Transformation and rescue of a flightless Drosophila tropomyosin mutant.

Abstract

In the Drosophila flightless mutant Ifin(3)3, a transposable element inserted into the alternatively spliced fourth exon of the tropomyosin I (TmI) gene prevents proper expression of Ifm-TmI, the tropomyosin isoform found in indirect flight muscle. We have rescued the flightless phenotype of Ifin(3)3 flies using P-element-mediated transformation with a segment of the Drosophia genome containing the wild-type TmI gene plus 2.5 kb of 5' flanking and 2 kb of 3' flanking DNA. The inserted TmI gene is expressed with the proper developmental and tissue specificity, although its level of expression vanes among the five transfonned lines examined. These conclusions are based on analyses of flight, myofibrillar morphology, and TmI RNA and protein levels. A minimum of two copies of the inserted TmI gene per cell is necessary to restore flight to most of the flies in each line. We also show that the Ifm-TmI isoform is expressed in the leg muscle of wild-type flies and is decreased in Ifn(3)3 leg muscle. Homozygous Ifin(3)3 mutants do not jump. The ability to jump can be restored with a single copy of the wild-type TmI gene per cell.

Small differences in Drosophila tropomyosin expression have significant effects on muscle function.

“…Expression of a second, non-IFM-TDT TmI isoform is unaffected by the Ifm(3)3 mutation. We demonstrated earlier that a cloned TmI gene introduced into Ifm(3)3 flies by P-element-mediated transformation restores TmI RNA and protein and the ability to jump and fly to nearly wild-type levels (19). Significantly, two copies of the inserted TmI gene per cell are necessary to restore flight, while one copy of the transgene restores the ability to jump.…”

“…Selected constructs were sequenced to determine the extent of the deletions. The deleted TmI genes A-1900, A-790, and A-452 were inserted between the Sacl and Sall sites of the P-element vector pUC13hsneo (17,19). All other TmI genes were inserted between the KpnI and XhoI sites of the P-element vector pW8 (8).…”

“…All other TmI genes were inserted between the KpnI and XhoI sites of the P-element vector pW8 (8). Transformations were carried out essentially as described previously (16,19). Ifm(3)3 flies obtained from Y. Hotta (11) were the recipients of plasmids derived from pUC13hsneo.…”

“…Only homozygous transformants containing a single, unrearranged copy of the transgene per haploid genome were analyzed in this study. In previous experiments, a flight tester was used to quantitate rescue of the Ifm(3)3 phenotype (19). Because of the large number of transformants generated in the experiments whose results are presented here, it was impractical to measure flight for all transformants by using the flight tester.…”

“…We focused on deletions in the region between positions -374 and -146, since this stretch of sequence contains four short elements similar to sequences present in the 5'-flanking DNA of the Drosophila tropomyosin II (Tmll) or actin88F gene (see Fig. 5 in reference 6) (19); remaining lanes, RNA from the transformants indicated in Table 1. N (jumpless and flightless), J (jump), and F (jump and fly) indicate the observed phenotypes.…”