Gene Deletions by Ends-In Targeting in Drosophila melanogaster

Xie, Heng; Golic, Kent G.

doi:10.1534/genetics.104.030882

Cited by 50 publications

(48 citation statements)

References 23 publications

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“…Extant p53 loss-of function alleles cannot be used to address this question because they disrupt multiple isoforms (Figure 1a). [43][44][45] We therefore began with an overexpression approach, using the GAL4/ UAS system to evaluate in parallel the ability of the A, B, E, and D protein isoforms to induce apoptosis when overexpressed in a variety of tissues. To construct these transgenes, PCR products that spanned the start to stop codons in the cDNAs were tagged on the N-terminus with six Myc epitope tags, fused to the UAS promoter, and inserted into flies at the same attP genomic docking site using phiC31 transformation.…”

Section: Resultsmentioning

confidence: 99%

The function of Drosophila p53 isoforms in apoptosis

et al. 2015

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The p53 protein is a major mediator of the cellular response to genotoxic stress and is a crucial suppressor of tumor formation. In a variety of organisms, p53 and its paralogs, p63 and p73, each encode multiple protein isoforms through alternative splicing, promoters, and translation start sites. The function of these isoforms in development and disease are still being defined. Here, we evaluate the apoptotic potential of multiple isoforms of the single p53 gene in the genetic model Drosophila melanogaster. Most previous studies have focused on the p53A isoform, but it has been recently shown that a larger p53B isoform can induce apoptosis when overexpressed. It has remained unclear, however, whether one or both isoforms are required for the apoptotic response to genotoxic stress. We show that p53B is a much more potent inducer of apoptosis than p53A when overexpressed. Overexpression of two newly identified short isoforms perturbed development and inhibited the apoptotic response to ionizing radiation. Analysis of physiological protein expression indicated that p53A is the most abundant isoform, and that both p53A and p53B can form a complex and co-localize to sub-nuclear compartments. In contrast to the overexpression results, new isoformspecific loss-of-function mutants indicated that it is the shorter p53A isoform, not full-length p53B, that is the primary mediator of pro-apoptotic gene transcription and apoptosis after ionizing radiation. Together, our data show that it is the shorter p53A isoform that mediates the apoptotic response to DNA damage, and further suggest that p53B and shorter isoforms have specialized functions.

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Section: Resultsmentioning

confidence: 99%

The function of Drosophila p53 isoforms in apoptosis

et al. 2015

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“…Southern blot analysis was used to confirm the expected targeted tandem duplication for 5 of the 14 lines (data not shown), 1 of which was used for the reduction step. A reduction step, as in Xie and Golic (2004), was then executed to leave a single copy of the Acp62F gene ( Figure 1B). One of the 14 third chromosome targeting events was used to screen for recombination events within the flanking regions of wildtype Acp62F and the adjacent Acp62F del .…”

Section: Methodsmentioning

confidence: 99%

“…One of the 14 third chromosome targeting events was used to screen for recombination events within the flanking regions of wildtype Acp62F and the adjacent Acp62F del . Males with the targeted allele were crossed to females carrying a hsp70-CreI transgene (Xie and Golic 2004) and heat-shocked to induce Cre-mediated recombination between flanking regions of the Acp62F tandem copies ( Figure 1C). Four reduction events leaving only the deleted copy of Acp62F were identified (Acp62F ), which are expected to be equivalent in their removal of Acp62F.…”

Section: Methodsmentioning

confidence: 99%

“…A two-step gene-targeting approach (Xie and Golic 2004) was used here to generate a precise deletion of the Acp62F coding region, which represents the definitive null allele needed for phenotypic characterization. Flies lacking Acp62F were tested for roles in receptivity to mating, egg laying, fertility, proteolytic processing, sperm competition, and the cost of mating.…”

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confidence: 99%

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Targeted Gene Deletion and Phenotypic Analysis of the Drosophila melanogaster Seminal Fluid Protease Inhibitor Acp62F

et al. 2008

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Internally fertilizing organisms transfer a complex assortment of seminal fluid proteins, a substantial fraction of which are proteolysis regulators. In mammals, some seminal protease inhibitors have been implicated in male infertility and these same molecular classes of protease inhibitors are also found in Drosophila seminal fluid. Here, we tested the reproductive functions of the Drosophila melanogaster seminal fluid protease inhibitor Acp62F by generating a precise deletion of the Acp62F gene. We did not detect a nonredundant function for Acp62F in modulating the egg laying, fertility, remating frequency, or life span of mated females. However, loss of Acp62F did alter a male's defensive sperm competitive ability, consistent with the localization of Acp62F to sperm storage organs. In addition, the processing of at least one seminal protein, the ovulation hormone ovulin, is slower in the absence of Acp62F.

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“…The wIw reporter construct has been described (Rong and Golic 2003 (Rong and Golic 2003) and used in combination with [wIw]2 in the homozygous assays. An X chromosome hsp70-driven FLP line (70FLP3F) has been described previously (Golic et al 1997 (Ghabrial et al 1998);sir2 2A-7-11 from Kent Golic at the University of Utah (Xie and Golic 2004); and mus309 D2 and mus309 D3 from Jeff Sekelsky at the University of North Carolina (Adams et al 2003). For deletion mapping of ssar, deficiencies from Bloomington's ''Deficiency Kit'' were used that cover the region from 85 to 91.…”

Section: Methodsmentioning

confidence: 99%

A Genetic Screen For DNA Double-Strand Break Repair Mutations in Drosophila

Wei

Rong

2007

Genetics

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The study of DNA double-strand break (DSB) repair has been greatly facilitated by the use of rarecutting endonucleases, which induce a break precisely at their cut sites that can be strategically placed in the genome. We previously established such a system in Drosophila and showed that the yeast I-SceI enzyme cuts efficiently in Drosophila cells and those breaks are effectively repaired by conserved mechanisms. In this study, we determined the genetic requirements for the repair of this I-SceI-induced DSB in the germline. We show that Drosophila Rad51 and Rad54 are both required for homologous repair by gene conversion, but are dispensable for single-strand annealing repair. We provided evidence suggesting that Rad51 is more stringently required than Rad54 for intersister gene conversion. We uncovered a significant role of DNA ligase IV in nonhomologous end joining. We conducted a screen for candidate mutations affecting DSB repair and discovered novel mutations in genes that include mutagen sensitive 206, single-strand annealing reducer, and others. In addition, we demonstrated an intricate balance among different repair pathways in which the cell differentially utilizes repair mechanisms in response to both changes in the genomic environment surrounding the break and deficiencies in one or the other repair pathways.

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Gene Deletions by Ends-In Targeting in Drosophila melanogaster

Cited by 50 publications

References 23 publications

The function of Drosophila p53 isoforms in apoptosis

The function of Drosophila p53 isoforms in apoptosis

Targeted Gene Deletion and Phenotypic Analysis of the Drosophila melanogaster Seminal Fluid Protease Inhibitor Acp62F

A Genetic Screen For DNA Double-Strand Break Repair Mutations in Drosophila

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