Winston Group Meeting

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Winston Group Meeting

• 09.19.06

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Overview

• 2 projects SAGA swap
• mtDNA rewrite
• justification
• design
• data
• next steps/next summer (

Cindy Kolodziejski Bittersweet Chocolate Drop,
2004 earthenware with metal support 23.5 x 5.0 x
9.5 in.
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mtDNA rewrite justification
Dedicated system for synthesis

• Ideally want system channel/chassis channel to be
• orthogonale.g. draw from different pools of
  reagents
• decouplede.g. run system independent of growth
  rate
• generice.g. run same system in different chassis

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mtDNA rewrite justification
(we often) imagine the mitochondrion as a
lonely participant in the cell, working
tirelessly to produce the energy required for
life. McBride et al Curr Biol 2006
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mtDNA rewrite justification
mt DNA 85,779 bps 8 verified protein encoding
genes 24 tRNA genes 2 rRNA genes 20 nucleic acid
processing factors encoded by introns
http//db.yeastgenome.org/cgi-bin/gbrowse/yeast/?n
amechrMito3A1..85779
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mtDNA rewrite design
Existing marker for mtDNA manipulation
2 other mtDNA markers GFP, BARSTAR
Steele et al PNAS (1996) 935253
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mtDNA rewrite design
New marker for mtDNA

• Considered
• MEL1
• xFP
• LYS12
• PUT1
• HEM1

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mtDNA rewrite design
New marker for mtDNA

• Considered
• MEL1
• xFP
• LYS12
• PUT1
• HEM1

5-aminolevulinate synthase
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mtDNA rewrite design
New marker for mtDNA

• Considered
• MEL1
• xFP
• LYS12
• PUT1
• HEM1

5-aminolevulinate synthase 1647 bp, 549
aa localized to mitochondrial matrix
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain
DSF160 from Tom Fox, Cornell MATalpha ade2-101
leu2? ura3-52 arg8? URA3 kar1-1 rho0
outgrow in YPD ON select on G418dALA
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain
5
6
MH339
4
DFS160
3
1
2
G418 dALA
G418 30 2d
Also checked by PCR with KanMX/dwstm primer And
will check by Western
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mtDNA rewrite design
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1
BBpre
BBsuff
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
Select for nuclear txn (Leu), screen for mt txn
(dALA indep)
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
Take 1
Visitor with Yasunori Hayashi and Ken Okamoto
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
Take 2
Visitor with Marc Vidal and Stu Milstein
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
Take 2
Visitor with Marc Vidal and Stu Milstein
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
Take 2
Visitor with Marc Vidal and Stu Milstein
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
Take 2
Visitor with Marc Vidal and Stu Milstein
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mtDNA rewrite data
Transformation results
with screen DFS160 DFS160 hem1KanMX No
DNA 0 6 (hole in plate) pRS415 0, 0
6, 6 pRS415, BBa_Y00100 nd 3, 4
no screen No DNA 25 (shot last)
0 pRS415 28, 90 58, 40 pRS415,
BBa_Y00100 nd 78, 35
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mtDNA rewrite data
Transformation results
with screen DFS160 DFS160 hem1KanMX No
DNA 0 6 (hole in plate) pRS415 0, 0
6, 6 pRS415, BBa_Y00100 nd 3, 4
-leudALA
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mtDNA rewrite data
Step 1 hem1KanMX deletion strain Step 2
synthesis of mitochondrially encoded HEM1 Step
3 biolistic transformation
Select for nuclear txn (Leu), screen for mt txn
(dALA indep)
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mtDNA rewrite data
Check for mtDNA by mating
x
ade2 leu2? hem1KanMX kar1-1 pRS415 rho0
mHEM1?
Desired strain will be red/Leu/G418R/dALA
indep/respiration deficient 2 of 7 show these
p-types
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mtDNA rewrite data
LEU2
LEU2 mHEM1
hem1
Mated cand 1
Mated cand 2
-leu dALA
-leu 30 3d
red/Leu/G418R/dALA indep/respiration
deficient
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mtDNA rewrite data
G418 dALA
G418 30 3d
red/Leu/G418R/dALA indep/respiration
deficient
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mtDNA rewrite data
YPEG dALA
YPEG 30 3d
red/Leu/G418R/dALA indep/respiration
deficient
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mtDNA rewrite data
mHEM1 seems to complement nuclear hem1? but
unclear why integration into mtDNA makes cells
more red why respiration requires dALA

• Follow up with
• Western.protocol for isolating of mt proteins?
• PCR of mtDNAprotocol for isolation mtDNA?)
• Microarray
• Other targets in mtDNA

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Thanks and see you next summer!