TRANSFORMATION OF UNICELLULAR GREEN-ALGA CHLAMYDOMONAS-REINHARDTII WITH THE AMINOGLYCOSIDE-3'-PHOSPHOTRANSFERASE GENE FROM STREPTOMYCES-RIMOSUS

Eukaryotic unicellular green alga Chlamydomonas reinhardtii was transf ormed with the aph gene for aminoglycoside-3'-phosphorilase (APH) from Streptomyces rimosus, which determined resistance to aminoglycoside a ntibiotics. The structural part of the aph gene was previously cloned into a pSU937 plasmid containing no eukaryotic regulatory elements. Th e method used to obtain stable transformants involved selection of cel ls that showed stable expression of heterologous genes controlled from chromosomal regulatory elements. The aph genes from S. rimosus and nu clear genome of Ch. reinhardtii are similar in nucleotide and codon se quences. The frequency of transformation of cells to paromomycin resis tance (Pm-R) was (1.3-1.9) x 10(-7) to 1.7 x 10(-7) i.e., was much hig her than that of spontaneous or transformation-induced Pm-R mutations (less than 1 x 10(-8)). Integration of the aph gene into the genome of Ch. reinhardtii was demonstrated by means of (1) DNA blot hybridizati on to a DNA probe containing the aph gene and (2) estimation of phosph otransferase activity in crude cell-free extracts by native gel electr ophoresis of proteins and thin-layer chromatography. The spectrum of a minoglycoside antibiotics to which the transformants were resistant co rresponded to substrate specificity of APH from S. rimosus. Thus, the phenotype of transformants was determined by the expressed aph gene.