The erect wing (ewg) locus ofDrosophila melanogaster encodes a vital function important for the development of the nervous system and the indirect flight muscles. In In recent years, many genes that participate in the formation of the nervous system have been identified in Drosophila melanogaster by genetic or molecular methods. It is reasonable to suggest that certain genes are important for the differentiation of all neurons, whereas others will be important for the enormous microheterogeneity found in the nervous system. Among the former category of genes, one expects to find genes that regulate common aspects of neuronal differentiation at the transcriptional and posttranscriptional levels (41) and those encoding other specialized components (12,33,45,48,50). We have been studying genes expressed in all neurons, as some are likely to play key roles in neuronal differentiation.Mutations of the D. melanogaster erect wing (ewg) locus exhibit both neural and muscle phenotypes; these include breaks in the central nervous system (CNS) commissures and longitudinal tracts and aberrant intersegmental axonal projection pathways in the embryo, aberrant giant fiber position in the adult fly (11), missing or reduced indirect flight muscles associated with erect wing posture (10), and adult hypoactivity (15). Certain ewg mutations cause embryonic lethality; however, genetic mosaic analysis has shown previously that the ewg function is not required for the viability of all cell types (17). Current evidence strongly suggests that the vital embryonic ewg function is necessary for proper neuronal development, as ewg transcripts are limited to, and distributed ubiquitously throughout, the developing nervous system of the 6-to 12-h embryos (16).The pattern of transcription from ewg is complex, with several overlapping transcripts that show temporal regulation during development (16). The neural expression of ewg transcripts, the pleiotropy of phenotypes associated with * Corresponding author. t Present address:

The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone-receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone-receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone-receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved byAlu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% ho mologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes. (4,5). DNA was isolated from frozen adult female worms or from microfilariae in whole blood by the proteinase K-digestion method of Emmons et al. (6) followed by phenol extraction. For the Southern blots and cloning experiments, the DNA was further purified by ethidium bromide/CsCl centrifugation (7), which removed a band of white material, possibly from the cuticle, that was more dense than the DNA.Cloning of Repeated DNA from B. malayi. All restriction endonucleases, methylases, ligases, linkers, and 35S-labeled dideoxy sequencing reagents used in these experiments were prepared at New England Biolabs and used as described by the supplier. B. malayi DNA was cleaved with Alu I, protected with EcoRI methylase, and then ligated to 5'-phosphorylated EcoRI linkers with T4 DNA ligase. Cohesive EcoRI ends were generated by digestion with EcoRI. The linkers were separated from the Brugia DNA by electrophoresis on a 1.5% low melt agarose gel stained with ethidium bromide. The 320-base-pair (bp) and 640-bp restriction fragments were cut out and the DNA isolated (8). In two separate reactions, either the 640-bp or the 320-bp fragments were ligated to EcoRI cleaved pBR322 that had been dephosphorylated by treatment with bacterial alkaline phosphatase (9).Escherichia coli RRI cells were transformed with the recombinant plasmids, selected for growth on ampicillin, and screened for repeated sequences by hybridization with radioactively labeled B....

In this report, we document an unusual mode of tissue-enriched gene expression that is primarily mediated by alternative and inefficient splicing. We have analyzed posttranscriptional regulation of the Drosophila erect wing gene, which provides a vital neuronal function and is essential for the formation of certain muscles. Its predominant protein product, the 116-kDa EWG protein, a putative transcriptional regulator, can provide all known erect wing-associated functions. Moreover, consistent with its function, the 116-kDa protein is highly enriched in neurons and is also observed transiently in migrating myoblasts. In contrast to the protein distribution, we observed that erect wing transcripts are present in comparable levels in neuron-enriched heads and neuron-poor bodies of adult Drosophila. Our analyses shows that erect wing transcript consists of 10 exons and is alternatively spliced and that a subset of introns are inefficiently spliced. We also show that the 116-kDa EWG protein-encoding splice isoform is head enriched. In contrast, bodies have lower levels of transcripts that can encode the 116-kDa protein and greater amounts of unprocessed erect wing RNA. Thus, the enrichment of the 116-kDa protein in heads is ensured by tissue-specific alternative and inefficient splicing and not by transcriptional regulation. Furthermore, this regulation is biologically important, as an increased level of the 116-kDa protein outside the nervous system is lethal.Most eukaryotic primary RNA transcripts undergo posttranscriptional processing requiring splicing of introns. The bestappreciated regulatory outcome of posttranscriptional processing is alternatively spliced transcripts that differ in the coding exons or have distinct 3Ј or 5Ј untranslated ends (reviewed in reference 32). A second consequence of posttranscriptional regulation is the modulation of amounts of specific transcripts dependent on differential splicing efficiencies of different splice sites. It is generally thought that differences in cell-type-specific splicing machineries result in cell type-enriched or -specific alternative splicing (5, 19). In addition, efficiency of splicing could play a major role in gene regulation as primary transcripts that are not completely processed are generally not transported to the cytoplasm and are unlikely to code functional proteins (6,21,23). We decided to investigate the role of alternative and inefficient splicing in the regulation of the Drosophila erect wing (ewg) gene, as previous studies indicated a complex transcript profile, intron-containing cDNAs, as well as poly(A) ϩ transcripts with retained introns (9). The Drosophila ewg gene provides a function that is vital in the nervous system and essential to the development of certain muscles (16). EWG protein contains an unusual DNA binding domain that is homologous to sea urchin P3A2 protein (4, 10), zebrafish Nrf (3), and mammalian transcription factors NRF-1 and initiation binding receptor (13,18,31). Our previous studies suggested that ewg primary transcrip...

The molecular study of the erect wing (ewg) locus was initiated by isolating DNA in the lA8-lB1 interval of the X chromosome. Previous developmental genetic analyses of the mutant alleles at the ewg locus have demonstrated that the wild-type ewg product is essential during embryogenesis and is required postembryonically at least for the development of indirect ffight muscle. To define the ewg-encoding DNA, chromosomal breakpoints that genetically flank the ewg locus were used. P-element-mediated transformation followed by subsequent rescue of the ewg-lethal alleles has defined a 11.5-kilobase genomic fragment as encoding the ewg locus. Northern blot analysis of transcription from this DNA has revealed a complex pattern of transcription with respect to both size and developmental profile. Tissue distribution of putative ewg transcription was examined by in situ hybridization to 6-to 14-h-old embryonic sections. These sections revealed that the expression of putative ewg messages is limited to the central nervous system-derived structures and not observed within the mesoderm during this developmental stage.In the fruit fly, Drosophila melanogaster, a large array of mutations have been discovered through phenotypic wing position abnormalities or flight abnormalities (13). Several mutants with impaired flight display defects in muscle structure or organization (8,18,23,26,27). A major fraction of these mutations are expected to reside in genes that specify muscle-specific proteins. This expectation is corroborated in the cases of a few genes whose wild-type products have been characterized (1,16,17,23,26,27). A second set of mutations may represent genes expressed in the muscle which also have expression in other tissues. Finally, a third class of mutations may identify an interesting group of genes whose expression lies outside of the mesodermal tissue but is essential to the proper development or function of muscle. The involvement of a functional nervous system in the proper development and maintenance of muscle has been recognized in vertebrate and invertebrate systems (3, 12, 14, 19, 25, 30). The recent work of Lawrence and Johnston (20) has revealed neural influence during the differentiation and development of insect musculature.We have been studying the erect wing (ewg) locus in Drosophila melanogaster because of the interesting phenotypes associated with mutations at this locus (11; R. J. Fleming, Ph.D. thesis, Brandeis University, Waltham, Mass., 1987 blastoderm, the area from which the mesoderm and the nervous system are derived (11). Thus, both the phenotypic defects associated with mutant ewg alleles and the genetic mosaic analyses are consistent with the suggestion that the ewg gene product may be required in the musculature or the nervous system.To directly address the question of tissue specificity of expression and function of the ewg gene, we have initiated a molecular characterization of the ewg-encoding DNA. The proximal to distal order for known genes in the 1A8-1B1-2 interval of the X chrom...