Abstract. We have isolated a Drosophila melanogaster alpha-actinin gene and partially characterized several mutant alleles. The Drosophila protein sequence is very similar (68 % identity) to those of chicken alpha-actinin isoforms, but less closely related (30% identity) to Dictyostelium alpha-actinin. The gene is within subdivision 2C of the X chromosome, coincident with 15 lethal (1)2Cb mutations. At least four alleles, l(1)2Cb l, l(1)2Cb 2, l(1)2Cb 4, and l(1)2Cb 5 are interrupted by rearrangement breakpoints and must be null. In all four cases, hemizygous mutants complete embryogenesis and do not die until the second day of larval growth, signifying that either the role of alphaactinin in nonmuscle cells is redundant or that a distinct and only distantly related gene encodes the nonmuscle isoform. Allelic but less severely affected ilia mutants are apparently due to point mutations, and develop into adults having thoracic muscle abnormalities. EM of mutant muscles reveals that Z discs and myofibrillar attachments are disrupted, whereas epithelial "tendon" cells are less affected. We discuss these phenotypes in the light of presumed in vivo alphaactinin functions.T o better understand muscle physiology and mechanical properties of the cytoskeleton, it is essential to learn in more detail how actin filaments are organized and anchored. The observation that distinct muscle types have rather differently arranged Z discs suggests that thin filament organization is a critical determinant of contractile properties (Auber and Couteaux, 1963;Goldstein et al., 1982;Yamaguchi et al., 1985). Likewise, considerable evidence suggests that proteins that anchor or cross-link actin filaments profoundly affect cytoskeletal functions (Smith, 1988).Probably the best characterized actin filament crosslinking protein is alpha-actinin. The 100,000-mol wt polypeptide occurs as antiparallel homodimers, which approximate rods 7 nm wide by 48 nm long . Both muscle and nonmuscle isoforms of alpha-actinin have been described (Endo and Masaki, 1982;Duhaiman and Bamburg, 1984). Skeletal muscle alpha-actinin is stably localized within Z discs (Masaki et al., 1967), whereas the distribution of nonmuscle isoforms is dynamic and complex, generally paralleling that of microfilaments. In avian fibroblasts, for example, alpha-actinin antibodies stain stress fibers in a periodic pattern and also decorate less orderly cortical actin filament arrays, especially in the vicinity of where cells are bound to the substrate (Lazarides and Burridge, 1975;Tokuyasu et al., 1981).Recent sequence analyses have generated a hypothesis for alpha-actinin structure and function (refer to the review of Blanchard et al., 1989). Alpha-actinins include three distinct domains, an NH2-terminal actin binding domain of ~220 residues, four 122-amino acid central repeats, and a carboxyterminal region containing two EF-hand-like sequences. The actin-binding regions, which interact most closely with residues 1-12 and 86-123 of actin, would be located at the ends of the rod-sh...
Abstract. We have investigated accumulation of a-actinin, the principal cross-linker of actin filaments, in four Drosophila fliA mutants . A single gene is variably spliced to generate one nonmuscle and two muscle isoforms whose primary sequence differences are confined to a peptide spanning the actin binding domain and first central repeat . In fliA3 the synthesis of an adult muscle-specific isoform is blocked in flight and leg muscles, while in fliA4 the synthesis of nonmuscle and both muscle-specific isoforms is severely reduced . Affected muscles are weak or paralyzed, and, T HE cellular and developmental roles of the spectrin superfamily ofproteins are being clarified by genetics. Hemolytic diseases of humans are known to result from spectrin defects that weaken the erythrocyte plasma membrane, predisposing the cells to collapse in the face of circulatory system shear forces (Knowles et al., 1983 ;Marchesi et al., 1987) . Analyses of humans, dogs, and mice having muscular dystrophies have correlated dystrophin defects with syndromes of muscle wasting (Hoffman et al ., 1987;Cooper et al., 1988). These results suggest that mutant dystrophins engender muscle necrosis by failing to localize or anchor sarcolemmal glycoproteins (Ervasti et al., 1990) . Genetic investigations of the third spectrin superfamily member and principal actin filament cross-linking protein, a-actinin, have not been as informative. Gene knockout experiments performed in Dictyostelium did not confer a detectable phenotype (Wallraff et al., 1986;Noegel and Witke, 1988;Schleicher et al., 1988). This result almost certainly signifies that the role of a-actinin in nonmuscle cells is largely redundant, but does not clarify the function of the protein . aActinin mutations in Drosophila engender either lethal or flightless phenotypes (Fyrberg et al ., 1990) . No nonmuscle cell phenotypes have been noted, but within muscles disruptions of Z-discs and attachments of muscle fibers to epithelial tendon cells are readily apparent (Fyrberg et al., 1990).Our in the case of fliA3, atrophic. Their myofibrils, while structurally irregular, are remarkably normal considering that they are nearly devoid of a major contractile protein . Also surprising is that no obvious nonmuscle cell abnormalities can be discerned despite the fact that both the fliA'-andfliA4-associated mutations perturb the nonmuscle isoform . Our observations suggest that a-actinin stabilizes and anchors thin filament arrays, rather than orchestrating their assembly, and further imply that a-actinin function is redundant in both muscle and nonmuscle cells.Z-discs, where it has been proposed to cross-link ends of both parallel and antiparallel arrays of actin filaments (Suzuki et al., 1976;Endo and Masaki, 1982 ;Duhaiman and Bamburg, 1984; for review see Blanchard et al ., 1989) . In vitro studies have shown that at very low (0-4°C) temperatures, antiparallel dimers of a-actinin do indeed cross-link actin filaments (Goll et al ., 1972;Bennett et al., 1984), but the length of these cross-li...
We have isolated a Drosophila gene, DmGST-2, that encodes glutathione S-transferase, a homo- or heterodimeric enzyme thought to be involved in detoxification of xenobiotics, including known carcinogens. The encoded protein has a primary sequence that is more similar to mammalian placental and nematode GSTs than that of a previously described Drosophila GST gene, herein referred to as DmGST-1. We provide a physical map of the gene and show that it specifies at least two mRNAs, measuring 1.9 and 1.6 kb, which differ only in the lengths of their 3' untranslated regions. Both of the mRNAs are present during all developmental stages. In situ hybridization of the DmGST-2 gene to larval polytene chromosomes places it within the 53F subdivision of chromosome 2, and Southern blotting to chromosomal DNA indicates that the gene has no close relatives within the Drosophila genome. Our results make possible molecular genetic approaches for further elaborating the function of glutathione S-transferases in insect development and physiology, in the metabolism of plant toxins, and in conferring insecticide resistance.
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