20.500.12556/RUL-92502 Priprava gensko spremenjene kvasovke Saccharomyces cerevisiae kot modelnega organizma za študij metabolizma rakastih celic. Preparation of genetically modified yeast saccharomyces cerevisiae as model organism for study metabolism of cancer cells Humani modificirani encim Pfk-M, velikosti 47 kDa, je ključni dejavnik povečane aktivnosti glikolize v rakastih celicah. Encim Pfk-M, ki ga kodira konstruiran sfPFKM gen, je bolj dovzeten za aktivacijo z efektorji, kot nativni protein in odporen na povratno inhibicijo. Visoko aktiven encim Pfk-M dvigne nivo citosolnega NADH v rakastih celicah in transformantah S. cerevisae, ki imajo vstavljen humani sfPFKM gen. Pri transformantah S. cerevisiae z vstavljenim sfPFKM genom so opazili porušeno razmerje NADH/NADPH, ki preprečuje rast na maltozi. Namen dela je bil vnos MAEA gena, ki kodira citosolni NADP+ specifični malični encim v transformanto S. cerevisiae z sfPFKM. Spremenjeni bakterijski MAEA gen kodira protein, katerega vloga je popraviti ustrezno razmerje med NADH/NADPH nukleotidoma izboljšanje razmerja med nukleotidoma v celicah in s tem omogočiti rast sevu S. cerevisiae sfPFKM/MAEA na maltozi. Po uspešnem vnosu MAEA gena v S. cerevisiae/sfPFKM smo zaznali visoko aktivnost NADP+ specifičnega maličnega encima. Ugotovili smo, da imajo sevi najboljšo specifično hitrost rasti v gojišču z 0,05 % maltozo z dodanima aminokislinama, glutamatom in glutaminsko kislino, ter malatom. Spremljali smo specifično hitrost rasti, porabo maltoze in nastajanje etanola, glicerola ter acetata. Predvidevamo, da je vnos gena MAEA v S. cerevisiae omogočil rast seva S. cerevisiae/sfPFKM/ MAEA na maltozi zaradi popravljenega razmerja NADH/NADPH v citosolu. Human modified 47 kDa Pfk-M enzyme might be the pivotal factor of enhanced glycolysis in cancer cells. It is more susceptible to activation by specific effectors and resistant to feed-back inhibition. It can be encoded by artificially prepared sfPFKM gene. Highly active Pfk-M enzymes cause increased cytosolic NADH levels in cancer cells and in S. cerevisiae transformants with the inserted human sfPFKM gene. Imbalanced NADH/NADPH ratio was observed in the yeast sfPFKM transformant that prevented its growth in maltose medium. The purpose of this work was to insert the MAEA gene encoding cytosolic NADP+ dependent malic enzyme in the yeast sfPFKM transformant. The modified bacterial MAEA gene encoded the protein that was expected to improve the pyrimidine nucleotide ratios in the cells and consequently enable growth of sfPFKM/MAEA strain in maltose. After successful insert of MAEA gene into S. cerevisiae/sfPFKM, high NADP+ specific malic enzyme activities were detected. We found that the strains grew in maltose with the best specific growth coefficients detected in 0,05 % maltose media with added amino acids, glutamate and glutamic acid, and malate. We monitored specific growth rate, consumption of maltose and formation of ethanol, glycerol and acetate. To conclude the insertion of MAEA gene enabled growth of S. cerevisiae/sfPFKM/MAEA strain on maltose, most probably by improving NADH/NADPH ratio in the cytosol. Saccharomyces cerevisiae rak 6-fosfofrukto-1-kinaza malični encim Saccharomyces cerevisiae cancer 6-phosphofructo-1-kinase malic enzyme true false false [T. Ambrožič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2017-06-05 07:59:11 2017-06-05 07:59:12 2022-08-09 23:28:40 0000-00-00 00:00:00 2017 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.6:582.282.23:606:61(043.2) 130731 8728697 432.pdf 432.pdf 1 84CB59B5FEE1C44033D47C64B7BEFB39 1d081db35ab74208a42646d11260be707690f23425adb68b0e54f3e31d185e91 1061b982-a1b3-11eb-a523-00155dcfd717 20.500.12556/rul/04b4d5ab-4c43-4706-832c-81759d265780 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=99268 Biotehniška fakulteta 0 0 0