|Handler, Alfred - Al|
Submitted to: Molecular Insect Science International Symposium Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 5/1/2006
Publication Date: 5/21/2006
Citation: Xavier, N., Handler, A.M. 2006. Use Of Missense Proteasome Subunits For Conditional Lethality In the Tephritid Fruit Flies Anastrepha Suspensa And Ceratitis Capitata. 5th International Symposium on Molecular Insect Science, May 21, 2006, Tucson, Arizona. Interpretive Summary: N/A
Technical Abstract: Proteasomes play a critical role in eukaryote development by regulating protein degradation (see Covi et al., 1999). Ubiquinated proteins undergo proteolysis in a multi-subunit complex known as the 26S proteasome, which is comprised of a 20S core and 19S regulatory complexes. Mis-sense mutations in the 20S subunits lead to the production of dominant temperature-sensitive (DTS) “poison subunits” or antimorphs that disrupt proteasome function. DTS5 and DTS7 are two such mutations identified originally in Drosophila melanogaster that result in late larval or pupal lethality at 29'C. To study the potential of these genes to control the populations of tephritid fruit fly pests by conditional lethality, the D. melanogaster DTS5 mutation was genetically transformed into the medfly, Ceratitis capitata, and the caribfly, Anastrepha suspensa. When reared at 30'C transformed medflies homozygous for the transgene exhibited 90-95% late larval or pupal lethality, with lower lethality levels found in transformed caribflies (Handler, unpublished). To enhance the temperature sensitive lethal effect we propose the use of native mutated proteasome genes in these species. The proteasome '2 subunit corresponding to DTS7 was isolated from A. suspensa pupal cDNA library by gene amplification. Degenerate primers designed from the most conserved regions of insect DTS7 were used in combination with 5’ and 3’ adaptors. Subsequently DTS7 genomic DNA was isolated by gene amplification using gene specific primers. The A. suspensa DTS7 (AsDTS7) coding region contains 843 nts that potentially encodes a 281 amino acid protein. Residues 40 to 224 comprise the proteasome beta domain conserved among eukaryotes. At the amino acid level AsDTS7 shares 85.7% to D. melanogaster proteasome subunit. AsDTS7 transcript contains 1024 bp that is interrupted in the genome by 3 short introns ranging in size from 57-66 nts. Northern blot analysis indicates the presence of AsDTS7 transcript from embryonic through the adult stages with quantitative variations during development, with an apparent maternal contribution to embryos. In vitro mutagenesis will be used to introduce the missense mutation in AsDTS7 that corresponds to the DTS7 mutation in D. melanogaster.