extracting exon0 isolating a mec-8 isoform
DESCRIPTION
Extracting Exon0 Isolating a mec-8 Isoform. Sol Katzman. Zahler lab -- C. elegans “Alt splicing, all the time ”. Where’s the binf? Uncoordinated worms Mixing colorless liquids. from http://elegans.swmed.edu/. unc-52 exons 17,18 optional mediated by mec-8. mec-8 four exons +C43002. - PowerPoint PPT PresentationTRANSCRIPT
04/20/23 1
Extracting Exon0Isolating a mec-8 Isoform
Sol Katzman
Sol Katzman 04/20/23 2
Zahler lab -- C. elegans“Alt splicing, all the time” Where’s the binf? Uncoordinated worms Mixing colorless liquids
from http://elegans.swmed.edu/
Sol Katzman 04/20/23 3
unc-52 exons 17,18 optionalmediated by mec-8
Sol Katzman 04/20/23 4
mec-8 four exons +C43002
Sol Katzman 04/20/23 5
mec-8 zoom out to see Exon0A 6500bp intron in C. elegans?
Sol Katzman 04/20/23 6
mec-8 (Wormbase view)
Sol Katzman 04/20/23 7
5’ RACE procedures5’
mRNA3’
gsp1
reverse transcribe
3’cDNA
5’
purify
3’cDNA
5’
tail
3’cDNA
5’
PCR
CCCCCAAPgsp2
5’dsDNA
3’ AAPgsp2
Sol Katzman 04/20/23 8
trans-spliced isoforms and primers.known and unknownshould be at least 85bp difference
exon4 exon3 exon2 exon1
exon4 exon3 exon2 exon1 exon0
SL1
SL1?
SL1
SL1
AAP
AAP
3’
3’ 5’
5’tailed
tailed
ex0for
ex01revgsp2
gsp2
Sol Katzman 04/20/23 9
5’ RACE gel using AAP and SL1 forward primers
Sol Katzman 04/20/23 10
TOPO-TA cloning
Sol Katzman 04/20/23 11
TOPO clone vector
Sol Katzman 04/20/23 12
plasmid digestion after cloning
Sol Katzman 04/20/23 13
First clones: exon1 and 2, plus SL1
Sol Katzman 04/20/23 14
trans-spliced isoforms and primers.try exon0forward (and other RNA)
exon4 exon3 exon2 exon1
exon4 exon3 exon2 exon1 exon0
SL1
SL1?
SL1
SL1
AAP
AAP
3’
3’ 5’
5’tailed
tailed
ex0for
ex01revgsp2
gsp2
Sol Katzman 04/20/23 15
Exon0For primer on First Strand cDNA3 RNA samples: embryo, L4, mixed-stageFound something!
Sol Katzman 04/20/23 16
5’ RACE procedures5’
mRNA3’
gsp1
reverse transcribe
3’cDNA
5’
purify
3’cDNA
5’
tail
3’cDNA
5’
PCR
CCCCCAAPgsp2
5’dsDNA
3’ AAPgsp2
Sol Katzman 04/20/23 17
Exon0For primer: fading through 5’RACE procedure steps. (embryo RNA only)
Sol Katzman 04/20/23 18
trans-spliced isoforms and primers.try exon01reverse primer
exon4 exon3 exon2 exon1
exon4 exon3 exon2 exon1 exon0
SL1
SL1?
SL1
SL1
AAP
AAP
3’
3’ 5’
5’tailed
tailed
ex0for
ex01revgsp2
gsp2
Sol Katzman 04/20/23 19
SL1 forward - Exon01Rev primerson purified cDNA. Double band at 200bp
Sol Katzman 04/20/23 20
Clone and PCR sequences – BLAT.2 versions of exon0. SL1 leaders.Where’s the ORF?
Sol Katzman 04/20/23 21
To Do
find promoter and bash it investigate conservation regions in intron use the third worm -- C. remanei consider uORFs
Sol Katzman 04/20/23 22
Acknowledgements
Al Zahler Sam Gu Jen Kabat
Val, Brian, Mary Ann, Poojitha, Joy, Sergio U.C. Regents