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Winston Group Meeting

Winston Group Meeting. 09.19.06. 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. SAGA swap: justification.

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Winston Group Meeting

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  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) 15:199

  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. his4-917∂ lys2-173R2 SPT3 spt3∆ His- Lys+ phenotype His+ Lys- phenotype SAGA swap: justification Madison, Winston Yeast (1998) 14:409

  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. ORF of S.c. SGF# PCR from pRS406, select Ura+ URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# phenotype complementation? SAGA swap: expt’l design ura3-52 Starting strain Intermediate Final strain ura3-52 ura3-52

  10. SC+erg/tween SC-his+erg/tween ura3-52 his3∆200 MH176 NY350 30° 11days hypox SAGA swap: expt’l 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 sgf73::URA3

  11. YPEG 30° 4d SAGA swap: expt’l design MH176 ~wt FY2475 sgf73∆0::KanMX FY2474 sgf29∆0::KanMX

  12. conserved non-canonical Zn-finger sgf11 homology canonical Zn-finger 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. ORF of S.c. SGF# PCR from pRS406, select Ura+ ura3-52 URA3 ura3-52 SAGA swap: data sgf73::URA3 in MH176 used primers +/- 100 bp SGF73 gives 2kb URA3 gives 1kb 2 1

  19. ORF of S.c. SGF# PCR from pRS406, select Ura+ ura3-52 URA3 ura3-52 SAGA swap: data sgf73::URA3 in MH176 confirm w/ URA3 +dwstm primer SGF73 gives no product URA3 gives ~500bp 1

  20. SAGA swap: data MH176 sgf73:KMX sgf73:URA3 sgf29:KMX sgf73:URA3, sgf29:KMX sgf29:URA3, sgf73:KMX Phenotypes of intermediate strains YPD, 30° 4d YPD +3% form YPEG

  21. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data

  22. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data Round 1 tx’d 10 ul of PCR rxn YPD ON (plates and liquid) to FOA

  23. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data Round 2 cotxn 40 ul PCR/purif w/ 2ug pRS415 YPD ON (liquid) to -L ON replica to FOA

  24. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data Round 3 reamplify so 80 bp homology 200ul PCR/purified YPD ON (plates) replica to FOA

  25. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data Round 4 40 bp homology Cotx’d with 3 ug pRS415 500ul PCR/purified (lost some) YPD ON (plates) replica to -L+FOA

  26. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data Round 5 40 bp homology Cotx’d with 3 ug pRS415 500ul PCR/purified (lost some) Mark’s help -leu ON (plates), replica to FOA

  27. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data confirm w/ SpSgf73 +dwstm primer SpSgf73 gives ~ 1.2 kb URA3 gives no product SpSgf73 in MH176 1

  28. URA3 PCR from S.p. genomic, select FOAR ORF of S.p. SGF# SAGA swap: data

  29. SAGA swap: data sgf73::URA3 in MH176 S.p.Sgf73 candA S.p.Sgf73 candG S.p.Sgf73* candE S.p.Sgf73* candH S.p.Sgf73* P344A candD Phenotypes of final strains YPD, 30° 2d YPD +3% form YPG

  30. SAGA swap: data sgf73::URA3 in MH176 S.p.Sgf73 candA S.p.Sgf73 candG sgf73::URA3sgf29::KMX S.p.Sgf73 sgf29::KMX candA S.p.Sgf73 sgf29::KMX candD Phenotypes of final strains YPD, 30° 2d YPD +3% form YPG

  31. SAGA swap: data S.p.Sgf29* sgf73::KMX candG S.p.Sgf29* sgf73::KMX candH S.p.Sgf29* sgf73::KMX candI S.p.Sgf29* sgf73::KMX candJ S.p.Sgf29* sgf73::KMX candK S.p.Sgf29* sgf73::KMX candL Phenotypes of final strains 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 doesn’t 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 95:717 or Nature 2000 405:701). 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 tx’n rate of gene Robert et al MolCell 2004 16:199

  38. For Endy lab presentation: Fine mapping of occupancy at an active gene Robert et al MolCell 2004 16:199

  39. For Endy lab presentation: Occupancy correlates with activity black= uninduced, grey = induced Robert et al MolCell 2004 16:199

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