cir-2R TTAAAGACTTCATAGTTGTTCTT Primer for 3′-RACE PCR (Gene-specific primer) GSP-Mucor-1 F GATGGTCGTGCCTGGTCTATCCAAT Primer for 5′-RACE PCR (gene-specific primer) GSP-Mucor-2R CATTGTCTCTGGCACCGTATTGAGCAGC Primers for full-length cDNA and recombinant plasmids APMC-EcoNaeI-F ATGGAATTCGCCGGCGCTACTACTGATGCCACTGGTACTGTCCCCG APMC-F AGGAATTCTTCTCATTAGTCTCTTCTTG APMC-Met-F ATGGAATTCATGAAATTCTCATTAGTCTCTTCTTGTGTC MCAP-3 F TATCTCGAGaaaagaGCTCCCAGTGGTAGCAAGAA XhoI-N-MCAP-F TATCTCGAGaaaagaATGAAATTCTCATTAGTCTCTTCTTGTG APMC-NotI-R AAAGCGGCCGCGACAGATTTGGCAATTT APMC-Stop-R

GTGATTTATAGATAGATAGATGAAATGTACCAAA Primers to identify clones containing recombinant plasmids pGAP-F GTCCCTATTTCAATCAATTGAACAAC AOX1pGAP-Rev CAAATGGCATTCTGACATCCTC The underlined sequences (GAATTC; EcoRI, CTCGAG; XhoI and GCGGCCGC; NotI) represent the additional restriction LY3009104 mw sites at the 5′ ends of forward and reverse primers. The lowercase letters indicate the Kex2 cleavage sites. The primers (for First-strand cDNA

synthesis, 3′-RACE cDNA and 5′-RACE cDNA) provided in the SMART RG7112 mouse RACE cDNA Amplification Kit (Clontech) are not described in the table. The PCR reactions contained the following components each listed at their final concentrations: 1 × Advantage 2 PCR Buffer, 200 pmol μL-1 dNTPs, 2 pmol μL-1 of each primer (forward and reverse), 2.5 μL of 5′ first-strand cDNA (unknown concentration), 1 × Advantage 2 Polymerase Mix (Clontech, Palo Alto, CA, USA). PCR was carried out at an annealing temperature of 61°C. Amplification of the cDNA encoding MCAP To clone the full-length cDNA encoding MCAP in M. circinelloides, a partial sequence of genomic DNA of the acidic proteinase gene was first obtained. Non-specific

primers (12 ND-F and M.cir-2R) (Table 2) were designed using the conserved motifs of aspartic proteinases from different species of filamentous fungi (Figure 1). In this case, the amino acid sequence of the Mucor bacilliformis proteinase [12] and those of Rhizopus microspores var. rhizopodiformis (accession Cell Cycle inhibitor number CAA72511), Rhizopus niveus (accession number Q03700), Rhizopus microspores var. chinensis (accession number AAB59306), Rhizopus microsporus var. chinensis (accession number AAA33881), Rhizopus microsporus var. chinensis (accession Sitaxentan number AAA33879) and Syncephalastrum racemosum (accession number AAC69517) were downloaded from the GenBank and aligned with BLAST. Figure 1 Multiple alignment of the consensus motifs sequences NDIEYYG and FLKNNYVVFN of several fungal aspartic proteinases. Consensus motifs sequences are marked in black arrows. Asterisks indicate conserved amino acids. The number to the right of the amino acid sequence is based on the protein. After PCR, a 956 bp fragment was obtained. PCR amplification was carried out at an annealing temperature of 52°C using 1.25 U Taq DNA polymerase and 200 ng of genomic DNA.