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

2005 ◽  
Vol 25 (20) ◽  
pp. 9151-9164 ◽  
Author(s):  
Colin D. Malone ◽  
Alissa M. Anderson ◽  
Jason A. Motl ◽  
Charles H. Rexer ◽  
Douglas L. Chalker
Keyword(s):  
Dna Sequences ◽  
Small Rnas ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Protein S ◽  
Helicase Domain ◽  
H3k9 Methylation ◽  
Wild Type ◽  
Rnase Iii ◽  
Somatic Genome

ABSTRACT Abundant ∼28-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 (ΔDCL1) 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 ΔDCL1 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 ΔDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, ΔDCL1 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.

scholarly journals Elimination of Foreign DNA during Somatic Differentiation in Tetrahymena thermophila Shows Position Effect and Is Dosage Dependent

2005 ◽  
Vol 4 (2) ◽  
pp. 421-431 ◽  
Author(s):  
Yifan Liu ◽  
Xiaoyuan Song ◽  
Martin A. Gorovsky ◽  
Kathleen M. Karrer
Keyword(s):  
Transposable Elements ◽  
Dna Sequences ◽  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Position Effect ◽  
Dna Elimination ◽  
Internal Eliminated Sequences ◽  
Somatic Genome ◽  
Foreign Dna

ABSTRACT In the ciliate Tetrahymena thermophila, approximately 15% of the germ line micronuclear DNA sequences are eliminated during formation of the somatic macronucleus. The vast majority of the internal eliminated sequences (IESs) are repeated in the micronuclear genome, and several of them resemble transposable elements. Thus, it has been suggested that DNA elimination evolved as a means for removing invading DNAs. In the present study, bacterial neo genes introduced into the germ line micronuclei were eliminated from the somatic genome. The efficiency of elimination from two different loci increased dramatically with the copy number of the neo genes in the micronuclei. The timing of neo elimination is similar to that of endogenous IESs, and they both produce bidirectional transcripts of the eliminated element, suggesting that the deletion of neo occurred by the same mechanism as elimination of endogenous IESs. These results indicate that repetition of an element in the micronucleus enhances the efficiency of its elimination from the newly formed somatic genome of Tetrahymena thermophila. The implications of these data in relation to the function and mechanism of IES elimination are discussed.

scholarly journals A method for mapping germ line sequences in Tetrahymena thermophila using the polymerase chain reaction.

Genetics ◽  
1994 ◽  
Vol 137 (1) ◽  
pp. 95-106 ◽  
Author(s):  
D Cassidy-Hanley ◽  
M C Yao ◽  
P J Bruns
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Repetitive Sequences ◽  
Mapping Technique ◽  
Ciliated Protozoan ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Template Dna

Abstract A method for mapping DNA sequences to specific germinal chromosomes in the ciliated protozoan Tetrahymena thermophila has been developed. This mapping technique (PCR mapping) utilizes the polymerase chain reaction and template DNA derived from nullisomic strains to directly assign micronuclear DNA sequences to specific micronuclear chromosomes. Using this technique, a number of unique sequences and short repetitive sequences flanked by unique sequences have been mapped to four of the five germinal chromosomes.

scholarly journals Germ line-specific DNA sequences are present on all five micronuclear chromosomes in Tetrahymena thermophila.

1983 ◽  
Vol 3 (11) ◽  
pp. 1909-1919 ◽  
Author(s):  
K M Karrer
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Plasmid Vector ◽  
Ciliated Protozoan ◽  
Specific Sequence ◽  
Individual Probe ◽  
Vector Pbr322

The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp. involves the elimination of specific DNA sequences (M. C. Yao and M. Gorovsky, Chromosoma 48:1-18 1974). The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes. Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322. A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe. Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific. They were all members of families of repeated sequences. Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T. thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes. One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes.

scholarly journals Germ line-specific DNA sequences are present on all five micronuclear chromosomes in Tetrahymena thermophila

1983 ◽  
Vol 3 (11) ◽  
pp. 1909-1919
Author(s):  
K M Karrer
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequences ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Plasmid Vector ◽  
Ciliated Protozoan ◽  
Specific Sequence ◽  
Individual Probe ◽  
Vector Pbr322

The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp. involves the elimination of specific DNA sequences (M. C. Yao and M. Gorovsky, Chromosoma 48:1-18 1974). The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes. Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322. A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe. Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific. They were all members of families of repeated sequences. Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T. thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes. One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes.

scholarly journals Two GW Repeat Proteins Interact with Tetrahymena thermophila Argonaute and Promote Genome Rearrangement

2009 ◽  
Vol 29 (18) ◽  
pp. 5020-5030 ◽  
Author(s):  
Janna Bednenko ◽  
Tomoko Noto ◽  
Leroi V. DeSouza ◽  
K. W. Michael Siu ◽  
Ronald E. Pearlman ◽  
...  
Keyword(s):  
Genome Rearrangement ◽  
Developmental Stages ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Nuclear Bodies ◽  
Interacting Proteins ◽  
Heterochromatin Formation ◽  
Dna Elimination ◽  
Somatic Genome ◽  
Argonaute Family

ABSTRACT In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, ∼28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of ΔWAG1, ΔCnjB, and double ΔWAG1 ΔCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.

scholarly journals INDUCED RESISTANCE TO 6-METHYLPURINE AND CYCLOHEXIMIDE IN TETRAHYMENA. I. GERM LINE MUTANTS OF T. THERMOPHILA

Genetics ◽  
1978 ◽  
Vol 89 (4) ◽  
pp. 695-702
Author(s):  
Bruce C Byrne ◽  
Trudy B Brussard ◽  
Peter J Bruns
Keyword(s):  
Induced Resistance ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Sexual Cycle ◽  
Marker Genes ◽  
Critical Importance ◽  
Wild Type ◽  
Phenotypic Assortment ◽  
Mutant Lines ◽  
Dominant Genes

ABSTRACT Two new mutant lines of Tetrahymena thermophila (T. pyriformis, syngen 1), each conferring resistance to a different agent, are described. Resistance to cycloheximide and 6-methylpurine are each determined by dominant genes, ChxA2 and Mpr; the traits show phenotypic assortment. The method used to select these mutations, the critical importance of backcrossing to wild type following mutagenesis, and the utility of these marker genes in further mutagenic selection schemes and studies of the sexual cycle of Tetrahymena are noted.

scholarly journals A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

1994 ◽  
Vol 14 (9) ◽  
pp. 5939-5949 ◽  
Author(s):  
J M Wells ◽  
J L Ellingson ◽  
D M Catt ◽  
P J Berger ◽  
K M Karrer
Keyword(s):  
Dna Sequences ◽  
Inverted Repeat ◽  
Tandem Repeats ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Inverted Repeats ◽  
Dna Rearrangement ◽  
Dna Rearrangements ◽  
Developmentally Regulated ◽  
Small Family

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.

scholarly journals A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila

1994 ◽  
Vol 14 (9) ◽  
pp. 5939-5949
Author(s):  
J M Wells ◽  
J L Ellingson ◽  
D M Catt ◽  
P J Berger ◽  
K M Karrer
Keyword(s):  
Dna Sequences ◽  
Inverted Repeat ◽  
Tandem Repeats ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Inverted Repeats ◽  
Dna Rearrangement ◽  
Dna Rearrangements ◽  
Developmentally Regulated ◽  
Small Family

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.

scholarly journals Restricted epigenetic inheritance of H3K9 methylation

Science ◽  
2015 ◽  
Vol 348 (6230) ◽  
pp. 132-135 ◽  
Author(s):  
Pauline N. C. B. Audergon ◽  
Sandra Catania ◽  
Alexander Kagansky ◽  
Pin Tong ◽  
Manu Shukla ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Histone H3 ◽  
Epigenetic Inheritance ◽  
Histone Demethylase ◽  
Epigenetic Mark ◽  
H3k9 Methylation ◽  
Silent Chromatin ◽  
Wild Type ◽  
Heterochromatin Formation ◽  
Posttranslational Histone Modifications

Posttranslational histone modifications are believed to allow the epigenetic transmission of distinct chromatin states, independently of associated DNA sequences. Histone H3 lysine 9 (H3K9) methylation is essential for heterochromatin formation; however, a demonstration of its epigenetic heritability is lacking. Fission yeast has a single H3K9 methyltransferase, Clr4, that directs all H3K9 methylation and heterochromatin. Using releasable tethered Clr4 reveals that an active process rapidly erases H3K9 methylation from tethering sites in wild-type cells. However, inactivation of the putative histone demethylase Epe1 allows H3K9 methylation and silent chromatin maintenance at the tethering site through many mitotic divisions, and transgenerationally through meiosis, after release of tethered Clr4. Thus, H3K9 methylation is a heritable epigenetic mark whose transmission is usually countered by its active removal, which prevents the unauthorized inheritance of heterochromatin.

scholarly journals Mutations in Pdd1 Reveal Distinct Requirements for Its Chromodomain and Chromoshadow Domain in Directing Histone Methylation and Heterochromatin Elimination

2013 ◽  
Vol 13 (2) ◽  
pp. 190-201 ◽  
Author(s):  
Rachel M. Schwope ◽  
Douglas L. Chalker
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Histone H3 ◽  
Amino Acid Residues ◽  
Focus Formation ◽  
Content Type ◽  
Genome Wide ◽  
Somatic Genome ◽  
Low Levels

ABSTRACTPdd1, a specialized HP1-like protein, is required for genome-wide DNA rearrangements that restructure a previously silent germ line genome into an active somatic genome during macronuclear differentiation ofTetrahymena thermophila. We deleted or otherwise mutated conserved regions of the protein to investigate how its different domains promote the excision of thousands of internal eliminated sequences (IESs). Previous studies revealed that Pdd1 contributes to recognition of IES loci after they are targeted by small-RNA-guided methylation of histone H3 on lysine 27 (H3K27), subsequently aids the establishment of H3K9 methylation, and recruits proteins that lead to excision. The phenotypes we observed for different Pdd1 alleles showed that each of the two chromodomains and the chromoshadow domain (CSD) have distinct contributions during somatic genome differentiation. Chromodomain 1 (CD1) is essential for conjugation as either its deletion or the substitution of two key aromatic amino acid residues (the W97A W100A mutant) is lethal. These mutations caused mislocalization of a cyan fluorescent protein (CFP)-tagged protein, prevented the establishment of histone H3 dimethylated on K9 (H3K9me2), and abolished IES excision. Nevertheless, the requirement for CD1 could be bypassed by recruiting Pdd1 directly to an IES by addition of a specific DNA binding domain. Chromodomain 2 (CD2) was necessary for producing viable progeny, but low levels of H3K9me2 and IES excision still occurred. A mutation in the chromoshadow domain (CSD) prevented Pdd1 focus formation but still permitted ∼17% of conjugants to produce viable progeny. However, this mutant was unable to stimulate excision when recruited to an ectopic IES, indicating that this domain is important for recruitment of excision factors.

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