Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila

DNA deletion by site-specific chromosome breakage and rejoining occurs extensively during macronuclear development in the ciliate Tetrahymena thermophila. We have sequenced both the micronuclear (germ line) and rearranged macronuclear (somatic) forms of one region from which 1.1 kilobases of micronuclear DNA are reproducibly deleted during macronuclear development. The deletion junctions lie within a pair of 6-base-pair direct repeats. The termini of the deleted sequence are not inverted repeats. The precision of deletion at the nucleotide level was also characterized by hybridization with a synthetic oligonucleotide matching the determined macronuclear (rejoined) junction sequence. This deletion occurs in a remarkably sequence-specific manner. However, a very minor degree of variability in the macronuclear junction sequences was detected and was shown to be inherent in the mechanism of deletion itself. These results suggest that DNA deletion during macronuclear development in T. thermophila may constitute a novel type of DNA recombination and that it can create sequence heterogeneity on the order of a few base pairs at rejoining junctions.

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Role of Micronucleus-Limited DNA in Programmed Deletion of mse2.9 during Macronuclear Development of Tetrahymena thermophila

Eukaryotic Cell ◽

10.1128/ec.3.2.288-301.2004 ◽

2004 ◽

Vol 3 (2) ◽

pp. 288-301 ◽

Cited By ~ 6

Author(s):

Jeffrey S. Fillingham ◽

Ronald E. Pearlman

Keyword(s):

Epigenetic Regulation ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Sexual Cycle ◽

Ciliated Protozoan ◽

Dna Rearrangements ◽

Macronuclear Development ◽

Dna Deletion

ABSTRACT Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.

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A Developmentally Regulated Gene, ASI2, Is Required for Endocycling in the Macronuclear Anlagen of Tetrahymena

Eukaryotic Cell ◽

10.1128/ec.00089-10 ◽

2010 ◽

Vol 9 (9) ◽

pp. 1343-1353 ◽

Cited By ~ 11

Author(s):

Lihui Yin ◽

Susan T. Gater ◽

Kathleen M. Karrer

Keyword(s):

Signal Transduction ◽

Mitotic Cycle ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Ciliated Protozoa ◽

Dna Rearrangement ◽

Developmentally Regulated ◽

Content Type ◽

Molecular Events ◽

Multicellular Organisms

ABSTRACT Ciliated protozoa contain two types of nuclei, germ line micronuclei (Mic) and transcriptionally active macronuclei (Mac). During sexual reproduction, the parental Mac degenerates and a new Mac develops from a mitotic product of the zygotic Mic. Macronuclear development involves extensive endoreplication of the genome. The present study shows that endoreplication of macronuclear DNA in Tetrahymena is an example of endocyling, a variant of the mitotic cycle with alternating S and G phases in the absence of cell division. Thus, endocycling is conserved from ciliates to multicellular organisms. The gene ASI2 in Tetrahymena thermophila encodes a putative signal transduction receptor. ASI2 is nonessential for vegetative growth, but it is upregulated during development of the new Mac. Cells that lack ASI2 in the developing Mac anlagen are arrested in endoreplication of the DNA and die. This study shows that ASI2 is also transcribed in the parental Mac early in conjugation and that transcription of ASI2 in the parental Mac supports endoreplication of the DNA during early stages of development of the Mac anlagen. Other molecular events in Mac anlage development, including developmentally regulated DNA rearrangement, occur normally in matings between ASI2 knockouts, suggesting that ASI2 specifically regulates endocycling in Tetrahymena.

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An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.163-173.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 163-173

Author(s):

T Wang ◽

C D Allis

Keyword(s):

Cell Cycle ◽

Tetrahymena Thermophila ◽

Polyclonal Antibodies ◽

Dna Recombination ◽

High Mobility ◽

High Mobility Group ◽

Sexual Cycle ◽

Developmentally Regulated ◽

Early Stages ◽

Cycle Dependent

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

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A Novel Chromodomain Protein, Pdd3p, Associates with Internal Eliminated Sequences during Macronuclear Development inTetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.20.11.4128-4134.2000 ◽

2000 ◽

Vol 20 (11) ◽

pp. 4128-4134 ◽

Cited By ~ 47

Author(s):

Mikhail A. Nikiforov ◽

Martin A. Gorovsky ◽

C. David Allis

Keyword(s):

De Novo ◽

Germ Line ◽

Chromosome Breakage ◽

Specific Protein ◽

Developmentally Regulated ◽

Dna Elimination ◽

Internal Eliminated Sequences ◽

Subsequent Elimination ◽

Rearrangement Processes

ABSTRACT Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

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Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3947-3950.1988 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3947-3950

Author(s):

C F Austerberry ◽

M C Yao

Keyword(s):

Dna Sequences ◽

Base Pair ◽

Tetrahymena Thermophila ◽

Developmental Regulation ◽

Direct Repeat ◽

Similar Sequence ◽

Developmentally Regulated ◽

Dna Deletion ◽

Inverted Orientation ◽

The Right

Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C.F. Austerberry, C.D. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.

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An abundant high-mobility-group-like protein is targeted to micronuclei in a cell cycle-dependent and developmentally regulated fashion in Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.163 ◽

1993 ◽

Vol 13 (1) ◽

pp. 163-173 ◽

Cited By ~ 7

Author(s):

T Wang ◽

C D Allis

Keyword(s):

Cell Cycle ◽

Tetrahymena Thermophila ◽

Polyclonal Antibodies ◽

Dna Recombination ◽

High Mobility ◽

High Mobility Group ◽

Sexual Cycle ◽

Developmentally Regulated ◽

Early Stages ◽

Cycle Dependent

In this report, we have demonstrated for the first time that an abundant high-mobility-group (HMG)-like protein, HMG B, previously thought to be specific to macronuclei in Tetrahymena thermophila, is also present in micronuclei. Biochemical data document the fact that HMG B is extremely labile in micronuclei. Unless extreme precautions are taken during the isolation of nuclei (addition of 1% formaldehyde to the nucleus isolation buffer), HMG B is not detected in micronuclei. Using polyclonal antibodies highly selective for HMG B, immunoblotting and immunofluorescence analyses show that the presence of HMG B in micronuclei is dynamic, correlating well with known periods of micronuclear DNA replication. This is the case not only during the vegetative cell cycle but also during early stages of the sexual cycle, conjugation, when the presence of HMG B in micronuclei is also closely correlated with meiotic DNA recombination and repair. Since micronuclei are transcriptionally inactive during vegetative growth, our data lend support to the idea that HMG B does not function exclusively in the establishment of transcriptionally competent chromatin. However, micronuclei are transcriptionally active during early stages of conjugation. Evidence that HMG B is strongly synthesized and deposited into micronuclei during this stage is presented. Therefore, it is tempting to suggest that HMG B may play an important role in remodeling micronuclear chromatin into an "active," more open configuration. We favor a model wherein HMG B, like other abundant, low-specificity HMG box-containing proteins, functions to wrap DNA, presumably modulating higher-order chromatin structure for a broad range of biological processes, including transcription and replication.

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

Eukaryotic Cell ◽

10.1128/ec.2.4.678-689.2003 ◽

2003 ◽

Vol 2 (4) ◽

pp. 678-689 ◽

Cited By ~ 14

Author(s):

Jeffrey D. Wuitschick ◽

Kathleen M. Karrer

Keyword(s):

Family Member ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Dna Rearrangement ◽

Developmentally Regulated ◽

Elimination Process ◽

Dna Elimination ◽

Flanking Sequences ◽

Foreign Dna

ABSTRACT Tlr elements are a novel family of ∼30 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. Macronucleus-retained 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 ∼23-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.

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Lia1p, a Novel Protein Required during Nuclear Differentiation for Genome-Wide DNA Rearrangements in Tetrahymena thermophila

Eukaryotic Cell ◽

10.1128/ec.00157-07 ◽

2007 ◽

Vol 6 (8) ◽

pp. 1320-1329 ◽

Cited By ~ 27

Author(s):

Charles H. Rexer ◽

Douglas L. Chalker

Keyword(s):

Genome Rearrangement ◽

Fluorescent Protein ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Chromatin Modification ◽

Striking Similarity ◽

Macronuclear Development ◽

Genome Wide ◽

Green Fluorescent ◽

Novel Protein

ABSTRACT Extensive genome-wide rearrangements occur during somatic macronuclear development in Tetrahymena thermophila. These events are guided by RNA interference-directed chromatin modification including histone H3 lysine 9 methylation, which marks specific germ line-limited internal eliminated sequences (IESs) for excision. Several genes putatively involved in these developmental genome rearrangements were identified based on their proteins' localization to differentiating somatic nuclei, and here we demonstrate that one, LIA1, encodes a novel protein that is an essential component of the genome rearrangement machinery. A green fluorescent protein-Lia1 fusion protein exhibited dynamic nuclear localization during development that has striking similarity to that of the dual chromodomain-containing DNA rearrangement protein, Pdd1p. Coimmunoprecipitation experiments showed that Lia1p associates with Pdd1p and IES chromatin during macronuclear development. Cell lines in which we disrupted both the germ line and somatic copies of LIA1 (ΔLIA1) grew normally but were unable to generate viable progeny, arresting late in development just prior to returning to vegetative growth. These mutant lines failed to properly form Pdd1p-containing nuclear structures and eliminate IESs despite showing normal levels of H3K9 methylation. These data indicate that Lia1p is required late in conjugation for the reorganization of the Tetrahymena genome.

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The Highly Conserved Family of Tetrahymena thermophila Chromosome Breakage Elements Contains an Invariant 10-Base-Pair Core

Eukaryotic Cell ◽

10.1128/ec.5.4.771-780.2006 ◽

2006 ◽

Vol 5 (4) ◽

pp. 771-780 ◽

Cited By ~ 16

Author(s):

Eileen P. Hamilton ◽

Sondra Williamson ◽

Sandra Dunn ◽

Virginia Merriam ◽

Cindy Lin ◽

...

Keyword(s):

Nucleotide Sequence ◽

Sequence Variation ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Chromosome Breakage ◽

Whole Genome Sequence ◽

Unicellular Eukaryote ◽

Whole Genome ◽

Chromosome Fragmentation ◽

Necessary And Sufficient

ABSTRACT As a typical ciliate, Tetrahymena thermophila is a unicellular eukaryote that exhibits nuclear dimorphism: each cell contains a diploid, germ line micronucleus (MICN) and a polyploid, somatic macronucleus (MACN). During conjugation, when a new MACN differentiates from a mitotic descendant of the diploid fertilization nucleus, the five MICN chromosomes are site-specifically fragmented into 250 to 300 MACN chromosomes. The classic chromosome breakage sequence (CBS) is a 15-bp element (TAAACCAACCTCTTT) reported to be necessary and sufficient for chromosome breakage. To determine whether a CBS is present at every site of chromosome fragmentation and to assess the range of sequence variation tolerated, 31 CBSs were isolated without preconception as to the sequence of the chromosome breakage element. Additional CBS-related sequences were identified in the whole-genome sequence by their similarities to the classic CBS. Forty CBS elements behaved as authentic chromosome breakage sites. The CBS nucleotide sequence is more diverse than previously thought: nearly half of the CBS elements identified by unbiased methods have a variant of the classic CBS. Only an internal 10-bp core is completely conserved, but the entire 15-bp chromosome breakage sequence shows significant sequence conservation. Our results suggest that any one member of the CBS family provides a necessary and sufficient cis element for chromosome breakage. No chromosome breakage element totally unrelated to the classic CBS element was found; such elements, if they exist at all, must be rare.

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