Date of Award
Master of Science (MS)
School of Biological Sciences
Thomas M. Hammond
Erik D. Larson
In an attempt to neutralize transposable elements or retrovirus invasions Neurospora crassa will rely on one of its many genome defense mechanism, Meiotic Silencing by Unpaired DNA (MSUD). MSUD works in a two-step process that first detects unpaired sequences between homologous chromosomes followed by downstream silenced expression of the sequence. The ultimate silencing stage of MSUD is widely accepted to operate through an RNAi-like system. However, the mechanics of the detection step of MSUD remains elusive. The research presented attempts to elaborate on how the initial stage of MSUD occurs and its specifics. First, a genetic approach is utilized to answer what kind of genomic distance limitations are placed on the homology searching procedure of this mechanism. In these experiments, we have inserted a genetic phenotypic marker at different locations on N.crassa's chromosome VII. Many combinations of strains were crossed to create varying distances of the marker between homologs during a sexual cross. Interestingly, we observed mixed phenotypes when markers were physically unpaired by as small a distance as 13.9kB. This suggested that MSUD was only partial detecting the unpairing events. Overall, the experimental crosses expressed an interesting trend that illustrated a positive correlation between increasing distance between markers and MSUD activity. We concluded that the searching process is effected by distance and may not search in a linear manner.
Secondly, biochemical attempts were conducted to involve the first recognized nuclear MSUD protein, SAD-5. We inserted the sad-5 gene into a pET15b expression vector and attempted to express the protein in Lemo21 E.coli cells. After multiple rounds of unsuccessful purification using many techniques to try to alleviate the protein from the insoluble fraction, we decided to move our vector to a different cell line. The new cell line, ArcticExpress, was believed to be more suitable because of its modified ability to produce charperonin proteins to aid in folding of the recombinant protein. ArcticExress cells are also adapted to grow at lower temperatures which is also thought to support proper protein folding. However, expressing our recombinant protein in this cell line failed to solubilize the SAD-5 protein. It is unfortunate that all attempts were unsuccessful, but many alternative methods have still yet to be tested. One potential alternative would be to move our eukaryotic protein to a eukaryotic system. Once we have successfully purified the protein, protein binding assays can be accomplished to determine SAD-5's binding preferences to different substrate allowing insight into the protein's function. The research outlined is only the beginning of our understanding of how MSUD's detection processes operates. As our lab continues to investigate this phenomenon, we may find that the characteristics of MSUD that we discover may elaborate on current problems in biological research and medicine such as RNAi treatments for cancer or retroviral detection.
Sauls, Pegan A., "Investigating the Initial Detection Stage of Meiotic Silencing by Unpaired Dna in the Model Organism Neurospora Crassa" (2015). Theses and Dissertations. 396.