Gene Expression new frontiers

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Gene Expressionnew frontiers

• the processes by which information contained in
  genes and genomes is decoded by cells, in order
  to produce molecules that determine the
  phenotypes observed in organisms,
• transcription is controlled so that the correct
  DNA sequences are expressed as mRNA in the right
  cells, at the right time, and in the right amount.

- and, now we are learning -
- translation of mRNA is further controlled
(through RNA/Protein complexes), via ancient,
conserved processes.
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Central Dogma addendum
DNA
RNA
Transcription Factors
Protein
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Transcriptional Network(cell cycle)
(example)
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Central Dogma addendums
DNA
TGS Transcriptional Gene Silencing
RNA
Transcription Factors
Protein
PTGS Post Transcriptional Gene
Silencing
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tiny RNAs (20-to-25 nt RNAs)

• in eukaryotic cells, tiny RNAs function as
  post-transcriptional regulators of gene
  expression in (at least) two distinct pathways,
• small interfering RNAs (siRNAs) direct RNA
  destruction via the RNA interference (RNAi)
  pathway,
• micro (miRNAs) regulate RNA translation.

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Ancient History (1)
Cell 75, 843 (1993)
Some development timing genes code for short
anti-sense molecules, appeared to be unique to
C. elegans.
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miRNAmicro-RNA
How would you express this?

• Post-transcriptional regulatory genes,
• contain 22 nucleotides (processed),
• are cleaved from somewhat larger double stranded
  RNA (dsRNA) precursors - by a protein called
  Dicer
• are expressed in certain cell types and at
  certain times during differentiation (also
  called short temporal (stRNA).

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Anti-Sense Blocking of Translation
Why use RNA to block mRNA function?
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miRNAs

• Conserved amongst eukaryotic cells,
• Often associated with hetrochronic genes,
• difficult to identify in genomic sequences
  because they dont have long ORFs,

How might you locate them?
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Ancient History (II)(co-suppression)

• Transgene expression often decreases as the copy
  number of transgenes increased.

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Co-suppression Modes

• ...Transcriptional Gene Silencing (TGS),
• RNA functions in the methylation of promoters and
  structural elements of genes,
• ...Post-Transcritional Gene Silencing (PTGS),
• involves the specific degradation of mRNA via a
  double-stranded RNA intermediate, dsRNA.

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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.

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RNAi

• ...siRNA control of gene expression by RNA
  processing is now considered a common element in
  eukaryotic cells,
• defense against viruses,
• control of transposable elements,
• regulate gene expression?

• useful for doing Reverse Genetic studies,
• dsRNA triggers gene specific degradation of
  complementary mRNAs.

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New PhenomenonBiochemistry/Genetics/Genomics

• Grind em, find em.

Nature makes em, we breaks em.
insight
Genomics
Broader Understanding
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Nature, 404, pp 293
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Drosophila S2 Cells(cell tissue cultures)

• ...loss-of-function phenotypes?
• targeted degradation of exogenous RNA,
• targeted degradation of endogenous RNA,
• RNAi () activity, RNAi(-) activity.

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Figure 1a.
What does this figure establish?

• Transient Expression of lacZ?
• Transfection?
• CD8 ds RNA?
• lacZ dsRNA?

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Figure 1b.
What does this figure establish?

• Endogenous Gene Silencing?
• Count?
• PI?

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Figure 1b.
What does this figure establish?

• Endogenous Gene Silencing?
• Count?
• PI?

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Figure 1c.
What does this figure illustrate?

• Northern blot?
• Whats bound to the membrane?
• Whats labelled (probe)?
• Treatment?

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Figure 2a.
What does this figure establish?

• Cellular extracts, from cells treated how (MM)?
• Synthetic mRNA transcripts?
• Whats bound to the membrane?
• Whats the probe?

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Figure 2b.
What does this figure show?

• Whats the extract?
• Whats the target?
• Whats bound to the membrane?
• Whats the probe?

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Figure 2c.
What does this figure show?

• Whats the extract?
• Whats the target?
• Whats bound to the membrane?
• Whats the probe?

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Figure 3.
What does this figure establish?

• Whats a Ca-dependent nuclease?
• What does microccocal nuclease do?
• What does EGTA do?
• Why assay DNAse I activilty in vitro?

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Figure 4a.
What does this figure establish?

• Whats the probe?
• why the sense strand?
• Whats bound to the membrane?
• what steps did the RNA and associated molecules
  go though prior to gel electrophoresis? Why?
• Why is there so much signal?

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Figure 4b.
What does this figure establish?

• Whats the probe?
• Whats bound to the membrane?
• Whats different in each lane?

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?
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Please read for Friday.
Cell, 1999, 99, pp 123-132
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Midterm III(107 / 2 3.5 pt curve)

• Average (37.5 pts, 75 )
• 45 -
• 40 -
• 35 -
• 30 -
• 20 -