Daniel Bopp

Structure of the segmentation gene paired and the Drosophila PRD gene set as part of a gene network

The sequence of paired, a pair-rule gene required for segmentation in Drosophila, is presented. A search for genes with domains homologous to the paired gene was initiated and three homologues from a set of 12 were characterized with respect to temporal or spatial expression and sequence homologies. All four are transcribed in early development, one in the oocyte and during cleavage stages in the form of a gradient. In addition to the prd-specific his-pro repeat, some of the 12 genes contain M-repeats and two new types of homeo boxes not detectable by hybridization with the two known classes of homeo boxes. The observed linking of gene sets through combinations of homologies coding for protein domains is consistent with a general network concept of gene action.

Conservation of the paired domain in metazoans and its structure in three isolated human genes.

Sequences homologous to the paired domain of Drosophila melanogaster have been conserved in species as distantly related as nematodes, sea urchins, or man. In particular, paired domains of three human genes, HuP1, HuP2 and HuP48, have been isolated and sequenced. Together with four Drosophila paired domains, they fall into two separate paired domain classes named according to their Drosophila members, paired–gooseberry and P29 class. The P29 class includes the mouse Pax 1 and the human HuP48 gene which are nearly identical in their sequenced portions and hence might be true homologues. In addition to the paired domain, the two human genes HuP1 and HuP2 share the highly conserved octapeptide HSIAGILG with the two gooseberry genes of Drosophila. Possible functions of the paired domain are discussed in the light of a predicted helix‐turn‐helix structure in its carboxy‐terminal portion.

Isolation of two tissue-specific Drosophila paired box genes, Pox meso and Pox neuro.

Two new paired domain genes of Drosophila, Pox meso and Pox neuro, are described. In contrast to the previously isolated paired domain genes, paired and gooseberry, which contain both a paired and a homeo‐domain (PHox genes), Pox meso and Pox neuro possess no homeodomain. Evidence suggesting that the new genes encode tissue‐specific transcriptional factors and belong to the same regulatory cascade as the other paired domain genes includes (i) tissue‐specific expression of Pox meso in the somatic mesoderm and of Pox neuro in the central and peripheral nervous system, (ii) nuclear localization of their proteins, (iii) dependence on prd activity and (iv) presence of the paired domain in genes of known regulatory activity. While no mutant phenotypes of Pox meso and Pox neuro have yet been discovered, a murine gene with a paired domain closely homologous to that of Pox meso has recently been identified with the undulated mutant. Both Pox meso and undulated are expressed in tissues derived from the somatic mesoderm. The five known Drosophila paired domains fall into three classes: (i) the prd,gsb‐class, (ii) the Pox meso, undulated‐class and (iii) the Pox neuro‐class which probably includes the paired domain of the murine gene Pax 2.

Genome of the house fly, Musca domestica L., a global vector of diseases with adaptations to a septic environment

BackgroundAdult house flies, Musca domestica L., are mechanical vectors of more than 100 devastating diseases that have severe consequences for human and animal health. House fly larvae play a vital role as decomposers of animal wastes, and thus live in intimate association with many animal pathogens.ResultsWe have sequenced and analyzed the genome of the house fly using DNA from female flies. The sequenced genome is 691 Mb. Compared with Drosophila melanogaster, the genome contains a rich resource of shared and novel protein coding genes, a significantly higher amount of repetitive elements, and substantial increases in copy number and diversity of both the recognition and effector components of the immune system, consistent with life in a pathogen-rich environment. There are 146 P450 genes, plus 11 pseudogenes, in M. domestica, representing a significant increase relative to D. melanogaster and suggesting the presence of enhanced detoxification in house flies. Relative to D. melanogaster, M. domestica has also evolved an expanded repertoire of chemoreceptors and odorant binding proteins, many associated with gustation.ConclusionsThis represents the first genome sequence of an insect that lives in intimate association with abundant animal pathogens. The house fly genome provides a rich resource for enabling work on innovative methods of insect control, for understanding the mechanisms of insecticide resistance, genetic adaptation to high pathogen loads, and for exploring the basic biology of this important pest. The genome of this species will also serve as a close out-group to Drosophila in comparative genomic studies.

Molecular Characterization of the Key Switch F Provides a Basis for Understanding the Rapid Divergence of the Sex-Determining Pathway in the Housefly

The housefly, Musca domestica, is an excellent model system to study the diversification of the pathway that specifies the sexual fate. A number of different mechanisms have been described in the housefly, which reflects in part the broad diversity of sex-determining strategies used in insects. In this study we present the molecular identification and characterization of F, which acts as the master switch in the housefly pathway. We provide evidence that F corresponds to the transformer ortholog in Musca (Mdtra), which, as a result of alternative processing, expresses functional products only in individuals committed to the female fate. We demonstrate that, once activated, a self-sustaining feedback loop will maintain the female-promoting functions of Mdtra. Absence of Mdtra transcripts in eggs of Arrhenogenic (Ag) mutant females suggests that maternally deployed Mdtra activity initiates this self-sustaining loop in the zygote. When an M factor is paternally transmitted to the zygote, the establishment of the loop is prevented at an early stage before cellularization and splicing of Mdtra shifts irreversibly to the male nonproductive mode. On the basis of the analysis of two mutant alleles we can explain the different sex-determining systems in the housefly largely as deviations at the level of Mdtra regulation. This plasticity in the housefly pathway may provide a suitable framework to understand the evolution of sex-determining mechanisms in other insect species. For instance, while sex determination in a close relative, the tsetse fly Glossina morsitans, differs at the level of the instructive signal, we find that its tra ortholog, Gmtra, is regulated in a mode similar to that of Mdtra.

Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac.

The piggyBac transposable element was successfully used for stable genetic transformation of the housefly Musca domestica. The construct contains the EGFP marker under the control of Pax‐6 binding sites, which can drive eye‐specific expression in insect species as distantly related as Drosophila melanogaster and Tribolium castaneum[ Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999)Nature 402: 370–371]. We obtained seven independent integration events among 41 fertile G0Musca flies. Most of the transformed lines contained two or more chromosomal insertions of the EGFP marker which were stably inherited over more than 15 generations. piggyBac‐mediated transposition was verified by identifying the characteristic TTAA duplication at the insertion sites. This first report of stable transmission of a genetic marker in Musca confirms the use of this vector‐marker system for effective gene transfer in a broad range of insect species.

Rapid restructuring of bicoid-dependent hunchback promoters within and between Dipteran species: implications for molecular coevolution

SUMMARY Interacting genetic elements need to coevolve if their joint function is to be maintained; for example, the correct binding of transcriptional regulators to defined binding sites in gene promoters needs to be maintained during evolution to ensure proper function. As part of a wider investigation into the molecular coevolution of the Dipteran homeodomain‐bearing regulator bicoid (bcd) and Bcd‐dependent promoters, we present data on the functional, structural, and sequence differences between the promoters of the segmentation gene hunchback (hb), in several species of Cyclorrhaphan (higher) Diptera. The result of phenocopying hb mutations using RNA interference (RNAi) in Musca domestica shows broadly similar functions to the hb gene in Drosophila melanogaster. However, the Bcd‐binding sites in the hb promoters of Drosophila, Musca, and the two blowfly species Lucilia sericata and Calliphora vicina differ in copy number, sequence, orientation, and spacing. Furthermore, all promoters are subject to rapid turnover by slippage‐like processes leading to high densities of short repetitive motifs. A study of polymorphism among six strains of M. domestica reveals that turnover by slippage also occurs in the promoter, untranslated leader, and exonic coding sequences of hb, but to different extents. We discuss these results in terms of the known interspecific differences in bcd and the potential coevolution of selected compensatory mutations in trans and cis in response to continuous promoter restructuring.

The transformer2 gene in Musca domestica is required for selecting and maintaining the female pathway of development

We present the isolation and functional analysis of a transformer2 homologue Mdtra2 in the housefly Musca domestica. Compromising the activity of this gene by injecting dsRNA into embryos causes complete sex reversal of genotypically female individuals into fertile males, revealing an essential function of Mdtra2 in female development of the housefly. Mdtra2 is required for female-specific splicing of Musca doublesex (Mddsx) which structurally and functionally corresponds to Drosophila dsx, the bottom-most regulator in the sex-determining pathway. Since Mdtra2 is expressed in males and females, we propose that Mdtra2 serves as an essential co-factor of F, the key sex-determining switch upstream of Mddsx. We also provide evidence that Mdtra2 acts upstream as a positive regulator of F supporting genetic data which suggest that F relies on an autocatalytic activity to select and maintain the female path of development. We further show that repression of male courtship behavior by F requires Mdtra2. This function of F and Mdtra2 appears not to be mediated by Mddsx, suggesting that bifurcation of the pathway at this level is a conserved feature in the genetic architecture of Musca and Drosophila.

Sexual Behavior: How Sex Peptide Flips the Postmating Switch of Female Flies

Drosophila male Sex Peptide elicits an amazing variety of postmating responses in mated females, some of which are transmitted via a receptor on specific neurons of the female genital tract. New work shows that neurons expressing the sex-determination gene doublesex (dsx) play a pivotal role in the female postmating switch.

A New Component of the Nasonia Sex Determining Cascade Is Maternally Silenced and Regulates Transformer Expression

Although sex determination is a universal process in sexually reproducing organisms, sex determination pathways are among the most highly variable genetic systems found in nature. Nevertheless, general principles can be identified among the diversity, like the central role of transformer (tra) in insects. When a functional TRA protein is produced in early embryogenesis, the female sex determining route is activated, while prevention of TRA production leads to male development. In dipterans, male development is achieved by prevention of female-specific splicing of tra mRNA, either mediated by X-chromosome dose or masculinizing factors. In Hymenoptera, which have haplodiploid sex determination, complementary sex determination and maternal imprinting have been identified to regulate timely TRA production. In the parasitoid Nasonia, zygotic transformer (Nvtra) expression and splicing is regulated by a combination of maternal provision of Nvtra mRNA and silencing of Nvtra expression in unfertilized eggs. It is unclear, however, if this silencing is directly on the tra locus or whether it is mediated through maternal silencing of a trans-acting factor. Here we show that in Nasonia, female sex determination is dependent on zygotic activation of Nvtra expression by an as yet unknown factor. This factor, which we propose to term womanizer (wom), is maternally silenced during oogenesis to ensure male development in unfertilized eggs. This finding implicates the upstream recruitment of a novel gene in the Nasonia sex determining cascade and supports the notion that sex determining cascades can rapidly change by adding new components on top of existing regulators.