Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae

Winckler, Thomas; Trautwein, Christine; Tschepke, C.; Neuhäuser, Christin; Zündorf, Ilse; Beck, Peter; Vogel, Günter; Dingermann, Theo

doi:10.1006/jmbi.2000.4341

Cited by 17 publications

(40 citation statements)

References 24 publications

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“…This observation correlated well with the growth characteristics of CbfA-deficient cells, including a decreased phagocytosis rate, slow growth on bacteria, and the generation of giant cells containing multiple nuclei (15). We performed a qRT-PCR analysis of 10 of these genes on RNA from three independently grown cultures to confirm the RNA-seq data.…”

Section: Resultssupporting

confidence: 61%

“…A gene fragment spanning amino acids 724 to 1000 of the D. discoideum CbfA protein was cloned as a BamHI fragment into the pDXA-GFP2 plasmid (21). The resulting plasmid, pDXA-DdCbfA-CTD, was later transformed into JH.D cells (15). The corresponding sequence of P. pallidum CbfA was isolated by PCR and cloned as mentioned above.…”

Section: Dictyostelidsmentioning

confidence: 99%

“…As an alternative, a cbfA gene knock-in mutant was generated in which the codon at position 455 was replaced with a TAG (amber) stop codon (15). Because of the low frequency of amber codons in the D. discoideum genome, the expression of an amber suppressor tRNA gene does not produce a phenotype (16) but rather allows readthrough translation of the full-length protein from the cbfA (amber) mRNA.…”

mentioning

confidence: 99%

“…Because of the low frequency of amber codons in the D. discoideum genome, the expression of an amber suppressor tRNA gene does not produce a phenotype (16) but rather allows readthrough translation of the full-length protein from the cbfA (amber) mRNA. Because of the low efficacy of amber suppression, the JH.D strain produces less than 5% of the full-length CbfA protein (15).…”

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confidence: 99%

“…JH.D cells are defective in phagocytosis, cytokinesis (15), and multicellular development (17). In mutant cells, the expression of the aggregation-specific adenylyl cyclase ACA is strongly reduced; therefore, cyclic-AMP (cAMP)-induced gene expression is absent.…”

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confidence: 99%

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Conserved Gene Regulatory Function of the Carboxy-Terminal Domain of Dictyostelid C-Module-Binding Factor

Schmith¹,

Groth²,

Ratka³

et al. 2013

Eukaryot Cell

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e C-module-binding factor A (CbfA) is a jumonji-type transcription regulator that is important for maintaining the expression and mobility of the retrotransposable element TRE5-A in the social amoeba Dictyostelium discoideum. CbfA-deficient cells have lost TRE5-A retrotransposition, are impaired in the ability to feed on bacteria, and do not enter multicellular development because of a block in cell aggregation. In this study, we performed Illumina RNA-seq of growing CbfA mutant cells to obtain a list of CbfA-regulated genes. We demonstrate that the carboxy-terminal domain of CbfA alone is sufficient to mediate most CbfAdependent gene expression. The carboxy-terminal domain of CbfA from the distantly related social amoeba Polysphondylium pallidum restored the expression of CbfA-dependent genes in the D. discoideum CbfA mutant, indicating a deep conservation in the gene regulatory function of this domain in the dictyostelid clade. The CbfA-like protein CbfB displays ϳ25% sequence identity with CbfA in the amino-terminal region, which contains a JmjC domain and two zinc finger regions and is thought to mediate chromatin-remodeling activity. In contrast to CbfA proteins, where the carboxy-terminal domains are strictly conserved in all dictyostelids, CbfB proteins have completely unrelated carboxy-terminal domains. Outside the dictyostelid clade, CbfA-like proteins with the CbfA-archetypical JmjC/zinc finger arrangement and individual carboxy-terminal domains are prominent in filamentous fungi but are not found in yeasts, plants, and metazoans. Our data suggest that two functional regions of the CbfAlike proteins evolved at different rates to allow the occurrence of species-specific adaptation processes during genome evolution.

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Section: Resultssupporting

confidence: 61%

Section: Dictyostelidsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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