User talk:Nkuldell/mtDNA pt3

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Experimental Details

deoxycycline-regulation of RNase expression

plasmid map for pCM224
  • regulation of expression from deoxycline-induced promoter, described in Herrero Yeast 1998 14(12):1127-38
  • sequence file for pCM224 which encodes tTA and CYC1-repressible promoter due to 2 copies of tetO
  • can excise relevant regulatory module (tTA driven by ADH1 and CYC1 with 2XtetO) on RI, BamHI fragment

pRS416

plasmid map for pRS416 with single cutters indicated
  • sequence file for pRS416
  • can clone tTA/CYC1tetO2/mtRNT1 into RI, BamHI bkb

mtRnt1

Steps to target RNT1 to mitochondrial matrix

  • 1. downloaded genomic sequence of RNT1 from SGD. RNT1/YMR239C on chromosome XIII from coordinates 749676 to 748261 on Crick (bottom) strand.
  • 2. removed EcoRI site from coding sequence of RNT1. Changed GAA(bp721-723)TTC(bp724-726) at Glu240Phe241 to GAG TTC which removed RI but keeps GluPhe.
    • sequence file for RNT1 genomic DNA with silent mutation to remove EcoRI
RNTI with silent mutations to remove EcoRI; restriction with favorite enzymes
    • Favorite Enzymes: AvaI, BamHI, BglII, EcoRI, HincII, HindIII, PstI, SmaI, XbaI, XmnI
  • 3. added mitochondrial targeting sequence from HEM1 to 5' end of RNT1
    • described as sufficient for mt localization of b-gal
    • does not change restriction analysis with favorite enzymes
    • sequence ATG CAA CGC TCC ATT TTT GCG AGG TTC/ Met Gln Arg Ser Ile Phe Ala Arg Phe
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