Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning

Wiegand, Stephan; Kruse, Janis; Gronemann, Sina; Hammann, Christian

doi:10.1016/j.ygeno.2011.02.001

Cited by 32 publications

(25 citation statements)

References 15 publications

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“…To study the impact of RdRPs on DIRS-1 expression in D. discoideum in detail, we made use of a recently introduced series of strains (9), with the genes of the RdRPs in all possible combinations deleted by a one-step cloning strategy (38) that is based on the Cre-lox system (27). Unlike in the originally used gene disruption strains (21), both functional domains of each rrp gene (Supplementary Figure S1) were removed completely (9), resulting in three single, three double and one triple rrp gene deletion strain.…”

Section: Resultsmentioning

confidence: 99%

The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

Wiegand¹,

Meier²,

Seehafer³

et al. 2013

Nucleic Acids Research

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Dictyostelium intermediate repeat sequence 1 (DIRS-1) is the founding member of a poorly characterized class of retrotransposable elements that contain inverse long terminal repeats and tyrosine recombinase instead of DDE-type integrase enzymes. In Dictyostelium discoideum, DIRS-1 forms clusters that adopt the function of centromeres, rendering tight retrotransposition control critical to maintaining chromosome integrity. We report that in deletion strains of the RNA-dependent RNA polymerase RrpC, full-length and shorter DIRS-1 messenger RNAs are strongly enriched. Shorter versions of a hitherto unknown long non-coding RNA in DIRS-1 antisense orientation are also enriched in rrpC– strains. Concurrent with the accumulation of long transcripts, the vast majority of small (21 mer) DIRS-1 RNAs vanish in rrpC– strains. RNASeq reveals an asymmetric distribution of the DIRS-1 small RNAs, both along DIRS-1 and with respect to sense and antisense orientation. We show that RrpC is required for post-transcriptional DIRS-1 silencing and also for spreading of RNA silencing signals. Finally, DIRS-1 mis-regulation in the absence of RrpC leads to retrotransposon mobilization. In summary, our data reveal RrpC as a key player in the silencing of centromeric retrotransposon DIRS-1. RrpC acts at the post-transcriptional level and is involved in spreading of RNA silencing signals, both in the 5′ and 3′ directions.

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Section: Resultsmentioning

confidence: 99%

The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

Wiegand¹,

Meier²,

Seehafer³

et al. 2013

Nucleic Acids Research

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“…RrpA and RrpB share 94% sequence identity on the protein level over the entire length, while RrpC is more distinct with 35% identity over 65% of the protein sequence [19]. We generated deletion strains of all three genes using a recently developed one-step protocol [23] that exploits the loxP system for the generation of multiple gene deletion strains [25]. The gene deletion cassettes were designed such that in each gene, the sequences encoding the two domains were completely removed after homologous recombination (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

“…The drnB – strain was described recently [21]. Deletion of the rrp genes was performed as described using constructs generated by combinatorial cloning [23]. In brief, in each of the used constructs, the two arms for homologous recombination were placed in front of the Helicase domain and after the RdRP domain, respectively to ensure the generation of loss-of-function mutant strains (Figure 1B).…”

Section: Methodsmentioning

confidence: 99%

“…Ten µg of linearized vector were transformed into A×2 D. discoideum cells by electroporation [24]. Single clone transformants were isolated on Klebsiella aerogenes and analyzed initially by PCR and deletions were confirmed by Southern blot (Figure 1), as described recently [23]. Upon excision of the BS (r) cassette by transient expression of the Cre recombinase [25] the gene deletion cassettes could be used again in single knock out strains, yielding the double mutants rrpA – : rrpB – , rrpA – : rrpC – and rrpB – : rrpC – .…”

Section: Methodsmentioning

confidence: 99%

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The 5′ Spreading of Small RNAs in Dictyostelium discoideum Depends on the RNA-Dependent RNA Polymerase RrpC and on the Dicer-Related Nuclease DrnB

Wiegand

Hammann

2013

PLoS ONE

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RNA interference (RNAi) is a gene-regulatory mechanism in eukarya that is based on the presence of double stranded RNA and that can act on both, the transcription or post-transcriptional level. In many species, RNA-dependent RNA polymerases (RdRPs) are required for RNAi. To study the function of the three RdRPs in the amoeba Dictyostelium discoideum, we have deleted the encoding genes rrpA, rrpB and rrpC in all possible combinations. In these strains, expression of either antisense or hairpin RNA constructs against the transgene lacZ resulted in a 50% reduced β-Galactosidase activity. Northern blots surprisingly revealed unchanged lacZ mRNA levels, indicative of post-transcriptional regulation. Only in rrpC knock out strains, low levels of β-gal small interfering RNAs (siRNAs) could be detected in antisense RNA expressing strains. In contrast to this, and at considerably higher levels, all hairpin RNA expressing strains featured β-gal siRNAs. Spreading of the silencing signal to mRNA sequences 5′ of the original hairpin trigger was observed in all strains, except when the rrpC gene or that of the dicer-related nuclease DrnB was deleted. Thus, our data indicate that transitivity of an RNA silencing signal exists in D. discoideum and that it requires the two enzymes RrpC and DrnB.

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“…Dictyostelium discoideum depends on efficient motility during its developmental cycle and its vegetative amoeboid stage 52 and provides a model for the study of chemotaxis in the absence of the complexity imposed by the in vivo microenvironment of animals 152–154 . One of its main benefits is that when grown in culture these cells exist as homogenous chemotactic phagocytes 48,49 , which can be grown in high-density suspension cultures to generate kilogram quantities of proteins for biochemical analysis 155 .…”

Section: Mechanisms Of Chemotaxis In Tumour Cellsmentioning

confidence: 99%

Chemotaxis in cancer

2011

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Chemotaxis of tumour cells and stromal cells in the surrounding microenvironment is an essential component of tumour dissemination during progression and metastasis. This Review summarizes how chemotaxis directs the different behaviours of tumour cells and stromal cells in vivo, how molecular pathways regulate chemotaxis in tumour cells and how chemotaxis choreographs cell behaviour to shape the tumour microenvironment and to determine metastatic spread. The central importance of chemotaxis in cancer progression is highlighted by discussion of the use of chemotaxis as a prognostic marker, a treatment end point and a target of therapeutic intervention.

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Efficient generation of gene knockout plasmids for Dictyostelium discoideum using one-step cloning

Cited by 32 publications

References 15 publications

The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

The Dictyostelium discoideum RNA-dependent RNA polymerase RrpC silences the centromeric retrotransposon DIRS-1 post-transcriptionally and is required for the spreading of RNA silencing signals

The 5′ Spreading of Small RNAs in Dictyostelium discoideum Depends on the RNA-Dependent RNA Polymerase RrpC and on the Dicer-Related Nuclease DrnB

Chemotaxis in cancer

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