what i wanted to do this summer evaluate inducible inactivation system designed to regulate...
TRANSCRIPT
What I wanted to do this summer
Evaluate inducible inactivation system designed to regulate mitochondrial gene expression
Examine unintended consequences of TAP-tagging
What I really did this summer
Inducible gene inactivation: using guide RNAs
“Short RNA guides cleavage by eukaryotic RNase
III.” PLoS ONE. (2007) 2(5):e472
mt gene
Inducible inactivation system for mitochondrial gene expression
Part1: mtRNT1
FY2068 MAT ura3-52 his3200 leu2 1 lys2-128
mt gene
Inducible inactivation system for mitochondrial gene expression
Part1: mtRNT1
Is mtRNT1 induced +dox?
Is mtRNT1 localized to mt?
Are there consequences of induction/localization?
anti-Rnt1 Western
1 Marker2 - mtRNT, - dox3 + mtRNT, - dox4 - mtRNT, + dox5 +mtRNT, + dox
Is mtRNT1 induced + dox?
1 2 3 4 5
“- mtRNT”
“+ mtRNT”
50 kDa
75
Is mtRNT1 localized to mt?
“A cotranscriptional model for 3'-end processing of the Saccharomyces cerevisiae pre-ribosomal RNA precursor” RNA (2004) 10(10):1572
“The results presented so far indicate that Rnt1p is a nuclear enzyme distributed throughout the nucleus that also strongly accumulates within a nucleolar structure corresponding to the site of transcription of the rDNAs.”
Is mtRNT1 localized to mt?
“A cotranscriptional model for 3'-end processing of the Saccharomyces cerevisiae pre-ribosomal RNA precursor” RNA (2004) 10(10):1572
= - mtRNT +dox = + mtRNT +dox
Agilent 4x44K spotted array, 3DNA labeling
Are there consequences of induction/localization?
mtRNT: most affected genes, pt 1
higher no mtRNT
I-SceII, encoded by intron within the mitochondrial COX1 gene
mfold.bioinfo.rpi.edu/
mtRNT: most affected genes, pt 1
higher no mtRNT
Dubious ORF in mt genome within RPM1 gene
mfold.bioinfo.rpi.edu/
mtRNT: most affected genes, pt 1
higher no mtRNT
I-SceIV, encoded by intron within the mitochondrial COX1 gene
Sequence is 9350 bases…too long for mfold (limit 800)
mtRNT: most affected genes, pt 1
higher no mtRNT
F0-ATP synthase subunit 9 encoded on the mitochondrial genome
mfold.bioinfo.rpi.edu/
Ask: OligomycinR?