Transfer RNA Gene-targeted Retrotransposition of Dictyostelium TRE5-A into a Chromosomal UMP Synthase Gene Trap

Beck, Peter; Dingermann, Theo; Winckler, Thomas

doi:10.1016/s0022-2836(02)00097-9

Cited by 21 publications

(58 citation statements)

References 37 publications

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“…Plasmid pGEM-pyr5-6(cbfA/Val UAC ) contains the pyr5-6 gene of D. discoideum including its own promoter and terminator. The pyr5-6 gene is tagged with the intron of the D. discoideum cbfA gene (2). This reporter gene can be equipped with any suitable pol III gene to attract TREs for integration (referred to as the "TRE trap").…”

Section: Methodsmentioning

confidence: 99%

“…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%

“…The TRE5 elements insert exclusively 48 Ϯ 3 bp upstream of the first coding nucleotide of the targeted tRNA gene. All as-yet-analyzed chromosomal TRE5 insertions occurred in an orientation-specific manner, with the 5Ј ends of the retrotransposons facing the targeted tRNA genes (2). On the other hand, TRE3 elements are found exclusively in a region ϳ100 bp downstream of tRNA genes (37).…”

mentioning

confidence: 99%

“…Furthermore, we have not yet succeeded in cloning an auton-omous TRE that would allow for the analysis of retrotransposition of tagged versions of the element, a strategy that is very successfully applied to study retrotransposable elements in mammalian cells (12,24,29). Taking advantage of the strong preference of TRE5-A to integrate at tRNA gene loci, we have recently established an in vivo selection system that allows for the isolation of new integrations of naturally active TRE5-A copies from a population of D. discoideum cells (2). The "TRE trap" is a plasmid-borne pyr5-6 gene, which encodes UMP synthase, tagged with an intron carrying a D. discoideum tRNA gene (see Fig.…”

mentioning

confidence: 99%

“…These cells are sensitive to the cytostatic drug 5-fluoroorotic acid (5-FOA). If the pyr5-6 gene is disrupted by mutation, e.g., by targeted integration of a TRE near the "bait" tRNA gene within the TRE trap, then the affected cells gain resistance to 5-FOA and grow out as clones (2).…”

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confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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An NIH Model Organism for Biopharmaceutical and Biomedical Research: The Lower EukaryoteDictyostelium discoideum

Winckler¹,

Zündorf²,

Dingermann³

2005

Modern Biopharmaceuticals

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Dictyostelium mobile elements: strategies to amplify in a compact genome

Winckler

Dingermann

Glöckner³

2002

Cellular and Molecular Life Sciences (CMLS)

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Dictyostelium discoideum is a eukaryotic microorganism that is attractive for the study of fundamental biological phenomena such as cell-cell communication, formation of multicellularity, cell differentiation and morphogenesis. Large-scale sequencing of the D. discoideum genome has provided new insights into evolutionary strategies evolved by transposable elements (TEs) to settle in compact microbial genomes and to maintain active populations over evolutionary time. The high gene density (about 1 gene/2.6 kb) of the D. discoideum genome leaves limited space for selfish molecular invaders to move and amplify without causing deleterious mutations that eradicate their host. Targeting of transfer RNA (tRNA) gene loci appears to be a generally successful strategy for TEs residing in compact genomes to insert away from coding regions. In D. discoideum, tRNA gene-targeted retrotransposition has evolved independently at least three times by both non-long terminal repeat (LTR) retrotransposons and retrovirus-like LTR retrotransposons. Unlike the nonspecifically inserting D. discoideum TEs, which have a strong tendency to insert into preexisting TE copies and form large and complex clusters near the ends of chromosomes, the tRNA gene-targeted retrotransposons have managed to occupy 75% of the tRNA gene loci spread on chromosome 2 and represent 80% of the TEs recognized on the assembled central 6.5-Mb part of chromosome 2. In this review we update the available information about D. discoideum TEs which emerges both from previous work and current large-scale genome sequencing, with special emphasis on the fact that tRNA genes are principal determinants of retrotransposon insertions into the D. discoideum genome.

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Transfer RNA Gene-targeted Retrotransposition of Dictyostelium TRE5-A into a Chromosomal UMP Synthase Gene Trap

Cited by 21 publications

References 37 publications

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

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

An NIH Model Organism for Biopharmaceutical and Biomedical Research: The Lower EukaryoteDictyostelium discoideum

Dictyostelium mobile elements: strategies to amplify in a compact genome

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