Higher order RNA structures can mask splicing signals, loop out exons, or constitute riboswitches all of which contributes to the complexity of splicing regulation. We identified a G to A substitution between branch point (BP) and 3' splice site (3'ss) of Saccharomyces cerevisiae COF1 intron, which dramatically impaired its splicing. RNA structure prediction and in-line probing showed that this mutation disrupted a stem in the BP-3'ss region. Analyses of various COF1 intron modifications revealed that the secondary structure brought about the reduction of BP to 3'ss distance and masked potential 3'ss. We demonstrated the same structural requisite for the splicing of UBC13 intron. Moreover, RNAfold predicted stable structures for almost all distant BP introns in S. cerevisiae and for selected examples in several other Saccharomycotina species. The employment of intramolecular structure to localize 3'ss for the second splicing step suggests the existence of pre-mRNA structure-based mechanism of 3'ss recognition.
- MeSH
- Ascomycota genetika MeSH
- fungální RNA chemie MeSH
- introny * MeSH
- kofilin 1 genetika MeSH
- konformace nukleové kyseliny MeSH
- místa sestřihu RNA * MeSH
- molekulární sekvence - údaje MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvence nukleotidů MeSH
- sestřih RNA * MeSH
- teplota MeSH
- ubikvitin konjugující enzymy genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- COF1 protein, S cerevisiae MeSH Prohlížeč
- fungální RNA MeSH
- kofilin 1 MeSH
- místa sestřihu RNA * MeSH
- Saccharomyces cerevisiae - proteiny MeSH
- UBC13 protein, S cerevisiae MeSH Prohlížeč
- ubikvitin konjugující enzymy MeSH
May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.
- MeSH
- lidé MeSH
- missense mutace * MeSH
- molekulární motory chemie genetika MeSH
- mutační analýza DNA metody MeSH
- těžké řetězce myosinu chemie genetika MeSH
- tranzitní teplota MeSH
- trombocytopenie krev genetika MeSH
- trombocyty patologie MeSH
- velikost buňky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- molekulární motory MeSH
- MYH9 protein, human MeSH Prohlížeč
- těžké řetězce myosinu MeSH
The main regulatory mechanism of smooth muscle contraction involves Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin (CDPM), by myosin light chain kinase (MLCK). It is also known that the increase in intracellular Ca2+ and phosphorylation of myosin occurs within a short time under physiological conditions, but the muscle tension may persist for a longer period of time. However, the mechanism of this phenomenon is still not clear. We hypothesize that MLCK also phosphorylates myosin in a Ca2+/CaM-independent manner (CIPM). The difference between CIPM and CDPM are as follows. Firstly, the extent of CIPM by MLCK was temperature-independent, whereas CDPM by MLCK was apparently decreasing with increasing temperature. Secondly, in contrast to the decreased extent of CDPM, the prolongation of incubation time did not decrease the extent of CIPM. Thirdly, a high concentration of K+ influences CIPM less than CDPM. Furthermore, the MLCK inhibitor ML-9 significantly inhibited CDPM by MLCK but not CIPM by MLCK. Lastly, arachidonic acid selectively increased CIPM by MLCK but not CDPM by MLCK. Finally, the activity of Mg2+-ATPase of myosin followed the sequence as this: CDPM>CIPM>unphosphorylated myosin. Our results revealed some primary features of CIPM by MLCK.
- MeSH
- azepiny farmakologie MeSH
- časové faktory MeSH
- chlorid draselný MeSH
- fosforylace MeSH
- inhibitory enzymů farmakologie MeSH
- kalmodulin metabolismus MeSH
- kinasa lehkého řetězce myosinu antagonisté a inhibitory metabolismus MeSH
- kur domácí MeSH
- kyselina arachidonová farmakologie MeSH
- lehké řetězce myosinu chemie metabolismus MeSH
- myosiny metabolismus MeSH
- osmolární koncentrace MeSH
- teplota MeSH
- vápník metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- azepiny MeSH
- chlorid draselný MeSH
- inhibitory enzymů MeSH
- kalmodulin MeSH
- kinasa lehkého řetězce myosinu MeSH
- kyselina arachidonová MeSH
- lehké řetězce myosinu MeSH
- ML 9 MeSH Prohlížeč
- myosiny MeSH
- vápník MeSH
We developed a simple and reliable technique for immunofluorescence detection of F-actin on microtome sections of plant tissues. For the first time, large numbers of plant cells from various tissues that pass through their developmental stages could be consistently visualized on one section from plant organs. n-Maleimidobenzoic acid N-hydroxysuccinimide ester-pretreated and formalin-fixed segments of plant roots and shoots were embedded in low melting point ester wax at 37C and sectioned on a microtome. After dewaxing and rehydration, microfilaments were visualized by indirect immunofluorescence technique with a monoclonal anti-actin antibody. The technique has been successfully used for visualization of tissue- and development-specific F-actin arrays in cells of Zea mays and Lepidium sativum root tips and of maize stem nodes.
- MeSH
- aktiny analýza MeSH
- fixace tkání MeSH
- fluorescenční protilátková technika nepřímá * MeSH
- kořeny rostlin chemie MeSH
- kukuřice setá MeSH
- mikrofilamenta chemie ultrastruktura MeSH
- stonky rostlin chemie MeSH
- teplota MeSH
- zalévání tkání MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktiny MeSH
The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts of Saccharomyces cerevisiae DBY 1693 containing the act1-1 allele. Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons. In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized. After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth. This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature. Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts. These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope. A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of beta-(1-->3)-D-glucan wall microfibrils in yeast cells.
- MeSH
- aktiny genetika metabolismus MeSH
- buněčná stěna metabolismus ultrastruktura MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- fungální proteiny genetika metabolismus MeSH
- leptání mrazem MeSH
- molekulární modely MeSH
- protoplasty metabolismus ultrastruktura MeSH
- Saccharomyces cerevisiae genetika růst a vývoj ultrastruktura MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktiny MeSH
- fungální proteiny MeSH
A temperature-sensitive, conditionally lethal actin mutant of Saccharomyces cerevisiae, DBY 1693, was used to study, using light and electron microscopy, dysfunction of the actin cytoskeleton in the morphogenesis of the cell wall. Cells of this mutant strain survived at least 24 h at the restrictive temperature (37 degrees C). These cells showed isodiametric growth. Mutant cells accumulated vesicles, probably as a consequence of chaotic secretory transport caused by loss of polarity. A conspicuous morphological response to the dysfunction of actin was the formation of an aberrant wall over the whole surface of the isodiametrically-growing cell. This wall was of loose texture with protruding glucan microfibrils incompletely masked with amorphous matrix. It resembled the regenerating cell wall on the surfaces of yeast protoplasts. The localization of wall synthesis over the whole surface of temperature sensitive actin mutant cells was in accordance with an even distribution of submembranous actin in the form of patches (similarly to regenerating protoplasts). Delocalization of finger-like invaginations of the plasma membrane from the bud region to the whole surface of the growing cell was also found in mutant cells.
- MeSH
- aktiny genetika metabolismus ultrastruktura MeSH
- buněčná stěna metabolismus ultrastruktura MeSH
- buněčné dělení MeSH
- buněčné jádro ultrastruktura MeSH
- buněčný cyklus MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- elektronová mikroskopie MeSH
- mrazové lámání MeSH
- mutace MeSH
- polarita buněk MeSH
- Saccharomyces cerevisiae genetika metabolismus ultrastruktura MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aktiny MeSH
The conformational stability of erythrocyte spectrin and brain spectrin-like protein (fodrin) has been studied by circular dichroism. In agreement with previous reports the circular dichroism spectra of both proteins in the peptide region were almost identical. The essential differences, on the other hand, were found in the near u.v. range, most probably due to differences in the conformation of intrachain disulphide bonds. Heat denaturation curves, relating to the level of secondary structure (ellipticity at 221 nm) showed that fodrin is more stable than spectrin: curves of reversible as well as irreversible denaturation are shifted to higher temperatures and also the amount of alpha-helices in the denatured state is higher. Spectrin conformation was found to be very sensitive to the presence of water-soluble organic solvents; the denaturation curves exhibit maxima and minima not typical of protein isothermic denaturation. The observed low conformational stability of spectrin is discussed in the context of its molecular environment and function in the red cell membrane.
- MeSH
- cirkulární dichroismus MeSH
- denaturace proteinů MeSH
- disulfidy MeSH
- konformace proteinů MeSH
- lidé MeSH
- mikrofilamentové proteiny chemie MeSH
- mozek - chemie MeSH
- proteiny nervové tkáně chemie MeSH
- skot MeSH
- spektrin chemie MeSH
- teplota MeSH
- transportní proteiny chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- disulfidy MeSH
- fodrin MeSH Prohlížeč
- mikrofilamentové proteiny MeSH
- proteiny nervové tkáně MeSH
- spektrin MeSH
- transportní proteiny MeSH
