Analysis of the Aspergillus nidulans PRP8 intein
|dc.identifier.citation||Islam, S. (2012). Analysis of the Aspergillus nidulans PRP8 intein (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/2569||en|
|dc.description.abstract||Inteins are naturally-occurring intervening sequences that are translated along with their host protein. The intein insertion is excised from its host protein post-translationally in an autocatalytic process known as intein splicing. Full-length inteins encode a second homing endonuclease (HEase) function which allows their genetic mobility. In higher organisms, intein homing occurs during meiosis. In a cell that is heterozygous for the intein+/intein- allele of the host gene, the HEase causes a double-stranded break (DSB) in the intein- allele. The resulting homologous repair by host organism cellular machinery results in the duplication of the intein+ allele. In meiosis, homing results in the super-Mendelian inheritance of the intein sequence with all meiotic progeny carrying the intein+ allele. This should lead to the spread of the intein through the population. The nuclear intein found in the essential PRP8 gene is the most widespread among fungi. This study describes the analysis of the full-length PRP8 intein – AniPRP8 – found in the genome of the filamentous fungus Aspergillus nidulans. AniPRP8 was characterized with regard to its evolutionary history, HEase and splicing activities. It was found that the PRP8 gene in A. nidulans is saturated with the intein+ allele. Three distinct intein types are present corresponding to three varieties of this fungus. Sequence analysis indicates that the HEases of all three intein types are active. The distribution of AniPRP8 within A. nidulans suggests that the evolution of this sequence cannot be easily explained by the currently accepted model of intein evolution. An alternative evolutionary scenario is offered. Two independent experimental avenues were utilized to demonstrate that the HEase encoded by AniPRP8 – PI-AniI – is active. The first of these employed an A. nidulans-based fungal system, which was constructed and characterized to enable the examination of PI-AniI activity. The endonuclease activity of this HEase was demonstrated in vivo in its native A. nidulans context. In a second avenue confirming PI-AniI endonuclease activity, it was established that the purified enzyme mediates DSBs of its substrate DNA in vitro. HEase activity in vitro requires the presence of the divalent cation Mn2+ as a co-factor. The design of an in vivo assay in E. coli for the gene conversion events mediated by the HEase PI-AniI was investigated. The splicing reaction mediated by inteins is required to produce the mature, functional form of the host protein. Inhibition of intein splicing in the essential genes of pathogenic organisms presents an attractive drug target. AniPRP8 splicing was demonstrated in a foreign extein context in an in vivo LacZ-based E. coli system. The assay can distinguish splicing and non-splicing variants of the intein.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.subject||Active Homing Endonuclease|
|dc.subject||in vitro assay|
|dc.title||Analysis of the Aspergillus nidulans PRP8 intein|
|thesis.degree.discipline||Department of Biochemistry|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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