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M Bownes

Publications and source records attributed to M Bownes.

At least 73 records · Page 4Linked to original sources
PubMed1990

Preferential insertion of P elements into genes expressed in the germ-line of Drosophila melanogaster.

A Drosophila transposable element, the P element, containing the bacterial gene encoding beta-galactosidase is widely used to search for tissue-specific enhancers. This lac-Z-containing P element (P-lacZ ry+) can be moved around the genome by a number of techniques. When it comes to lie close to a tissue-specific enhancer, blue staining results in particular tissues of the fly. Many different patterns of expression have already been obtained with the long-term aim of cloning the nearby genes that these enhancers normally regulate. Whilst analysing a set of flies containing these P-lacZ inserts for sex-specific expression in the adult, a preference was noticed for insertion into regions of DNA generating expression of beta-galactosidase in the male gonad. Since the transposition events generating these flies occurs in the male germ-line, it seemed possible that there was preferential insertion of the element into DNA which was being transcribed. To test this, transpositions were generated of the same P-lacZ ry+ in both the male and female germ-lines. The results are compatible with the above hypothesis. This finding has important implications for the type of enhancers likely to be found by this method, and may also be relevant to those using P elements as mutagens in Drosophila and for the study of the mechanism of P transposition.

Animals↗
PubMed1990

Sex determination in Drosophila melanogaster.

The understanding of sex determination is a fundamental goal in the study of eukaryotic developmental genetics. The mechanisms governing the generation of sexual dimorphism have been well characterized in Drosophila because of its amenability to both genetic manipulation and the application of the techniques of modern molecular genetics. By using classical genetics to search for sex-transforming mutations and by analysing their phenotypes and how they interact, a picture has emerged involving a cascade of regulatory genes. The primary sex determining signal--the ratio of the number of X chromosomes to the number of sets of autosomes--sets this cascade into motion. Genetic evidence has suggested that the intervening genes in this pathway are active in females but not in males, whereas the final gene has active but opposing roles in the two sexes. This bifunctional locus is responsible for the repression of female differentiation genes in males and male differentiation genes in females. The cloning of the key genes of the regulatory cascade and the study of their transcription patterns have revealed that their different functional states in the two sexes do not result from control at the transcriptional level, as might have been expected. Instead, common primary transcripts are produced in male and female flies; these are then differentially spliced to encode sex-specific gene products. In this paper we focus on the contributions of molecular genetics to the understanding of sex determination. Sufficient background is included for the reader to see how the models of the Drosophila sex determination system were first developed. We then show how the application of new technology has complemented the genetic approach and refined our understanding of the system. Current intensive research in this area should lead within the next few years to definitive knowledge at the molecular level of the cascade of differential splicing of regulatory genes, and how this hierarchy ultimately gives rise to the appropriate sex-specific patterns of structural gene expression that underlie sexual dimorphism.

Animals↗
PubMed1989

cricklet: A locus regulating a number of adult functions of Drosophila melanogaster.

During a screen for mutations in trans-acting genes regulating yolk protein synthesis in Drosophila melanogaster, we have isolated a mutant (cricklet, clt) that is defective in yolk protein synthesis, histolysis of the larval fat body, vitellogenesis, and synthesis of larval serum protein 2 in the adult, larval synthesis occurring normally. Larval serum protein 2 was previously thought to be synthesized only in the third-instar larva. We suggest that the clt locus may encode a protein essential for mediating the response of adult tissues to juvenile hormone.

Journal Article↗
PubMed1988

Evidence that insect embryogenesis is regulated by ecdysteroids released from yolk proteins.

That the yolk proteins (YPs), or vitellins, stored in the oocytes of insects are a nutritional store for subsequent embryogenesis has long been assumed. Exhaustive data base searching programs revealed highly significant sequence similarity between the three YPs of Drosophila melanogaster and part of the triacylglycerol lipase of the domestic pig. Based upon time of degradation of YPs during embryogenesis, existence of maternally stored ecdysteroid conjugates in embryos, location of these conjugates in locust embryos, and the fact that free active ecdysteroid hormones are released at a specific time in embryogenesis to trigger cuticle deposition, we postulate that the similarity reflects a common property of Drosophila YPs--the ability to bind the fatty acid ecdysteroid conjugates. Our finding of conjugated ecdysteroids tightly bound to purified Drosophila YP supports this prediction.

Amino Acid Sequence↗
PubMed1988

Dietary components modulate yolk protein gene transcription in Drosophila melanogaster.

The three yolk proteins of Drosophila melanogaster begin to be synthesized at eclosion. Transcription of the genes is regulated by the genes tra, tra-2 and dsx and also by the insect hormones, juvenile hormone and 20-hydroxyecdysone. We show that there is yet another level of control which is dependent upon feeding. Females that are starved from eclosion show a basal level of yolk protein gene transcription, which is rapidly increased when a complete diet is supplied. We show that the effect is not due to incorrect development of the fat body and is unlikely to be solely due to a general effect on protein synthesis. Later in development, cessation of feeding leads to selective inhibition of yolk protein synthesis and hence egg production. The effects of starvation can be partially overcome by 20-hydroxyecdysone, juvenile hormone, casein, amino acid mix or sucrose, but only a complete medium or live yeast brings about total recovery. Using yp1-Adh fusions (fusions of the promoter region of yp1 to the structural gene for Adh), the DNA sequence required for this diet-enhanced transcription has been located within an 890 bp fragment upstream of the yp1 gene. The insect hormones do not operate on this same DNA fragment.

Animals↗
PubMed1987

The titre of juvenile hormone during the pupal and adult stages of the life cycle of Drosophila melanogaster.

Using combined gas chromatography/selected-ion mass spectroscopy, the titer of juvenile hormone was determined for whole-body extracts at various morphologically defined stages of the life cycle of Drosophila melanogaster. Only juvenile hormone III (JH-III) was detected. JH-III is present in early metamorphosis but by mid-metamorphosis it is below the level of detection (0.05 pmol/g). It then increases as the pharate adult matures and rises dramatically, beginning just prior to eclosion and reaching 5-7 pmol/g shortly after eclosion. The titer then begins to fall again as the adults mature in both males and females, though the decrease is more rapid in females. Preliminary studies show that low levels of JH-III are present during all the larval instars but are absent from eggs.

Animals↗
PubMed1987

Identification of a female-sterile mutation affecting yolk protein 2 in Drosophila melanogaster.

The three yolk proteins (YP1, YP2 and YP3) of Drosophila melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated in the developing oocytes to provide a nutrient source for embryogenesis. We have described the phenotype of a temperature-sensitive female-sterile mutant, fs (1) K313, nad characterised its yolk proteins. This mutation affects the secretion of YP2 and is the first mutation affecting YP2 to be described. Using genetic and molecular tests we argue that the female-sterile phenotype results, at least in part, from the abnormal secretion of YP2 perturbing the follicle cell secretory pathway in general and thus causing defects in chorion protein secretion. The gene coding for YP2 in fs (1) K313 has been cloned and sequenced. Two amino acid substitutions have been found which probably cause the abnormal secretion of YP2 and the resulting female-sterile phenotype.

Animals↗
PubMed1987

Separate DNA sequences are required for normal female and ecdysone-induced male expression of Drosophila melanogaster yolk protein 1.

Drosophila melanogaster flies were transformed with a yp1-Adh fusion gene with 890 bp of yp1 5' flanking sequence. In an Adh- background these flies show a stage, tissue and sex-specific pattern of alcohol dehydrogenase (ADH) activity characteristic of yolk protein genes. ADH activity is not present in dsxD/dsx pseudomales indicating that this fragment contains sites where the dsx gene product exerts its effect. Transformed male flies do not exhibit ADH activity when injected with 20-hydroxyecdysone while synthesis of native yolk proteins is induced. Thus the hormone inducibility and sex regulation have been separated in this construct.

Alcohol Dehydrogenase↗
PubMed1987

The nucleotide sequence of the gene coding for Drosophila melanogaster yolk protein 3.

The entire sequence of the Drosophila melanogaster yolk protein 3 (YP3) gene (yp3), including 1822 nucleotides (nt) of 5'- and 834 nt of 3'-flanking DNA, has been determined. In addition, the 5' and 3' ends of the mRNA and the two introns have been mapped. The predicted amino acid sequence of YP3 has considerable homology (43%) to the other two yolk proteins of D. melanogaster. The nucleotide sequence of yp3 was compared to the other two yolk protein genes which have the same developmental pattern of expression. In addition to extensive homology between the protein coding regions, we found two small regions of homology between yp3 flanking sequences and a segment of DNA required for normal expression of the yolk protein 1 gene in adult female fat bodies.

Adipose Tissue↗
PubMed1987

The use of an inhibitor of protein synthesis to investigate the roles of ecdysteroids and sex-determination genes on the expression of the genes encoding the Drosophila yolk proteins.

The three yolk-protein genes of Drosophila are normally expressed only in adult female fat bodies and ovaries. 20-hydroxyecdysone can affect the transcription of these genes in males and females, as can mutations in the sex-determining genes tra, tra-2, ix and dsx. We have asked a number of basic questions about how these genes are regulated, using an inhibitor of protein synthesis (cycloheximide), labelling RNA in vivo, a temperature-sensitive sex-determination mutant (tra-2ts1), and 20-hydroxyecdysone. We have found that the yolk-protein genes are continuously transcribed in the fat bodies of adult females and that maintenance of this transcription requires protein synthesis. Hormone induction in males is also inhibited by cycloheximide, suggesting that the products of other genes are essential both for 20-hydroxyecdysone to be able to switch on the genes, and for their continuous transcription in the female fat body. The products of the tra-2 gene are also required for continuous transcription of the yolk-protein genes, suggesting that the pathway inhibited by the cycloheximide is that of the sex-determination hierarchy. 20-hydroxyecdysone can override the sex-determination system and induce yolk protein synthesis in normal males and tra-2ts reared and maintained at the restrictive temperature.

Animals↗
PubMed1986

Reduced stability of RNA coding for yolk polypeptide 3 in Drosophila melanogaster ovary.

In Drosophila three yolk polypeptides (YP1, YP2 and YP3) are synthesized at two sites in the adult female: in the fat body tissue, from which they are transported via the haemolymph to the ovary, and in the ovarian follicle cells which surround the developing oocytes. All three yolk polypeptides are synthesized at equal levels in the fat body. In this paper we show that the steady-state level of YP3 RNA is significantly reduced in the ovary in comparison with the fat body, and that none of the yolk protein genes is amplified either in the fat body or the follicle cells. In order to determine the basis of the reduced level of YP3 RNA in the ovary, which could result from a lower rate of transcription or through a decreased stability of the RNA, we have devised an in vivo method of determining relative rates of gene transcription. In both the fat body and the ovary all three yolk proteins are transcribed at similar rates. Thus we infer that YP3 RNA is destabilised in the ovary, accounting for the reduction in its steady-state level.

Drosophila melanogaster↗
PubMed1985

Pattern regulation in fragments of Drosophila wing discs which show variable wound healing.

When complementary fragments of the imaginal wing disc of Drosophila are cultured for several days prior to inducing metamorphosis, usually one fragment will regenerate while the second duplicates. It has been proposed that wound healing plays an important part in disc regulation by initiating cell proliferation and determining the mode of regulation (regeneration/duplication). To test the latter proposal 15 types of wing disc fragments were examined for variability both in the mode of wound healing and the mode of pattern regulation. Two modes of wound healing were observed, regular - the two wound edges heal with each other, and irregular - each wound edge heals with itself. When cultured separately fragments that healed regularly regenerated, while fragments that healed irregularly duplicated. This suggests that the mode of wound healing determines the mode of pattern regulation.

Animals↗
PubMed1983

The regulation of yolk protein gene expression in Drosophila melanogaster.

The three genes, located in the X chromosome, which code for the three yolk polypeptides (YPs) of Drosophila melanogaster are expressed in the fat bodies and ovarian follicle cells of adult females. Both juvenile hormone and ecdysone are involved in regulating their expression. The yolk protein genes (YP genes), normally not transcribed in males, become expressed when males are injected with or fed 20-hydroxyecdysone. Superimposed on this hormonal regime is a sex determination mechanism which ensures that normally YP gene expression is female-specific. There are a series of autosomal genes in D. melanogaster which ensure that individual cells follow a male or female developmental pathway. When they are mutant, flies with two X chromosomes, which would normally be female, can become intersexual in phenotype or transformed into sterile males and flies with one X and one Y chromosome can become intersexual. It has been found that the YPs are part of the set of female characteristics controlled by these sex genes. The YP genes are expressed in female and intersexual flies, regardless of the X chromosome constitution, but not in males or pseudomales. Transcript levels of yolk proteins have been measured in female and intersexual flies by hybridization to cloned YP DNA sequences. It is suggested that transcription of the YP genes is under the cell-autonomous control of the sex genes and that the sex genes do not exert their effect by modulating the levels of steroid hormones in adults.

Age Factors↗
PubMed1983

Expression of the yolk-protein genes in the mutant doublesex dominant (dsxD) of Drosophila melanogaster.

Adult flies mutant for doublesex dominant (dsxD) are intermediate in phenotype between males and females. The dsxD mutation acts in the heterozygous state to transform only flies with two X chromosomes into intersexes, XY flies are unaffected by the mutation. Yolk-protein synthesis, which normally occurs in the ovaries and fat bodies of females, but not in males unless stimulated with 20-hydroxy-ecdysone, is reduced. The dsxD fat body synthesizes less yolk proteins throughout adult life, and the gonads rarely make yolk proteins. Using cloned yolk-protein genes as probes for measuring transcript levels we have shown that expression of these genes in dsxD is regulated both transcriptionally and post-transcriptionally. We suggest that the dsxD locus regulates the expression of the yolk-protein genes from within the fat body cells and does not operate by modulating ecdysteroid titres in the adults.

Animals↗
PubMed1983

20-hydroxyecdysone stimulates tissue-specific yolk-protein gene transcription in both male and female Drosophila.

The yolk polypeptides of Drosophila are normally synthesized in the fat body and ovarian follicle cells of adult females. In response to 20-hydroxyecdysone males synthesize yolk polypeptides. The actual level of yolk polypeptides synthesized in males is not always a direct reflection of the YP-transcripts present. Initially YP-transcripts are efficiently translated into polypeptides whereas later they are not and the YP-transcripts can have a half-life of less than 8 h in males. We suggest that the expression of the genes coding for the yolk polypeptides in males may be regulated at transcriptional and translational levels. Treatment of females with 20-hydroxyecdysone leads to a transient increase in YP-transcript accumulation, but the response is difficult to assess in whole flies due to the high variability in transcript levels during normal development. Analysing the response to 20-hydroxyecdysone at the level of specific tissues shows that transcript accumulation is dramatically increased in body walls (fat-body cells, epidermis and oenocytes) of both males and females. Gut, Malpighian tubules, testis and ovaries are not affected. Treatment of females with 20-hydroxyecdysone followed by measuring YP-transcript accumulation over the next 24 h in ovaries and body walls separately, confirms that only body walls respond to the hormone. There is an increase in yolk-polypeptide synthesis during the period of increased YP-transcript accumulation in females. We conclude that the response of the YP-genes to 20-hydroxyecdysone is tissue-, but not sex-specific.

Animals↗
PubMed1982

Hormonal and genetic regulation of vitellogenesis in Drosophila.

Morphological, genetic, and hormonal studies of the process of vitellogenesis, whereby yolk is accumulated in the developing oocytes, have been going on for many years in Drosophila. Recently there has been a resurgence of interest in vitellogenesis, for it provides a model system for investigating how eukaryotic genes are regulated. The proteins found in the yolk are regulated in a tissue-specific, stage-specific and sex-limited fashion. Molecular studies have identified the major proteins concerned and their genes have been cloned. This has facilitated a new approach to how this fascinating process is controlled. In this review current understanding of the factors required for normal vitellogenesis in Drosophila is analyzed. The nature of the proteins themselves, their sites of synthesis, and the organization and characterization of the genes and transcripts that code for them are emphasized. The manner in which the expression of these genes is regulated by the insect hormones, ecdysone and juvenile hormone, is described and, finally, an analysis is made of how various mutants that disrupt vitellogenesis can contribute further to our understanding of vitellogenesis regulation in Drosophila.

Adipose Tissue↗
PubMed1982

Ovarian and fat-body vitellogenin synthesis in Drosophila melanogaster.

The ovary and the fat body of Drosophila melanogaster both synthesise vitellogenins in vivo. The ovary contributes nearly as much vitellogenin to the yolk of an oocyte as does the fat body. Densitometry of fluorographs and gels has been used to compare the amount of the smallest vitellogenin polypeptide, yolk protein 3, synthesised by each tissue. Cell-free translations indicate that the ovary, in contrast to the fat body, contains a much reduced level of the mRNA for yolk protein 3 compared with the mRNAs for the other vitellogenin polypeptides. However, if tissues are cultured in vitro, the underproduction of this protein by the ovary is not significant. Because young embryos have levels of this polypeptide which are expected if the ovary has a low level of its corresponding mRNA, we argue that the ovary genuinely underproduces this protein in vivo and that the relative levels synthesised by the ovary in vitro are an artefact. Egg chambers of previtellogenic stages can synthesise vitellogenins, but the maximum level of vitellogenin synthesis occurs in egg chambers of the early vitellogenic stages. We conclude that the expression of the vitellogenin genes is subject to different controls at each site of synthesis. The possible cell types responsible for ovarian vitellogenin synthesis are discussed; the follicle epithelial cells are tentatively nominated for this role. We also suggest that a specific repression mechanism for vitellogenin gene expression exists in the ovary.

Animals↗