<i>Dictyostelium discoideum</i> plasmid containing an AP‐endonuclease upstream sequence: Bleomycin induction of a luciferase reporter

Ling, Angela Z.; Guyer, Robert B.; Deering, R. A.

doi:10.1002/em.1078

Cited by 3 publications

(1 citation statement)

References 21 publications

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“…An A module of TRE5-A.1 was amplified by PCR from plasmid pB3 (18) and inserted into pGEM-T (Promega). A firefly luciferase gene, including the downstream cotB (sp70) terminator, was isolated as a HindIII/SpeI fragment from plasmid pVTL-AL (12) and then ligated into the primer-derived HindIII site downstream of the A module and the vector-derived SpeI site of pGEM-T. The resulting vector was named pGEM-A-luc.…”

Section: In Vivo Retrotransposition Assay (Tre Trap Assaymentioning

confidence: 99%

The C-Module-Binding Factor Supports Amplification of TRE5-A Retrotransposons in the Dictyostelium discoideum Genome

et al. 2011

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Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery.

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Section: In Vivo Retrotransposition Assay (Tre Trap Assaymentioning

confidence: 99%

The C-Module-Binding Factor Supports Amplification of TRE5-A Retrotransposons in the Dictyostelium discoideum Genome

et al. 2011

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Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events

Kolb

Hsu

Wallis

et al. 2018

Methods in Molecular Biology

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The amoeba Dictyostelium discoideum is a single-cell organism that can undergo a simple developmental program, making it an excellent model to study the molecular mechanisms of cell motility, signal transduction, and cell-type differentiation. A variety of human genes that are absent or show limited conservation in other invertebrate models have been identified in this organism. This includes ADP-ribosyltransferases, also known as poly-ADP-ribose polymerases (PARPs), a family of proteins that catalyze the addition of single or poly-ADP-ribose moieties onto target proteins. The genetic tractability of Dictyostelium and its relatively simple genome structure makes it possible to disrupt PARP gene combinations, in addition to specific ADP-ribosylation sites at endogenous loci. Together, this makes Dictyostelium an attractive model to assess how ADP-ribosylation regulates a variety of cellular processes including DNA repair, transcription, and cell-type specification. Here we describe a range of techniques to study ADP-ribosylation in Dictyostelium, including analysis of ADP-ribosylation events in vitro and in vivo, in addition to approaches to assess the functional roles of this modification in vivo.

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Investigation of DNA Repair Pathway Activity

Couto

Lakin

Pears

2013

Methods in Molecular Biology

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DNA is constantly being damaged from endogenous and exogenous sources and efficient repair of different types of DNA lesions is essential for the survival of the organism. Dictyostelium is highly resistant to DNA damage and its genome sequence has revealed the presence of multiple repair pathways conserved with vertebrates but lost in other genetically tractable invertebrate models. As such, Dictyostelium is a powerful model organism to study selected human DNA repair pathways and may provide insights into the molecular basis of how cells become resistant to DNA damage. Here we describe a range of assays used to study DNA repair in Dictyostelium. Genes required for repair of DNA damage can be identified and analyzed by comparing the ability of control or mutant cells to survive exposure to genotoxic agents that induce different types of DNA lesion. We also describe assays that assess the presence of markers for DNA repair within chromatin either in the form of posttranslational modification of proteins at sites of damage or the recruitment of repair factors to DNA lesions. Finally, we also describe more direct assays to assess repair of DNA double-strand breaks by either homologous recombination or non-homologous end joining.

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Dictyostelium discoideum plasmid containing an AP‐endonuclease upstream sequence: Bleomycin induction of a luciferase reporter

Cited by 3 publications

References 21 publications

The C-Module-Binding Factor Supports Amplification of TRE5-A Retrotransposons in the Dictyostelium discoideum Genome

The C-Module-Binding Factor Supports Amplification of TRE5-A Retrotransposons in the Dictyostelium discoideum Genome

Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events

Investigation of DNA Repair Pathway Activity

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