Viruses, retrotransposons, and insertion sequences put the genomic integrity of all organisms at risk. Fungi are arguably under even greater jeopardy than plants and animals because they are organized coenocytically: a macroscopic "individual" may contain millions of nuclei sharing a continuous cytoplasm that is undivided by septa or cell membranes. Unsurprisingly, fungi have at least three sophisticated mechanisms for fighting back: quelling, RIP, and meiotic silencing by unpaired DNA (MSUD). The research that is the subject of this project is aimed at understanding the mechanism of MSUD and its role in the overall biology of one experimental organism, Neurospora crassa. Earlier work showed that during the very brief diploid phase of this organism, presumably when homologs are paired in the pachytene stage of meiosis, any sizeable stretch of unpaired DNA is "seen" by the organism as a foreign sequence, one that was not present in both parents at the same locus, and hence having the capacity for mischief. A signal in the form of double-stranded RNA is generated. Ultimately, this silences the expression not only of the unpaired DNA, but also any other DNA of the same sequence, whether paired or unpaired. Selecting and screening for suppressor mutations which fail in this silencing process has already been rewarding in terms of insight gained; one called Sad-1 (suppressor of ascus dominance) encodes a sexual-phase specific RNA-directed RNA polymerase, and other Sad mutants will be sought and studied. A major focus of the research will be on the role of two paralogous genes called dicer-1 and dicer-2 thought to be involved in quelling. In many organisms, and in the vegetative phase of fungal growth, these aptly-names genes are thought to dice up long double-stranded RNA molecules into short, double-stranded pieces which, in turn, give rise to single-stranded molecules. These then direct the destruction of mRNA homologous to them, resulting in "silencing". It is not clear that they have a similar role in MSUD, and a sizeable effort will be expended in trying to illuminate this issue. Finally, it has become clear that functional Sad genes are necessary for the completion of meiosis. This suggests that some of the machinery of silencing, presumably involving double-stranded RNA and its cleavage, is shared with the machinery of meiosis. A search for the key molecules is an additional aim of this research.
All organisms are under continuous challenge by "selfish" DNA sequences or molecular parasites that are able to reproduce themselves at the expense of the host organism. Often the host has sophisticated machinery for fighting back, either by attacking and mutating the parasitic DNA to a harmless condition, or by preventing the parasitic DNA from making molecules necessary for its multiplication. An understanding of how these silencing mechanisms work would make it possible to prevent not only the reproduction of viruses, but also to prevent other cellular genes from making undesired products, or from making benign products in excessive amounts.
