RESUMO
Target sequence cleavage is the essential step for intron invasion into an intronless allele. DNA cleavage at a specific site is performed by an endonuclease, termed a homing enzyme, which is encoded by an open reading frame within the intron. The recognition properties of them have only been analyzed in vitro, using purified, recombinant homing enzyme and various mutated DNA substrates, but it is unclear whether the homing enzyme behaves similarly in vivo. To answer this question, we determined the recognition properties of I-CsmI in vivo. I-CsmI is a homing enzyme encoded by the open reading frame of the alpha-group I-intron, located in the mitochondrial apocytochrome b gene of the green alga Chlamydomonas smithii. The in vivo recognition properties of it were determined as the frequency of intron invasion into a mutated target site. For this purpose, we utilized hybrid diploid cells developed by crossing alpha-intron-plus C. smithii to intron-minus C. reinhardtii containing mutated target sequences. The intron invasion frequency was much higher than the expected from the in vitro cleavage frequency of the respective mutated substrates. Even the substrates that had very little cleavage in the in vitro experiment were efficiently invaded in vivo, and were accompanied by a large degree of coconversion. Considering the ease of the homing enzyme invading into various mutated target sequences, we propose that the principle bottleneck for lateral intron transmission is not the sequence specificity of the homing enzyme, but instead is limited by the rare occurrence of inter-specific cell fusion.
