Cloning and sequence analysis of the Mucor circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase: use of pyrG for homologous transformation

Abstract

A 3.2-kb BamHI genomic DNA fragment containing the pyrG gene of Mucor circinelloides was isolated by heterologous hybridization using a pyrG cDNA clone of Phycomyces blakesleeanus as the probe. The complete nucleotide sequence of the M. circinelloides pyrG gene encoding orotidine-5'-monophosphate decarboxylase (OMPD) was determined and the transcription start points (tsp) were mapped by primer extension analysis. The predicted amino acid sequence showed homology with the OMPD sequences reported from other filamentous fungi, with 96% similarity with the OMPD of P. blakesleeanus. Analysis of the sequence revealed the presence of two short introns whose length and location were confirmed by sequencing a cDNA clone and comparing this with its genomic counterpart. The intron splice sites and the 5'- and 3'-noncoding flanking regions show general features of fungal genes. Northern-blot hybridization revealed the pyrG transcript to be approx. 1.0 kb. The M. circinelloides pyrG cDNA clone was able to complement the pyrF::Mu-1 mutation of Escherichia coli when inserted between bacterial expression signals. Additionally, the genomic clone complemented the M. circinelloides pyrG4 mutation. When an M. circinelloides autonomous replication sequence was included in the transforming plasmid, the average transformation frequency obtained was 600 to 800 transformants per micrograms DNA and per 10(6) viable protoplasts.