2. HerrickLab Jon SegerKevin WilliamsTom DoakDavid Witherspoon I. Understanding de novo telomere formation in Oxytricha macronuclear development by analyses of cis-acting sequences II. Developmental transcription of transposons of Oxytricha trifallax: old data in the light of ciliate RNAi phenomena

3. Oxytricha trifallax SEM A ciliated protozoan. All ciliates are covered with cilia. –in this hypotrichous ciliate, bundles of cilia are used to walk on the substrate, in fresh water.

4. DAPI-stained vegetative cell All ciliates also have: a specialized gene expression organelle, the macronucleus [MAC]. We study –development of the “somatic” macronucleus –from a copy of the “germline” micronucleus [Mi] –after sexual conjugation. Cilia, and this nuclear dimorphism— the two major taxonomic characters that define the clade, ciliates…

5. Baldauf tree Baldauf et al. 2000. Science 290:972.

6. Ciliates diverged from other eukaryotes maybe 1.1-1.2 BYA. Re: Baldauf tree Features in common between ciliates and us are especially rich to study in ciliates, because shared features have been conserved and are probably important. We will consider ciliate developmental chromosome breakage and de novo telomere formation. This process occurs massively during macronuclear development. Telomeres [“end bodies”] cap the ends of eukaryotic chromosomes and make them inert. The failure of telomere function appears to be basic to metazoan cell senescence and oncogenic initiation.

7. Conjugation –Meiosis: 2N => 1N –Gametic nuclei exchange –Zygosis: 2N+1N=2N Replace old MAC with new MAC –Destroy old MAC –Duplicate zygotic 2N nucleus One copy is new MIC Edit other copy =>new MAC New MAC => mRNAs MAC development takes ~3 days. A rich program. Nuclear dimorphism: Relationship between nuclei Clonal proliferation by binary fission

8. Macronuclear development from a mitotic sister of the new MIC Polytene chromatids ~95% p Chromatid breakage and concerted de novo telomere formation Macronuclear development –from a mitotic sister of the new MIC Telomeres –40,000,000/MAC Rich biochem source of –telomere DNA –telomere proteins First studied in Oxytricha & Tetrahymena –Created de novo in a few hours, by telomerase Exconjugants a rich source Large RiboNucleoProtein –A reverse transcriptase protein –Carries its own RNA template Polymerizes GT-rich repeats onto 3’ OH ends ≥20,000 genes All ~12,000 TBE transposons, by precise excision –Replication leaves 5’termini Recessed 5’ phosphorylated — important for TAS mapping

9. Cell Pairing Meiosis and Nuclear Exchange Nuclear Fusion and Duplication of the Zygotic Nucleus Macronuclear Development and Nuclear Degeneration MIC MAC Conjugation and Macronuclear Development Modified from Larry Klobutcher & Carolyn Jahn Ann. Review Microbiology, 2002 Polytenization Chromatid breakage De novo telomere formation

10. Four Telomere Addition Site regions of the 81 MAC family locus 4 TAS regions of the 81 MAC locus “CR-L”“CR-R” Model for generation of family by alternative processing of polytene chromatids. Complete cutting of all chromatids at the arm ends… but incomplete cutting at the CR borders, de novo telomere formation on MAC ends, Three chromosomes, comprised from three segments: –Each segment carries a protein coding gene. –All chromosomes share a “common region” (“CR”). MAC III = CR+telomeres MACs I & II –have arms appended to their CRs. –have two genes each: “gene-sized” NOT! generates MACs III’s, II’s, & I’s