Douglas R. Drummond

Characterisation of missense mutations in the Act88F gene of Drosophila melanogaster

SummaryWe have created missense mutations in the indirect flight muscle (IFM)-specific Act88F actin gene of Drosophila melanogaster by random in vitro mutagenesis. Following P element-mediated transformation into wild-type flies and subsequent transfer of the inserts into Act88F null strains, the effects of the actin mutants on the structure and function of the IFMs were examined. All of the mutants were antimorphic for flight ability. E316K and G368E formed muscle with only relatively small defects in structure whilst the others produced IFMs with large amounts of disruption. E334K formed filaments but lacked Z discs. V339I formed no muscle structure in null flies and did not accumulate actin. E364K and G366D both had relatively stable actin but did not form myofibrils. Using an in vitro polymerisation assay we found no significant effects on the ability of the mutant actins to polymerise. E364K and G366D also caused a strong induction of heat shock protein (hsp) synthesis at normal temperatures and accumulated large amounts of hsp22 which, together with the mutant actin, was resistant to detergent extraction. Both E316K and E334K caused a weak induction of hsp synthesis. We discuss how the stability, structure and function of the different mutant actins affects myofibril assembly and function, and the induction of hsps.

Protein engineering and the study of muscle contraction in Drosophila flight muscles

Summary We describe an experimental approach to the use of genetics to study muscle contraction in Drosophila melanogaster. Mutations induced by in vitro mutagenesis are inserted into the genome of flies using P-element mediated transformation, permitting the effects of the mutant genes to be studied in vivo in the indirect flight muscles (IFMs). Details of how mechanical experiments can be performed on skinned IFMs, despite their small size, are provided. The effects of two in vitro actin mutations, G368E and E316K, are described. The problems of performing biochemical and biophysical experiments on the IFMs and their myofibrillar proteins are described, together with indications as to how these may be overcome.