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Isolation, characterization and transformation, by autonomous replication, ofMucor circinelloides OMPdecase-deficient mutants

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Abstract

Pyrimidine auxotrophs ofMucor circinelloides were isolated after mutagenesis with nitrosoguanidine and selected for resistance to 5-fluoroorotate. These mutants were genetically and biochemically characterized and found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase) activity or in orotate phosphoribosyl transferase (OPRTase) activity. Different circular DNA molecules containing the homologouspyrG gene were used to transform a representative OMPdecase-deficient strain to uracil prototrophy. Southern analysis, as well as mitotic stability analysis of the transformants, showed that the transforming DNA is always maintained extrachromosomally. The smallest fragment tested that retained both the capacity to complement thepyrG4 mutation and the ability to be maintained extrachromosomally when cloned in a suitable vector is a 1.85 kbM. circinelloides genomic DNA fragment. This fragment consists of thepyrG coding region flanked by 606 nucleotides at the 5′ and 330 nucleotides at the 3′ ends, respectively. Sequence analysis reveals that it does not share any element in common with anotherM. circinelloides genomic DNA fragment which also promotes autonomous replication in this organism, except those related to transcription. Furthermore, it differs from elements which have been shown to be involved in autonomous replication in other fungal systems. An equivalent plasmid harbouring the heterologousPhycomyces blakesleeanus pyrG gene yielded lower transformation rates, but the transforming DNA was also maintained extrachromosomally. Our results suggest that autonomous replication inM. circinelloides may be driven by elements normally present in nuclear coding genes.

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Author notes

  1. Ernesto P. Benito

    Present address: Department of Phytopathology, Wageningen Agricultural University, Wageningen, The Netherlands

Authors and Affiliations

  1. Area de Genética, Dpto. de Microbiología y Genética, Universidad de Salamanca,Edificio Departmental, Avd. del Campo Charro, S/N. 37007, Salamanca, Spain

    Ernesto P. Benito, Victoria Campuzano, M. Angeles Lôpez-Matas, José I. De Vicente & Arturo P. Eslava

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  1. Ernesto P. Benito
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  2. Victoria Campuzano
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  3. M. Angeles Lôpez-Matas
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  4. José I. De Vicente
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  5. Arturo P. Eslava
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Communicated by C. A. M. J. J. van den Hondel

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Benito, E.P., Campuzano, V., Lôpez-Matas, M.A. et al. Isolation, characterization and transformation, by autonomous replication, ofMucor circinelloides OMPdecase-deficient mutants. Molec. Gen. Genet. 248, 126–135 (1995). https://doi.org/10.1007/BF02190793

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  • DOI: https://doi.org/10.1007/BF02190793

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