Search

We present the isolation and functional analysis of atransformer2homologueMdtra2in the houseflyMusca 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 ofMdtra2in female development of the housefly.Mdtra2is required for female-specific splicing ofMusca doublesex(Mddsx) which structurally and functionally corresponds toDrosophila dsx, the bottom-most regulator in the sex-determining pathway. SinceMdtra2is expressed in males and females, we propose thatMdtra2serves as an essential co-factor ofF, the key sex-determining switch upstream ofMddsx. We also provide evidence thatMdtra2acts upstream as a positive regulator ofFsupporting genetic data which suggest thatFrelies on an autocatalytic activity to select and maintain the female path of development. We further show that repression of male courtship behavior byFrequiresMdtra2. This function ofFandMdtra2appears not to be mediated byMddsx, suggesting that bifurcation of the pathway at this level is a conserved feature in the genetic architecture ofMuscaandDrosophila. [ABSTRACT FROM AUTHOR]

Sex-determining cascades are supposed to have evolved in a retrograde manner from bottom to top. Wilkins’ 1995 hypothesis finds support from our comparative studies in Drosophila melanogaster and Musca domestica, two dipteran species that separated some 120 million years ago. The sex-determining cascades in these flies differ at the level of the primary sex-determining signal and their targets, Sxl in Drosophila and F in Musca. Here we present evidence that they converge at the level of the terminal regulator, doublesex (dsx), which conveys the selected sexual fate to the differentiation genes. The dsx homologue in Musca, Md-dsx, encodes male-specific (MdDSXM) and female-specific (MdDSXF) protein variants which correspond in structure to those in Drosophila. Sex-specific regulation of Md-dsx is controlled by the switch gene F via a splicing mechanism that is similar but in some relevant aspects different from that in Drosophila. MdDSXF expression can activate the vitellogenin genes in Drosophila and Musca males, and MdDSXM expression in Drosophila females can cause male-like pigmentation of posterior tergites, suggesting that these Musca dsx variants are conserved not only in structure but also in function. Furthermore, downregulation of Md-dsx activity in Musca by injecting dsRNA into embryos leads to intersexual differentiation of the gonads. These results strongly support a role of Md-dsx as the final regulatory gene in the sex-determining hierarchy of the housefly. [ABSTRACT FROM AUTHOR]

We have isolated and sequenced a cDNA encoding a predicted 524 amino acid protein from a Drosophilamelanogaster ovarian library. Sequence comparisons suggest that this protein encodes a sodium-dependent inorganic phosphate co-transporter similar to a sequence isolated from a rat brain library. In situ hybridisation to messenger RNA in ovaries shows strong expression in germarium at stage 2 of oogenesis. Expression is then weak in follicle cells until stage 10, when high transcript levels are seen in the nurse cells and transferred to the oocyte. This presumably reflects functions in oogenesis and the production of stored mRNAs for use in embryogenesis. [ABSTRACT FROM AUTHOR]

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2× L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). Fibroblast-like cells formed confluent monolayers in modified 2× L-15 medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media. [ABSTRACT FROM AUTHOR]

Differential screening was used to isolate a genomic clone, λ89Ba located at 89B that hybridised preferentially with female cDNA. On further investigation, a 3.1-kb subfragment, 89Ba(3.1), was shown to contain a gene with male germline-specific expression (Mst89B) flanked by two genes (Mat89Ba and Mat89Bb) expressed predominantly in the ovaries and embryo of Drosophila melanogaster. Mat89Bb is separated from Mst89B by at most 100 bp; Mst89B and Mat89Ba are convergently transcribed and their 3′ untranslated regions (UTRs) overlap by a minimum of 85 bp. Database searches with either the 89Ba(3.1) genomic DNA sequence or conceptual translations of Mst89B or Mat89Bb cDNAs failed to reveal any significant similarities with database entries. Using in situ hybridisation to ovaries, Mat89Ba and Mat89Bb were shown to be expressed in nurse cells at stages nine and ten of oogenesis and exported to the oocyte. In addition, Mat89Bb transcripts were detected in the follicle cells surrounding the oocyte. Mst89B transcripts were present throughout spermatogenesis in germline-derived cells, consistent with northern analysis which showed that they were absent in the offspring of tudor flies that lack a germline. The absence of Mst89B transcripts at the tip of the testis suggested that the somatic cells in this region do not express Mst89B. Two 12-bp sequences were identified in the 5′ UTR of Mst89B with a strong similarity to translational control elements (TCEs) originally identified in the CGP gene family. This suggests that TCEs may be present in a wider class of testis transcripts. [ABSTRACT FROM AUTHOR]

A genomic fragment was cloned from a DNA library constructed from a Drosophila enhancer trap line in which reporter gene expression was observed at the anterior-most tip of the ovaries and testes. This genomic clone was identified as the L-repeat of the Drosophila melanogaster histone gene cluster. Northern blotting and in situ hybridisation to RNA in tissues with individual cDNAs and PCR-generated probes for each histone confirmed that gene expression was greatest at the anterior portion of each ovariole, in the germarium, and was also elevated in a few individual nurse cells and somatic follicle cells within the egg chamber during early developmental stages. Histone H1 and each of the core histones had a similar expression pattern which was correlated to cell division. Maternal stores of histone transcripts were also transported to the mature oocyte from the nurse cells at a later stage of oogenesis (stage 10), when virtually all the nurse cells contained high levels of histone transcripts. The results are consistent with expression of the somatic histone gene cluster during oogenesis as a co-ordinate unit. There does not seem to be a reduced level of somatic type H1 in the germ-line, as is observed in some other species. The relationship between the P[lacZ] expression pattern in the germarium and the overall expression of the histone cluster suggests there are specific regulatory elements for germ-line expression. [ABSTRACT FROM AUTHOR]

We have investigated the conservation of regulatory elements for sex- and tissue-specific gene expression in three dipteran species , Drosophila melanogaster, Musca domestica and Calliphora erythrocephala, using the yolk protein (yp) genes. Yolk proteins of the fruitfly, medfly, housefly and blowfly are very well conserved both in their sequence and their expression in ovarian follicle cells and in fat bodies of adult females. Furthermore, yp regulation by both hormonal and nutritional factors shows similar features in all four species. To study conservation of yp regulation in dipteran insects, we tested 5′ flanking regions from one Musca yp gene and one Calliphora yp gene for enhancer functions in D. melanogaster. Two fragments of 823 and 1046 bp isolated from Musca and Calliphora yp genes, respectively, are able to direct correct expression of a reporter gene in the ovarian follicle cells of transformed Drosophila at specific stages during oogenesis. Surprisingly, these enhancers do not confer sex-specific reporter gene expression in the fat body, as expression was found in both sexes of the transformed flies. None-the-less by in vitro DNA/protein interaction assays, a 284-bp DNA region from the Musca yp enhancer was able to bind the Drosophila DOUBLESEX (DSX) protein, which in D.melanogaster confers sex-specific expression of yp. We speculate that the sex-determining pathway is not directly involved in yp regulation in Musca or Calliphora adult females, but depends instead on hormonal controls to achieve sex-specific expression of yp genes in the adult. [ABSTRACT FROM AUTHOR]

When complementary fragments of an imaginal disc of Drosophila are cultured for several days prior to metamorphosis, usually one fragment will regenerate while the other will duplicate. It has been proposed that wound healing plays an important part in disc regulation (French et al. 1976; Reinhardt et al. 1977) by initiating cell proliferation and determining the mode of regulation. We tried to delay the wound healing process by leaving a region of dead cells between the wound edges. In '06' fragments (Bryant 1975a) wound healing has occurred after 1-2 days of culture and the regeneration of missing structures after 2-4 days of culture. We observed that leaving a region of dead cells between the wound edges delays both wound healing and the regeneration of missing structures by 2 days. When disc fragments are cultured in female abdomens and then exposed toH-thymidine to label replicating cells, then the label is found to be localised around the wound. We observed that delaying wound healing does not delay this localisation of labelled nuclei indicating that wound healing may not be required to initiate DNA replication. [ABSTRACT FROM AUTHOR]

It has been known for many years that when a wing disc of Drosophila is bisected, and the fragments cultured in adult females, regulation occurs and either a complete disc is regenerated or the fragment is duplicated. We have investigated how this regeneration process occurs. To establish which cells contribute to the regenerate, and thus determine if regeneration is the result of epimorphic regulation, fragments of discs, after culture in an adult for one to five days, were exposed toH-thymidine to label replicating cells. Imaginal discs, both whole and as regenerating fragments, undergo some DNA replication which is distributed throughout the disc, but cut discs frequently show clusters of labelled cells around the wound, indicating that regeneration is probably epimorphic. [ABSTRACT FROM AUTHOR]

The yolk protein genes ( yps) are expressed in a temporal, tissue- and sex-specific fashion in Drosophila melanogaster. Here we report the sequence of two related genes in Calliphora erythrocephala. The predicted Calliphora yolk protein (YP) sequences are well conserved, especially at the C-terminal end when compared to those of D. melanogaster and Ceratitis capitata. Database searches with the Calliphora yolk protein B (CeYPB) sequence identify the vertebrate lipase similarity reported for the YPs of Drosophila and Ceratitis. Moreover, sequences with identity to divalent ion-binding sites were observed, which colocalized with putative tyrosine sulfation sites. Calliphora oogenesis differs from Drosophila in that it is cyclic in response to a meat feed. The Calliphora yp genes are expressed in the follicle cells of the egg chamber during vitellogenesis, as shown by in situ hybridization, and the yp message levels correlate with YP synthesis. The synthesis of the yp transcripts in ovaries of Calliphora occurs in the same pattern as that for ovarian transcripts in Drosophila. In the carcass, yp transcript levels are correlated with the production of a batch of eggs. [ABSTRACT FROM AUTHOR]

Yolk proteins are transported from the hemolymph into the oocytes of insects during vitellogenesis by receptor-mediated endocytosis. Since other hemolymph proteins, both native and foreign, are not accumulated in the oocyte, the process of uptake is selective for yolk proteins. Peptide domains within the yolk proteins must therefore be involved in receptor recognition. With the longterm aim of identifying these domains and to open the possibility of understanding the molecular basis of receptor-mediated endocytosis of yolk proteins, we began investigating how well this mechanism has been conserved in evolution. We studied the uptake of yolk proteins from 13 different Drosophila species and five other dipteran species, namely, Calliphora erythrocephala, Sarcophaga argyrostoma, Musca domestica, Lucilia servicata, and Protophormia terrae-novae, into the ovaries of Drosophila melanogaster and Drosophila funebris. The results from these experiments showed that in all cases the foreign yolk proteins were taken up by the host ovaries, indicating that the mechanism and peptide domains of the yolk proteins involved in recognition of the receptor have been well conserved in dipteran evolution. [ABSTRACT FROM AUTHOR]

A Drosophila gene ( az2), mapping to a cluster of embryonic lethals at 43BC on the polytene chromosomes, has been sequenced and found to encode a predicted protein with six consecutive C2H2 zinc finger domains. The carboxy-terminus of az2 is related to a number of Drosophila and mammalian transcription factors. The 5’ end of the gene is unrelated to genes in the databases. The gene is expressed in the adult female, in both the carcass and ovary, but is most abundant in the ovary. It is expressed in the nurse cells and transported to the oocyte. [ABSTRACT FROM AUTHOR]