A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Patil, Namrata; Karrer, Kathleen M.

doi:10.1093/nar/28.6.1465

Cited by 26 publications

(17 citation statements)

References 26 publications

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“…Tflank-series rearrangement constructs were derived from pHWT.cam, which consists of the previously described WT.cam construct (48) cloned between two NotI sites in the polylinker region of bacterial plasmid vector pHSS6 (49 Once assembled, pHTflank-series and pHCflank-series plasmid constructs were excised from pHSS6 with NotI and ligated to the unique NotI site in Tetrahymena ribosomal DNA (rDNA) vector pD5H8 (22). The structures of the resulting pDTflank-series and pDCflank-series plasmids, respectively, were verified by DNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…The 825-bp terminal inverted repeat of Tlr1 is located near, but not at, the rearrangement junctions. Obliteration of the macronucleusdestined DNA flanking the right side of Tlr1 results in aberrant processing of the element (48). Thus, it seems likely that this region contains cis-acting DNA signals that control the position of the adjacent elimination junction, analogous to the regulatory sequences located outside the M, R, and mse2.9 elements.…”

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confidence: 99%

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Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in Tetrahymena thermophila

Wuitschick

Karrer

2003

Eukaryot Cell

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Tlr elements are a novel family of ϳ30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleusretained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ϳ23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA.Ciliates are unusual among eukaryotes in that each cell contains two structurally and functionally distinct nuclei. This phenomenon of nuclear dualism has apparently evolved to separate the two primary functions of the genome: transmission and expression of genetic information (14). The micronucleus, which is transcriptionally silent in vegetatively growing cells, is the germ line nucleus in ciliates. It contains an intact copy of the complete genome of the organism and participates primarily in the reciprocal exchange of gametic nuclei that occurs during conjugation, the sexual phase of the ciliate life cycle. In contrast, the somatic macronucleus contains a highly processed subset of the micronuclear genome and is responsible for essentially all nuclear transcription in vegetatively growing cells. In each sexual generation, the macronucleus is degraded, and a replacement is generated from a mitotic copy of the zygotic micronucleus formed during conjugation. As the new macronucleus differentiates, it undergoes dramatic genome reorganization involving chromosomal fragmentation and extensive DNA elimination (14).The ciliate Tetrahymena thermophila provides a particularly useful system for the molecular analysis of programmed DNA elimination events (33). In the developing macronucleus, the five germ line-derived T. thermophila chromosomes are fragmented at 50 to 250 distinct chromosome breakage sequences, and telomeres are added de novo to the resulting subchromosomal molecules. In addition, an estimated 6,000 stretches of micronucleus-limited DNA are removed through progr...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in Tetrahymena thermophila

Wuitschick

Karrer

2003

Eukaryot Cell

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“…These germ line-limited DNA segments range in size from a few hundred base pairs to more than 20 kbp and are comprised of both unique sequences as well as repetitive elements. While flanking regulatory sequences that demarcate the boundaries of specific deletion events have been identified (8,17,21,22,45), identification of any consensus sequences that are required to promote these DNA rearrangements has remained elusive. The heterogeneity of the sequences eliminated, together with the lack of a defined consensus sequence, has provided a challenge in describing a simple model for the control of this process.…”

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confidence: 99%

Germ Line Transcripts Are Processed by a Dicer-Like Protein That Is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila

Malone

Anderson

Motl

et al. 2005

Molecular and Cellular Biology

156

252

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Abundant ϳ28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (⌬DCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These ⌬DCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation ⌬DCL1 cells die without eliminating the germ linelimited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicerrelated gene is required for programmed DNA rearrangements. Surprisingly, ⌬DCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.RNA interference (RNAi) describes an array of related mechanisms involved in diverse biological processes including defense against RNA viruses, specification of centromeric heterochromatin structure, and developmental control of gene expression (reviewed in reference 25). These mechanisms share the use of small RNAs to target specific effector protein complexes to homologous sequences via base-pairing interactions. The use of small, homologous RNAs as specificity factors imparts tremendous flexibility of targets on a single protein complex. These targeting RNAs are generated by RNase III enzymes, collectively called Dicer ribonucleases, that cleave longer, double-stranded RNA (dsRNA) into ϳ20-to 26-nucleotide (nt) species that are incorporated into the effector complexes (3, 24, 27, 30; reviewed in reference 6). The genomes of many eukaryotes encode multiple Dicer-related proteins, and the specific Dicer used to generate the small RNAs can determine the downstream pathway that they enter. For instance, in Arabidopsis thaliana, the Dicer-like 3 (Dcl3) gene product is required to produce endogenous short interfering RNAs (siRNAs), A. thaliana Dcl2 is necessary for accumulation of siRNAs in response to RNA virus infections, and A. thaliana Dcl1 is necessary to generate micro-RNAs (miRNAs) involved in the control of flower development (31,53). Similarly, th...

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“…We have demonstrated that microinjection of cloned Paramecium IESs into mated cells can be used to analyze the cis-acting sequence requirements for developmentally controlled DNA excision. Similar methods were previously developed for T. thermophila (29), and studies of eliminated sequences in this organism have shown that flanking DNA plays an important role in these events (3,8,14,20). In the case of the Tetrahymena M element, it is clear that a 10-bp sequence (A 5 G 5 ) located 50 bp outside the deleted region is sufficient to specify one end of the DNA splice junction (9).…”

Section: Discussionmentioning

confidence: 89%

Developmentally Regulated Excision of a 28-Base-Pair Sequence from the Paramecium Genome Requires Flanking DNA

Mayer²,

Forney

2000

Molecular and Cellular Biology

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The micronuclear DNA of Paramecium tetraurelia is estimated to contain over 50,000 short DNA elements that are precisely removed during the formation of the transcriptionally active macronucleus. Ciliated protozoa provide a unique biological system for the study of DNA rearrangements. During sexual reproduction, a transcriptionally active macronucleus is formed from the germ line DNA in the micronucleus. The precise details of the process vary among different ciliates, but common features include fragmentation of germ line chromosomes, elimination of specific DNA elements, and amplification of the macronucleusdestined linear fragments (reviewed in references 2, 13, and 21). Elimination of relatively small regions of the genome (14 bp to several kilobases) followed by rejoining of the adjacent sequences has been observed in a wide variety of ciliate species. These excised DNA elements are commonly referred to as internal eliminated sequences (IESs) to distinguish them from elimination that results from fragmentation events.In Paramecium tetraurelia, the micronuclear genome contains relatively short IESs (26 bp to about 1 kb) that always begin and end with a 5Ј-TA-3Ј dinucleotide. Excision results in precise removal of the element, leaving a single TA within the macronuclear DNA (4,5,15,23,26,27). No significant open reading frames are encoded by these elements, and comparison of evolutionarily related IESs within the variable surface antigen genes of P. tetraurelia revealed substantial variation in the size and sequence of an IES relative to the adjacent macronuclear DNA (23). The ends of the element generally include a perfect inverted repeat that includes the TA and extends either into the IES or out toward the macronucleusdestined DNA (26). Statistical analysis of 20 IESs from P. tetraurelia identified an 8-bp consensus inverted terminal repeat that includes the invariant TA dinucleotide (12). This consensus repeat is inside the IES and therefore does not necessarily include the perfect inverted repeats. The functional significance of the consensus repeat is supported by the analysis of Paramecium mutant cell lines defective in IES excision.Isolated cell lines that are unable to excise a specific IES contain single nucleotide mutations in the consensus region (16,17).The structure of a Paramecium IES can be complex. There are at least two examples in which one IES is located inside a larger IES (6,16,17). In this report (which focuses on one of the complex IESs), we will refer to the smaller IES located inside another as an internal IES. The enzymatic machinery responsible for IES excision has not been identified, but analysis of a pleiotropic mutant line has shown that excision of one IES (or a small subclass) is inhibited by a mutation in an unlinked locus (19).Paramecium is not the only ciliate that contains TA IESs (IESs bounded by TA repeats). Euplotes crassus contains IESs bounded by 5Ј-TA-3Ј direct repeats, and they have similar consensus terminal inverted repeats (reviewed in reference 11). Interesting...

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A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Cited by 26 publications

References 26 publications

Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in Tetrahymena thermophila

Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in Tetrahymena thermophila

Germ Line Transcripts Are Processed by a Dicer-Like Protein That Is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila

Developmentally Regulated Excision of a 28-Base-Pair Sequence from the Paramecium Genome Requires Flanking DNA

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