Lecture 5-6.pdf - Lecture 5 Complete protein sequencing is...

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Lecture 5 Complete protein sequencing is slow, hard, & expensive work (usually unrewarding) Most investigators are impatient for sequence information Complete cDNA sequencing is fast, & inexpensive work (usually unrewarding & unreliable) Short cut: combine partial protein sequence & cDNA sequence
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Silica deposition vesicle Cell membrane Silica deposition vesicle SDV silicic acid transport channel Nascent frustule Si(OH) 4 Silaffins polyamines Internal pooling transport to SDV Secretion as frustule Condensation to silica Frustulin scaffold
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Frustulin 75 kDa Select confident sequence such as T-3 n.b. 2 unknowns Design oligos for known ends AVPG FFDY Sense antisense GCN-GTN-CCN-GG TTY-TTY-GAY-TAY AAR-AAR-CTR-ATR RTA-RTC-RAA-RAA T-3: AVPG X SGGAQDSSFFDY X VR
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Apply to mRNA AAAA N x PCR Isolate & size on agarose gel Insert & clone in plasmid pUC18 Stability, amplification, screening*, sequencing α -complementation Sequence DNA - Sanger or Gilbert/Maxam RT 1st amplification 2nd amplification
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Genetic code T-3: AVPG X SGGAQDSSFFDY X VR Design oligos for T-3 fourX2 AVPG FFDY Sense antisense GCN-GTN-CCN-GG TTY-TTY-GAY-TAY AAR-AAR-CTR-ATR RTA-RTC-RAA-RAA
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Wobble preference - % Wobble base Drosophila C. elegans Mytilus pCOL PRO A 44 0 76 G 34 2 1 U 11 95 11 C 10 4 12 ALA A 61 35 62 G 31 41 1 U 2 4 20 C 7 24 17 GLY A 38 0 63 G 27 0 1 U 37 98 24 C 0 2 12 For three 90k proteins
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Partial cDNA AAAAAA AAAAAA AAAAAA AAAAAA AAAAAA RT-PCR mRNAs cDNA library AVPG FFDY degenerate oligos & PCR 40 cycles melt/anneal specific partial cDNA 5’ 3’ 3’ 5’
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Compare sequences Knew T-3: AVPG X SGGAQDSSFFDY X VR Now know: AVPGC SGGA Q DS S F FDY as well as nondegenerate cDNA Want complete nondegenerate sequence: use T-3 cDNA and do 3’ 5’ RACE 3’ PCR: AAAA TTTT-XYZ AAAA-X’Y’Z’ sense TTTT-XYZ etc, etc 5’ PCR: AAAA AS etc, etc AS 5’ 3’ GCCGTTCCAGGA TGCTCTGGAGGT GCG CAG GATTCCTCG TTCTTCGATTAC AS AAAA XYZ-TTTTT Add polyA
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Vectors: pUC18, pUC19 identical with pc sites having an opposite orientation HIGH COPY NUMBER pUC18 2.69 kB lacZ lacI origin AMP’ PC site Polycloning sites in PC P lac N-terminal frag of β -gal-dase
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Plasmid insertion pUC18 SAL I S I nuclease pUC18 w/ insert SAL I SMA I Polycloning site 3’ race product * adaptor extension * WHY: additional amplification, α -complementation, sequencing ligase why insert? for SalI SmaI excision LAC gene complement: detection 4K fold more amplification orientational insertion compatible with sequencing
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α -complementation Screening for recombinants Host chromosome β -galactosidase (C) inactive β -galactosidase (N) inactive Mutant lac Z (N) gene Mutant lac Z (C) gene plasmid Inducer: isopropylthio- β -galactoside (IPTG) + ACTIVE SUBSTRATE + ENZYME PRODUCT X-GAL + B-GAL-C B-GAL-N bluish H N O Br Cl Colonies transfected by plasmids w/o insertions Bluish Colonies transfected by plasmids w/ insertions Clear N H O Br Cl OH OH HO O CH 2 OH X-GAL = 5-bromo-4-chloro-3-indolyl-b-D-galactoside X X Repressor (blocks Lac expression)
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