TopicNext 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 orthologsUse sequence to design primers to clone cDNA
IssuesAll genes that have been cloned and expressed have acidic pH optima: but Trinchant found bacteroid with pH 8 optOxalate oxidase belongs to the cupin superfamily •functionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signature
Cupinsfunctionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signatureProposal: you each clone one safe and one risky oxalate ox
Safe = known Oxalate oxidase or decarboxylaseRisky = based on homology
Cupinsfunctionally diverse proteins that have a highly conserved pattern of two histidine-containing motifs: the cupin signatureProposal: you each clone one safe and one risky oxalate oxSafe = 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•Ceriporiopsis subvermispora oxalate oxidase: AJ746414•B. subtilis oxalate decarboxylase: O34714•White rot oxalate decarboxylase: AF200683
Risky = based on homology, eg to rice OXO1SORBIDRAFT_01g011370 [Sorghum bicolor]Sequence ID: ref|XP_002464052.1| Length: 225Expect Identities Positives Gaps1e-123 171/216(79%) 196/216(90%) 0/216(0%)
LOC100272932 precursor [Zea mays]Sequence ID: ref|NP_001140856.1| Length: 225Expect Identities Positives Gaps3e-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 Gaps4e-114 168/222(76%) 191/222(86%) 4/222(1%)
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%)
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%)
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%)
How to proceed?
Kinetic and Spectroscopic Studies of Bicupin OxalateOxidase and Putative Active Site Mutants
Using the genomeBisulfite sequencing to detect C methylationChIP-chip or ChIP-seq to detect chromatin modifications: 17 mods are associated with active genes in CD-4 T cells
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
Histone codeModifications tend to group together: genes with H3K4me3 also have H3K9acCytosine methylation is also associated with repressed genes
Generating the histone codeHistone acetyltransferases add acetic acid
Generating the histone codeHistone acetyltransferases add acetic acidMany HAT proteins: mutants are very sick!
Generating the histone codeHistone acetyltransferases add acetic acidMany HAT proteins: mutants are very sick!HATs are part of many complexes
Generating the histone codeBromodomains specifically bind acetylated lysines
Generating the histone codeBromodomains specifically bind acetylated lysinesFound in transcriptional activators & general TFs
Generating the histone codeacetylated lysinesDeacetylases “reset” by removing the acetate
Generating the histone codeacetylated lysinesDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!
Generating the histone codeDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!Many drugs are histone deacetylase inhibitors
Generating the histone codeDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!Many drugs are histone deacetylase inhibitorsSAHA = suberanilohydroxamic acid = vorinostatMerck calls it Zolinza, treats cutaneous T cell lymphoma
Generating the histone codeDeacetylases “reset” by removing the acetateDeacetylase mutants are sick!Many drugs are histone deacetylase inhibitorsSAHA = suberanilohydroxamic acid = vorinostatMerck calls it Zolinza, treats cutaneous T cell lymphomaBinds HDAC active site & chelates Zn2+
Generating the histone codeWhen coupled SAHA to PIPS (pyrrole-imidazole Polyamides) got gene-specific DNA binding & gene activation
Generating the histone codeOther drugs are activators of histone deacetylasesResveratrol increases Sirtuin 1 expression and activity, possibly by enhancing its binding toLamin A
