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Topic Next step: deciding which genes to clone Problem = correlating enzymes with genes

Topic 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. Issues

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Topic Next step: deciding which genes to clone Problem = correlating enzymes with genes

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  1. Topic • 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

  2. Issues • All genes that have been cloned and expressed have acidic pH optima: but Trinchant found bacteroid with pH 8 opt • Oxalate oxidase belongs to the cupin superfamily • functionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signature

  3. Cupins • functionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signature • Proposal: you each clone one safe and one risky oxalate ox • Safe = known Oxalate oxidase or decarboxylase • Risky = based on homology

  4. Cupins • functionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signature • Proposal: you each clone one safe and one risky oxalate ox • Safe = known Oxalate oxidase or decarboxylase • Wheat oxalate oxidase GF-2.8: P15290http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1987392/ • Barley oxalate oxidase: CAA74595 • Rice oxalate oxidase: Os03g0693700 or Os03g0694000 • Ceriporiopsissubvermisporaoxalate oxidase: AJ746414 • B. subtilisoxalate decarboxylase: O34714 • White rot oxalate decarboxylase: AF200683

  5. Risky = based on homology, eg to rice OXO1 SORBIDRAFT_01g011370 [Sorghum bicolor] Sequence ID: ref|XP_002464052.1| Length: 225 Expect Identities Positives Gaps 1e-123 171/216(79%) 196/216(90%) 0/216(0%) LOC100272932 precursor [Zea mays] Sequence ID:  ref|NP_001140856.1| Length: 225 Expect Identities Positives Gaps 3e-122 173/228(76%) 203/228(89%) 4/228(1%) oxalate oxidase GF-2.8-like [Brachypodium distachyon] Sequence ID: ref|XP_003561035.1| Length: 226 Expect Identities Positives Gaps 4e-114 168/222(76%) 191/222(86%) 4/222(1%)

  6. Risky = based on homology, eg to Bacillus subtilis oxdC as query Accession: NP_391204.1oxalate decarboxylase oxdC [Neurospora crassa OR74A]Sequence ID: ref|XP_964781.1|Length: 455Expect   Identities       Positives       Gaps5e-150   211/356(59%)    269/356(75%)    2/356(0%)Oxalate decarboxylase [Mesorhizobium sp. LNHC209A00]Sequence ID: ref|WP_023795524.1|Length: 377Expect   Identities       Positives       Gaps8e-150   210/367(57%)     259/367(70%)    4/367(1%)hypothetical protein BJ6T_62210 [Bradyrhizobium japonicum]Sequence ID: ref|YP_005611058.1|Length: 357Expect   Identities       Positives       Gaps 3e-180   244/355(69%)    283/355(79%)    1/355(0%)

  7. Risky = based on homology, eg to Agrobacterium oxalate decarboxylase Accession: NP_355894.1OxdD [Rhizobium sp. IRBG74]Sequence ID: ref|YP_008649166.1|Length: 415Expect  Identities       Positives       Gaps0.0     402/415(97%)    406/415(97%)    0/415(0%)cupin [Rhizobium lupini]Sequence ID: ref|WP_006700522.1|Length: 415Expect  Identities       Positives       Gaps0.0     404/415(97%)    407/415(98%)    0/415(0%)Oxalate decarboxylase [Bradyrhizobium sp.] Sequence ID: gb|EJZ30017.1|Length: 406Expect  Identities       Positives       Gaps0.0     267/403(66%)     320/403(79%)     2/403(0%)oxalate decarboxylase [Bradyrhizobium japonicum USDA 6]Sequence ID: ref|YP_005611126.1|Length: 415Expect  Identities       Positives       Gaps0.0     264/413(64%)     321/413(77%)     3/413(0%)

  8. Risky = based on homology, eg to Agrobacterium oxalate decarboxylase Accession: NP_355894.1 uncharacterized protein LOC102383685 [Alligator sinensis] XP_006018933.1 [XM_006018871] Length = 341 Expect = 5e-12, Identities = 67/277 (24%), Positives = 119/277 (42%), Gaps = 28/277 (10%) uncharacterized protein C18orf54 [Bos taurus] XP_005224336.1 [XM_005224279] Length = 453 Expect = 7e-12,Identities = 78/312 (25%), Positives = 132/312 (42%), Gaps = 36/312 (11%)

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

  10. Using the genome Bisulfite sequencing to detect C methylation ChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells

  11. Using the Genome • Acetylation, egH3K9Ac, is associated with active genes • Phosphorylation shows condensation • Ubiquitination of H2A and H2B shows repression • Methylation is more complex: • H3K36me3 = on • H3K27me3 = off • H3K4me1 = off • H3K4me2 = primed • H3K4me3 = on

  12. Histone code • Modifications tend to group together: genes with H3K4me3 also have H3K9ac • Cytosine methylation is also associated with repressed genes

  13. Generating the histone code • Histone acetyltransferases add acetic acid

  14. Generating the histone code • Histone acetyltransferases add acetic acid • Many HAT proteins: mutants are very sick!

  15. Generating the histone code • Histone acetyltransferases add acetic acid • Many HAT proteins: mutants are very sick! • HATs are part of many complexes

  16. Generating the histone code • Bromodomains specifically bind acetylated lysines

  17. Generating the histone code • Bromodomains specifically bind acetylated lysines • Found in transcriptional activators & general TFs

  18. Generating the histone code • acetylated lysines • Deacetylases “reset” by removing the acetate

  19. Generating the histone code • acetylated lysines • Deacetylases “reset” by removing the acetate • Deacetylase mutants are sick!

  20. Generating the histone code • Deacetylases “reset” by removing the acetate • Deacetylase mutants are sick! • Many drugs are histone deacetylase inhibitors

  21. Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors SAHA = suberanilohydroxamic acid = vorinostat Merck calls it Zolinza, treats cutaneous T cell lymphoma

  22. Generating the histone code Deacetylases “reset” by removing the acetate Deacetylase mutants are sick! Many drugs are histone deacetylase inhibitors SAHA = suberanilohydroxamic acid = vorinostat Merck calls it Zolinza, treats cutaneous T cell lymphoma Binds HDAC active site & chelates Zn2+

  23. Generating the histone code • When coupled SAHA to • PIPS (pyrrole-imidazole • Polyamides) got gene- • specific DNA binding & • gene activation

  24. Generating the histone code Other drugs are activators of histone deacetylases Resveratrol increases Sirtuin 1 expression and activity, possibly by enhancing its binding to Lamin A

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