Next step: deciding which genes to clone

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.ncb
  i.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

5
Risky based on homology, eg to rice
OXO1 SORBIDRAFT_01g011370 Sorghum
bicolor Sequence ID refXP_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 
refNP_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 refXP_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 OR74ASequence ID
refXP_964781.1Length 455Expect   Identities
      Positives       Gaps5e-150  
211/356(59)    269/356(75)    2/356(0)Oxalate
decarboxylase Mesorhizobium sp.
LNHC209A00Sequence ID refWP_023795524.1Length
377Expect   Identities       Positives
      Gaps8e-150   210/367(57)    
259/367(70)    4/367(1)hypothetical protein
BJ6T_62210 Bradyrhizobium japonicumSequence
ID refYP_005611058.1Length 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. IRBG74Sequence ID
refYP_008649166.1Length 415Expect  Identities
      Positives       Gaps0.0    
402/415(97)    406/415(97)    0/415(0)cupin
Rhizobium lupiniSequence ID
refWP_006700522.1Length 415Expect  Identities
      Positives       Gaps0.0    
404/415(97)    407/415(98)    0/415(0)Oxalate
decarboxylase Bradyrhizobium sp. Sequence ID
gbEJZ30017.1Length 406Expect  Identities    
  Positives       Gaps0.0    
267/403(66)     320/403(79)    
2/403(0)oxalate decarboxylase Bradyrhizobium
japonicum USDA 6Sequence ID refYP_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