BLAST searches of the sequences of all positives were performed against EMBL and Swiss Prot/Translated EMBL (SPTR) databases and Unigene clusters. Care was taken to check whether the nucleotide sequence was correctly orientated and in the coding region of the gene matches, and for protein searches that the protein was in the correct frame. The results of the BLAST searches are summarised in Tables 5.4 a and b.
C olony O rie n ta tio n C o d in g / n o n -c o d in g
re g io n of g e n e
EMBL SP T R F ram e A n a ly sis
1 + non-coding mm myelin expression factor 3-like protein No significant hits
In non-coding region 4 + c o d in g
m m HMG box tra n s c rip tio n fa cto r
Sox-13
SOX-13 p ro tein , SOX-12, SOX-6, SOX 5 +
P o s s ib le in te ra c to r
5 -
Various IMAGE clones Coatom er alpha subunit
-
Incorrect orientation/ fram e 6 + c o d in g
m m h o m eo d o m a in in te rac tin p ro tein k in a s e -1
m m h o m e o d o m a in in te ra c tin p ro te in k in a s e -1, 2A a n d 2B + P o s s ib le in te ra c to r 8 -
Various IMAGE clones No significant hits Incorrect orientation/ fram e
9 +
mm cDNA clones. IMAGE 5043975 and others
Proline rich protein expressed
in brain +
Unknown significance 10 + c o d in g
m m HMG box tra n s c rip tio n fa c to r Sox-13 SOX-13 p ro tein + P o s s ib le in te ra c to r 11 -
Various IMAGE clones Tyrosine PK transforming
protein -
Incorrect orientation/ fram e 12 - coding mm intermediate filament protein nestin
mm intermediate filament
protein nestin -
Incorrect orientation/ fram e
13 -
Various IMAGE clones C oatom er alpha subunit
-
Incorrect orientation/ fram e 14 + c o d in g
m m HMG tx)x tra n s c rip tio n fa cto r Sox-13
SOX-13 p ro tein , SOX-12, SOX-6, SOX 5 + P o s s ib le in te ra c to r 15 + c o d in g m m h o m eo d o m a in in te ra c tin p ro tein k in a s e -1 mm h o m e o d o m a in in te ra c tin p ro te in k in a s e -1, 2A a n d 2B + P o s s ib le in te ra c to r 16 + coding
Human monocytic leukaemia Zn finger protein
Human monocytic leukaemia Zn finger protein +
Known common false positive class
19 +
mm cDNA clones: IMAGE 4985915 and others
partial protein fragment
+
Unknown significance 20 + coding ELAV-like neuronal protein-1 ELAV-like neuronal protein- la n d 2 (hel-NI) + False positive (NS specific)
22 +
mm cDNA clone H3046D 11 and others
No significant hits Unknown significance 24 + coding mm pumilio 1 (P um m i) pumilio 1RNA BP, translational repressor + P ossiblesignificance 26 - coding sem aphorin sem aphorin precursor -
Incorrect orientation/ frame 27 - coding mm intermediate filament protein nestin mm intermediate filament protein nestin - Incorrect orientation/ frame Table 5.4 a
BLA ST searches o f positives from library screen 3 using exon 5/6a bait again st EM BL an d Swiss P rot/T ranslated EM BL (SP T R ) databases. T he possible and probable interactors w ith SHOX are h ig h lig h ted in red. m m ^ mus muscularis.
C olony O rien tatio n C o d in g / n o n -c o d in g
re g io n of g e n e
EMBL SP TR F ram e A n a ly sis
28 +
mm cDNA clones: IMAGE 3215605
No significant hits Unknown significance 29 + coding ELAV-like neuronal protein-1 ELAV-like neuronal protein-1 +
F alse positive (NS specific)
30 + coding Splicing factor prpB mRNA Splicing factor prpB mRNA + Unlikely sgnificance 31 + coding Splicing factor prpB mRNA Splicing factor prpB mRNA + Unlikely sgnificance
32 + coding
mm pumilio 1 (P u m m i) pumilio 1RNA BP,
translational repressor +
Possible significance
33 -
mm cDNA clones; IMAGE 500B022
No significant hits Incorrect orientation/ frame 38 - mm cDNA clones:IMAGE 3592920 Protein fragment - Incorrect orientation/ frame 39 + mm cDNA clones:
IMAGE 502B92B and others
Pyruvate kinase
+ False positive 40 + coding mm ubiquitin specific pro tease ubiquitin hydrolase + False positive 41 + c o d in g Dlxin-1 Dlxin-1 + P ro b a b lein te ra c to r
42 -
mm cDNA clones:
IMAGE 3500151 and others
No significant hits Incorrect orientation/ frame 46 + coding Phosphatidylserine sy n th ase 2 Phosphatidylserine sy nthase 2 -F False positive 47 + mm cDNA clones: 49B5915 and others
Partial protein fragment
+ Unknownsignificance 48 + coding R etinoblastom a binding protein 2 Retinoblastoma binding protein 2 -F Possiblesignificance 50 + non-coding mm myelin expression factor 3-like protein No significant hits
In non-coding region 51 + c o d in g m m h o m e o d o m a in in te ra c tin p ro te in k in a s e -1 m m h o m e o d o m a in in te rac tin p ro te in k in a s e -1, 2A, 2 8 + P o s s ib le in te ra c to r Table 5.4 a co n tin u ed
BLA ST searches o f positives from library screen 3 using exon 5/6a bait ag ain st EM B L and Swiss P ro t/T ran slated E M B L (S P T R ) databases. T h e possible and probable interactors with SHOX are high lig h ted in red. m m = mus muscularis.
C lo n e O rie n ta tio n C o d in g / n o n c o d in g re g io n o f g e n e EMBL S P T R F ram e A n a ly s is 1 - - - coding
V ascular endothelial zinc finger 1
V ascular endothelial zinc
finger 1 - - Incorrectorientation/ frame 2 + mm cDNA clonesilMAGE
621639 and others
SNRNA activating protein
complex +
Unknown significance 3 No significant hits No signifcant hits Unknownsignificance
8 - m .m us clone R P23-277F5
No signifcant hits Unknown significance 9 - IMAGE clone 1513337, similar to 60S ribosomal protein 60S ribosomal protein - False positive 10 + IMAGE clone 3496608, similar to KIAA0144 Hypothetical protein KIAA0144 + Unknown significance 11 - coding Peroxisom e proliferator- activated receptor (PPAR) interacting-protein PPA R interacting-prot - Incorrect orientation/ frame 12 + coding m .m us mRNA for LIM
kinase 2b
LIM domain kinase
+ F alse positive (cytoplasmic) 15 + mm cDNA clones; IMAGE 4458123 and others
Protein fragm ent KIAA0532 +
Unknown significance
16 +
Enactin-2 Enactin-2 F alse positive (basem ent m em brane protein)
18 - coding
Vascular endothelial zinc finger
V ascular endothelial zinc
finger -
Incorrect orientation/ frame 19 + coding m .m us mRNA for pyruvate kinase m .m us mRNA for pyruvate kinase False positive
21 +
mm cDNA clone:IMAGE 5012102
Methionyl tRNA sy n th etase
+ False positive (cytoplasmic) 22 + mm cDNA clone:IMAGE 3499933 Hypothetical 66.5 kD protein + Unknown significance 23 +
mm cDNA clone: IMAGE 697720 similar to guanine nucleotide-binding protein beta subunit 2
G uanine nucleotide-binding protein beta subunit 2
+
F alse positive
24 +
mm cDNA clone: IMAGE 4221824 putative adenosylhom ocysteinase + F alse positive 25 + mm mRNA for deadenylation factor EDEN-BP
deadenylation factor EDEN-
BP +
F alse positive
26 + mm cDNA clone: IMAGE 4976148 Hypotheical 24.1 kD protein +
Unknown significance
30 -
mm cDNA clone: IMAGE:4978093
E nolase 1, alpha non-neuron -
Incorrect orientation/ frame
Table 5.4 b
BLAST searches of positives from library screen 3 using full length-bait against EMBL and Swiss Prot/Translated EMBL (SPTR) databases. The possible and probable interactors with SHOX are highlighted in red. mm= mus muscularis.
C lo n e O rien tatio n C o d in g / n o n c o d in g re g io n o f g e n e EMBL S P T R A n a ly s is
31 + mm cDNA clone: IMAGE 5135713 2 oxo-isovalerate dehydrogenase a subunit + F a lse positive (mitochondrial) 32 - - - - mm cDNA,clone: IMAGE 4221901 Peroxidasin precursor - Incorrect orientation/ fram e 33 + coding mm fibrillin 2 g e n e Fibrillin 2 precursor + F alse positive
(cytoplasmic) 34 + mm cDNA clone: IMAGE
1125003
No significant hits Unknown significance 35 + mm cDNA clone: IMAGE
3383356
No significant hits Unknown significance 36 + mm cDNA clone: IMAGE
5029189
Elongation factor 1-alpha
+ F alse positive (cytoplasmic) 37 No significant hits No significant hits Unknown
significance
38 +
mm cDNA clone: IMAGE 1531557
Putative RNA binding protein -
Incorrect orientation/ fram e
39 -
mm cDNA clone: IMAGE 4873718
Nuclear envelope pore
m em brane protein -
Incorrect orientation/ fram e 40 + mm mRNA for pyruvate
kinase
pyruvate kinase M2 isozyme
+ F a lse positive (cytoplasmic) 42 + mm cDNA clone: IMAGE
4458337
pyruvate kinase M2 isozyme
+ F a lse positive (cytoplasmic) 43 + mm cDNA clone: IMAGE
1511623 Hypothetical protein KIAA1181 + Unknown significance 44 +
mm cDNA clone IMAGE 1279495 similar to laminin receptor Laminin receptor (67kD, ribosomal protein) + F alse positive (cytosolic) 45 + mm genomic clone No significant hits Unknown
significance 47 + mm cDNA clone H3032A04 G uanine nucleotide- ex ch an g e factor + P ossible significance 48 + coding mm laminin b eta 2 g e n e Laminin t>eta 2 chain
precursor +
F alse positive (cytosolic) 50 - mm cDNA clone IMAGE 5025078 No significant hits Unknownsignificance
51 +
mm cDNA clone IMAGE 1365913 similar to type IV collagen
Collagen alpha 1 (IV) chain
p recursor +
F a lse positive 52 No significant hits No significant hits Unknown
significance 54 + mm cDNA clone IMAGE
4317312
No significant hits Unknown significance 55 + mm clone RP23-104L6 No significant hits Unknown
significance 56 + non-coding mm myelin expression
factor-3-like
No significant hits In non-coding region 57 + non-coding mm myelin expression
factor-3-like
No significant hits In non-coding region 58 - mm mRNA for protein
kinase C
Protein kinase 0
- Incorrect fram e /orientation 59 + mm mRNA for pyruvate
kinase
Pyruvate kinase M2
isoenzym e +
F a lse positive (cytoplasmic) 60 + non-coding mm myelin expression
factor-3-like
No significant hits In non-coding region
Table 5.4 b continued
BLAST searches of positives from library screen 3 using fiill-length bait against EMBL and Swiss Prot/Translated EMBL (SPTR) databases. mm= mus muscularis.
C lo n e O rien tatio n C o d in g / n o n c o d in g reg io n of
g e n e
EMBL SP TR F ram e A n a ly sis
61
- mm cDNA clone IMAGE 5039126 T ropomyosin - /orientationIncorrect frame 62
+
mm IMAGE clone 5006774 and mm mRNA for pyruvate kinase
Pyruvate kinase M2
isoenzyme 4-
F alse positive (cytoplasmic) 63
+ coding mm lim kinase LIM domain kinase 4- F alse positive (cytoplasmic) 64 No significant hits No significant hits Unknown
significance 65
+
mm cDNA clone IMAGE 1261008 similar to fibronectin precursor Fibronectin precursor 4- Unlikely significance 68
- mm IMAGE clone 3500151 No significant hits Unknownsignificance 69
4- coding □yen in heavy chain Dyenin heavy chain F alse positive (cytoplasmic) 70
+ coding MyoD family inhibitor MyoD family inhibitor 4- Unlikely significance 72
+ mm cDNA IMAGE 1481238
No significant hits Unknown significance 74
+ mm cDNA clone IMAGE 5153066
Envelope protein
4- P ossible significance 75
4- coding Hyaluronidase Hyaluronidase 4- F alse positive (lysosomal) 76
4- coding GCN2 eukaryotic initiation factor GCN2 eukaryotic initiation factor 4- Unknown significance 77 + c o d in g Dlxin-1 Dlxin-1 4- P ro b a b lein te ra c to r 78 4- c o d in g Dlxin-1 Dlxin-1 4- P ro b a b le in te ra c to r 79 4- coding Similar to tyrosine p hosphatase Protein tyrosine phosp h atase kappa precursor 4- F alse positive (m em brane protein, brain) 80 4-
mm cDNA clone IMAGE 4489198
Myotonic dystrophy kinase
related binding kinase 4-
F alse positive (brain) 81
4- 4489346mm cDNA clone IMAGE G am m a tubulin complex protein 2 4- F alse positive (pancreas) 85 4- coding mm arsenite-translocating AT R ase mm arsenite-translocating A T P ase 4- F alse positive 86 No significant hits No significant hits Unknown
significance 88
4- non-coding M -S em a F (factor in neural networking) No significant hits In non-coding region 89
4- non-coding mm myelin expression factor 3-like
No significant hits In non-coding region 93
- mm cDNA clone IMAGE 5132622 No significant hits significanceUnknown
Table 5.4 b continued
BLAST searches of positives from library screen 3 using full-length bait against EMBL and Swiss Prot/Translated EMBL (SPTR) databases. The possible and probable interactors with SHOX are highlighted in red. mm= mus muscularis.