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Submitted to: Molecular Genetics and Genomics
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 9/17/2002 Publication Date: 10/17/2002 Citation: SKORY, C.D. HOMOLOGOUS RECOMBINATION AND DOUBLE-STRAND BREAK REPAIR IN THE TRANSFORMATION OF RHIZOPUS ORYZAE. MOLECULAR GENETICS AND GENOMICS. 2002. V. 268. P. 397-406. Interpretive Summary: Rhizopus is a filamentous fungus with an intriguing history of use in the production of fermented foods, industrial enzymes, organic acids, and corticosteroids; all the while having the unfortunate reputation of being a food spoilage organism, a plant pathogen, and an opportunistic human pathogen. Despite its importance, the techniques for genetic manipulation of this remarkable organism are still limited 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 studied the repair systems found in Rhizopus and ascertained ways to establish stable replication of the modified DNA. The results of this study are expected to provide the needed tools to allow a more rapid progression of the industrial utilization of this valuable organism. Technical Abstract: Genetic transformation of the Mucorales fungi has been problematic, since transformed DNA rarely integrates and usually is mitotically unstable in the absence of selective pressure. In this study, transformation of Rhizopus oryzae was investigated to determine if the fate of transformed DNA could be predicted based on double-strand break repair and recombination mechanisms found in other fungi. A transformation system was developed with uridine auxotrophs of R. oryzae that could be complemented with the pyrG gene isolated in this work. DNA transformed as uncut plasmid was maintained extrachromosomally in a high MW (>23 kb) concatenated arrangement. Type-I crossover integration into the pyrG locus and type-III pyrG gene replacement events occurred in approximately 1-5% of transformants. Linearization of plasmid pPyr225 with a single restriction enzyme that cleaves within the vector sequence almost always resulted in isolates with replicating concatenated plasmids that were repaired by end-joining recombination that restored the restriction site. The addition of a 40-bp direct repeat on either side of this cleavage site shifted repair to homologous recombination between the repeated sequences on the plasmid, resulting in loss of the restriction site. When plasmid pPyr225 was digested with two different enzymes that cleave within the vector sequence to release the pyrG containing fragment, only pyrG gene replacement recombination occurred in transformants. Linearization of plasmid pPyr225 within the pyrG gene gave the highest percentage, 20%, of type-I integration at the pyrG locus. However, end-joining repair and gene replacement events were still the predominant types of recombination for transformations with this plasmid topology. |
