attached to the cytoplasmic membrane.
- MeSH
- bílkoviny řas metabolismus MeSH
- Chlamydomonas metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fluorescenční protilátková technika MeSH
- imunoblotting MeSH
- králíci MeSH
- plektin MeSH
- proteiny intermediálních filament metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bílkoviny řas MeSH
- plektin MeSH
- proteiny intermediálních filament MeSH
