Characterization of a leuA gene and an ARS element from Mucor circinelloides.

A 4.4-kb PstI restriction endonuclease fragment of Mucor circinelloides DNA has previously been shown to both complement a leuA- mutation, and to enable the autonomous replication of plasmids within this organism. The complete nucleotide (nt) sequence of this fragment has been determined and an open reading frame of 1935 bp with no introns has been identified, which exhibits significant similarity (75% at the nt level) with 114 bp of the 5' coding region of the Saccharomyces cerevisiae LEU1 gene. Based on this and on the fact that the fragment weakly complements a leu1 auxotroph of S. cerevisiae, we concluded that the Mucor leu gene encodes alpha-isopropylmalate (alpha-IPM) isomerase and designated it leuA+ accordingly. Primer extension analysis of leuA mRNA and Northern-blot hybridization, indicated the leuA transcript to be approx. 2.3 kb in size, with 5'- and 3'-untranslated regions of 16-20 nt and approx. 450 nt, respectively. Specific Mucor ARS sequence(s) were not identified, although the general location of ARS was indicated by subcloning experiments. Nucleotide sequences are present within this region, which show some similarity with the core consensus of the S. cerevisiae ARS; however, any functional homology is doubtful, since insertion of the 4.4-kb PstI fragment into YIp5 did not increase the transformation frequency of S. cerevisiae with such a vector.