Analysis of localization of cell-cycle regulators in Neurospora crassa.

Most fungi are multinucleated organisms. In some fungi, they have asynchronous nuclei in the same cytoplasm. We analyzed a cell-cycle regulation mechanism using a model fungus Neurospora crassa, which can make heterokaryon cells. G1/S cyclin CLN-1 and cyclin-dependent kinase CDC-2 were tagged with different fluorescence in different strains and expressed. By forming a heterokaryon strain of these, two different fluorescence-tagged proteins were expressed in the same cytoplasm. CDC-2 was localized in all nuclei, whereas CLN-1 was not detected in most of the nuclei and was dispersed in the cytoplasm with small granular clusters. This indicates that in multinucleated fungi, cell-cycle regulators, similar to other proteins, are shared around the nuclei regardless of different cell-cycle stages. Moreover, each nucleus can select and use a special cell-cycle regulator only when it is necessary. Fungal nuclei may have a novel pickup mechanism of necessary proteins from their cytoplasm at the point of use.

Phenotypic analysis of newly isolated short-lifespan Neurospora crassa mutant deficient in a high mobility group box protein.

To elucidate genetic mechanisms affecting the lifespan of the filamentous fungus Neurospora crassa, we attempted to identify a gene of which a defect causes a short-lifespan. By screening a Neurospora knockout library, provided by the Fungal Genetics Stock Center at Kansas State University, several KO strains with a short-lifespan were isolated. FGSC#11693 is one of these, which shows similar phenotypes to known Neurospora short-lifespan mutants as follows: 1) hyphal growth ceases after about 2weeks of cultivation, despite that of the wild-type continuing for over 2years, 2) viability of conidia is lower than that of the wild-type, and 3) high sensitivity to mutagens such as methyl methanesulfonate, ultraviolet radiation, and hydroxyl urea is exhibited. The NCU number of the knocked-out gene in the KO strain is NCU02695, and recovery from the short-lifespan and mutagen sensitivity was achieved by the introduction of this gene from the wild-type. The putative amino acid sequence of the knocked-out gene contains two high mobility group box domains and a mitochondrial localization signal is found at the N-terminal of this sequence. Upon analyzing the subcellular localization of the gene product fused with GFP, GFP signals were detected in mitochondria. From these observations, the gene and KO strain were named mitochondrial high mobility group box protein 1 (MHG1) and mhg1KO strain, respectively. The amount of mtDNA relative to the nuclear amount was lower in the mhg1KO strain than in the wild-type. mtDNA aberration was also observed in the mhg1KO strain. These results suggest that the MHG1 protein plays an important role in the maintenance of mitochondrial DNA, and mitochondrial abnormality caused by mtDNA aberration is responsible for the short-lifespan of the mhg1KO strain.