Avtor/Urednik		Pečar, Urša
Naslov		Priprava sistemov za izražanje steroidne-1-dehidrogenaze v bakteriji Escherichia coli, kvasovki Pichia pastoris in rastlini Solanum laciniatum
Tip		monografija
Kraj izdaje		Ljubljana
Založnik		Medicinska fakulteta
Leto izdaje		2000
Obseg		str. 79
Jezik		slo
Abstrakt		Many steroid compounds which are used for local anti-inflammatory treatment possess double bond between C1 and C2 atoms of steroid skeleton. Double bond is introduced by the enzyme steroid-1-dehydrogenase. We introduced a gene for steroid-1-dehydrogenase from Nocardia opaca into 3 different expression vectors: into pB3 with which we transformed bacteria Escherichia coli, into pPIC9 which we electroporated in yeast Pichia pastoris, and in pBINplus-pUCCAB which we electroporated in bacteria Agrobacterium tumefaciens. The presence of gene was shown in all expression vectors with PCR reaction, restriction and nucleotide sequence analysis. After introduction of vectors into living organisms we obtained positive clones only in E. coli which showed highly different phenotype compared with control cells. The cells with target gene grew slowly on plates and formed smaller colonies and in liquid medium sedimented faster than control counterparts. After induction of growing cells no differences in protein expression occured between cells that contain the target gene and cells without it. We prepared nodium cultures of plants Solanum laciniatum and showed that those which were obtained from New Zealand grow more effectively than those from Australia, therefore they are preferred for further transformation experiments. We determined the nucleotide and deduced amino acid sequence of the steroid-1-dehydrogenase gene.
Deskriptorji		ANTI-INFLAMMATORY AGENTS, STEROIDAL STEROID HYDROXYLASES GENE EXPRESSION REGULATION GENETIC VECTORS GENE TRANSFER ESCHERICHIA COLI NOCARDIA PICHIA AGROBACTERIUM TUMEFACIENS POLYMERASE CHAIN REACTION BASE SEQUENCE VEGETABLES TRANSFORMATION, GENETIC