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Gladfelter Laboratory

Summary:

We study how the cell cycle evolved to function in multinucleated cells. Single cells with many nuclei are found in bones and muscles, in fungal pathogens and in many cancers. We use two evolutionarily related fungi, the uninucleated budding yeast (S. cerevisiae) and a filamentous, multinucleated fungus (A. gossypii) to identify how the cell cycle machinery may have diverged to support accurate division within the spatial requirements of a multinucleated cell. These two related organisms are an excellent pair for such studies because while the genomes share about 95% of the same genes, approximately 100 million years have passed since their common ancestor allowing for significant divergence between homologues. We employ a broad range of experimental approaches including in vivo timelapse microscopy, cell biology, mathematical modeling, biochemistry and genetics to explore how cell cycle networks direct nuclear division within the unique geometry found in cells where many nuclei share one cytoplasm.

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Organisms and Viruses

  • Ashbya gossypii ΔleuΔthr (wild type) ( Eremothecium gossypii )

    Kingdom: Fungi
    Phylum: Ascomycota
    Subphylum: Saccharomycotina
    Class: Saccharomycetes
    Order: Saccharomycetales
    Family: Saccharomycetaceae
    Genus: Eremothecium
    Species: A. gossypii
    Subspecies: ATCC 10895, FDAG Ashbya is a filamentous fungus that has a dual role as a model multinucleated cell in the lab and as a cotton and citrus pathogen in the natural world. The whole genome sequence of Ashbya (carried out in the laboratory of Peter Philippsen, the pioneer of Ashbya cell biology, in collaboration with Syngenta) demonstrated that it diverged from a common ancestor with the budding yeast about 100 million years ago. Unlike budding yeast, the Ashbya genome did not undergo detectable duplication events giving it a smaller (4.2Mb and 4700 ORFs) and more compact genome that lacks much of the redundancy that remains in the yeast genome. Synteny or gene order is conserved for over 90% of the Ashbya genome making it a powerful tool for studying how whole systems, such as the cell cycle machinery, evolved to function within a long multinucleated, hyphae as opposed to uninucleated, spherical yeast cells. Ashbya cells are constitutively filamentous and multinucleated. Each nucleus contains one complete haploid genome and nuclei of different genotypes can reside within one single cell. Cells with genetically different nuclei are called heterokaryons. These may arise in the wild due to DNA damage that is limited to a subset of nuclei and in the lab are formed when Ashbya cells are transformed with either plasmids or linear pieces of DNA for gene targeting. Gene deletions or gene fusions are readily made with PCR fragments containing 45-50bp homology to the loci of interest and a dominant selectable marker encoding genes that supply resistance to compounds such as G418. Heterokaryons can be sporulated to generate asexual spores with a single nucleus and thus a single genotype which are called homokaryons. The phenotypes of mutants are studied in this homokaryon stage. Ashbya has been exploited as a model organism for understanding novel mechanisms of cell cycle control and polarized growth.

  • Sep7-GFP::GEN ( Eremothecium gossypii )

    Kingdom: Fungi
    Phylum: Ascomycota
    Subphylum: Saccharomycotina
    Class: Saccharomycetes
    Order: Saccharomycetales
    Family: Saccharomycetaceae
    Genus: Eremothecium
    Species: A. gossypii
    Subspecies: ATCC 10895, FDAG Ashbya is a filamentous fungus that has a dual role as a model multinucleated cell in the lab and as a cotton and citrus pathogen in the natural world. The whole genome sequence of Ashbya (carried out in the laboratory of Peter Philippsen, the pioneer of Ashbya cell biology, in collaboration with Syngenta) demonstrated that it diverged from a common ancestor with the budding yeast about 100 million years ago. Unlike budding yeast, the Ashbya genome did not undergo detectable duplication events giving it a smaller (4.2Mb and 4700 ORFs) and more compact genome that lacks much of the redundancy that remains in the yeast genome. Synteny or gene order is conserved for over 90% of the Ashbya genome making it a powerful tool for studying how whole systems, such as the cell cycle machinery, evolved to function within a long multinucleated, hyphae as opposed to uninucleated, spherical yeast cells. Ashbya cells are constitutively filamentous and multinucleated. Each nucleus contains one complete haploid genome and nuclei of different genotypes can reside within one single cell. Cells with genetically different nuclei are called heterokaryons. These may arise in the wild due to DNA damage that is limited to a subset of nuclei and in the lab are formed when Ashbya cells are transformed with either plasmids or linear pieces of DNA for gene targeting. Gene deletions or gene fusions are readily made with PCR fragments containing 45-50bp homology to the loci of interest and a dominant selectable marker encoding genes that supply resistance to compounds such as G418. Heterokaryons can be sporulated to generate asexual spores with a single nucleus and thus a single genotype which are called homokaryons. The phenotypes of mutants are studied in this homokaryon stage. Ashbya has been exploited as a model organism for understanding novel mechanisms of cell cycle control and polarized growth.

  • Tub4-Cherry::NAT ( Eremothecium gossypii )

    Kingdom: Fungi
    Phylum: Ascomycota
    Subphylum: Saccharomycotina
    Class: Saccharomycetes
    Order: Saccharomycetales
    Family: Saccharomycetaceae
    Genus: Eremothecium
    Species: A. gossypii
    Subspecies: ATCC 10895, FDAG Ashbya is a filamentous fungus that has a dual role as a model multinucleated cell in the lab and as a cotton and citrus pathogen in the natural world. The whole genome sequence of Ashbya (carried out in the laboratory of Peter Philippsen, the pioneer of Ashbya cell biology, in collaboration with Syngenta) demonstrated that it diverged from a common ancestor with the budding yeast about 100 million years ago. Unlike budding yeast, the Ashbya genome did not undergo detectable duplication events giving it a smaller (4.2Mb and 4700 ORFs) and more compact genome that lacks much of the redundancy that remains in the yeast genome. Synteny or gene order is conserved for over 90% of the Ashbya genome making it a powerful tool for studying how whole systems, such as the cell cycle machinery, evolved to function within a long multinucleated, hyphae as opposed to uninucleated, spherical yeast cells. Ashbya cells are constitutively filamentous and multinucleated. Each nucleus contains one complete haploid genome and nuclei of different genotypes can reside within one single cell. Cells with genetically different nuclei are called heterokaryons. These may arise in the wild due to DNA damage that is limited to a subset of nuclei and in the lab are formed when Ashbya cells are transformed with either plasmids or linear pieces of DNA for gene targeting. Gene deletions or gene fusions are readily made with PCR fragments containing 45-50bp homology to the loci of interest and a dominant selectable marker encoding genes that supply resistance to compounds such as G418. Heterokaryons can be sporulated to generate asexual spores with a single nucleus and thus a single genotype which are called homokaryons. The phenotypes of mutants are studied in this homokaryon stage. Ashbya has been exploited as a model organism for understanding novel mechanisms of cell cycle control and polarized growth.

Reagents

  • pAGT 248 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrineA206R.

  • pAGT 353 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker tdTomato.

  • pAGT100 ( Plasmid )

    Ashbya tagging plasmid.

  • pAGT101 ( Plasmid )

    Ashbya tagging plasmid. Expresses green fluorescent protein (GFP).

  • pAGT102 ( Plasmid )

    Ashbya tagging plasmid. Expresses cyan fluorescent protein (CFP).

  • pAGT103 ( Plasmid )

    Ashbya tagging plasmid. Expressed yellow fluorescent protein (YFP).

  • pAGT104 ( Plasmid )

    Ashbya tagging plasmid. Expresssed red fluorescent protein, RedStar2.

  • pAGT105 ( Plasmid )

    Ashbya tagging plasmid. Contains 6HA marker.

  • pAGT107 ( Plasmid )

    Ashbya tagging plasmid Expresses marker Venus

  • pAGT108 ( Plasmid )

    Ashbya tagging plasmid. Expresses tag: Cerulean.

  • pAGT120 ( Plasmid )

    Ashbya tagging plasmid

  • pAGT121 ( Plasmid )

    Ashbya tagging plasmid. Expressed green fluorescent protein (GFP).

  • pAGT122 ( Plasmid )

    Ashbya tagging plasmid Expresses Cyan fluorescent protein (CFP).

  • pAGT123 ( Plasmid )

    Ashbya tagging plasmid. Expresses yellow fluorescent protein (YFP).

  • pAGT124 ( Plasmid )

    Ashbya tagging plasmid. Expresses yeast-optimized red-fluorescent RedStar2 tag.

  • pAGT125 ( Plasmid )

    Ashbya tagging plasmid.

  • pAGT126 ( Plasmid )

    Ashbya tagging plasmid. Expresses 13Myc tag.

  • pAGT127 ( Plasmid )

    Ashbya tagging plasmid. Expresses Venus marker.

  • pAGT128 ( Plasmid )

    Ashbya tagging plasmid. Expressed Cerulean marker.

  • pAGT140 ( Plasmid )

    Ashbya tagging plasmid.

  • pAGT142 ( Plasmid )

    Ashbya tagging plasmid. Expresses yellow fluorescent protein (YFP).

  • pAGT143 ( Plasmid )

    Ashbya tagging plasmid. Expresses yellow fluorescent protein (YFP).

  • pAGT144 ( Plasmid )

    Ashbya tagging plasmid. Expresses yeast-optimized red-fluorescent RedStar2 tag.

  • pAGT145 ( Plasmid )

    Ashbya tagging plasmid. Expresses 6HA marker.

  • pAGT146 ( Plasmid )

    Ashbya tagging plasmid. Expresses 13Myc marker.

  • pAGT147 ( Plasmid )

    Ashbya tagging plasmid. Expresses Venus marker.

  • pAGT148 ( Plasmid )

    Ashbya tagging plasmid. Expresses Cerulean marker.

  • pAGT149 ( Plasmid )

    Ashbya tagging plasmid. Expresses GST.

  • pAGT201 ( Plasmid )

    Ashbya tagging plasmid. Expresses yEGFP.

  • pAGT202 ( Plasmid )

    Ashbya tagging plasmid. Expresses green fluorescent protein (yEGFP).

  • pAGT203 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFPA206R.

  • pAGT204 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP. Expresses marker 3HA.

  • pAGT205 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP. Expresses marker 13Myc.

  • pAGT206 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEVenus.

  • pAGT207 ( Plasmid )

    Ashbya tagging plasmid Expresses marker yECitrine.

  • pAGT208 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEmCitrine.

  • pAGT209 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrine. Expresses marker 3HA.

  • pAGT210 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrine. Expresses marker 13Myc.

  • pAGT211 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker mCherry.

  • pAGT212 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker mStrawberry.

  • pAGT213 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker tdTomato.

  • pAGT221 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEGFP1.

  • pAGT222 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP.

  • pAGT223 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFPA206R.

  • pAGT224 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP. Expresses marker 3HA.

  • pAGT225 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP. Expresses marker 13Myc.

  • pAGT226 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEVenus.

  • pAGT227 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrine.

  • pAGT228 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrineA206R.

  • pAGT229 ( Plasmid )

    Ashbya tagging plasmid Expresses marker yECitrineA206R. Expresses marker 3HA.

  • pAGT230 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrine. Expresses marker 13Myc.

  • pAGT241 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEGFP.

  • pAGT242 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP.

  • pAGT243 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFPA206R.

  • pAGT244 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP. Expresses marker 13Myc.

  • pAGT245 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECFP. Expresses marker 13Myc.

  • pAGT246 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEVenus.

  • pAGT247 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrine.

  • pAGT249 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yECitrine. Expresses marker 3HA.

  • pAGT250 ( Plasmid )

    Ashbya tagging plasmid Expresses marker yECitrine. Expresses marker 13Myc.

  • pAGT341 ( Plasmid )

    Ashbya tagging plasmid Expresses marker yEGFP.

  • pAGT342 ( Plasmid )

    Ashbya tagging plasmid Expresses marker yECFP.

  • pAGT343 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEmCFP.

  • pAGT346 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEVenus.

  • pAGT347 ( Plasmid )

    Ashbya tagging plasmid. Expresses marker yEmCitrine.

  • pAGT348 ( Plasmid )

    Ashbya tagging plasmid. Contains marker yEmCitrine.

  • pAGT351 ( Plasmid )

    Ashbya tagging plasmid. Expresses fluorescent protein, mCherry.

  • pAGT352 ( Plasmid )

    Ashbya tagging plasmid. Expresses red fluorescent protein, mStrawberry.


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Last updated: 2011-04-29T12:31:41.627-04:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016