Download Sequence ID: ref|WP_006700522.1

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
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
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
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/PMC1
987392/
• 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 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%)
Risky = based on homology, eg to Bacillus subtilis oxdC as query
Accession: NP_391204.1
oxalate decarboxylase oxdC [Neurospora crassa OR74A]
Sequence ID: ref|XP_964781.1|Length: 455
Expect Identities
Positives
Gaps
5e-150 211/356(59%) 269/356(75%) 2/356(0%)
Oxalate decarboxylase [Mesorhizobium sp. LNHC209A00]
Sequence ID: ref|WP_023795524.1|Length: 377
Expect Identities
Positives
Gaps
8e-150 210/367(57%) 259/367(70%) 4/367(1%)
hypothetical protein BJ6T_62210 [Bradyrhizobium japonicum]
Sequence ID: ref|YP_005611058.1|Length: 357
Expect 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_355894.1
OxdD [Rhizobium sp. IRBG74]
Sequence ID: ref|YP_008649166.1|Length: 415
Expect Identities
Positives
Gaps
0.0
402/415(97%) 406/415(97%) 0/415(0%)
cupin [Rhizobium lupini]
Sequence ID: ref|WP_006700522.1|Length: 415
Expect Identities
Positives
Gaps
0.0
404/415(97%) 407/415(98%) 0/415(0%)
Oxalate decarboxylase [Bradyrhizobium sp.]
Sequence ID: gb|EJZ30017.1|Length: 406
Expect Identities
Positives
Gaps
0.0
267/403(66%) 320/403(79%)
2/403(0%)
oxalate decarboxylase [Bradyrhizobium japonicum USDA 6]
Sequence ID: ref|YP_005611126.1|Length: 415
Expect Identities
Positives
Gaps
0.0
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 Oxalate
Oxidase and Putative Active Site Mutants
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
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 code
Modifications tend to group together: genes with
H3K4me3 also have H3K9ac
Cytosine methylation is also associated with repressed
genes
Generating the histone code
Histone acetyltransferases add acetic acid
Generating the histone code
Histone acetyltransferases add acetic acid
Many HAT proteins: mutants are very sick!
Generating the histone code
Histone acetyltransferases add acetic acid
Many HAT proteins: mutants are very sick!
HATs are part of many complexes
Generating the histone code
Bromodomains specifically bind acetylated lysines
Generating the histone code
Bromodomains specifically bind acetylated lysines
Found in transcriptional activators & general TFs
Generating the histone code
acetylated lysines
Deacetylases “reset” by removing the acetate
Generating the histone code
acetylated lysines
Deacetylases “reset” by removing the acetate
Deacetylase mutants are sick!
Generating the histone code
Deacetylases “reset” by removing the acetate
Deacetylase mutants are sick!
Many drugs are histone deacetylase inhibitors
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
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+
Generating the histone code
When coupled SAHA to
PIPS (pyrrole-imidazole
Polyamides) got genespecific DNA binding &
gene activation
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