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

2010 ◽  
Vol 9 (9) ◽  
pp. 1343-1353 ◽  
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.

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 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.

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 SUMOylation Is Developmentally Regulated and Required for Cell Pairing during Conjugation in Tetrahymena thermophila

2014 ◽  
Vol 14 (2) ◽  
pp. 170-181 ◽  
Author(s):  
Amjad M. Nasir ◽  
Qianyi Yang ◽  
Douglas L. Chalker ◽  
James D. Forney
Keyword(s):  
Sexual Reproduction ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Covalent Attachment ◽  
Sexual Life ◽  
Dynamic Regulation ◽  
Content Type ◽  
Sumo Pathway

ABSTRACT The covalent attachment of s mall u biquitin-like mo difier (SUMO) to target proteins regulates numerous nuclear events in eukaryotes, including transcription, mitosis and meiosis, and DNA repair. Despite extensive interest in nuclear pathways within the field of ciliate molecular biology, there have been no investigations of the SUMO pathway in Tetrahymena . The developmental program of sexual reproduction of this organism includes cell pairing, micronuclear meiosis, and the formation of a new somatic macronucleus. We identified the Tetrahymena thermophila SMT3 (SUMO) and UBA2 (SUMO-activating enzyme) genes and demonstrated that the corresponding green fluorescent protein (GFP) tagged gene products are found predominantly in the somatic macronucleus during vegetative growth. Use of an anti-Smt3p antibody to perform immunoblot assays with whole-cell lysates during conjugation revealed a large increase in SUMOylation that peaked during formation of the new macronucleus. Immunofluorescence using the same antibody showed that the increase was localized primarily within the new macronucleus. To initiate functional analysis of the SUMO pathway, we created germ line knockout cell lines for both the SMT3 and UBA2 genes and found both are essential for cell viability. Conditional Smt3p and Uba2p cell lines were constructed by incorporation of the cadmium-inducible metallothionein promoter. Withdrawal of cadmium resulted in reduced cell growth and increased sensitivity to DNA-damaging agents. Interestingly, Smt3p and Uba2p conditional cell lines were unable to pair during sexual reproduction in the absence of cadmium, consistent with a function early in conjugation. Our studies are consistent with multiple roles for SUMOylation in Tetrahymena , including a dynamic regulation associated with the sexual life cycle.

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 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 Role of ATG8 and Autophagy in Programmed Nuclear Degradation in Tetrahymena thermophila

2012 ◽  
Vol 11 (4) ◽  
pp. 494-506 ◽  
Author(s):  
Ming-Liang Liu ◽  
Meng-Chao Yao
Keyword(s):  
Fluorescent Protein ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Critical Role ◽  
Nuclear Periphery ◽  
Unicellular Eukaryote ◽  
Knockout Mutant ◽  
Content Type ◽  
Nuclear Degradation

ABSTRACT Autophagy is an evolutionarily conserved mechanism for the degradation of cellular components, but its role in enucleation during differentiation has not been established. Tetrahymena thermophila is a unicellular eukaryote with two functionally distinct nuclei, the somatic (macro-) and the germ line (micro-) nuclei. These nuclei are produced during sexual reproduction (conjugation), which involves differentiation and selective degradation of several specific nuclei. To examine the role of autophagy in nuclear degradation, we studied the function of two ATG8 genes in Tetrahymena . Through fluorescent protein tagging, we found that both proteins are targeted to degrading nuclei at specific stages, with some enrichment on the nuclear periphery, suggesting the formation of autophagosomes surrounding these nuclei. In addition, ATG8 knockout mutant cells showed a pronounced delay in nuclear degradation without apparently preventing the completion of other developmental events. This evidence provided direct support for a critical role for autophagy in programmed nuclear degradation. The results also showed differential roles for two ATG8 genes, with ATG8-65 playing a more significant role in starvation than ATG8-2 , although both are important in nuclear degradation.

scholarly journals Non-Mendelian, heritable blocks to DNA rearrangement are induced by loading the somatic nucleus of Tetrahymena thermophila with germ line-limited DNA.

1996 ◽  
Vol 16 (7) ◽  
pp. 3658-3667 ◽  
Author(s):  
D L Chalker ◽  
M C Yao
Keyword(s):  
Copy Number ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
High Copy Number ◽  
Dna Rearrangement ◽  
Somatic Nucleus ◽  
Limited Region ◽  
Dna Deletion ◽  
Genes Encoding ◽  
Transformed Cell Lines

Site-specific DNA deletion occurs at thousands of sites within the genome during macronuclear development of Tetrahymena thermophila. These deletion elements are usually not detected in macronuclear chromosomes. We have interfered with the normal deletion of two of these elements, the adjacent M and R elements, by loading vegetative macronuclei with these elements prior to sexual conjugation. Transformed cell lines containing the exogenous M or R element, carried on high-copy-number vectors containing genes encoding rRNA within parental (old) macronuclei, consistently failed to excise chromosomal copies of the M or R element during formation of new macronuclei. Little or no interference with the deletions of adjacent elements or of unlinked elements was observed. The micronucleus (germ line)-limited region of each element was sufficient to inhibit specific DNA deletion. This interference with DNA deletion usually is manifested as a cytoplasmic dominant trait: deletion elements present in the old macronucleus of one partner of a mating pair were sufficient to inhibit deletion occurring in the other partner. Remarkably, the failure to excise these elements became a non-Mendelian, inheritable trait in the next generation and did not require the high copy number of exogenously introduced elements. The introduction of exogenous deletion elements into parental macronuclei provides us with an epigenetic means to establish a heritable pattern of DNA rearrangement.

Sign in / Sign up

Export Citation Format

Share Document