AGTHR4, A NEW SELECTION MARKER FOR TRANSFORMATION OF THE FILAMENTOUS FUNGUS ASHBYA-GOSSYPII, MAPS IN A 4-GENE CLUSTER THAT IS CONSERVED BETWEEN ASHBYA-GOSSYPII AND SACCHAROMYCES-CEREVISIAE

Single-read sequence analysis of the termini of eight randomly picked clones of Ashbya gossypii genomic DNA revealed seven sequences with ho mology to Saccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-termi nus of threonine synthase, the product of the S. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the comp lete putative AgTHR4 gene of A. gossypii. It comprises 512 codons, two less than the S. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids d isplaying complete identity between these two fungal threonine synthas es presumably contains the pyridoxal phosphate attachment site. Disrup tion of the A. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying t he AgTHR4 gene and various S. cerevisiae ARS elements. Using these pla smids only very weak complementation of a S. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to the AgTHR4 gene i dentified three additional ORFs. Surprisingly, the order and orientati on of these four ORFs is conserved in A. gossypii and S. cerevisiae.