title: Tracking chromosome dynamics in live yeast cells: coordinated movement of rDNA homologs and anaphase disassembly of the nucleolus during meiosis.
abstract: A prerequisite for determination of chromosome dynamics in live cells is development of a method for staining or marking the chromosome of interest. We describe here a unique chromosome-tracking system that differentially marks two large chromosome segments from homologs in the budding yeast Saccharomyces cerevisiae. Using yeast genetics and the special features at the repetitive ribosomal RNA (rRNA) gene cluster, we incorporated arrays of the tet operator and the lac operator into each repeat of the two rDNA homologs by homologous recombination. Expression of tet repressor-fused green fluorescent protein and lac repressor-fused red fluorescent protein in engineered cells led to the differential labeling of rDNA homologs. Using live-cell three-dimensional fluorescence microscopy, we showed that homologs undergo contraction and expansion cycles in an actin-dependent manner during meiosis and that chromosome mobility appears to be correlated with nuclear positioning. Our observations further revealed that, in contrast to mitosis, in meiosis the yeast nucleolus, the site of rRNA processing, was disassembled upon anaphase onset, suggesting a differential regulation of the rDNA array during meiotic chromosome segregation. Because rRNA genes are highly conserved, a similar chromosome-engineering approach may be adaptable in other eukaryotes for functional assays of chromosome organization in live cells.
authors.affiliation: Department of Biological Science, The Florida State University, Tallahassee, FL 32306-4370, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Jin
authors.forename: Margaret L
authors.initials: ML
authors.lastname: Hoang
authors.forename: Hong-Guo
authors.initials: HG
authors.lastname: Yu
mesh_terms: D000705:Anaphase / Q000235:genetics*; D002466:Cell Nucleolus / Q000235:genetics* / Q000378:metabolism; D020090:Chromosome Segregation / Q000235:genetics; D015825:Chromosomes, Fungal / Q000235:genetics* / Q000378:metabolism; D004275:DNA, Ribosomal / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics; D059765:Homologous Recombination / Q000235:genetics; D056968:Lac Repressors / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008540:Meiosis; D008856:Microscopy, Fluorescence; D015202:Protein Engineering / Q000379:methods; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D004275:DNA, Ribosomal; D049452:Green Fluorescent Proteins; D056968:Lac Repressors; D008164:Luminescent Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D000097573:Red Fluorescent Protein; C034811:tetracycline resistance-encoding transposon repressor protein
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