A SITE-SPECIFIC DNA ENDONUCLEASE SPECIFIED BY ONE OF 2 ORF ENCODED BYA GROUP-I INTRON IN DICTYOSTELIUM-DISCOIDEUM MITOCHONDRIAL-DNA

The second intron (DdOX1/2.2) of Dictyostelium discoideum cytochrome o xidase subunit 1/2 fused gene has two free-standing ORF genes (Dd ai2a and Dd ai2b) in a loop, which have similar amino acid sequences and a re homologous to aI4 DNA endonuclease (I-SceII) of Saccharomyces cerev isiae. To elucidate the functions of these ORFs, we cloned the ORFs in to an expression vector and introduced the composite vectors into E. c oli. The expression of Dd ai2a in E. coli caused growth inhibition and degradation of the E. coli genomic DNA. To determine whether Dd ai2a protein is a homing type DNA endonuclease, the ability to cleave the h oming site of its intron in vivo was examined. Dd ai2a cleaved only on e strand of intronless DNA sequence at the site which coincides with t he I-SceII cleavage recognition site. We suppose that Dd ai2a function s actually as a homing type DNA endonuclease in D. discoideum mitochon dria by virtue of other factors. To obtain further information about t he relationship between tile existence of introns and the mating syste m, we carried out in vitro self-splicing assay and polymerase chain re action analysis using 13 strains of the cellular slime mold. (C) 1997 Elsevier Science B.V.