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.

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

A complete chitin synthase gene and one chitin synthase gene fragment of the zygomycete Mucor circinelloides have been cloned and analyzed. Both genes encode zymogenic Class II chitin synthases. Hybridization analysis showed that there must exist at least another Class II chitin synthase gene in M. circinelloides highly homologous to the cloned Mcchs1 and Mcchs2. The expression of these genes during the dimorphic growing stages was analyzed. Northern hybridizations showed that Mcchs1 transcript accumulates only during the exponentially growing hyphal stage, while no expression could be detected in the yeast form. Expression of Mcchs2 could not be detected at any stage. Accumulation of Mcchs1 transcript was not influenced by visible light. The existence of a multigene chitin synthase family and the observation that Mcchs1 transcription depends upon the dimorphic stage indicate that various chitin synthase activities may have different roles in the dimorphic growth of M. circinelloides.