Abstract
The unicellular red alga Cyanidioschyzon merolae 10D has simple cell structure, which contains one nucleus, one mitochondrion and one plastid. C. merolae is considered to be closest to the origin of eukaryotic cells. In order to use this organism as a model cell, it is indispensable to develop reverse-genetical techniques to facilitate functional genomics.
We demonstrated previously that the URA3 mutant (frameshift mutation in URA3 gene, uracil auxotroph) incorporated an exogenous DNA fragment coding for wild type URA3 (Minoda et al. (2004) PCP 45:667-671). However it was hard to distinguish between transformation and reversion mutation. In this study, we performed transformation experiments using modified URA3 gene, which contains two base substitutions unaccompanied by amino acid change. As a result, appearance of uracil prototroph transformant which contained substituted bases, suggests incorporation of exogenous DNA by double-crossover. This result strongly suggests that development of proper marker genes enables gene disruption for reverse genetics.
