Nonsense suppression in <i>Dictyostelium discoideum</i>

Dingermann, Theo; Reindl, Norbert; Brechner, Thomas; Werner, Herbert; Nerke, Käthe

doi:10.1002/dvg.1020110514

Cited by 20 publications

(13 citation statements)

References 30 publications

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“…The A-module promoter by itself seems fairly strong, about 30% as active as the well-studied actin 6 promoter from D. discoideum (12,13). A-module promoter activity appears to be enhanced slightly further if a tRNA gene is located upstream of the element.…”

Section: Methodsmentioning

confidence: 85%

Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

Schumann¹,

Zündorf²,

Hofmann³

et al. 1994

Mol. Cell. Biol.

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The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10-to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 ± 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.Retrotransposable Dictyostelium repetitive elements (DREs) are present in approximately 150 to 200 copies in the genomes of different Dictyostelium discoideum strains (28-31).Most remarkably, this element always integrates at a distance of 50 ± 4 nucleotides upstream from tRNA genes but in the opposite transcriptional orientation. Upon integration, a 14 + 2-nucleotide target site duplication is created (29). The 5.7-kb DREs contain an internal coding portion with two open reading frames (ORFs) which is flanked by nonidentical terminal repeats (TRs) (see Fig. IA). These TRs are composed of three distinct modules, termed A, B, and C. The 5' TR consists of one or several A modules followed by a 290-bp B module which provides the AUG translation initiation codon. The 3' TR consists of a B module followed by a terminal C module.Approximately half of the DREs in NC4-derived strains show a variant organization (30). They are only 2.4 kb long and are termed DREbs, as opposed to the 5.7-kb DREas (Fig. IA). DREbs carry an extensive internal deletion encompassing nearly the entire ORF2. They also contain several small deletions within the right TR and a variant A module (termed Ab), which always differs from the Aa module of the 5.7-kb DREas by the same three distinct point mutations (30). A similar situation has been described for jockey elements of Drosophila melanogaster, which occur as full-size (5-kb) copies

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Section: Methodsmentioning

confidence: 85%

Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

Schumann¹,

Zündorf²,

Hofmann³

et al. 1994

Mol. Cell. Biol.

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“…This reporter gene can be equipped with any suitable pol III gene to attract TREs for integration (referred to as the "TRE trap"). The Val UAC tRNA gene used in this and our previous study (2) (10) was amplified by PCR from plasmid pGTET ϩ 1 (40) using primers Glu-01 (5Ј-GGAATTCTCCTCATTGGTGTAGTCGGTAA CAC-3Ј) and Glu-02 (5Ј-GGAATTCTAATTTTGGTCGGAATAAAAACCTC C-3Ј). The resulting PCR fragment was inserted as an EcoRI fragment into the TRE trap.…”

Section: Methodsmentioning

confidence: 99%

Role of RNA Polymerase III Transcription Factors in the Selection of Integration Sites by the Dictyostelium Non-Long Terminal Repeat Retrotransposon TRE5-A

Siol

Boutliliss

Chung

et al. 2006

Molecular and Cellular Biology

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In the compact Dictyostelium discoideum genome, non-long terminal repeat (non-LTR) retrotransposons known as TREs avoid accidental integration-mediated gene disruption by targeting the vicinity of tRNA genes. In this study we provide the first evidence that proteins of a non-LTR retrotransposon interact with a target-specific transcription factor to direct its integration. We applied an in vivo selection system that allows for the isolation of natural TRE5-A integrations into a known genomic location upstream of tRNA genes. TRE5-A frequently modified the integration site in a way characteristic of other non-LTR retrotransposons by adding nontemplated extra nucleotides and generating small and extended target site deletions. Mutations within the B-box promoter of the targeted tRNA genes interfered with both the in vitro binding of RNA polymerase III transcription factor TFIIIC and the ability of TRE5-A to target these genes. An isolated B box was sufficient to enhance TRE5-A integration in the absence of a surrounding tRNA gene. The RNA polymerase III-transcribed ribosomal 5S gene recruits TFIIIC in a B-box-independent manner, yet it was readily targeted by TRE5-A in our assay. These results suggest a direct role of an RNA polymerase III transcription factor in the targeting process.

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“…This organism provides a wellestablished genetic system (38), including suppressor genetics (18). As a reporter for nonsense suppression, a mutated version of the lacZ gene from Escherichia coli which can be expressed in D. discoideum as controlled by a homologous actin 6 promoter is available (18,19 MATERIALS AND METHODS Plasmid DNAs. Plasmid pDneoA6PTR was obtained after in-frame fusion of the tetracycline repressor gene to the N-terminal coding region of the actin 6 gene contained on pDneo2 (56).…”

mentioning

confidence: 99%

“…Transcription termination is controlled by the actin 8 terminator from D. discoideum. A GAG glutamic acid codon in the N-terminal region of the actin 6::1acZ fusion gene was changed into a UAG amber codon by primer-directed mutagenesis (18). A HindIII fragment containing the actin 6::1acZ(Am) fusion was isolated and ligated into the three pGTET plasmids, yielding pGTETR+1, pGTETR-7, and pGTETR-46 (see Fig.…”

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confidence: 99%

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

Dingermann¹,

Werner²,

Schütz³

et al. 1992

Mol. Cell. Biol.

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We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO_ operator fragment was inserted at three different positions in front of a tRNAGrU(Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a 1-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 ,g/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in f-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.tRNA suppressors are a classic means of regulating the expression of a protein. In the absence of a suppressor tRNA, the mRNA derived from a gene which has been mutated to contain a stop codon will be incompletely translated. When a tRNA suppressor gene is introduced into the cell, an authentic protein is synthesized, provided the suppressor tRNA inserts the same amino acid as the cognate tRNA of the unmutated codon. This is a powerful tool for analysis of the function of any gene but has been relegated primarily to procaryotes because of the difficulty of manipulating tRNA expression in eukaryotic cells. Eukaryotic tRNA genes contain gene-internal polymerase III (polIII) promoters (for reviews, see references 23 and 46). These transcriptional control regions become part of the mature tRNA coding sequence and are indispensable for tRNA function. As a consequence, these regions are difficult to manipulate. Another consequence is that other than in the case of polII genes, polIII gene promoters cannot be replaced by regulated polII promoters, such as the heat shock and metallothionein promoters.In addition to the gene-internal control elements, 5'-flanking regions of tRNA genes frequently exert a modulatory influence on gene expression (1-3, 10, 15, 17, 27, 37, 41, 47, 49). Although the mechanism of tRNA gene modulation by 5'-flanking regions is not understood, we recently demonstrated that stable binding of a protein near a tRNA gene strongly inhibits its expression (39). This inhibition apparently resulted from masking of the initiation site for tRNA gene transcription by the bound protein and from a strong interaction of the protein with the RNA polIII transcription complex. These findings suggested th...

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Nonsense suppression in Dictyostelium discoideum

Cited by 20 publications

References 30 publications

Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

Internally located and oppositely oriented polymerase II promoters direct convergent transcription of a LINE-like retroelement, the Dictyostelium repetitive element, from Dictyostelium discoideum.

Role of RNA Polymerase III Transcription Factors in the Selection of Integration Sites by the Dictyostelium Non-Long Terminal Repeat Retrotransposon TRE5-A

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

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