The developmental consequences of alternate splicing in sex determination and differentiation in Drosophila.
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Alternative Splicing↗
Biomedical subjects
M Bownes
Publications and source records attributed to M Bownes.
The developmental consequences of RNA processing. Introduction.
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Animals↗
Two independent cis-acting elements regulate the sex- and tissue-specific expression of yp3 in Drosophila melanogaster.
In Drosophila, the three yolk protein (yp) genes are transcribed in a sex-, tissue- and developmentally specific manner, providing an ideal system in which to investigate the factors involved in their regulation. The yolk proteins are synthesized in the fat body of adult females, and in the ovarian follicle cells surrounding the developing oocyte during stages 8-10 of oogenesis. We report here an analysis of the yolk protein 3 (yp3) gene and its flanking sequences by means of P-element mediated germ-line transformation and demonstrate that a 747 bp promoter region is sufficient to direct sex-specific expression in the female fat body and both the temporal- and cell-type-specificity of expression during oogenesis. Two elements that independently govern yp3 transcription in these tissues have been separated and no other sequences in the upstream, downstream or coding regions have been identified that are autonomously involved in yp3 expression.
Animals↗
Specificity and localization of lipolytic activity in adult Drosophila melanogaster.
The triacylglycerol lipases present in adult Drosophila melanogaster have been investigated. Different lipase activities are present in various tissues in the fly. In particular, an abundant lipase activity is present in the male accessory gland. An esterase null mutant was used to confirm that the enzyme activity was due to a distinct lipase and not non-specific activity from esterase 6 which is also abundant in accessory glands. The properties of the accessory-gland lipase were investigated, and pH optima and substrate utilization suggest that it has some similarities to vertebrate bile-salt-stimulated lipase. Lipase activity is significantly reduced in males and increased in females shortly after mating. This finding suggests that lipase activity is transferred to the female and may be important in mating and reproduction in Drosophila.
Animals↗
The regulation of the yolk protein genes, a family of sex differentiation genes in Drosophila melanogaster.
There are many obvious morphological and behavioural differences between male and female Drosophila, whose differing phenotypes are produced by a hierarchy of sex determination genes. These genes have been well characterised at the genetic and molecular level. Similarly, a number of sex-specific differentiation genes have been characterised, such as the chorion and vitelline membrane genes in females and the sex peptide and other accessory gland proteins in males. Despite the depth of these parallel studies, there is only one example of a direct link between the sex determination pathway and the downstream sex differentiation genes, namely the regulation of the female-specific yolk protein genes. The yolk proteins are synthesised in the fat body and ovarian follicle cells of the adult female and are subsequently transported to the oocyte where they are stored for utilization during embryogenesis. The expression of the yolk protein genes is not entirely controlled by the sex determination hierarchy, as several different regulatory pathways must interact to direct their correct sexual, temporal and spatial regulation during development.
Adipose Tissue↗
The sequence and expression pattern of the Calliphora erythrocephala yolk protein A and B genes.
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.
Amino Acid Sequence↗
Cloning and analysis of expression of a ubiquitin carboxyl terminal hydrolase expressed during oogenesis in Drosophila melanogaster.
Using the enhancer trap approach we have searched for genes with important functions in oogenesis. We selected a line of flies with a P insertion, carrying the Escherichia coli lac Z gene, which showed beta-galactosidase expression in the nurse cell nuclei during oogenesis. Surrounding the P insertion we discovered a cluster of transcription units with enriched expression in the ovary. One of these encodes a protein with extensive sequence similarity to the human and yeast ubiquitin carboxyl terminal hydrolase. Analysis of a fusion protein including the putative ubiquitin carboxyl terminal hydrolase indicated that this protein does have the appropriate enzyme activity, and the gene was assigned the name ubiquitin carboxyl terminal hydrolase uch-D. The expression of this gene is enriched in the nurse cells and transcripts are transported to the embryo. Transcripts are abundant for the first few hours of development. The transcripts are found to be enriched on the ventral side of the oocyte and nurse cells. Rather little is known about the ubiquitin pathway in Drosophila and the discovery of this gene enables us to make predictions as to the roles it may play during early embryogenesis.
Amino Acid Sequence↗
Cis-regulatory sequences leading to female-specific expression of yolk protein genes 1 and 2 in the fat body of Drosophila melanogaster.
The three yolk protein genes (yp) of Drosophila melanogaster are transcribed in a sex- and tissue-limited fashion. We have searched for cis-regulatory sequences in regions flanking yp1 and yp2 to identify the elements that confer female-specific expression in the fat body. One such 127 bp element has previously been identified in this region. We show here the existence of two additional regions which confer female fat body-specific expression on an Adh reporter gene and on the native yp2 gene, respectively. This suggests some redundancy in the regulation of expression of the yp genes. Computer searches for putative binding sites for the DSX protein, which regulates sex-specific expression of the yp genes, revealed several such sites in our constructs. However, the significance of these is unclear since many such sites also occur in genes which one would not expect to be regulated in a sex-specific manner (e.g. Adh, Actin 5C). We suggest that DSX acts in concert with other proteins to mediate sex- and tissue-specific expression of the yp genes.
Alcohol Dehydrogenase↗
Ultrastructural analysis of Drosophila ovarian follicles differing in yolk polypeptide (yps) composition.
Drosophila ovarian follicles were examined ultrastructurally to study the vesicular traffic in the cortical ooplasm. The endocytic pathway leading to the production of yolk spheres was visualized following in vivo or in vitro exposure to peroxidase. The Golgi apparatus and the yolk spheres of wild-type ovarian follicles were preferentially labelled by fixation with osmium zinc iodide (OZI). Labelling of wild-type ovarian follicles was compared to that of several mutant follicles--L186/Basc, fs(2)A17 and ap4--which are defective in vitellogenesis. In these mutants, the Golgi apparatus and the vesicles nearby were either scantly labelled or not labelled at all. In oocytes from flies homozygous for the gene fs(1)1163, the Golgi apparatus was labelled as in the controls, but no yolk spheres appeared to be labelled with OZI at any of the developmental stages. In several Drosophila strains, the pattern of OZI label in the cortical ooplasm was seen to vary in relation to the number of yp structural genes. In starved Drosophila females, OZI labelling of the cortical ooplasm appeared restricted to the Golgi apparatus and to an extended tubular network. A similar labelling pattern was also detected in in vitro cultured vitellogenic follicles. Refeeding, topical application of juvenile hormone analogue to starved females or hormone addition to the culture medium, all caused the yolk spheres to become labelled with OZI and to incorporate peroxidase. These observations prove that impairing endocytic uptake by either mutation or lack of juvenile hormone prevents fusion of coated vesicles and tubules with the yolk spheres and leads them instead to form an intermediate cell compartment with Golgi-derived vesicles.
Animals↗
Sequence and expression of a Drosophila melanogaster cDNA encoding a putative ribosomal protein.
An abundant ovarian cDNA from Drosophila melanogaster has been cloned and sequenced. The predicted protein sequence is similar to that of the ribosomal protein 1024 of Dictyostelium discoideum, the 40S ribosomal protein ys11 of Saccharomyces cerevisiae and a 22 kd protein from Trypanosoma brucei. It seems, therefore, that the Drosophila cDNA also encodes a ribosomal protein. Transcripts are found at all stages of the life cycle but are especially abundant in the ovary, suggesting that this mRNA is maternally stored for utilization in embryogenesis to enable the rapid production of ribosomal proteins and assembly of ribosomes.
Amino Acid Sequence↗
The specificity of yolk protein uptake in cyclorrhaphan diptera is conserved through evolution.
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 long-term 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.
Animals↗
Mutant yolk proteins lead to female sterility in Drosophila.
Specific mutations in the yolk protein genes, yp1 and yp2, of Drosophila melanogaster cause the yolk proteins (YPs) they encode to precipitate, ultimately resulting in female sterility. YPs of the yp1 mutant fs(1)1163 are secreted normally but then precipitate as globules and occasionally as crystalline fibers in the subbasement membrane space of the fat body (Butterworth et al., 1991, J. Cell Biol. 112, 727-737). The present ultrastructural and immunological studies of the fat body of the yp2 mutant fs(1)K313 show that YP also precipitates as globules in the same tissue compartment. The globules are also incapable of passing into the hemolymph but they are morphologically distinct from those of fs(1)1163. Similar analyses were performed on developing oocytes in wild type and both mutant strains. YP-containing aggregates, ultrastructurally similar to those in the fat body of each respective mutant, were found in the space between the plasmalemma and the vitelline membrane and embedded within the membrane itself. The evidence suggests that the precipitates interfere with the correct assembly of the eggshell membranes, leading to the sterile phenotype. Immunogold studies demonstrate that newly synthesized YPs in the normal and mutant strains share secretory vesicles with putative, vitelline membrane proteins and that the translocation of follicle cell YP is not through the membrane along the interfollicular spaces but directly through the plasmalemma facing the oocyte. Further the YP precipitates in the mutants permit visualization of the polarity of exocytosis of YP from the follicle cells.
Animals↗
Why is there sequence similarity between insect yolk proteins and vertebrate lipases?
The major proteins stored in the yolk of developing oocytes are thought to provide a nutritional store for utilization during embryogenesis. They seem to fall into two major families of proteins. The first are called vitellogenins and are found in frog, chicken, nematode, fish, and some insects such as the boll weevil. The other group are called yolk proteins and are found in dipteran insects such as fruitfly, housefly, fleshfly, and blue-bottles. Both groups are the major proteins found in the oocyte and are female-specific proteins endocytosed from the serum or hemolymph. The yolk protein group were found to have sequence similarity to the triacylglycerol lipases and lipoprotein lipases of vertebrates, including rat, pig, and human. The yolk proteins do not have lipase activity, but the sequences conserved between yolk proteins and lipases surround the active site where there are interactions with lipids. The likely reason for the presence of this domain in the yolk proteins is to bind a steroid hormone in a storage form conjugated to lipids. This permits the storage of the hormone in an inactive form until the yolk proteins are degraded, when it can be released from its conjugate to induce developmental decisions in embryogenesis. They may also transport lipids into the oocyte for use in embryogenesis. Whilst the vitellogenin family of proteins do not share this homology with the lipases they do have similarity to the human serum protein, apolipoprotein B, which also has a role in binding lipids. These findings are discussed in relation to the evolution and functions of lipases, apolipoproteins, vitellogenins, and yolk proteins. Experiments aimed at isolating genes encoding lipases in insects and at further elucidating the function of the yolk proteins are suggested.
Amino Acid Sequence↗
Characterization, molecular cloning and sequencing of YP3s1, a fertile yolk protein 3 mutant in Drosophila.
The three yolk proteins (YP1, YP2 and YP3) of Drosophila melanogaster are synthesized in two tissues of the adult female, the fat body and ovarian follicle cells. The YPs are selectively accumulated in the oocyte to provide nutrients for embryogenesis. We describe a female-sterile mutant, fs(1) A1526, which lacks YP3 in the haemolymph. The female sterility mutation mapped some distance away from the yp3 gene on the X chromosome and we were able to separate the YP3 defect from the female sterility by recombination, thus producing a fertile line of flies having no YP3 in the eggs. This shows that YP3 is not essential for embryogenesis. The mutant line is to be known as YP3s1. Investigation of yp3 transcription in the mutant females revealed that the gene is transcribed but yp3s1 mRNA levels are reduced relative to wild type. Transcription of the mutant yp3 gene can be induced in males by ecdysone. Investigation of the yolk proteins in YP3s1 females suggested that the YP3s1 polypeptide is synthesized in the fat body but not secreted. The mutant YP3 protein shows an increase in apparent molecular weight of approximately 1 kDa. The mutant yp3 gene was cloned and the DNA sequence determined. The sequence differences between the mutant and wild-type genes include an amino acid substitution in the leader sequence. We suggest that this may be responsible for the failure of YP3 secretion in the mutant YP3s1, and speculate on the cause of the reduction seen in the steady-state level of yp3 mRNA.
Animals↗
Egg production and fertility in Drosophila depend upon the number of yolk-protein gene copies.
The yolk proteins of Drosophila melanogaster comprise a family of three related yolk polypeptides each encoded by a single-copy gene. We show by genetic crosses that each gene makes an equivalent contribution to the fecundity and fertility of the female and they do not individually provide unique functions to the embryo. We show that the number of eggs laid by a female depends upon the number of genes encoding yolk polypeptides present in the genome and furthermore that the probability of an egg hatching into an adult also critically depends upon the number of yolk protein genes present in the mother. This suggests that the three yolk protein-encoding genes in Drosophila melanogaster may have arisen by duplication, then been maintained for quantitative reasons because they increased egg production and fertility, rather than each protein evolving a different function as is the case with most small gene families, such as tubulins and collagen genes.
Animals↗
Investigation of cis-acting sequences regulating expression of the gene encoding yolk protein 3 in Drosophila melanogaster.
The regulatory sequences leading to the ovarian and fat body expression of yolk proteins 1 and 2 (YP1 and 2) of Drosophila melanogaster have been characterised in some detail. These genes (yp1 and yp2) share many enhancer elements, and some important regulatory sequences lie within the coding regions. We have begun to investigate the cis-regulation of the gene encoding yolk protein 3 (yp3). We describe a system for P element transformation using the complete and unaltered yp3 gene rather than reporter genes and describe sequences conferring correct expression in the ovary and carcass.
Animals↗
Genetically modified yolk proteins precipitate in the adult Drosophila fat body.
Ultrastructural and genetic studies were carried out on the fat body of a female sterile mutant fs(1)1163 to ascertain why yolk protein 1 (YP1) is not secreted from this tissue. Earlier molecular studies demonstrated that (a) normally yolk protein is synthesized in the fat body, secreted into the hemolymph and taken up by the ovary, (b) the 1163 mutation causes a single amino acid substitution in YP1, and (c) females homozygous for the mutation, or heterozygous females raised at 29 degrees C, retain YP1 in the fat body. Ultrastructural analysis in this paper shows that the fat body of these females contains masses of electron-dense material deposited in the subbasement membrane space. This subbasement membrane material (SBMM), which occasionally has a crystalline-like, fibrous component, is found in females whose genotypes include at least one copy of the mutant 1163 gene. These strains include a deletion strain that is hemizygous for the 1163 gene and two strains that are transgenic for the mutant gene. Immunogold studies indicate that SBMM contains yolk protein. We propose that the mutant protein is secreted into the subbasement membrane space, but because of the amino acid substitution in YP1, the oligomers containing YP1 condense into SBMM, which cannot penetrate the basement membrane. The similarity of SBMM and deoxyhemoglobin S fibers is discussed.
Animals↗
Differential control of yolk protein gene expression in fat bodies and gonads by the sex-determining gene tra-2 of Drosophila.
We studied the regulation of the yolk protein (YP) genes in the somatic cells of the gonads, using temperature sensitive mutations (tra-2ts) of transformer-2, a gene required for female sexual differentiation. XX;tra-2ts mutant animals were raised at the permissive temperature so that they developed as females and were then shifted to the restrictive male-determining temperature either 1-2 days before or 0-2 h after eclosion. These animals formed vitellogenic ovaries. Likewise, mutant gonads transplanted into either normal female hosts or normal male hosts, kept at the restrictive temperature, underwent vitellogenesis. Thus, the ovarian follicle cells can mature and express their YP genes in the absence of a functional product of the tra-2 gene. Although the gonadal somatic cells of ovary and testis may derive from the same progenitor cells, the testicular cells of XX;tra-2ts pseudomales did not express their YP genes nor take up YP from the haemolymph at the permissive female-determining temperature. We conclude that in the somatic cells of the gonad, the YP genes are no longer under direct control of the sex-determining genes, but instead are regulated by tissue specific factors present in the follicle cells. It is the formation of follicle cells which requires the activity of tra-2.
Adipose Tissue↗