1887

Abstract

High-copy-number amplification of the AUD1 element is frequently associated with the large chromosomal deletions responsible for genetic instability in TK64. Five ORFs were found in a 7 kb region directly adjacent to AUD1. The putative products of ORF1, ORF2 and ORF3 showed similarities to ATP-binding cassette (ABC) sugar transporters, the deduced protein sequence of ORF4 displayed similarities to α-glucosidases whilst no homology to proteins with known functions was found for ORF5. ORF4 (renamed ) was expressed in and the protein purified and characterized. An α-glucosidase activity was detected using the synthetic α-glucoside -nitrophenyl α-D-glucopyranoside. Of the many oligosaccharides tested, only sucrose was hydrolysed at a measurable rate [specific activity 324 units (mg protein)] but no growth of TK64 on sucrose was observed. A strain in which was disrupted showed the same low α-glucosidase activity as strain TK64 and in both strains no stimulation of activity was seen by sucrose, trehalose or maltose; dextrin increased α-glucosidase activity about 10-fold. This probably resulted from induction of a second α-glucosidase-encoding gene. The AUD1 element contains three 1 kb repeats which encode DNA-binding proteins necessary for high-frequency amplification. In strains with a unique 1 kb repeat, disruption of the repeat led to a significant increase in the α-glucosidase activity. These results strongly suggest that the 1-kb-repeat-encoded proteins of AUD1 have a dual function: they are the repressors of the genes and they promote amplification of AUD1.

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2000-04-01
2021-07-29
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