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Generating the histone code CDK8 kinases histones to repress transcription.

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Presentation on theme: "Generating the histone code CDK8 kinases histones to repress transcription."— Presentation transcript:

1 Generating the histone code CDK8 kinases histones to repress transcription

2 Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription

3 Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription Polycomb proteins ubiquitinate histone H2A to silence genes

4 Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription Phosphorylation of Histone H3 correlates with activation of heat shock genes!

5 Generating the histone code CDK8 kinases histones to repress transcription Appears to interact with mediator to block transcription Phosphorylation of Histone H3 correlates with activation of heat shock genes! Phosphatases reset the genes

6 Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription

7 Generating the histone code Rad6 proteins ubiquitinate histone H2B to repress transcription Polycomb proteins ubiquitinate histone H2A to silence genes A TFTC/STAGA module mediates histone H2A and H2B deubiquitination, coactivates nuclear receptors, and counteracts heterochromatin silencing

8 Generating the histone code Many proteins methylate histones: highly regulated!

9 Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity

10 Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy!

11 Generating the histone code Many proteins methylate histones: highly regulated! Methylation status determines gene activity Mutants (eg Curly leaf) are unhappy! Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red)

12 Generating the histone code Chromodomain protein HP1 can tell the difference between H3K9me2 (yellow) & H3K9me3 (red) Histone demethylases have been recently discovered

13 Next step: deciding which genes to clone Problem = correlating enzymes with genes Who matches the pH? Who localizes where? Which isoform if alternatively spliced? Clone several, using one known to work to find orthologs Use sequence to design primers to clone cDNA

14 How to proceed? Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

15 Primer/probe design Crucial for successful DNA & RNA analysis! Main source of specificity for PCR good primers only bind your sequence

16 Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity: only want them to bind at one place Main concern: 3’ end should not bind

17 Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Hairpins homoduplexes heteroduplexes may not melt May be extended by DNA polymerase

18 Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Should match! Every site calculates them differently!

19 Primer/probe design Also important for microarrays, sequencing, Southerns Concerns Specificity Complementarity: Melting T Targeting specific locations amplifying specific sequences creating mutations: need mismatch towards 5’ end so 3’ end binds well Add restriction sites at 5’ end: may need to reamplify an amplicon

20 Choosing enzyme Template Fidelity Taq from Thermus aquaticus has no proof-reading goes faster, but error freq of 1 in 3000 Vent from Thermococcus litoralis has error frequency of 1 in 30,000 Pfu from Pyrococcus furiosus has error frequency of 1 in 400,000 Genetically engineered proof-reading Phusion from NEB has error frequency of 1 in 2,000,000

21 Choosing enzyme Template Fidelity Temperature stability E.coli DNA polymerase I denatures at 75˚ C T 1/2 of 95˚ C is 0.9 hours, < ˚ C T 1/2 of 96˚ C is >6 hours, 2 98˚ C T 1/2 of 95˚ C is 6.7 hours, ˚ C T 1/2 of Deep Vent from Pyrococcus species GB-D 104˚ C)is 23 95˚ C, 8 100˚ C

22 Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Phusion is 10x more processive than Pfu, 2x more than Taq lets you make longer amplicons in shorter time Taq = 8 kb max cf 40 kb for Phusion

23 Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km dNTP: 13 µM for Taq, 60 µM for Vent DNA: 2 nM for Taq, 0.01 nM for Deep Vent Vmax: >1,000 nt/s when attached Binding is limiting, processivity determines actual rate 1000 bp/min is good for PCR Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion

24 Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Dirty DNA: in general, non-proofreading polymerases tolerate dirtier DNA than proof-readers except Phusion dNTP analogs or modified dNTP non-proofreading polymerases do better, but varies according to the modification [Mg]: Vent is more sensitive to [Mg] and needs 2x more than Taq

25 Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends Non-proof-readers (eg Taq) give a mix of blunt & 3’A Can use 3’A for t:A cloning GAATTCAtcgca CTTAAGtagcgt

26 Choosing enzyme Template Fidelity Temperature stability Processivity (how far does it go before falling off) Km Vmax Tolerance of imperfect conditions Fragment ends: proof-readers (eg Vent) give blunt ends NEB: Taq = $59.00 for 400 units Vent = $62.00 for 200 units Deep Vent = $90.00 for 200 units Phusion = $ for 100 units

27 Optimizing PCR [enzyme] [Template] [Mg 2+ ] Annealing Temperature Denaturation temperature

28 Optimizing PCR [enzyme] units/100 µl for proofreaders : start with 1

29 Optimizing PCR [enzyme] units/100 µl for proofreaders : start with units/100 µl for non-proofreaders : start with 3

30 Optimizing PCR [enzyme] [Template] 1-10 ng/100 µl reaction for plasmids ng/100 µl reaction for genomic DNA Excess DNA can give extra bands, also brings more contaminants

31 Optimizing PCR [enzyme] [Template] 1-10 ng/100 µl reaction for plasmids ng/100 µl reaction for genomic DNA Excess DNA can give extra bands, also brings more contaminants [dNTP] µM for Taq: start with 200, lower increases fidelity, higher increases yield µM for proof-readers: if too low start eating

32 Optimizing PCR [enzyme] [Template] [Mg 2+ ] mM for Taq: start with 1.5; lower if extra bands, raise if low yield

33 Optimizing PCR [enzyme] [Template] [Mg 2+ ] mM for Taq: start with 1.5; lower if extra bands, raise if low yield 1- 8 mM for proofreaders: start with 2, lower if extra bands, raise if low yield

34 Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Go as high as you can w/o killing enzyme before end 94˚C for Taq 96-98˚C for Vent 98˚C for Deep Vent & Phusion

35 Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm

36 Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm Adjust up and down as needed Use temperature gradient feature to find best T

37 Optimizing PCR [enzyme] [Template] [Mg 2+ ] Denaturation Temperature Annealing Temperature Start 5 ˚C below lowest primer Tm Adjust up and down as needed # cycles: raise if no bands, lower if OK yield but extra bands

38 Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles

39 Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles Can try “PCR enhancers” to overcome dirty DNA Use Ammonium SO 4 in buffer cf KCl Use molecules that alter Tm eg DMSO & formamide Use molecules that stabilise Taq eg Betaine & BSA

40 Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles If extra bands persist, use Taq bound to antibody Inactive until denature antibody 7’ at 94˚ C

41 Optimizing PCR Most common problems = wrong [DNA], dirty DNA, [Mg 2+ ] annealing temperature & # cycles If extra bands persist, use Taq bound to antibody Inactive until denature antibody 7’ at 94˚ C Alternatively, try touch-down: start too high & lower 1˚ C each cycle ( binds correct target first)


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