“…Although MNase can degrade both DNA and RNA (Cuatrecasas et al, 1967), Tetrahymena MAC (Karrer, 2012), with high transcription activities, numerous nucleoli, and abundant rRNA, may present a particular challenge. To address this concern, RNase A treatment was carried out after MNase digestion ( Figure 3E).…”
Section: Optimization Of Mnase Digestion For Macmentioning
confidence: 99%
“…Like most ciliates, Tetrahymena exhibits nuclear dimorphism, containing one micronucleus (MIC) and one macronucleus (MAC) in the same cell compartment ( Figure 1F) (Karrer, 2012). MIC is the germline nucleus that ensures the transmission of genetic information from generation to generation, while MAC is the somatic nucleus that provides "house-keeping" func- ) indicates the membranelles.…”
Section: Introductionmentioning
confidence: 99%
“…MIC is diploid and transcriptionally silent except during sexual development (conjugation), whereas MAC is polyploidy (about 45×) and transcriptionally active during vegetative growth (Karrer, 2012). The zygotic nucleus, developed from parent MIC, differentiates into MAC during conjugation (Greider and Blackburn, 1985).…”
Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus (MIC) and macronucleus (MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease (MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of ~200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.
“…Although MNase can degrade both DNA and RNA (Cuatrecasas et al, 1967), Tetrahymena MAC (Karrer, 2012), with high transcription activities, numerous nucleoli, and abundant rRNA, may present a particular challenge. To address this concern, RNase A treatment was carried out after MNase digestion ( Figure 3E).…”
Section: Optimization Of Mnase Digestion For Macmentioning
confidence: 99%
“…Like most ciliates, Tetrahymena exhibits nuclear dimorphism, containing one micronucleus (MIC) and one macronucleus (MAC) in the same cell compartment ( Figure 1F) (Karrer, 2012). MIC is the germline nucleus that ensures the transmission of genetic information from generation to generation, while MAC is the somatic nucleus that provides "house-keeping" func- ) indicates the membranelles.…”
Section: Introductionmentioning
confidence: 99%
“…MIC is diploid and transcriptionally silent except during sexual development (conjugation), whereas MAC is polyploidy (about 45×) and transcriptionally active during vegetative growth (Karrer, 2012). The zygotic nucleus, developed from parent MIC, differentiates into MAC during conjugation (Greider and Blackburn, 1985).…”
Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus (MIC) and macronucleus (MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease (MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of ~200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.
“…Since the micronucleus is transcriptionally silent before meiosis and there is no IES in the transcriptionally active macronucleus, RNA is very unlikely to play any roles here. There is no cytosine methylation in Tetrahymena, while adenine methylation is most likely excluded from the micronucleus (Karrer 2012). That leaves only histone modifications associated with heterochromatin, which is paradoxically connected with ncRNA transcription.…”
Section: Coding or Noncoding That Is The Questionmentioning
confidence: 99%
“…Like most ciliated protozoa, Tetrahymena contains in the same cytoplasmic compartment a somatic macronucleus that is transcriptionally active and a germline micronucleus that is transcriptionally inert in asexually dividing cells (Karrer 2012). Dramatic genome rearrangement occurs when the micronucleus differentiates into the macronucleus during conjugation, the sexual phase of the Tetrahymena life cycle.…”
There is growing evidence to support the notion that small RNAs derived from noncoding RNAs (ncRNAs) are mobile carriers of epigenetic information in diverse eukaryotic systems. However, challenges remain in defining what messages are being sent and how. In the August 1, 2012, issue of Genes & Development, Schoeberl and colleagues (pp. 1729–1742) reported a detailed analysis of the turnover of small RNAs during the sexual reproduction of the ciliated protozoan Tetrahymena. The results revealed surprisingly complicated roles played by small RNAs in shaping the communication between the germline and the soma.
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