SMART MODE:

NORMAL GENOMIC

• Walker JJ, Lee KK, Desai RN, Erickson JW
• The Drosophila melanogaster sex determination gene sisA is required in yolk nuclei for midgut formation.
• Genetics. 2000; 155: 191-202
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• During sex determination, the sisterlessA (sisA) gene functions as one of four X:A numerator elements that set the alternative male or female regulatory states of the switch gene Sex-lethal. In somatic cells, sisA functions specifically in sex determination, but its expression pattern also hints at a role in the yolk cell, a syncytial structure believed to provide energy and nutrients to the developing embryo. Previous studies of sisA have been limited by the lack of a null allele, leaving open the possibility that sisA has additional functions. Here we report the isolation and molecular characterization of four new sisA alleles including two null mutations. Our findings highlight key aspects of sisA structure-function and reveal important qualitative differences between the effects of sisA and the other strong X:A numerator element, sisterlessB, on Sex-lethal expression. We use genetic, expression, clonal, and phenotypic analyses to demonstrate that sisA has an essential function in the yolk nuclei of both sexes. In the absence of sisA, endoderm migration and midgut formation are blocked, suggesting that the yolk cell may have a direct role in larval gut development. To our knowledge, this is the first report of a requirement for the yolk nuclei in Drosophila development.

• Kamashev DE, Balandina AV, Karpov VL
• Tramtrack protein-DNA interactions. A cross-linking study.
• J Biol Chem. 2000; 275: 36056-61
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• Interaction of the Tramtrack protein from Drosophila melanogaster with DNA was analyzed by a cross-linking method. Tramtrack residues cross-linkable to the partially depurinated DNA were identified by direct sequencing. The N-terminal alpha-amino group of the protein DNA-binding domain was found to be the major product of cross-linking. The location of the N terminus on the DNA was determined by identification of the DNA bases that were cross-linked to the protein alpha-amino group. We conclude that accessory N-terminal peptide preceding the first zinc finger of Tramtrack directly interacts with DNA, both in specific and nonspecific DNA-protein complexes. Our finding explains the role in the protein binding of the DNA bases outside of the direct interaction with the zinc fingers.

• Mohr SE, Boswell RE
• Zimp encodes a homologue of mouse Miz1 and PIAS3 and is an essential gene in Drosophila melanogaster.
• Gene. 1999; 229: 109-16
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• The related mouse proteins Miz1 and PIAS3, which have predicted zinc finger domains, interact with the transcription factors Msx2 and STAT3, modulating the ability of Msx2 and STAT3 to regulate transcription. Here, we describe a Drosophila gene, zimp, that encodes a protein with similarity to Miz1 and PIAS3. The zimp gene appears to be post-transcriptionally regulated, as three alternatively spliced forms are detected in a cDNA library screen and on an RNA blot. In addition, all three zimp transcripts are detected in embryonic mRNA, but only two of the transcripts are detected in adult mRNA. The three transcripts have the ability to encode two proteins, of 554 and 522 amino acids. The two Zimp amino acid sequences share an amino-terminal 515-amino-acid region and differ in their carboxy-termini. These proteins and related proteins in other organisms, including mammals, C. elegans, yeast, and plants, share a highly conserved region predicted to form a zinc finger. Deletion of the zimp gene or P-element insertion in zimp is lethal; thus, zimp is an essential gene in Drosophila. These data underscore the potential importance of Zimp-related proteins cross-species, and conservation of the putative zinc finger domain suggests that it is functionally important.

• Yi W, Zarkower D
• Similarity of DNA binding and transcriptional regulation by Caenorhabditis elegans MAB-3 and Drosophila melanogaster DSX suggests conservation of sex determining mechanisms.
• Development. 1999; 126: 873-81
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• Although most animals occur in two sexes, the molecular pathways they employ to control sexual development vary considerably. The only known molecular similarity between phyla in sex determination is between two genes, mab-3 from C. elegans, and doublesex (dsx) from Drosophila. Both genes contain a DNA binding motif called a DM domain and they regulate similar aspects of sexual development, including yolk protein synthesis and peripheral nervous system differentiation. Here we show that MAB-3, like the DSX proteins, is a direct regulator of yolk protein gene transcription. We show that despite containing different numbers of DM domains MAB-3 and DSX bind to similar DNA sequences. mab-3 mutations deregulate vitellogenin synthesis at the level of transcription, resulting in expression in both sexes, and the vitellogenin genes have potential MAB-3 binding sites upstream of their transcriptional start sites. MAB-3 binds to a site in the vit-2 promoter in vitro, and this site is required in vivo to prevent transcription of a vit-2 reporter construct in males, suggesting that MAB-3 is a direct repressor of vitellogenin transcription. This is the first direct link between the sex determination regulatory pathway and sex-specific structural genes in C. elegans, and it suggests that nematodes and insects use at least some of the same mechanisms to control sexual development.

• Allen BL et al.
• DNA recognition properties of the N-terminal DNA binding domain within the large subunit of replication factor C.
• Nucleic Acids Res. 1998; 26: 3877-82
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• Replication Factor C (RFC) is a five-subunit protein complex required for eukaryotic DNA replication and repair. The large subunit within this complex contains a C-terminal DNA binding domain which provides specificity for PCNA loading at a primer-template and a second, N-terminal DNA binding domain of unknown function. We isolated the N-terminal DNA binding domain from Drosophila melanogaster and defined the region within this polypeptide required for DNA binding. The DNA determinants most efficiently recognized by both the Drosophila minimal DNA binding domain and the N-terminal half of the human large subunit consist of a double-stranded DNA containing a recessed 5' phosphate. DNA containing a recessed 5' phosphate was preferred 5-fold over hairpined DNA containing a recessed 3' hydroxyl. Combined with existing data, these DNA binding properties suggest a role for the N-terminal DNA binding domain in the recognition of phosphorylated DNA ends.

• Raymond CS et al.
• Evidence for evolutionary conservation of sex-determining genes.
• Nature. 1998; 391: 691-5
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• Most metazoans occur as two sexes. Surprisingly, molecular analyses have hitherto indicated that sex-determining mechanisms differ completely between phyla. Here we present evidence to the contrary. We have isolated the male sexual regulatory gene mab-3 from the nematode Caenorhabditis elegans and found that it is related to the Drosophila melanogaster sexual regulatory gene doublesex (dsx)2. Both genes encode proteins with a DNA-binding motif that we have named the 'DM domain'. Both genes control sex-specific neuroblast differentiation and yolk protein gene transcription; dsx controls other sexually dimorphic features as well. The form of DSX that is found in males can direct male-specific neuroblast differentiation in C. elegans. This structural and functional similarity between phyla suggests a common evolutionary origin of at least some aspects of sexual regulation. We have identified a human gene, DMT1, that encodes a protein with a DM domain and find that DMT1 is expressed only in testis. DMT1 maps to the distal short arm of chromosome 9, a location implicated in human XY sex reversal. Proteins with DM domains may therefore also regulate sexual development in mammals.

• Lee CC, Beall EL, Rio DC
• DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro.
• EMBO J. 1998; 17: 4166-74
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• P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.

• Brown JL, Mucci D, Whiteley M, Dirksen ML, Kassis JA
• The Drosophila Polycomb group gene pleiohomeotic encodes a DNA binding protein with homology to the transcription factor YY1.
• Mol Cell. 1998; 1: 1057-64
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• Genes of the Polycomb group (PcG) of Drosophila encode proteins necessary for the maintenance of transcriptional repression of homeotic genes. PcG proteins are thought to act by binding as multiprotein complexes to DNA through Polycomb group response elements (PREs); however, specific DNA binding has not been demonstrated for any of the PcG proteins. We have identified a sequence-specific DNA binding protein that interacts with a PRE from the Drosophila engrailed gene. This protein (PHO) is a homolog of the ubiquitous mammalian transcription factor Yin Yang-1 and is encoded by pleiohomeotic, a known member of the PcG. We propose that PHO acts to anchor PcG protein complexes to DNA.

• Kozlova T, Pokholkova GV, Tzertzinis G, Sutherland JD, Zhimulev IF, Kafatos FC
• Drosophila hormone receptor 38 functions in metamorphosis: a role in adult cuticle formation.
• Genetics. 1998; 149: 1465-75
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• DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

• Shearman DC, Frommer M
• The Bactrocera tryoni homologue of the Drosophila melanogaster sex-determination gene doublesex.
• Insect Mol Biol. 1998; 7: 355-66
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• A homologue of the bifunctional sex-determining gene, doublesex (dsx), has been identified in the tephritid fruit fly, Bactrocera tryoni, and has been found to be expressed in a sex-specific manner in adult flies. The male- and female-specific cDNAs are identical at their 5' ends but differ at their 3' ends and appear to be the products of alternate splicing. The level of identity of the sex-specific DSX proteins of B. tryoni with the D. melanogaster DSX proteins, across the region corresponding to the DNA binding domain and the oligomerization domains, is greater than 85%. Four sequence motifs which are ten to thirteen bases identical to the TRA/TRA-2 binding sites (thirteen-nucleotide repeat sequences) are present in the female-specific exon of the B. tryoni dsx gene.

• Cho S, Wensink PC
• Linkage between oligomerization and DNA binding in Drosophila doublesex proteins.
• Biochemistry. 1998; 37: 11301-8
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• The doublesex gene of Drosophila melanogaster encodes DSXM protein in males and DSXF protein in females. Dimers of each protein bind a DNA site from which DSXM represses and DSXF activates transcription. Amino acids 1-397 are identical between the proteins and include a domain (DBD) for both DNA binding and protein oligomerization. The remaining nonhomologous and therefore sex-specific C-termini include an essential part of a second oligomerization domain. We have used mobility shift assays to investigate the effects these three oligomerization domains (DBD and two sex-specific) have on DSX dimerization and DNA binding. The intrinsic DNA binding affinities of DSXM and DSXF dimers are indistinguishable from each other (0.17 +/- 0.04 nM) and slightly lower than that of DBD dimers (0.48 nM). In contrast, the dimerization dissociation constants of DSXM (0.05 +/- 0.02 nM) and DSXF (0.16 +/- 0.05 nM) are slightly different, but 4 orders of magnitude lower than that of DBD (430 nM). Thus sequences outside of DBD, presumably the sex-specific oligomerization domains, have substantial effects on apparent DNA binding affinity through thermodynamically linked effects on dimerization of full-length proteins. Further, when two DNA binding sites are adjacent, DBD dimers show no binding cooperativity, whereas full-length dimers bind with 2-fold different cooperativity (DSXF, k12 = 2.6; DSXM, k12 = 5.4). This suggests that the sex-specific domains may have a second effect on DNA binding, namely, an effect on binding cooperativity that depends on the number and arrangement of DNA sites.

• Cho S, Wensink PC
• DNA binding by the male and female doublesex proteins of Drosophila melanogaster.
• J Biol Chem. 1997; 272: 3185-9
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• Drosophila yolk protein genes are regulated by doublesex male protein (DSXM) in males and doublesex female protein (DSXF) in females. Both proteins bind to the same DNA sites from which DSXM represses and DSXF activates transcription. The proteins are identical through 397 NH2-terminal amino acids that include domains for oligomerization and DNA binding. The remaining COOH termini are sex-specific and include an essential part of a second oligomerization domain. We report here mobility shift assays that examine the DNA binding properties of purified DSXM and DSXF. Dimers of DSXM and DSXF bind to a regulatory site, dsxA, with the same affinity (Kapp = 0.2 nM), specificity (specific/nonspecific approximately 1.2 x 10(4)), and dependence on monovalent and divalent cations. The DNA association rate constants also are indistinguishable (kon = 4.6 x 10(6) M-1 s-1) as are the several terms of the dissociation reaction. Dissociation has an intrinsic rate of koff = 5.1 x 10(-4) s-1 and other rate terms that depend on the free concentration of specific DNA binding sites (2.4 x 10(4) M-1 s-1) or nonspecific binding sites (2.4 M-1 s-1). This first order dependence on unbound DNA suggests that a direct transfer between DNAs is likely to occur when DSX proteins search for specific sites in the many short open DNA regions of chromatin. Overall, dimer binding to individual DNA sites appears to be determined by the sex-nonspecific part of the two proteins. We infer that the sex-specific oligomerization domains play roles in binding cooperativity to multiple DNA sites or in other protein:protein interactions.

• Payre F, Buono P, Vanzo N, Vincent A
• Two types of zinc fingers are required for dimerization of the serendipity delta transcriptional activator.
• Mol Cell Biol. 1997; 17: 3137-45
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• The serendipity (sry) delta zinc finger protein controls bicoid gene expression during Drosophila melanogaster oogenesis. In addition, sry delta mutants display various zygotic phenotypes, ranging from abnormal embryogenesis to sex-biased adult lethality. We report here that sry delta is a sequence-specific transcriptional activator. A single sry delta consensus binding site (SDCS), in either orientation, is sufficient to promote transcription activation in cell culture, and multiple SDCSs mediate a strong synergistic activation, reflecting the cooperativity of sry delta binding to DNA. Further, several lines of evidence strongly suggest that sry delta binds to DNA as a dimer. While each of three point mutations located in the third zinc finger of sry delta drastically reduces its DNA binding affinity, a fourth mutation, located in the N-terminal region of the protein, specifically affects the cooperativity of DNA binding. This mutation reveals the functional importance of a putative Cys2/Cys2 zinc finger motif of a novel type, located outside the DNA binding domain. A systematic deletion analysis shows that interaction between this proposed Cys2/Cys2 motif and a classical Cys2/His2 zinc finger mediates homodimerization, which is required for DNA binding cooperativity.

• Shestopal SA, Makunin IV, Belyaeva ES, Ashburner M, Zhimulev IF
• Molecular characterization of the deep orange (dor) gene of Drosophila melanogaster.
• Mol Gen Genet. 1997; 253: 642-8
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• Mutations of the dor gene of Drosophila melanogaster cause defects in different stages of development. Heterozygotes for lethal or viable dor alleles and the rearrangement T(1;2)dor(var7), which causes position effect variegation of dor, exhibit traits such as rough eyes, reduction of bristles on the thorax and scutellum and wavy wings. The dor gene was mapped to the proximal part of the 2B3-5 band or in the interband between 2B3-5 and 2B6 and localised within an interval of 5 kb on the physical map of the cloned 2B region. The 3.0-3.1 kb dor transcript was detected by Northern hybridization at all stages of development and is expressed in salivary glands of third instar larve. This RNA was not expressed in the dor mutants with insertions in the 5' part of the gene. The sequence of the 3180 bp (dor cDNA predicts a 115.3 kDa protein that contains a cysteine- and histidine-rich zinc finger-like motif CX2CX13CXHX2HX2CX2H at the C-terminus. The protein sequence reveals 23% identity to the Saccharomyces cerevisiae PEP3 protein. The most significant homology (57%) similarity and 32%, identity) between the DOR and PEP3 proteins is observed at the C-termini of the proteins.

• Lyman LM, Copps K, Rastelli L, Kelley RL, Kuroda MI
• Drosophila male-specific lethal-2 protein: structure/function analysis and dependence on MSL-1 for chromosome association.
• Genetics. 1997; 147: 1743-53
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• MSL-2 is required for the male-specific assembly of a dosage compensation regulatory complex on the X chromosome of Drosophila melanogaster. We found that MSL-2 binds in a reproducible, partial pattern to the male X chromosome in the absence of MLE or MSL-3, or when ectopically expressed at a low level in females. Moreover, the pattern of MSL-2 binding corresponds precisely in each case to that of MSL-1, suggesting that the two proteins function together to associate with the X. Consistent with this hypothesis, we isolated EMS-induced loss of function msl-1 and msl-2 alleles in a screen for suppressors of the toxic effects of MSL-2 expression in females. We also used site-directed mutagenesis to determine the importance of the MSL-2 RING finger domain and second cysteine-rich motif. The mutations, including those in conserved zinc coordinating cysteines, confirm that the RING finger is essential for MSL-2 function, while suggesting a less stringent requirement for an intact second motif.

• Granadino B, Penalva LO, Sanchez L
• The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster.
• Mol Gen Genet. 1996; 253: 26-31
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• In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2) d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.

• Gregory SL, Kortschak RD, Kalionis B, Saint R
• Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.
• Mol Cell Biol. 1996; 16: 792-9
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• We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others.

• Ma C, Liu H, Zhou Y, Moses K
• Identification and characterization of autosomal genes that interact with glass in the developing Drosophila eye.
• Genetics. 1996; 142: 1199-213
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• The glass gene encodes a zinc finger, DNA-binding protein that is required for photoreceptor cell development in Drosophila melanogaster. In the developing compound eye, glass function is regulated at two points: (1) the protein is expressed in all cells' nuclei posterior to the morphogenetic furrow and (2) the ability of the Glass protein to regulate downstream genes is largely limited to the developing photoreceptor cells. We conducted a series of genetic screens for autosomal dominant second-site modifiers of the weal allele glass3, to discover genes with products that may regulate glass function at either of these levels. Seventy-six dominant enhancer mutations were recovered (and no dominant suppressors). Most of these dominant mutations are in essential genes and are associated with recessive lethality. We have assigned these mutations to 23 complementation groups that include multiple alleles of Star and hedgehog as well as single alleles of Delta, roughened eye, glass and hairy. Mutations in 18 of the complementation groups are embryonic lethals, and of these, 13 show abnormal adult retinal phenotypes in homozygous clones, usually with altered numbers of photoreceptor cells in some of the ommatidia.

• Alexandre E et al.
• The Drosophila teashirt homeotic protein is a DNA-binding protein and modulo, a HOM-C regulated modifier of variegation, is a likely candidate for being a direct target gene.
• Mech Dev. 1996; 59: 191-204
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• The Drosophila teashirt (tsh) gene has an homeotic function which, in combination with HOM-C genes, determines thoracic and abdominal (trunk) identities. Analysis of TSH protein distribution during embryogenesis using a specific polyclonal antibody shows that it is nuclear. The protein is present with regional modulation in several tissues within the trunk, suggesting additional tsh functions to those already studied. We identified a candidate tsh target shared with some HOM-C genes, the modifier of variegation gene modulo (mod). The TSH zinc-finger protein recognizes in vitro two specific sites within a 5' control element of the mod gene which responds in vivo to tsh activity. TSH is therefore a DNA binding protein and might directly control mod expression.

• An W, Cho S, Ishii H, Wensink PC
• Sex-specific and non-sex-specific oligomerization domains in both of the doublesex transcription factors from Drosophila melanogaster.
• Mol Cell Biol. 1996; 16: 3106-11
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• The doublesex gene of Drosophila melanogaster encodes the alternatively spliced, sex-specific transcription factors DSXM and DSXF. These factors regulate male- and female-specific transcription of many genes. For example, female-specific transcription of the yolk protein 1 gene is regulated by DSXM repression in males and DSXF activation in females. In this study we used in vitro interaction assays and the in vivo yeast two-hybrid method to identify and examine oligomerization domains of the DSX proteins. A 66-amino-acid segment common to both proteins (amino acids 39 to 104) contains a sequence-specific DNA binding domain and an oligomerization domain (OD1). The OD1 domain oligomerizes up to at least a pentamer, but only dimers bound to a palindromic regulatory site in the yolk protein 1 gene are detected. Both subunits of the OD1 dimer are in contact with DNA. Another segment of each protein (amino acids 350 to 412 for DSXF and 350 to 427 for DSXM) contains a second oligomerization domain (OD2F and OD2M, respectively). The OD2 domains have both sex-specific and non-sex-specific sequences which are necessary for oligomerization. On the basis of sequence analysis, we predict that OD2 oligomerizes through coiled-coil interactions. We speculate that the common function of OD1 and OD2 is to oligomerize the full-length proteins, whereas their specialized functions are to form a dimeric DNA binding unit and a sex-specific transcriptional activation or repression unit.

• Cho S, Wensink PC
• Purification and physical properties of the male and female double sex proteins of Drosophila.
• Proc Natl Acad Sci U S A. 1996; 93: 2043-7
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• The double sex gene (dsx) encodes two proteins, DSX(M) and DSX(F), that regulate sex-specific transcription in Drosophila. These proteins bind target sites in DNA from which the male-specific DSX(M) represses and the female-specific DSX(F) activates transcription of yolk protein (Yp) genes. We investigated the physical properties of these DSX proteins, which are identical in their amino-terminal 397 residues but are entirely different in their carboxyl-terminal sequences (DSX(F), 30 amino acids; DSX(M), 152 amino acids). DSX(M) and DSX(F) were overexpressed in cultured insect cells and purified to near homogeneity. Gel filtration chromatography and glycerol gradient sedimentation showed that at low concentrations both proteins are dimers of highly asymmetrical shape. The axial ratios are approximately 18:1 (DSX(M), 860 X 48 angstroms; DSX(F), 735 X 43 angstroms). At higher concentrations, the proteins form tetramers. Through use of a novel, double crosslinking assay (protein-DNA plus protein-protein), we demonstrated that a DNA regulatory site binds to both monomers of the DSX dimer and to only two monomers of the tetramer. Furthermore, binding another DNA molecule to what we presume is the second and identical site in the tetramer dramatically shifts the equilibrium from tetramers to dimers. These oligomerization and DNA binding properties are indistinguishable between the male and female proteins.

• Liu Y, Belote JM
• Protein-protein interactions among components of the Drosophila primary sex determination signal.
• Mol Gen Genet. 1995; 248: 182-9
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• Sex determination in Drosophila melanogaster is initiated in the early embryo by a signal provided by three types of genes: (1) X-linked numerator elements [e.g., sisterless-a (sis-a) and sisterless-b (sis-b)], (2) autosomally linked denominator elements [e.g., deadpan (dpn)], and (3) maternal factors [e.g., daughterless (da)]. This signal acts to stimulate transcription from an embryo-specific promoter of the master regulatory gene Sex-lethal (Sxl) in embryos that have two X chromosomes (females), while it fails to activate Sxl in those with only one X (males). It has been previously proposed that competitive dimerizations among the components of this signal might provide the molecular basis for this sex specificity. Here, we use the yeast two-hybrid system to demonstrate specific protein-protein interactions among the above-mentioned factors, and to delimit their interacting domains. These results support and extend the model of the molecular basis of the X/A ratio signal.

• Urness LD, Thummel CS
• Molecular analysis of a steroid-induced regulatory hierarchy: the Drosophila E74A protein directly regulates L71-6 transcription.
• EMBO J. 1995; 14: 6239-46
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• Steroid hormones orchestrate the growth and development of higher organisms by directing spatially and temporally coordinated programs of gene expression. These changes in gene activity can be visualized in Drosophila by virtue of its giant salivary gland polytene chromosomes. A small set of early puffs are induced directly by the steroid hormone ecdysone. The proteins encoded by these puffs appear to induce many late secondary-response puffs as the animal begins to undergo metamorphosis. Here we report that the ETS domain DNA-binding protein encoded by the E74A early gene directly induces L71-6 late gene transcription. We identify four strong E74A binding sites within the 5' region of L71-6 demonstrate that these sites are essential for proper L71-6 induction at puparium formation. These studies provide a direct link between a steroid-induced transcription factor and the activation of a secondary-response promoter, indicating that steroid signals in higher organisms can be transduced and amplified through regulatory hierarchies.

• Chen W, Zollman S, Couderc JL, Laski FA
• The BTB domain of bric a brac mediates dimerization in vitro.
• Mol Cell Biol. 1995; 15: 3424-9
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• The gene bric a brac (bab) is required for the proper development of the limbs and ovary in Drosophila melanogaster. bab encodes a BTB domain (also called a POZ domain), an approximately 115-amino-acid conserved motif found primarily in the N termini of zinc finger proteins. In this paper, we show that the BTB domain of bab can mediate protein dimerization in vitro. In addition, we demonstrate that the first 51 amino acids of the bab BTB domain are sufficient for dimerization, and we identify amino acids within this region that are required for binding.

• Kai M, Takahashi T, Todo T, Sakaguchi K
• Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.
• Nucleic Acids Res. 1995; 23: 2600-7
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• Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.

• An W, Wensink PC
• Three protein binding sites form an enhancer that regulates sex- and fat body-specific transcription of Drosophila yolk protein genes.
• EMBO J. 1995; 14: 1221-30
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• Transcription of the Drosophila yolk protein (Yp) genes is regulated by the somatic sex determination pathway. A gene at the bottom of this pathway, doublesex, encodes the female-specific DSXF and male-specific DSXM proteins that bind to and regulate transcription from several sites in the Yp genes. We report site-directed mutagenesis, protein binding and germline transformation experiments that identify and characterize the activity of a single binding site (dsxA) for the doublesex proteins and two binding sites for other regulatory proteins. A single copy of the three sites is sufficient to direct the sex and fat body specificities of Yp transcription. The sites form an enhancer with two strongly synergistic enhancer elements. One element (22 bp) consists of dsxA and an overlapping site, bzip1, that binds the DmC/EBP (slbo) protein, a member of the bZIP family of transcriptional activators. The other element is an 11 bp binding site (ref1) for an unknown protein. Tissue-specific activation requires strong cooperation between the ref1 site and the bzip1 or dsxA sites. Sex specificity is regulated exclusively by the dsxA site which connects the sex determination pathway to the target gene through DSXM repression and DSXF activation.

• Franke A, Messmer S, Paro R
• Mapping functional domains of the polycomb protein of Drosophila melanogaster.
• Chromosome Res. 1995; 3: 351-60
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• In Drosophila the Polycomb group (Pc-G) proteins are responsible for the stable and heritable silencing of genes. The Pc-G apparently uses heterochromatin-like mechanisms to transcriptionally inactivate developmental regulators such as the homeotic genes. The Polycomb (Pc) protein is part of a large multimeric complex composed of other members of the Pc-G. We have identified functionally relevant domains of the Pc protein by sequencing different Pc alleles. Additionally, using a Pc-beta gal fusion protein with deleted internal histidine repeats, we found that this mutant protein cannot bind to four particular target loci, but otherwise does not change the remaining overall binding pattern. We show that, in contrast to the dotted subnuclear localization of the wild-type protein, the nuclear distribution of mutant proteins becomes homogeneous. Surprisingly, in Pc mutants the polyhomeotic protein, another member of the Pc-G, is also redistributed in the nucleus. Our results indicate that the appropriate subnuclear localization of the two proteins is critical for the silencing function of the Pc-G complex.

• Karch F, Galloni M, Sipos L, Gausz J, Gyurkovics H, Schedl P
• Mcp and Fab-7: molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster.
• Nucleic Acids Res. 1994; 22: 3138-46
• Display abstract

• A very large cis-regulatory region of approximately 300 kb is responsible for the complex patterns of expression of the three homeotic genes of the bithorax complex Ubx, abd-A and Abd-B. This region can be subdivided in nine parasegment-specific regulatory subunits. Recent genetic and molecular analysis has revealed the existence of two novel cis-regulatory elements Mcp and Fab-7. Mcp is located between iab-4 and iab-5, the parasegment-specific regulatory subunits which direct Abd-B in parasegments 9 and 10. Similarly, Fab-7 is located between iab-6 and iab-7, the parasegment 11 and 12-specific regulatory units. Mcp and Fab-7 appear to function as domain boundaries that separate adjacent cis-regulatory units. We report the analysis of two new Mcp mutant deletions (McpH27 and McpB116) that allow us to localize sequences essential for boundary function to a approximately 0.4 kb DNA segment. These essential sequences closely coincide to a approximately 0.3 kb nuclease hypersensitive region in chromatin. We also show that sequences contributing to the Fab-7 boundary appear to be spread over a larger stretch of DNA, but like Mcp have an unusual chromatin structure.

• Zeng C, Pinsonneault J, Gellon G, McGinnis N, McGinnis W
• Deformed protein binding sites and cofactor binding sites are required for the function of a small segment-specific regulatory element in Drosophila embryos.
• EMBO J. 1994; 13: 2362-77
• Display abstract

• How each of the homeotic selector proteins can regulate distinct sets of DNA target elements in embryos is not understood. Here we describe a detailed functional dissection of a small element that is specifically regulated by the Deformed homeotic protein. This 120 bp element (module E) is part of a larger 2.7 kb autoregulatory enhancer that maintains Deformed (Dfd) transcription in the epidermis of the maxillary and mandibular segments of Drosophila embryos. In vitro binding assays show that module E contains only one Dfd protein binding site. Mutations in the Dfd binding site that increase or decrease its in vitro affinity for Dfd protein generate parallel changes in the regulatory activity of module E in transgenic embryos, strong evidence that the in vitro-defined binding site is a direct target of Dfd protein in embryos. However, a monomer or multimer of the Dfd binding region alone is not sufficient to supply Dfd-dependent, segment-specific reporter gene expression. An analysis of a systematic series of clustered point mutations in module E revealed that an additional region containing an imperfect inverted repeat sequence is also required for the function of this homeotic protein response element. The Dfd binding site and the putative cofactor binding site(s) in the region of the inverted repeat are both necessary and in combination sufficient for the function of module E.

• Georgiev PG
• Identification of mutations in three genes that interact with zeste in the control of white gene expression in Drosophila melanogaster.
• Genetics. 1994; 138: 733-9
• Display abstract

• Three previously described genes, enhancer of yellow, 1, 2 and 3, are shown to cooperate with the zeste gene in the control of white gene expression. The mutations e(y)1u1, e(y)3u1, and to a lesser extent e(y)2u1, enhance the effect of the zeste null allele zv77h. Different combinations of e(y)1u1, e(y)2u1 and e(y)3u1 mutations with several other z alleles also enhance the white mutant phenotype, but only to levels characteristic of white alleles containing a deletion of the upstream eye enhancer. Loss of zeste protein binding sites from the white locus does not eliminate the effect of e(y)1u1 and e(y)3u1 mutations, suggesting that the products of these genes interact with some other nucleotide sequences. Combinations of either e(y)1u1 or e(y)2u1 mutations with e(y)3u1 are lethal. The products of these three genes may represent, together with zeste, a group of proteins involved in the organization of long-distance interactions between DNA sequences.

• Hanes SD, Riddihough G, Ish-Horowicz D, Brent R
• Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen.
• Mol Cell Biol. 1994; 14: 3364-75
• Display abstract

• We examined DNA site recognition by Bicoid and its importance for pattern formation in developing Drosophila embryos. Using altered DNA specificity Bicoid mutants and appropriate reporter genes, we show that Bicoid distinguishes among related DNA-binding sites in vivo by a specific contact between amino acid 9 of its recognition alpha-helix (lysine 50 of the homeodomain) and bp 7 of the site. This result is consistent with our earlier results using Saccharomyces cerevisiae but differs from that predicted by crystallographic analysis of another homeodomain-DNA interaction. Our results also demonstrate that Bicoid binds directly to those genes whose transcription it regulates and that the amino acid 9 contact is necessary for Bicoid to direct anterior pattern formation. In both Drosophila embryos and yeast cells, Bicoid requires multiple binding sites to activate transcription of target genes. We find that the distance between binding sites is critical for Bicoid activation but that, unexpectedly, this critical distance differs between Drosophila and S. cerevisiae. This result suggests that Bicoid activation in Drosophila might require an ancillary protein(s) not present in S. cerevisiae.

• Strand D et al.
• The Drosophila lethal(2)giant larvae tumor suppressor protein forms homo-oligomers and is associated with nonmuscle myosin II heavy chain.
• J Cell Biol. 1994; 127: 1361-73
• Display abstract

• Inactivation of the Drosophila lethal(2)giant larvae (l(2)gl) gene causes malignant tumors in the brain and the imaginal discs and produces developmental abnormalities in other tissues, including the germline, the ring gland and the salivary glands. Our investigations into the l(2)gl function have revealed that the gene product, or p127 protein, acts as a cytoskeletal protein distributed in both the cytoplasm and on the inner face of lateral cell membranes in a number of tissues throughout development. To determine whether p127 can form oligomers or can stably interact with other proteins we have analyzed the structure of the cytosolic form of p127. Using gel filtration and immunoaffinity chromatography we found that p127 is consistently recovered as high molecular weight complexes that contain predominantly p127 and at least ten additional proteins. Blot overlay assays indicated that p127 can form homo-oligomers and the use of a series of chimaeric proteins made of segments of p127 fused to protein A, which alone behaves as a monomer, showed that p127 contains at least three distinct domains contributing to its homo-oligomerization. Among the proteins separated from the immuno-purified p127 complexes or isolated by virtue of their affinity to p127, we identified one of the proteins by microsequencing as nonmuscle myosin II heavy chain. Further blot overlay assay showed that p127 can directly interact with nonmuscle myosin II. These findings confirm that p127 is a component of a cytoskeletal network including myosin and suggest that the neoplastic transformation resulting from l(2)gl gene inactivation may be caused by the partial disruption of this network.

• Ekker SC, Jackson DG, von Kessler DP, Sun BI, Young KE, Beachy PA
• The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins.
• EMBO J. 1994; 13: 3551-60
• Display abstract

• The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. We compare here the DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity. We further show that amino acid residues within the N-terminal arm are responsible for the sequence specificity differences between the Ultrabithorax and Abdominal-B homeodomains. Similarities and differences at the corresponding positions within the N-terminal arms are conserved in the vertebrate Abdominal-B-like HOM proteins, which play critical roles in limb specifications as well as in regional specification along the anterior-posterior axis. This and other patterns of residue conservation suggest that differential DNA sequence recognition may play a role in HOM protein function in a wide range of organisms.

• Han W, Yu Y, Altan N, Pick L
• Multiple proteins interact with the fushi tarazu proximal enhancer.
• Mol Cell Biol. 1993; 13: 5549-59
• Display abstract

• The expression of the Drosophila segmentation gene fushi tarazu (ftz) is controlled at the level of transcription. The proximal enhancer, located approximately 3.4 kb upstream of the transcription start site, directs lacZ fusion gene expression in a ftz-like seven-stripe pattern in transgenic fly embryos. We have taken a biochemical approach to identify DNA-binding proteins that regulate ftz gene expression through the proximal enhancer. DNase I footprinting and methylation interference experiments with staged Drosophila embryo nuclear extracts identified nine protein binding sites in the proximal enhancer. Ten different sequence-specific DNA-binding complexes that interact with eight of these sites were identified. Some interact with multiple sites, while others bind to single sites in the enhancer. Two of the complexes that interact with multiple sites appear to contain the previously described ftz regulators, FTZ-F1 and TTK/FTZ-F2. These in vitro studies allowed us to narrow down the proximal enhancer to a 323-bp DNA fragment that contains all of the protein binding sites. Expression directed by this minimal enhancer element in seven ftz-like stripes in transgenic embryos is identical to that directed by the full-length enhancer. Internal deletions of several sites abolish reporter gene expression in vivo. Thus, the ftz proximal enhancer, like other cell-type-specific eukaryotic enhancers, interacts with an array of proteins that are expected to mediate the establishment, maintenance, and repression of transcription of the ftz gene in seven stripes in the developing embryo.

• Coschigano KT, Wensink PC
• Sex-specific transcriptional regulation by the male and female doublesex proteins of Drosophila.
• Genes Dev. 1993; 7: 42-54
• Display abstract

• The somatic sexual phenotype of Drosophila is regulated by the sexual differentiation pathway. Male (DSXM) and female (DSXF) proteins encoded by doublesex (dsx), a gene at the end of this pathway, bind to three sites within a 127-bp enhancer that directs sex- and tissue-specific transcription of Yolk protein genes. We describe mutagenesis of these binding sites and the resulting effects on DSXM and DSXF binding in vitro and on gene regulation in wild-type and dsx mutant flies. The results demonstrate that DSXM represses and DSXF activates transcription from the two strongest binding sites. Thus, the pathway regulates sex-specific transcription through the male and female dsx proteins that act directly on the target gene, but with opposite effects.

• Segal D
• Prospects of using Drosophila for insect neuroendocrine research.
• Arch Insect Biochem Physiol. 1993; 22: 199-231
• Display abstract

• Classical and in vitro approaches for the analysis of the molecular components of neuroendocrine systems often disrupt their close interaction with other bodily systems, which is a crucial aspect of their function in vivo. "Genetic dissection" is an alternative, noninvasive approach which involves the systematic generation of mutations in individual genes, followed by in vivo analysis of the phenotypic effects of altering a single protein at a time avoiding extraneous disruptions. Among insects Drosophila melanogaster is the most suitable model for this approach. This paper explores the application of genetic and molecular techniques available in Drosophila for studying its neuroendocrine system with special emphasis on the production of ecdysone and juvenile hormone. Strategies are described for the generation and identification of endocrine mutations, especially those affecting hormone synthesis and regulation. Once identified by a specific mutation, a gene in Drosophila can be cloned either by chromosomal microdissection and "chromosomal walk" or by transposon tagging. Methods for molecular analysis of the structure and function of a cloned gene and of the protein it encodes are available for further study. Alternatively, a gene can be cloned using heterologous DNA probes or oligonucleotides designed according to the amino acid sequence of a protein. Genes may also be cloned via their pattern of expression (using stage- or tissue-specific cDNA libraries or through transposon-mediated "enhancer detection." Anti-sense RNA, the replacement of the gene by in vitro manipulated versions, or mutagenesis of its endogenous copies can then be used for studying its function in vivo. Information about endocrine genes in Drosophila as well as material such as cloned genes and antibodies should be useful for the analysis of endocrine systems in other insects which are not amenable to genetic manipulations. Such information should be helpful in designing novel means for pest control based on the specific intervention with endocrine systems regulating insect development and reproduction.

• Chen JD, Pirrotta V
• Multimerization of the Drosophila zeste protein is required for efficient DNA binding.
• EMBO J. 1993; 12: 2075-83
• Display abstract

• The Drosophila zeste protein forms multimeric species in vitro through its C-terminal domain. Multimerization is required for efficient binding to DNA containing multiple recognition sequences and increasing the number of binding sites stimulates binding in a cooperative manner. Mutants that can only form dimers still bind to a dimeric site, but with lower affinity. Mutations or progressive deletions from the C-terminal show that when even dimer formation is prevented, DNA-binding activity is lost. Surprisingly, binding activity is regained with larger deletions that leave only the DNA-binding domain. Additional protein sequences apparently inhibit DNA binding unless they permit multimerization. The DNA-binding domain peptides bind strongly even to isolated recognition sequences and they bind as monomers. The ability of various zeste peptides to stimulate white gene expression in vivo shows that multimeric forms are the functional species of the zeste product in vivo. The DNA-binding domain peptide binds well to DNA in vitro, but it cannot stimulate white gene expression in vivo. This failure may reflect the need for an activation domain or it may be caused by indiscriminate binding of this peptide to non-functional isolated sites. Multimerization increases binding specificity, selecting only sites with multiple recognition sequences.

• Jursnich VA, Burtis KC
• A positive role in differentiation for the male doublesex protein of Drosophila.
• Dev Biol. 1993; 155: 235-49
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• The doublesex (dsx) locus encodes male-specific and female-specific polypeptides that are essential for the proper differentiation of sexually dimorphic somatic features of Drosophila melanogaster. Ectopic expression of the male-specific dsx polypeptide was obtained by P-element-mediated transformation of flies with a construct bearing a fusion between the hsp70 heat shock promoter and dsx male-specific cDNA sequences. Heat shock-induced expression of the male cDNA in either sex resulted in three novel phenotypes: transformation of bristles on all legs toward a sex comb-like morphology, pigmentation of dorsal spinules and ventral setae in third-instar larvae, and lethality. These results were not predicted by previous models of dsx function, and provide evidence that the role of the male dsx protein includes activation of some aspects of male differentiation as well as repression of female differentiation.

• Hill RJ, Segraves WA, Choi D, Underwood PA, Macavoy E
• The reaction with polytene chromosomes of antibodies raised against Drosophila E75A protein.
• Insect Biochem Mol Biol. 1993; 23: 99-104
• Display abstract

• The steroid insect molting hormone ecdysone rapidly induces a small number of polytene chromosome puffs in Drosophila. The Ashburner model proposes that the corresponding early genes encode proteins involved in both the induction of the late genes and the repression of the early genes. The Drosophila E75 early gene has been isolated and two of its products, E75A and E75B, have been shown to be members of the steroid receptor superfamily. We have now prepared antisera directed against A- and B-specific regions of the E75 proteins. Antisera and a monoclonal antibody raised against E75A, the major larval protein product of the E75 gene, bind to discrete sites in native salivary gland chromosomes. These sites are closely correlated with early and late ecdysone responsive loci.

• Schier AF, Gehring WJ
• Functional specificity of the homeodomain protein fushi tarazu: the role of DNA-binding specificity in vivo.
• Proc Natl Acad Sci U S A. 1993; 90: 1450-4
• Display abstract

• The mechanisms determining the functional specificity of Drosophila homeodomain proteins are largely unknown. Here, the role of DNA-binding specificity for the in vivo function of the homeodomain protein fushi tarazu (ftz) is analyzed. We find that specific DNA binding is an important but not sufficient determinant of the functional specificity of ftz in vivo: The ftz DNA-binding specificity mutant ftzQ50K retains partial ftz wild-type activity in gene activation and phenotypic rescue assays. Furthermore, specificity mutations in a ftz-in vivo binding site only partially reduce enhancer activity as compared to null mutations of this site. Despite bicoid-like DNA-binding specificity ftzQ50K does not activate natural or artificial bcd target genes in the realms of ftz. These results are discussed in the light of recent observations on the mechanism of action of the yeast homeodomain protein alpha 2.

• Liaw GJ, Steingrimsson E, Pignoni F, Courey AJ, Lengyel JA
• Characterization of downstream elements in a Raf-1 pathway.
• Proc Natl Acad Sci U S A. 1993; 90: 858-62
• Display abstract

• At the poles of the Drosophila embryo, cell fate is established by a pathway that begins with the activation of a membrane-associated tyrosine kinase (the torso gene product); this then leads to activation of a serine/threonine kinase (Drosophila Raf-1). Activated Raf-1 then leads, by an undefined mechanism, to the transcriptional activation of the tailless (tll) gene; the tll gene product, itself a transcription factor, subsequently regulates the expression of an array of target genes. To further define this pathway, we have utilized sequence comparison between Drosophila melanogaster and Drosophila virilis to identify conserved elements in the tll promoter region. As assessed by DNase I footprinting and promoter dissection experiments, two of these elements are potential regulatory targets of Raf-1-activated transcription factors. Sequence comparison also reveals that the unique residues in the DNA-binding domain of the tll protein, the next component in the pathway, are conserved. One of these residues, the alanine after the last cysteine in the first zinc finger, may be responsible for part of the difference between the tll protein DNA binding site and the closely related half-site of the retinoid/estrogen receptors. Consistent with the rapid turnover of the tll protein, it contains a PEST sequence (rich in proline, glutamate and aspartate, serine, and threonine) that is also conserved.

• Albrecht EB, Salz HK
• The Drosophila sex determination gene snf is utilized for the establishment of the female-specific splicing pattern of Sex-lethal.
• Genetics. 1993; 134: 801-7
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• The Drosophila snf gene is a positive regulator of the sex determination gene Sex-lethal in both the germline and the soma. Its role in the soma is only evident when the probability of Sex-lethal activation has been reduced. For instance, in an otherwise wild-type background, females homozygous for a weak snf mutation produce both male and female progeny; however, when mated to males hemizygous for a null allele of Sex-lethal, they produce only male progeny. We demonstrate that the lack of female progeny is due to aberrant Sex-lethal regulation in late embryogenesis. In these mutant embryos, there is little accumulation of the late female-specific spliced RNAs and proteins. In contrast, in early embryogenesis, Sex-lethal regulation is not affected. The accumulation of both the early Sex-lethal transcripts and proteins is normal. These results suggest that the wild-type product of snf plays an important role in establishing the female-specific RNA splicing pattern of Sex-lethal. Whether snf influences the female-specific splice site choice directly or indirectly remains to be determined.

• Martin-Blanco E, Kornberg TB
• DR-78, a novel Drosophila melanogaster genomic DNA fragment highly homologous to the DNA-binding domain of thyroid hormone-retinoic acid-vitamin D receptor subfamily.
• Biochim Biophys Acta. 1993; 1216: 339-41
• Display abstract

• Degenerate oligodeoxyribonucleotides were designed for both ends of the DNA-binding domain of members of the nuclear receptor superfamily. PCR amplified Drosophila melanogaster DNA was purified and cloned (DR plasmids). Genomic lambda DASH clones were identified at high stringency with an amplified DR-78 plasmid DNA and isolated. The partial sequence shows a very probable open reading frame which would encode a peptide highly homologous to members of the thyroid hormone-retinoic acid-vitamin D receptor subfamily. The fragment corresponds to a single copy gene and was mapped at position 78D of chromosome three by in situ hybridization.

• Chen JD, Chan CS, Pirrotta V
• Conserved DNA binding and self-association domains of the Drosophila zeste protein.
• Mol Cell Biol. 1992; 12: 598-608
• Display abstract

• The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.

• Burtis KC, Coschigano KT, Baker BS, Wensink PC
• The doublesex proteins of Drosophila melanogaster bind directly to a sex-specific yolk protein gene enhancer.
• EMBO J. 1991; 10: 2577-82
• Display abstract

• The doublesex (dsx) gene of Drosophila melanogaster encodes both male-specific and female-specific polypeptides, whose synthesis is regulated by alternative sex-specific splicing of the primary dsx transcript. The alternative splicing of the dsx mRNA is the last known step in a cascade of regulatory gene interactions that involves both transcriptional and post-transcriptional mechanisms. Genetic studies have shown that the products of the dsx locus are required for correct somatic sexual differentiation of both sexes, and have suggested that each dsx product functions by repressing expression of terminal differentiation genes specific to the opposite sex. However, these studies have not shown whether the dsx gene products function directly to regulate the expression of target genes, or indirectly through another regulatory gene. We report here that the male- and female-specific DSX proteins, expressed in E.coli, bind directly and specifically in vitro to three DNA sequences located in an enhancer region that regulates female-specific expression of two target genes, the yolk protein genes 1 and 2. This result suggests strongly that dsx is a final regulatory gene in the hierarchy of regulatory genes controlling somatic sexual differentiation.

• Van Doren M, Ellis HM, Posakony JW
• The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes.
• Development. 1991; 113: 245-55
• Display abstract

• In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three 'E box' consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

• Bickel S, Pirrotta V
• Self-association of the Drosophila zeste protein is responsible for transvection effects.
• EMBO J. 1990; 9: 2959-67
• Display abstract

• The zeste gene product is required for transvection effects that imply the ability of regulatory elements on one chromosome to affect the expression of the homologous gene in a somatically paired chromosome. The z1 mutation causes a pairing dependent inhibition of the expression of the white gene. Both of these phenomena can be explained by the tendency of zeste protein, expressed in bacteria or in flies, to self-associate, forming complexes of several hundred monomers. These large aggregates bind to DNA and are found in nuclear matrix preparations, probably because they co-sediment with the matrix. The principal determinants of this self-association are located in the C-terminal half of the protein but some limited aggregation is obtained also with the N-terminal half, which contains the DNA binding domain. The z1 and zop2 mutant proteins aggregate to the same degree as the wild type but the z11G3 product, a pseudorevertant of z1, has a reduced tendency to aggregate. This mutation, which in vivo is antagonistic to z1 and does not support transvection effects, can be made to revert its phenotype when the mutant protein is over-produced under the control of the heat shock promoter. These results indicate that both the zeste-white interaction and transvection effects require the formation of high order aggregates. When the z1 protein is over-produced in vivo, it reduces the expression of an unpaired copy of white, indicating that the normal requirement for chromosome pairing is simply a device to increase the size of the aggregate bound to the white regulatory region.

• Nagoshi RN, Baker BS
• Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns.
• Genes Dev. 1990; 4: 89-97
• Display abstract

• Sex-specific alternative RNA splicing of the doublesex (dsx) pre-mRNA results in sex-specific polypeptides that regulate both male and female somatic sexual differentiation in Drosophila melanogaster. We have molecularly characterized a class of dsx mutations that act in cis to disrupt the regulation of dsx RNA processing, causing the dsx pre-mRNA to be spliced in the male-specific pattern regardless of the chromosomal sex of the fly. These dsx mutations are associated with rearrangements in the female-specific exon just 3' to the female-specific splice acceptor. The mutations do not affect the female-specific splice sites or intron that are identical to wild-type sequences. These results indicate that sequences in the female-specific exon are important for the regulation of sex-specific RNA splicing, perhaps by acting as sites of interaction with trans-acting regulators. Furthermore, the data suggest that female-specific regulation of dsx RNA processing occurs by promoting the usage of the female splice acceptor site, rather than by repressing the usage of the alternative male-specific splice acceptor.

• Fitzpatrick VD, Ingles CJ
• The Drosophila fushi tarazu polypeptide is a DNA-binding transcriptional activator in yeast cells.
• Nature. 1989; 337: 666-8
• Display abstract

• Many of the regulatory genes controlling the developmental pattern of segmentation during embryonic development in Drosophila melanogaster encode nuclear proteins containing either homoeobox or 'zinc-finger' domains with putative or demonstrated sequence-specific DNA-binding properties. One of these Drosophila homoeobox-containing proteins is encoded by the fushi tarazu (ftz) gene. The expression of ftz is spatially restricted during embryogenesis and the ftz polypeptide has an important role in different stages of development. To determine whether the ftz polypeptide is a sequence-specific DNA-binding activator of transcription, we expressed portions of ftz as fusions with the yeast transcription factor GAL4 in yeast cells. Chimaeric GAL4/ftz proteins, like GAL4 itself, activated the transcription of a GAL4-dependent reporter gene. With reporter constructs containing Drosophila-derived chromosomal DNA sequences as transcriptional elements, the ftz polypeptide acted as a sequence-specific DNA-binding transcriptional activator. In Drosophila, the ftz product may therefore be a positive regulator of transcription.

• Mansukhani A, Crickmore A, Sherwood PW, Goldberg ML
• DNA-binding properties of the Drosophila melanogaster zeste gene product.
• Mol Cell Biol. 1988; 8: 615-23
• Display abstract

• The ability of the zeste moiety of beta-galactosidase-zeste fusion proteins synthesized in Escherichia coli to bind specific DNA sequences was examined. Such fusion proteins recognize a region of the white locus upstream of the start of transcription; this region has previously been shown to be required for genetic interaction between the zeste and white loci. Another strong binding site was localized to a region between 50 and 205 nucleotides before the start of the Ubx transcriptional unit; expression of the bithorax complex is also known to be influenced by the zeste locus. Weaker binding sites were also seen in the vicinity of the bxd and Sgs-4 genes, but it is currently unclear whether these binding sites play a role in transvection effects. The DNA-binding activity of the zeste protein is restricted to a domain of approximately 90 amino acids near the N terminus. This domain does not appear to contain homeobox or zinc finger motifs found in other DNA-binding proteins. The DNA-binding domain is not disrupted by any currently characterized zeste mutations.

• Chen HZ, Hoey T, Zubay G
• Purification and properties of the Drosophila zen protein.
• Mol Cell Biochem. 1988; 79: 181-9
• Display abstract

• The zen protein is encoded by the zerknullt gene required for normal early development in Drosophila. Like many regulatory proteins of this type, zen contains a 60 amino acid homeobox sequence. We have purified the zen protein and studied its solution behavior and its interaction with DNA. The zen protein exists as a monomer in solution with a molecular weight of about 40,000. It binds specifically to a site about 900 bases upstream from the zen gene. Within this binding site DNase protection experiments indicate that binding is confined to two regions approximately 11 and 14 bases in length that are separated by about 30 base pairs. The protein concentration dependence of the binding curve suggests that protein binding is non cooperative.

• Benson M, Pirrotta V
• The product of the Drosophila zeste gene binds to specific DNA sequences in white and Ubx.
• EMBO J. 1987; 6: 1387-92
• Display abstract

• Three different segments of the zeste coding sequence were inserted in an expression vector and antibodies were raised against the resulting zeste-beta galactosidase hybrid proteins. The antibodies were used to analyse the zeste protein produced in bacteria from a different expression vector containing the entire zeste coding region. The major products made in bacteria as well as the products of in vitro translation of zeste RNA migrate anomalously upon SDS--acrylamide gel electrophoresis. Specific DNA fragments from the white and Ubx gene co-immunoprecipitate with zeste protein. At least two independent zeste binding sites are found in a 250-bp interval of the white regulatory region that contains also the sites of wsp mutations, which are known to be deficient in zeste interaction.

• Laughon A, Scott MP
• Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins.
• Nature. 1984; 310: 25-31
• Display abstract

• Mutations in the fushi tarazu (ftz) locus of Drosophila result in embryos with half the usual number of body segments. The sequences of the wild-type gene, a temperature-sensitive allele and a dominant mutant allele are presented. A portion of the conserved protein domain present in ftz and several homoeotic genes resembles the DNA-binding region of prokaryotic DNA-binding proteins, and is also similar to products of the yeast mating-type locus.

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