Download de novo

HerrickLab
Jon Seger
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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.
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Ciliates diverged from other
eukaryotes maybe 1.1-1.2 BYA.
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.
Re: Baldauf tree
Nuclear dimorphism:
Relationship between nuclei
• Clonal proliferation by
binary fission
• 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.
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Macronuclear development
Macronuclear
– from a mitotic sister of
the new MIC
development
from a mitotic sister
Polytene chromatids
Telomeres
of the new MIC
– 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
All ~12,000 TBE
transposons, by
precise excision
~95%
Chromatid breakage
and
concerted
de novo telomere
formation
• 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
– Replication leaves 5’termini
• Recessed
• 5’ phosphorylated
— important for TAS mapping
p
Conjugation and Macronuclear Development
MIC
MAC
Cell
Pairing
Meiosis and
Nuclear Exchange
Nuclear Fusion and
Duplication of the
Zygotic Nucleus
Macronuclear Development
and Nuclear Degeneration
Polytenization
Chromatid breakage
De novo telomere formation
Modified from
Larry Klobutcher & Carolyn Jahn
Ann. Review Microbiology, 2002
Four Telomere Addition Site regions of the 81 MAC family locus
“CR-L”
“CR-R”
4 TAS regions of the 81 MAC
locus• Model for generation of family
by alternative processing of
polytene chromatids.
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Three chromosomes,
comprised from three segments:
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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!
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Complete cutting of all chromatids at the
arm ends…
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but incomplete cutting at the CR borders,
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de novo telomere formation on MAC ends,
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generates MACs III’s, II’s, & I’s