scholarly journals Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila.

1989 ◽  
Vol 9 (3) ◽  
pp. 1092-1099 ◽  
Author(s):  
M C Yao ◽  
C H Yao
Keyword(s):  
Developmental Stages ◽  
Transformation Efficiency ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Rrna Genes ◽  
Somatic Nucleus ◽  
Dna Breakage ◽  
Cis Acting ◽  
Palindromic Structure ◽  
Rdna Amplification

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.

Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila

1989 ◽  
Vol 9 (3) ◽  
pp. 1092-1099
Author(s):  
M C Yao ◽  
C H Yao
Keyword(s):  
Developmental Stages ◽  
Transformation Efficiency ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Rrna Genes ◽  
Somatic Nucleus ◽  
Dna Breakage ◽  
Cis Acting ◽  
Palindromic Structure ◽  
Rdna Amplification

The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome. These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus). In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T. thermophila. Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA. Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation. The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers. Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T. thermophila.

scholarly journals Transformation of Tetrahymena thermophila with hypermethylated rRNA genes.

1988 ◽  
Vol 8 (4) ◽  
pp. 1664-1669 ◽  
Author(s):  
K M Karrer ◽  
M C Yao
Keyword(s):  
Cell Lines ◽  
Transformation Efficiency ◽  
Tetrahymena Thermophila ◽  
Host Cells ◽  
Rrna Genes ◽  
Major Constituent ◽  
Clonal Cell ◽  
Clonal Cell Lines

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

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 The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila

1989 ◽  
Vol 9 (2) ◽  
pp. 452-460
Author(s):  
P C Yaeger ◽  
E Orias ◽  
W L Shaiu ◽  
D D Larson ◽  
E H Blackburn
Keyword(s):  
Inbred Strain ◽  
Southern Blot Analysis ◽  
Tetrahymena Thermophila ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Wild Type ◽  
Recombinational Event ◽  
Somatic Nucleus ◽  
Somatic Recombination ◽  
General Method

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.

scholarly journals The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila.

1989 ◽  
Vol 9 (2) ◽  
pp. 452-460 ◽  
Author(s):  
P C Yaeger ◽  
E Orias ◽  
W L Shaiu ◽  
D D Larson ◽  
E H Blackburn
Keyword(s):  
Inbred Strain ◽  
Southern Blot Analysis ◽  
Tetrahymena Thermophila ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Wild Type ◽  
Recombinational Event ◽  
Somatic Nucleus ◽  
Somatic Recombination ◽  
General Method

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.

scholarly journals Transformation of Tetrahymena thermophila with hypermethylated rRNA genes

1988 ◽  
Vol 8 (4) ◽  
pp. 1664-1669
Author(s):  
K M Karrer ◽  
M C Yao
Keyword(s):  
Cell Lines ◽  
Transformation Efficiency ◽  
Tetrahymena Thermophila ◽  
Host Cells ◽  
Rrna Genes ◽  
Major Constituent ◽  
Clonal Cell ◽  
Clonal Cell Lines

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5'-NAT-3', the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations.

scholarly journals A GENETIC LOCUS HAVING TRANS AND CONTIGUOUS CIS FUNCTIONS THAT CONTROL THE DISPROPORTIONATE REPLICATION OF RIBOSOMAL RNA GENES IN DROSOPHILA MELANOGASTER

Genetics ◽  
1978 ◽  
Vol 88 (1) ◽  
pp. 67-79
Author(s):  
James D Procunier ◽  
Kenneth D Tartof
Keyword(s):  
Ribosomal Rna ◽  
Germ Line ◽  
Genetic Locus ◽  
Position Effect ◽  
Ribosomal Rna Genes ◽  
Rrna Genes ◽  
Position Effect Variegation ◽  
Compensatory Response ◽  
Cis Acting ◽  
Rna Genes

ABSTRACT The results of deficiency mapping experiments reveal the presence of a compensatory response (c r +) locus that is located distal to the cluster of ribosomal RNA (rRNA) genes and is responsible for disproportionately replicating these genes when cr+ locus is present in a single dose, as in X/O males or X / SC4 - Sc8 females. The cr+ locus is novel in that it exhibits both trans and contiguous cis acting properties in somatic cells. It acts in trans to detect the presence of its partner locus in the opposite homolog, and if that partner locus is absent, it acts in cis to drive the disproportionate replication of those rRNA genes (rDNA) that are contiguous with it. The ability of cr+ to function is independent of the number of ribosomal RNA genes present. Furthermore, it can be shown that the cr+ locus is not required for the magnification or reduction of germ line rDNA. Finally, the implications of cr+ for position-effect variegation and the apparent reversion of the abnormal oocyte (abo) phenotype are discussed.

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.

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.

Elimination of micronuclear specific DNA sequences early in anlagen development

1985 ◽  
Vol 5 (1) ◽  
pp. 93-98
Author(s):  
C F Brunk ◽  
R K Conover
Keyword(s):  
Developmental Stage ◽  
Dna Sequences ◽  
Developmental Stages ◽  
Tetrahymena Thermophila ◽  
Southern Analysis ◽  
Limited Sequence

After conjugation in Tetrahymena thermophila, the old macronuclei degenerate, and new macronuclei (anlagen) develop. During anlagen development a number of DNA sequences found in the micronuclear genome (micronuclear limited sequences) are eliminated from the anlagen. A cloned copy of a repetitive micronuclear limited sequence has been used to determine the developmental stage at which micronuclear limited sequences are eliminated. DNAs from anlagen of various developmental stages were examined by Southern analysis. It was found that micronuclear limited sequences are present in 4C anlagen and essentially absent in 8C and 16C anlagen. The precipitous loss of these sequences in the 8C anlagen rules out under-replication as the mechanism for the loss and suggests that these sequences are specifically degraded early during anlagen development.

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