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Submitted to: Society of Industrial Microbiology Annual Meeting
Publication Type: Abstract Only Publication Acceptance Date: 8/14/2003 Publication Date: 8/14/2003 Citation: Skory, C.D. 2003. Mutation repair by synthesis dependent strand annealing in Rhizopus oryzae [abstract]. Society of Industrial Microbiology. p. 97. Interpretive Summary: Technical Abstract: Rhizopus is a filamentous fungus used for production of fermented foods, industrial enzymes, organic acids, and corticosteroids, while also being a food spoilage organism, a plant pathogen, and an opportunistic human pathogen. Despite this importance, techniques for genetic manipulation are still in an early stage of development compared to many other fungi. This dilemma is common among Mucorales fungi, due to a lack of understanding regarding recombination and replication mechanisms that affect the fate of introduced DNA. We previously showed that DNA transformed into the host rarely integrates and is replicated in a high MW concatenated arrangement. Linearization of the vector prior to transformation increases integration frequency, but repair of the double strand break by non-homologous end-joining is still the predominant event. We recently attempted to circumvent replication of the plasmid by introducing mutations in the selectable pyrG on the vector. These mutations were situated such that homologous integration into the non-functional chromosomal pyrG would result in two copies of the gene; one functional, the other not. Analysis of over 100 transformants revealed that integration had not occurred in any isolates transformed with linear or circular plasmid. In all cases, transformed DNA replicated autonomously in a concatenated arrangement. Sequence analysis showed that prototrophic growth was restored by repair of the non-functional pyrG sequence in the chromosome and/or plasmid. It is hypothesized that this repair occurred by synthesis dependent strand annealing. |
