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M Bownes

Publications and source records attributed to M Bownes.

At least 37 records · Page 2Linked to original sources
PubMed1999

Class VI unconventional myosin is required for spermatogenesis in Drosophila.

We have identified partial loss of function mutations in class VI unconventional myosin, 95F myosin, which results in male sterility. During spermatogenesis the germ line precursor cells undergo mitosis and meiosis to form a bundle of 64 spermatids. The spermatids remain interconnected by cytoplasmic bridges until individualization. The process of individualization involves the formation of a complex of cytoskeletal proteins and membrane, the individualization complex (IC), around the spermatid nuclei. This complex traverses the length of each spermatid resolving the shared membrane into a single membrane enclosing each spermatid. We have determined that 95F myosin is a component of the IC whose function is essential for individualization. In wild-type testes, 95F myosin localizes to the leading edge of the IC. Two independent mutations in 95F myosin reduce the amount of 95F myosin in only a subset of tissues, including the testes. This reduction of 95F myosin causes male sterility as a result of defects in spermatid individualization. Germ line transformation with the 95F myosin heavy chain cDNA rescues the male sterility phenotype. IC movement is aberrant in these 95F myosin mutants, indicating a critical role for 95F myosin in IC movement. This report is the first identification of a component of the IC other than actin. We propose that 95F myosin is a motor that participates in membrane reorganization during individualization.

Actins↗
PubMed1999

The function of the broad-complex during Drosophila melanogaster oogenesis.

The Broad-Complex (BR-C) is an early ecdysone response gene that functions during metamorphosis and encodes a family of zinc-finger transcription factors. It is expressed in a dynamic pattern during oogenesis. Its late expression in the lateral-dorsal-anterior follicle cells is related to the morphogenesis of the chorionic appendages. All four zinc-finger isoforms are expressed in oogenesis, which is consistent with the abnormal appendage phenotypes resulting from their ectopic expression. We investigated the mechanism by which the BR-C affects chorion deposition by using BrdU to follow the effects of BR-C misexpression on DNA replication and in situ hybridization to ovarian mRNA to evaluate chorion gene expression. Ectopic BR-C expression leads to prolonged endoreplication and to additional amplification of genes, besides the chorion genes, at other sites in the genome. The pattern of chorion gene expression is not affected along the anterior-posterior axis, but the follicle cells at the anterior of the oocyte fail to migrate correctly in an anterior direction when BR-C is misexpressed. We conclude that the target genes of the BR-C in oogenesis include a protein essential for endoreplication and chorion gene amplification. This may provide a link between steroid hormones and the control of DNA replication during oogenesis.

Alleles↗
PubMed1999

A targeted gene silencing technique shows that Drosophila myosin VI is required for egg chamber and imaginal disc morphogenesis.

We report that Drosophila unconventional myosin VI, encoded by Myosin heavy chain at 95F (Mhc95F), is required for both imaginal disc and egg chamber morphogenesis. During oogenesis, Mhc95F is expressed in migrating follicle cells, including the border cells, which migrate between the nurse cells to lie at the anterior of the oocyte; the columnar cells that migrate over the oocyte; the centripetal cells that migrate between the oocyte and nurse cells; and the dorsal-anterior follicle cells, which migrate to secrete the chorionic appendages. Its function during development has been studied using a targeted gene silencing technique, combining the Gal4-UAS targeted expression system and the antisense RNA technique. Antibody staining shows that the expression of myosin 95F is greatly decreased in follicle cells when antisense Mhc95F RNA is expressed. Interfering with expression of Drosophila myosin VI at various developmental stages frequently results in lethality. During metamorphosis it results in adult flies with malformed legs and wings, indicating that myosin VI is essential for imaginal disc morphogenesis. During oogenesis, abnormal follicle cell shapes and aberrant follicle cell migrations are observed when antisense Mhc95F is expressed in follicle cells during stages 9 to 10, suggesting that the Drosophila myosin VI is required for follicle cell epithelial morphogenesis.

Animals↗
PubMed1998

Identification of an essential gene encoding a class-V unconventional myosin in Drosophila melanogaster.

Class-V myosins are a unique type of myosin motor with roles in intracellular transport. The mouse dilute gene was the first member of this class to be cloned, with mutations resulting in lightening of the coat colour or neurogenic defects leading to early death. Further examples of class-V myosins have been described in yeast, chicken and rat. Here, we report the cloning of the first class-V myosin from Drosophila. We show that expression of this myosin is predominantly in the adult germ line and early embryo and that the transcript is localised in the oocyte during oogenesis. Genetic and in situ hybridisation experiments have determined that this gene is located in the 43C region. We have evidence that it maps to a mutation in this region with an embryonic lethal phenotype.

Amino Acid Sequence↗
PubMed1998

The expression of histone genes during Drosophila melanogaster oogenesis.

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.

Animals↗
PubMed1998

Cloning and expression of az2, a putative zinc finger transcription factor from Drosophila melanogaster.

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.

Amino Acid Sequence↗
PubMed1998

A testis-specifically expressed gene is embedded within a cluster of maternally expressed genes at 89B in Drosophila melanogaster.

Differential screening was used to isolate a genomic clone, lambda89Ba 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.

3' Untranslated Regions↗
PubMed1998

The RNA product of the Doc retrotransposon is localized on the Drosophila oocyte cytoskeleton.

The Doc retrotransposon of Drosophila melanogaster encodes an RNA with two open reading frames (ORFs). ORF1 encodes an RNA-binding protein and overlaps with ORF2, which encodes a reverse transcriptase. Retrotransposons transpose to new positions in the genome via a full-length RNA intermediate. Because they do not encode a coat protein, transposition is limited to single cell lineages. In this paper we present evidence that shows that the highest levels of Doc transcripts are found in the adult male and female germ-line. The Doc RNA is localized in the region of the cytoskeleton of the Drosophila oocyte. The biological significance of this is unknown, but we discuss some of the possibilities.

Animals↗
PubMed1997

3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase.

BACKGROUND: The activation of protein kinase B (PKB, also known as c-Akt) is stimulated by insulin or growth factors and results from its phosphorylation at Thr308 and Ser473. We recently identified a protein kinase, termed PDK1, that phosphorylates PKB at Thr308 only in the presence of lipid vesicles containing phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3) or phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2). RESULTS: We have cloned and sequenced human PDK1. The 556-residue monomeric enzyme comprises a catalytic domain that is most similar to the PKA, PKB and PKC subfamily of protein kinases and a carboxy-terminal pleckstrin homology (PH) domain. The PDK1 gene is located on human chromosome 16p13.3 and is expressed ubiquitously in human tissues. Human PDK1 is homologous to the Drosophila protein kinase DSTPK61, which has been implicated in the regulation of sex differentiation, oogenesis and spermatogenesis. Expressed PDK1 and DSTPK61 phosphorylated Thr308 of PKB alpha only in the presence of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2. Overexpression of PDK1 in 293 cells activated PKB alpha and potentiated the IGF1-induced phosphorylation of PKB alpha at Thr308. Experiments in which the PH domains of either PDK1 or PKB alpha were deleted indicated that the binding of Ptdlns(3,4,5)P3 or Ptdlns(3,4)P2 to PKB alpha is required for phosphorylation and activation by PDK1. IGF1 stimulation of 293 cells did not affect the activity or phosphorylation of PDK1. CONCLUSIONS: PDK1 is likely to mediate the activation of PKB by insulin or growth factors. DSTPK61 is a Drosophila homologue of PDK1. The effect of Ptdlns(3,4,5)P3/Ptdlns(3,4)P2 in the activation of PKB alpha is at least partly substrate directed.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1997

Capillary electrophoresis assay for ubiquitin carboxyl-terminal hydrolases with chemically synthesized ubiquitin-valine as substrate.

Ubiquitin is expressed in eukaryotic cells as precursors, fused via its carboxyl terminus either to other ubiquitin sequences in linear polyubiquitin arrays or to specific ribosomal proteins. In some of the polyubiquitin fusions a single amino acid (e.g., valine in humans) is attached to the carboxyl terminus. These gene products are rapidly (probably cotranslationally) cleaved by ubiquitin carboxyl-terminal hydrolase (UCH) enzymes; therefore, although ubiquitin precursors are suitable substrates for assays of UCH activity, they are difficult to isolate from nucleated cells. While the recombinant approach allows the production of ubiquitin precursors in prokaryotic cells (which do not contain the ubiquitin system), proteins produced in this manner require purification and may also be susceptible to modification by bacterial enzymes, e.g., adventitious proteolysis. As an alternative we have chemically synthesized human ubiquitin-valine. In the assay described here the cleavage of ubiquitin-valine to ubiquitin (77 and 76 residue proteins, respectively) by a purified recombinant Drosophila UCH was monitored by capillary electrophoresis. Mass spectrometry verified the precise cleavage of ubiquitin-valine, confirming that this synthetic protein is a UCH substrate. Synthetic ubiquitin-valine may serve as a generic substrate for UCHs allowing the purification and identification of new members of this enzyme family.

Animals↗
PubMed1997

Mating and sex peptide stimulate the accumulation of yolk in oocytes of Drosophila melanogaster.

Mating elicits two reactions in many insect females: egg deposition is increased and receptivity to males is reduced. Central to the control of receptivity and oviposition in Drosophila melanogaster is the sex peptide (SP), a 36-amino-acid peptide sex pheromone synthesized in the male accessory glands and transferred to the female during copulation. To identify regulatory mechanisms involved in the maintenance of the oviposition response, we have compared the effects of mating and SP application with respect to oogenesis. The distribution of the various stages of oogenesis in the ovary, yolk protein (YP) synthesis by the fat body, as well as YP content, uptake and synthesis by the ovary were investigated. Transcripts of the yolk protein genes (yp) were quantified by Northern blotting. Based on our results, we conclude that mating and SP injection into virgin females stimulate yp gene transcription in the fat body only moderately above the background level. However, uptake into the ovary and transcription of the yp genes in the ovary is strongly enhanced after either mating or SP injection. These data are supported by the finding that the abundance of the vitellogenic stage 10 oocytes is also increased. In contrast, early vitellogenic stages 8 and 9 of oogenesis are present in the same numbers in virgin, mated, and SP-injected females, which suggests a control point at about stage 9 determining vitellogenic oocyte progression. The finding that SP can elicit equally all changes observed after copulation suggests that in the sexually mature female it is the major component controlling and stimulating oogenesis after mating.

Animals↗
PubMed1997

Cloning and characterization of three Musca domestica yolk protein genes.

The yolk protein (yp) genes encode the major nutritional polypeptides deposited in developing oocytes for subsequent utilization during embryogenesis, and represent a highly conserved family of genes in higher Diptera. Originally isolated from Drosophila melanogaster, they are expressed in a temporal-, tissue- and sex-specific manner in all species in which they have been identified. We report here the isolation of cDNAs encoding three independent yolk proteins from the common housefly, Musca domestica. Expression of the three M. domestica yp genes is analysed both by Northern and in-situ hybridization. We discuss in an evolutionary context both the significance of the expression patterns, and regions of apparent polypeptide sequence divergence.

Amino Acid Sequence↗
PubMed1997

Analysis of P[gal4] insertion lines of Drosophila melanogaster as a route to identifying genes important in the follicle cells during oogenesis.

We report the analysis of a number of lines of Drosophila melanogaster containing insertions of the yeast gal4 gene. By crossing a UAS-lacZ fusion gene as a reporter into these lines, we analysed the expression patterns of beta-galactosidase during oogenesis. Since there is no expression of GAL4 in the germ-line in these experiments, this is an ideal system for the analysis of expression patterns in sub-sets of follicle cells. These lines provide ideal markers for sets of follicle cells, e.g. anterior or posterior polar cells for studying genetic interactions in oogenesis; however, they can also be used in the same way as conventional enhancer traps to clone nearby genes with similar expression patterns. The advantages of this dual gal4/UAS system over conventional enhancer trapping includes the possibility of GAL4-directed misexpression and antisense expression studies to establish the function of the genes we identified during follicle cell determination and differentiation. These studies could lead to the isolation of homologous genes crucial in mammalian oogenesis. Understanding how the somatic cells and germ cells interact to promote growth and maturation of the mammalian follicle and oocyte could well be crucial for improving the fertility of eggs used for in-vitro fertilization programmes, and could provide methods for assessing the quality of eggs.

Animals↗
PubMed1997

Two signalling pathways specify localised expression of the Broad-Complex in Drosophila eggshell patterning and morphogenesis.

The Drosophila eggshell, which has a pair of chorionic appendages (dorsal appendages) located asymmetrically along both the anterior/posterior and dorsal/ventral axes, provides a good model to study signal instructed morphogenesis. We show that the Broad-Complex, a gene encoding zinc-finger transcription factors, is essential for the morphogenesis of dorsal appendages and is expressed in a bilaterally symmetrical pattern in the lateral-dorsal-anterior follicle cells during late oogenesis. This is induced and specified along the dorsoventral axis by an epidermal growth factor receptor signalling pathway, which includes a localised transforming growth factor-alpha like molecule, Gurken, in the oocyte and the Drosophila EGF receptor homologue, Torpedo, in the surrounding somatic follicle cells. Furthermore, the precisely localised expression of BR-C along the AP axis requires a separate signalling pathway, initiated by a transforming growth factor-beta homologue, Decapentaplegic, in nearby follicle cells. These two signalling pathways, one from the oocyte and the other from the follicle cells, co-ordinately specify patches of follicle cells to express the Broad-Complex in a unique position in respect to both major axes, which in turn directs the differentiation of the dorsal appendages in the correct position on the eggshell.

Animals↗
PubMed1996

20-Hydroxyecdysone, but not juvenile hormone, regulation of yolk protein gene expression can be mapped to cis-acting DNA sequences.

The three yolk protein genes (yps) of Drosophila melanogaster are expressed in the ovary and fat body of the adult female. Their levels of expression in the fat body depend upon both juvenile hormone (JH) and 20-hydroxyecdysone (20E). Using transformed lines of flies with various flanking sequences from the yp genes and lacZ, Adh, or native yp genes as reporters, the regulation of the three yp genes by 20E and the JH analogue ZR515 (methoprene) was investigated. For 20E, induction of reporter gene expression in males was assayed and, for JH, upregulation of the genes in nutritionally deprived females, which express yolk proteins (YPs) at very low levels, was followed. We were able to map 20E inducible sites upstream of yp3 and sites located 3' and within the coding sequence or introns of yp3 which can interact to respond to 20E. There are also sites in the intergenic spacer between yp1 and yp2. Evidence for repressors was also found upstream of the yp genes, suggesting downstream 20E inducible elements may be important in vivo. There appears to be a difference in the response to 20E in the fat body of the thorax and abdomen between different constructs in males. It is not clear whether those sequences which respond to 20E are genuine ecdysone response elements (i.e., binding sites for the ecdysone receptor) or if the effect is indirect. Methoprene upregulation of YPs, however, was only ever observed using native yp genes as reporters, suggesting that this hormone may act on intron sequences or yp coding sequences, or perhaps by influencing stability of the yp mRNA.

Animals↗
PubMed1995

The raspberry locus encodes Drosophila inosine monophosphate dehydrogenase.

Investigation of an enhancer-trap line exhibiting testis-specific beta-galactosidase expression led to the isolation of the Drosophila gene encoding inosine monophosphate dehydrogenase (IMPD), the rate-limiting enzyme in guanine nucleotide synthesis, which has been implicated in cell cycle control and malignant transformation. Northern and in situ hybridization analysis demonstrated that the gene has a complex expression pattern involving several independently regulated transcripts. Two ubiquitous, but highly ovary enriched, transcripts of 2.5 and 1.9 kb are expressed in the nurse cells and delivered to the oocyte, whilst a 0.9 kb transcript is found exclusively in the testis. The 2.5 kb transcript encodes a 58 kDa protein, which is highly similar in length and sequence to mouse and human IMPDs and is presumably required for GTP synthesis during early embryogenesis. Over-expression of this cDNA in Escherichia coli yielded a product of the predicted size, which was demonstrated to possess IMPD activity in a spectrophotometric assay. The coding capacity of the other transcripts is currently uncertain. We present evidence that IMPD is the product of the raspberry (ras) locus at 9E and the functions of the gene are discussed in relation to the phenotypes of ras mutants.

Amino Acid Sequence↗
PubMed1995

Nutritional response in a Drosophila yolk protein gene promoter.

The yolk protein genes (yps) of Drosophila melanogaster are only expressed in the ovary and fat body of female flies if they are supplied with proteinaceous food. This nutritional response is specific to the yp genes. We have used transgenic flies transformed with a series of constructs bearing deletions in the upstream region of the yp1 and yp2 genes attached to a reporter gene to search for DNA sequences responsible for the nutritional induction specific for yp1 and yp2 genes. Several regions were shown independently of each other to confer nutritional regulation on the expression of the yp1 and yp2 genes. This regulation can be induced both on the yp promoter and the heterologous Drosophila heat-shock 70 (hsp 70) promoter. The redundancy of sequences conferring a nutritional response on the yp genes is similar to that observed for the female specificity of these genes and suggest that several DNA binding proteins interact to provide the correct regulation of these genes. These results suggest that nutrition acts to modify the level of a trans-acting factor in the fat body. Northern blot analysis showed that the transcript levels from the dsx gene are not affected by nutrition, indicating that the response is not mediated via the dsx gene.

Alcohol Dehydrogenase↗