Characterization of the proteasomebeta2 subunit gene and its mutant allele in the tephritid fruit fly pest,...

In Drosophila melanogaster the beta2 proteasome subunit gene, Prosbeta2, was first identified as a dominant temperature sensitive mutant, DTS-7, that causes pupal lethality at 29 degrees C but allows survival to adulthood at 25 degrees C. To explore the use of proteasome mutations for a conditional lethal system in insect pests, we identified and isolated the beta2 subunit gene of the 20S proteasome from the Caribbean fruit fly, Anastrepha suspensa. The caribfly ortholog AsProsbeta2 was isolated from pupal cDNA by 5' and 3' RACE. The AsProsbeta2 protein has high amino acid sequence similarity to predicted insect Prosbeta2 subunits and homologs from yeast and mammals, and it contains the well conserved amino acids that confer catalytic activity and substrate specificity. AsProsbeta2 is a single copy gene and its RNA accumulates throughout all developmental stages of the caribfly. For functional studies a point mutation, analogous to the Prosbeta2(1) mutation in D. melanogaster, was introduced into AsProsbeta2 to create an aberrant protein with a Gly170Arg substitution. Consistent with the DTS-7 mutation, transgenic insects carrying the mutant allele undergo normal metamorphosis at the permissive temperature (25 degrees C) but at the non-permissive temperature (29 degrees C) they exhibit effective pupal lethality. This is the first report of a functional characterization of a Prosbeta2 cognate based on the creation of a dominant temperature-sensitive mutation. This type of temperature-dependent lethality could be used for biological control, where transgenic insects are reared to adulthood at 25 degrees C or lower and then released into the field where ambient temperatures averaging 29 degrees C or greater cause lethality in their progeny.