Development of an indirect flight muscle in a muscle-specific mutant of Drosophila melanogaster

Stripe (sr) is a highly specific mutant affecting only one of the indirect flight muscles, the dorsal longitudinal muscle (DLM). In the homozygous condition the DLM is reduced in size. In the hemizygous condition (sr/Df(3)sr) no DLM is present in the adult, though all other thoracic muscles are present. In the early stages of pupation, DLM development in sr/Df(3)sr is no different from that in wild type. Adult myocytes collect around target larval muscles and fuse to form myotubes; myofilaments are synthesized. Subsequently (35-hr pupa) the DLM commences to degenerate, forming random clumps of vacuolated muscle tissue. Adjacent muscles are unaffected and develop normally. In the adult a neuroma-like mass of nerve tissue is maintained where the DLM would normally be located. In this mass many abnormal synapses (hemisynapses) are seen: presynaptic specializations occur in the absence of any postsynaptic structure. Small remnants (less than 16-microns diameter) of muscle tissue are sometimes found in the neuroma-like mass. Such remnants resemble slow muscle, not the normal fast type of DLM. These data suggest a possible muscle origin from primary and secondary myotubes. The DLM motor axons are present in the neuroma-like mass, persisting even with the virtual degeneration of their end target. Thus, motoneurons and presynaptic specializations can survive independently of postsynaptic targets.     

Drosophila ACT88F indirect flight muscle-specific actin is not N-terminally acetylated: a mutation in N-terminal processing affects actin function

Many eukaryotic proteins are co and post-translationally modified at their N termini by removal of one or two amino acid residues and N(alpha)-acetylation. Actins show two different forms of N-terminal processing dependent on their N-terminal sequence. In class II actins, which include muscle actins, the common primary sequence of Met-Cys-Asp-actin is processed to acetyl-Asp-actin. The functional significance of this in vivo is unknown. We have studied the indirect flight muscle-specific actin, ACT88F, of Drosophila melanogaster. Our results show that ACT88F is N-terminally processed in vivo as a class II actin by removal of the first two amino acid residues (Met and Cys), but that uniquely the N terminus is not acetylated. In addition we show that ACT88F is methylated, probably at His73.Flies carrying the mod(-) mutation fail to complete post-translational processing of ACT88F. We propose that the mod gene product is normally responsible for removing N-acetyl-cysteine from actin. The biological significance of this process is demonstrated by observations that retention of the N-acetyl-cysteine in ACT88F affects the flight muscle function of mod(-) flies. This suggests that the extreme N terminus affects actomyosin interactions in vivo, a proposal we have examined by in vitro motility assays of ACT88F F-actin from mod(-) flies. The mod(-) actin only moves in the presence of methylcellulose, a viscosity-enhancing agent, where it moves at velocities slightly, but significantly, reduced compared to wild-type. These data confirm that N-acetyl-cysteine at the N terminus affects actomyosin interactions, probably by reducing formation of the initial actomyosin collision complex, a process known to involve the actin N terminus.     

A nonsense mutation within the act88F actin gene disrupts myofibril formation in Drosophila indirect flight muscles

We have investigated the molecular basis of muscle abnormalities in the flightless Drosophila mutant lfm(3)7. This EMS-induced, semi-dominant allele was isolated by Mogami and Hotta (1981) and was shown to disrupt the organization of myofibrils in indirect flight muscles. Here we demonstrate that lfm(3)7 contains a nonsense mutation within codon 355 of the act88F actin gene. A single G greater than A transition converts a tryptophan (TGG) codon to an opal (TGA) terminator, thus deleting the carboxy-terminal 20 amino acids of an actin isoform that accumulates only in thoracic flight muscles. The truncated actin polypeptide is stable, and retains antigenicity to at least two anti-Drosophila actin monoclonal antibodies. We suggest that abnormalities in lfm(3)7 flight muscles result from incorporation of the mutant actin isoform into assembling myofibrils.     

Protein synthesis during flight muscle development in normal and wupB indirect flight muscles of Drosophila melanogaster

We have looked at protein synthesis in Drosophila pupae during normal and abnormal development of indirect flight muscle. Abnormal development was followed in the dominant flightless mutant wupB isolated by Hotta and Benzer (Genetic Mechanisms of Development, pp. 129–167. Academic Press, New York, 1972). The mutant muscles in adult wupB flies have abnormal morphology and disorganized myofibrils. Protein synthesis in developing muscle was followed on SDS-polyacrylamide gels. During early stages of development (55–60 h) protein synthesis patterns are similar in the mutant and the wild-type. However, at 61 h, the mutant shows a transient increase in synthesis of the 68 and 70 kDa heat shock proteins. This is followed at about 70 h by a divergence of the patterns of synthesis of other proteins seen in the mutant and wild type. These results suggest that induction of heat shock protein synthesis is an early event in abnormal morphogenesis in this mutant.     

In vivo x-ray diffraction of indirect flight muscle from Drosophila melanogaster

Small-angle x-ray diffraction from isolated muscle preparations is commonly used to obtain time-resolved structural information during contraction. We extended this technique to the thoracic flight muscles of living fruit flies, at rest and during tethered flight. Precise measurements at 1-ms time resolution indicate that the myofilament lattice spacing does not change significantly during oscillatory contraction. This result is consistent with the notion that a net radial force maintains the thick filaments at an equilibrium interfilament spacing of approximately 56 nm throughout the contractile cycle. Transgenic flies with amino-acid substitutions in the conserved phosphorylation site of the myosin regulatory light chain (RLC) exhibit structural abnormalities that can explain their flight impairment. The I(20)/I(10) equatorial intensity ratio of the mutant fly is 35% less than that of wild type, supporting the hypothesis that myosin heads that lack phosphorylated RLC remain close to the thick filament backbone. This new experimental system facilitates investigation of the relation between molecular structure and muscle function in living organisms.     

Stereotaxic map of the muscle fibers in the indirect flight muscles of Drosophila melanogaster

It is possible to monitor the electrical activity of the motor neurons of Drosophila by recording the electrical activity of the muscle fibers. We have found that it is possible to specify the location of the subcuticular terminations and to describe the orientation within the thorax for the individual muscle fibers, because of the large size of the fibers and because the surface anatomy of Drosophila is known in detail. A map has been made to indicate the location of the muscle fibers with respect to superficial landmarks. The importance of the stereotaxic map for physiological studies is discussed.     

A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression.

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.     

Transgenic expression and purification of myosin isoforms using the Drosophila melanogaster indirect flight muscle system

Biophysical and structural studies on muscle myosin rely upon milligram quantities of extremely pure material. However, many biologically interesting myosin isoforms are expressed at levels that are too low for direct purification from primary tissues. Efforts aimed at recombinant expression of functional striated muscle myosin isoforms in bacterial or insect cell culture have largely met with failure, although high level expression in muscle cell culture has recently been achieved at significant expense. We report a novel method for the use of strains of the fruit fly Drosophila melanogaster genetically engineered to produce histidine-tagged recombinant muscle myosin isoforms. This method takes advantage of the single muscle myosin heavy chain gene within the Drosophila genome, the high level of expression of accessible myosin in the thoracic indirect flight muscles, the ability to knock out endogenous expression of myosin in this tissue and the relatively low cost of fruit fly colony production and maintenance. We illustrate this method by expressing and purifying a recombinant histidine-tagged variant of embryonic body wall skeletal muscle myosin II from an engineered fly strain. The recombinant protein shows the expected ATPase activity and is of sufficient purity and homogeneity for crystallization. This system may prove useful for the expression and isolation of mutant myosins associated with skeletal muscle diseases and cardiomyopathies for their biochemical and structural characterization.     

Development of the indirect flight muscles of Drosophila.

We have followed the pupal development of the indirect flight muscles (IFMs) of Drosophila melanogaster. At the onset of metamorphosis larval muscles start to histolyze, with the exception of a specific set of thoracic muscles. Myoblasts surround these persisting larval muscles and begin the formation of one group of adult indirect flight muscles, the dorsal longitudinal muscles. We show that the other group of indirect flight muscles, the dorsoventral muscles, develops simultaneously but without the use of larval templates. By morphological criteria and by patterns of specific gene expression, our experiments define events in IFM development.     

Functional and phylogenetic analyses of a putative Drosophila melanogaster UDP-glycosyltransferase gene

Glucosidation plays a major role in the inactivation and excretion of a great variety of both endogenous and exogenous compounds. The recent determination of the complete genome sequence of Drosophila melanogaster has revealed the presence of over 30 putative UDP-glucosyltransferase (UGT) genes in this organism. We report here the molecular cloning and functional characterisation of one of these genes, named DmUgt37a1. The predicted protein comprises 525 amino acids and has about 30% overall amino acid identity with vertebrate members of the UGT family. The phylogenetic relationships of DmUgt37a1 with other members of the UGT family from D. melanogaster are discussed. DmUgt37a1 was expressed in lepidopteran insect cells and the ability of the enzyme to conjugate 38 potential substrates belonging to diverse chemical groups was assessed using UDP-glucose as sugar-donor. However, no activity was detected with any compound under the conditions used and thus, the substrate specificity of the enzyme remains unknown.     

Interacting proteins identified by genetic interactions: a missense mutation in alpha-tubulin fails to complement alleles of the testis-specific beta-tubulin gene of Drosophila melanogaster.

In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.     

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster

Summary Previous studies have demonstrated that the expression of the -amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the -amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.Offprint requests to: D.A. Hickey     

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster

A mutant gene, which we have designated AdhnB, codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by AdhnB is approximately 2000 Mr smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of AdhnB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the AdhnB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the AdhnB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in AdhnB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. AdhnB is the first reported nonsense mutant in Drosophila.     

The TATA-dependent and TATA-independent promoters of the Drosophila melanogaster actin 5C-encoding gene

The major cytoskeletal actin of Drosophila melanogaster, actin 5C, is encoded by a gene (act5C) that has two promoters which are differentially controlled and possess distinct sets of regulatory elements. The distal basal promoter has a TATA motif, but the proximal does not. The distal strong positive domain, centered at nucleotide -290, can be shifted and fused directly to the distal basal promoter without losing its activity. It can also activate heterologous basal promoters containing either TATAAAT or TATTTAA signal when directly fused to them, but cannot activate the basal proximal promoter, which is TATA-less. When the entire distal regulatory region, which includes a remote enhancer-like region, is fused to the proximal promoter, it does not increase the proximal promoter activity. Fusion of the distal strong negative domain to the proximal promoter does not inhibit activity. Thus, all the three major strong regulatory domains of the distal promoter are unable to act on the proximal promoter.     

Screen for new mutations on the 2nd chromosome involved in indirect flight muscle development in Drosophila melanogaster.

An extensive ethylmethanesulfonate mutagenesis of Drosophila melanogaster was undertaken to isolate the stronger alleles of 3 indirect flight-muscle mutations. We isolated 17 strong mutant lines, with nearly complete penetrance and expressivity, using direct screening under polarized light, from more than 1700 mutagenized chromosomes. On complementation, we found 11 of these 17 mutant lines to be alleles of 3 indirect flight-muscle mutations (Ifm(2)RU1, 3 noncomplementing lines; ifm(2)RU2, 6 alleles; ifm(2)RU3, 2 alleles) of the previously isolated 8 complementation groups (Ifm(2)RU1to ifm(2)RU8). In addition, we found 6 new complementation groups with strong defects in adult-muscle morphology; we named these ifm(2)RS1 to ifm(2)RS6. All mutant lines were mapped by meiotic recombination, and 5 of the 6 new complementation lines were mapped using chromosome deficiencies. ifm(2)RS1 maps to a region that harbors ifm(2)RU4 (a mutation that was isolated previously); however, theses are not alleles because each complements the other mutation, and the mutant-muscle phenotype is very different. We used direct screening under polarized light to find recessive mutations; although this method was labor intensive, it can be used to identify recessive genes involved in myogenesis, unlike screens for flightlessness or wing-position defects. This screen identifies regions on the second chromosome that harbor probable genes that are likely expressed in the mesoderm and are thought to be involved in myogenesis. This screen has generated valuable resources that will help us to understand the role of many molecular players involved in myogenesis.