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

Abstract

Pyrimidine auxotrophs of Mucor 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 homologous pyrG 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 the pyrG4 mutation and the ability to be maintained extrachromosomally when cloned in a suitable vector is a 1.85 kb M. circinelloides genomic DNA fragment. This fragment consists of the pyrG 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 another M. 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 heterologous Phycomyces blakesleeanus pyrG gene yielded lower transformation rates, but the transforming DNA was also maintained extrachromosomally. Our results suggest that autonomous replication in M. circinelloides may be driven by elements normally present in nuclear coding genes.

Complementation Analysis of Carotenogenic Mutants ofMucor circinelloides

“…Vegetative spore suspensions (about 10 6 spores/ml) of different strains of M. circinelloides were treated with N-methyl-NЈ-nitro-N-nitrosoguanidine at a final concentration of 0.1 mg/ml in accordance with Benito et al (1995). A single round of mutagenesis was carried out with each strain.…”

“…The carotenoid mutants were isolated by visual inspection of the colonies grown after plating treated spores on YNB pH 3 medium, as described by RuizHidalgo et al (1995). The procedure for the isolation and characterization of uracil auxotrophic mutants is a positive selection method which has been described in detail elsewhere (Boeke et al, 1984;Benito et al, 1995). 5-fluoroorotic acid (5-FOA) 2 is a toxic analog of orotic acid.…”

“…The related fungus Mucor circinelloides is being used in both basic and applied areas of biological research (for a review see Orlowsky, 1991) and shows high enough transformation efficiencies to isolate genes by complementation with a DNA library (Van Heeswijck and Roncero, 1984;Anaya and Roncero, 1991). Recently we have found that Phycomyces genes can be expressed in M. circinelloides, revealing a common genetic architecture in both fungi (Iturriaga et al, 1992;Benito et al, 1995). The genetics of M. circinelloides is poorly characterized, but the successful application of molecular techniques compensates, in part, for the lack of sexual genetic analysis in this fungus.…”

In VivoAddition of Telomeric Repeats to Foreign DNA Generates Extrachromosomal DNAs in the Taxol-Producing FungusPestalotiopsis microspora

“…This may explain why, in F. oxysporum, a minority of transformants contain extrachromosomal DNAs, whereas in the other systems most transformants harbor these DNAs. Indeed, it is clear that sequence requirements for DNA replication in fungi vary (Benito et al, 1995;Broach et al, 1982). Perhaps P. microspora has relaxed sequence requirements for replication, or terminal telomeric sequences are sufficient, as is believed to be the case for H. capsulatum (Woods and Goldman, 1993).…”

Blakeslea trispora Genes for Carotene Biosynthesis

“…1). pLeu4 (2,27) and pEPM9 (8) were used as M. circinelloides shuttle vectors. General procedures for plasmid DNA purification, cloning, and transformation of E. coli were those of Sambrook et al (30).…”