Assignment 2

1
Lecture 7

• Assignment 2
• RT-PCR process and principles
• Etiolation
• DET1
• Discussion of Article 4

2
Assignment 2

• Sample 1 General Hans Canis
• Sample 2 Field Marshall Heinrich Rattus
• Sample 3 Mrs. Heidi Rattus
• Sample 4 Miss. Gertrude Musca
• Sample 5 Mr. Ivan Apis
• Sample 6 Blood on the envelope
• Sample 7 Hair found in Gertrudes apartment.

3
Assignment 2
Assignment 2 due Nov 10, 2008
4
Experimental set up
5

• 1. wild-type grown in the light
• 2. wild-type grown in the dark
• 3. det1 grown in the light
• 4. det1 grown in the dark

What are the expression levels of light regulated
genes (chlorophyll a/b binding proteins CHAB and
CLAB) using tubulin as loading control.
CHAB
CLAB
det1-light
WT-dark
det1-dark
det1-light
det1-dark
WT-light
WT-dark
Ladder
WT-light
-Extract RNA -RT mRNA -PCR cDNA
CHAB or CLAB
TUB
Picture of Plants (WT and mutant)
6
Trizol Method

• Why do we grind in liquid N2?
• What does the Guanidium Thiocyanate do?
• What does the Phenol do?
• What does the Chloroform do?
• What does the isopropanol do?

7
Isolation of RNA using Commercial Trizol Reagent
Trizol-mono-phasic solution of phenol and
guanidine isothiocyanate -lyses cells and
dissolves cell components while maintaining
integrity of RNA
Phenol Chloroform
75 EtOH
Liquid N2 Trizol
Isopropyl alcohol
Aq
Org
Homogenize
Ppt RNA
DEPC H2O
RNA
Chloroform separate aqueous and organic phases
(RNA is in the top aqueous phase) Isopropanol
Precipitates RNA 75 Ethanol Wash RNA
pellet DEPC (Diethylpyrocarbonate) water
Resuspend RNA pellet in RNAse-free water DEPC
interacts with the active site histidine residue
of RNAse and irreversibly inactivates RNAses.
8
RNA preparation Considerations

• Yield What controls yield?
• RNases What do we need to control this?
• Quality of mRNA What do we mean?
• DNA contamination Why is this important in
  RT-PCR?

9
Dangers this week

• TRIzol is very dangerous and can cause severe
  chemical burns.
• Liquid N2 is very cold and can burn the skin be
  careful and no fooling around.

10
Original Northern blot
Pepper et al., 1994 Cell 78, 109-116
11
RT-PCR reverse transcriptase
12
COMPONENTS TO ADD FOR REVERSE TRANSCRIPTION
13
COMPONENTS TO ADD FOR REVERSE TRANSCRIPTION
Add 0.5 µl Superscript reverse transcriptase.
14
What do you think is in the RT buffer?
15
Multiplex PCR

• What is multiplex PCR?
• What are the top two considerations when
  performing a multiplex PCR?

16
What is in our multiplex PCR?

• What is the control and what are light regulated?

17
What is Multiplex PCR? (Combinatorial PCR)
Amplifying various genes simultaneously (One
template with multiple primer pairs)
CLAB
CHAB
det1-light
WT-light
WT-dark
det1-dark
det1-light
det1-dark
WT-light
WT-dark
Ladder
CHAB or CLAB
TUB
Why are we amplifying (CHAB or CLAB) and tubulin
simultaneously?
18
RT-PCR

• What is the problem with having DNA
  contamination?
• Why is a set of exon primers that span an intron
  a good idea?

19
Hint!!!
20
What will be the outcome of an RT-PCR experiment
if there is DNA contamination?
21
Nice figure taken from Ann. Rev. Cell Dev. Biol
13, 203
22
Etiolated stage

• Long hypocotyl (primary stem)
• Apical hook
• Unopened cotyledons (embryonic leaves)

23
De-etiolated

• Hypocotyl growth decreases
• Apical hook opens
• Cotyledons expand
• Chloroplasts develop
• A new gene expression program is induced

24
Comparison of dark-grown (etiolated) and
light-grown (de-etiolated) seedlings

25

• Etiolation and de-etiolation are normal stages of
  plant development.
• After germination there may be a lack of light
  and the seedling grows to find the light.
  Etiolated.
• When sufficient light is found the seedling
  de-etiolates a developmental process that
  optimizes the body plan for photosynthesis.

26
Picture of DET1 function
DET1 is a nuclear protein with a nuclear
localization sequence.
DET1 is part of a protein complex with COP10 and
DDB1 called CDD
CDD stimulates ubiquitin-conjugating
enzymes. Ubiquitination of HY5 (required for
normal light development) -targets it for
degradation.

• Genes and Dev. 18 2172-2181(2004)

27
DET1 function in light and dark
28
Why have det1 mutants lost this response?

• No DET1 HY5 is not degraded and can not initiate
  etiolated growth.

29
Det1 in Humans?

• Human de-etiolated-1 regulates c-Jun by
  assembling a CUL4A ubiquitin ligase. Science
  303, 1371-1374 (2004).
• Jun is an oncogene!

30
Discussion of Article 4
31
(No Transcript)
32
(No Transcript)