Winston Group Meeting

1
Winston Group Meeting

• 09.19.06

2
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.
3
SAGA swap justification
4
SAGA swap justification

• Bacterial Device Engineering

5
SAGA swap justification
Define conserved functions of SAGA
subunits Understand role/mech. of SAGA-regulated
gene expression
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
Wu Mol Cell (2004) 15199
6
SAGA swap justification
Define conserved functions of SAGA
subunits Understand role/mech. of SAGA-regulated
gene expression
Ada1 Ada2 Ada3 Gcn5
?
SPCC61.02 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
7
SAGA swap justification
Madison, Winston Yeast (1998) 14409
8
SAGA swap justification
Ada1 Ada2 Ada3 Gcn5
Spt3 Spt7 Spt8 Spt20
Taf5 Taf6 Taf9 Taf10 Taf12
Tra1
Sgf73 Sgf29 Sgf11 Ubp8 Sus1
SPCC126.04c
SPBC1921.07c
9
SAGA swap exptl design

• Starting strain
• Intermediate
• Final strain

ura3-52
ura3-52
ura3-52
10
SAGA swap exptl design
Phenotype sgf73 fails to induce hypoxic gene
expression
MH176 MATa ura3?0 his3?200 leu2?0 lys2-128? HAP1
DAN3-HIS3
NY350 MATa ura3?0 his3?200 leu2?0 lys2-128? HAP1
DAN3-HIS3 sgf73URA3
11
SAGA swap exptl design
MH176 wt FY2475 sgf73?0KanMX FY2474
sgf29?0KanMX
12
SAGA swap design

• S.p. Sgf73 into S.c.
• cerevisiae pombe
• gene SGF73 SPCC126.04c
• 1973 bp 1106 bp (unspliced)
• 1035 bp (spliced)

13
SAGA swap design

• S.p. Sgf29 into S.c.
• cerevisiae pombe
• gene SGF29 SPBC1921.07c
• 779 bp 836 bp (unspliced)
• 735 bp (spliced)

14
SAGA swap design

• S.p. Sgf73 codon usage in S.c.

Total 15/345 red 37/345 grey
15
SAGA swap design

• S.p. Sgf29 codon usage in S.c.

Total 9/244 red 32/244 grey
16
SAGA swap design
17
SAGA swap design
18
SAGA swap data
sgf73URA3 in MH176
used primers /- 100 bp SGF73 gives 2kb URA3
gives 1kb
2 1
19
SAGA swap data
sgf73URA3 in MH176
confirm w/ URA3 dwstm primer SGF73 gives no
product URA3 gives 500bp
1
20
SAGA swap data

• Phenotypes of intermediate strains

MH176 sgf73KMX sgf73URA3 sgf29KMX sgf73URA3,
sgf29KMX sgf29URA3, sgf73KMX
YPD, 30 4d
YPD 3 form
YPEG
21
SAGA swap data
22
SAGA swap data
Round 1 txd 10 ul of PCR rxn YPD ON (plates and
liquid) to FOA
23
SAGA swap data
Round 2 cotxn 40 ul PCR/purif w/ 2ug pRS415 YPD
ON (liquid) to -L ON replica to FOA
24
SAGA swap data
Round 3 reamplify so 80 bp homology 200ul
PCR/purified YPD ON (plates) replica to FOA
25
SAGA swap data
Round 4 40 bp homology Cotxd with 3 ug
pRS415 500ul PCR/purified (lost some) YPD ON
(plates) replica to -LFOA
26
SAGA swap data
Round 5 40 bp homology Cotxd with 3 ug
pRS415 500ul PCR/purified (lost some) Marks
help -leu ON (plates), replica to FOA
27
SAGA swap data
confirm w/ SpSgf73 dwstm primer SpSgf73 gives
1.2 kb URA3 gives no product
SpSgf73 in MH176
1
28
SAGA swap data
29
SAGA swap data

• Phenotypes of final strains

sgf73URA3 in MH176 S.p.Sgf73 candA S.p.Sgf73
candG S.p.Sgf73 candE S.p.Sgf73
candH S.p.Sgf73 P344A candD
YPD, 30 2d
YPD 3 form
YPG
30
SAGA swap data

• Phenotypes of final strains

sgf73URA3 in MH176 S.p.Sgf73 candA S.p.Sgf73
candG sgf73URA3 sgf29KMX S.p.Sgf73
sgf29KMX candA S.p.Sgf73 sgf29KMX candD
YPD, 30 2d
YPD 3 form
YPG
31
SAGA swap data

• Phenotypes of final strains

S.p.Sgf29 sgf73KMX candG S.p.Sgf29 sgf73KMX
candH S.p.Sgf29 sgf73KMX candI S.p.Sgf29
sgf73KMX candJ S.p.Sgf29 sgf73KMX
candK S.p.Sgf29 sgf73KMX candL
YPD, 30 2d
YPD 3 form
YPG
32
SAGA swap next

• Verify seq? epitope tag?
• Other consequences DAN3-HIS3? microarray?
  optimized vs genomic? protein expressed?
• Chimeras, Doubles, Triples, etc
• Others S.p. Sgf11, Ubp8, Sus1? teaching module?

33
Thank you
34
SGF73 SGF29
S. cerevisiae
S. pombe
35
Device Engineering
Gene expression devices
Building a robot that works involves building a
robot that doesnt work and then figuring out
what is wrong with it. -Benjamin Irwin
36

• For Endy lab presentation
• Bacterial cells DNA not nucleosome-bound..but euk
  cells DNA 3m long w/o
• Several chromatin modifiers exist that either
• covalent modif nucleosomes (Ac, deAc, CH3, Ubiq)
  changes interaction with regulators and wrapping
  with DNA (e.g. SAGA NuA4)
• reposition in ATP-dependent way (e.g. RSC)
• For Device Engineering may want standardized
  chrom. remodeling complex
• SAGA present at promoters of all protein coding
  genes, at level that correlates with gene
  activity (Young MolCell04)
• gcn5 or spt20 mutants reveal SAGA complex needed
  for robust gene expression at some genes (Cell
  1998 95717 or Nature 2000 405701). Not all?
  some redundant HATs confound analysis.

37
For Endy lab presentation SAGA representative
Gcn5 NuA4 representative Esa1
ChIP occupancy of indicated proteins relative to
txn rate of gene
Robert et al MolCell 2004 16199
38
For Endy lab presentation
Fine mapping of occupancy at an active gene
Robert et al MolCell 2004 16199
39
For Endy lab presentation
Occupancy correlates with activity black
uninduced, grey induced
Robert et al MolCell 2004 16199
40
(No Transcript)