Identification of chromosomal deficiencies that modify Drosophila mastermind mutant phenotypes.

Mastermind (Mam) is a component of Notch pathway signaling. In combination with the intracellular domain of Notch and Suppressor of Hairless, Mam forms a transcriptional activation complex. We have initiated a genetic approach to identify other loci involved in Mam function. The screen utilizes engineered mutations in Mam that derive from GAL4-UAS-directed expression of dominant negative constructs. When driven at the wing margin, truncated versions of Mam phenocopy Notch pathway mutations. Correlated with these phenotypes is depression of Notch pathway target expression. Strains expressing truncated versions of Mam were tested for genetic interactions with a large collection of chromosomal deficiencies. Genomic segments that enhanced and suppressed the dominant wing phenotype were identified. These regions may contain uncharacterized loci involved in Notch pathway function.

A human protein with sequence similarity to Drosophila mastermind coordinates the nuclear form of notch and a CSL protein to build a transcriptional activator complex on target promoters.

Mastermind (Mam) has been implicated as an important positive regulator of the Notch signaling pathway by genetic studies using Drosophila melanogaster. Here we describe a biochemical mechanism of action of Mam within the Notch signaling pathway. Expression of a human sequence related to Drosophila Mam (hMam-1) in mammalian cells augments induction of Hairy Enhancer of split (HES) promoters by Notch signaling. hMam-1 stabilizes and participates in the DNA binding complex of the intracellular domain of human Notch1 and a CSL protein. Truncated versions of hMam-1 that can maintain an association with the complex behave in a dominant negative fashion and depress transactivation. Furthermore, Drosophila Mam forms a similar complex with the intracellular domain of Drosophila Notch and Drosophila CSL protein during activation of Enhancer of split, the Drosophila counterpart of HES. These results indicate that Mam is an essential component of the transcriptional apparatus of Notch signaling.

Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function.

The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind (mam) gene have implicated it as a component of the Notch signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these questions, we have constructed truncated versions of the Mam protein that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of the role of Mam within the Notch pathway.

Sequence and expression analysis of a Drosophila gene product related to the tre oncogene.

We report the sequence and expression of a Drosophila cDNA that shows similarity to a portion of the tre oncogene. The deduced encoded protein, DRN-TRE, contains residues that are highly conserved across a wide phylogenetic spectrum. The temporal and spatial transcription pattern of DRN-TRE suggest that it functions in a broad range of tissues.

The nuclear protein encoded by the Drosophila neurogenic gene mastermind is widely expressed and associates with specific chromosomal regions.

The Drosophila neurogenic loci encode a diverse group of proteins that comprise an inhibitory signal transduction pathway. The pathway is used throughout development in numerous contexts. We have examined the distribution of the neurogenic locus mastermind protein (Mam). Mam is expressed through all germlayers during early embryogenesis, including ectodermal precursors to both neuroblasts and epidermoblasts. Mam is subsequently down-regulated within the nervous system and then reexpressed. It persists in the nervous system through late embryogenesis and postembryonically. Mam is ubiquitously expressed in wing and leg imaginal discs and is not down-regulated in sensory organ precursor cells of the wing margin or notum. In the eye disc, Mam shows most prominent expression posterior to the morphogenetic furrow. Expression of the protein during oogenesis appears limited to follicle cells. Immunohistochemical detection of Mam on polytene chromosomes revealed binding at > 100 sites. Chromosome colocalization studies with RNA polymerase and the groucho corepressor protein implicate Mam in transcriptional regulation.

Transcription of the neurogenic gene mastermind during Drosophila development.

The neurogenic loci of Drosophila encode the components of a cell communication pathway that operates during multiple developmental stages and in numerous tissues. Activation of the pathway is required for inhibitory interactions, during partitioning of cells into alternative pathways of differentiation. Genetic studies of these loci have demonstrated numerous interactions, suggesting a close relationship among the gene products; molecular studies have corroborated some of these ideas. The mastermind (mam) locus shows genetic interactions with several neurogenic loci, yet its role in this pathway is unknown. We have analyzed mam transcription and further characterized the phenotype associated with mam alleles. mam is widely expressed in patterns overlapping those of other neurogenic loci during embryonic and postembryonic development; embryonic transcription is not dependent upon function of neurogenic genes. mam transcription is widespread during most of embryogenesis; however, late embryonic expression appears limited to the nervous system. Central nervous system expression persists at high levels during larval and pupal stages. Widespread transcription is also observed in ovaries and imaginal discs, with enhanced levels just anterior to the morphogenetic furrow of the eye disc. Phenotypic analyses of mam mutations demonstrate a broad phenotypic range and suggest that particular alleles disturb features of the CNS unrelated to neural overgrowth.

Drosophila Notch receptor activity suppresses Hairless function during adult external sensory organ development.

The neurogenic Notch locus of Drosophila encodes a receptor necessary for cell fate decisions within equivalence groups, such as proneural clusters. Specification of alternate fates within clusters results from inhibitory communication among cells having comparable neural fate potential. Genetically, Hairless (H) acts as an antagonist of most neurogenic genes and may insulate neural precursor cells from inhibition. H function is required for commitment to the bristle sensory organ precursor (SOP) cell fate and for daughter cell fates. Using Notch gain-of-function alleles and conditional expression of an activated Notch transgene, we show that enhanced signaling produces H-like loss-of-function phenotypes by suppressing bristle SOP cell specification or by causing an H-like transformation of sensillum daughter cell fates. Furthermore, adults carrying Notch gain of function and H alleles exhibit synergistic enhancement of mutant phenotypes. Over-expression of an H+ transgene product suppressed virtually all phenotypes generated by Notch gain-of-function genotypes. Phenotypes resulting from over-expression of the H+ transgene were blocked by the Notch gain-of-function products, indicating a balance between Notch and H activity. The results suggest that H insulates SOP cells from inhibition and indicate that H activity is suppressed by Notch signaling.

Drive-selection equilibrium: homopolymer evolution in the Drosophila gene mastermind.

Interspecific sequence comparison of the highly repetitive Drosophila gene mastermind (mam) reveals extensive length variation in homopolymer domains. The length variation in homopolymers is due to nucleotide misalignment in the underlying triplet repeats, which can lead to slippage mutations during DNA replication or repair. In mam, the length variation in repetitive regions appears to be balanced by natural selection acting to maintain the distance between two highly conserved charge clusters. Here we report a statistical test of the null hypothesis that the similarity in the amino acid distance between the charge clusters of each species arose by chance. The results suggest that at mam there is a juxtaposition of length variability due to molecular drive and length conservation maintained by natural selection. The analysis of mam allows the extension of current theories of drive-selection interaction to encompass homopolymers. Our model of drive-selection equilibrium suggests that the physical flexibility, length variability, and abundance of homopolymer domains provide an important source of genetic variation for natural populations.

Homopolymer length variation in the Drosophila gene mastermind.

Runs of identical amino acids encoded by triplet repeats (homopolymers) are components of numerous proteins, yet their role is poorly understood. Large numbers of homopolymers are present in the Drosophila melanogaster mastermind (mam) protein surrounding several unique charged amino acid clusters. Comparison of mam sequences from D. virilis and D. melanogaster reveals a high level of amino acid conservation in the charged clusters. In contrast, significant divergence is found in repetitive regions resulting from numerous amino acid replacements and large insertions and deletions. It appears that repetitive regions are under less selective pressure than unique regions, consistent with the idea that homopolymers act as flexible spacers separating functional domains in proteins. Notwithstanding extensive length variation in intervening homopolymers, there is extreme conservation of the amino acid spacing of specific charge clusters. The results support a model where homopolymer length variability is constrained by natural selection.

Interspecific comparison of the unusually repetitive Drosophila locus mastermind.

The mastermind gene of Drosophila melanogaster encodes a novel, highly repetitive nuclear protein required for neural development. To identify functionally important regions we have initiated an interspecific comparison of the gene in Drosophila virilis. Mastermind transcription and genomic organization are similar in both species and sequence analysis reveals significant conservation in a major cluster of charged amino acids. In contrast, extensive variation is noted in homopolymer domains that immediately flank the acidic cluster. Distinct patterns of evolutionary change can be identified: the major difference between unique regions are occasional amino acid substitutions whereas the repetitive areas are characterized by numerous large in-frame insertions/deletions and a nearly threefold higher rate of amino acid replacement. Conservation of the acidic domain suggests that it has an important functional role whereas the hypervariable homopolymer regions appear to be under less selective constraints than adjacent unique areas.

Early ventral expression of the Drosophila neurogenic locus mastermind.

The neurogenic locus mastermind (mam) of Drosophila is required for the segregation of epidermal from neural cell lineages. Previous studies have shown that during neurogenesis mam appears to be expressed throughout the ectoderm, mesoderm, and neuroblast layer of the germ band. Here it is demonstrated that during early embryogenesis mam is expressed ubiquitously; however, the predominant domains of accumulation of mam RNA and protein during gastrulation are along the ventral longitudinal surface, including cells of the mesoderm, endoderm, mesectoderm, and neuroectoderm. The regions of elevated mam accumulation coincide with the realm of action of dorsoventral patterning genes.

The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers.

The neurogenic loci of Drosophila are required for proper partitioning of ectodermal cells into epidermal versus neural lineages. The loci appear to encode components of a developmental pathway involving cellular communication. In an effort to understand the role of the neurogenic locus mastermind in these processes, we have characterized its expression and sequence. The locus produces a number of transcripts that accumulate ubiquitously during early embryogenesis but more specifically in the central nervous system during later stages. Sequence analysis of a major cDNA product predicts an unusual protein containing an abundance of amino acid homopolymers and charge clusters typical of regulatory molecules. Nearly half of the mass of the predicted protein derives from only three amino acids: glutamine, glycine, and asparagine. Immunohistochemical studies of the protein in cell culture and early embryos show that the protein accumulates predominantly in the nucleus.

Molecular analysis of the neurogenic locus mastermind of Drosophila melanogaster.

The neurogenic loci comprise a small group of genes which are required for proper division between the neural and epidermal pathways of differentiation within the neuroectoderm. Loss of neurogenic gene function results in the misrouting of prospective epidermal cells into neuroblasts. A molecular analysis of the neurogenic locus mastermind (mam) has been initiated through transposon tagging with P elements. Employing the Harwich strain as the source of P in a hybrid dysgenesis screen, 6000 chromosomes were tested for the production of lethal mam alleles and eight mutations were isolated. The mam region is the site of residence of a P element in Harwich which forms the focus of a chromosome breakage hotspot. Hybrid dysgenic induced mam alleles elicit cuticular and neural abnormalities typical of the neurogenic phenotype, and in five of the eight cases the mutants appear to retain a P element in the cytogenetic region (50CD) of mam. Utilizing P element sequence as probe, mam region genomic DNA was cloned and used to initiate a chromosome walk extending over 120 kb. The physical breakpoints associated with the hybrid dysgenic alleles fall within a 60-kb genomic segment, predicting this as the minimal size of the mam locus barring position effects. The locus contains a high density of repeated elements of two classes; opa (CAX)n and (dC-dA)n.(dG-dT)n. A preliminary study of the transcriptional activity of the mam region is presented.

The molecular genetics of the Notch locus in Drosophila melanogaster.

The Notch locus of Drosophila melanogaster profoundly affects differentiation of the central nervous system in the early embryo. Previous molecular studies suggested that the locus spans 40 kb of DNA and encodes a 10.5-kb poly(A)+ RNA. The results of genetic, cytogenetic, and molecular studies of newly induced and preexisting Notch alleles are reported. Molecular analysis of 5' flanking mutations defines a distal limit for the locus, and transcriptional activity of Notch in relation to that of flanking transcription units provides evidence regarding the proximal limit. Examination of a set of mutable alleles implicates a foldback transposable element as the basis of chromosomal instability, and shows that insertion sequences within the locus do not necessarily result in a mutant phenotype. The results are discussed with regard to existing developmental and genetic analyses, and a molecular model is proposed that attempts to explain the pleiotropic action of Notch.

opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster.

The principal transcription product of Notch, a locus involved in the neurogenesis of D. melanogaster, is a developmentally regulated poly(A)+ RNA approximately 10.5 kb in length. Analysis of the structure of this RNA has revealed a 93 bp repeated sequence that is shared by many other developmentally regulated transcription units. Nucleotide sequence analysis of the repeat shows an unusual structure consisting predominantly of the triplets CAG and CAA, both of which can code for the amino acid Gln. We present evidence indicating that the Notch repeat is a member of a novel family of repetitive elements, which we term the opa family. Our data suggest that some of these elements may be not only transcribed but also translated. We compare opa with other known transcribed repeats and speculate on its functional significance.

Molecular cloning of Notch, a locus affecting neurogenesis in Drosophila melanogaster.

The Notch locus is one of the best characterized loci in Drosophila melanogaster in terms of its genetic structure and developmental effects. Mutations in this locus profoundly affect the differentiation of the early embryo. Using an inversion involving the Notch locus and previously cloned sequences, we have isolated chromosomal segments from the Notch region (3C7) encompassing 80 kilobases (kb) of DNA. Based on comparison between mutant and wild-type DNA, we have positioned cloned sequences within the Notch genetic map; furthermore, we have defined a region of approximately 40 kb within which the structural lesions correlating with all Notch alleles mapped to date appear to reside. We have examined the transcriptional activity of the cloned sequences during ontogeny and find a single size class of poly(A)+ RNA, 10.5 kb long, that is homologous to sequences within this 40-kb region. We conclude that DNA sequences belonging to the Notch locus have been cloned and that the 10.5-kb poly(A)+ RNA is essential for wild-type Notch function. We discuss these structural and transcriptional data in light of the existing genetic and developmental characterization of the Notch locus.