Nidulans Cytokinesis SIN Way Back Adjustment Of Genes

Cell divison is the main way of vegetative propagation for fungi and failure of cell division may be lethal for both, mother and daughter cells. Study on mechanism of this process has a very important significance for the control of medical and agricultural harmful fungi proliferation or promote industry beneficial fungi growth.Cytokinesis is the process by which a cell splits its cytoplasm, accomplished by the contraction of a contractile actin ring, to produce two daughter cells. Accordingly, cytokinesis is the final step in cell division after the nuclear division of mitosis. Numerous studies have identified that mitotic exit requires the activation of the conserved signalling network, termed the mitotic exit network (MEN), in budding yeast and the septation initiation network (SIN) in fission yeast. Although organisms of different kingdoms have developed unique mechanisms to execute cytokinesis, signals that trigger the onset of cytokinesis are evolutionarily conserved. Unlike yeast, the filamentous fungus Aspergillus nidulans contains a mycelium of multinucleate cells that are partitioned by septa. During the germination in A. nidulans, the conidiospores undergo multiple rounds of nuclear division to produce eight or16nuclei in germlings, but they do not undergo septation until the cell reaches an appropriate size/volume, and then forms the first septum near the neck between spore and germ tube. Therefore, as a whole, inter-compartment development and mitosis in the mycelium becomes asynchronous.The serine/threonine protein kinase SEPH in A. nidulans is a Cdc7p orthologue from fission yeast which was first cloned in a screen for temperature-sensitive cytokinesis mutants. It has been identified that SEPH plays a central part in the initiation of septation. However, little is known about how the SEPH kinase cascade is regulated by other components, or whether there exist any of the negative regulators that act antagonistically to others in the SIN. To gain insight into the regulatory mechanisms that underlie septation,116mutants that suppressed the defects of sepH in A. nidulans were isolated by UV mutation in previous study. By cross, backcross, complementation test and sequencing we identified a gene Anprs1(AN6711.4).In this thesis, we first coloned Anprsl using autonomous plasmid replication vector prg3-AMAl-Not I and transformed to strain Sin110. Results showed the same phenotype with previous study. This means phenotype of Sin110was caused by Anprsl. To further confirm how AnPRS1functions, we used a conditional strain in which the Anprsl gene was under the control of the inducible/repressible alcA promoter, TEM, deletion and C-deletion strains creation, and by qRT-PCR, AnPRS activity assay and Y2H to test AnPRS family function during cytokinesis in Aspergillus nidulans. GFP-AnPRS1appeared at the predicted septation site possibly prior to a detectable septum formation. AnPRS1may function in the cytosol and septation sites. When depressed, it displayed a number of phenotypic similarities to that of the Sinl10and transmission electron microscopy showed that, when the repression of AnPRS1showed the aberrant formation of delocalized septa. Anprsl full ORF deletion mutants display normal timing of cytokinesis while a C-terminal truncated displayed the same phenotype to Sinl10. Biochemical assays to test PRPP synthetase activity were performed and results indicated that measurable PRPP synthetase activities in extracts from Sinl10caused a mostly severe defect. Accordingly, normal cytokinesis seems to depend on a normal level of PRPP synthetase activity. While no mutation was found in Anprs genes in Sinl10, we wondered whether the decreased AnPRS activity in Sinl10was related to the transcription of the Anprs family. As expected, by real-time qPCR, three members of the Anprs family in A. nidulans were dramatically downregulated in Sinl10. In response to these results, we cloned ORFs of Anprsl, Anprs2and Anprs3into the AMA1vector to make the plasmids pAMA1-Anprs1, pAMA1-Anprs2and pAMAl-Anprs3respectively. The defects of Sinl10in growth and septation can be dramatically rescued.Lastly, based on the role of PP2A (protein phosphatases2A) negatively regulation SIN in yeast and transferring pi group, we searched its two regulatory subunits, par A and pabA. Though they can’t surppress sepH mutation, they are essential for morphogenesis of Aspergillus nidulans.This study will provide an important theoretic foundation for us in practice promote or inhibit fungal asexual reproduction, the medicine control and provide a strong theoretical basis for treatment of tumor.