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item Skory, Christopher - Chris

Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/13/2004
Publication Date: 3/9/2004
Citation: Skory, C.D. 2004. Repair of plasmid dna used for transformation of rhizopus oryzae by synthesis dependent strand annealing. Current Genetics. 45:302-310.

Interpretive Summary: Rhizopus is a valuable filamentous fungus that is used for production of fermented foods, industrial enzymes (e.g., glucoamylase and lipase), organic acids (e.g., lactate and fumarate), and corticosteroids, while also being a food spoilage organism and a plant pathogen. Yet even with such importance, the techniques for genetic manipulation of this remarkable organism are still in an early stage of development compared to those used for many other fungi. This void of understanding impedes the ability to study the organism and to exploit its valuable traits. Furthermore, many other related fungi of industrial interest share this same dilemma mainly due to a lack of knowledge regarding the mechanisms that control the replication and repair of DNA. A cornerstone of genetic engineering involves being able to put modified genes back into the organism of interest. In order to be useful, the fungus must be able to replicate this DNA and repair any damage that might occur during this manipulation process. This work established how Rhizopus is able to repair mutations that were introduced into the gene. The results of this study will allow new strategies to be developed that can exploit this discovery and allow more rapid progression of the industrial utilization of this valuable organism.

Technical Abstract: The techniques for genetic manipulation of the filamentous fungus Rhizopus have been hampered due to a lack of understanding regarding the recombination and replication mechanisms that affect the fate of introduced DNA. The ability to target chromosomal integration of a plasmid has been difficult because DNA transformed into Rhizopus rarely integrates and is autonomously replicated in a high molecular weight concatenated arrangement (i.e., series or chain). Linearization of the plasmid prior to transformation at a site having homology with the genomic DNA yields the highest frequency of integration, but repair of the double strand break by end-joining is still the predominant event. We recently attempted to circumvent replication of the plasmid by introducing frameshift mutations in the R. oryzae orotidine 5'-monophosphate (OMP) decarboxylase, pyrG, gene used for selection of the vector. It was hypothesized that autonomous replication of the mutated plasmids would be incapable of restoring prototrophic growth, since the genomic pyrG also contained a mutation. However, homologous integration of the plasmid will result in duplication of the pyrG gene, which can create a functional copy of the pyrG, if both the genomic and plasmid mutations are paired on the same duplicate copy. While this event was detected in one of the isolates, it represented less than 8% of the total transformants. The majority of transformants contained plasmid replicating autonomously in a concatenated arrangement. Sequence analysis showed that prototrophic growth was restored by repairing the non-functional pyrG sequence in plasmid, while the genomic pyrG gene was unaltered. The lack of integration in this study further suggests that Rhizopus does not rely primarily on double strand break induced recombination as a response to cleavage of the DNA. Instead, it is hypothesized that repair of the pyrG on the plasmid occurs mainly by synthesis dependent strand annealing.