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David A. Campbell - One of the best experts on this subject based on the ideXlab platform.

  • the promoter and transcribed regions of the leishmania tarentolae spliced leader RNA Gene array are devoid of nucleosomes
    BMC Microbiology, 2007
    Co-Authors: Robert A Hitchcock, David A. Campbell, Sean Thomas, Nancy R. Sturm
    Abstract:

    The spliced leader (SL) RNA provides the 5' m7G cap and first 39 nt for all nuclear mRNAs in kinetoplastids. This small nuclear RNA is transcribed by RNA polymerase II from individual promoters. In Leishmania tarentolae the SL RNA Genes reside in two multi-copy tandem arrays designated MINA and MINB. The transcript accumulation from the SL promoter on the drug-selected, episomal SL RNA Gene cassette pX-tSL is ~10% that of the genomic array in uncloned L. tarentolae transfectants. This disparity is neither sequence- nor copy-number related, and thus may be due to interference of SL promoter function by epiGenetic factors. To explore these possibilities we examined the nucleoplasmic localization of the SL RNA Genes as well as their nucleosomal architecture. The genomic SL RNA Genes and the episome did not co-localize within the nucleus. Each genomic repeat contains one nucleosome regularly positioned within the non-transcribed intergenic region. The 363-bp MINA array was resistant to micrococcal nuclease digestion between the -258 and -72 positions relative to the transcription start point due to nucleosome association, leaving the promoter elements and the entire transcribed region exposed for protein interactions. A pattern of ~164-bp protected segments was observed, corresponding to the amount of DNA typically bound by a nucleosome. By contrast, nucleosomes on the pX-tSL episome were randomly distributed over the episomal SL cassette, reducing transcription factor access to the episomal promoter by approximately 74%. Cloning of the episome transfectants revealed a range of transcriptional activities, implicating a mechanism of epiGenetic heredity. The disorganized nucleosomes on the pX episome are in a permissive conformation for transcription of the SL RNA cassette approximately 25% of the time within a given parasite. Nucleosome interference is likely the major factor in the apparent transcriptional repression of the SL RNA Gene cassette. Coupled with the requirement for run-around transcription that drives expression of the selectable drug marker, transcription of the episomal SL may be reduced even further due to sub-optimal nucleoplasmic localization and initiation complex disruption.

  • trypanosomatid biodiversity in costa rica genotyping of parasites from heteroptera using the spliced leader RNA Gene
    Parasitology, 2004
    Co-Authors: Scott J Westenberger, David A. Campbell, Nancy R. Sturm, D Yanega, Sergei A Podlipaev, R Zeledon, Dmitri A Maslov
    Abstract:

    SUMMARY The biodiversity of insect trypanosomes is largely unknown, resulting in significant gaps in the understanding of pathogen evolution. A culture-independent preliminary survey of trypanosomatid fauna was conducted for the parasites of Heteroptera (Hemiptera) from several localities in Costa Rica. Trypanosomatid infections were detected by light microscopy of smeared gut contents. Out of 257 insects representing 6 families, infections were found in 62 cases ; cultures were obtained for 29 new isolates. Gut material from infected hosts was preserved in the field using an SDS–EDTA buffer solution for subsequent DNA extraction in the laboratory. PCR amplification of the trypanosomatid-specific spliced leader (SL) RNA Gene repeats was successful for 60 field samples. Eighteen distinct SL RNA typing units were identified in a set of 28 samples analysed in detail. Cluster analysis indicated that these typing units were unique and thus could represent new species and, in some cases, new Genera. This study reveals only a minor fraction of the trypanosomatid biodiversity, which is anticipated to be high.

  • two distinct functional spliced leader RNA Gene arrays in leishmania tarentolae are found in several lizard leishmania species
    International Journal for Parasitology, 2002
    Co-Authors: Cristina T Orlando, Nancy R. Sturm, Liang Zhou, Mako R Saito, Lucile Maria Floeterwinter, David A. Campbell
    Abstract:

    A second distinct array of spliced leader RNA Genes has been found in several Leishmania species particular to lizards. This is the first report of two non-allelic arrays of spliced leader RNA Genes within a species cell line. The arrays are identical to each other in their transcribed spliced leader RNA Gene sequences, but variable in their non-transcribed spacer sequences. In the two arrays from Leishmania tarentolae UC strain the promoter regions are similar, but not identical, at positions shown previously to be critical for spliced leader RNA transcription. These arrays contain similar numbers of Genes and are both transcribed in L. tarentolae in vitro transcription extract as well as in vivo. The -66/-58 regions of both Genes, which contain an element of the spliced leader RNA Gene promoter, bind proteins likely to be transcription factors in a specific manner. A survey of lizard Leishmania spp. revealed a second spliced leader RNA Gene array in three of four species. PhyloGenetic analyses of these sequences with each other and with the spliced leader RNA Gene sequences of non-lizard Leishmania spp. and their near-relatives showed that the lizard groups are more closely related to each other than to arrays from other Leishmania spp. As the transcripts of the two arrays are identical, they may co-exist to fulfil the substantial requirement for spliced leader RNA production; however, they have the potential for differential usage modulated by their distinct promoter elements. The presence of two distinct spliced leader RNA Gene arrays within a single cell type may represent dissociated evolution of two redundant loci, or a previously unsuspected level of control in the post-transcriptional Gene expression within some kinetoplastids.

  • two sequence classes of kinetoplastid 5s ribosomal RNA Gene revealed among bodonid spliced leader RNA Gene arrays
    Fems Microbiology Letters, 2001
    Co-Authors: Debora M Santana, Nancy R. Sturm, Julius Lukes, David A. Campbell
    Abstract:

    The spliced leader RNA Genes of Bodo saltans, Cryptobia helicis and Dimastigella trypaniformis were analyzed as molecular markers for additional taxa within the suborder Bodonina. The non-transcribed spacer regions were distinctive for each organism, and 5S rRNA Genes were present in Bodo and Dimastigella but not in C. helicis. Two sequence classes of 5S rRNA were evident from analysis of the bodonid Genes. The two classes of 5S rRNA Genes were found in other Kinetoplastids independent of co-localization with the spliced leader RNA Gene.

  • transcription of the kinetoplastid spliced leader RNA Gene
    Parasitology Today, 2000
    Co-Authors: David A. Campbell, Nancy R. Sturm, Michael C Yu
    Abstract:

    In recent years, much has been learned about the cis-elements controlling transcription of the kinetoplastid spliced leader (SL) RNA Gene. The SL RNA Gene contains the first 39 nucleotides that are trans-spliced on to all nuclear-derived mRNAs in these organisms. Transcription initiation is determined by two precisely spaced upstream elements and transcription termination is directed by the downstream poly-T tract, although the RNA polymerase responsible for SL RNA synthesis is still questioned. In this article, David Campbell, Nancy Sturm and Michael Yu review the field of kinetoplastid SL RNA Gene transcription, address past proposals in light of current data and discuss some of the differences that appear in the literature.

Nancy R. Sturm - One of the best experts on this subject based on the ideXlab platform.

  • the promoter and transcribed regions of the leishmania tarentolae spliced leader RNA Gene array are devoid of nucleosomes
    BMC Microbiology, 2007
    Co-Authors: Robert A Hitchcock, David A. Campbell, Sean Thomas, Nancy R. Sturm
    Abstract:

    The spliced leader (SL) RNA provides the 5' m7G cap and first 39 nt for all nuclear mRNAs in kinetoplastids. This small nuclear RNA is transcribed by RNA polymerase II from individual promoters. In Leishmania tarentolae the SL RNA Genes reside in two multi-copy tandem arrays designated MINA and MINB. The transcript accumulation from the SL promoter on the drug-selected, episomal SL RNA Gene cassette pX-tSL is ~10% that of the genomic array in uncloned L. tarentolae transfectants. This disparity is neither sequence- nor copy-number related, and thus may be due to interference of SL promoter function by epiGenetic factors. To explore these possibilities we examined the nucleoplasmic localization of the SL RNA Genes as well as their nucleosomal architecture. The genomic SL RNA Genes and the episome did not co-localize within the nucleus. Each genomic repeat contains one nucleosome regularly positioned within the non-transcribed intergenic region. The 363-bp MINA array was resistant to micrococcal nuclease digestion between the -258 and -72 positions relative to the transcription start point due to nucleosome association, leaving the promoter elements and the entire transcribed region exposed for protein interactions. A pattern of ~164-bp protected segments was observed, corresponding to the amount of DNA typically bound by a nucleosome. By contrast, nucleosomes on the pX-tSL episome were randomly distributed over the episomal SL cassette, reducing transcription factor access to the episomal promoter by approximately 74%. Cloning of the episome transfectants revealed a range of transcriptional activities, implicating a mechanism of epiGenetic heredity. The disorganized nucleosomes on the pX episome are in a permissive conformation for transcription of the SL RNA cassette approximately 25% of the time within a given parasite. Nucleosome interference is likely the major factor in the apparent transcriptional repression of the SL RNA Gene cassette. Coupled with the requirement for run-around transcription that drives expression of the selectable drug marker, transcription of the episomal SL may be reduced even further due to sub-optimal nucleoplasmic localization and initiation complex disruption.

  • trypanosomatid biodiversity in costa rica genotyping of parasites from heteroptera using the spliced leader RNA Gene
    Parasitology, 2004
    Co-Authors: Scott J Westenberger, David A. Campbell, Nancy R. Sturm, D Yanega, Sergei A Podlipaev, R Zeledon, Dmitri A Maslov
    Abstract:

    SUMMARY The biodiversity of insect trypanosomes is largely unknown, resulting in significant gaps in the understanding of pathogen evolution. A culture-independent preliminary survey of trypanosomatid fauna was conducted for the parasites of Heteroptera (Hemiptera) from several localities in Costa Rica. Trypanosomatid infections were detected by light microscopy of smeared gut contents. Out of 257 insects representing 6 families, infections were found in 62 cases ; cultures were obtained for 29 new isolates. Gut material from infected hosts was preserved in the field using an SDS–EDTA buffer solution for subsequent DNA extraction in the laboratory. PCR amplification of the trypanosomatid-specific spliced leader (SL) RNA Gene repeats was successful for 60 field samples. Eighteen distinct SL RNA typing units were identified in a set of 28 samples analysed in detail. Cluster analysis indicated that these typing units were unique and thus could represent new species and, in some cases, new Genera. This study reveals only a minor fraction of the trypanosomatid biodiversity, which is anticipated to be high.

  • two distinct functional spliced leader RNA Gene arrays in leishmania tarentolae are found in several lizard leishmania species
    International Journal for Parasitology, 2002
    Co-Authors: Cristina T Orlando, Nancy R. Sturm, Liang Zhou, Mako R Saito, Lucile Maria Floeterwinter, David A. Campbell
    Abstract:

    A second distinct array of spliced leader RNA Genes has been found in several Leishmania species particular to lizards. This is the first report of two non-allelic arrays of spliced leader RNA Genes within a species cell line. The arrays are identical to each other in their transcribed spliced leader RNA Gene sequences, but variable in their non-transcribed spacer sequences. In the two arrays from Leishmania tarentolae UC strain the promoter regions are similar, but not identical, at positions shown previously to be critical for spliced leader RNA transcription. These arrays contain similar numbers of Genes and are both transcribed in L. tarentolae in vitro transcription extract as well as in vivo. The -66/-58 regions of both Genes, which contain an element of the spliced leader RNA Gene promoter, bind proteins likely to be transcription factors in a specific manner. A survey of lizard Leishmania spp. revealed a second spliced leader RNA Gene array in three of four species. PhyloGenetic analyses of these sequences with each other and with the spliced leader RNA Gene sequences of non-lizard Leishmania spp. and their near-relatives showed that the lizard groups are more closely related to each other than to arrays from other Leishmania spp. As the transcripts of the two arrays are identical, they may co-exist to fulfil the substantial requirement for spliced leader RNA production; however, they have the potential for differential usage modulated by their distinct promoter elements. The presence of two distinct spliced leader RNA Gene arrays within a single cell type may represent dissociated evolution of two redundant loci, or a previously unsuspected level of control in the post-transcriptional Gene expression within some kinetoplastids.

  • two sequence classes of kinetoplastid 5s ribosomal RNA Gene revealed among bodonid spliced leader RNA Gene arrays
    Fems Microbiology Letters, 2001
    Co-Authors: Debora M Santana, Nancy R. Sturm, Julius Lukes, David A. Campbell
    Abstract:

    The spliced leader RNA Genes of Bodo saltans, Cryptobia helicis and Dimastigella trypaniformis were analyzed as molecular markers for additional taxa within the suborder Bodonina. The non-transcribed spacer regions were distinctive for each organism, and 5S rRNA Genes were present in Bodo and Dimastigella but not in C. helicis. Two sequence classes of 5S rRNA were evident from analysis of the bodonid Genes. The two classes of 5S rRNA Genes were found in other Kinetoplastids independent of co-localization with the spliced leader RNA Gene.

  • transcription of the kinetoplastid spliced leader RNA Gene
    Parasitology Today, 2000
    Co-Authors: David A. Campbell, Nancy R. Sturm, Michael C Yu
    Abstract:

    In recent years, much has been learned about the cis-elements controlling transcription of the kinetoplastid spliced leader (SL) RNA Gene. The SL RNA Gene contains the first 39 nucleotides that are trans-spliced on to all nuclear-derived mRNAs in these organisms. Transcription initiation is determined by two precisely spaced upstream elements and transcription termination is directed by the downstream poly-T tract, although the RNA polymerase responsible for SL RNA synthesis is still questioned. In this article, David Campbell, Nancy Sturm and Michael Yu review the field of kinetoplastid SL RNA Gene transcription, address past proposals in light of current data and discuss some of the differences that appear in the literature.

Alan P. Wolffe - One of the best experts on this subject based on the ideXlab platform.

  • histones h2a h2b inhibit the interaction of transcription factor iiia with the xenopus borealis somatic 5s RNA Gene in a nucleosome
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Jeffrey J. Hayes, Alan P. Wolffe
    Abstract:

    Abstract A Xenopus borealis somatic 5S RNA Gene was assembled with either the complete octamer of histones, (H2A/H2B/H3/H4)2, or the (H3/H4)2 tetramer of histones that comprises the central protein kernel of the nucleosome. Gel-mobility shifts, DNase I protection, and immunoblotting assays demonstrate that the class III transcription factor IIIA (TFIIIA) readily interacts with 5S DNA associated with the tetramer but that little or no binding is detected when 5S DNA is associated with the full octamer of histones. Thus, the presence of histones H2A and H2B in the 5S nucleosome significantly inhibits the interaction of TFIIIA with its cognate binding site within the 5S RNA Gene. We propose that either the depletion of histones H2A and H2B from preexisting nucleosomes or the staged assembly of chromatin after replication in which a tetramer of histones H3/H4 associates with DNA before histones H2A/H2B will facilitate the binding of transcription factors to their cognate DNA sequences.

Joel M Gottesfeld - One of the best experts on this subject based on the ideXlab platform.

  • definition of the binding sites of individual zinc fingers in the transcription factor iiia 5s RNA Gene complex
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Karen R Clemens, Xiubei Liao, Veronica Wolf, Peter E Wright, Joel M Gottesfeld
    Abstract:

    Abstract A series of polypeptides containing increasing numbers of zinc fingers of Xenopus transcription factor IIIA has been Generated and binding to the 5S RNA Gene inteRNAl control region has been studied in order to elucidate the mode of interaction of the individual fingers with DNA. By using a combination of DNase I footprinting, methylation interference, and differential binding to mixtures of DNA fragments differing in length by single base pairs, the binding sites for individual fingers have been defined. These results have led to a model for the interaction of transcription factor IIIA with the inteRNAl control region in which fingers 1-3 bind in the major groove of the promoter C block, fingers 7-9 bind in the major groove of the A block, and finger 5 binds in the major groove of the intermediate element. Fingers 4 and 6 each bind across the minor groove, spanning these promoter elements.

Debora M Santana - One of the best experts on this subject based on the ideXlab platform.