A novel strategy for identifying mutations that sensitize Drosophila eye development to caffeine and hydroxyurea

This report describes a novel strategy for isolating Drosophila mutants with conditional eye phenotypes that should be generally applicable for identifying genes required for cellular responses to specific drugs. To test the strategy, we screened 3 of the 5 major chromosome arms for hydroxyurea- and (or) caffeine-sensitive (huc) mutants, and isolated mutations affecting 5 different complementation groups. Most of these were represented by single alleles; however, we also isolated multiple alleles of huc29DE gene, an essential gene that is also associated with a nonconditional pupal lethal phenotype. We also identified huc95E mutants, which are extremely sensitive to caffeine. Although huc95E is a nonessential gene, mutant imaginal disc cells undergo caffeine-dependent apoptosis, and huc95E gene function is required for the viability of the organism when mutant larvae are exposed to levels of caffeine that controls can easily tolerate. We have mapped the cytological positions of huc29D and huc95E as a first step toward molecularly characterizing the relevant genes.

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Polar Effects of Transposon Insertion into a Minimal Bacterial Genome

Journal of Bacteriology ◽

10.1128/jb.00185-19 ◽

2019 ◽

Vol 201 (19) ◽

Cited By ~ 3

Author(s):

Clyde A. Hutchison ◽

Chuck Merryman ◽

Lijie Sun ◽

Nacyra Assad-Garcia ◽

R. Alexander Richter ◽

...

Keyword(s):

Gene Function ◽

Essential Gene ◽

Global Analysis ◽

Bacterial Genome ◽

Transposon Mutagenesis ◽

Protein Coding ◽

Transposon Insertion ◽

Nonessential Gene ◽

Intergenic Regions

ABSTRACT Global transposon mutagenesis is a valuable tool for identifying genes required for cell viability. Here we present a global analysis of the orientation of viable Tn5-Puror (Tn5-puromycin resistance) insertions into the near-minimal bacterial genome of JCVI-syn2.0. Sixteen of the 478 protein-coding genes show a noticeable asymmetry in the orientation of disrupting insertions of Tn5-Puror. Ten of these are located in operons, upstream of essential or quasi-essential genes. Inserts transcribed in the same direction as the downstream gene are favored, permitting read-through transcription of the essential or quasi-essential gene. Some of these genes were classified as quasi-essential solely because of polar effects on the expression of downstream genes. Three genes showing asymmetry in Tn5-Puror insertion orientation prefer the orientation that avoids collisions between read-through transcription of Tn5-Puror and transcription of an adjacent gene. One gene (JCVISYN2_0132 [abbreviated here as “_0132”]) shows a strong preference for Tn5-Puror insertions transcribed upstream, away from the downstream nonessential gene _0133. This suggested that expression of _0133 due to read-through from Tn5-Puror is lethal when _0132 function is disrupted by transposon insertion. This led to the identification of genes _0133 and _0132 as a toxin-antitoxin pair. The three remaining genes show read-through transcription of Tn5-Puror directed downstream and away from sizable upstream intergenic regions (199 bp to 363 bp), for unknown reasons. In summary, polar effects of transposon insertion can, in a few cases, affect the classification of genes as essential, quasi-essential, or nonessential and sometimes can give clues to gene function. IMPORTANCE In studies of the minimal genetic requirements for life, we used global transposon mutagenesis to identify genes needed for a minimal bacterial genome. Transposon insertion can disrupt the function of a gene but can also have polar effects on the expression of adjacent genes. In the Tn5-Puror construct used in our studies, read-through transcription from Tn5-Puror can drive expression of downstream genes. This results in a preference for Tn5-Puror insertions transcribed toward a downstream essential or quasi-essential gene within the same operon. Such polar effects can have an impact on the classification of genes as essential, quasi-essential, or nonessential, but this has been observed in only a few cases. Also, polar effects of Tn5-Puror insertion can sometimes give clues to gene function.

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A Yeast taf17 Mutant Requires the Swi6 Transcriptional Activator for Viability and Shows Defects in Cell Cycle-Regulated Transcription

Genetics ◽

10.1093/genetics/154.4.1561 ◽

2000 ◽

Vol 154 (4) ◽

pp. 1561-1576

Author(s):

Neil Macpherson ◽

Vivien Measday ◽

Lynda Moore ◽

Brenda Andrews

Keyword(s):

Cell Cycle ◽

Transcriptional Activation ◽

Essential Gene ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Synthetic Lethal ◽

Cycle Arrest ◽

A Cell ◽

Allelism Tests ◽

Complementation Groups

Abstract In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle. Although SBF is required for cell viability, SWI6 is not an essential gene. We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10. We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Δ and slm8-1 swi6Δ double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6. Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes. Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex. Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region. The cell cycle and transcriptional defects caused by taf17slm7-1 are consistent with the role of TAFIIs as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

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High temperature-induced changes in morphology in imaginal disc cells of a temperature sensitive lethal mutant of Drosophila melanogaster

Hereditas ◽

10.1111/j.1601-5223.1980.tb01058.x ◽

2009 ◽

Vol 93 (1) ◽

pp. 169-176 ◽

Cited By ~ 4

Author(s):

EVA FEKETE ◽

ANDREW LAMBERTSSON

Keyword(s):

Drosophila Melanogaster ◽

High Temperature ◽

Imaginal Disc ◽

Temperature Sensitive ◽

Lethal Mutant ◽

Disc Cells ◽

Induced Changes

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Expanded: a gene involved in the control of cell proliferation in imaginal discs

Development ◽

10.1242/dev.118.4.1291 ◽

1993 ◽

Vol 118 (4) ◽

pp. 1291-1301 ◽

Cited By ~ 15

Author(s):

M. Boedigheimer ◽

A. Laughon

Keyword(s):

Cell Proliferation ◽

Protein Interactions ◽

Essential Gene ◽

Spontaneous Mutation ◽

Growth Control ◽

Imaginal Discs ◽

Apical Surface ◽

Protein Protein Interactions ◽

Acid Protein ◽

Disc Cells

The expanded gene was first identified by a spontaneous mutation that causes broad wings. We have identified an enhancer-trap insertion within expanded and used it to generate additional mutations, including one null allele. expanded is an essential gene, necessary for proper growth control of imaginal discs and, when mutant, causes either hyperplasia or degeneration depending on the disc. Wing overgrowth in expanded hypermorphs is limited to specific regions along the anterior-posterior and dorsal-ventral axis. expanded encodes a novel 1429 amino acid protein that is localized to the apical surface of disc cells and contains three potential SH3-binding sites. Together, these observations suggest that the Expanded protein engages in protein-protein interactions regulating cell proliferation in discs.

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A Transient Cell Cycle Shift in Drosophila Imaginal Disc Cells Precedes Multipotency

Cell ◽

10.1016/j.cell.2004.12.008 ◽

2005 ◽

Vol 120 (3) ◽

pp. 383-393 ◽

Cited By ~ 58

Author(s):

Anne Sustar ◽

Gerold Schubiger

Keyword(s):

Cell Cycle ◽

Imaginal Disc ◽

Disc Cells

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The tumor suppressor gene, lethal(2)giant larvae (1(2)g1), is required for cell shape change of epithelial cells during Drosophila development

Development ◽

10.1242/dev.122.7.2283 ◽

1996 ◽

Vol 122 (7) ◽

pp. 2283-2294 ◽

Cited By ~ 7

Author(s):

P. Manfruelli ◽

N. Arquier ◽

W.P. Hanratty ◽

M. Semeriva

Keyword(s):

Epithelial Cells ◽

Cell Shape ◽

Imaginal Disc ◽

Biochemical Characterization ◽

Shape Change ◽

Follicle Cells ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Egg Chambers ◽

Disc Cells

Inactivation of the lethal(2)giant larvae (l(2)gl) gene results in malignant transformation of imaginal disc cells and neuroblasts of the larval brain in Drosophila. Subcellular localization of the l(2)gl gene product, P127, and its biochemical characterization have indicated that it participates in the formation of the cytoskeletal network. In this paper, genetic and phenotypic analyses of a temperature-sensitive mutation (l(2)glts3) that behaves as a hypomorphic allele at restrictive temperature are presented. In experimentally overaged larvae obtained by using mutants in the production of ecdysone, the l(2)glts3 mutation displays a tumorous potential. This temperature-sensitive allele of the l(2)gl gene has been used to describe the primary function of the gene before tumor progression. A reduced contribution of both maternal and zygotic activities in l(2)glts3 homozygous mutant embryos blocks embryogenesis at the end of germ-band retraction. The mutant embryos are consequently affected in dorsal closure and head involution and show a hypertrophy of the midgut. These phenotypes are accompanied by an arrest of the cell shape changes normally occurring in lateral epidermis and in epithelial midgut cells. l(2)gl activity is also necessary for larval fife and the critical period falls within the third instar larval stage. Finally, l(2)gl activity is required during oogenesis and mutations in the gene disorganize egg chambers and cause abnormalities in the shape of follicle cells, which are eventually internalized within the egg chamber. These results together with the tumoral phenotype of epithelial imaginal disc cells strongly suggest that the l(2)gl product is required in vivo in different types of epithelial cells to control their shape during development.

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Development and differentiation in vitro of Drosophila imaginal disc cells from dissociated early embryos

Development ◽

10.1242/dev.33.2.487 ◽

1975 ◽

Vol 33 (2) ◽

pp. 487-498

Author(s):

Andreas Dübendorfer ◽

Glen Shields ◽

James H. Sang

Keyword(s):

Imaginal Disc ◽

Larval Instar ◽

Cell Types ◽

The Third ◽

Early Embryos ◽

Disc Cells ◽

Development And Differentiation ◽

Pattern Specification

Embryos of Drosophila melanogaster, 6–8 h after oviposition, were dissociated and the cells cultured in vitro. Besides larval cell types, imaginal disc cells, assembled and growing in bloated monolayered vesicles, were obtained. The cells of these vesicles become competent to differentiate adult structures when treated with α-ecdysone or ecdysterone in vitro. Recognizable patterns of the adult fly are not formed though. If metamorphosis of imaginal cell vesicles from in vitro-cultures is induced in vivo by transplantation into host larvae of various ages within the third larval instar, recognizable patterns can differentiate provided the host larva does not metamorphose prior to 2 days after transplantation. The frequency of specific patterns in the implants can be increased by providing 9 days of culture in vivo (adult host flies) before metamorphosis. Passage through the third larval instar is not essential for these cells to produce identifiable patterns since culture in adult flies alone can achieve this. The quality of the differentiated pattern is not correlated with the extent of cell proliferation in the cultured tissues. The problem of pattern specification in vitro and in vivo is discussed.

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A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

mSphere ◽

10.1128/msphere.00086-20 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Michelle Spoto ◽

Changhui Guan ◽

Elizabeth Fleming ◽

Julia Oh

Keyword(s):

Gene Function ◽

Large Scale ◽

Essential Gene ◽

Bacterial Species ◽

Bacterial Genome ◽

Model Organisms ◽

Design Parameters ◽

Bacterial Genomes ◽

Wide Range ◽

User Friendly

ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

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The Singularity of the Drosophila Male Germ Cell Centriole: The Asymmetric Distribution of Sas4 and Sas6

Cells ◽

10.3390/cells9010115 ◽

2020 ◽

Vol 9 (1) ◽

pp. 115 ◽

Cited By ~ 2

Author(s):

Veronica Persico ◽

Massimo Migliorini ◽

Giuliano Callaini ◽

Maria Giovanna Riparbelli

Keyword(s):

Germ Cell ◽

Fluorescence Intensity ◽

Imaginal Disc ◽

Structural Features ◽

Asymmetric Distribution ◽

Cell Type ◽

Immunofluorescence Analysis ◽

Mother And Daughter ◽

Disc Cells ◽

A Cell

Drosophila spermatocytes have giant centrioles that display unique properties. Both the parent centrioles maintain a distinct cartwheel and nucleate a cilium-like region that persists during the meiotic divisions and organizes a structured sperm axoneme. Moreover, the parent centrioles are morphologically undistinguishable, unlike vertebrate cells in which mother and daughter centrioles have distinct structural features. However, our immunofluorescence analysis of the parent centrioles in mature primary spermatocytes revealed an asymmetric accumulation of the typical Sas4 and Sas6 proteins. Notably, the fluorescence intensity of Sas4 and Sas6 at the daughter centrioles is greater than the intensity found at the mother ones. In contrast, the centrioles of wing imaginal disc cells display an opposite condition in which the loading of Sas4 and Sas6 at the mother centrioles is greater. These data underlie a subtle asymmetry among the parent centrioles and point to a cell type diversity of the localization of the Sas4 and Sas6 proteins.

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