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