scholarly journals Regulation of the Vitellogenin Receptor during Drosophila melanogaster Oogenesis

2000 ◽  
Vol 11 (2) ◽  
pp. 511-521 ◽  
Author(s):  
Christopher P. Schonbaum ◽  
John J. Perrino ◽  
Anthony P. Mahowald
Keyword(s):  
Germ Line ◽  
Early Stage ◽  
Environmental Cues ◽  
Multivesicular Bodies ◽  
Wild Type ◽  
Yolk Proteins ◽  
Vitellogenin Receptor ◽  
Protein Receptor ◽  
Rna In Situ Hybridization

In many insects, development of the oocyte arrests temporarily just before vitellogenesis, the period when vitellogenins (yolk proteins) accumulate in the oocyte. Following hormonal and environmental cues, development of the oocyte resumes, and endocytosis of vitellogenins begins. An essential component of yolk uptake is the vitellogenin receptor. In this report, we describe the ovarian expression pattern and subcellular localization of the mRNA and protein encoded by theDrosophila melanogaster vitellogenin receptor geneyolkless (yl). yl RNA and protein are both expressed very early during the development of the oocyte, long before vitellogenesis begins. RNA in situ hybridization and lacZ reporter analyses show that ylRNA is synthesized by the germ line nurse cells and then transported to the oocyte. Yl protein is evenly distributed throughout the oocyte during the previtellogenic stages of oogenesis, demonstrating that the failure to take up yolk in these early stage oocyte is not due to the absence of the receptor. The transition to the vitellogenic stages is marked by the accumulation of yolk via clathrin-coated vesicles. After this transition, yolk protein receptor levels increase markedly at the cortex of the egg. Consistent with its role in yolk uptake, immunogold labeling of the receptor reveals Yl in endocytic structures at the cortex of wild-type vitellogenic oocytes. In addition, shortly after the inception of yolk uptake, we find multivesicular bodies where the yolk and receptor are distinctly partitioned. By the end of vitellogenesis, the receptor localizes predominantly to the cortex of the oocyte. However, during oogenesis in yl mutants that express full-length protein yet fail to incorporate yolk proteins, the receptor remains evenly distributed throughout the oocyte.

The sisterless-b function of the Drosophila gene scute is restricted to the stage when the X:A ratio determines the activity of Sex-lethal

Development ◽  
1991 ◽  
Vol 113 (2) ◽  
pp. 715-722 ◽  
Author(s):  
M. Torres ◽  
L. Sanchez
Keyword(s):  
In Situ Hybridization ◽  
Heat Shock ◽  
Dosage Compensation ◽  
Early Stage ◽  
Dual Function ◽  
Transcriptional Level ◽  
Wild Type ◽  
Blastoderm Stage ◽  
State Of Activity

The gene scute (sc) has a dual function: the scute function which is involved in neurogenesis and the sisterless-b function which is involved in generating the X:A signal that determines the state of activity of Sxl, a gene that controls sex determination and dosage compensation. We show here that the lethal phase of sc- females is embryonic and caused by the lack of Sxl function. We also analyze the time in development when sc and Sxl interact by means of (a) determining the thermosensitive phase (TSP) of the interaction between Sxl and sc and (b) a chimeric gene in which sc is under the control of a heat-shock promoter (HSSC-3). Pulses of sc expression from the HSSC-3 activate Sxl only at a very specific and early stage in development, which coincides with the TSP of the interaction between sc and Sxl. It corresponds to the syncytial blastoderm stage and coincides with the time when the X:A signal regulates Sxl. At this stage sc undergoes a homogeneous transient expression in wild-type flies. We conclude that the sc expression at the syncytial blastoderm is responsible for its sisterless-b function. Since sc expression from the HSSC-3 fully suppresses the sisterless-b phenotype, we further conclude that the sisterless-b function is exclusively provided by the sc protein. Finally, we have analyzed, by in situ hybridization, the effect of sc and sis-a mutations on the embryonic transcription of Sxl. Our results support the view that the control of Sxl by the X:A signal occurs at the transcriptional level.

An extensive 3′ cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-beta family in Drosophila

Development ◽  
1991 ◽  
Vol 111 (3) ◽  
pp. 657-666 ◽  
Author(s):  
R.K. Blackman ◽  
M. Sanicola ◽  
L.A. Raftery ◽  
T. Gillevet ◽  
W.M. Gelbart
Keyword(s):  
Germ Line ◽  
Regulatory Region ◽  
Imaginal Disk ◽  
Tgf Beta ◽  
Signalling Molecule ◽  
Rna In Situ Hybridization ◽  
Imaginal Disks ◽  
Beta Galactosidase

The decapentaplegic (dpp) gene in Drosophila melanogaster encodes a TGF-beta-like signalling molecule that is expressed in a complex and changing pattern during development. One of dpp's contributions is to proximal-distal outgrowth of the adult appendages, structures derived from the larval imaginal disks. Appendage specific mutations of dpp fall in a 20 kb interval 3′ to the known dpp transcripts. Here, we directly test the hypothesis that these mutations define an extended 3′ cis-regulatory region. By analysis of germ-line transformants expressing a reporter gene, we show that sequences from this portion of the gene, termed the dppdisk region, are capable of directing expression comparable to that defined by RNA in situ hybridization. We localize two intervals of the dppdisk region that appear to account for much of the dpp spatial pattern in imaginal disks and discuss the positions of these important elements in terms of the genetics of dpp. Finally, we provide evidence to suggest that one of our constructs expresses beta-galactosidase in the early imaginal disk primordia in the embryo, at approximately the time when they are set aside from surrounding larval epidermal tissues. Thus, dpp may be involved directly in the determination of the imaginal disks.

scholarly journals Genetic and molecular analyses of vgal: a spontaneous and unstable mutation at the vestigial locus in Drosophila melanogaster

1991 ◽  
Vol 57 (3) ◽  
pp. 235-243 ◽  
Author(s):  
Claude Bazin ◽  
Françoise Lemeunier ◽  
Georges Periquet ◽  
Joël Silber
Keyword(s):  
Drosophila Melanogaster ◽  
Low Temperature ◽  
Germ Line ◽  
The Other ◽  
Wild Type ◽  
Male Germ Line ◽  
Molecular Analyses ◽  
Dna Insertion ◽  
Unstable Mutation

SummaryWe describe herein, a new unstable mutant of the vestigial locus, isolated from a French natural population. From this mutant vestigialalmost (vgal) wild-type flies (vgal+) and extreme vg phenotypes (vge) arose spontaneously without genomic shock. The occurrence of vgal+ or vge alleles depends mostly on the breeding temperature; vgal+ revertants arose principally at low temperature (21 °C) and vge at 28 °C. These events occur mainly in the male germ line and the phenomenon appears to be premeiotic. Our results with in situ hybridization experiments and Southern blots show that the vgal mutation is due to a 2 kb DNA insertion, which is a deleted hobo element. Genetic and molecular analyses show that two distinct events may underly the wild-type revertants. One is the excision of the resident hobo element, the other a further deletion (about 300 bp in the example characterized herein). The vge mutation is probably due to a deletion of vestigial sequences flanking the hobo insertion.

scholarly journals Disruption of Brachypodium Lichenase Alters Metabolism of Mixed-linkage Glucan and Starch

2021 ◽  
Author(s):  
Mingzhu Fan ◽  
Jacob Krüger Jensen ◽  
Starla Zemelis-Durfee ◽  
Sang-Jin Kim ◽  
Jia-Yi Chan ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Brachypodium Distachyon ◽  
Fold Increase ◽  
Wild Type ◽  
Gene Encoding ◽  
Vegetative Tissues ◽  
Hybridization Data ◽  
Rna In Situ Hybridization ◽  
Germinating Seeds

Mixed-linkage glucan (MLG), which is widely distributed in grasses, is a polysaccharide highly abundant in cell walls of grass endosperm and young vegetative tissues. Lichenases are enzymes that hydrolyze MLG first identified in MLG-rich lichens. In this study, we identify a gene encoding a lichenase we name Brachypodium distachyon LICHENASE 1 (BdLCH1), which is highly expressed in the endosperm of germinating seeds and coleoptiles and at lower amounts in mature shoots. RNA in situ hybridization showed that BdLCH1 is primarily expressed in chlorenchyma cells of mature leaves and internodes. Disruption of BdLCH1 resulted in an eight-fold increase in MLG content in senesced leaves. Consistent with the in situ hybridization data, immunolocalization results showed that MLG was not removed in chlorenchyma cells of lch1 mutants as it was in wild type and implicate the BdLCH1 enzyme in removing MLG in chlorenchyma cells in mature vegetative tissues. We also show that MLG accumulation in lch1 mutants was resistant to dark induced degradation, and eight-week-old lch1 plants showed a faster rate of starch breakdown than wild type in darkness. Our results suggest a role for BdLCH1 in modifying the cell wall to support highly metabolically active cells.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

scholarly journals Characterization of neuroendocrine tumors in heterozygous mutant MENX rats: a novel model of invasive medullary thyroid carcinoma

2018 ◽  
Vol 25 (2) ◽  
pp. 145-162 ◽  
Author(s):  
Sara Molatore ◽  
Andrea Kügler ◽  
Martin Irmler ◽  
Tobias Wiedemann ◽  
Frauke Neff ◽  
...  
Keyword(s):  
Neuroendocrine Tumors ◽  
Early Stage ◽  
Pituitary Tumors ◽  
Transcriptome Profiling ◽  
Thyroid Neoplasms ◽  
Recessive Trait ◽  
Wild Type ◽  
Incurable Cancer ◽  
Medullary Thyroid ◽  
Adrenal Pheochromocytoma

Rats affected by the MENX syndrome spontaneously develop multiple neuroendocrine tumors (NETs) including adrenal, pituitary and thyroid gland neoplasms. MENX was initially reported to be inherited as a recessive trait and affected rats were found to be homozygous for the predisposingCdkn1bmutation encoding p27. We here report that heterozygous MENX-mutant rats (p27+/mut) develop the same spectrum of NETs seen in the homozygous (p27mut/mut) animals but with slower progression. Consequently, p27+/mut rats have a significantly shorter lifespan compared with their wild-type (p27+/+) littermates. In the tumors of p27+/mut rats, the wild-typeCdkn1ballele is neither lost nor silenced, implying that p27 is haploinsufficient for tumor suppression in this model. Transcriptome profiling of rat adrenal (pheochromocytoma) and pituitary tumors having different p27 dosages revealed a tissue-specific, dose-dependent effect of p27 on gene expression. In p27+/mut rats, thyroid neoplasms progress to invasive and metastatic medullary thyroid carcinomas (MTCs) accompanied by increased calcitonin levels, as in humans. Comparison of expression signatures of late-stage vs early-stage MTCs from p27+/mut rats identified genes potentially involved in tumor aggressiveness. The expression of a subset of these genes was evaluated in human MTCs and found to be associated with aggressive RET-M918T-positive tumors. Altogether, p27 haploinsufficiency in MENX rats uncovered a novel, representative model of invasive and metastatic MTC exploitable for translational studies of this often aggressive and incurable cancer.

scholarly journals SNARE phosphorylation: a control mechanism for insulin-stimulated glucose transport and other regulated exocytic events

2017 ◽  
Vol 45 (6) ◽  
pp. 1271-1277 ◽  
Author(s):  
Kamilla M.E. Laidlaw ◽  
Rachel Livingstone ◽  
Mohammed Al-Tobi ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Membrane Fusion ◽  
Molecular Mechanisms ◽  
Membrane Receptors ◽  
Multivesicular Bodies ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Plasma Membrane Receptors ◽  
Regulated Trafficking ◽  
Receptor Family

Trafficking within eukaryotic cells is a complex and highly regulated process; events such as recycling of plasma membrane receptors, formation of multivesicular bodies, regulated release of hormones and delivery of proteins to membranes all require directionality and specificity. The underpinning processes, including cargo selection, membrane fusion, trafficking flow and timing, are controlled by a variety of molecular mechanisms and engage multiple families of lipids and proteins. Here, we will focus on control of trafficking processes via the action of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of proteins, in particular their regulation by phosphorylation. We will describe how these proteins are controlled in a range of regulated trafficking events, with particular emphasis on the insulin-stimulated delivery of glucose transporters to the surface of adipose and muscle cells. Here, we focus on a few examples of SNARE phosphorylation which exemplify distinct ways in which SNARE machinery phosphorylation may regulate membrane fusion.

Endothelial dysfunction and arterial pressure regulation during early diabetes in mice: roles for nitric oxide and endothelium-derived hyperpolarizing factor

2007 ◽  
Vol 293 (2) ◽  
pp. R707-R713 ◽  
Author(s):  
Sharyn M. Fitzgerald ◽  
Barbara K. Kemp-Harper ◽  
Helena C. Parkington ◽  
Geoffrey A. Head ◽  
Roger G. Evans
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Arterial Pressure ◽  
Early Stage ◽  
Mesenteric Arteries ◽  
No Production ◽  
Wild Type ◽  
Early Diabetes ◽  
Diabetes In Mice ◽  
Vehicle Treatment

We determined whether nitric oxide (NO) counters the development of hypertension at the onset of diabetes in mice, whether this is dependent on endothelial NO synthase (eNOS), and whether non-NO endothelium-dependent vasodilator mechanisms are altered in diabetes in mice. Male mice were instrumented for chronic measurement of mean arterial pressure (MAP). In wild-type mice, MAP was greater after 5 wk of Nω-nitro-l-arginine methyl ester (l-NAME; 100 mg·kg−1·day−1 in drinking water; 97 ± 3 mmHg) than after vehicle treatment (88 ± 3 mmHg). MAP was also elevated in eNOS null mice (113 ± 4 mmHg). Seven days after streptozotocin treatment (200 mg/kg iv) MAP was further increased in l-NAME-treated mice (108 ± 5 mmHg) but not in vehicle-treated mice (88 ± 3 mmHg) nor eNOS null mice (104 ± 3 mmHg). In wild-type mice, maximal vasorelaxation of mesenteric arteries to acetylcholine was not altered by chronic l-NAME or induction of diabetes but was reduced by 42 ± 6% in l-NAME-treated diabetic mice. Furthermore, the relative roles of NO and endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced vasorelaxation were altered; the EDHF component was enhanced by l-NAME and blunted by diabetes. These data suggest that NO protects against the development of hypertension during early-stage diabetes in mice, even in the absence of eNOS. Furthermore, in mesenteric arteries, diabetes is associated with reduced EDHF function, with an apparent compensatory increase in NO function. Thus, prior inhibition of NOS results in endothelial dysfunction in early diabetes, since the diabetes-induced reduction in EDHF function cannot be compensated by increases in NO production.

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