RNAi living-cell microarrays for loss-of-function screens in Drosophila melanogaster cells

2004 ◽  
Vol 1 (2) ◽  
pp. 127-132 ◽  
Author(s):  
Douglas B Wheeler ◽  
Steve N Bailey ◽  
David A Guertin ◽  
Anne E Carpenter ◽  
Caitlin O Higgins ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Living Cell ◽  
Loss Of Function ◽  
Cell Microarrays

scholarly journals Genome-scale RNAi on living-cell microarrays identifies novel regulators of Drosophila melanogaster TORC1-S6K pathway signaling

2011 ◽  
Vol 21 (3) ◽  
pp. 433-446 ◽  
Author(s):  
R. A. Lindquist ◽  
K. A. Ottina ◽  
D. B. Wheeler ◽  
P. P. Hsu ◽  
C. C. Thoreen ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Living Cell ◽  
Cell Microarrays ◽  
Genome Scale

scholarly journals Somatic production of reactive oxygen species does not predict its production in sperm cells across Drosophila melanogaster lines

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Biz R. Turnell ◽  
Luisa Kumpitsch ◽  
Anne-Cécile Ribou ◽  
Klaus Reinhardt
Keyword(s):  
Reactive Oxygen Species ◽  
Drosophila Melanogaster ◽  
Reactive Oxygen ◽  
Future Research ◽  
Ros Production ◽  
Oxygen Species ◽  
Loss Of Function ◽  
Wild Type ◽  
Ros Scavenging ◽  
Transport Chain

Abstract Objective Sperm ageing has major evolutionary implications but has received comparatively little attention. Ageing in sperm and other cells is driven largely by oxidative damage from reactive oxygen species (ROS) generated by the mitochondria. Rates of organismal ageing differ across species and are theorized to be linked to somatic ROS levels. However, it is unknown whether sperm ageing rates are correlated with organismal ageing rates. Here, we investigate this question by comparing sperm ROS production in four lines of Drosophila melanogaster that have previously been shown to differ in somatic mitochondrial ROS production, including two commonly used wild-type lines and two lines with genetic modifications standardly used in ageing research. Results Somatic ROS production was previously shown to be lower in wild-type Oregon-R than in wild-type Dahomey flies; decreased by the expression of alternative oxidase (AOX), a protein that shortens the electron transport chain; and increased by a loss-of-function mutation in dj-1β, a gene involved in ROS scavenging. Contrary to predictions, we found no differences among these four lines in the rate of sperm ROS production. We discuss the implications of our results, the limitations of our study, and possible directions for future research.

scholarly journals Trans-acting Factors Required for Inclusion of Regulated Exons in the Ultrabithorax mRNAs of Drosophila melanogaster

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1517-1529 ◽  
Author(s):  
James M Burnette ◽  
Allyson R Hatton ◽  
A Javier Lopez
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Splicing ◽  
P Element ◽  
Splicing Factors ◽  
Loss Of Function ◽  
Large Collection ◽  
Splicing Pattern ◽  
Alternatively Spliced ◽  
Hypomorphic Alleles

Abstract Alternatively spliced Ultrabithorax mRNAs differ by the presence of internal exons mI and mII. Two approaches were used to identify trans-acting factors required for inclusion of these cassette exons. First, mutations in a set of genes implicated in the control of other alternative splicing decisions were tested for dominant effects on the Ubx alternative splicing pattern. To identify additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested first for dominant enhancement of the haploinsufficient Ubx haltere phenotype and second for effects on the splicing pattern. Inclusion of the cassette exons in Ubx mRNAs was reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing. Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII. Possible roles of identified factors are discussed in the context of the resplicing strategy for generation of alternative Ubx mRNAs.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

scholarly journals Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila melanogaster.

1996 ◽  
Vol 16 (5) ◽  
pp. 1966-1977 ◽  
Author(s):  
T Dick ◽  
K Ray ◽  
H K Salz ◽  
W Chia
Keyword(s):  
Drosophila Melanogaster ◽  
Light Chain ◽  
Blot Analysis ◽  
Cytoplasmic Dynein ◽  
P Element ◽  
Female Sterility ◽  
Dynein Light Chain ◽  
Loss Of Function ◽  
Light Chain Gene

We report the molecular and genetic characterization of the cytoplasmic dynein light-chain gene, ddlc1, from Drosophila melanogaster. ddlc1 encodes the first cytoplasmic dynein light chain identified, and its genetic analysis represents the first in vivo characterization of cytoplasmic dynein function in higher eucaryotes. The ddlc1 gene maps to 4E1-2 and encodes an 89-amino-acid polypeptide with a high similarity to the axonemal 8-kDa outer-arm dynein light chain from Chlamydomonas flagella. Developmental Northern (RNA) blot analysis and ovary and embryo RNA in situ hybridizations indicate that the ddlc1 gene is expressed ubiquitously. Anti-DDLC1 antibody analyses show that the DDLC1 protein is localized in the cytoplasm. P-element-induced partial-loss-of-function mutations cause pleiotropic morphogenetic defects in bristle and wing development, as well as in oogenesis, and hence result in female sterility. The morphological abnormalities found in the ovaries are always associated with a loss of cellular shape and structure, as visualized by a disorganization of the actin cytoskeleton. Total-loss-of-function mutations cause lethality. A large proportion of mutant animals degenerate during embryogenesis, and the dying cells show morphological changes characteristic of apoptosis, namely, cell and nuclear condensation and fragmentation, as well as DNA degradation. Cloning of the human homolog of the ddlc1 gene, hdlc1, demonstrates that the dynein light-chain 1 is highly conserved in flies and humans. Northern blot analysis and epitope tagging show that the hdlc1 gene is ubiquitously expressed and that the human dynein light chain 1 is localized in the cytoplasm. hdlc1 maps to 14q24.

Genetic characterization of ms (3) K81, a paternal effect gene of Drosophila melanogaster.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 219-229 ◽  
Author(s):  
G K Yasuda ◽  
G Schubiger ◽  
B T Wakimoto
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Stages ◽  
Normal Function ◽  
Loss Of Function ◽  
Male Sterile ◽  
Specific Product ◽  
Paternal Effect ◽  
Developmental Arrest ◽  
Effect Gene

Abstract The vast majority of known male sterile mutants of Drosophila melanogaster fail to produce mature sperm or mate properly. The ms(3) K81(1) mutation is one of a rare class of male sterile mutations in which sterility is caused by developmental arrest after sperm entry into the egg. Previous studies showed that males homozygous for the K81(1) mutation produce progeny that arrest at either of two developmental stages. Most embryos arrest during early nuclear cycles, whereas the remainder are haploid embryos that arrest at a later stage. This description of the mutant phenotype was based on the analysis of a single allele isolated from a natural population. It was therefore unclear whether this unique paternal effect phenotype reflected the normal function of the gene. The genetic analysis and initial molecular characterization of five new K81 mutations are described here. Hemizygous conditions and heteroallelic combinations of the alleles were associated with male sterility caused by defects in embryogenesis. No other mutant phenotypes were observed. Thus, the K81 gene acted as a strict paternal effect gene. Moreover, the biphasic pattern of developmental arrest was common to all the alleles. These findings strongly suggested that the unusual embryonic phenotype caused by all five new alleles was due to loss of function of the K81+ gene. The K81 gene is therefore the first clear example of a strict paternal effect gene in Drosophila. Based on the embryonic lethal phenotypes, we suggest that the K81+ gene encodes a sperm-specific product that is essential for the male pronucleus to participate in the first few embryonic nuclear divisions.

DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster Abelson proto-oncogene homolog

1988 ◽  
Vol 8 (2) ◽  
pp. 843-853
Author(s):  
M J Henkemeyer ◽  
R L Bennett ◽  
F B Gertler ◽  
F M Hoffmann
Keyword(s):  
Drosophila Melanogaster ◽  
Tyrosine Kinase ◽  
Dna Sequence ◽  
Kinase Activity ◽  
Essential Gene ◽  
Sequence Similarity ◽  
Homologous Region ◽  
Loss Of Function ◽  
Tyrosine Kinase Activity ◽  
Stage Of Development

We report our molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type lb 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.

Sol–Gel-Derived Materials for Production of Pin-Printed Reporter Gene Living-Cell Microarrays

2013 ◽  
Vol 85 (24) ◽  
pp. 12108-12117 ◽  
Author(s):  
Xin Ge ◽  
Nikolas M. Eleftheriou ◽  
Si Amar Dahoumane ◽  
John D. Brennan
Keyword(s):  
Reporter Gene ◽  
Living Cell ◽  
Sol Gel ◽  
Cell Microarrays

scholarly journals Headcase is a Repressor of Lamellocyte Fate in Drosophila melanogaster

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 173 ◽  
Author(s):  
Gergely I. B. Varga ◽  
Gábor Csordás ◽  
Gyöngyi Cinege ◽  
Ferenc Jankovics ◽  
Rita Sinka ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cell Differentiation ◽  
Blood Cell ◽  
Cell Fate ◽  
Blood Cells ◽  
Mutational Analysis ◽  
Genetic Interaction ◽  
Lymph Gland ◽  
Loss Of Function ◽  
Hematopoietic Stem

Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.

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