U.S. patent application number 15/206627 was filed with the patent office on 2017-01-05 for avian colony stimulating factor 1 receptor binding proteins.
The applicant listed for this patent is The University Court of the University of Edinburgh. Invention is credited to Dave Burt, Valerie Garceau, David Arthur Hume, Bob Paton, David Sester, Jacqueline Smith.
Application Number | 20170002051 15/206627 |
Document ID | / |
Family ID | 40972646 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170002051 |
Kind Code |
A1 |
Hume; David Arthur ; et
al. |
January 5, 2017 |
AVIAN COLONY STIMULATING FACTOR 1 RECEPTOR BINDING PROTEINS
Abstract
The present invention provides avian CSF1 genes encoding
proteins which bind avian colony stimulating factor 1 receptor
(CSF1R) and which exhibit immunomodulatory properties.
Inventors: |
Hume; David Arthur; (Roslin,
GB) ; Burt; Dave; (Roslin, GB) ; Sester;
David; (Roslin, GB) ; Garceau; Valerie;
(Roslin, GB) ; Smith; Jacqueline; (Roslin, GB)
; Paton; Bob; (Roslin, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University Court of the University of Edinburgh |
Edinburgh |
|
GB |
|
|
Family ID: |
40972646 |
Appl. No.: |
15/206627 |
Filed: |
July 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13380185 |
Mar 12, 2012 |
9403893 |
|
|
PCT/GB2010/001221 |
Jun 22, 2010 |
|
|
|
15206627 |
|
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|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/55522
20130101; C12N 15/09 20130101; A61K 2039/55527 20130101; C12Q
1/6888 20130101; C07K 14/54 20130101; A61K 38/00 20130101; C12Q
2600/158 20130101; A61K 39/39 20130101; A61P 29/00 20180101; C12Q
2600/156 20130101; A61K 38/193 20130101; C12N 15/63 20130101; C07K
14/53 20130101 |
International
Class: |
C07K 14/53 20060101
C07K014/53; C12Q 1/68 20060101 C12Q001/68; C07K 14/54 20060101
C07K014/54; A61K 39/39 20060101 A61K039/39 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 23, 2009 |
GB |
0910833.3 |
Claims
1-9. (canceled)
10. A method of modulating avian growth and/or organ development
comprising administering an effective amount of an avian CSF1
and/or IL34 gene and/or protein to an avian subject.
11-15. (canceled)
16. A method of modulating the avian immune system comprising
administering an immunomodulatory amount of an avian CSF1 and/or
IL34 gene and/or protein to an avian subject.
17. A vaccine adjuvant comprising an avian CSF1 and IL34
protein.
18. The vaccine adjuvant of claim 17, wherein the avian CSF1
protein, comprises an amino acid sequence at least 60% identical or
homologous to the amino acid sequences of SEQ ID NOS:3 or 5.
19. An immunogenic composition, comprising an avian CSF1 and/or
IL34 protein.
20. A method of treating conditions, such as inflammatory diseases,
resulting from or associated with aberrant CSF1/IL34 gene/protein
expression, said method comprising an effective amount of a
compound capable of inhibiting CSF1/IL34 gene expression or protein
production to an avian subject.
21. The method of claim 20, wherein the compound is selected from
the group consisting of: a sense or antisense nucleic acid
molecule; a fragment, portion or derivative of an avian CSF1/IL34
gene; and an antibody.
22. A method of screening avian species, particularly
agriculturally significant or important avian species for potential
inclusion in breeding programs said method comprising the steps of:
a) providing a nucleic acid sample from an avian species; and b)
comparing the level of expression of the CSF1 and/or IL34 gene(s)
with a reference nucleic acid sample or value; wherein avian
species which exhibit and increased level of CSF1 and/or IL34 gene
expression relative to a that present in a reference nucleic acid
sample or value, may be selected for inclusion in breeding
programs.
23. An avian CSF1 gene comprising a nucleic acid sequence at least
60% identical or homologous to the nucleic acid sequences of SEQ ID
NOS: 1, 2 or 4.
24. A fragment, analogue, portion, mutant, variant, derivative
and/or homologue/orthologue of the gene of claim 23.
25. An avian IL34 gene comprising a nucleic acid sequence at least
60% identical or homologous to the nucleic acid sequence of SEQ ID
NO:6.
26. A fragment, analogue, portion, mutant, variant, derivative
and/or homologue/orthologue of the gene of claim 25.
27. A substantially purified avian CSF1 protein, comprising an
amino acid sequence at least 60% identical or homologous to the
amino acid sequences of SEQ ID NOS:3 or 5.
28. A fragment, analogue, portion, mutant, variant, derivative
and/or homologue/orthologue of the protein of claim 27.
29. A substantially purified avian IL34 protein, comprising an
amino acid sequence at least 60% identical or homologous to the
amino acid sequence of SEQ ID NO:6.
30. A fragment, analogue, portion, mutant, variant, derivative
and/or homologue/orthologue of the protein of claim 29.
31. An expression vector comprising an avian CSF1 gene sequence or
fragment or portion thereof.
32. The expression vector of claim 31, wherein the avian gene
sequence comprises a nucleic acid sequence at least 60% identical
or homologous to the nucleic acid sequences of SEQ ID NOS:1, 2 or
4.
33. An expression vector comprising an avian IL34 gene sequence or
fragment or portion thereof.
34. The expression vector of claim 33, wherein the avian IL34 gene
sequence comprises a nucleic acid sequence at least 60% identical
or homologous to the nucleic acid sequence of SEQ ID NO:6.
35. A host cell transformed with the expression vector of claim
31.
36. A host cell transformed with the expression vector of claim
32.
37. A host cell transformed with the expression vector of claim 33.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/380,185, filed Mar. 12, 2012, now allowed, which is a 35
U.S.C. .sctn.371 national phase entry of PCT Application
PCT/GB2010/001221, filed Jun. 22, 2010, and published in English on
Dec. 29, 2010, as International Publication No. WO 2010/149960 A1,
and which claims the priority to United Kingdom Application No.
0910833.3, filed Jun. 23, 2009, the disclosure of each of which is
incorporated herein by reference in its entirety.
STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
[0002] A Sequence Listing in ASCII text format, submitted under 37
C.F.R. .sctn.1.821, entitled 9013-115TSCT_ST25.txt, 18,021 bytes in
size, generated on Jul. 8, 2016, and filed via EFS-Web, is provided
in lieu of a paper copy. This Sequence Listing is hereby
incorporated by reference into the specification for its
disclosures.
FIELD OF THE INVENTION
[0003] The present invention relates to newly identified avian
genes which encode proteins which bind avian colony stimulating
factor 1 receptor (CSF1R). Through binding to CSF1R, these proteins
exhibit immunomodulatory properties which may be exploited to
modulate the avian immune system as well as effects on cell growth,
differentiation and/or proliferation, organ/tissue development and
vaccine efficacy.
BACKGROUND OF THE INVENTION
[0004] Mononuclear phagocytes are derived from progenitor cells in
the bone marrow which differentiate into blood monocytes and then
enter the tissues to occupy specific niches (Hume et al. 2002).
They constitute the first line of defense against pathogens,
maintain homeostasis, and have trophic functions ranging from bone
morphogenesis to neuronal patterning in sexual development, from
angiogenesis to adipogenesis (Pollard 2009). Macrophage
colony-stimulating factor (CSF1) is required for normal
differentiation, proliferation and survival of macrophage lineage
cells (Sweet and Hume 2003; Chitu and Stanley 2006; Bonifer and
Hume 2008). Mice and rats bearing mutations in the CSF1 gene (i.e.
op/op mice and tl/tl rats) show a mononuclear phagocyte deficiency,
and their important developmental abnormalities, such as reduced
somatic growth, perinatal mortality, osteopetrosis, neurological
and reproductive defects, highlight many of the macrophage trophic
roles mentioned above (Marks et al. 1992; Pollard 1997, 2009; Ryan
et al. 2001).
[0005] Although CSF-1 exists in a number of isoforms, most
biochemical studies have focused on the minimal biologically active
fragment i.e. the 154 N-terminal amino acids, common to all
isoforms. In a full-length CSF1 molecule, this receptor-binding
region is preceded by a 32-amino-acid signal peptide, and followed
by a variable spacer region, a 24-amino-acid transmembrane region
and a 35-amino-acid cytoplasmic tail. The tertiary structure of the
active fragment of CSF1 forms a short-chain four-helical bundle (A,
B, C and D) with small regions of beta-sheet (1 and 2). The helices
are paired into A-C and B-D by intrachain disulfide bonds, while
one interchain disulfide bond generates a mature homodimer with a
two-fold rotation axis (Pandit et al. 1992). The crystal structure
of mouse CSF1 bound to its receptor, CSF1R, was recently solved to
a resolution of 2.4 .ANG., showing that CSF1 N-terminal segment
(residues 6-15), helix B (residues 55-66) and helix C (residues
79-85) are implicated in receptor binding. (PDB code 3EJJ) (Chen et
al. 2008).
[0006] All CSF1 effects are mediated through binding to the CSF1
receptor (CSF1R), a glycoprotein of 165 kDa that is encoded by the
c-fms proto-oncogene (Dai et al. 2002). CSF1R is a member of the
type III protein tyrosine kinase family, along with PDGFRA, PDGFRB
and c-kit, and shares with other family members a characteristic
extracellular region of five immunoglobulin-like domains (D1 to
D5), a single transmembrane helix and a intracellular tyrosine
kinase domain (Rosnet and Birnbaum 1993). CSF-1 associates tightly
with the receptor (K.sub.D=0.4 nM at 37.degree. C.) in a 2:2
stoichiometry (Guilbert and Stanley 1986). This binding involves
the CD loop (residues 141-151) and the EF loop (residues 168-173)
of D2, as well as the BC and DE loops (residues 231-232 and 250-257
respectively) of D3 (Chen et al. 2008).
[0007] The expression of CSF1R on the cell surface is amongst the
earliest events in macrophage lineage commitment, and is mostly
restricted to these cells throughout embryonic development as well
as in adults (Lichanska et al. 1999) Like other myeloid promoters,
the proximal CSF1R promoter lacks the classic TATA box and GC-rich
sequences but contains recognition sites for AML1 transcription
factors, and for transcription factors of the C/EBP and Ets
families, including the myeloid-restricted transcription factor
PU.1 (Reddy et al. 1994; Himes et al. 2005; Bonifer and Hume 2008).
Expression of CSF1R is also controlled by FIRE (Fms Intronic
Regulatory Element) a highly-conserved enhancer element in the
first intron (Himes et al. 2001; Sasmono et al. 2003).
[0008] Most of the phenotypic defects seen in the op/op mice
including reproductive defects and perturbations in organ
development, are even more severe in the CSF1R knockout mice (Dai
et al. 2002). First attributed to the availability of
maternal-derived CSF1, these observations can now be explained by
the recent discovery of a second ligand for the human CSF1R,
designated IL34, with an activity on monocyte viability. IL34 was
purified as a homodimer composed of 241-amino acid monomers, and
shown to be expressed mostly in the brain but also in many other
tissues including heart, spleen, lung, liver, kidney and thymus
(Lin et al. 2008).
[0009] WO2008/031172 describes the use of CSF1 to promote organ
development in warm-blooded animals and in particular premature
human foetuses/embryos. Furthermore, WO03/028752 describes methods
and compositions for modulating immune responses in animals, said
methods comprising modulating CSF1 activity.
[0010] The chicken has been used widely in studies of early
embryonic myelopoiesis (Lichanska et al. 1999; Lichanska and Flume
2000), but compared to our knowledge of the mammalian mononuclear
phagocyte system, our knowledge of avian systems is rather limited
and neither WO2008/031172 or WO03/028752 describe the existence of
an avian CSF1 gene. Indeed, there are only two characterized
colony-stimulating factors in chickens. Chicken GM-CSF (CSF2) has
been cloned and shown to drive the proliferation of chicken bone
marrow cells (Avery et al. 2004). The other described chicken CSF,
myelomonocytic growth factor, has recently been shown to be the
chicken ortholog of G-CSF (CSF3) (Gibson et al. 2009). Apparent
orthologs of the GM-CSF receptor alpha chain, and the beta chain,
shared with the IL3 receptor, are annotated in the chicken genome
(Ensembl). So far, no function for the CSF1R has been demonstrated
in birds, CSF1 was believed to be absent from avian genomes and
IL34 had not been recognized (Kaiser 2007).
[0011] CSF1 and IL34 in humans were reported to have little obvious
homology, and in mammals at least, CSF1 has evolved rather rapidly.
The existence of two ligands for a single receptor is difficult to
maintain across evolution if they evolve independently of each
other and of the receptor.
SUMMARY OF THE INVENTION
[0012] The present invention concerns the identification of novel
avian genes encoding proteins which bind avian colony stimulating
factor 1 receptor (CSF1R). In particular, the invention describes,
for the first time, the existence of avian genes encoding colony
stimulating factor 1 (CSF1) and interleukin 34 (IL34). It should be
noted that these findings contradict the statement by Kaiser (2007)
that there is no CSF1 equivalent present in the avian genome.
[0013] Furthermore, the inventors have surprisingly discovered that
the avian CSF1R is expressed by macrophages--this is in contrast to
other species, for example fish, where the CSF1R is expressed in
other cell types.
[0014] In view of the avian CSF1R expression profile, the inventors
have discovered that through binding to CSF1R, avian CSF1 and IL34
exhibit immunomodulatory properties which may be exploited to
modulate the avian immune system as well as effects on cell growth,
differentiation and/or proliferation, organ/tissue development and
vaccine efficacy.
[0015] It should be understood that references to the terms "avian"
or "avian species" should be taken to encompass all species within
the Class, Ayes and in particular those species classified as
belonging to the Order, Galliformes and/or the Genus, Gallus. Avian
species now known to harbour CSF1 and IL34 genes may include, for
example, Gallus gallus otherwise known as the domestic chicken
and/or other poultry or fowl species. In other embodiments, the
term "avian" may be construed as including species classified
within the Order, Passeriformes, such as, for example, finches, in
particular, the zebra finch. In general, the terms "avian CSF1
gene", "avian IL34 gene", "avian IL34 protein" and "avian CSF1
protein" encompass CSF1 and/or IL34 genes, proteins (and fragments
thereof) present in, or encoded by, the genomes of the avian
species described herein.
[0016] The inventors have ascertained the sequences of certain
avian forms of CSF1 and the cDNA sequence of an exemplary CSF1
gene, present in the Gallus gallus genome, is given below as SEQ ID
NO: 1:
TABLE-US-00001 SEQ ID NO: 1
ATAAAGGGCAGCGCGGCGGCGACGGCGGACTCAGCCCGGCCCCGCTCCGC
CGCCTTCTCCCGCACCGCCCGACCCGCCGCAGCCCCGGCCCCACGGCAGC
CCCCATGCCCCGCCTCGGATCCCAGGTGTCCCTGTTCCGCTGCACCCTGC
TCTCGTCCCTCCTCCTCGTCTGCAGCATCCATGAGACGGAGCAGAACAGC
TACTGCCAGCAGATCATCACCGAGCGGCACCTGGACCACCTGCAGGAGCT
GGCGGACACGCAGATGCAGCAGCCGGGCACAGTGTCCTTCAGATTCATCA
GCAAGATGCGGCTGAGCGACTCTGTCTGCTACGTGAAAGCCGCCTTCCCT
TTGCTGGGCACCATCCTGAACAGGACGACGTTCAAGGAGAACTCAACAAA
CGCCAACAAGATGAAGACGGTGCGCAAGATGTACGAAAACATCGATGAGA
ACGTGGACCCCTGCATCAGGGACGAGGATGACAAGGAGCACGCGCTGTCC
GAAATGTGCTTTGAGGAGTTCACCACGTCCCCCTACGAGATGCTGGTGCT
GGTGAGGCAGTTCTTCCAGGACATCAAACAGCTGCTGCAGAACAAGGAGA
CCTTCGAGAAGGACTGCAGCCAGGTGTACCGCAGTGCGTGCGCGGGGCCC
CGGCAGCACAGCTCCTCCCCAGGTGTGGGGACAGATCCTGACTGCAATTG
CCTGTCCCCTGCCCTCCCTTCTGCCACCCAGCCCTCCCTCTCCGCTGCCA
CCCGTGCCGGCAGGGACGTGGCGCCCGCTAGCACCAGGGTCCCTTACCGC
CAGCTCGGTGGCATCCTGGCTGAGTTAGGCAGCAGTGCCCCGTCCGAGCC
CCCCAGTAGCGTGGAGGGCAGCTCGGGGGCCGAGGAACTGCCAGGAGCCG
GGCTCGGCGACGCGTCGGCGCCGTCCCCCACCATGCAGCAGACGCTTGGA
GCCCTCCTGGATCCAGCCGCGAGCGCCGGCCCGAAGGCTGAGGACGTATC
CATCCCGTCCCACGGGATGCCGGAGGAGGGCGCCGGGACCCCCGCCCTCC
CACATCGGCTCCCTTCGCCGCGAGGGATCAGCGCGGCGATGCCGGCGGCG
GTCCCCAGCAGCGGCTCTGCGCAGCGCCGCGGGGTCGGGCGCCGTCCCAC
CGAGAGCCCCGAGCGGGTCACGCAGCTCCGCTTCCCCAGGATGGCTCCGC
CGTTGCGGGGCCGGGCGGAGGGCGGCCCCGGGGACGGGGCGAGGGCGCGA
GGCTGGGGGCTGAGCCGGCTGCGGGAGCCCGAGGACGGCGGGGCCGGACC
CAGCTTTGATTCGAGCTTTGTTCTGAGCGCAGAGCAGCGCAGGAAGGAGC
CGCCAGCCGCCAGCGGGGGGCACCGGGAGCTCCTGGTGTACGTCACGGTG
GCCAGCGTGGTGGCCGTGCTGCTGGCCATGGGCGGGCTGCTCTTCTACAA
GTATAAGTCCAAGGTCCTGCAGCGGGGAGCAGCGCTAAAAGAGGGGGGCT
GCGACCCCGAGGAGCCGGAGAGCAGGGCGCTGCAGGGAGCGCAGGGCTGC
GCGGAGCTGGAGACGCAGGAGCTGTGAGGGCCCCCTGCGGGACGTGATGC
TGCTCGGGGGGACGGACGGGGACGCTCCTCGCTGGGCGACGGACGGCTGC
TGCTCGGCCTCCCCCCGCCGCGATGACCCCCAGGCCCTGTCCTGCAGCTG
CAACCCACGGGTGAGGATGGCAGGACGGGGCGGTGCAGCCCTGCAGGACC
CCGGCGATGGGGCGGATGGCACCGAGGGGCTCCACGGGGACGGCATTGGG
TGCCGCGAGTGGAACATCTCCCCCCACCCATCCACGGTTCCCGTTGCTCC
TCTCCCACCCCTGGCACGGGGGGACCCCCGGCGCCCCATGGGGGGACCCC
TCCCGCATCCCACCGGTGCCGAGGACCCAACGCCCGGCCTGCAAAGGGGG
AAACCCTCACACTGTGAATATTTAAGACCCGTGGTGCCGTCCCCATCCCG
CGATCCCAAGCTGGCCTTGGGAGCTGCCCGGCGCCGCTCTGCGCAGGAAG
GCTCTCCACGAACGCGGTGGATAAACGCTTTTATCCAACAAATGCACTTG
GGGGGGGGGGTTCCCCCCTCCCTGCAGGGTTATTGCTGCGAGCTGGCCTC
GCCCCAGACTGGATTTTGTTGCTGGAGCACAGCACGGCAATGGGGCCGTG
GCTGCAGTGTGGGGTTTGGGGGCTCAGCGGTACCCGGACTGCGTCCCACC
CCACACGGCATCCCTGCCCAGCGCCGCTCCCGGGGGGTCGGAAGTGTTAT
TTTTATATTACATGAGATGCAAACGGGACGGAGCACATTGGGGTGTGGTG
GGGTTTTGTTTTTTAAAGCATTAGTATTGATTTTGGGGTTTTTTTTTCTA
TGCGTATTTATGGACTGCCAAAAAAAGAGGCGTTTCCTGGGGGTGATGGG
GGGGGGGGTGGAAGTGGGGTGCAGAGCCGGGCTGGGGCCGGAGCTGGTGC
TGGCTCAGTATGTGGGGTGTGGGTGAGGGGGGTTGGGGGGGGGGCAGCTT
TTGGAGCTCTTTCTGCCTCTGTTGTCTCATTTTTTGTACAGTGAAATGGT
GAAATATTTTATACAAAGTCATTTAAAGAAGTCTATTTAAGGAAAATAAT
AGAAAACAGCTTGTATATTTAATATTATTAATAAAGATGGACGTGCAAAA AAAAAAAAAAA
[0017] The native CSF1 Gallus gallus sequence, comprises eight
exons and yields two transcripts, one encoding a protein of 490
amino acids, the other a protein of 270 amino acids. The transcript
encoding the longer, 490 amino acid protein is provided below as
SEQ ID NO: 2
TABLE-US-00002 SEQ ID NO: 2
ATGCCCCGCCTCGGATCCCAGGTGTCCCTGTTCCGCTGCACCCTGCTCTC
GTCCCTCCTCCTCGTCTGCAGCATCCATGAGACGGAGCAGAACAGCTACT
GCCAGCAGATCATCACCGAGCGGCACCTGGACCACCTGCAGGAGCTGGCG
GACACGCAGATGCAGCAGCCGGGCACAGTGTCCTTCAGATTCATCAGCAA
GATGCGGCTGAGCGACTCTGTCTGCTACGTGAAAGCCGCCTTCCCTTTGC
TGGGCACCATCCTGAACAGGACGACGTTCAAGGAGAACTCAACAAACGCC
AACAAGATGAAGACGGTGCGCAAGATGTACGAAAACATCGATGAGAACGT
GGACCCCTGCATCAGGGACGAGGATGACAAGGAGCACGCGCTGTCCGAAA
TGTGCTTTGAGGAGTTCACCACGTCCCCCTACGAGATGCTGGTGCTGGTG
AGGCAGTTCTTCCAGGACATCAAACAGCTGCTGCAGAACAAGGAGACCTT
CGAGAAGGACTGCAGCCAGGTGTACCGCAGTGCGTGCGCGGGGCCCCGGC
AGCACAGCTCCTCCCCAGGTGTGGGGACAGATCCTGACTGCAATTGCCTG
TCCCCTGCCCTCCCTTCTGCCACCCAGCCCTCCCTCTCCGCTGCCACCCG
TGCCGGCAGGGACGTGGCGCCCGCTAGCACCAGGGTCCCTTACCGCCAGC
TCGGTGGCATCCTGGCTGAGTTAGGCAGCAGTGCCCCGTCCGAGCCCCCC
AGTAGCGTGGAGGGCAGCTCGGGGGCCGAGGAACTGCCAGGAGCCGGGCT
CGGCGACGCGTCGGCGCCGTCCCCCACCATGCAGCAGACGCTTGGAGCCC
TCCTGGATCCAGCCGCGAGCGCCGGCCCGAAGGCTGAGGACGTATCCATC
CCGTCCCACGGGATGCCGGAGGAGGGCGCCGGGACCCCCGCCCTCCCACA
TCGGCTCCCTTCGCCGCGAGGGATCAGCGCGGCGATGCCGGCGGCGGTCC
CCAGCAGCGGCTCTGCGCAGCGCCGCGGGGTCGGGCGCCGTCCCACCGAG
AGCCCCGAGCGGGTCACGCAGCTCCGCTTCCCCAGGATGGCTCCGCCGTT
GCGGGGCCGGGCGGAGGGCGGCCCCGGGGACGGGGCGAGGGCGCGAGGCT
GGGGGCTGAGCCGGCTGCGGGAGCCCGAGGACGGCGGGGCCGGACCCAGC
TTTGATTCGAGCTTTGTTCTGAGCGCAGAGCAGCGCAGGAAGGAGCCGCC
AGCCGCCAGCGGGGGGCACCGGGAGCTCCTGGTGTACGTCACGGTGGCCA
GCGTGGTGGCCGTGCTGCTGGCCATGGGCGGGCTGCTCTTCTACAAGTAT
AAGTCCAAGGTCCTGCAGCGGGGAGCAGCGCTAAAAGAGGGGGGCTGCGA
CCCCGAGGAGCCGGAGAGCAGGGCGCTGCAGGGAGCGCAGGGCTGCGCGG
AGCTGGAGACGCAGGAGCTGTGA
[0018] The inventors have ascertained that SEQ ID NO: 2 encodes the
following amino acid sequence (given as SEQ ID NO: 3).
TABLE-US-00003 SEQ ID NO: 3
MPRLGSQVSLFRCTLLSSLLLVCSIHETEQNSYCQQIITERHLDHLQELA
DTQMQQPGTVSFRFISKMRLSDSVCYVKAAFPLLGTILNRTTFKENSTNA
NKMKTVRKMYENIDENVDPCIRDEDDKEHALSEMCFEEFTTSPYEMLVLV
RQFFQDIKQLLQNKETFEKDCSQVYRSACAGPRQHSSSPGVGTDPDCNCL
SPALPSATQPSLSAATRAGRDVAPASTRVPYRQLGGILAELGSSAPSEPP
SSVEGSSGAEELPGAGLGDASAPSPTMQQTLGALLDPAASAGPKAEDVSI
PSHGMPEEGAGTPALPHRLPSPRGISAAMPAAVPSSGSAQRRGVGRRPTE
SPERVTQLRFPRMAPPLRGRAEGGPGDGARARGWGLSRLREPEDGGAGPS
FDSSFVLSAEQRRKEPPAASGGHRELLVYVTVASVVAVLLAMGGLLFYKY
KSKVLQRGAALKEGGCDPEEPESRALQGAQGCAELETQEL
[0019] The second transcript encoding the shorter, 270 amino acid
protein is given below as SEQ ID NO: 4:
TABLE-US-00004 SEQ ID NO: 4
ATGCCCCGCCTCGGATCCCAGGTGTCCCTGTTCCGCTGCACCCTGCTCTC
GTCCCTCCTCCTCGTCTGCAGCATCCATGAGACGGAGCAGAACAGCTACT
GCCAGCAGATCATCACCGAGCGGCACCTGGACCACCTGCAGGAGCTGGCG
GACACGCAGATGCAGCAGCCGGGCACAGTGTCCTTCAGATTCATCAGCAA
GATGCGGCTGAGCGACTCTGTCTGCTACGTGAAAGCCGCCTTCCCTTTGC
TGGGCACCATCCTGAACAGGACGACGTTCAAGGAGAACTCAACAAACGCC
AACAAGATGAAGACGGTGCGCAAGATGTACGAAAACATCGATGAGGACGT
GGACCCCTGCATCAGGGACGAGGATGACGAGGAGCACGCGCTGTCCGAAA
TGTGCTTTGAGGAGTTCACCACGTCCCCCTACGAGATGCTGGTGCTGGTG
AGGCAGTTCTTCCAGGACATCAAACAGCTGCTGCAGAACAAGGAGACCTT
CGAGAAGGACTGCAGCCAGGTGTACCGCAGTGCGTGCGCGGGGCCCCGGC
AGCACAGCTCCTCCCCAGAGCAGCGCAGGAAGGAGCCGCCAGCCGCCAGC
GGGGGGCACCGGGAGCTCCTGGTGTACGTCACGGTGGCCAGCGTGGTGGC
CGTGCTGCTGGCCATGGGCGGGCTGCTCTTCTACAAGTATAAGTCCAAGG
TCCTGCAGCGGGGAGCAGCGCTAAAAGAGGGGGGCTGCGACCCCGAGGAG
CCGGAGAGCAGGGCGCTGCAGGGAGCGCAGGGCTGCGCGGAGCTGGAGAC
GCAGGAGCTGTGA
[0020] SEQ ID NO: 4 encodes the following amino acid sequence
(given as SEQ ID NO: 5):
TABLE-US-00005 SEQ ID NO: 5
MPRLGSQVSLFRCTLLSSLLLVCSIHETEQNSYCQQIITERHLDHLQELA
DTQMQQPGTVSFRFISKMRLSDSVCYVKAAFPLLGTILNRTTFKENSTNA
NKMKTVRKMYENIDEDVDPCIRDEDDEEHALSEMCFEEFTTSPYEMLVLV
RQFFQDIKQLLQNKETFEKDCSQVYRSACAGPRQHSSSPEQRRKEPPAAS
GGHRELLVYVTVASVVAVLLAMGGLLFYKYKSKVLQRGAALKEGGCDPEE
PESRALQGAQGCAELETQEL
[0021] In addition to the above, the inventors have ascertained the
sequences of the avian IL34 gene and the sequence of an exemplary
IL34 gene, present in the Gallus gallus genome, is given below as
SEQ ID NO: 6:
TABLE-US-00006 SEQ ID NO: 6
ATGCACCAGGGCTGCGCGGCTGTCCTCTGTGTCCTGGCCGTGCTGGGGCT
GGAGGTGGCTGCGCTGGGGGAATGCGAGCTCGCCCGCCTGCTGCAGGACA
AGCTGCGGTATGAGATGCGCCTGCAGTACATGAAGCACAACTTCCCCATT
GACTACACTCTCCGGGTGCAGCACGAGGAGGTGCTGCGGACCGCCAACGT
CACCCGCCTGCGTGATGGGAAGGTGTCGGAGGCGTCGCTGCGCTACCTGT
GGTTCCACGCCTGCTCCCAGGCGGTGCTGCACATCCTCGAGGTGCTGCCG
GAGAAGCACCCGTCCCGTGGGTACACGCAGGAGCTGAGCCAGCTTTTGGA
TGCCCTGGGCGTGGAGTACAGTGGGTACCGGCAGAGCGATGTGGACGCGG
TGGTGGCCGACCTGGTGAAGCAGCTGCACAGCGGCGATAGCCGGCAGAAG
GCCGTGCGCCCCAAAGCACTGCTGGACAACTGCCTCAAGGTCCTGCGGAT
GCTCTTCGGGGCACACTGTCGGTGGGACTCCGCT
[0022] SEQ ID NO: 6 encodes the following amino acid sequence
(given as SEQ ID NO: 7):
TABLE-US-00007 SEQ ID NO: 7
MHQGCAAVLCVLAVLGLEVAALGECELARLLQDKLRYEMRLQYMKHNFPI
DYTLRVQHEEVLRTANVTRLRDGKVSEASLRYLWFHACSQAVLHILEVLP
EKHPSRGYTQELSQLLDALGVEYSGYRQSDVDAVVADLVKQLHSGDSRQK
AVRPKALLDNCLKVLRMLFGAHCRWDSA
[0023] As such, in a first aspect, the present invention relates to
the sequences designated SEQ ID NOS: 1-7, encoding avian CSF1 genes
(SEQ ID NOS: 1, 2 and 4), the avian IL34 gene (SEQ ID NO: 6), avian
CSF1 proteins (SEQ ID NOS: 3 and 5) and the avian IL34 protein (SEQ
ID NO: 7). In a further embodiment, the present invention provides
fragments, analogues, portions, mutants, variants, derivatives
and/or homologues/orthologues of any of the sequences described
herein. Advantageously, the fragments, analogues, portions,
mutants, variants, derivatives and/or homologues/orthologues
provided by this invention might be functional or active--that is,
they retain the function of the wild type avian CSF1 and IL34
genes/proteins.
[0024] The term "mutants" may encompass naturally occurring nucleic
acid or protein mutants or those artificially created by the
introduction of one or more nucleic acid or amino acid additions,
deletions, substitutions or inversions.
[0025] Sequences homologous to the avian CSF1 and IL34 nucleic
acid/protein sequences detailed above may be found in a number of
different avian species, including each of those species belonging
to the various Classes and Orders described above. One of skill
will appreciate that homologous sequences may exhibit as little as
approximately 20 or 30% sequence homology or identity however, in
other cases, homologous sequences may exhibit at least 40, 50, 60,
65 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% homology
or identity to the various sequences given above. As such,
homologous forms of other avian species are to be included within
the scope of this invention.
[0026] Using the various nucleic acid and amino acid sequences
described herein, one of skill in the art could readily identify
related sequences in other avian species. For example, nucleic acid
obtained from a particular species may be probed using the
fragments or portions of the sequences described herein, for
homologous or closely related sequences. In other methods,
antibodies specific to or selective for the CSF1 or IL34 proteins
described herein may be used to probe for or bind homologous
proteins in other avian species. Such antibodies are described in
more detail below.
[0027] Natural variations due to, for example, polymorphisms, may
exist between the sequences of CSF1 and IL34 genes or proteins
isolated from any given species; these variants may manifest as
proteins and/or genes which exhibit one or more amino acid/nucleic
acid substitutions, additions, deletions and/or inversions relative
to a reference sequence (for example any of the sequences described
above). It is to be understood that all such variants, especially
those which are functional and/or display the desired activity, are
to be included within the scope of this invention.
[0028] Additionally, or alternatively, analogues of the various
amino acid sequences (i.e. proteins or peptides) described herein
may be made by introducing one or more conservative amino acid
substitutions into the primary sequence. One of skill in this field
will understand that the term "conservative substitution" is
intended to embrace the act of replacing one or more amino acids of
a protein or peptide with an alternate amino acid with similar
properties and which does not substantially alter the
physio-chemical properties and/or structure or function of the
native (or wild type) protein. Analogues of this type are also
encompassed with the scope of this invention.
[0029] As is well known in the art, the degeneracy of the genetic
code permits substitution of one or more bases in a codon without
changing the primary amino acid sequence. Consequently, although
the sequences described in this application are known to encode
avian CSF1 and IL34 proteins, the degeneracy of the code may be
exploited to yield variant nucleic acid sequences which encode the
same primary amino acid sequences.
[0030] The provision of certain avian CSF1 and IL34 gene and/or
protein sequences renders it possible for one skilled in this field
to express and/or purify avian CSF1 and IL34 genes and/or proteins.
By way of example, standard laboratory techniques may be used to
express and/or purify recombinant avian CSF1 and IL34 genes and/or
proteins. In one embodiment, the CSF1 and IL34 nucleic acid
sequences described herein (or indeed any fragments or portions
thereof) may be introduced into vector systems which can, inturn,
be introduced into prokaryotic and/or eukaryotic cells for
expression--such vectors may be known as eukaryotic/prokaryotic
expression vectors. The methods and vectors which may be used in
such techniques are described in detail by Sambrook et al.,
(Molecular Cloning: A Laboratory Manual, CSHL, 1989). By way of
example, the avian CSF1 and IL34 genes (or fragments thereof)
described herein may be cloned into a variety of vectors including,
for example, plasmids, bacteriophages, cosmids, viral vectors,
yeast artificial chromosomes and/or bacterial artificial
chromosomes.
[0031] CSF1 and IL34 genes and/or proteins can be expressed with or
without a fused heterologous sequence. CSF1 or IL34 fusion proteins
may be generated and expressed using pET and pGEX type vectors
which comprise short nucleic acid sequences encoding heterologous
peptide sequences, such as, for example, peptide tags, immediately
downstream of the cloning site. Vectors of this type are
particularly useful for generating tagged CSF1 and IL34 proteins
which can easily be removed, isolated or purified from
heterogeneous protein mixtures by, for example, affinity
purification procedures. Suitable methods for isolating tagged
(fusion) proteins, such as, for example, those tagged or fused to
short peptides comprising, for example histidine or glutathione
s-transferase moieties, include the use of nickel columns or
glutathione-sephaprose substrates. Other techniques are detailed in
Sambrook et al., (1989).
[0032] Vectors into which CSF1 or IL34 genes (or fragments thereof)
have been cloned, may be introduced or transfected into cells using
a variety of techniques--such techniques may otherwise be referred
to as transfection protocols. Transfection protocols utilise
conditions which render cell membranes permeable to compounds such
as nucleic acids. By way of example, it may be possible to
facilitate the transfection of vectors, including expression
vectors, into cells using electroporation, heat shock, chemical
compounds such, for example, calcium phosphate, stronitium
phosphate, microinjection techniques and/or gene guns.
[0033] As such, in a second aspect, the present invention provides
a vector comprising an avian CSF1 and/or IL34 gene or a fragment
thereof. In one embodiment, the vector may be an expression vector
containing elements for driving expression in a host cell.
[0034] In a third aspect, the invention provides a cell transfected
with a vector according to this invention. By way of example, the
host cell may be a prokaryotic or a eukaryotic cell such as, for
example a bacterial cell such as one selected from the group
consisting of E. coli, Pseudomonas sp. and Bacillus sp. or, in
another embodiment, a yeast, fungal, insect, plant or animal
cell.
[0035] In certain embodiments, the fused or unfused recombinant
avian CSF1 and/or IL34 proteins provided by this invention may be
used to generate antibodies which exhibit an affinity for, or are
specific to, or selective for, a recombinant avian CSF1/IL34 or a
fragment thereof. In particular, the recombinant avian CSF1 and
IL34 proteins may be used to immunize animals (for example rodents
and the like) to generate polyclonal sera, or with hybridomas to
generate monoclonal antibodies. As such, a fourth aspect of this
invention provides binding agents, for example antibodies, that
bind specifically (or selectively) to one or more epitopes present
in an avian CSF1 or IL34 protein, such as, for example, those
described herein.
[0036] The avian CSF1/IL34 genes/proteins described herein may be
exploited to execute a variety of immunological effects in the
avian host. For example, methods or compounds which modulate avian
CSF1/IL34 gene expression and/or protein levels may be used to
modulate avian cell, for example monocyte/macrophage, production,
proliferation, survival and/or differentiation. In addition,
methods or compounds which modulate avian CSF1/IL34 gene expression
and/or protein levels may be used to modulate the growth,
proliferation and/or survival of tissues and/organs.
[0037] Modulation of CSF1/IL34 genes/proteins may result in
modulation of mature avian macrophage/monocyte function. In
particular, avian macrophage phagocytic and our tumoricidal
activity may be enhanced by the methods or compounds described
herein.
[0038] In other embodiments, methods or compositions which modulate
avian CSF1/IL34 gene/protein expression/levels may be used to
regulate primary avian immune responses and/or prime immune system
cells for subsequent activation stimuli. By way of example, the
production of cytokines, for example, pro-inflammatory cytokines,
may be modulated by the methods/uses which exploit the avian
CSF1/IL34 genes/proteins described herein or compounds which
modulate the expression or levels of CSF1/IL34 genes/proteins.
[0039] In view of the above, a fifth aspect of this invention
provides a method of modulating avian growth and/or organ
development, said method comprising the step of modulating the
expression of the avian CSF1 or IL34 genes and/or the level of CSF1
or IL34 protein.
[0040] In a sixth aspect, the present invention provides the use of
an avian CSF1 or IL34 gene and/or protein for modulating avian
growth and/or organ development.
[0041] In mammalian systems, the production of circulating
monocytes and tissue macrophages from the bone marrow is dependent
upon the activity of CSF1 and IL34. The discovery that the avian
genome also encodes CSF1 and IL34 genes, provides a means by which
avian monocyte/macrophage development may be modulated.
[0042] As such, in a seventh aspect, the present invention provides
a method of modulating the avian immune system, said method
comprising the step of modulating the expression of the avian CSF1
and/or IL34 genes and/or the level of the CSF1 and/or IL34
proteins.
[0043] In an eighth aspect, the present invention provides the use
of an avian CSF1 and/or IL34 gene and/or avian CSF1 and/or IL34
protein for modulating the avian immune system.
[0044] One of skill in this field will appreciate that by
increasing the level of avian CSF1 or IL34 gene expression, it may
be possible to increase macrophage development. Similarly, by
increasing the in vivo level of CSF1 or IL34, it may be possible to
modulate monocyte/macrophage development.
[0045] Commercial and domestic farming is reliant on effective
vaccines to ensure avian stocks, for example poultry/fowl species,
remain healthy while farmed. It is well established that the
efficacy of a vaccine can be enhanced or improved with the use of
an adjuvant. Adjuvants which can be used in combination with
vaccines for use in farming are particularly useful and in this
regard, avian CSF1 and IL34 proteins, for example the Gallus gallus
CSF1 and IL34 proteins (or fragments thereof) described herein, may
be used as vaccine adjuvants.
[0046] As such, a ninth aspect of this invention provides avian
CSF1 and IL34 proteins, or fragments thereof, for use as vaccine
adjuvants.
[0047] In a tenth aspect, the present invention provides
immunogenic compositions, potentially useful as avian vaccines,
said immunogenic compositions comprising an avian CSF1 and/or IL34
protein.
[0048] The ability of CSF1 or IL34 proteins to promote the
development of myeloid cells provides a further use for the avian
CSF1 and/or IL34 proteins described herein as agents which can be
used to induce or promote the growth of macrophages and/or other
myeloid cells in vitro. By using avian CSF1 or IL34 proteins to
facilitate the generation of populations of myeloid cells, it may
be possible to produce populations of myeloid cells for use in
research.
[0049] One of skill will appreciate that levels of gene expression
can be modulated by administering one or more copies of the gene to
a subject. By way of example, copies of the avian CSF1 and/or IL34
genes (or functional fragments thereof) may be administered to an
avian subject in the form of an expressible vector such as those
described above. In other embodiments, purified avian CSF1 and/or
IL34 proteins (perhaps a recombinant avian CSF1 or IL34 proteins)
may be administered to avians to increase the amount of circulating
CSF1 and/or IL34. In one embodiment, an avian CSF1 or IL34 protein
or gene (or functional fragment thereof) may be added or
administered directly to a particular cell type, tissue or organ.
For example, an avian CSF1 or IL34 protein or gene may be
administered directly to avian bone marrow.
[0050] It should be understood that the terms "modulate" or
"modulating" refer to an increase or decrease in the level of CSF1
or IL34 gene expression and/or CSF1 or IL34 protein levels,
relative to, for example, the level of expression or protein
observed in a normal, healthy (wild-type) avian subject. In one
embodiment, the level of CSF1 or IL34 gene expression or levels of
CSF1 or IL34 protein, may be modulated in vivo.
[0051] In other embodiments, compounds which modulate the
expression of the avian CSF1 and/or IL34 genes and/or level of
CSF1/IL34 proteins may be used to achieve any of the effects
detailed above or in any of the described methods. Such compounds
may include, for example, small organic molecules, nucleic acids
(including sense and antisense DNA/RNA sequences) and antibodies
and/or antigen binding fragments thereof. In one embodiment,
compounds capable of inhibiting lactoferrin concentration and/or
expression may include, for example, DNA or RNA oligonucleotides,
preferably antisense oligonucleotides. Oligonucleotides useful as
modulators of avian CSF1/IL34 gene and/or protein expression may be
RNA molecules known to those skilled in this field as small/short
interfering and/or silencing RNA and which will be referred to
hereinafter as siRNA. Such siRNA oligonucleotides may take the form
of native RNA duplexes or duplexes which have been modified in some
way (for example by chemical modification) to be nuclease
resistant. Additionally, or alternatively, the siRNA
oligonucleotides may take the form of short hairpin RNA (shRNA)
expression or plasmid constructs.
[0052] The skilled man will readily understand that antisense
oligonucleotides may be used to modulate (for example, inhibit,
down-regulate or substantially ablate) the expression of any given
gene. Accordingly, (antisense) oligonucleotides provided by this
invention may be designed to modulate, i.e. inhibit or neutralize,
the expression and/or function of avian CSF1/IL34 genes and/or the
protein products thereof.
[0053] By analysing native or wild-type lactoferrin sequences and
with the aid of algorithms such as BIOPREDsi, one of skill in the
art could easily determine or computationally predict nucleic acid
sequences that have an optimal knockdown effect for these genes.
Accordingly, the skilled man may generate and test an array or
library of different oligonucleotides to determine whether or not
they are capable of modulating the expression or function of avian
CSF1/IL34 lactoferrin genes and/or proteins.
[0054] The identification of genes which bind the avian CSF1R to
bring about increased myeloid cell production, proliferation and/or
differentiation, may be exploited in methods for screening avians
for individuals which exhibit increased levels of CSF1 and/or IL34
gene expression or increased levels of CSF1 and/or IL34 protein.
Avians with these phenotypes may be particularly useful in, for
example, breeding programs.
[0055] As such, an eleventh aspect the present invention provides a
method of screening avian species, particularly agriculturally
significant or important avian species (such as, for example, those
classified as poultry or fowl) for potential inclusion in breeding
programs said method comprising the steps of: [0056] a) providing a
nucleic acid sample from an avian species; and [0057] b) comparing
the level of expression of the CSF1 and/or IL34 gene(s) with a
reference nucleic acid sample or value;
[0058] wherein avian species which exhibit and increased level of
CSF1 and/or IL34 gene expression relative to a that present in a
reference nucleic acid sample or value, may be selected for
inclusion in breeding programs.
[0059] In one embodiment, the reference nucleic acid sample may be
derived from an avian individual which exhibits normal or wild type
CSF1/IL34 gene expression. In other embodiments, the reference
nucleic acid value may represent the mean, average or median level
of avian CSF1/IL34 gene expression.
[0060] In a twelfth aspect, the present invention provides a
compound capable of inhibiting CSF1/IL34 gene expression or protein
production for treating conditions, such as inflammatory diseases,
resulting from or associated with aberrant CSF1/IL34 gene/protein
expression. The present invention may also extend to uses of such
compounds for the manufacture of medicaments and methods of
treating avian diseases resulting from or associated with aberrant
CSF1/IL34 gene/protein expression. The term "compounds" may include
small organic molecules, fragments of native avian CSF1/IL34
proteins, CSF1/IL34 binding agents (for example antibodies or
antigen binding fragments thereof) or nucleic acids, for example
antisense sequences designed to inhibit the expression of avian
CSF1/IL34 genes.
[0061] The present invention will now be described in more detail
with reference to the following Figures which show:
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] FIGS. 1A and 1B: Structure-based sequence analysis of
chicken CSF1. FIG. 1A--Ribbons representation of the chicken (left)
and mouse (right) CSF1 structures. The PDB file for the chicken
CSF1 was generated using 3D-Jigsaw with the mouse CSF1 structure as
template (PDB 3ejj). The figures were rendered by PyMol in
Polyview-3D. Residues are colored according to hydrophilicity where
yellow is used for hydrophobic residues (A, C, F, G, I, L, M, P,
V), dark yellow for amphipathic residues (H, W, Y), orange for
polar residues (N, Q, S, T), red for residues charged negatively
(D, E) and brown for residues charged positively (R, K). The
cysteine forming the inter-chain disulfide bond is labelled on the
mouse structure. The loop created by the presence of additional
amino acids in the chicken sequence is marked by a black arrow. The
red arrows show non-conserved substitutions within the binding site
1 to CSF1R as described in the mouse (Chen et al. 2008). FIG.
1B--Dimer interface of chicken CSF1 (left) and SCF (right).
Residues are colored as in A. Amino acids present at the homodimer
interface are highlighted by rendering their atoms as spheres.
[0063] FIGS. 2A and 2B: Structure-based sequence analysis of the
chicken IL34. FIG. 2A--Ribbons representation of the chicken IL34
(left). Structures of the chicken CSF1 (center) and chicken SCF
(right) are shown for comparison. The PDB file for the chicken IL34
was generated using 3D-Jigsaw with the mouse CSF1 structure as
template (PDB 3ejj). The PDB file for the chicken SCF was generated
using 3D-Jigsaw with the human SCF structure as template (PDB
1exz). The figures were rendered by PyMol in Polyview-3D. Residues
are colored as in FIG. 1A. The red arrows highlight the
corresponding residues composing the receptor binding site 1 in
CSF1. FIG. 2B--Dimer interface of chicken IL34. Residues are
colored as in FIG. 1A. Amino acids present at the homodimer
interface are highlighted by rendering their atoms as spheres.
[0064] FIGS. 3A-3F: Characterization of the avian CSF1R. FIG.
3A--Ribbons representation of CSF1-binding site 1 of the zebra
finch (left) and chicken (right) CSF1R. The PDB files for the
chicken and zebra finch CSF1R were created with 3D-Jigsaw using the
mouse CSF1R structure as template (PDB 3ejj). The figures were
rendered by PyMol in Polyview-3D. Residues are colored as in FIG.
1A. Arrows point at some of the non-conserved amino acid
substitutions. FIG. 3B--Superimposition of the zebra finch (left)
and chicken (right) D1-D3 domains of CSF1R with their respective
CSF1 ligand. The two CSF1R structures are viewed from the same
angle as in FIG. 3A. The chicken CSF1 structure from FIG. 1A is
here rendered as surface in Polyview-3D. The PDB file for the zebra
finch CSF1 structure was created with 3D-Jigsaw using the mouse
CSF1 as template (PDB 3ejj) and rendered with Polyview-3D. Residues
are colored as in FIG. 1A. FIG. 3C--Pustell DNA matrix alignment of
the 5' ends of the avian CSF1R genes. The two genes align up to 2.5
kb upstream and through the first intron. FIG. 3D--Sequence
alignment of the promoter-exon 1 region of the zebra finch and
chicken CSF1R gene. Alignment was performed using MacVector.
Binding sites for PU.1, C/EBP and AML1 are identified. FIG.
3E--Alignment of the highly-conserved segment in the intron of the
zebra finch and chicken CSF1R gene. Alignment was performed as in
D. Binding sites for PU.1, C/EBP and AML1 are identified. FIG.
3F--Localization of CSF1R mRNA in chick embryo at 20HH by whole
mount In Situ. The figure shows the antisense (left) and the sense
control ISH (right).
[0065] FIGS. 4A and 4B: Expression and activity of the chicken CSF1
and IL34. FIG. 4A--Expression and secretion of chicken CSF1 and
IL34 by HEK293T transfected with pEF6-cCSF1, pEF6-cIL34 or empty
pEF6 vector. The cells were transfected using Lipofectamine and
incubated for 72 hours. The cell lysates and supernatants were run
on SDS-page gel in non-reducing and reducing conditions, and probed
with an anti-V5 tag antibody. The arrows show the double bands for
the chicken CSF1 (ca. 60 kD) and IL34 (ca. 25 kDa) in which the
upper bands are the glycosylated forms of these proteins, whereas
the lower bands constitute de non-glycosylated forms. FIG.
4B--Chicken bone marrow derived macrophages at Day 10 of
differentiation using 20% supernatant from pEF6 (left), pEF6-cCSF1
(centre) and pEF6-cIL34 (right) transfected HEK293T.
[0066] FIGS. 5A and 5B: Co-evolution of CSF1, IL34 and CSF1R. FIG.
5A--Diagram summarizing the main results of the co-evolution
analyses performed by CAPS, a PERL-based software (Fares and
McNally 2006a). The co-evolving residues between IL34 (left, in
pink) and CSF1R extracellular domain (right, in blue) are shown as
identified by CAPS 2Q (Fares and McNally 2006a). The correlation
coefficient is indicated by the color of the line between two
coevolving amino acids, and measures the correlated evolutionary
variation at these sites. The residue numbers refer to the human
sequences. FIG. 5B--Speculated IL34 binding mode of CSF1R based on
the co-evolution analysis. The ribbons representation of the
chicken CSF1R D1-D5 was created by superimposing the chicken CSF1R
D1-D3 model produced for FIG. 3 and a chicken CSF1R D4-D5 model
made using the human KIT D1-D5 (PDB 2e9w) as template in 3D-Jigsaw.
The chicken IL34 model is the same as in FIG. 2 in a slightly
different angle. All the models were rendered in Polyview-3D. The
residues are colored as in FIG. 1A with the co-evolving homologous
residues in chicken highlighted in blue.
[0067] FIG. 6: Bis-Tris NuPAGE analysis of IPTG induced protein
expression
[0068] FIG. 7: SDS-PAGE analysis of the concentrated monomeric
chicken CSF-1 protein
[0069] FIG. 8: Dose-Reponses data for all three CSF1 preparation on
the parental BaF/3 cell line.
[0070] FIG. 9: Dose-Reponses data for all three CSF1 preparation on
chicken CSF1R-expressing BaF/3 cells.
[0071] FIG. 10: Dose-Reponses data for all three CSF1 preparation
on Porcine CSF1R-expressing BaF/3 cells.
DETAILED DESCRIPTION
Methods
Bioinformatic Analysis
[0072] Sequences were identified using the databases at NCBI and
the genome resources from the University of Santa Cruz and
Ensemble. The translation of the zebra finch CSF1R gene was
predicted using the GeneWise program and the chicken IL34 EST was
analysed using ESTscan
Cloning of Chicken and Zebra Finch cDNA Genes
[0073] RNA from chicken stage 20 embryo and zebra finch brain was
extracted using TRIzol reagent, as described by the manufacturers
(Invitrogen, Paisley, UK). cDNAs were cloned via RT-PCR using
Superscript III reverse transcriptase and the TOPO TA cloning kit
for sequencing (Invitrogen, Paisley, UK). 5' Rapid amplification of
cDNA ends (5' RACE) of chicken and zebra finch CSF1, and zebra
finch IL34, was carried out using the First Choice RLM-RACE Kit
(Ambion, Warrington, UK). PCR products were cloned using the TOPO
TA cloning kit for sequencing (Invitrogen, Paisley, UK). The 3' end
of chicken CSF1 was cloned using a modified 3' Rapid amplification
of cDNA ends (3' RACE) technique, 3' RACE LaNe (Park 2004).
Isolation of Chicken BACs Containing CSF1
[0074] A chicken probe for CSF1 was prepared using the Prime-a-gene
kit (Promega, Southampton, UK) and hybridized to the chicken
CHORI-261 BAC library overnight at 65.degree. C. in 10% PEG8000; 7%
SDS; 1.5.times.SSC. Filters were then washed twice in 2.times.SSC;
0.1% SDS and once in 0.5.times.SSC; 0.1% SDS at 65.degree. C. and
exposed to autoradiographic film for 1 week at room temperature.
Six positive clones were identified: 44-116, 68-D10, 22-M13,
171-K3, 171-N10 and 172-O23. 44-116, 68-D10 and 171-N10 were
confirmed by PCR. These clones were all end-sequenced and shown by
Blat to map to Chr26.
Sequence Analysis
[0075] Clones were sequenced using BigDye Terminator v3.1 Cycle
sequencing kit (Applied Biosystems, Foster City, Calif., USA) on an
ABI 3730x1 sequencer.
[0076] Genetic Mapping of Chicken CSF1
[0077] A 437 by genomic fragment of chicken CSF1 was amplified by
the primers: ckcsfex4-for1: GCGACTCTGTCTGCTACGTG (SEQ ID NO: 8) and
ckcsfex5-rev1: CGAAGGTCTCCTTGTTCTGC (SEQ ID NO: 9). Sequencing of
the parental DNA from the East Lansing reference population
(Crittenden et al. 1993) identified a SNP in exon4 (G/A in Red
Jungle Fowl male; A/A in White Leghorn female) and sequencing of 52
backcross DNAs from confirmed CSF1 as mapping to Chr26. Linkage
analysis was carried out using the Map Manager program (Manly and
Olsen, 1999).
Phylogenetic Analyses
[0078] Amino acid sequences of annotated CSF1, IL34 and CSF1R genes
from various taxa were aligned using the ClustalW software
(Thompson et al. 1994) (Gonnet protein weight matrix, no ends gaps
inactivated, default parameters). The secondary structures shown in
the structure-based alignments were predicted using PSIPRED (Jones
1999), and the alignments performed by Domain Fishing
(Contreras-Moreira and Bates 2002).
Whole Mount In Situ Hybridization
[0079] Fertilized White Leghorn eggs were collected weekly and
incubated at 38.degree. C. for between 3-6 days of development.
Embryos were dissected into cold DEPC-PBS, staged as per (Hamburger
and Hamilton 1992), and fixed immediately in 4% PFA/DEPC-PBS
overnight, dehydrated into 100% methanol through graded
methanol/PBS steps and stored at -20.degree. C. Whole-mount ISH on
embryos were carried out as per (Nieto et al. 1996). The CSF1R
probe was made using the Ark-Genomics (Roslin, UK) clone 654 for
template.
Chicken CSF1 and IL34 Expression
[0080] HEK293T cells (ATCC) and were cultured in DMEM (Sigma)
supplemented with 10% heat inactivated (HI)-FCS, 2 mM L-glutamine,
0.1 mM non-essential amino acids and antibiotics (100 ug/ml
penicillin, 100 ug/ml streptomycin). One day before transfection,
8.times.10.sup.5 HEK293T cells were plated in 2 ml growth medium
without antibiotics in a 6-well plate. The cells were transfected
with Lipofectamoine 2000 as per product instructions. Cells were
then incubated at 37.degree. C. in a CO.sub.2 incubator for 24 hr
and transferred into 25 cm.sup.2 dishes each containing 7 ml of
growth medium (without antibiotics) for another 48 hr prior to
harvesting the supernatant and lysing the cells in 2% SDS-10 mM
Tris buffer. The cell extracts and supernatants were then mixed
with Laemli buffer with or without DTT (5 mM final) or
B-mercaptoethanol (5% final), run on a 4-12% gradient SDS-PAGE gel
and transferred on PVDF membrane as per Bio-Rad apparatus
instructions. The membrane was blotted using a mouse anti-v5 tag
antibody (AbD Serotec) and an anti-mouse IgG HRP-conjugated (Cell
Signaling Technology).
Bone Marrow Differentiation
[0081] Chicken bone marrow cells were obtained by flushing the
marrow from 2 femurs and 2 tibias with PBS using a syringe and a
blunt needle. For each condition, 1/250 of total cells was pelleted
and resuspended in 4 ml of complete RPMI (supplemented with 10%
heat inactivated (HI)-FCS, 2 mM L-glutamine, 100 ug/ml penicillin,
100 ug/ml streptomycin) containing 20% supernatant from empty
pEF6-, pEF6-cCSF1- or pEF6-cIL34-transfected HEK293T. Cells were
plated in 60 mm Bacteriological plates and incubated at 37.degree.
C. in a CO.sub.2 incubator for 12 days.
Intra- and Inter-Molecular Co-Evolution Analysis
[0082] To identify co-evolutionary patterns we used the parametric
method based on correlated evolutionary patterns among amino acid
sites previously published (Fares and Travers 2006b). To perform
the analysis we used the software implementing this method CAPS
version 1.0 (Fares and McNally 2006b). This method has proved to be
successful in yielding meaningful results in several case studies,
including those aimed at identifying co-evolution of membrane
proteins (Fuchs et al. 2007), HIV gp120 and gp41 proteins (Travers
et al. 2007) as well as Hsp70-Hop-Hsp90 system (Travers and Fares
2007). To estimate the probabilities and significance of the
correlated evolutionary patterns among amino acid sites we used a
large number of random samplings (1 million and 10 million random
samples) and a small alpha value (0.001) to minimize false positive
rate (type I error). CAPS also implements the step-down
permutational procedure as described previously (Westfall and Young
1993; Travers and Fares 2007) to correct for multiple testing. The
scores for the amino acid substitutions were obtained using the
appropriate blocks substitution matrix (BLOSUM80) (Henikoff and
Henikoff 1992) depending on the similarity of our protein
sequences. All amino acid sites reported in the co-evolutionary
analyses present the positions in the protein from the reference
sequence (human).
Visualization of Co-Evolutionary Networks
[0083] We used the software Cytoscape (version 2.6.1) (Shannon et
al. 2003) to visualize the co-evolutionary networks identified by
CAPS. Cytoscape was originally designed to visualize bimolecular
interaction networks. This tool however can be used to visualize
any data that describes interactions between objects. CAPS can
produce four files containing information of co-evolutionary
networks and compensatory mutations. We used this program to
generate the networks of correlation between co-evolving amino
acids and used the correlation coefficients generated in CAPS to
determine the coloring patterns of the linking lines between nodes
(amino acid residues).
Cloning of Chicken CSF-1 Gene
[0084] The sequence corresponding to the active fragment of chicken
CSF-1 (NSYCQQIITERHLDHLQELADTQMQQPGTVSFRFTSKMRLSDSVCYVKAAFPLLGTI
LNRTTFKENSTNANKMKTVRKMYENIDENVDPCIRDEDDKEHALSEMCFEEFTTSPY
EMLVLVRQFFQDIKQLLQNKETFEKDCSQVYRSACAGPRQHSSSP, SEQ ID NO: 10) was
codon optimized for expression in E. coli and synthesized by Blue
Heron Biotechnologies (WA, USA). The sequence was engineered with a
BspHI restriction site at the 5' end and an EcoRI restriction site
at the 3' end and cloned into the expression plasmid pET-28(b)
using the complimentary restriction sites NcoI and EcoRI. The
resulting plasmid, pTLW54, was transformed into MAX Efficiency.RTM.
DH5.alpha..TM. Chemically Competent E. coli according to the
manufacturer's protocol (Invitrogen, CA, USA). A kanamycin
resistant transformant was selected and the plasmid sequenced to
verify the error-free ORF. The pTLW54 plasmid was isolated via
QIAprep.RTM. spin miniprep kit (Qiagen, CA, USA) according to the
manufacturer's recommendations and transformed into One Shot.RTM.
BL21 Star.TM. Chemically Competent E. coli (Invitrogen, CA, USA).
The chicken CSF-1 gene within pTLW54 was again sequenced to verify
error-free ORF.
Expression of Chicken CSF-1 Protein
[0085] An overnight LB/Kan.sup.50 broth of pTLW54/One Shot.RTM.
BL21 Star.TM. E. coli incubating at 37.degree. C. with 225 rpm
shaking was refreshed 1:10 into 1 L of LB/Kan.sup.50 broth. The
refreshed culture was incubated with 225 rpm shaking at 37.degree.
C. for two hours to ensure mid-log phase growth. Protein expression
was induced with 1 mM IPTG, final concentration, with incubation
conditions continued at 37.degree. C. and 225 rpm shaking. After 2
hours induction, the culture was centrifuged and the E. coli pellet
was stored at -80.degree. C. Prior to centrifugation, aliquots were
analyzed for expression of protein compared to a non-induced
control. Soluble and non-soluble protein fractions were analyzed on
a 4-12% Bis-Tris NuPAGE gel run in MES buffer. As shown in the FIG.
6, the induced culture produced a band of approximately 18.7 kDa in
the non-soluble protein as expected.
Purification and Processing of Chicken CSF-1 Protein
[0086] Frozen cell pellets were broken and inclusion bodies were
washed to near homogeneity. Monomeric chicken CSF-1 protein was
purified using a 2.6.times.60 cm Superose 12 size exclusion
chromatography column (SEC) in a 50 mM Tris, pH 8.5, 5 mm EDTA, 7M
guanidine. The eluted chicken CSF-1 was diluted 10-fold in the 7M
guanidine buffer and allowed to refold via sequential dialysis by
addition of 50 mM Tris, pH 8.5, 100 mM NaCl, 5 mM EDTA, 1 mM
oxidized glutathione, 2 mM reduced glutathione buffer through 8
steps resulting in a final guanidine concentration of 0.15 M. The
refolded chicken CSF-1 was concentrated and the monomeric species
was purified using a 1.times.30 cm Superose SEC column run in PBS.
The monomeric protein was concentrated to 0.58 mg/ml and analyzed
by 16% SDS-PAGE with and without BME as shown in the FIG. 7.
Aliquots of purified chicken CSF-1 were stored at -80.degree.
C.
Results
Identification of Avian CSF1 Genes
[0087] There is currently no annotated CSF1 gene in the chicken
genome, but the region containing the mouse CSF1 gene displayed
synteny with the chicken suggesting there was a gap in the chicken
genome assembly. Based upon privileged access to the zebra finch
genomic sequence, it was possible to identify a clear CSF1 ortholog
(starts in exon3) [Chr26:24187-27419, July 2008 assembly]
(GQ249405). This, in turn, led to the identification of a partial
CSF1 sequence in the chicken EST collection at the Roslin
Institute. A complete ORF was obtained by 5' and 3' RACE to
determine the full CDS. CSF1--containing BACs were also identified
and end-sequenced to confirm mapping to Chr.26. The CSF1 locus is
indeed predicted to be in a gap in the chicken genome assembly,
since in the zebra finch the flanking gene order is the same as in
mammals. The newly-identified avian CSF1 genes each contain 8
exons. The zebra finch gene encodes a protein of 489 amino acids.
Two transcripts have been identified in the chicken--one encoding a
protein of 490 amino acids, and the other comprising 270 amino
acids (GQ249403 and GQ249404). The shorter transcript is missing a
substantial part of Exon 6. In mammals, this exon encodes a large
domain that contains a proteolytic cleavage site which permits the
release of CSF1 from a membrane-anchored precursor. The shorter
transcript would encode the membrane-anchored cell surface form of
CSF1, which cannot be cleaved. The exon 6 of chicken CSF1 also
contains the unique glycosaminoglycan (chondroitin sulfate)
addition site (SGXG/A, SEQ ID NO: 11) found in the mammalian genes.
Hence, the basic biology of CSF1, involving secreted and
membrane-anchored forms with variable post-translational
modification and distinct functions (Dai et al. 2004; Jang et al.
2006; Nandi et al. 2006) appears to be conserved in the
chicken.
Conserved Structure of Avian CSF1
[0088] In order to assess whether the avian CSF1 sequences
identified are functional orthologs of mammalian CSF1, a multiple
alignment of deduced amino acid sequences across species was
performed using the ClustalW software (Thompson et al. 1994) (Table
1). The six cysteine residues responsible for the three
intra-molecular disulfide bonds (Pandit et al. 1992) are conserved
in birds, but the cysteine forming the inter-chain disulfide bond
that is located at the dimer interface and conserved through all
species including zebrafish and gold fish (Hanington et al. 2007)
is not conserved in birds. The alignment of the cysteines and
predicted helices highlight the contact residues for CSF1 bound to
CSF1R deduced from the co-crystal in mouse (Chen et al. 2008), some
of which are clearly divergent in birds. In particular, the Asp91
and 94, Gln113, Glu114, and Asn117 (position numbers referring to
the mouse sequence in Table 1) are not conserved or have
semi-conservative substitutions. Immediately downstream of these
binding sites, the bird sequences have additional amino acids that
are not present in the mammalian sequences. This alignment also
highlights the substitution of R111 in mouse, with Q in human,
which could explain why the human ligand works on mouse cells, but
not vice versa (Bonifer and Hume 2008).
[0089] To verify avian CSF1 structure predictions, 3D-models in PDB
format were generated with 3D-Jigsaw using structure-based
alignments (performed by Domain Fishing) (Bates et al. 2001). The
PDB files obtained were viewed in FirstGlance in Jmol and the
models rendered by PyMol using Polyview-3D (Porollo and Meller
2007). The avian CSF1 are predicted to have the same four-helix
bundle structure as the well described mammalian CSF1 (Pandit et
al. 1992). In FIG. 1A, the chicken CSF1 model is compared with the
published mouse structure (Chen et al. 2008). Although the overall
topology is conserved from mammals through birds, all the
differences found in the sequence alignment translate into
structural changes. Hence, the CSF1R-binding site 1 of chicken CSF1
comprises different charges from the mouse binding site 1 as the
non-conserved amino acid substitutions are precisely positioned to
contact with the receptor (red arrows). Moreover, the extra
residues found in the chicken sequence are predicted to create a
protuberance making the positively charged Arg122 stick out between
the binding sites 1 and 2 (black arrow). As previously mentioned,
the cysteine forming the inter-chain disulfide bond in mammals
(labelled Cys on the mouse structure) is absent from the chicken
CSF1, resulting in non-covalently associated homodimers. This is
also the case for the closely related Stem cell factor (SCF) in
mammals as well as in birds (Arakawa et al. 1991). These proteins
can form stable dimers and induce receptor dimerization because of
the large contact surface area between the monodimers (Jiang et al.
2000). Interestingly, the contact surface between the chicken CSF1
dimers is very similar to that of chicken SCF. They both contain
exposed hydrophobic residues, as shown in FIG. 1B where the amino
acids present on the dimer interface were rendered as
spacefill.
Identification of Avian IL34 Genes
[0090] There is an obvious chicken ortholog of human IL34 in the
chicken genome, and we have identified a chicken cDNA within the
Ark-Genomics (Roslin, UK) collection which maps to chromosome 11
and contains the full-length IL34 CDS (Genbank accession no.
BX931154). This chicken sequence allowed for the identification of
the orthologous zebra finch gene within the genome sequence [Chr11:
5,575,502-5,577,560; July 2008 assembly] (GQ249406). The avian
genes each contain 6 exons, the chicken gene encoding a protein of
178 amino acids, and the zebra finch gene encoding 180 amino
acids.
Conserved Structure of IL34
[0091] At the amino acid sequence level, IL34 is considerably
better conserved across species than CSF1 (Table 2). Although the
paper describing human IL34 claimed that it was structurally novel
(Lin et al. 2008), our own analysis suggests that IL34 is a
four-helix bundle protein just like the well-described CSF1 (Pandit
et al. 1992; Taylor et al. 1994; Chen et al. 2008). It is true that
IL34 and CSF1 can be aligned with each other with only weak primary
homology but they have a very similar predicted topology. A
3D-model of the chicken IL34 was generated with 3D-Jigsaw using
structure-based alignments (performed by Domain Fishing) (Bates et
al. 2001). The resulting PDB file was viewed in FirstGlance in Jmol
and the model rendered by PyMol using Polyview-3D (Porollo and
Meller 2007) (FIG. 2A). For structural comparison, a model for the
chicken SCF was created following the same procedure, and the
chicken CSF1 model shown in FIG. 1 was also included in the figure.
This structural analysis of the derived protein sequence predicts a
molecule that lacks all the seven strategically positioned
cysteines. IL34 contains some generally conserved cysteine
residues, but these are not positioned and matched together in
order to form intra-chain disfulfide bonds. The cysteine required
for the formation of an inter-chain disulphide bridge is not found
on the dimer interface as in mammalian CSF1, but that region
contains exposed hydrophobic residues (FIG. 2B). The corresponding
amino acids that constitute the receptor-binding site 1 on CSF1 are
pointed out by red arrows. These residues are totally different
from those in IL34, immediately suggesting an alternate binding
mechanism from that of CSF1 protein.
Identification of the Zebra Finch CSF1R Gene
[0092] In the chicken genome on chromosome 13, there is an
annotated CSF1R gene (Ensembl ID: ENSGALG00000005725), occupying
the same position as in mammals, immediately 3' of the
closely-related PDGFRB gene (Ensembl ID: ENSGALG00000021313). The
availability of the chicken sequence allowed us to identify the
orthologous sequence in the zebra finch genome [Chr13:
6,954,381-6,972,446; July 2008 assembly] (GQ249407). This gene
contains 21 exons like the chicken CSF1R, and codes for a protein
of 967 amino acids, which again is the same length as the chicken
CSF1R.
Evolutionary Conservation of CSF1R
[0093] A new multiple alignment of amino acid sequences across
species was performed using the ClustalW software to examine the
homology between the bird CSF1R and the mammalian orthologs (Table
3). As expected, the intracellular tyrosine kinase domain (aa 540
to 977) of the receptor is extremely conserved through all species,
including birds. Amongst the residues that are not conserved
however, is the cysteine 665 (indicated by an arrow in table 3)
which is substituted for an arginine only in birds. This
substitution may underlie our finding that chicken CSF1-induced
macrophage growth is not blocked by a kinase inhibitor, GW580 (V
Garceau, unpublished) that inhibits mammalian CSF1R activity
(Irvine et al. 2006). Most of the residues composing the
CSF1-binding sites 1 and 2 (labeled respectively with "+" and
".degree." symbols in table 3) deduced from the mouse CSF1R/CSF1
co-crystal are not conserved between the two birds. Some of the
amino acid substitutions observed between the two birds are
illustrated in FIG. 3A, where 3D-models of the CSF1-binding site 1
on the D2 domain of the zebra finch (left) and the chicken (right)
CSF1R are presented. As with the ligands, the models were created
using 3D-Jigsaw and Polyview-3D. A more general view of the D1-D3
domains of these same receptors with a superimposed structure of
their respective CSF1 molecule is presented in FIG. 3B. The
receptors are shown from the same angle as in FIG. 3A, and the
chicken CSF1 structure is the same model as in FIG. 1A rendered as
surface instead of cartoon, with the dimer interface labeled for
orientation. The zebra finch CSF1 structure was produced using the
same settings, and the superimposition angle is based on the
co-structure of the mouse CSF1:CSF1R complex (Chen et al. 2008). In
this figure, the amino acid substitutions in the zebra finch (left)
and chicken (right) receptors are put in context with those present
in their respective CSF1 ligand. This high level of divergence is
not surprising since zebra finches and chickens are not part of the
same taxonomic order. As with primate and rodent CSF1, we predict
that the zebra finch CSF1 will not activate the chicken receptor,
or vice versa, due to the substantial changes in charge density in
the binding sites.
Transcriptional Regulation of Avian CSF1R
[0094] The mammalian CSF1R loci contain a conserved
macrophage-specific promoter region, and a remarkably conserved
enhancer (FIRE) in the first intron that is required for
macrophage-specific expression of a transgene (Sasmono et al.
2003). These elements are not evidently conserved in birds.
However, the transcription factors required for macrophage gene
expression, such as PU.1, have clear chicken orthologs. To assess
the likelihood of CSF-1R being a macrophage regulator in birds, we
first assessed the transcriptional regulation of the chicken and
zebrafinch genes. Passeriformes and galliformes are separated by
around 100M years of evolution, about the same as rodents and
humans. Because of this distance, evolutionary conserved,
non-coding regions provide strong indications of the location of
functional promoters and enhancers; in mammals the FIRE (fms
intronic regulatory element) is more conserved than any of the
exons (Himes et al. 2001). The intron-exon structure of avian CSF1R
is the same as in mammals, with the ATG start codon located in the
first exon. Like the mammalian CSF1R promoters, the two avian CSF1R
proximal promoters are not highly conserved, but both are
purine-rich and TATA-less. In both bird species, there are two very
highly conserved regions, upstream and downstream of the promoter.
This is shown in the Pustell DNA matrix alignment of FIG. 3C. The
downstream element is in the same relative location within the
first intron as the mammalian FIRE (Himes et al. 2001). The
candidate avian FIRE sequence cannot be aligned with the mammalian
FIRE, but contains the same basic elements. These regions contain
multiple repeats of consensus sequences in common with mammalian
CSF1R and FIRE, including candidate binding sites for PU.1 and
other Ets factors, AP1, C/EBP, Sp1 and AML1 (FIGS. 3D and 3E). Each
of these transcription factors is expressed in haematopoietic cells
in chickens (Faust et al. 1999; Bakri et al. 2005). These findings
suggest that avian CSF1R is controlled in basically the same manner
as in mammals, despite the reassortment of the cis-acting
individual elements.
Expression Pattern of CSF1R in Chick Embryo
[0095] The analysis of conserved regions identifies candidate
enhancers of the chicken CSF1R locus, and suggests that the gene is
likely to be expressed specifically in macrophages. To confirm that
prediction, we carried out whole mount in situ hybridization of
chicken embryos at 20HH or 3.5 days of development, the wing bud
stage (FIG. 3F). This stage corresponds to 11.5 dpc in the mouse
embryo. As in the mouse (Lichanska et al. 1999), and consistent
with macrophage distributions in Xenopus (Tomlinson et al. 2008),
the chicken csflr mRNA is expressed in a speckled pattern all over
the embryo, consistent with restriction to the numerous macrophages
in every organ of the body (left panel).
Activation of the Chicken CSF1R by CSF1 and IL34
[0096] The expression of CSF1R on the cell surface is amongst the
earliest events in macrophage lineage commitment (Tagoh et al.
2002), and CSF1 is commonly used to grow pure populations of
macrophages from mouse bone marrow (Bonifer and Hume 2008). Both
ligands were therefore tested for differentiation of chicken bone
marrow cells. The chicken CSF1 (first six exons) and IL34 genes
were cloned in the pEF6 vector, providing them with a C-terminal
tag for detection, and transfected into HEK293T cells. The
expressed proteins within the cell and supernatant were detected by
Western blotting (FIG. 4). In the case of CSF1, three apparent
molecular entities are seen in the non-reduced supernatant, and the
lowest apparent MR (ca. 60 kD) is mostly present in the cells;
suggestive that the secreted protein is processed in the ER and
glycosylated, maybe even as a proteoglycan. In the case of IL34,
the protein was detected in the cells and supernatant as a doublet,
with the higher apparent MR being more abundant than the lower MR,
suggesting that IL34 is glycosylated in this system. The amount of
epitope-tagged cIL34 detected in the supernatant was considerably
less than in the cell lysates. This might be due to some
proteolytic cleavage of the C-terminus by trypsin-like proteases in
the secretory pathway or medium as a string of basic amino acids
are found at the C-terminal of IL34, just upstream of the v5 tag.
The supernatant from the transfected HEK293T cells, or cells
transfected with the vector only, was added to bone marrow cells
obtained by flushing the marrow from a chicken femur, and the cells
were incubated for ten days. Around Day 3, cells growing in the
presence of chicken CSF1 or IL34 became adherent, and by Day 12,
the dishes were confluent. No bone marrow cells survived in the
control dish containing supernatant from empty pEF6-transfected
HEK. Hence, both CSF1 and IL34 offer the possibility of growing
macrophages from chicken marrow for functional studies; bone
marrow-derived macrophages have been a mainstay of macrophage
functional studies in the mouse (Bonifer and Hume 2008). Together,
the data indicate that the function of CSF1R and its two ligands is
conserved in birds.
Co-Evolution of CSF1R, CSF1 and IL34
[0097] The extracellular domain of CSF1R is quite divergent among
species, as is CSF1. Even between mice and rats, IL34 is
considerably better conserved, and yet activates the same receptor
as CSF1. The divergence of CSF1/CSF1R could be related to the fact
that (a) CSF1 is massively inducible in the circulation in response
to innate immune stimuli such as LPS, and (b) the CSF1R
extracellular domain is cleaved from the cell surface in cells
responding to a range of TLR agonist, through the actions of
ADAM14/TACE (Sester et al. 1999; Rovida et al. 2001). So, one might
argue that CSF1 is required for effective innate immune responses
and is therefore under immune selection. Indeed, Epstein-Barr virus
encodes a soluble CSF1R antagonist, BARF1 (Strockbine et al. 1998).
This raises the interesting evolutionary question of exactly how
two ligands could evolve with a single receptor. In theory, and
provided the overall structure is conserved, alterations in contact
residues in the ligand should be compensated by alterations in the
receptor to preserve binding affinity, and within a sufficiently
large sample, there should be a correlation matrix between amino
acids on the partners. Incorporating the alignments presented in
Tables 1, 2 and 3, and published PDB structures for CSF1/CSF1R it
was possible to distinguish functional co-evolution from
phylogenetic or random co-variation in order to calculate a
correlation coefficient (Fares and Travers 2006a). The CAPS
software was used to identify amino acid sites having a strong
correlation coefficient (Fares and McNally 2006a), and the networks
of these co-evolving residues arc shown in FIG. 5A. The strength of
the correlation coefficient for specific site pairs is indicated by
the color of the line connecting them, and the amino acid positions
numbering follows the human sequences as reference. Unexpectedly,
the only significant correlations were found between CSF1R and the
more conserved of the two ligands, IL34. Moreover, no sign of
intra-protein co-evolution was found in any of the three
proteins.
IL34 Binding Mode of CSF1R
[0098] None of the co-evolving amino acid positions identified by
CAPS in CSF1R are located within the CSF1-binding sites which are
in D2 and D3 domains (Chen et al. 2008). Instead, they are
concentrated at the junction between D3 and D4. In a similar
manner, the co-evolving residue positions in IL34 are located
outside the corresponding CSF1R-binding site of CSF1. All the
co-evolving residues appearing in the networks of FIG. 5A were
mapped on the chicken CSF1R and IL34 structures (FIG. 5B). The
chicken CSF1R D1-D5 structure was generated as a chimera of two PDB
files generated by 3D-Jigsaw for the chicken receptor: D1-D3 using
the mouse CSF1R structure as template (3ejj), and D1-D5 using the
human KIT structure as template (2e9w). The two models were then
superimposed to create the chicken CSF1R D1-D5 structure. The
chicken IL34 model is the same one as in FIG. 2, viewed in a
slightly different angle. The corresponding co-evolving residue
positions in chicken were deduced from the alignment in Table 2,
then highlighted in blue using Polyview-3D. The co-evolution of
specific sites on CSF1R and IL34 can be interpreted as a possible
binding mode between this uncharacterized new ligand and the
receptor. Hence, we can speculate that IL34:CSF1R binding site
interface consists of the CD loop of IL34 and the region around the
D3-D4 junction of CSF1R.
Assessment of Bioactivity Via BaF/3 Cell Based Assay
[0099] Recombinant chicken CSF1 has demonstrable specific
bioactivity as demonstrated when titrated on to factor-dependent
parental BaF/3 cells and BaF/3 cells expressing chicken CSF1R or
porcine CSF1R. Parental BaF/3 cells, chicken CSF1R expressing BaF/3
cells or porcine CSF1R expressing BaF/3 cells that had been
cultured in RPMI 1640 media+10% HI FBS+GlutaMax+pen/strep (20 U/mL
and 20 micrograms/mL) and 10 ng/mL recombinant murine IL-3 were
collected, washed and plated in 96-well microtitre plates at 20K
cells per well and cultured overnight. The following day,
recombinant chicken CSF1 was titrated against all three cell lines.
As controls, recombinant porcine CSF1 and recombinant human CSF1
(Sigma 6518) were also titrated against all three cell lines. Cell
assays were then incubated for a further 48 hrs afterwhich cell
survival/proliferation was assessed using the TireGlo assay system
(Cell TiterGlo; Promega G7571). None of the CSF1 preparations
promoted survival or proliferation in the BaF/3 parental line (FIG.
8). Whilst porcine and human CSF1 preparations did not promote
survival/proliferation of BaF/3 cells expressing the chicken CSF1R,
the chicken CSF1 preparation did show bioactivity (EC50=ca. 20
ng/mL) through the chicken CSF1R as demonstrated by robust survival
and proliferation when titrated onto the chicken CSF1R expressing
BaF/3 line (FIG. 9). Both Porcine and human CSF1 preparation were
bioactive as they had equimolar bioactivity (EC50=30 ng/mL) on the
porcine CSF1R-expressing BaF/3 cell line as demonstrated in FIG.
10.
DISCUSSION
[0100] The structural basis of the interaction between CSF-1, IL34
and CSF-1R presents a scientifically interesting phenomenon from an
evolutionary point of view. Current genomics efforts now provide
sequences from more than fifteen species for both ligands and the
receptor. From these sequences, it is apparent that the interaction
is conserved evolutionarily back as far as fish. By aligning the
sequences, and examining the tolerance of different residues at
significant positions, it is possible to identify particular amino
acids in the receptor that vary in conjunction with a given residue
of CSF-1 or IL34. The ability of CSF-1 from one species to induce
growth and survival of macrophages (or cells transfected with the
receptor) from another species adds weight to predictions based on
evolutionary conservation. For instance, mouse CSF-1 cannot bind
human CSF-1R, yet human CSF-1 can bind and activate mouse CSF-1R
(Koths 1997). In fact, human CSF-1 can activate CSF-1R from all
species for which it has been tested (human, mouse, feline, sheep
and dog), whereas mouse CSF-1 can activate all non-primate CSF-1R
tested (mouse, feline, sheep and pig), but not human CSF-1R
(Stanley and Guilbert 1981; Woolford et al. 1988; Tamura et al.
1990; Francey et al. 1992; Ramsoondar et al. 1993; Yoshihara et al.
1998; Abrams et al. 2003). The only contact amino acid that is not
conserved in mammals is mouse R111, which is Q in humans, and
varies in other species. Bovine CSF-1 causes growth of murine bone
marrow macrophages, presumably through activation of murine CSF-1R
(Yoshihara et al. 1998). Chicken (or at last M-CSF bioactivity in
chicken cell conditioned medium) and feline CSF-1, conversely, are
unable to activate the human and mouse CSF-1R, and are restricted
to activating the receptor of their own species (Tamura et al.
1990; Tamura et al. 1991).
[0101] Recent studies identified the CSF1 genes of several fish
species, and provided evidence of primitive duplications of the
gene in these species (Wang et al. 2008). All of the piscine CSF1
genes had almost complete divergence of the mammalian contact
residues implied from the mouse CSF1/CSF1R co-crystal structure. In
the current study, we have identified and expressed the chicken
CSF1 and IL34 genes, provided evidence that CSF1R is expressed in
chicken macrophages as it is in mammals (and most likely controlled
in a similar manner (FIGS. 3C-3F), and that recombinant factors can
produce pure macrophage cultures from bone marrow precursors.
Hence, the biology of the CSF1/IL34/CSF1R triad is also conserved
in another class of vertebrates, the birds. Molecular modeling
suggests that CSF1 has a conserved structure in birds and mammals,
and in contrast the published study (Lin et al. 2008) suggests that
IL34 also shares that topology characterized by a four-helix bundle
(Pandit et al. 1992). Although the fact that IL34 lacks all the
cysteines forming the distinctive intra-chain disulfide bonds in
CSF1, other growth factors such as SCF, GM-CSF and GH all have only
one or two intra-chain bonds, and yet have that same four-helix
topology (Pandit et al. 1992).
[0102] It was already known that IL34 binds CSF1R (Lin et al.
2008), and the finding of a structure similar to that of CSF1 could
have lead to the conclusion that they were both sharing the same
binding sites on CSF1R. This is not compatible with the sequence of
IL34, wherein the contact points identified from the CSF1/CSF1R
co-crystal structure are completely variant. The co-evolution study
performed here revealed a new perspective. When identified on the
3D structure of the proteins, the co-evolving residue positions
uncovered by CAPS are grouped together in distinctive regions. On
IL34, most of them are located at the end distal to the dimer
interface, in particular on a flexible loop between helix C and
helix D. None of the co-evolving residues are situated in the
corresponding binding site on CSF1. On CSF1R, the majority of them
are positioned at the junction of D3 and D4. If brought together,
these two binding sites would naturally fit with each other.
Moreover, it is known that the CSF1R D4 lacks the characteristic
disulfide bond of Ig-like domains that connects the beta-sheets,
and is likely to have greater flexibility (Blechman et al. 1995).
Even the recently published model for CSF1R dimerization and
activation could accommodate such an alternative binding mode for
IL34 (Chen et al. 2008). Interestingly, this binding mode is
reminiscent of the binding of other 4-helix bundle factors (GH,
GM-CSF, EPO) to their receptors, involving sites between helices,
and binding within an intra-domain cleft that is rather closer to
the plasma membrane than the domain D2/3 cleft of CSF1R. Activation
models of CSF1/CSF1R suggest that dimerization permits interactions
between the two domain D4s, leading to a conformational change that
generates signaling (Chen et al. 2008). Could IL34 thereby generate
a distinct signal? In the case of the GHR, distinct mutations in
the extracellular domain that alter conformation can lead to
selective loss of particular signaling pathways in transfected
FDCP1 cells (Rowlinson et al. 2008). The extracellular domain of
CSF1R linked to the intracellular domain of GHR can provide a
CSF1-dependent growth-promoting signal to the same factor-dependent
cells (M J Waters, D A Hume, unpublished). So, it is conceivable
that the two ligands could signal through the same receptor to
generate signals that only partly overlap.
[0103] The different binding mode of CSF1 and IL34 is implied by
the fact that, even though changes of charge in CSF1 binding sites
are matched by changes of opposite charges in the receptor binding
sites, there is no significant correlation coefficient between
them. The weak binding of CSF1 to CSF1R is based on salt bridges,
and simply requires the presence of opposite charges at the site to
occur. This does not have to involve a strict co-evolution of
matching amino acids. The binding of IL34 to CSF1R, however, could
be based on hydrogen bonds necessitating perfectly complementarity,
thus co-evolving, residues.
[0104] In addition to suggesting alternative binding sites for IL34
and CSF1R, the co-evolution study gives us a hint about a
functional difference between CSF1 and IL34. Indeed, the fact that
a correlated evolutionary co-variation with CSF1R can only be
detected for IL34 means that the latter is subjected to stronger
selective constraints than CSF1. In other words, a change in the
genetic composition of IL34 would necessarily involve a reciprocal
evolutionary change in the receptor as the consequences of any loss
of activity would be more dramatic than a similar loss for CSF1.
Furthermore, it also suggests that CSF1 is free to evolve more
quickly than IL34, and without the receptor coevolving with it.
These interpretations, taken together with the better conservation
of IL34 and the rather different expression pattern of IL34
compared to CSF1 suggest that IL34 might perform a more trophic
role.
[0105] Macrophages have many apparent roles in embryonic
development (Lichanska and Hume 2000; Rae et al. 2007) but studies
of their function in mammals have been constrained by the
inaccessibility of the embryo, and the fact that CSF1 is produced
by the mother and transmitted across the placenta. We have now
identified the key regulators that are likely to control avian
myelopoiesis and will be able to take advantage of the
accessibility of chicken development in ovo to manipulate
expression and function of these genes.
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Sequence CWU 1
1
1012711DNAGallus gallus 1ataaagggca gcgcggcggc gacggcggac
tcagcccggc cccgctccgc cgccttctcc 60cgcaccgccc gacccgccgc agccccggcc
ccacggcagc ccccatgccc cgcctcggat 120cccaggtgtc cctgttccgc
tgcaccctgc tctcgtccct cctcctcgtc tgcagcatcc 180atgagacgga
gcagaacagc tactgccagc agatcatcac cgagcggcac ctggaccacc
240tgcaggagct ggcggacacg cagatgcagc agccgggcac agtgtccttc
agattcatca 300gcaagatgcg gctgagcgac tctgtctgct acgtgaaagc
cgccttccct ttgctgggca 360ccatcctgaa caggacgacg ttcaaggaga
actcaacaaa cgccaacaag atgaagacgg 420tgcgcaagat gtacgaaaac
atcgatgaga acgtggaccc ctgcatcagg gacgaggatg 480acaaggagca
cgcgctgtcc gaaatgtgct ttgaggagtt caccacgtcc ccctacgaga
540tgctggtgct ggtgaggcag ttcttccagg acatcaaaca gctgctgcag
aacaaggaga 600ccttcgagaa ggactgcagc caggtgtacc gcagtgcgtg
cgcggggccc cggcagcaca 660gctcctcccc aggtgtgggg acagatcctg
actgcaattg cctgtcccct gccctccctt 720ctgccaccca gccctccctc
tccgctgcca cccgtgccgg cagggacgtg gcgcccgcta 780gcaccagggt
cccttaccgc cagctcggtg gcatcctggc tgagttaggc agcagtgccc
840cgtccgagcc ccccagtagc gtggagggca gctcgggggc cgaggaactg
ccaggagccg 900ggctcggcga cgcgtcggcg ccgtccccca ccatgcagca
gacgcttgga gccctcctgg 960atccagccgc gagcgccggc ccgaaggctg
aggacgtatc catcccgtcc cacgggatgc 1020cggaggaggg cgccgggacc
cccgccctcc cacatcggct cccttcgccg cgagggatca 1080gcgcggcgat
gccggcggcg gtccccagca gcggctctgc gcagcgccgc ggggtcgggc
1140gccgtcccac cgagagcccc gagcgggtca cgcagctccg cttccccagg
atggctccgc 1200cgttgcgggg ccgggcggag ggcggccccg gggacggggc
gagggcgcga ggctgggggc 1260tgagccggct gcgggagccc gaggacggcg
gggccggacc cagctttgat tcgagctttg 1320ttctgagcgc agagcagcgc
aggaaggagc cgccagccgc cagcgggggg caccgggagc 1380tcctggtgta
cgtcacggtg gccagcgtgg tggccgtgct gctggccatg ggcgggctgc
1440tcttctacaa gtataagtcc aaggtcctgc agcggggagc agcgctaaaa
gaggggggct 1500gcgaccccga ggagccggag agcagggcgc tgcagggagc
gcagggctgc gcggagctgg 1560agacgcagga gctgtgaggg ccccctgcgg
gacgtgatgc tgctcggggg gacggacggg 1620gacgctcctc gctgggcgac
ggacggctgc tgctcggcct ccccccgccg cgatgacccc 1680caggccctgt
cctgcagctg caacccacgg gtgaggatgg caggacgggg cggtgcagcc
1740ctgcaggacc ccggcgatgg ggcggatggc accgaggggc tccacgggga
cggcattggg 1800tgccgcgagt ggaacatctc cccccaccca tccacggttc
ccgttgctcc tctcccaccc 1860ctggcacggg gggacccccg gcgccccatg
gggggacccc tcccgcatcc caccggtgcc 1920gaggacccaa cgcccggcct
gcaaaggggg aaaccctcac actgtgaata tttaagaccc 1980gtggtgccgt
ccccatcccg cgatcccaag ctggccttgg gagctgcccg gcgccgctct
2040gcgcaggaag gctctccacg aacgcggtgg ataaacgctt ttatccaaca
aatgcacttg 2100gggggggggg ttcccccctc cctgcagggt tattgctgcg
agctggcctc gccccagact 2160ggattttgtt gctggagcac agcacggcaa
tggggccgtg gctgcagtgt ggggtttggg 2220ggctcagcgg tacccggact
gcgtcccacc ccacacggca tccctgccca gcgccgctcc 2280cggggggtcg
gaagtgttat ttttatatta catgagatgc aaacgggacg gagcacattg
2340gggtgtggtg gggttttgtt ttttaaagca ttagtattga ttttggggtt
tttttttcta 2400tgcgtattta tggactgcca aaaaaagagg cgtttcctgg
gggtgatggg ggggggggtg 2460gaagtggggt gcagagccgg gctggggccg
gagctggtgc tggctcagta tgtggggtgt 2520gggtgagggg ggttgggggg
ggggcagctt ttggagctct ttctgcctct gttgtctcat 2580tttttgtaca
gtgaaatggt gaaatatttt atacaaagtc atttaaagaa gtctatttaa
2640ggaaaataat agaaaacagc ttgtatattt aatattatta ataaagatgg
acgtgcaaaa 2700aaaaaaaaaa a 271121473DNAGallus gallus 2atgccccgcc
tcggatccca ggtgtccctg ttccgctgca ccctgctctc gtccctcctc 60ctcgtctgca
gcatccatga gacggagcag aacagctact gccagcagat catcaccgag
120cggcacctgg accacctgca ggagctggcg gacacgcaga tgcagcagcc
gggcacagtg 180tccttcagat tcatcagcaa gatgcggctg agcgactctg
tctgctacgt gaaagccgcc 240ttccctttgc tgggcaccat cctgaacagg
acgacgttca aggagaactc aacaaacgcc 300aacaagatga agacggtgcg
caagatgtac gaaaacatcg atgagaacgt ggacccctgc 360atcagggacg
aggatgacaa ggagcacgcg ctgtccgaaa tgtgctttga ggagttcacc
420acgtccccct acgagatgct ggtgctggtg aggcagttct tccaggacat
caaacagctg 480ctgcagaaca aggagacctt cgagaaggac tgcagccagg
tgtaccgcag tgcgtgcgcg 540gggccccggc agcacagctc ctccccaggt
gtggggacag atcctgactg caattgcctg 600tcccctgccc tcccttctgc
cacccagccc tccctctccg ctgccacccg tgccggcagg 660gacgtggcgc
ccgctagcac cagggtccct taccgccagc tcggtggcat cctggctgag
720ttaggcagca gtgccccgtc cgagcccccc agtagcgtgg agggcagctc
gggggccgag 780gaactgccag gagccgggct cggcgacgcg tcggcgccgt
cccccaccat gcagcagacg 840cttggagccc tcctggatcc agccgcgagc
gccggcccga aggctgagga cgtatccatc 900ccgtcccacg ggatgccgga
ggagggcgcc gggacccccg ccctcccaca tcggctccct 960tcgccgcgag
ggatcagcgc ggcgatgccg gcggcggtcc ccagcagcgg ctctgcgcag
1020cgccgcgggg tcgggcgccg tcccaccgag agccccgagc gggtcacgca
gctccgcttc 1080cccaggatgg ctccgccgtt gcggggccgg gcggagggcg
gccccgggga cggggcgagg 1140gcgcgaggct gggggctgag ccggctgcgg
gagcccgagg acggcggggc cggacccagc 1200tttgattcga gctttgttct
gagcgcagag cagcgcagga aggagccgcc agccgccagc 1260ggggggcacc
gggagctcct ggtgtacgtc acggtggcca gcgtggtggc cgtgctgctg
1320gccatgggcg ggctgctctt ctacaagtat aagtccaagg tcctgcagcg
gggagcagcg 1380ctaaaagagg ggggctgcga ccccgaggag ccggagagca
gggcgctgca gggagcgcag 1440ggctgcgcgg agctggagac gcaggagctg tga
14733490PRTGallus gallus 3Met Pro Arg Leu Gly Ser Gln Val Ser Leu
Phe Arg Cys Thr Leu Leu 1 5 10 15 Ser Ser Leu Leu Leu Val Cys Ser
Ile His Glu Thr Glu Gln Asn Ser 20 25 30 Tyr Cys Gln Gln Ile Ile
Thr Glu Arg His Leu Asp His Leu Gln Glu 35 40 45 Leu Ala Asp Thr
Gln Met Gln Gln Pro Gly Thr Val Ser Phe Arg Phe 50 55 60 Ile Ser
Lys Met Arg Leu Ser Asp Ser Val Cys Tyr Val Lys Ala Ala 65 70 75 80
Phe Pro Leu Leu Gly Thr Ile Leu Asn Arg Thr Thr Phe Lys Glu Asn 85
90 95 Ser Thr Asn Ala Asn Lys Met Lys Thr Val Arg Lys Met Tyr Glu
Asn 100 105 110 Ile Asp Glu Asn Val Asp Pro Cys Ile Arg Asp Glu Asp
Asp Lys Glu 115 120 125 His Ala Leu Ser Glu Met Cys Phe Glu Glu Phe
Thr Thr Ser Pro Tyr 130 135 140 Glu Met Leu Val Leu Val Arg Gln Phe
Phe Gln Asp Ile Lys Gln Leu 145 150 155 160 Leu Gln Asn Lys Glu Thr
Phe Glu Lys Asp Cys Ser Gln Val Tyr Arg 165 170 175 Ser Ala Cys Ala
Gly Pro Arg Gln His Ser Ser Ser Pro Gly Val Gly 180 185 190 Thr Asp
Pro Asp Cys Asn Cys Leu Ser Pro Ala Leu Pro Ser Ala Thr 195 200 205
Gln Pro Ser Leu Ser Ala Ala Thr Arg Ala Gly Arg Asp Val Ala Pro 210
215 220 Ala Ser Thr Arg Val Pro Tyr Arg Gln Leu Gly Gly Ile Leu Ala
Glu 225 230 235 240 Leu Gly Ser Ser Ala Pro Ser Glu Pro Pro Ser Ser
Val Glu Gly Ser 245 250 255 Ser Gly Ala Glu Glu Leu Pro Gly Ala Gly
Leu Gly Asp Ala Ser Ala 260 265 270 Pro Ser Pro Thr Met Gln Gln Thr
Leu Gly Ala Leu Leu Asp Pro Ala 275 280 285 Ala Ser Ala Gly Pro Lys
Ala Glu Asp Val Ser Ile Pro Ser His Gly 290 295 300 Met Pro Glu Glu
Gly Ala Gly Thr Pro Ala Leu Pro His Arg Leu Pro 305 310 315 320 Ser
Pro Arg Gly Ile Ser Ala Ala Met Pro Ala Ala Val Pro Ser Ser 325 330
335 Gly Ser Ala Gln Arg Arg Gly Val Gly Arg Arg Pro Thr Glu Ser Pro
340 345 350 Glu Arg Val Thr Gln Leu Arg Phe Pro Arg Met Ala Pro Pro
Leu Arg 355 360 365 Gly Arg Ala Glu Gly Gly Pro Gly Asp Gly Ala Arg
Ala Arg Gly Trp 370 375 380 Gly Leu Ser Arg Leu Arg Glu Pro Glu Asp
Gly Gly Ala Gly Pro Ser 385 390 395 400 Phe Asp Ser Ser Phe Val Leu
Ser Ala Glu Gln Arg Arg Lys Glu Pro 405 410 415 Pro Ala Ala Ser Gly
Gly His Arg Glu Leu Leu Val Tyr Val Thr Val 420 425 430 Ala Ser Val
Val Ala Val Leu Leu Ala Met Gly Gly Leu Leu Phe Tyr 435 440 445 Lys
Tyr Lys Ser Lys Val Leu Gln Arg Gly Ala Ala Leu Lys Glu Gly 450 455
460 Gly Cys Asp Pro Glu Glu Pro Glu Ser Arg Ala Leu Gln Gly Ala Gln
465 470 475 480 Gly Cys Ala Glu Leu Glu Thr Gln Glu Leu 485 490
4813DNAGallus gallus 4atgccccgcc tcggatccca ggtgtccctg ttccgctgca
ccctgctctc gtccctcctc 60ctcgtctgca gcatccatga gacggagcag aacagctact
gccagcagat catcaccgag 120cggcacctgg accacctgca ggagctggcg
gacacgcaga tgcagcagcc gggcacagtg 180tccttcagat tcatcagcaa
gatgcggctg agcgactctg tctgctacgt gaaagccgcc 240ttccctttgc
tgggcaccat cctgaacagg acgacgttca aggagaactc aacaaacgcc
300aacaagatga agacggtgcg caagatgtac gaaaacatcg atgaggacgt
ggacccctgc 360atcagggacg aggatgacga ggagcacgcg ctgtccgaaa
tgtgctttga ggagttcacc 420acgtccccct acgagatgct ggtgctggtg
aggcagttct tccaggacat caaacagctg 480ctgcagaaca aggagacctt
cgagaaggac tgcagccagg tgtaccgcag tgcgtgcgcg 540gggccccggc
agcacagctc ctccccagag cagcgcagga aggagccgcc agccgccagc
600ggggggcacc gggagctcct ggtgtacgtc acggtggcca gcgtggtggc
cgtgctgctg 660gccatgggcg ggctgctctt ctacaagtat aagtccaagg
tcctgcagcg gggagcagcg 720ctaaaagagg ggggctgcga ccccgaggag
ccggagagca gggcgctgca gggagcgcag 780ggctgcgcgg agctggagac
gcaggagctg tga 8135270PRTGallus gallus 5Met Pro Arg Leu Gly Ser Gln
Val Ser Leu Phe Arg Cys Thr Leu Leu 1 5 10 15 Ser Ser Leu Leu Leu
Val Cys Ser Ile His Glu Thr Glu Gln Asn Ser 20 25 30 Tyr Cys Gln
Gln Ile Ile Thr Glu Arg His Leu Asp His Leu Gln Glu 35 40 45 Leu
Ala Asp Thr Gln Met Gln Gln Pro Gly Thr Val Ser Phe Arg Phe 50 55
60 Ile Ser Lys Met Arg Leu Ser Asp Ser Val Cys Tyr Val Lys Ala Ala
65 70 75 80 Phe Pro Leu Leu Gly Thr Ile Leu Asn Arg Thr Thr Phe Lys
Glu Asn 85 90 95 Ser Thr Asn Ala Asn Lys Met Lys Thr Val Arg Lys
Met Tyr Glu Asn 100 105 110 Ile Asp Glu Asp Val Asp Pro Cys Ile Arg
Asp Glu Asp Asp Glu Glu 115 120 125 His Ala Leu Ser Glu Met Cys Phe
Glu Glu Phe Thr Thr Ser Pro Tyr 130 135 140 Glu Met Leu Val Leu Val
Arg Gln Phe Phe Gln Asp Ile Lys Gln Leu 145 150 155 160 Leu Gln Asn
Lys Glu Thr Phe Glu Lys Asp Cys Ser Gln Val Tyr Arg 165 170 175 Ser
Ala Cys Ala Gly Pro Arg Gln His Ser Ser Ser Pro Glu Gln Arg 180 185
190 Arg Lys Glu Pro Pro Ala Ala Ser Gly Gly His Arg Glu Leu Leu Val
195 200 205 Tyr Val Thr Val Ala Ser Val Val Ala Val Leu Leu Ala Met
Gly Gly 210 215 220 Leu Leu Phe Tyr Lys Tyr Lys Ser Lys Val Leu Gln
Arg Gly Ala Ala 225 230 235 240 Leu Lys Glu Gly Gly Cys Asp Pro Glu
Glu Pro Glu Ser Arg Ala Leu 245 250 255 Gln Gly Ala Gln Gly Cys Ala
Glu Leu Glu Thr Gln Glu Leu 260 265 270 6534DNAGallus gallus
6atgcaccagg gctgcgcggc tgtcctctgt gtcctggccg tgctggggct ggaggtggct
60gcgctggggg aatgcgagct cgcccgcctg ctgcaggaca agctgcggta tgagatgcgc
120ctgcagtaca tgaagcacaa cttccccatt gactacactc tccgggtgca
gcacgaggag 180gtgctgcgga ccgccaacgt cacccgcctg cgtgatggga
aggtgtcgga ggcgtcgctg 240cgctacctgt ggttccacgc ctgctcccag
gcggtgctgc acatcctcga ggtgctgccg 300gagaagcacc cgtcccgtgg
gtacacgcag gagctgagcc agcttttgga tgccctgggc 360gtggagtaca
gtgggtaccg gcagagcgat gtggacgcgg tggtggccga cctggtgaag
420cagctgcaca gcggcgatag ccggcagaag gccgtgcgcc ccaaagcact
gctggacaac 480tgcctcaagg tcctgcggat gctcttcggg gcacactgtc
ggtgggactc cgct 5347178PRTGallus gallus 7Met His Gln Gly Cys Ala
Ala Val Leu Cys Val Leu Ala Val Leu Gly 1 5 10 15 Leu Glu Val Ala
Ala Leu Gly Glu Cys Glu Leu Ala Arg Leu Leu Gln 20 25 30 Asp Lys
Leu Arg Tyr Glu Met Arg Leu Gln Tyr Met Lys His Asn Phe 35 40 45
Pro Ile Asp Tyr Thr Leu Arg Val Gln His Glu Glu Val Leu Arg Thr 50
55 60 Ala Asn Val Thr Arg Leu Arg Asp Gly Lys Val Ser Glu Ala Ser
Leu 65 70 75 80 Arg Tyr Leu Trp Phe His Ala Cys Ser Gln Ala Val Leu
His Ile Leu 85 90 95 Glu Val Leu Pro Glu Lys His Pro Ser Arg Gly
Tyr Thr Gln Glu Leu 100 105 110 Ser Gln Leu Leu Asp Ala Leu Gly Val
Glu Tyr Ser Gly Tyr Arg Gln 115 120 125 Ser Asp Val Asp Ala Val Val
Ala Asp Leu Val Lys Gln Leu His Ser 130 135 140 Gly Asp Ser Arg Gln
Lys Ala Val Arg Pro Lys Ala Leu Leu Asp Asn 145 150 155 160 Cys Leu
Lys Val Leu Arg Met Leu Phe Gly Ala His Cys Arg Trp Asp 165 170 175
Ser Ala 820DNAArtificialPrimer 8gcgactctgt ctgctacgtg
20920DNAArtificialPrimer 9cgaaggtctc cttgttctgc 2010159PRTGallus
gallus 10Asn Ser Tyr Cys Gln Gln Ile Ile Thr Glu Arg His Leu Asp
His Leu 1 5 10 15 Gln Glu Leu Ala Asp Thr Gln Met Gln Gln Pro Gly
Thr Val Ser Phe 20 25 30 Arg Phe Ile Ser Lys Met Arg Leu Ser Asp
Ser Val Cys Tyr Val Lys 35 40 45 Ala Ala Phe Pro Leu Leu Gly Thr
Ile Leu Asn Arg Thr Thr Phe Lys 50 55 60 Glu Asn Ser Thr Asn Ala
Asn Lys Met Lys Thr Val Arg Lys Met Tyr 65 70 75 80 Glu Asn Ile Asp
Glu Asn Val Asp Pro Cys Ile Arg Asp Glu Asp Asp 85 90 95 Lys Glu
His Ala Leu Ser Glu Met Cys Phe Glu Glu Phe Thr Thr Ser 100 105 110
Pro Tyr Glu Met Leu Val Leu Val Arg Gln Phe Phe Gln Asp Ile Lys 115
120 125 Gln Leu Leu Gln Asn Lys Glu Thr Phe Glu Lys Asp Cys Ser Gln
Val 130 135 140 Tyr Arg Ser Ala Cys Ala Gly Pro Arg Gln His Ser Ser
Ser Pro 145 150 155
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