Uravnavanje genske ekspresije s stereoidi in uporaba genske tehnologije P450 v medicini (Slovene)

a. The mechanism of induction of steroid inducible enzyme systems in filamentous fungus R.nigricans was studied on DNA level and on the level of membrane receptors. It was confirmed that induction process could involve membrane bound G-proteins and phosphorylation of cellular proteins. The presence of progesterone receptors and G-proteins was demonstrated in the plasma membrane fraction . The GTPase activity of G-proteins increased after treatment with progesterone. The involvement of protein kinases was studied indirectly using genistein the inhibitor of protein kinases. Fungal cells tested for the induction ability in the presence of kinase inhibitor exhibited significantly lower hydroxylation activity ( 43%) when compared to the control samples. Phosphorylation of cellular proteins was studied also directly using radioactively labelled ATP. Phosphorylated proteins were analysed by PAGE method and it was shown that the detection of radioactively labelled phosphorylated proteins was possible but the additional methodological improvements are needed. Concerning the studies on DNA level we isolated and characterised two different cDNA clones containing information for cytocolic stress proteins from Hsp70 family. It was also shown on RNA level that expression of the above stress proteins increased by the action of testosterone and deoxycorticosterone. The expression was also influenced by temperature stress and oxidative stress. b. The detoxification and activation of many xenobiotics in man are catalysed by polymorphic cytochrome P450 enzymes. The polymorphism of corresponding genes was studied in correlation with the genes responsible for activating as well as detoxifying enzymes. Interindividual variability due to genetic polymorphism was used to evaluate susceptibility to human cancer caused by chemical carcinogenesis.