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

2000 ◽  
Vol 20 (11) ◽  
pp. 4128-4134 ◽  
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.

scholarly journals Developmentally Regulated Chromosome Fragmentation Linked to Imprecise Elimination of Repeated Sequences in Paramecia

2003 ◽  
Vol 2 (5) ◽  
pp. 1076-1090 ◽  
Author(s):  
Anne Le Mouël ◽  
Alain Butler ◽  
François Caron ◽  
Eric Meyer
Keyword(s):  
De Novo ◽  
Germ Line ◽  
Single Copy ◽  
Mendelian Inheritance ◽  
Direct Repeats ◽  
Developmentally Regulated ◽  
Chromosome Fragmentation ◽  
Dna Elimination ◽  
Flanking Sequences ◽  
Identical Cell

ABSTRACT The chromosomes of ciliates are fragmented at reproducible sites during the development of the polyploid somatic macronucleus, but the mechanisms involved appear to be quite diverse in different species. In Paramecium aurelia, the process is imprecise and results in de novo telomere addition at locally heterogeneous positions. To search for possible determinants of chromosome fragmentation, we have studied an ∼21-kb fragmentation region from the germ line genome of P. primaurelia. The mapping and sequencing of alternative macronuclear versions of the region show that two distinct multicopy elements, a minisatellite and a degenerate transposon copy, are eliminated by an imprecise mechanism leading either to chromosome fragmentation and the formation of new telomeres or to the rejoining of flanking sequences. Heterogeneous internal deletions occur between short direct repeats containing TA dinucleotides. The complex rearrangement patterns produced vary slightly among genetically identical cell lines, show non-Mendelian inheritance during sexual reproduction, and can be experimentally modified by transformation of the maternal macronucleus with homologous sequences. These results suggest that chromosome fragmentation in Paramecium is the consequence of imprecise DNA elimination events that are distinct from the precise excision of single-copy internal eliminated sequences and that target multicopy germ line sequences by homology-dependent epigenetic mechanisms.

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

1987 ◽  
Vol 7 (1) ◽  
pp. 435-443 ◽  
Author(s):  
C F Austerberry ◽  
M C Yao
Keyword(s):  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Dna Recombination ◽  
Chromosome Breakage ◽  
Base Pairs ◽  
Developmentally Regulated ◽  
Junction Sequence ◽  
Macronuclear Development ◽  
Dna Deletion ◽  
Minor Degree

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.

scholarly journals The SUMO Pathway Is Developmentally Regulated and Required for Programmed DNA Elimination in Paramecium tetraurelia

2006 ◽  
Vol 5 (5) ◽  
pp. 806-815 ◽  
Author(s):  
Atsushi Matsuda ◽  
James D. Forney
Keyword(s):  
Sexual Reproduction ◽  
Germ Line ◽  
Developmental Regulation ◽  
Dna Amplification ◽  
Northern Analysis ◽  
Paramecium Tetraurelia ◽  
Developmentally Regulated ◽  
Gene Encoding ◽  
Dna Elimination ◽  
Sumo Pathway

ABSTRACT Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.

scholarly journals Role of Histone Deacetylation in Developmentally Programmed DNA Rearrangements in Tetrahymena thermophila

2002 ◽  
Vol 1 (2) ◽  
pp. 293-303 ◽  
Author(s):  
Sandra Duharcourt ◽  
Meng-Chao Yao
Keyword(s):  
Molecular Mechanisms ◽  
Germ Line ◽  
Trichostatin A ◽  
Chromosome Breakage ◽  
Histone Deacetylation ◽  
Subnuclear Localization ◽  
Dna Elimination ◽  
Deacetylase Inhibitor ◽  
Histone Deacetylase Inhibitor Trichostatin

ABSTRACT In Tetrahymena, as in other ciliates, development of the somatic macronucleus during conjugation involves extensive and reproducible rearrangements of the germ line genome, including chromosome fragmentation and excision of internal eliminated sequences (IESs). The molecular mechanisms controlling these events are poorly understood. To investigate the role that histone acetylation may play in the regulation of these processes, we treated Tetrahymena cells during conjugation with the histone deacetylase inhibitor trichostatin A (TSA). We show that TSA treatment induces developmental arrests in the early stages of conjugation but does not significantly affect the progression of conjugation once the mitotic divisions of the zygotic nucleus have occurred. Progeny produced from TSA-treated cells were examined for effects on IES excision and chromosome breakage. We found that TSA treatment caused partial inhibition of excision of five out of the six IESs analyzed but did not affect chromosome breakage at four different sites. TSA treatment greatly delayed in some cells and inhibited in most the excision events in the developing macronucleus. It also led to loss of the specialized subnuclear localization of the chromodomain protein Pdd1p that is normally associated with DNA elimination. We propose a model in which underacetylated nucleosomes mark germ line-limited sequences for excision.

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

2003 ◽  
Vol 2 (4) ◽  
pp. 678-689 ◽  
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.

scholarly journals THE ABSENCE OF SOMATIC EFFECTS OF P-M HYBRID DYSGENESIS IN DROSOPHILA MELANOGASTER

Genetics ◽  
1986 ◽  
Vol 113 (4) ◽  
pp. 897-918
Author(s):  
M Catharine McElwain
Keyword(s):  
Drosophila Melanogaster ◽  
Size Distribution ◽  
De Novo ◽  
Germ Line ◽  
Adaptive Significance ◽  
Hybrid Dysgenesis ◽  
De Novo Mutation ◽  
Anatomical Distribution ◽  
Parallel Processes

ABSTRACT The wings and abdomens of dysgenic and nondysgenic control flies were scored for the presence of clones of cells mutant for first and third chromosome markers. These exceptional clones can arise from mitotic recombination, de novo mutation or deletion, and P-M hybrid dysgenesis has been shown to increase the frequency of parallel processes occurring in germ-line cells. Particular attention was given to careful genetic and molecular characterization of all stocks and to providing adequate and appropriate controls so that even very small increases in somatic clone frequency due to P-M hybrid dysgenesis would be detected. No difference was found in the frequency, size distribution or anatomical distribution of mutant somatic clones correlated to hybrid dysgenesis, confirming previous indications. The potential adaptive significance of a germ-line restriction of P-M hybrid dysgenesis is discussed.

scholarly journals Zygotic Expression of the Double-Stranded RNA Binding Motif Protein Drb2p Is Required for DNA Elimination in the Ciliate Tetrahymena thermophila

2011 ◽  
Vol 10 (12) ◽  
pp. 1648-1659 ◽  
Author(s):  
Jason A. Motl ◽  
Douglas L. Chalker
Keyword(s):  
Rna Binding ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Rna Binding Proteins ◽  
Chromosome Breakage ◽  
Binding Motif ◽  
Double Stranded Rna ◽  
Content Type ◽  
Dna Elimination ◽  
Genes Encoding

ABSTRACTDouble-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1andDRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliateTetrahymena thermophila.Both proteins are expressed throughout growth and development but exhibit distinct peaks of expression, suggesting different biological roles. In support of this, we show that expression ofDRB2is essential for vegetative growth whileDRB1expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking allDRB1copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ lineDRB2copies, which thus cannot express Drb2p zygotically, fail to produce progeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize the essential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination and chromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclear structures, which are necessary for reorganization of the somatic genome.

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

1987 ◽  
Vol 7 (1) ◽  
pp. 435-443 ◽  
Author(s):  
C F Austerberry ◽  
M C Yao
Keyword(s):  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Dna Recombination ◽  
Chromosome Breakage ◽  
Base Pairs ◽  
Developmentally Regulated ◽  
Junction Sequence ◽  
Macronuclear Development ◽  
Dna Deletion ◽  
Minor Degree

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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