6/27/20151 Extracting Exon0 Isolating a mec-8 Isoform Sol Katzman

Sol Katzman 6/27/20152 Zahler lab -- C. elegans “Alt splicing, all the time” Where’s the binf? Uncoordinated worms Mixing colorless liquids from

Sol Katzman 6/27/20153 unc-52 exons 17,18 optional mediated by mec-8

Sol Katzman 6/27/20154 mec-8 four exons +C43002

Sol Katzman 6/27/20155 mec-8 zoom out to see Exon0 A 6500bp intron in C. elegans?

Sol Katzman 6/27/20156 mec-8 (Wormbase view)

Sol Katzman 6/27/ ’ RACE procedures 5’ mRNA 3’ gsp1 reverse transcribe 3’ cDNA 5’ purify 3’ cDNA 5’ tail 3’ cDNA 5’ PCR CCCCC AAP gsp2 5’ dsDNA 3’ AAP gsp2

Sol Katzman 6/27/20158 trans-spliced isoforms and primers. known and unknown should be at least 85bp difference exon4exon3exon2exon1 exon4exon3exon2exon1 exon0 SL1 SL1? SL1 AAP 3’ 5’ tailed ex0for ex01revgsp2

Sol Katzman 6/27/ ’ RACE gel using AAP and SL1 forward primers

Sol Katzman 6/27/ TOPO-TA cloning

Sol Katzman 6/27/ TOPO clone vector

Sol Katzman 6/27/ plasmid digestion after cloning

Sol Katzman 6/27/ First clones: exon1 and 2, plus SL1

Sol Katzman 6/27/ trans-spliced isoforms and primers. try exon0forward (and other RNA) exon4exon3exon2exon1 exon4exon3exon2exon1 exon0 SL1 SL1? SL1 AAP 3’ 5’ tailed ex0for ex01revgsp2

Sol Katzman 6/27/ Exon0For primer on First Strand cDNA 3 RNA samples: embryo, L4, mixed-stage Found something!

Sol Katzman 6/27/ ’ RACE procedures 5’ mRNA 3’ gsp1 reverse transcribe 3’ cDNA 5’ purify 3’ cDNA 5’ tail 3’ cDNA 5’ PCR CCCCC AAP gsp2 5’ dsDNA 3’ AAP gsp2

Sol Katzman 6/27/ Exon0For primer: fading through 5’RACE procedure steps. (embryo RNA only)

Sol Katzman 6/27/ trans-spliced isoforms and primers. try exon01reverse primer exon4exon3exon2exon1 exon4exon3exon2exon1 exon0 SL1 SL1? SL1 AAP 3’ 5’ tailed ex0for ex01revgsp2

Sol Katzman 6/27/ SL1 forward - Exon01Rev primers on purified cDNA. Double band at 200bp

Sol Katzman 6/27/ Clone and PCR sequences – BLAT. 2 versions of exon0. SL1 leaders. Where’s the ORF?

Sol Katzman 6/27/ To Do find promoter and bash it investigate conservation regions in intron use the third worm -- C. remanei consider uORFs

Sol Katzman 6/27/ Acknowledgements Al Zahler Sam Gu Jen Kabat  Val, Brian, Mary Ann, Poojitha, Joy, Sergio U.C. Regents