Browsing by Subject "actin"
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(2016)Actin is known as abundant cytoplasmic protein, which functions as a component of the cytoskeleton and in cell motility together with motor protein myosins. However, actin is also present in the nucleus, where it has been shown to take part in the control of gene expression, both independently and as part of chromatin remodeling complexes. An important aspect in the study of nuclear actin is to identify other nuclear proteins interacting with actin and to confirm these interactions in biochemical experiments. Through these interactions actin can be linked to various nuclear processes. The aim of this master’s thesis study was to express and purify five nuclear proteins that have been suggested to bind actin and to study the binding in detail both with actin filaments and monomers. The proteins of interest include four actin-related proteins (ARPs) Arp4, Arp5, Arp6 and Arp8 as well as RNA polymerase II subunit Rpb8. Out of the proteins selected for this study, the expression and purification of Arp4 and Rpb8 was successful. Neither one did bind monomeric actin with high affinity, but interestingly Rpb8 did bind actin filaments. On the other hand, Rpb8 did not have any effect on actin polymerization. These results provide new insights into nuclear actin function. It has been suggested earlier that Arp4 would form a heterocomplex with actin, but the results of this study do not support this. The binding between Rpb8 and actin in RNA polymerase II complex has not been extensively studied before, so the results provide new information about the function of actin in the polymerase complex.
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(2022)Abstract Background. Faba bean (Vicia faba L.,) is a protein-rich grain crop and a good source of many nutrients. However, the productivity and composition of this crop can be limited by several biotic and abiotic stresses. Acid soil leads to a reduction in water uptake, and influences on major biological and chemical processes. However, some plants can manage stress. Mostly, low pH reduces plant vegetative organs and a has a negative effect on physiological processes such as gas exchange, photosynthesis, chlorophyll concentration, and transpiration. The aim of this study was to find homolog genes in close spp. and o test selected gene response as a factor in resilience to rootzone acidity in Vicia faba L. Methods. Four faba bean genotypes were selected for our experiment based on germplasm screening in aquaponic conditions from a previous study. According to obtained results of morpho-physiological measures from a previous experiment, IG11204 and Bourdon showed tolerance to acid only. DULUC is tolerant to acid and Al treatments. In contrast, Nora38 demonstrated susceptibility to both stresses. Selected genotypes were grown under the same experimental conditions in order to proceed with samples for further analysis (total RNA extraction, cDNA synthesis, RT-PCR, gel-electrophoresis, validation of selected gene by RT-qPCR). Results. Selected genes have been identified as hub genes in A. thaliana (alternative oxidase 1A, glutamate dehydrogenase 1, zinc fingers superfamily protein), which demonstrated an increase in expression levels under acidic pH stress. We have found homolog genes in the Hedin cultivar. Selected homolog gene were used to check its expression in four genotypes under Al3+ and acidic stresses using RT-qPCR. Finally, up-regulation of the GDH1 was detected by acid and also occurred by Al3+ treatments. Discussion. Our analysis of gene expression revealed that GDH1 was expressed differently between control and treated groups. However, its activity was detected for both stresses. The expression of GDH1 transcripts in this study, also concurs with findings where GDH1 in A. thaliana were expressed under Al3+ stress. Furthermore, it was confirmed that they are novel Al3+ resistance genes, which are regulated by the transcription factor STOP1 (sensitive-to-proton-rhizotoxicity1). GDHs are involved in stress tolerance, and pH-regulated metabolic pathways – GABA shunt. It participates in the regulation of cytosolic pH, carbon fluxes into the TCA cycle, nitrogen metabolism, deterrence of insects, protection against oxidative stress, osmoregulation and signaling in plant cells under different stress conditions. The GABA shunt is enhanced by stresses, which cause cytosol acidification. Genes expressed in conditions of low pH partly overlap with those expressed in plants exposed to Al3+ stress. Generally, acidic soil is associated with the co-occurrence of Al3+ and H+ rhizotoxicities.
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