Avtor/Urednik		Kunič, Barbara
Naslov		Genes for cytochrome P450 reductase from fungus Rhizopus nigricans - expression cloning in yeast and characterisation
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		2001
Obseg		str. 200
Jezik		eng
Abstrakt		NADPH-cytochrome P450 reductase and cytochrome b5 are the major cytochrome P450 redox-associated proteins. The role of NADPH-cytochrome P450 reductase and cytochrome b5 and their differential regulations upon progesterone treatment of filamentous fungus Rhizopus nigricans were investigated. RT-PCR amplification of progesterone induced fungal mRNAs by using degenerated primers designed based on conserved FAD and FMN binding regions was performed. It resulted in the cloning of CPR1-CS and CPR2-CS cDNAs, which were highly homologous to the central regions of known NADPH-cytochrome P450 reductases. Alternatively, expression cloning involving suppression of ketoconazole hypersensitivity of a cyb5-disrupted and cpr1 conditionally disrupted Saccharomyces cerevisiae strain led to identification of a longer reductase cDNA (CPR1-FL) and a cytochrome b5 coding cDNA (CYBS-1) CPR1-FL was found to extend the CPR1-CS sequence. Northern blot analysis revealed that the CPR1-FL and CPR2-CS cDNAs hybridised respectively to transcripts of similar lengths (2.1 kbp). The CPR1-FL hybridising mRNA was found strongly inducible (8-fold) by progesterone while CPR2-CS and CYBS-1 mRNAs were not. Subcellular localisation and function of CPR1-FL product was analysed by expression in cpr1-disrupted yeast strain. This indicated that the full CPR1-FL cDNA and its derivative (CPR1-S) truncated at the most 5'-ATG-codon gave functionally equivalent products exhibiting mixed (cytosolic and microsomal) localisation. In contrast, addition of an in-frame initiation-codon immediately upstream the CPR1-FL cDNA (CPR1-L) restricted corresponding product activity to microsomes, improved ketoconazole resistance but decreased global level of NADPH-cytochrome c reductase activity. CYBS-1 cDNA was reformatted and expressed in Escherichia coli. (Abstract truncated at 2000 characters).
Deskriptorji		RHIZOPUS CYTOCHROME P-450 ESCHERICHIA COLI CYTOCHROME B5 SACCHAROMYCES CEREVISIAE CLONING, MOLECULAR GENE EXPRESSION TRANSFORMATION, GENETIC POLYMERASE CHAIN REACTION BASE SEQUENCE NADPH-FERRIHEMOPROTEIN REDUCTASE