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Kathleen M. Karrer - One of the best experts on this subject based on the ideXlab platform.

  • Alternate junctions and microheterogeneity of Tlr1, a developmentally regulated DNA Rearrangement in Tetrahymena thermophila
    The Journal of eukaryotic microbiology, 1997
    Co-Authors: Namrata S. Patil, Paula M. Hempen, Rupa A Udani, Kathleen M. Karrer
    Abstract:

    A large number of developmentally regulated DNA Rearrangements occur during the development of the macronucleus in Tetrahymena thermophila. Tlr1 is a deletion element which has large inverted repeats near the Rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 Rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate Rearrangement junctions. We have mapped and sequenced two additional Rearrangement variants and find that both the left and right junctions can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 Rearrangement product.

  • A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA Rearrangement in Tetrahymena thermophila.
    Molecular and cellular biology, 1994
    Co-Authors: John M. Wells, Jay Lee Edward Ellingson, Diana M. Catt, Patricia J. Berger, Kathleen M. Karrer
    Abstract:

    Extensive DNA Rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA Rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal Rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA Rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a Rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA Rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.

Kenneth Wolfe - One of the best experts on this subject based on the ideXlab platform.

  • The yeast mating-type switching endonuclease HO is a domesticated member of an unorthodox homing genetic element family
    eLife, 2020
    Co-Authors: Aisling Coughlan, Lisa Lombardi, Stephanie Braun-galleani, Alexandre Ar Martos, Frederic Bigey, Sylvie Dequin, Kevin Byrne, Kenneth Wolfe
    Abstract:

    The mating-type switching endonuclease HO plays a central role in the natural life cycle of Saccharomyces cerevisiae, but its evolutionary origin is unknown. HO is a recent addition to yeast genomes, present in only a few genera close to Saccharomyces. Here we show that HO is structurally and phylogenetically related to a family of unorthodox homing genetic elements found in Torulaspora and Lachancea yeasts. These WHO elements home into the aldolase gene FBA1, replacing its 3' end each time they integrate. They resemble inteins but they operate by a different mechanism that does not require protein splicing. We show that a WHO protein cleaves Torulaspora delbrueckii FBA1 efficiently and in an allele-specific manner, leading to DNA repair by gene conversion or NHEJ. The DNA Rearrangement steps during WHO element homing are very similar to those during mating-type switching, and indicate that HO is a domesticated WHO-like element.

Douglas L Chalker - One of the best experts on this subject based on the ideXlab platform.

  • nongenic bidirectional transcription precedes and may promote developmental DNA deletion in tetrahymena thermophila
    Genes & Development, 2001
    Co-Authors: Douglas L Chalker
    Abstract:

    A large number of DNA segments are excised from the chromosomes of the somatic nucleus during development of Tetrahymena thermophila. How these germline-limited sequences are recognized and excised is still poorly understood. We have found that many of these noncoding DNAs are transcribed during nuclear development. Transcription of the germline-limited M element occurs from both DNA strands and results in heterogeneous transcripts of 1 kb. Transcripts are most abundant when developing micro- and macronuclei begin their differentiation. Transcription is normally restricted to unrearranged DNA of micronuclei and/or developing nuclei, but germline-limited DNAs can induce their own transcription when placed into somatic macronuclei. Brief actinomycin D treatment of conjugating cells blocked M-element excision, providing evidence that transcription is important for efficient DNA Rearrangement. We propose that transcription targets these germline-limited sequences for elimination by altering chromatin to ensure their accessibility to the excision machinery.

  • flanking regulatory sequences of the tetrahymena r deletion element determine the boundaries of DNA Rearrangement
    Molecular and Cellular Biology, 1999
    Co-Authors: Douglas L Chalker, Antonietta La Terza, Allison Wilson, Christopher D Kroenke, Mengchao Yao
    Abstract:

    In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA Rearrangements. It is unclear whether Rearrangement of the many different DNA elements occurs via a single mechanism or via multiple Rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the Rearrangement of the 1.1-kbp R deletion element. We found that Rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the Rearrangement boundary. When we moved the location of the left cis-acting sequences closer to the eliminated region, we observed a rightward shift of the Rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the Rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect Rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the Rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its Rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymena Rearrangement.

  • non mendelian heritable blocks to DNA Rearrangement are induced by loading the somatic nucleus of tetrahymena thermophila with germ line limited DNA
    Molecular and Cellular Biology, 1996
    Co-Authors: Douglas L Chalker
    Abstract:

    Site-specificDNAdeletionoccursatthousandsofsiteswithinthegenomeduringmacronucleardevelopment ofTetrahymena 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. Programmed DNA Rearrangements occur in a wide variety of organisms and play significant roles in cell differentiation. Some of the most remarkable examples of these processes are found in ciliated protozoa (reviewed in reference 24). The ciliates exhibit nuclear dualism. Polyploid macronuclei are active in gene expression, providing for the somatic functions of cells. Diploid micronuclei are transcriptionally silent in vegetative growth but provide germ line functions during sexual reproduction. This dualism is one of the simplest separations of the soma from the germ line. The conjugation process results in the loss of parental macronuclei, and new macronuclei differentiate from zygotic nuclei formed by the fusion of micronucleus-derived gametes. Macronuclear differentiation involves extensive genome reorganization (reviewed in references 24 and 33). Chromosomes are fragmented and amplified to 45 to 50 copies per macronucleus. In Tetrahymena thermophila, the ribosomal RNA gene, rDNA, exemplifies the Rearrangementprocess.TheTetrahymenarDNAispresentasa single-copy sequence in the micronuclear genome (35) and is flanked by chromosomal breakage sequences. During macronuclear development, this copy is cut out of the chromosomal DNA and is converted into a unique, head-to-head palindromicmoleculewithtelomericDNAaddedtothelinearends.

Y Kobayashi - One of the best experts on this subject based on the ideXlab platform.

James W. Golden - One of the best experts on this subject based on the ideXlab platform.

  • Programmed DNA Rearrangement of a cyanobacterial hupL gene in heterocysts
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Claudio D. Carrasco, Jill A. Buettner, James W. Golden
    Abstract:

    Abstract Programmed DNA Rearrangements that occur during cellular differentiation are uncommon and have been described in only two prokaryotic organisms. Here, we identify the developmentally regulated Rearrangement of a hydrogenase gene in heterocysts of the cyanobacterium Anabaena sp. strain PCC 7120. Heterocysts are terminally differentiated cells specialized for nitrogen fixation. Late during heterocyst differentiation, a 10.5-kb DNA element is excised from within the hupL gene by site-specific recombination between 16-bp direct repeats that flank the element. The predicted HupL polypeptide is homologous to the large subunit of [NiFe] uptake hydrogenases. hupL is expressed similarly to the nitrogen-fixation genes; hupL message was detected only during the late stages of heterocyst development. An open reading frame, named xisC, identified near one end of the hupL DNA element is presumed to encode the element's site-specific recombinase. The predicted XisC polypeptide is homologous with the Anabaena sp. strain PCC 7120 site-specific recombinase XisA. Neither XisC nor XisA shows sequence similarity to other proteins, suggesting that they represent a different class of site-specific recombinase.

  • Independent regulation of nifHDK operon transcription and DNA Rearrangement during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.
    Journal of bacteriology, 1991
    Co-Authors: James W. Golden, L L Whorff, D R Wiest
    Abstract:

    The filamentous cyanobacterium Anabaena sp. strain PCC 7120 expresses the genes required for nitrogen fixation in terminally differentiated cells called heterocysts. The nifHDK operon encodes the nitrogenase polypeptides and is expressed at high levels in heterocysts. During heterocyst differentiation, an 11-kb DNA element is excised from the nifD gene by site-specific recombination. The xisA gene, located on the 11-kb element, is required for the excision of the element. Transcription and DNA Rearrangement of the nifHDK operon both occur late during heterocyst differentiation, about 18 to 24 h after induction, suggesting that the regulation of these events might be coupled. We show that heterocyst-specific transcription and DNA Rearrangement of the nifHDK operon are independent of one another. Northern (RNA) analysis of the xisA mutant strain DW12-2.2, which cannot excise the nifD 11-kb element or fix nitrogen, showed that the nifH and nifD genes are transcribed on unrearranged chromosomes. The nifK gene was not transcribed in DW12-2.2, indicating that its expression is dependent on the nifH promoter and excision of the 11-kb element from the operon. A 1.68-kb DNA fragment containing the nifH promoter was deleted from the chromosome to produce the mutant strain LW1. LW1 formed heterocysts but did not grow on nitrogen-free medium and showed no transcription through nifD. Southern analysis of LW1 showed normal excision of the 11-kb element from the nifHDK operon, indicating that transcription from the nifH promoter is not required for the developmentally regulated DNA Rearrangement.