The Drosophila somatic sex-determination regulatory pathway has been well studied, but little is known about the target genes that it ultimately controls. In a differential screen for sex-specific transcripts expressed in fly heads, we identified a highly male-enriched transcript encoding Takeout, a protein related to a superfamily of factors that bind small lipophilic molecules. We show that sex-specific takeout transcripts derive from fat body tissue closely associated with the adult brain and are dependent on the sex determination genes doublesex (dsx) and fruitless (fru). The male-specific Doublesex and Fruitless proteins together activate Takeout expression, whereas the female-specific Doublesex protein represses takeout independently of Fru. When cells that normally express takeout are feminized by expression of the Transformer-F protein, male courtship behavior is dramatically reduced, suggesting that male identity in these cells is necessary for behavior. A loss-of-function mutation in the takeout gene reduces male courtship and synergizes with fruitless mutations, suggesting that takeout plays a redundant role with other fru-dependent factors involved in male mating behavior. Comparison of Takeout sequences to the Drosophila genome reveals a family of 20 related secreted factors. Expression analysis of a subset of these genes suggests that the takeout gene family encodes multiple factors with sex-specific functions.

Mating behavior in Drosophila depends critically on the sexual identity of specific regions in the brain, but several studies have identified courtship genes that express products only outside the nervous system. Although these genes are each active in a variety of non-neuronal cell types, they are all prominently expressed in the adult fat body, suggesting an important role for this tissue in behavior. To test its role in male courtship, fat body was feminized using the highly specific Larval serum protein promoter. We report here that the specific feminization of this tissue strongly reduces the competence of males to perform courtship. This effect is limited to the fat body of sexually mature adults as the feminization of larval fat body that normally persists in young adults does not affect mating. We propose that feminization of fat body affects the synthesis of male-specific secreted circulating proteins that influence the central nervous system. In support of this idea, we demonstrate that Takeout, a protein known to influence mating, is present in the hemolymph of adult males but not females and acts as a secreted protein.

The pre-mRNA splicing machinery recognizes exons and joins them together with remarkable precision to form mRNAs with intact translational reading frames. Splicing requires canonical sequences at the intron/exon border, and mutation of these sequences may cause abnormal splicing patterns that affect gene expression and cause disease. Recent studies indicate that distinct sequence elements that are distant from the splice sites are also needed for normal splicing. These elements can affect splice-site recognition during constitutive splicing and also play important roles in directing alternative splicing, a common phenomenon in which multiple mRNAs, encoding functionally distinct proteins, are generated by use of different combinations of splice junctions, according to developmentally regulated or tissue-specific programs. A number of auxiliary splicing elements required for cell-specific modulation of alternative splicing have been found within introns that flank alternative exons. A second set of splicing elements, exonic splicing enhancers (ESEs), are found within both coding and noncoding exons. These enhancers direct the specific recognition of splice sites during constitutive and alternative splicing. The prevalence of alternative splicing as a mechanism for regulation of gene expression makes it a likely target for alterations leading to human disease. Below we summarize what is known about various sequences that affect splice-site selection and illustrate how changes in alternative splicing may lead either to disease or, conversely, to an amelioration of the effects of certain genetic lesions. Intronic Splicing Elements and Splicing RegulatorsAlthough modulation of the nuclear concentrations of constitutive RNA processing factors causes some al-ternative splicing events (Takagaki et al. 1996), this mechanism is unlikely to account for all regulated splicing observed in vertebrates. There are many examples in which different regulatory programs run concurrently within the same cell, suggesting that different alternatively spliced pre-mRNAs are regulated by distinct programs that use different sets of cis elements and transacting factors. Strong evidence that cell-specific factors are responsible for alternative splicing comes from studies on intronic elements that mediate cell-specific splicing (Guo et al. 1991;Tacke and Goridis 1991;Black 1992;Gooding et al. 1994;Huh and Hynes 1994;Ryan and Cooper 1996). One model system in which such elements have been identified is the cardiac troponin T (cTNT) gene (Ryan and Cooper 1996). Figure 1A shows a diagram of cTNT exons 4-6, in which the alternative exon 5 is included in embryonic striated muscle and is skipped in the adult. Exon inclusion in embryonic muscle requires intronic elements, referred to as "musclespecific splicing enhancers" (MSEs), located a short distance upstream and downstream of the exon (shown as small boxes in fig. 1A). Evidence from transient transfection into embryonic muscle and nonmuscle cell cultures suggests that these elements regulate ...

Regulation of gene expression through alternative pre-mRNA splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila.Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mRNA from the sex determination gene doublesex (dsx). Here we report the identification of htra-2a, a human homologue of tra-2. Two alternative types of htra-2a cDNA clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one hTRA-2a isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2a to regulate d&x is femalespecific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2a has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions.Despite the fact that a large fraction of identified cellular pre-mRNAs undergo alternative splicing, few vertebrate factors have been identified that affect splicing patterns. Of those factors so far studied, the evidence for a role in the regulation of splicing is best for the SR proteins, a family of RNA binding proteins that contain extensive regions rich in arginine and serine (RS domains) (for review, see ref. 1). Several lines of evidence suggest these domains may facilitate interactions with other RNA binding proteins. While SR proteins are known to play vital roles during constitutive pre-mRNA splicing both in the initiation of spliceosome assembly and in interactions between small nuclear ribonucleoproteins they have also been shown to affect the selection of alternative 5' splice sites in a variety of artificial and natural substrates in a concentration dependent manner. In addition, several SR proteins are known to interact with the purine-rich splicing enhancer elements found in some vertebrate exons. While these observations strongly suggest a role for SR proteins in regulating splicing, there is still little direct evidence that vertebrate SR proteins normally direct the developmentally specific alternative splicing of any particular cellular pre-mRNAs in vivo.Proteins with established roles in developmental regulation of splicing have been identified in Drosophila through genetic analysis (2-7). Many of these proteins form a cascade of splicing factors that directs sexual differentiation in the fly. Two SR-related proteins, encoded by the transformer (tra) and transformer-2 (tra-2) genes, play a central role in this pathway (8). However, unlike the vertebrate SR proteins described above, these SR-related splicing factors...

Alternative mRNA splicing directed by SR proteins and the splicing regulators TRA and TRA2 is an essential feature of Drosophila sex determination. These factors are highly phosphorylated, but the role of their phosphorylation in vivo is unclear. We show that mutations in the Drosophila LAMMER kinase, Doa, alter sexual differentiation and interact synergistically with tra and tra2 mutations. Doa mutations disrupt sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination, by affecting the phosphorylation of one or more proteins in the female-specific splicing enhancer complex. Examination of pre-mRNAs regulated similarly to dsx shows that the requirement for Doa is substrate specific. These results demonstrate that a SR protein kinase plays a specific role in developmentally regulated alternative splicing.

The transformer-2 (tra-2) gene of Drosophila melanogaster plays essential roles in both sexual differentiation in the female soma and spermatogenesis in the male germ line. In the female soma, tra-2 is known to act with other genes in the sex determination regulatory cascade to control the sex-specific alternative splicing of transcripts from the doublesex gene. Here, we determine whether or not any sex-specific tra-2 products are expressed that may account for either of these sex-specific activities. Sequence analysis of the tra-2 gene and 10 tra-2 cDNA clones coupled with nuclease protection analysis reveals a variety of alternatively spliced tra-2 mRNAs that each encode one of four distinct but overlapping polypeptides. Three of the encoded polypeptides contain both a ribonucleoprotein consensus sequence and arginine/serine-rich regions, suggesting a direct role for these products in RNA splicing. We show that although two transcripts are expressed male specifically in the germ line, the tra-2 transcripts expressed in the soma are not sex-specific. The translation of products from a tra-2-1acZ fusion gene in both sexes suggests that the female-specific functioning of tra-2 in somatic tissues is not attributable to a translational mechanism. We suggest that tra-2 activity in somatic tissues is regulated through a post-translational sex-specific interaction with the product of the tra gene rather than through the expression of a female-specific tra-2 polypeptide.

The Drosophila transformer-2 gene uses alternative promoters and splicing patterns to generate four different mRNAs that together encode three putative RNA-binding polypeptides. The transformer-2 products expressed in somatic tissues function to regulate the RNA splicing of the sex determination gene doublesex, whereas products expressed in the male germ line play an unknown, but essential, role in spermatogenesis. Two alternatively spliced transformer-2 transcripts, each encoding a different putative RNA-binding protein, are found only in the male germ line. These male germ line-specific mRNAs differ from each other by the presence or absence of a single intron called M1. We show that Ml-containing transcripts make up a majority of transformer-2 germ-line transcripts in wild-type males but fail to accumulate in males homozygous for transformer-2 null mutations. Germ-line transformation experiments using a variety of reporter gene constructs demonstrate that specific polypeptide products of the transformer-2 gene itself normally repress M1 splicing in the male germ line. Thus, in addition to its role in the sex-specific control of doublesex RNA splicing in somatic tissues, the transformer-2 gene also regulates the splicing of its own transcripts in the male germ line. We propose that this autoregulatory function may serve in negative feedback control of transformer-2 activity during spermatogenesis. The finding that transformer-2 controls multiple splicing decisions suggests that a variety of different alternative splicing choices could be regulated by a relatively limited number of trans-acting factors.

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybridselected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain gene in D. melanogaster; its chromosomal location is region 98B. This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.Myosin light chains are proteins which occur abundantly and in a defined stoichiometry in myofibrils. They are members of an evolutionarily related group of calciumbinding proteins known as the troponin C superfamily, which includes calmodulin, troponin C, and the myosin alkali and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chains. The primary amino acid sequence has been determined for at least one vertebrate example of each of these polypeptides (2). The principal sequence homology between these proteins resides in the putative Ca-+-binding domains, which are known as EF hands (14). The roles of all of these proteins, except for the myosin alkali light chain, in muscle function have been determined (10. 13, 29; R. A. Murphy, M. 0. Askoy, P. F. Dillon, W. T. Gerthoffer, and K. E. Kanim, Fed. Proc. 42:51-57, 1983).The skeletal muscle myosin alkali light chains are so named because of the high pH required to dissociate them from the myosin heavy chain (39). For vertebrate muscles, they are sometimes called MLC-1 and MLC-3. The two skeletal muscle alkali light chains of mammals and chickens, which have molecular weights of about 21,000 (MLC-1) and 17,000 (MLC-3), are virtually identical in sequence over their C-terminal 141 residues, but diverge in sequence at the amino terminus. MLC-1, depending upon the tissue from which it is isolated, has an additional alanine-proline-or alanine-lysine-rich sequence of 40 amino acids at its amino terminus. There is evidence that in rats the two proteins are encoded by a single gene (L. Garfinkel, R. Gubits, B. NadalGinard, and N. Davidson, manuscript in preparation). At one time, these peptides were thought to be essential for the actin-activated adenosine triphosphatase activity of myosin (16,32,38)