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Alternativní telomerické kompenzační mechanismy jako způsob adaptace telomer vůči stresovým podmínkám v průběhu evoluce hmyzu
KRŮČEK, Tomáš
Telomeres are special nucleoprotein structures at the ends of eukaryotic chromosomes. Because of incomplete DNA replication telomere length becomes shorter with each cell cycle. Several mechanisms of telomere maintenance have been identified. Although the most common mechanism is the activity of telomerase that synthesizes short telomeric sequences onto chromosome ends, telomere length might be elongated by alternative ways such as the retrotransposition of special telomeric mobile elements targeted to chromosome ends, found in Drosophila, and homologous recombination. Homologous recombination extends satelite sequences in some dipteran species and also serves in vertebrates as the alternative mechanism to telomerase. The (TTAGG)n sequence was designated as the insect telomeric sequence. Although the (TTAGG)n sequence was found at telomeres of most of the tested insect orders in previous studies, there are numerous insect species showing the (TTAGG)n absence. Diptera is the large insect order, which shows the lost of the (TTAGG)n sequence together with telomerase system and its replacement by the transposition of telomeric elements in Drosophila or homologous recombination in mosquitoes or midges. The (TTAGG)n sequence was reported as telomeric sequence in most insect orders, however, the (TTAGG) specific telomerase activity was not tested in these orders.Therefore, I first speculated that non-telomerase systems in insects may not be limited only to Diptera and the presence of non-telomerase systems in insect might be underestimated. When I tested a distribution of the TTAGG-specific telomerase activity I confirmed that the telomerase system is the most comon telomere length compensation mechanism in insect, however, it is not present in Zygentoma, Orthoptera and Phasmida, which are insect orders with a previously reported presence of the (TTAGG)n telomeric motif. Data of the thesis showed that the activity of telomeric retrolements at Drosophila is up-regulated by mild-levels of free radical species, enhancing telomere extension. The role of non-telomerase mechanisms to overcome stress conditions during evolution of insect telomeres is hypothesized.
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Studies on the sericin 3 of \kur{Bombyx mori} and cloned sericin into \kur{Escherichia coli}
KRŮČEK, Tomáš
The spun-out silk fiber consists of two fibroin filaments that are cemented together by sericin coating. The serine-rich sericins, which make 20-30% of the cocoon silk proteins in Bombyx mori, are dissolved in hot water during silk fiber reeling from the cocoon. The sericin extract is usually discarded. Only small amounts are currently used in cosmetics and lately also as replacement of bovine serum products in the cell culture media. The use in culture media is hindered by poor standardization of the extracts. To overcome this problem, we attempted preparation of several recombinant proteins based on partial sequences of one out of the three sericin genes of Bombyx mori, sericin 3. Translation product of the Ser3 gene is extremely serine-rich with a relatively high representation of the aspartate, asparagin, glutamate, and glutamine. Using specific primers we have prepared a cDNAs of 267, 279, 525, 672, and 528 bp, respectively, derived from the Ser3 gene. The cDNAs were cloned and expressed as fusion proteins with hexahistidine in Escherichia coli. The proteins were purified by affinity chromatography and analyzed by acrylamide electrophoresis. Ser3 gene contain repetitive motifs rich in serine and including some charged amino acids.
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