Construction of a Saccharomyces cerevisiae strain expressing the Leishmania major nucleoside hydrolase gene.
Nucleoside hydrolase (NH) (EC 3.2.2.3) is an essential enzyme in the purine-pyrimidine salvage pathway utilised by many protozoan parasites and may be a useful drug target. However, the search for NH inhibitors has been hampered by the lack of suitable in vitro screens. We have constructed a Saccharomyces cerevisiae strain that requires expression of the Leishmania major nucleoside hydrolase (LmNH) enzyme for growth and that may be suitable as a screen for NH inhibitors. The gene encoding LmNH was amplified using polymerase chain reaction with L. major genomic DNA as the template, cloned into an expression vector and used to transform a yeast mutant unable to grow on uridine as the sole source of uracil. Expression of LmNH yielded an ca. 35.6kDa protein, which was shown to be functional as the mutant strain was able to grow on medium containing uridine as the sole source of uracil. Importantly, this work has resulted in a strain that can be used to screen compounds as potential inhibitors of this essential Leishmania enzyme.
Miller
Tamara K
TK
MycoLogics, 12635 E. Montview Boulevard, Suite 215, Aurora, CO 80010, USA.
Patel
Champa
C
Selitrennikoff
Claude P
CP
D000818:Animals; D015153:Blotting, Western; D003300:Copper / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors / Q000235:genetics; D018320:Leishmania major / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*; D014498:Uracil / Q000378:metabolism; D014529:Uridine / Q000378:metabolism
D016271:Proto-Oncogene Proteins c-myc; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014498:Uracil; D003300:Copper; D009699:N-Glycosyl Hydrolases; D014529:Uridine