The transformer-2 (tra-2) locus is one of a set of regulatory loci that control sex determination in Drosophila melanogaster. Temperature-shift experiments with temperature-sensitive tra-2 mutants demonstrate that within single cell lineages tra-2+ function is required at several times, and probably continuously, during development for the occurrence of a series of determinative decisions necessary for female sexual differentiation. Analysis of the effects of tra-2 in the genital disc demonstrates that the tra-2+ function is necessary in females both to prevent male sexual differentiation and to permit female differentiation. These and other results support the model that the tra-2+ and tra+ loci act to control the expression of the bifunctional doublesex (dsx) locus.

Female sex determination in the germ line of Drosophila melanogaster is regulated by genes functioning in the soma as well as genes that function within the germ line. Genes known or suspected to be involved in germ-line sex determination in Drosophila melanogaster have been examined to determine if they are required upstream or downstream of Sex-lethal+, a known germ-line sex determination gene. Seven genes required for female-specific splicing of germ-line Sex-lethal+ pre-mRNA are identified. These results together with information about the tissues in which these genes function and whether they control sex determination and viability or just sex determination in the germ line have been used to deduce the genetic hierarchy regulating female germ-line sex determination. This hierarchy includes the somatic sex determination genes transformer+, transformer-2+ and doublesex+ (and by inference Sex-lethal+), which control a somatic signal required for female germ-line sex determination, and the germ-line ovarian tumor genes fused+, ovarian tumor+, ovo+, sans fille+, and Sex-lethal+, which are involved in either the reception or interpretation of this somatic sex determination signal. The fused+, ovarian tumor+, ovo+ and sans fille+ genes function upstream of Sex-lethal+ in the germ line.

Many of the genes in the regulatory hierarchy controlling sex determination in Drosophila melanogaster are known. Here we examine how this regulatory hierarchy controls the expression of the structural genes encoding the female-specific yolk polypeptides. Temperature shift experiments with a temperature-sensitive allele of the sex determination regulatory gene transformer-2 (tra-2) showed that tra-2+ function is required in the adult for both the sex-specific initiation and maintenance of YP synthesis. Control of the YP genes by this regulatory hierarchy is at the level of transcription, or transcript stability. The results of temperature shift experiments with abdomens isolated from tra-2ts homozygotes support the notion that the tra-2+ function acts in a cell-autonomous manner to control YP synthesis. These results provide a paradigm for the way this regulatory hierarchy controls the terminal differentiation functions for sexually dimorphic development.

In species where males and females differ in number of sex chromosomes, the expression of sex-linked genes is equalized by a process known as dosage compensation. In Drosophila melanogaster, dosage compensation is mediated by the binding of the products of the male-specific lethal (msl) genes to the single male X chromosome. Here we report that the sex- and chromosome-specific binding of three of the msl proteins (MSLs) occurs in other drosophilid species, spanning four genera. Moreover, we show that MSL binding correlates with the evolution of the sex chromosomes: in species that have acquired a second X chromosome arm because of an X-autosome translocation, we observe binding of the MSLs to the 'new' (previously autosomal) arm of the X chromosome, only when its homologue has degenerated. Moreover, in Drosophila miranda, a Y-autosome translocation has produced a new X chromosome (called neo-X), only some regions of which are dosage compensated. In this neo-X chromosome, the pattern of MSL binding correlates with the known pattern of dosage compensation.

The maleless (mle) gene is one of four known regulatory loci required for increased transcription (dosage compensation) of X-linked genes in D. melanogaster males. A predicted mle protein (MLE) contains seven short segments that define a superfamily of known and putative RNA and DNA helicases. MLE, while present in the nuclei of both male and female cells, differs in its association with polytene X chromosomes in the two sexes. MLE is associated with hundreds of discrete sites along the length of the X chromosome in males and not in females. The predominant localization of MLE to the X chromosome in males makes it a strong candidate to be a direct regulator of dosage compensation.

In Drosophila dosage compensation increases the rate of transcription of the male's X chromosome and depends on four autosomal male-specific lethal genes. We have cloned the msl-2 gene and shown that MSL-2 protein is co-localized with the other three MSL proteins at hundreds of sites along the male polytene X chromosome and that this binding requires the other three MSL proteins. msl-2 encodes a protein with a putative DNA-binding domain: the RING finger. MSL-2 protein is not produced in females and sequences in both the 5' and 3' UTRs are important for this sex-specific regulation. Furthermore, msl-2 pre-mRNA is alternatively spliced in a Sex-lethal-dependent fashion in its 5' UTR.

In Drosophila, dosage compensation occurs by increasing the transcription of the single male X chromosome. Four trans-acting factors encoded by the male-specific lethal genes are required for this process. Dosage compensation is restricted to males by the splicing regulator Sex-lethal, which functions to prevent the production of the MSL-2 protein in females by an unknown mechanism. In this report, we provide evidence that Sex-lethal acts synergistically through sequences in both the 5' and 3' untranslated regions of MSL-2 to mediate repression. We also provide evidence that the repression of MSL-2 is directly regulated by Sex-lethal at the level of translation.

The D. melanogaster transformer-2 (tra-2) gene regulates somatic sexual differentiation in females and is necessary for spermatogenesis in males. Wild-type tra-2 function is required for the female-specific splicing of the pre-mRNA of the next known gene (doublesex) downstream of tra-2 in the sex determination regulatory hierarchy. The tra-2 gene was cloned, and P element-mediated transformation was used to demonstrate that a 3.9 kb genomic fragment contains all sequences necessary for tra-2 function. A 1.7 kb transcript was shown to be the product of the tra-2 locus based on its reduced level in flies containing a tra-2 mutant allele. The sequence of a cDNA corresponding to this transcript indicates that it encodes a polypeptide with strong similarity to a family of RNA binding proteins that includes proteins found associated with hnRNPs and snRNPs, suggesting that the tra-2 product may directly regulate the processing of the double-sex pre-mRNA in females.

It has been proposed that dosage compensation in Drosophila males occurs by binding of two core proteins, MSL-1 and MSL-2, to a set of 35-40 X chromosome "entry sites" that serve to nucleate mature complexes, termed compensasomes, which then spread to neighboring sequences to double expression of most X-linked genes. Here we show that any piece of the X chromosome with which compensasomes are associated in wild-type displays a normal pattern of compensasome binding when inserted into an autosome, independently of the presence of an entry site. Furthermore, in chromosomal rearrangements in which a piece of X chromosome is inserted into an autosome, or a piece of autosome is translocated to the X chromosome, we do not observe spreading of compensasomes to regions of autosomes that have been juxtaposed to X chromosomal material. Taken together these results suggest that spreading is not involved in dosage compensation and that nothing distinguishes an entry site from the other X chromosome sites occupied by compensasomes beyond their relative affinities for compensasomes. We propose a new model in which the distribution of compensasomes along the X chromosome is achieved according to the hierarchical affinities of individual binding sites.