Homologous Recombination Range

1
Homologous Recombination Range

• Yes...
• Mice, many well characterized mammalian cells,
• bacteria, yeast,
• No (maybe)...
• C. elegans,
• Arabidopsis (done once, not repeated),
• Drosophila (shown in principle, not repeated),
• the rest?

2
Epigenetics

• ...is the study of heritable changes in gene
  expression that occur without a change in DNA
  sequence,
• DNA methylation,
• chromosome remodeling,
• RNA turnover.

3
Genome Surveilance

• ...epigenetic control of gene expression is now
  considered a component of normal development,
• stable repression of genes not required by
  specific cell types,
• defense against viruses,
• control of transposable elements,
• others?

4
Co-suppression(RNA turnover)

• ...gene silencing was first observed in plants,
• transgene expression often decreases as the copy
  number of transgenes increased.

5
Modes

• ...Translational Gene Silencing (TGS),
• mRNA may potentiate the methylation of promoters,
• ...Post-Translational Gene Silencing (PTGS),
• appears to involve the specific degradation of
  mRNA via a double-stranded RNA intermediate,
  dsRNA.

6
RNAiRNA interference

• ...while attempting to do anti-sense KO of gene
  expression in C. elegans, Guo and Kemphues, Cell
  81, 611 (1995) observed that sense and anti-sense
  strands worked equally,
• in an anti-sense experiment, a gene is
  constructed so that it produces a complementary
  strand to an expressed transcript,
• the goal is to complement, thus inactivate the
  mRNA.
• ...following up, other researchers found that
  dsRNA worked at least an order of magnitude
  better that either sense or anti-sense strands.

7
dsRNA
8
dsRNA Delivery

• ...dsRNA can move across cell boundaries,
• through the gut of C. elgans,
• injected,
• ingested, E. coli expressing a dsRNA,
• soaking,
• through the vascular system of plants,
• ...or deliver as a heritable transgene.

9
amplification
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11
It Works

• Arabidopsis Homeobox Genes

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Next Week

• RNAi
• Genetics,
• Biochemistry.