The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Sandra Duharcourt - One of the best experts on this subject based on the ideXlab platform.
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Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements
BMC Genomics, 2017Co-Authors: Frédéric Guérin, Nicole Boggetto, Cyril Denby Wilkes, Linda Sperling, Eric Meyer, Olivier Arnaiz, Sandra DuharcourtAbstract:Background DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic Macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic Macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. Results We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia . We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM -silenced cells reveals that the latter does not provide a faithful representation of the germline genome. Conclusions We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
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flow cytometry sorting of nuclei enables the first global characterization of paramecium germline dna and transposable elements
BMC Genomics, 2017Co-Authors: Frédéric Guérin, Nicole Boggetto, Linda Sperling, Eric Meyer, Olivier Arnaiz, Cyril Denby Wilkes, Sandra DuharcourtAbstract:DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic Macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic Macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
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Developmental genome rearrangements in ciliates: a natural genomic subtraction mediated by non-coding transcripts.
Trends in Genetics, 2009Co-Authors: Sandra Duharcourt, Gersende Lepère, Eric MeyerAbstract:Several classes of non-protein-coding RNAs have recently been identified as epigenetic regulators of developmental genome rearrangements in ciliates, providing an interesting insight into the role of genome-wide transcription. In these unicellular eukaryotes, extensive rearrangements of the germline genome occur during the development of a new somatic Macronucleus from the germline micronucleus. Rearrangement patterns are not dictated by the germline sequence, but reproduce the pre-existing rearrangements of the maternal somatic genome, implying a homology-dependent global comparison of germline and somatic genomes. We review recent evidence showing that this is achieved by a natural genomic subtraction, computed by pairing interactions between meiosis-specific, germline scnRNAs (small RNAs that resemble metazoan piRNAs) and longer non-coding transcripts from the somatic genome. We focus on current models for the RNA-based mechanisms enabling the cell to recognize the germline sequences to be eliminated from the somatic genome and to maintain an epigenetic memory of rearrangement patterns across sexual generations.
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Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia.
Genes & Development, 2008Co-Authors: Gersende Lepère, Mireille Bétermier, Eric Meyer, Sandra DuharcourtAbstract:The germline genome of ciliates is extensively rearranged during the development of a new somatic Macronucleus from the germline micronucleus, after sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) are precisely excised from coding sequences and intergenic regions. For a subset of IESs, introduction of the IES sequence into the maternal Macronucleus specifically inhibits excision of the homologous IES in the developing zygotic Macronucleus, suggesting that epigenetic regulation of excision involves a global comparison of germline and somatic genomes. ScanRNAs (scnRNAs) produced during micronuclear meiosis by a developmentally regulated RNAi pathway have been proposed to mediate this transnuclear cross-talk. In this study, microinjection experiments provide direct evidence that 25-nucleotide (nt) scnRNAs promote IES excision. We further show that noncoding RNAs are produced from the somatic maternal genome, both during vegetative growth and during sexual events. Maternal inhibition of IES excision is abolished when maternal somatic transcripts containing an IES are targeted for degradation by a distinct RNAi pathway involving 23-nt siRNAs. The results strongly support a scnRNA/macronuclear RNA scanning model in which a natural genomic subtraction, occurring during meiosis between deletion-inducing scnRNAs and antagonistic transcripts from the maternal Macronucleus, regulates rearrangements of the zygotic genome.
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rna mediated programming of developmental genome rearrangements in paramecium tetraurelia
Molecular and Cellular Biology, 2004Co-Authors: Olivier Garnier, Sandra Duharcourt, Vincent Serrano, Eric MeyerAbstract:The germ line genome of ciliates is extensively rearranged during development of the somatic Macronucleus. Numerous sequences are eliminated, while others are amplified to a high ploidy level. In the Paramecium aurelia group of species, transformation of the maternal Macronucleus with transgenes at high copy numbers can induce the deletion of homologous genes in sexual progeny, when a new Macronucleus develops from the wild-type germ line. We show that this trans-nuclear effect correlates with homology-dependent silencing of maternal genes before autogamy and with the accumulation of approximately 22- to 23-nucleotide (nt) RNA molecules. The same effects are induced by feeding cells before meiosis with bacteria containing double-stranded RNA, suggesting that small interfering RNA-like molecules can target deletions. Furthermore, experimentally induced macronuclear deletions are spontaneously reproduced in subsequent sexual generations, and reintroduction of the missing gene into the variant Macronucleus restores developmental amplification in sexual progeny. We discuss the possible roles of the approximately 22- to 23-nt RNAs in the targeting of deletions and the implications for the RNA-mediated genome-scanning process that is thought to determine developmentally regulated rearrangements in ciliates.
Masahiro Fujishima - One of the best experts on this subject based on the ideXlab platform.
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draft genome sequences of three holospora species holospora obtusa holospora undulata and holospora elegans endonuclear symbiotic bacteria of the ciliate paramecium caudatum
Fems Microbiology Letters, 2014Co-Authors: Hideo Dohra, Masahiro Fujishima, Tomohiro Suzuki, Kenya Tanaka, Haruo SuzukiAbstract:We present draft genome sequences of three Holospora species, hosted by the ciliate Paramecium caudatum ; that is, the Macronucleus-specific H. obtusa and the micronucleus-specific H. undulata and H. elegans . We investigate functions of orthologous core genes conserved across the three Holospora species, which may be essential for the infection and survival in the host nucleus.
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micronucleus specific bacterium holospora elegans irreversibly enhances stress gene expression of the host paramecium caudatum
Journal of Eukaryotic Microbiology, 2008Co-Authors: Manabu Hori, Kimiko Fujii, Masahiro FujishimaAbstract:The bacterium Holospora is an endonuclear symbiont of the ciliate Paramecium. Previously, we reported that paramecia bearing the macronuclear-specific symbiont Holospora obtusa survived better than symbiont-free paramecia, even under high temperatures unsuitable for growth. The paramecia with symbionts expressed high levels of hsp70 mRNAs even at 25 degrees C, a usual growth temperature. We report herein that paramecia bearing the micronuclear-specific symbiont Holospora elegans also acquire the heat-shock resistance. Even after the removal of the bacteria from the hosts by treatment with penicillin, the resulting aposymbiotic paramecia nevertheless maintained their heat shock-resistant nature for over 1 yr. Like symbiotic paramecia, these aposymbiotic paramecia also expressed high levels of both hsp60 and hsp70 mRNAs even at 25 degrees C. Moreover, analysis by fluorescent in situ hybridization with a probe specific for Holospora 16S rRNA revealed that the 16S rRNA of H. elegans was expressed around the nucleoli of the Macronucleus in the aposymbiotic cells. This result suggests the possible transfer of Holospora genomic DNA from the micronucleus into the Macronucleus in symbiotic paramecia. Perhaps this exogenous DNA could trigger the aposymbiotic paramecia to induce a stress response, inducing higher expression of Hsp60 and Hsp70, and thus conferring heat-shock resistance.
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fates of the endonuclear symbiotic bacteria holospora obtusa and holospora undulata injected into the Macronucleus of paramecium caudatum
European Journal of Protistology, 2001Co-Authors: Ilya Skovorodkin, Masahiro Fujishima, Sergei I FokinAbstract:Gram-negative endosymbiotic bacteria Holospora obtusa and H. undulata infect the Macronucleus and micronucleus of the ciliate Paramecium caudatum, respectively. Microsurgical transfers of infectious and reproductive forms of these symbionts were carried out from the donor nuclei to the Macronucleus of aposymbiotic P. caudatum. The reproductive forms of the micronucleus-specific H. undulata initially grew in the Macronucleus, but were eventually eliminated from the nucleus within five days after the injection. On the other hand, the infectious forms of both Holospora species did not differentiate into the reproductive forms in the recipient Macronucleus, and were expelled from the nucleus into the cytoplasm. These results indicate that the infectious form has no ability to differentiate into the reproductive form unless it infects the nucleus through the host digestive vacuole. It was found that injection of holosporas into the recipient Macronucleus induced unusual vesicular structures in the nucleus.
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invasion of the Macronucleus of paramecium caudatum by the bacterium holospora obtusa fates of the bacteria and timings of invasion steps
European Journal of Protistology, 2000Co-Authors: Miki Kawai, Masahiro FujishimaAbstract:Summary Holospora obtusa is a Macronucleus-specific bacterium of the ciliate Paramecium caudatum. The infectious form of this bacterium (about 13 μm in length) infects the host Macronucleus through the host digestive vacuole, forms constrictions and eventually separates into the reproductive form (1.5–2 μm in length) in the host Macronucleus. Indirect immunofluorescence microscopy with a monoclonal antibody specific for the outer membrane of H. obtusa showed that the first constriction and the first single reproductive-form appeared between 32–34 h and between 34–36 h, respectively, after the infection at 25°C Furthermore, it was found that a short infectious form and a long infectious form, which are about 1/4–1/2 the length and about 1.5 times the length of the normal infectious form, were able to infect the Macronucleus. In the early infection process, infectious forms, which had failed to escape from the host digestive vacuole, were digested in the vacuole. It was found that some bacteria degraded soon after infecting the Macronucleus.
Eric Meyer - One of the best experts on this subject based on the ideXlab platform.
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Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements
BMC Genomics, 2017Co-Authors: Frédéric Guérin, Nicole Boggetto, Cyril Denby Wilkes, Linda Sperling, Eric Meyer, Olivier Arnaiz, Sandra DuharcourtAbstract:Background DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic Macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic Macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. Results We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia . We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM -silenced cells reveals that the latter does not provide a faithful representation of the germline genome. Conclusions We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
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flow cytometry sorting of nuclei enables the first global characterization of paramecium germline dna and transposable elements
BMC Genomics, 2017Co-Authors: Frédéric Guérin, Nicole Boggetto, Linda Sperling, Eric Meyer, Olivier Arnaiz, Cyril Denby Wilkes, Sandra DuharcourtAbstract:DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic Macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic Macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
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Developmental genome rearrangements in ciliates: a natural genomic subtraction mediated by non-coding transcripts.
Trends in Genetics, 2009Co-Authors: Sandra Duharcourt, Gersende Lepère, Eric MeyerAbstract:Several classes of non-protein-coding RNAs have recently been identified as epigenetic regulators of developmental genome rearrangements in ciliates, providing an interesting insight into the role of genome-wide transcription. In these unicellular eukaryotes, extensive rearrangements of the germline genome occur during the development of a new somatic Macronucleus from the germline micronucleus. Rearrangement patterns are not dictated by the germline sequence, but reproduce the pre-existing rearrangements of the maternal somatic genome, implying a homology-dependent global comparison of germline and somatic genomes. We review recent evidence showing that this is achieved by a natural genomic subtraction, computed by pairing interactions between meiosis-specific, germline scnRNAs (small RNAs that resemble metazoan piRNAs) and longer non-coding transcripts from the somatic genome. We focus on current models for the RNA-based mechanisms enabling the cell to recognize the germline sequences to be eliminated from the somatic genome and to maintain an epigenetic memory of rearrangement patterns across sexual generations.
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Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia.
Genes & Development, 2008Co-Authors: Gersende Lepère, Mireille Bétermier, Eric Meyer, Sandra DuharcourtAbstract:The germline genome of ciliates is extensively rearranged during the development of a new somatic Macronucleus from the germline micronucleus, after sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) are precisely excised from coding sequences and intergenic regions. For a subset of IESs, introduction of the IES sequence into the maternal Macronucleus specifically inhibits excision of the homologous IES in the developing zygotic Macronucleus, suggesting that epigenetic regulation of excision involves a global comparison of germline and somatic genomes. ScanRNAs (scnRNAs) produced during micronuclear meiosis by a developmentally regulated RNAi pathway have been proposed to mediate this transnuclear cross-talk. In this study, microinjection experiments provide direct evidence that 25-nucleotide (nt) scnRNAs promote IES excision. We further show that noncoding RNAs are produced from the somatic maternal genome, both during vegetative growth and during sexual events. Maternal inhibition of IES excision is abolished when maternal somatic transcripts containing an IES are targeted for degradation by a distinct RNAi pathway involving 23-nt siRNAs. The results strongly support a scnRNA/macronuclear RNA scanning model in which a natural genomic subtraction, occurring during meiosis between deletion-inducing scnRNAs and antagonistic transcripts from the maternal Macronucleus, regulates rearrangements of the zygotic genome.
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Nowa1p and Nowa2p: Novel Putative RNA Binding Proteins Involved in trans-Nuclear Crosstalk in Paramecium tetraurelia
Current Biology, 2005Co-Authors: Mariusz Nowacki, Wlodzimierz Zagorski-ostoja, Eric MeyerAbstract:Summary Background: The germline genome of ciliates is extensively rearranged during development of a new somatic Macronucleus from the germline micronucleus, a process that follows sexual events. In Paramecium tetraurelia , single-copy internal eliminated sequences (IESs) and multicopy transposons are eliminated, whereas cellular genes are amplified to ∼800 n. For a subset of IESs, introduction of the IES sequence into the maternal (prezygotic) Macronucleus specifically inhibits excision of the homologous IES in the developing zygotic Macronucleus. This and other homology-dependent maternal effects have suggested that rearrangement patterns are epigenetically determined by an RNA-mediated, trans -nuclear comparison, involving the RNA interference pathway, of germline and somatic genomes. Results: We report the identification of novel developmentally regulated RNA binding proteins, Nowa1p and Nowa2p, which are required for the survival of sexual progeny. Green fluorescent protein (GFP) fusions show that Nowa1p accumulates into the maternal Macronucleus shortly before meiosis of germline micronuclei and is later transported to developing macronuclei. Nowa1p/2p depletion impairs the elimination of transposons and of those IESs that are controlled by maternal effects, confirming the existence of distinct IES classes. Conclusions: The results indicate that Nowa proteins are essential components of the trans -nuclear-crosstalk mechanism that is responsible for epigenetic programming of genome rearrangements. We discuss implications for the current models of genome scanning in ciliates, a process related to the formation of heterochromatin by RNA interference in other eukaryotes.
Martin A Gorovsky - 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, Robert S Coyne, Mary T. Couvillion, Kyungah Hong, Eileen P Hamilton, Josephine Bowen, Martin A Gorovsky, Eduardo Orias, 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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Analysis of a piwi-Related Gene Implicates Small RNAs in Genome Rearrangement in Tetrahymena
Cell, 2002Co-Authors: Kazufumi Mochizuki, Noah A. Fine, Toshitaka Fujisawa, Martin A GorovskyAbstract:During development of the somatic Macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres. We identified a gene, TWI1, in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination. We also found that small RNAs were specifically expressed prior to chromosome rearrangement during conjugation. These RNAs were not observed in TWI1 knockout cells and required PDD1, another gene required for rearrangement, for expression. We propose that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing.
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germline and somatic transformation of mating tetrahymena thermophila by particle bombardment
Genetics, 1997Co-Authors: Donna Cassidyhanley, Josephine Bowen, Martin A Gorovsky, Eric S Cole, Lynn A Verplank, Jacek Gaertig, Peter J BrunsAbstract:Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development. Both macronuclear and micronuclear transformants were recovered. Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the Macronucleus. Evidence is given for transient retention of the introduced plasmid. Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome.
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germ line knockout heterokaryons of an essential α tubulin gene enable high frequency gene replacement and a test of gene transfer from somatic to germ line nuclei in tetrahymena thermophila
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Martin A GorovskyAbstract:The haploid Tetrahymena thermophila genome contains a single α-tubulin (ATU) gene. Using biolistic transformation, we disrupted one of the two copies of the ATU gene in the diploid germ-line micronucleus. The heterozygous germ-line transformants were made homozygous in the micronucleus by mating to a star strain containing a defective micronucleus. This mating, known as round 1 genomic exclusion, resulted in two heterokaryon clones of different mating types which have both copies of the ATU gene knocked out in the micronucleus but only wild-type genes in the polycopy somatic Macronucleus. When these heterokaryons were mated, the exconjugant progeny cells did not grow because the new somatic macronuclei do not have any α-tubulin genes. However, when these conjugants were transformed with a functional marked ATU gene, viable transformants were obtained that contained the transforming ATU gene at the homologous locus in the new Macronucleus. The exconjugant progeny could be rescued at a high efficiency (900 transformants per μg of DNA) with a wild-type ATU gene. Unlike previous macronuclear transformation protocols, this strategy should allow introduction of highly disadvantageous (but viable) mutations into Tetrahymena, providing a powerful tool for molecular and functional studies of essential genes. These knockout heterokaryons were used to demonstrate that gene transfer from somatic macronuclei to germ-line micronuclei occurs rarely if at all.
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strategies for the isolation of ciliary motility and assembly mutants in tetrahymena
Methods in Cell Biology, 1995Co-Authors: David G. Pennock, Martin A GorovskyAbstract:Publisher Summary A screen for the isolation of mutations affecting ciliary regeneration in Tetrahyrnena thermophila has been deveoped. The development of transformation and gene replacement techniques in Tetrahymena should lead to increased interest in genetic analyses of ciliary function and assembly. Cells homozygous for the chp (cell cycle, heat shock, and phosphorylation defect) mutation fail to initiate ciliogenesis after deciliation and incubation at the restrictive temperature. Researchers showed that chp mutants are defective in the heat shock response. Cell maintenance medium includes 2% proteose peptone, 0.1% yeast extract, 0.2% glucose. All ingredients, bring to volume with water, aliquot approximately 6 ml into capped culture tubes, and autoclave are dissolved. Cells can be stored for 2-3 weeks between transfers. The chapter provides a clear description of the procedures and equipment for performing genetic analyses on Tetrahymena . Although the micronucleus is diploid, three ways are commonly used to bring recessive micronuclear mutations to expression in the Macronucleus in Tetrahymena after mutagenesis and a single round of mating. One method is to induce cytogamy in mating pairs of Tetrahymena . To induce cytogamy, mating pairs of heterokaryons are subjected to an osmotic shock during mating. This prevents nuclear exchange and causes the production of whole-genome homozygotes in some fraction of the pairs.
James D. Forney - One of the best experts on this subject based on the ideXlab platform.
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depletion of ubc9 causes nuclear defects during the vegetative and sexual life cycles in tetrahymena thermophila
Eukaryotic Cell, 2015Co-Authors: Qianyi Yang, Robert S Coyne, Amjad M Nasir, James D. ForneyAbstract:Ubc9p is the sole E2-conjugating enzyme for SUMOylation, and its proper function is required for regulating key nuclear events such as transcription, DNA repair, and mitosis. In Tetrahymena thermophila, the genome is separated into a diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid somatic Macronucleus (MAC) that divides amitotically. This unusual nuclear organization provides novel opportunities for the study of SUMOylation and Ubc9p function. We identified the UBC9 gene and demonstrated that its complete deletion from both MIC and MAC genomes is lethal. Rescue of the lethal phenotype with a GFP-UBC9 fusion gene driven by a metallothionein promoter generated a cell line with CdCl2-dependent expression of green fluorescent protein (GFP)-Ubc9p. Depletion of Ubc9p in vegetative cells resulted in the loss of MICs, but MACs continued to divide. In contrast, expression of catalytically inactive Ubc9p resulted in the accumulation of multiple MICs. Critical roles for Ubc9p were also identified during the sexual life cycle of Tetrahymena. Cell lines that were depleted for Ubc9p did not form mating pairs and therefore could not complete any of the subsequent stages of conjugation, including meiosis and macronuclear development. Mating between cells expressing catalytically inactive Ubc9p resulted in arrest during macronuclear development, consistent with our observation that Ubc9p accumulates in the developing Macronucleus.
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Non-Mendelian inheritance of macronuclear mutations is gene specific in Paramecium tetraurelia
Molecular and Cellular Biology, 1994Co-Authors: J. M. Scott, C L Leeck, K. Mikami, James D. ForneyAbstract:Paramecium tetraurelia contains two types of nuclei, a diploid germinal micronucleus and a large transcriptionally active Macronucleus. The macronuclear genome is formed from the micronuclear DNA during sexual reproduction. Previous studies have shown that the processing of the A-type variable surface protein gene during formation of a new Macronucleus is dependent on the presence of the A gene in the old Macronucleus. It is not clear if this is a general feature that controls the formation of the Paramecium macronuclear genome or a unique feature of the A locus. Using micronuclear transplantation, we have constructed a strain that has a wild-type micronucleus but has macronuclear deletions of the A- and B-type surface protein genes. Neither the A nor the B gene is incorporated into the new Macronucleus after sexual reproduction. Macronuclear transformation of this strain with the B gene rescues the B-gene deletion after formation of the next Macronucleus but has not effect on the A deletion. Similarly, transformation with the A gene shows gene-specific rescue for A but not B. The effect of the old Macronucleus on the processing of the new Macronucleus results in a pattern of non-Mendelian inheritance of both macronuclear deletions. Progeny from the wild-type exconjugant are all wild type, and progeny from the A- B- exconjugant are mutant. The features of this A- B- non-Mendelian mutant demonstrate that the regulation of macronuclear DNA processing is gene specific, and our results open the possibility that this type of regulation affects many regions of the Paramecium genome.
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macronuclear transformation with specific dna fragments controls the content of the new macronuclear genome in paramecium tetraurelia
Molecular and Cellular Biology, 1991Co-Authors: Yun You, Karl J Aufderheide, J Morand, K Rodkey, James D. ForneyAbstract:A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the Macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type Macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 Macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the Macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.
