RESEARCH ARTICLE Yohsuke Moriyama Æ Tomokazu Yamazaki Hideo Nomura Æ Narie Sasaki Æ Shigeyuki Kawano Early zygote-specific nuclease in mitochondria of the true slime mold Physarum polycephalum Received: 1 June 2005 / Revised: 22 August 2005 / Accepted: 31 August 2005 Ó Springer-Verlag 2005 Abstract The active, selective digestion of mtDNA from one parent is a possible molecular mechanism for the uniparental inheritance of mtDNA. In Physarum poly- cephalum, mtDNA is packed by DNA-binding protein Glom, which packs mtDNA into rod-shaped mt-nucle- oids. After the mating, mtDNA from one parent is selectively digested, and the Glom began to disperse. Dispersed Glom was retained for at least 6 h after mtDNA digestion, but disappeared completely by about 12 h after mixing two strains. We identified two novel nucleases using DNA zymography with native-PAGE and SDS-PAGE. One is a Ca 2+ -dependent, high- molecular-weight nuclease complex (about 670 kDa), and the other is a Mn 2+ -dependent, high-molecular- weight nuclease complex (440–670 kDa); the activity of the latter was detected as a Mn 2+ -dependent, 13-kDa DNase band on SDS-PAGE. All mitochondria isolated from myxamoebae had mt-nucleoids, whereas half of the mitochondria isolated from the zygotes at 12 h after mixing had lost the mt-nucleoids. The activity of the Mn 2+ -dependent nuclease in the isolated mitochondria was detected at least 8 h after mixing of two strains. The timing and localization of the Mn 2+ -dependent DNase activity matched the selective digestion of mtDNA. Key words Isogamous mating Æ Uniparental inheritance Æ Mitochondrial transmission genetics Æ Myxomycetes Introduction Mitochondria contain their own genomes, which are transmitted either uniparentally or maternally in diverse taxa of sexual eukaryotes, including higher plants, mosses, ferns, algae, fungi, and animals, including hu- mans (reviewed by Birky 1995). However, the molecular mechanism underlying uniparental inheritance remains unclear. Recently, the selective destruction of sperm mito- chondria in zygotes was reported for mammals, and the possible involvement of ubiquitin in the destruction of sperm mitochondria in fertilized cow and monkey eggs was suggested (Sutovsky et al. 1999, 2000). Previously, we showed that the selective digestion of mtDNA occurs before the destruction of mitochondria in the zygote of the true slime mold Physarum polycephalum (Moriyama and Kawano 2003). In a cross between myxamoeba strains AI35 and DP246, AI35 is consistently the mito- chondrial donor strain and DP246 is the recipient strain. About 5 h after mating, the mtDNA in the mitochon- dria from the recipient strain was digested synchro- nously. The mitochondrial inner and outer membranes from the recipient strain were destroyed long after digestion of the mtDNA, at about 36 h after mating. The active digestion of organelle DNA has also been reported in chloroplast inheritance. Kuroiwa et al. (1982) found that DAPI-stained chloroplast DNA (cpDNA) from mating type minus strains disappeared preferentially in young zygotes of the isogamous green algae Chlamydomonas reinhardtii within 50 min of mating. Recently, a nuclease that is potentially involved in chloroplast inheritance has been investigated in terms of a mating type plus (mt + )-specific DNase (MDN, Nishimura et al. 2002). This nuclease accumulates in the cytoplasm of mt + cells during gamete maturation. After mating, it is imported into mating type minus (mt À ) chloroplasts. Based on these results, MDN has been proposed as a possible force driving the uniparental inheritance of cpDNA in C. reinhardtii. Communicated by L. Tomaska Y. Moriyama Æ T. Yamazaki Æ H. Nomura Æ S. Kawano (&) Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg. FSB-601, 5-1-5 Kashiwanoha, Kashiwa, 277-8562 Chiba, Japan E-mail: kawano@k.u-tokyo.ac.jp N. Sasaki Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, 112-8610 Tokyo, Japan Curr Genet (2005) DOI 10.1007/s00294-005-0025-2