Schedule

1
Schedule

• Weds. (6/2)
• Functional genomic analysis of cell divisions in
  C. elegans using RNAi of genes on chromosome III,
  Gonczy et al. Nature 408 331-336,
• Question and Answer Session ES100 12 - 1 pm,
• Friday (6/4) Question and Answer Session,
• Monday (6/7) Midterm IV,
• 1030 -1230, RNAi papers (3), general class
  concepts.

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(cont.)
Today
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Figure 1b.
What does Gated figure establish?
GFP co-transfected Shows transfection alone does
not inhibit cell cycle.
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Figure 3.
What does this figure establish?
AndSequence-specific nuclease activity,
however, did require protein (data not shown).

• Whats a Ca-dependent nuclease?
• What does microccocal nuclease do?
• What does EGTA do?
• Why assay DNAse I activilty in vitro?
• Why tRNA (why the controls)?

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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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?
What did we learn?
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New Phenomenon

• Grind em, find em.

Nature makes em, we breaks em.
insight
Genomics
Broader Understanding
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dsRNA
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Caenorhabditis elegansA small, free-living
bacteriovorous soil nematode.
The Model Organism

• bacteriovorous in the lab, C. elegans is fed on
  E. coli.
• sex and self-fertilization adult C. elegans are
  male or protandrous hermaphrodites.
• protandrous the hermaphrodite gonad first
  produces germ cells which differentiate as sperm
  (circa 250 sperm are produced) and then produces
  eggs,
• or, males can fertilize hermaphrodite eggs,
• C. elegans is diploid and has five pairs of
  autosomal chromosomes (named I, II, III, IV and
  V) and one sex chromosome (X).
• Gender is determined by an XX (hermaphrodite) -
  XO (male) chromosomal system.
• males are found rarely (about 0.05 of normal lab
  populations).

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Making dsRNA(examples)
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dsRNA Delivery(pick your poison)

• Eaten
• Head-to-Head promoter on a plasmid in E. coli,

• Injected
• Microinjection to the intestines of the worm.

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Fig 1a
Eat dsRNA from where?
Selecting for Mutants
Fig. 1
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Complementation Groups and Mapping(self quiz)

• 7 mutants
• 4 complementation groups
• rde-1 (3) , rde-2, rde-3 and rde-4 (2)

Fig. 1
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mut-2, mut-7

• ...exhibit high rates of transposon mobilization
  compared to wild type,
• ...are also RNAi deficient.

How was RNAi deficiency confirmed for the mut-2
locus?
How was RNAi deficiency confirmed for mut-7 locus?
Sensitivity to RNAi was tested in the following
strains during the course of this work. MT3126
mut-2(r459) dpy-19(n1347), TW410 mut-2(r459)
sem-4(n1378), NL917 mut-7(pk204), SS552 mes-
2(bn76) rol- (e91)/mnC1, SS449 mes-3(bn88)
dpy-5(e61) hDp20, SS268 dpy-11(e224)
mes-4(bn23) unc-76(e911)/nT1, SS360 mes-6(bn66)
dpy-20(e1282)/nT1, CB879 him-1(e879). A non-Unc
mut-strain used was derived from RW7096,
mut-6(st702) unc-st192Tc1), due to the loss of
Tc1 insertion in unc-22.
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fig 2a
Microinjected dsRNA

• What does this figure tell you?

Fig. 2
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fig 2b
Microinjected dsRNA
Where are pos-1 and par-2 expressed?

• What does this figure tell you?

Fig. 2
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fig 2c
Microinjected dsRNA
Where is sqt-3 expressed?
Who is treated, how?

• What does this figure tell you?

Fig. 2
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DsRNA Delivery(pick your poison)

• Transposons
• Tested in Mutator lines, mutants that have
  higher frequency of transposon mobilizations,

• Transgenes
• GFP transgenes in a variety of constructs.

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tab 1
What is unc-22 (r765Tc4)?
What has happened when non-Unc worms appear?

• What does this table tell you?

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tab 2
What is transgene silencing?
mes mutants?
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fig 3
Why rde-1 and rde-4?
dsRNA uptake, transport, stability?

• What does this figure demonstrate?

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tab21
cell specific, RNAi activity ?
reporter gene

• rde-1 and rde-4 show RNAi activity?

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Cloning rde-1

• ...co-transfect rde1 worms with YACs or cosmids
  (containing wild-type genomic DNA), and plasmids
  that produce unc-22(RNAi),

rde1 unc-22(RNAi)
rde1 unc-22(RNAi) RDE1
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How did they locate RDE-1? Confirmation?
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Pfam PAZ PIWI Domains.
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Wednesday

• Please read.

Nature 408 331 - 336