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Brian J Haas - One of the best experts on this subject based on the ideXlab platform.
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refined annotation and assembly of the tetrahymena thermophila genome sequence through est analysis comparative genomic hybridization and targeted gap closure
BMC Genomics, 2008Co-Authors: Robert S Coyne, Mathangi Thiagarajan, Kristie M Jones, Jennifer R Wortman, Luke J Tallon, Brian J HaasAbstract:Background Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a Germline Micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.
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Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure
BMC Genomics, 2008Co-Authors: Robert S Coyne, Mathangi Thiagarajan, Kristie M Jones, Jennifer R Wortman, Luke J Tallon, Brian J Haas, Donna M Cassidy-hanley, Emily A Wiley, Joshua J Smith, Kathleen CollinsAbstract:Background Tetrahymena thermophila , a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a Germline Micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena 's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing. Results We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified. Conclusion We report here significant progress in genome closure and reannotation of Tetrahymena thermophila . Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.
Hans J. Lipps - One of the best experts on this subject based on the ideXlab platform.
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A permissive chromatin structure is adopted prior to site-specific DNA demethylation of developmentally expressed genes involved in macronuclear differentiation
Epigenetics & Chromatin, 2013Co-Authors: Aneta Bulic, Jan Postberg, Franziska Jönsson, Andreas Fischer, Günter Reuter, Hans J. LippsAbstract:Background DNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood. We exploit the minimalistic biological ciliate system to understand the crosstalk between DNA modification and chromatin structure. In the macronucleus of these cells, the DNA is fragmented into individual short DNA molecules, each representing a functional expression and replication unit. Therefore, long range epigenomic interaction can be excluded in this system. Results In the stichotrichous ciliate Stylonychia lemnae , cytosine methylation occurs in a small subset of macronuclear nanochromosomes expressed only during sexual reproduction. Methylation pattern shows similarity to that observed in fungi and Drosophila . Cytosine methylation correlates with gene activity and chromatin structure. Upon gene activation, cytosines become demethylated and a redistribution of histone post-translational modifications (PTMs) takes place. Evidence is presented that the formation of a permissive chromatin structure in the vicinity of the 5meCs precedes cytosine methylation and is probably a necessary prerequisite for their demethylation. Shortly after demethylation of cytosines occurs, the parental macronucleus degenerates, a new macronucleus is formed from a micronuclear derivative and the specific methylation pattern is transmitted from the Germline Micronucleus to the new macronucleus. Conclusions We show that very few, or even only one, discrete methylated cytosines are required to assign regulatory functions at a specific locus. Furthermore, evidence is provided that a permissive chromatin structure is probably a necessary prerequisite for the demethylation of specific cytosines. Our results allow us to propose a mechanistic model for the biological function of cytosine methylation in the ciliate cell and its regulation during the cell cycle.
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Copy number variations of 11 macronuclear chromosomes and their gene expression in Oxytricha trifallax
Gene, 2012Co-Authors: Ke Xu, Hans J. Lipps, Thomas G. Doak, Jingmei Wang, Estienne C. Swart, Wei-jen ChangAbstract:Abstract Ciliated protozoa are peculiar for their nuclear dimorphism, wherein two types of nuclei divide nuclear functions: a Germline Micronucleus (MIC) is transcriptionally inert during vegetative growth, but serves as the genetic blueprint for the somatic macronucleus (MAC), which is responsible for all transcripts supporting cell growth and reproduction. While all the advantages/disadvantages associated with nuclear dimorphism are not clear, an essential advantage seems to be the ability to produce a highly polyploid MAC, which then allows for the maintenance of extremely large single cells — many ciliate cells are larger than small metazoa. In some ciliate classes, chromosomes in the MAC are extensively fragmented to create extremely short chromosomes that often carry single genes, and these chromosomes may be present in different copy numbers, resulting in different ploidies. While using gene copy number to regulate gene expression is limited in most eukaryotic systems, the extensive fragmentation in some ciliate classes provides this opportunity to every MAC gene. However, it is still unclear if this mechanism is in fact used extensively in these ciliates. To address this, we have quantified copy numbers of 11 MAC chromosomes and their gene expression in Oxytricha trifallax (CI: Spirotrichea). We compared copy numbers between two subpopulations of O. trifallax, and copy numbers of 7 orthologous genes between O. trifallax and the closely related Stylonychia lemnae. We show that copy numbers of MAC chromosomes are variable, dynamic, and positively correlated to gene expression. These features might be conserved in all spirotrichs, and might exist in other classes of ciliates with heavily fragmented MAC chromosomes.
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Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development.
Epigenetics & Chromatin, 2008Co-Authors: Jan Postberg, Katharina Heyse, Marion Cremer, Thomas Cremer, Hans J. LippsAbstract:Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the Germline Micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained.
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Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development
Epigenetics & Chromatin, 2008Co-Authors: Jan Postberg, Katharina Heyse, Marion Cremer, Thomas Cremer, Hans J. LippsAbstract:Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the Germline Micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained. Results: We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP) experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic Germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo . ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization. Conclusion: Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis.
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The pathway to detangle a scrambled gene.
PLOS ONE, 2008Co-Authors: Matthias Möllenbeck, Yi Zhou, Andre R. O. Cavalcanti, Franziska Jönsson, Brian P. Higgins, Wei-jen Chang, Stefan Juranek, Thomas G. Doak, Grzegorz Rozenberg, Hans J. LippsAbstract:Background Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20–30% of the genes are estimated to be scrambled in the Germline Micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development. Methodology/Principal Findings To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways. Conclusions/Significance This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.
Kathleen Collins - One of the best experts on this subject based on the ideXlab platform.
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Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci.
RNA, 2017Co-Authors: Brian M. Farley, Kathleen CollinsAbstract:: Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as Tetrahymena have a phenotypically silent Germline Micronucleus and an expressed somatic macronucleus, which is differentiated anew from a Germline genome copy in sexual reproduction. We show that the nuclear-localized Tetrahymena Piwi protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the Germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent Germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.
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Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure
BMC Genomics, 2008Co-Authors: Robert S Coyne, Mathangi Thiagarajan, Kristie M Jones, Jennifer R Wortman, Luke J Tallon, Brian J Haas, Donna M Cassidy-hanley, Emily A Wiley, Joshua J Smith, Kathleen CollinsAbstract:Background Tetrahymena thermophila , a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a Germline Micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena 's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing. Results We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified. Conclusion We report here significant progress in genome closure and reannotation of Tetrahymena thermophila . Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.
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two classes of endogenous small rnas in tetrahymena thermophila
Genes & Development, 2006Co-Authors: Kathleen CollinsAbstract:In diverse eukaryotes from parasitic protozoa to humans, RNA interference (RNAi) pathways regulate gene expression, establish heterochromatin, and/or protect the genome from viruses and mobile DNA elements (Matzke and Birchler 2005; Sontheimer and Carthew 2005). Although the biological function of RNAi varies, central to all pathways are ∼21–30-nucleotide (nt) small noncoding RNAs (sRNAs) that provide specificity for RNA or DNA targets. In multicellular organisms, three major classes of endogenous sRNAs have been characterized in detail: micro RNAs (miRNAs), repeat-associated small interfering RNAs (rasiRNAs), and trans-acting small interfering RNAs (ta-siRNAs) (Bartel 2005; Sontheimer and Carthew 2005). The miRNAs and tasiRNAs direct translational repression and/or degradation of messenger RNAs. The rasiRNAs, derived from repetitive DNA elements such as transposons and centromeres, function to promote heterochromatin formation, DNA methylation, and/or RNA degradation. Less-well-characterized sRNAs include those with precise complementarity to protein-coding genes, pseudogenes, and intergenic regions (e.g., see Ambros et al. 2003). The biogenesis of diverse sRNAs depends on an RNaseIII family nuclease called Dicer (Tomari and Zamore 2005). The Dicer substrates for miRNA production are single-stranded RNAs with stem-loop structures, while precursors to ta-siRNAs and most rasiRNAs are double-stranded RNAs (dsRNAs) resulting from bidirectional transcription or RNA-dependent RNA polymerase activity. Dicer processing of precursors yields short sRNA duplexes of homogeneous length. One strand of each sRNA duplex is stabilized by assembly into an effector ribonucleoprotein (RNP) containing a Piwi/PAZ domain (PPD) protein of the Argonaute family. Multicellular eukaryotes express multiple paralogs of RNAi pathway components that are specialized in function. In contrast to the diversity of sRNAs in multicellular organisms, unicellular eukaryotes are only known to express rasiRNA-like sRNAs (Djikeng et al. 2001; Reinhart and Bartel 2002; Chicas et al. 2004; Ullu et al. 2005). In the free-living ciliated protozoan Tetrahymena thermophila, RNAs ∼26–31 nt in length direct developmentally programmed DNA elimination (Mochizuki and Gorovsky 2004b). T. thermophila, like other ciliates, has nuclear dualism, with a diploid, Germline Micronucleus (MIC) that remains phenotypically silent and a polyploid, transcriptionally active, somatic macronucleus (MAC). When starved for nutrients, T. thermophila ceases to divide vegetatively and becomes competent to reproduce sexually by conjugation. In conjugating cells, new MACs are developed from mitotic siblings of the zygotic MIC in a process involving site-specific chromosome fragmentation and deletion of ∼6000 internally eliminated sequences (IESs). The IESs are single-copy elements or moderately repetitive, transposon-like sequences that together account for ∼15% of the MIC genome (Yao and Chao 2005). DNA elimination occurs under epigenetic regulation: Sequences in the parental MAC can protect corresponding sequences in the developing MAC from elimination. Normal MAC development and the conjugation-induced accumulation of ∼26–31-nt sRNAs require the PPD-containing TWI1 and the Dicer-like DCL1 (Mochizuki et al. 2002; Malone et al. 2005; Mochizuki and Gorovsky 2005). Bidirectional nongenic transcription in the MIC during conjugation (Chalker and Yao 2001) is proposed to provide dsRNA precursors that are processed by Dcl1p into sRNAs (Yao et al. 2003; Mochizuki and Gorovsky 2004b). Northern blot assays have confirmed that a known MIC-limited IES is represented in the conjugation-induced sRNA population (Chalker et al. 2005). In addition, DNA hybridization studies using sRNAs isolated from conjugating cells have suggested that as conjugation progresses, the sRNA population becomes enriched for MIC-limited sequence (Mochizuki and Gorovsky 2004a). To account for this finding and provide a mechanism for the epigenetic influence of the parental MAC, the ∼26–31-nt sRNAs, termed the scan (scn)RNAs, are proposed to enter the parental MAC in association with Twi1p and scan for homologous sequence in a manner that results in degradation of MAC-cognate sRNAs. The sRNAs remaining after parental MAC subtraction are thought to then transit to the developing MAC where they guide the histone H3 Lys 9 (H3K9) methylation of MIC-limited chromatin, which likely marks IESs for subsequent elimination (Taverna et al. 2002; Liu et al. 2004). In this manner, sRNA-guided DNA elimination in T. thermophila is similar to rasiRNA-guided heterochromatin formation in Schizosaccharomyces pombe (Matzke and Birchler 2005). The recently sequenced MAC genome of T. thermophila encodes multiple Dicer and PPD family members, implying the existence of additional RNAi pathways with roles other than DNA elimination. RasiRNA-like sRNAs derived from MIC centromeres may function in MIC maintenance in a manner dependent on DCL1 during vegetative growth (Mochizuki and Gorovsky 2005), although conflicting results have been reported (Malone et al. 2005). However, the full complexity of sRNAs in T. thermophila has not been examined. Here we present our analysis of sRNAs expressed in vegetatively growing, starving, and conjugating cells. We describe a second class of T. thermophila sRNAs with ubiquitous accumulation throughout the life cycle. These ∼23–24-nt sRNAs have features characteristic of sRNAs from other organisms but with interesting differences that suggest a novel biogenesis pathway distinct from those previously described for miRNAs, rasiRNAs, and ta-siRNAs. Analogous to the diversity of sRNAs found in multicellular organisms, the ∼27–30-nt sRNAs and the ∼23–24-nt sRNAs in T. thermophila represent coexisting yet genetically separable RNAi pathways.
Robert S Coyne - One of the best experts on this subject based on the ideXlab platform.
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genome scale analysis of programmed dna elimination sites in tetrahymena thermophila
G3: Genes Genomes Genetics, 2011Co-Authors: Joseph Fass, Eduardo Orias, Martin A. Gorovsky, Robert S Coyne, Eileen P. Hamilton, Mary T. Couvillion, Josephine Bowen, Kyungah Hong, Nikhil Joshi, Jonathan A. EisenAbstract:Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the Germline Micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3′ exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.
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Genome-Scale Analysis of Programmed DNA Elimination Sites in Tetrahymena thermophila
G3: Genes Genomes Genetics, 2011Co-Authors: Joseph Fass, Eduardo Orias, Martin A. Gorovsky, Robert S Coyne, Eileen P. Hamilton, Nikhil A. Joshi, Mary T. Couvillion, Josephine Bowen, Kyungah Hong, Jonathan A. EisenAbstract:Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the Germline Micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (
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refined annotation and assembly of the tetrahymena thermophila genome sequence through est analysis comparative genomic hybridization and targeted gap closure
BMC Genomics, 2008Co-Authors: Robert S Coyne, Mathangi Thiagarajan, Kristie M Jones, Jennifer R Wortman, Luke J Tallon, Brian J HaasAbstract:Background Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a Germline Micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.
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Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure
BMC Genomics, 2008Co-Authors: Robert S Coyne, Mathangi Thiagarajan, Kristie M Jones, Jennifer R Wortman, Luke J Tallon, Brian J Haas, Donna M Cassidy-hanley, Emily A Wiley, Joshua J Smith, Kathleen CollinsAbstract:Background Tetrahymena thermophila , a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a Germline Micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena 's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing. Results We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified. Conclusion We report here significant progress in genome closure and reannotation of Tetrahymena thermophila . Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.
Kristie M Jones - One of the best experts on this subject based on the ideXlab platform.
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refined annotation and assembly of the tetrahymena thermophila genome sequence through est analysis comparative genomic hybridization and targeted gap closure
BMC Genomics, 2008Co-Authors: Robert S Coyne, Mathangi Thiagarajan, Kristie M Jones, Jennifer R Wortman, Luke J Tallon, Brian J HaasAbstract:Background Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a Germline Micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.
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Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure
BMC Genomics, 2008Co-Authors: Robert S Coyne, Mathangi Thiagarajan, Kristie M Jones, Jennifer R Wortman, Luke J Tallon, Brian J Haas, Donna M Cassidy-hanley, Emily A Wiley, Joshua J Smith, Kathleen CollinsAbstract:Background Tetrahymena thermophila , a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a Germline Micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena 's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing. Results We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified. Conclusion We report here significant progress in genome closure and reannotation of Tetrahymena thermophila . Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.