HerrickLab
Jon Seger Kevin Williams Tom Doak David 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
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.
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…
Baldauf tree
Baldauf et al. 2000. Science 290:972.
• 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.
• 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
Macronuclear developmentfrom a mitotic sister of the new MIC
Polytene chromatids
~95%
p
Chromatid breakageand
concertedde 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
CellPairing
Meiosis andNuclear Exchange
Nuclear Fusion andDuplication of theZygotic Nucleus
Macronuclear Developmentand Nuclear Degeneration
MIC
MAC
Conjugation and Macronuclear DevelopmentConjugation and Macronuclear Development
Modified fromLarry Klobutcher & Carolyn JahnAnn. Review Microbiology, 2002
PolytenizationChromatid breakageDe novo telomere formation
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