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

2004 ◽  
Vol 3 (2) ◽  
pp. 288-301 ◽  
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.

scholarly journals A Non-Long Terminal Repeat Retrotransposon Family Is Restricted to the Germ Line Micronucleus of the Ciliated Protozoan Tetrahymena thermophila

2004 ◽  
Vol 3 (1) ◽  
pp. 157-169 ◽  
Author(s):  
Jeffrey S. Fillingham ◽  
Trine A. Thing ◽  
Nama Vythilingum ◽  
Alex Keuroghlian ◽  
Deanna Bruno ◽  
...  
Keyword(s):  
Long Terminal Repeat ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Terminal Repeat ◽  
Sexual Cycle ◽  
Northern Analysis ◽  
Ciliated Protozoan ◽  
Dna Rearrangements ◽  
Retrotransposon Family ◽  
The Relationship

ABSTRACT The ciliated protozoan Tetrahymena thermophila undergoes extensive programmed DNA rearrangements during the development of a somatic macronucleus from the germ line micronucleus in its sexual cycle. To investigate the relationship between programmed DNA rearrangements and transposable elements, we identified several members of a family of non-long terminal repeat (LTR) retrotransposons (retroposons) in T. thermophila, the first characterized in the ciliated protozoa. This multiple-copy retrotransposon family is restricted to the micronucleus of T. thermophila. The REP (Tetrahymena non-LTR retroposon) elements encode an ORF2 typical of non-LTR elements that contains apurinic/apyrimidinic endonuclease (APE) and reverse transcriptase (RT) domains. Phylogenetic analysis of the RT and APE domains indicates that the element forms a deep-branching clade within the non-LTR retrotransposon family. Northern analysis with a probe to the conserved RT domain indicates that transcripts from the element are small and heterogeneous in length during early macronuclear development. The presence of a repeated transposable element in the genome is consistent with the model that programmed DNA deletion in T. thermophila evolved as a method of eliminating deleterious transposons from the somatic macronucleus.

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 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 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 Abnormal Micronuclear Telomeres Lead to an Unusual Cell Cycle Checkpoint and Defects in Tetrahymena Oral Morphogenesis

2008 ◽  
Vol 7 (10) ◽  
pp. 1712-1723 ◽  
Author(s):  
Karen E. Kirk ◽  
Christina Christ ◽  
Jennifer M. McGuire ◽  
Arun G. Paul ◽  
Mithaq Vahedi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Cell Cycle Checkpoint ◽  
Dna Damage Checkpoint ◽  
Telomeric Dna ◽  
Spindle Apparatus ◽  
Cycle Checkpoint

ABSTRACT Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.

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 DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

2017 ◽  
Vol 114 (28) ◽  
pp. 7408-7413 ◽  
Author(s):  
Yan Jin ◽  
Yaohui Chen ◽  
Shimin Zhao ◽  
Kun-Liang Guan ◽  
Yuan Zhuang ◽  
...  
Keyword(s):  
Complex Formation ◽  
Germ Line ◽  
Mechanistic Explanation ◽  
Mass Spectrometry Analysis ◽  
Physical Interaction ◽  
Spectrometry Analysis ◽  
Culture Cells ◽  
Underlying Mechanisms

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA–PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA–PK components Ku70, Ku80, and DNA-PKcs. Overexpression or knockdown of DNA–PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA–PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA–PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA–PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA–PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.

scholarly journals Tudor Nuclease Genes and Programmed DNA Rearrangements in Tetrahymena thermophila

2007 ◽  
Vol 6 (10) ◽  
pp. 1795-1804 ◽  
Author(s):  
Rachel A. Howard-Till ◽  
Meng-Chao Yao
Keyword(s):  
Sexual Reproduction ◽  
Small Rna ◽  
Tetrahymena Thermophila ◽  
Minor Role ◽  
Dna Deletion ◽  
Genome Defense ◽  
Rna Pathways ◽  
A Minor

ABSTRACT Proteins containing a Tudor domain and domains homologous to staphylococcal nucleases are found in a number of eukaryotes. These “Tudor nucleases” have been found to be associated with the RNA-induced silencing complex (A. A. Caudy, R. F. Ketting, S. M. Hammond, A. M. Denli, A. M. Bathoorn, B. B. Tops, J. M. Silva, M. M. Myers, G. J. Hannon, and R. H. Plasterk, Nature 425:411-414, 2003). We have identified two Tudor nuclease gene homologs, TTN1 and TTN2, in the ciliate Tetrahymena thermophila, which has two distinct small-RNA pathways. Characterization of single and double KOs of TTN1 and TTN2 shows that neither of these genes is essential for growth or sexual reproduction. Progeny of TTN2 KOs and double knockouts occasionally show minor defects in the small-RNA-guided process of DNA deletion but appear to be normal in hairpin RNA-induced gene silencing, suggesting that Tudor nucleases play only a minor role in RNA interference in Tetrahymena. Previous studies of Tetrahymena have shown that inserted copies of the neo gene from Escherichia coli are often deleted from the developing macronucleus during sexual reproduction (Y. Liu, X. Song, M. A. Gorovsky, and K. M. Karrer, Eukaryot. Cell 4:421-431, 2005; M. C. Yao, P. Fuller, and X. Xi, Science 300:1581-1584, 2003). This transgene deletion phenomenon is hypothesized to be a form of genome defense. Analysis of the Tudor nuclease mutants revealed exceptionally high rates of deletion of the neo transgene at the TTN2 locus but no deletion at the TTN1 locus. When present in the same genome, however, the neo gene is deleted at high rates even at the TTN1 locus, further supporting a role for trans-acting RNA in this process. This deletion is not affected by the presence of the same sequence in the macronucleus, thus providing a counterargument for the role of the macronuclear genome in specifying all sequences for deletion.

scholarly journals Germline deletion reveals a non-essential role of the atypical MAPK6/ERK3

2018 ◽  
Author(s):  
N. Ronkina ◽  
K. Schuster-Gossler ◽  
F. Hansmann ◽  
H. Kunze-Schumacher ◽  
I. Sandrock ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Knockout Mice ◽  
Essential Role ◽  
Germ Line ◽  
Exon 2 ◽  
Signaling Complex ◽  
Cell Functions

AbstractMAPK6/ERK3 is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary disfunction and by defects in function, activation and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon3 flanked by LoxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing first evidence for the existence of the ERK3/MK5 signaling complex in vivo. In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile, do not display pulmonary hypoplasia, acute respiratory failure, abnormal T cell development, reduction of thymocyte numbers or altered T cells selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality, lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin-resistance-cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal suggesting redundant functions of both protein kinases.

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.

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