Nucleic acids encoding soluble human FLK-2 extracellular domain

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG. 1A (murine flk-2), FIG. 1B (human flk-2) and FIG. 2 (murine flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG. 1A, FIG. 1B and FIG. 2; ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

FIELD OF THE INVENTION 
The present invention relates to hematopoietic stem cell receptors, ligands 
for such receptors, and nucleic acid molecules encoding such receptors and 
ligands. 
BACKGROUND OF THE INVENTION 
The mammalian hematopoietic system comprises red and white blood cells. 
These cells are the mature cells that result from more primitive 
lineage-restricted cells. The cells of the hematopoietic system have been 
reviewed by Dexter and Spooncer in the Annual Review of Cell Biology 3, 
423-441 (1987). 
The red blood cells, or erythrocytes, result from primitive cells referred 
to by Dexter and Spooncer as erythroid burst-forming units (BFU-E). The 
immediate progeny of the erythroid burst-forming units are called 
erythroid colony-forming units (CFU-E). 
The white blood cells contain the mature cells of the lymphoid and myeloid 
systems. The lymphoid cells include B lymphocytes and T lymphocytes. The B 
and T lymphocytes result from earlier progenitor cells referred to by 
Dexter and Spooncer as preT and preB cells. 
The myeloid system comprises a number of cells including granulocytes, 
platelets, monocytes, macrophages, and megakaryocytes. The granulocytes 
are further divided into neutrophils, eosinophils, basophils and mast 
cells. 
Each of the mature hematopoietic cells are specialized for specific 
functions. For example, erythrocytes are responsible for oxygen and carbon 
dioxide transport. T and B lymphocytes are responsible for cell- and 
antibody-mediated immune responses, respectively. Platelets are involved 
in blood clotting. Granulocytes and macrophages act generally as 
scavengers and accessory cells in the immune response against invading 
organisms and their by-products. 
At the center of the hematopoietic system lie one or more totipotent 
hematopoietic stem cells, which undergo a series of differentiation steps 
leading to increasingly lineage-restricted progenitor cells. The more 
mature progenitor cells are restricted to producing one or two lineages. 
Some examples of lineage-restricted progenitor cells mentioned by Dexter 
and Spooncer include granulocyte/macrophage colony-forming cells (GM-CFC), 
megakaryocyte colony-forming cells (Meg-CFC), eosinophil colony-forming 
cells (Eos-CFC), and basophil colony-forming cells (Bas-CFC). Other 
examples of progenitor cells are discussed above. 
The hematopoietic system functions by means of a precisely controlled 
production of the various mature lineages. The totipotent stem cell 
possesses the ability both to self renew and to differentiate into 
committed progenitors for all hematopoietic lineages. These most primitive 
of hematopoietic cells are both necessary and sufficient for the complete 
and permanent hematopoietic reconstitution of a radiation-ablated 
hematopoietic system in mammals. The ability of stem cells to reconstitute 
the entire hematopoietic system is the basis of bone marrow transplant 
therapy. 
It is known that growth factors play an important role in the development 
and operation of the mammalian hematopoietic system. The role of growth 
factors is complex, however, and not well understood at the present time. 
One reason for the uncertainty is that much of what is known about 
hematopoietic growth factors results from in vitro experiments. Such 
experiments do not necessarily reflect in vivo realities. 
In addition, in vitro hematopoiesis can be established in the absence of 
added growth factors, provided that marrow stromal cells are added to the 
medium. The relationship between stromal cells and hematopoietic growth 
factors in vivo is not understood. Nevertheless, hematopoietic growth 
factors have been shown to be highly active in vivo. 
From what is known about them, hematopoietic growth factors appear to 
exhibit a spectrum of activities. At one end of the spectrum are growth 
factors such as erythropoietin, which is believed to promote proliferation 
only of mature erythroid progenitor cells. In the middle of the spectrum 
are growth factors such as IL-3, which is believed to facilitate the 
growth and development of early stem cells as well as of numerous 
progenitor cells. Some examples of progenitor cells induced by IL-3 
include those restricted to the granulocyte/macrophage, eosinophil, 
megakaryocyte, erythroid and mast cell lineages. 
At the other end of the spectrum is the hematopoietic growth factor that, 
along with the corresponding receptor, was discussed in a series of 
articles in the Oct. 5, 1990 edition of Cell. The receptor is the product 
of the W locus, c-kit, which is a member of the class of receptor protein 
tyrosine kinases. The ligand for c-kit, which is referred to by various 
names such as stem cell factor (SCF) and mast cell growth factor (MGF), is 
believed to be essential for the development of early hematopoietic stem 
cells and cells restricted to the erythroid and mast cell lineages in 
mice; see, for example, Copeland et al., Cell 63, 175-183 (1990). 
It appears, therefore, that there are growth factors that exclusively 
affect mature cells. There also appear to be growth factors that affect 
both mature cells and stem cells. The growth factors that affect both 
types of cells may affect a small number or a large number of mature 
cells. 
There further appears to be an inverse relationship between the ability of 
a growth factor to affect mature cells and the ability of the growth 
factor to affect stem cells. For example, the c-kit ligand, which 
stimulates a small number of mature cells, is believed to be more 
important in the renewal and development of stem cells then is IL-3, which 
is reported to stimulate proliferation of many mature cells (see above). 
Prior to the present specification, there have been no reports of growth 
factors that exclusively stimulate stem cells in the absence of an effect 
on mature cells. The discovery of such growth factors would be of 
particular significance. 
As mentioned above, c-kit is a protein tyrosine kinase (pTK). It is 
becoming increasingly apparent that the protein tyrosine kinases play an 
important role as cellular receptors for hematopoietic growth factors. 
Other receptor pTKs include the receptors of colony stimulating factor 1 
(CSF-1) and PDGF. 
The pTK family can be recognized by the presence of several conserved amino 
acid regions in the catalytic domain. These conserved regions are 
summarized by Hanks et al. in Science 241, 42-52 (1988), see FIG. 1 
starting on page 46 and by Wilks in Proc. Natl. Acad. Sci. U.S.A. 86, 
1603-1607 (1989), see FIG. 2 on page 1605. 
Additional protein tyrosine kinases that represent hematopoietic growth 
factor receptors are needed in order more effectively to stimulate the 
self-renewal of the totipotent hematopoietic stem cell and to stimulate 
the development of all cells of the hematopoietic system both in vitro and 
in vivo. Novel hematopoietic growth factor receptors that are present only 
on primitive stem cells, but are not present on mature progenitor cells, 
are particularly desired. Ligands for the novel receptors are also 
desirable to act as hematopoietic growth factors. Nucleic acid sequences 
encoding the receptors and ligands are needed to produce recombinant 
receptors and ligands. 
SUMMARY OF THE INVENTION 
These and other objectives as will be apparent to those with ordinary skill 
in the art have been met by providing isolated mammalian nucleic acid 
molecules encoding receptor protein tyrosine kinases expressed in 
primitive hematopoietic cells and not expressed in mature hematopoietic 
cells. Also included are the receptors encoded by such nucleic acid 
molecules; the nucleic acid molecules encoding receptor protein tyrosine 
kinases having the sequences shown in FIG. 1A (murine flk-2), FIG. 1B 
(human flk-2) and FIG. 2 (murine flk-1) (see SEQ ID. NOS. 1, 3, and 5, 
respectively); the receptor protein tyrosine kinases having the amino acid 
sequences shown in FIG. 1A, FIG. 1B, and FIG. 2 (see SEQ ID. NOS. 2, 4 and 
6, respectively); ligands for the receptors; nucleic acid sequences that 
encode the ligands; and methods of stimulating the proliferation of 
primitive mammalian hematopoietic stem cells comprising contacting the 
stem cells with a ligand that binds to a receptor protein tyrosine kinase 
expressed in primitive mammalian hematopoietic cells and not expressed in 
mature hematopoietic cells.

DETAILED DESCRIPTION OF THE INVENTION 
Receptors 
In one embodiment, the invention relates to an isolated mammalian nucleic 
acid molecule encoding a receptor protein tyrosine kinase expressed in 
primitive mammalian hematopoietic cells and not expressed in mature 
hematopoietic cells. 
The nucleic acid molecule may be a DNA, cDNA, or RNA molecule. The mammal 
in which the nucleic acid molecule exists may be any mammal, such as a 
mouse, rat, rabbit, or human. 
The nucleic acid molecule encodes a protein tyrosine kinase (pTK). Members 
of the pTK family can be recognized by the conserved amino acid regions in 
the catalytic domains. Examples of pTK consensus sequences have been 
provided by Hanks et al. in Science 241, 42-52 (1988); see especially FIG. 
1 starting on page 46 and by Wilks in Proc. Natl. Acad. Sci. U.S.A. 86, 
1603-1607 (1989); see especially FIG. 2 on page 1605. A methionine residue 
at position 205 in the conserved sequence WMAPES is characteristic of 
pTK's that are receptors. 
The Hanks et al article identifies eleven catalytic sub-domains containing 
pTK consensus residues and sequences. The pTKs of the present invention 
will have most or all of these consensus residues and sequences. 
Some particularly strongly conserved residues and sequences are shown in 
Table 1. 
TABLE 1 
______________________________________ 
Conserved Residues and Sequences in pTKs.sup.1 
Residue or 
Catalytic 
Position.sup.2 Sequence Domain 
______________________________________ 
50 G I 
52 G I 
57 V I 
70 A II 
72 K II 
91 E III 
166 D VI 
171 N VI 
184-186 DFG VII 
208 E VIII 
220 D IX 
225 G IX 
280 R XI 
______________________________________ 
1. See Hanks et al., Science 241, 42-52 (1988) 
2. Adjusted in accordance with Hanks et al., Id. 
A pTK of the invention may contain all thirteen of these highly conserved 
residues and sequences. As a result of natural or synthetic mutations, the 
pTKs of the invention may contain fewer than all thirteen strongly 
conserved residues and sequences, such as 11, 9, or 7 such sequences. 
The receptors of the invention generally belong to the same class of pTK 
sequences that c-kit belongs to. It has surprisingly been discovered, 
however, that a new functional class of receptor pTKs exists. The new 
functional class of receptor pTKs is expressed in primitive hematopoietic 
cells, but not expressed in mature hematopoietic cells. 
For the purpose of this specification, a primitive hematopoietic cell is 
totipotent, i.e. capable of reconstituting all hematopoietic blood cells 
in vivo. A mature hematopoietic cell is non-self-renewing, and has limited 
proliferative capacity--i.e., a limited ability to give rise to multiple 
lineages. Mature hematopoietic cells, for the purposes of this 
specification, are generally capable of giving rise to only one or two 
lineages in vitro or in vivo. 
It should be understood that the hematopoietic system is complex, and 
contains many intermediate cells between the primitive totipotent 
hematopoietic stem cell and the totally committed mature hematopoietic 
cells defined above. As the stem cell develops into increasingly mature, 
lineage-restricted cells, it gradually loses its capacity for 
self-renewal. 
The receptors of the present invention may and may not be expressed in 
these intermediate cells. The necessary and sufficient condition that 
defines members of the new class of receptors is that they are present in 
the primitive, totipotent stem cell or cells, and not in mature cells 
restricted only to one or, at most, two lineages. 
An example of a member of the new class of receptor pTKs is called fetal 
liver kinase 2 (flk-2) after the organ in which it was found. There is 
approximately 1 totipotent stem cell per 10.sup.4 cells in mid-gestation 
(day 14) fetal liver in mice. In addition to fetal liver, flk-2 is also 
expressed in fetal spleen, fetal thymus, adult brain, and adult marrow. 
For example, flk-2 is expressed in individual multipotential CFU-Blast 
colonies capable of generating numerous multilineage colonies upon 
replating. It is likely, therefore, that flk-2 is expressed in the entire 
primitive (i.e. self-renewing) portion of the hematopoietic hierarchy. 
This discovery is consistent with flk-2 being important in transducing 
putative self-renewal signals from the environment. 
It is particularly relevant that the expression of flk-2 mRNA occurs in the 
most primitive thymocyte subset. Even in two closely linked immature 
subsets that differ in expression of the IL-2 receptor, flk-2 expression 
segregates to the more primitive subset lacking an IL-2 receptor. The 
earliest thymocyte subset is believed to be uncommitted. Therefore, the 
thymocytes expressing flk-2 may be multipotential. flk-2 is the first 
receptor tyrosine kinase known to be expressed in the T-lymphoid lineage. 
The fetal liver mRNA migrates relative to 28S and 18S ribosomal bands on 
formaldehyde agarose gels at approximately 3.5 kb, while the brain message 
is considerably larger. In adult tissues, flk-2 m-RNA from both brain and 
bone marrow migrated at approximately 3.5 kb. 
A second pTK receptor is also included in the present invention. This 
second receptor, which is called fetal liver kinase 1 (flk-1), is not a 
member of the same class of receptors as flk-2, since flk-1 may be found 
in some more mature hematopoietic cells. The amino acid sequence of flk-1 
is given in FIG. 2. (See SEQ. ID. NO. 6) 
The present invention includes the flk-1 receptor as well as DNA, cDNA and 
RNA encoding flk-1. The DNA sequence of flk-1 is also given in FIG. 2. 
(See SEQ. ID. NO. 5) Flk-1 may be found in the same organs as flk-2, as 
well as in fetal brain, stomach, kidney, lung, heart and intestine; and in 
adult kidney, heart, spleen, lung, muscle, and lymph nodes. 
The receptor protein tyrosine kinases of the invention are known to be 
divided into easily found domains. The DNA sequence corresponding to the 
pTKs encode, starting at their 5'-ends, a hydrophobic leader sequence 
followed by a hydrophilic extracellular domain, which binds to, and is 
activated by, a specific ligand. Immediately downstream from the 
extracellular receptor domain, is a hydrophobic transmembrane region. The 
transmembrane region is immediately followed by a basic catalytic domain, 
which may easily be identified by reference to the Hanks et al. and Wilks 
articles discussed above. 
The present invention includes the extracellular receptor domain lacking 
the transmembrane region and catalytic domain. Preferably, the hydrophobic 
leader sequence is also removed from the extracellular domain. In the case 
of flk-2, the hydrophobic leader sequence includes amino acids 1-27. (See 
SEQ. ID. NOS. 2 and 4) 
These regions and domains may easily be visually identified by those having 
ordinary skill in the art by reviewing the amino acid sequence in a 
suspected pTK and comparing it to known pTKs. For example, referring to 
FIG. 1A, the transmembrane region of flk-2, which separates the 
extracellular receptor domain from the catalytic domain, is encoded by 
nucleotides 1663 (T) to 1722 (C). These nucleotides correspond to amino 
acid residues 545 (Phe) to 564 (Cys) (See SEQ. ID. NOS. 1-2). The amino 
acid sequence between the transmembrane region and the catalytic 
sub-domain (amino acids 618-623) identified by Hanks et al. as sub-domain 
I (i.e., GXGXXG) is characteristic of receptor protein tyrosine kinases. 
The extracellular domain may also be identified through commonly recognized 
criteria of extracellular amino acid sequences. The determination of 
appropriate criteria is known to those skilled in the art, and has been 
described, for example, by Hopp et al, Proc. Nat'l Acad. Sci. U.S.A. 78, 
3824-3828 (1981); Kyte et al, J. Mol. Biolo 157, 105-132 (1982); Emini, J. 
Virol. 55, 836-839 (1985); Jameson et al, CA BIOS 4, 181-186 (1988); and 
Karplus et al, Naturwissenschaften 72, 212-213 (1985). Amino acid domains 
predicted by these criteria to be surface exposed characteristic of 
extracellular domains. 
As will be discussed in more detail below, the nucleic acid molecules that 
encode the receptors of the invention may be inserted into known vectors 
for use in standard recombinant DNA techniques. Standard recombinant DNA 
techniques are those such as are described in Sambrook et al , "Molecular 
Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and 
by Ausubel et al., Eds, "Current Protocols in Molecular Biology," Green 
Publishing Associates and Wiley-Interscience, New York (1987). The vectors 
may be circular (i.e. plasmids) or non-circular. Standard vectors are 
available for cloning and expression in a host. The host may be 
prokaryotic or eucaryotic. Prokaryotic hosts are preferably E. coli. 
Preferred eucaryotic hosts include yeast, insect and mammalian cells. 
Preferred mammalian cells include, for example, CHO, COS and human cells. 
Ligands 
The invention also includes ligands that bind to the receptor pTKs of the 
invention. In addition to binding, the ligands stimulate the proliferation 
of additional primitive stem cells, differentiation into more mature 
progenitor cells, or both. 
The ligand may be a growth factor that occurs naturally in a mammal, 
preferably the same mammal that produces the corresponding receptor. The 
growth factor may be isolated and purified, or be present on the surface 
of an isolated population of cells, such as stromal cells. 
The ligand may also be a molecule that does not occur naturally in a 
mammal. For example, antibodies, preferably monoclonal, raised against the 
receptors of the invention or against anti-ligand antibodies mimic the 
shape of, and act as, ligands if they constitute the negative image of the 
receptor or anti-ligand antibody binding site. The ligand may also be a 
non-protein molecule that acts as a ligand when it binds to, or otherwise 
comes into contact with, the receptor. 
In another embodiment, nucleic acid molecules encoding the ligands of the 
invention are provided. The nucleic acid molecule may be RNA, DNA or cDNA. 
Stimulating Proliferation of Stem Cells 
The invention also includes a method of stimulating the proliferation 
and/or differentiation of primitive mammalian hematopoietic stem cells as 
defined above. The method comprises contacting the stem cells with a 
ligand in accordance with the present invention. The stimulation of 
proliferation and/or differentiation may occur in vitro or in vivo. 
The ability of a ligand according to the invention to stimulate 
proliferation of stem cells in vitro and in vivo has important therapeutic 
applications. Such applications include treating mammals, including 
humans, whose primitive stem cells do not sufficiently undergo 
self-renewal. Example of such medical problems include those that occur 
when defects in hematopoietic stem cells or their related growth factors 
depress the number of white blood cells. Examples of such medical problems 
include anemia, such as macrocytic and aplastic anemia. Bone marrow damage 
resulting from cancer chemotherapy and radiation is another example of a 
medical problem that would be helped by the stem cell factors of the 
invention. 
Functional Equivalents 
The invention includes functional equivalents of the pTK receptors, 
receptor domains, and ligands described above as well as of the nucleic 
acid sequences encoding them. A protein is considered a functional 
equivalent of another protein for a specific function if the equivalent 
protein is immunologically cross-reactive with, and has the same function 
as, the receptors and ligands of the invention. The equivalent may, for 
example, be a fragment of the protein, or a substitution, addition or 
deletion mutant of the protein. 
For example, it is possible to substitute amino acids in a sequence with 
equivalent amino acids. Groups of amino acids known normally to be 
equivalent are: 
(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G); 
(b) Asn(N) Asp(D) Glu(E) Gln(Q); 
(c) His(H) Arg(R) Lys(K); 
(d) Met(M) Leu(L) Ile(I) Val(V); and 
(e) Phe(F) Tyr(Y) Trp(W). 
Substitutions, additions and/or deletions in the receptors and ligands may 
be made as long as the resulting equivalent receptors and ligands are 
immunologically cross reactive with, and have the same function as, the 
native receptors and ligands. 
The equivalent receptors and ligands will normally have substantially the 
same amino acid sequence as the native receptors and ligands. An amino 
acid sequence that is substantially the same as another sequence, but that 
differs from the other sequence by means of one or more substitutions, 
additions and/or deletions is considered to be an equivalent sequence. 
Preferably, less than 25%, more preferably less than 10%, and most 
preferably less than 5% of the number of amino acid residues in the amino 
acid sequence of the native receptors and ligands are substituted for, 
added to, or deleted from. 
Equivalent nucleic acid molecules include nucleic acid sequences that 
encode equivalent receptors and ligands as defined above. Equivalent 
nucleic acid molecules also include nucleic acid sequences that differ 
from native nucleic acid sequences in ways that do not affect the 
corresponding amino acid sequences. 
ISOLATION OF NUCLEIC ACID MOLECULES AND PROTEINS 
Isolation of Nucleic Acid Molecules Encoding Receptors 
In order to produce nucleic acid molecules encoding mammalian stem cell 
receptors, a source of stem cells is provided. Suitable sources include 
fetal liver, spleen, or thymus cells or adult marrow or brain cells. 
For example, suitable mouse fetal liver cells may be obtained at day 14 of 
gestation. Mouse fetal thymus cells may be obtained at day 14-18, 
preferably day 15, of gestation. Suitable fetal cells of other mammals are 
obtained at gestation times corresponding to those of mouse. 
Total RNA is prepared by standard procedures from stem cell 
receptor-containing tissue. The total RNA is used to direct cDNA 
synthesis. Standard methods for isolating RNA and synthesizing cDNA are 
provided in standard manuals of molecular biology such as, for example, in 
Sambrook et al , "Molecular Cloning," Second Edition, Cold Spring Harbor 
Laboratory Press (1987) and in Ausubel et al., (Eds), "Current Protocols 
in Molecular Biology," Greene Associates/Wiley Interscience, New York 
(1990). 
The cDNA of the receptors is amplified by known methods. For example, the 
cDNA may be used as a template for amplification by polymerase chain 
reaction (PCR); see Saiki et al., Science, 239, 487 (1988) or Mullis et 
al., U.S. Pat. No. 4,683,195. The sequences of the oligonucleotide primers 
for the PCR amplification are derived from the sequences of known 
receptors, such as from the sequences given in FIGS. 1A and 1B for flk-2 
and in FIG. 2 for flk-1, respectively, preferably from flk-2 (See SEQ. ID. 
NOS. 1, 3 and 5, respectively). The oligonucleotides are synthesized by 
methods known in the art. Suitable methods include those described by 
Caruthers in Science 230, 281-285 (1985). 
In order to isolate the entire protein-coding regions for the receptors of 
the invention, the upstream oligonucleotide is complementary to the 
sequence at the 5' end, preferably encompassing the ATG start codon and at 
least 5-10 nucleotides upstream of the start codon. The downstream 
oligonucleotide is complementary to the sequence at the 3' end, optionally 
encompassing the stop codon. A mixture of upstream and downstream 
oligonucleotides are used in the PCR amplification. The conditions are 
optimized for each particular primer pair according to standard 
procedures. The PCR product is analyzed by electrophoresis for the correct 
size cDNA corresponding to the sequence between the primers. 
Alternatively, the coding region may be amplified in two or more 
overlapping fragments. The overlapping fragments are designed to include a 
restriction site permitting the assembly of the intact cDNA from the 
fragments. 
The amplified DNA encoding the receptors of the invention may be replicated 
in a wide variety of cloning vectors in a wide variety of host cells. The 
host cell may be prokaryotic or eukaryotic. The DNA may be obtained from 
natural sources and, optionally, modified, or may be synthesized in whole 
or in part. 
The vector into which the DNA is spliced may comprise segments of 
chromosomal, non-chromosomal and synthetic DNA sequences. Some suitable 
prokaryotic cloning vectors include plasmids from E. coli, such as colE1, 
pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include 
derivatives of phage DNA such as M13 and other filamentous single-stranded 
DNA phages. 
Isolation of Receptors 
DNA encoding the receptors of the invention are inserted into a suitable 
vector and expressed in a suitable prokaryotic or eucaryotic host. Vectors 
for expressing proteins in bacteria, especially E. coli, are known. Such 
vectors include the PATH vectors described by Dieckmann and Tzagoloff in 
J. Biol. Chem. 260, 1513-1520 (1985). These vectors contain DNA sequences 
that encode anthranilate synthetase (TrpE) followed by a polylinker at the 
carboxy terminus. Other expression vector systems are based on 
beta-galactosidase (pEX); lambda P.sub.L ; maltose binding protein (pMAL); 
and glutathione S-transferase (pGST)--see Gene 67, 31 (1988) and Peptide 
Research 3, 167 (1990). 
Vectors useful in yeast are available. A suitable example is the 2u 
plasmid. 
Suitable vectors for use in mammalian cells are also known. Such vectors 
include well-known derivatives of SV40, adenovirus, retrovirus-derived DNA 
sequences and shuttle vectors derived from combination of functional 
mammalian vectors, such as those described above, and functional plasmids 
and phage DNA. 
Further eukaryotic expression vectors are known in the art (e.g., P. J. 
Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S. Subramani 
et al, Mol. Cell. Biol. 1, 854-864 (1981); R. J. Kaufmann and P. A. Sharp, 
"Amplification And Expression Of Sequences Cotransfected with A Modular 
Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol. 159, 
601-621 (1982); R. J. Kaufmann and P. A. Sharp, Mol. Cell. Biol. 159, 
601-664 (1982); S. I. Scahill et al, "Expression And Characterization Of 
The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary 
Cells," Proc. Natl. Acad. Sci. U.S.A. 80, 4654-4659 (1983); G. Urlaub and 
L. A. Chasin, Proc. Natl. Acad. Sci. U.S.A. 77, 4216-4220, (1980). 
The expression vectors useful in the present invention contain at least one 
expression control sequence that is operatively linked to the DNA sequence 
or fragment to be expressed. The control sequence is inserted in the 
vector in order to control and to regulate the expression of the cloned 
DNA sequence. Examples of useful expression control sequences are the lac 
system, the trp system, the tac system, the trc system, major operator and 
promoter regions of phage lambda, the control region of fd coat protein, 
the glycolytic promoters of yeast, e.g., the promoter for 
3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., 
Pho5, the promoters of the yeast alpha-mating factors, and promoters 
derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the 
early and late promoters or SV40, and other sequences known to control the 
expression of genes of prokaryotic or eukaryotic cells and their viruses 
or combinations thereof. 
Vectors containing the receptor-encoding DNA and control signals are 
inserted into a host cell for expression of the receptor. Some useful 
expression host cells include well-known prokaryotic and eukaryotic cells. 
Some suitable prokaryotic hosts include, for example, E. coli, such as E. 
coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, 
E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus 
subtilis, and Streptomyces. Suitable eukaryotic cells include yeast and 
other fungi, insect, animal cells, such as COS cells and CHO cells, human 
cells and plant cells in tissue culture. 
The human homologs of the mouse receptors described above are isolated by a 
similar strategy. RNA encoding the receptors are obtained from a source of 
human cells enriched for primitive stem cells. Suitable human cells 
include fetal spleen, thymus and liver cells, and umbilical cord blood as 
well as adult brain and bone marrow cells. The human fetal cells are 
preferably obtained on the day of gestation corresponding to mid-gestation 
in mice. The amino acid sequences of the human flk receptors as well as of 
the nucleic acid sequences encoding them are homologous to the amino acid 
and nucleotide sequences of the mouse receptors. 
In the present specification, the sequence of a first protein, such as a 
receptor or a ligand, or of a nucleic acid molecule that encodes the 
protein, is considered homologous to a second protein or nucleic acid 
molecule if the amino acid or nucleotide sequence of the first protein or 
nucleic acid molecule is at least about 30% homologous, preferably at 
least about 50% homologous, and more preferably at least about 65% 
homologous to the respective sequences of the second protein or nucleic 
acid molecule. In the case of proteins having high homology, the amino 
acid or nucleotide sequence of the first protein or nucleic acid molecule 
is at least about 75% homologous, preferably at least about 85% 
homologous, and more preferably at least about 95% homologous to the amino 
acid or nucleotide sequence of the second protein or nucleic acid 
molecule. 
Combinations of mouse oligonucleotide pairs are used as PCR primers to 
amplify the human homologs from the cells to account for sequence 
divergence. The remainder of the procedure for obtaining the human flk 
homologs are similar to those described above for obtaining mouse flk 
receptors. The less than perfect homology between the human flk homologs 
and the mouse oligonucleotides is taken into account in determining the 
stringency of the hybridization conditions. 
Assay for expression of Receptors on Stem Cells 
In order to demonstrate the expression of flk receptors on the surface of 
primitive hematopoietic stem cells, antibodies that recognize the receptor 
are raised. The receptor may be the entire protein as it exists in nature, 
or an antigenic fragment of the whole protein. Preferably, the fragment 
comprises the predicted extra-cellular portion of the molecule. 
Antigenic fragments may be identified by methods known in the art. 
Fragments containing antigenic sequences may be selected on the basis of 
generally accepted criteria of potential antigenicity and/or exposure. 
Such criteria include the hydrophilicity and relative antigenic index, as 
determined by surface exposure analysis of proteins. The determination of 
appropriate criteria is known to those skilled in the art, and has been 
described, for example, by Hopp et al, Proc. Nat'l Acad. Sci. U.S.A. 78, 
3824-3828 (1981); Kyte et al, J. Mol. Biol. 157, 105-132 (1982); Emini, J. 
Virol. 55, 836-839 (1985); Jameson et al, CA BIOS 4, 181-186 (1988); and 
Karplus et al, Naturwissenschaften 72, 212-213 (1985). Amino acid domains 
predicted by these criteria to be surface exposed are selected 
preferentially over domains predicted to be more hydrophobic or hidden. 
The proteins and fragments of the receptors to be used as antigens may be 
prepared by methods known in the art. Such methods include isolating or 
synthesizing DNA encoding the proteins and fragments, and using the DNA to 
produce recombinant proteins, as described above. 
Fragments of proteins and DNA encoding the fragments may be chemically 
synthesized by methods known in the art from individual amino acids and 
nucleotides. Suitable methods for synthesizing protein fragments are 
described by Stuart and Young in "Solid Phase Peptide Synthesis," Second 
Edition, Pierce Chemical Company (1984). Suitable methods for synthesizing 
DNA fragments are described by Caruthers in Science 230, 281-285 (1985). 
If the receptor fragment defines the epitope, but is too short to be 
antigenic, it may be conjugated to a carrier molecule in order to produce 
antibodies. Some suitable carrier molecules include keyhole limpet 
hemocyanin, Ig sequences, TrpE, and human or bovine serum albumen. 
Conjugation may be carried out by methods known in the art. One such 
method is to combine a cysteine residue of the fragment with a cysteine 
residue on the carrier molecule. 
The antibodies are preferably monoclonal. Monoclonal antibodies may be 
produced by methods known in the art. These methods include the 
immunological method described by Kohler and Milstein in Nature 256, 
495-497 (1975) and Campbell in "Monoclonal Antibody Technology, The 
Production and Characterization of Rodent and Human Hybridomas" in Burdon 
et al., Eds, Laboratory Techniques in Biochemistry and Molecular Biology, 
Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well as by 
the recombinant DNA method described by Huse et al in Science 246, 
1275-1281 (1989). 
Polyclonal or monoclonal antisera shown to be reactive with 
receptor-encoded native proteins, such as with flk-1 and flk-2 encoded 
proteins, expressed on the surface of viable cells are used to isolate 
antibody-positive cells. One method for isolating such cells is flow 
cytometry; see, for example, Loken et al., European patent application 
317,156. The cells obtained are assayed for stem cells by engraftment into 
radiation-ablated hosts by methods known in the art; see, for example, 
Jordan et al., Cell 61, 953-963 (1990). 
Criteria for Novel Stem Cell Receptor Tyrosine Kinases Expressed in Stem 
Cells 
Additional novel receptor tyrosine kinase cDNAs are obtained by amplifying 
cDNAs from stem cell populations using oligonucleotides as PCR primers; 
see above. Examples of suitable oligonucleotides are PTK1 and PTK2, which 
were described by Wilks et al. in Proc. Natl. Acad. Sci. U.S.A. 86, 
1603-1607 (1989). Novel cDNA is selected on the basis of differential 
hybridization screening with probes representing known kinases. The cDNA 
clones hybridizing only at low stringency are selected and sequenced. The 
presence of the amino acid triplet DFG confirms that the sequence 
represents a kinase. The diagnostic methionine residue in the WMAPES motif 
is indicative of a receptor-like kinase, as described above. Potentially 
novel sequences obtained are compared to available sequences using 
databases such as Genbank in order to confirm uniqueness. Gene-specific 
oligonucleotides are prepared as described above based on the sequence 
obtained. The oligonucleotides are used to analyze stem cell enriched and 
depleted populations for expression. Such cell populations in mice are 
described, for example, by Jordan et al. in Cell 61, 953-956 (1990); Ikuta 
et al. in Cell 62, 863-864 (1990); Spangrude et al. in Science 241, 58-62 
(1988); and Szilvassy et al. in Blood 74, 930-939 (1989). Examples of such 
human cell populations are described as CD33.sup.- CD34.sup.+ by Andrews 
et al. in the Journal of Experimental Medicine 169, 1721-1731 (1989). 
Other human stem cell populations are described, for example, in Civin et 
al., European Patent Application 395,355 and in Loken et al., European 
Patent Application 317,156. 
Isolating Ligands and Nucleic Acid Molecules Encoding Ligands 
Cells that may be used for obtaining ligands include stromal cells, for 
example stromal cells from fetal liver, fetal spleen, fetal thymus and 
fetal or adult bone marrow. Cell lines expressing ligands are established 
and screened. 
For example, cells such as stromal (non-hematopoietic) cells from fetal 
liver are immortalized by known methods. Examples of known methods of 
immortalizing cells include transduction with a temperature sensitive SV40 
T-antigen expressed in a retroviral vector. Infection of fetal liver cells 
with this virus permits the rapid and efficient establishment of multiple 
independent cell lines. These lines are screened for ligand activity by 
methods known in the art, such as those outlined below. 
Ligands for the receptors of the invention, such as flk-1 and flk-2, may be 
obtained from the cells in several ways. For example, a bioassay system 
for ligand activity employs chimeric tagged receptors; see, for example, 
Flanagan et al., Cell 63, 185-194 (1990). One strategy measures ligand 
binding directly via a histochemical assay. Fusion proteins comprising the 
extracellular receptor domains and secretable alkaline phosphatase (SEAP) 
are constructed and transfected into suitable cells such as NIH/3T3 or COS 
cells. Flanagan et al. refer to such DNA or amino acid constructs as APtag 
followed by the name of the receptor--i.e. APtag-c-kit. The fusion 
proteins bind with high affinity to cells expressing surface-bound ligand. 
Binding is detectable by the enzymatic activity of the alkaline 
phosphatase secreted into the medium. The bound cells, which are often 
stromal cells, are isolated from the APtag-receptor complex. 
For example, some stromal cells that bind APtag-flk1 and APtag-flk2 fusion 
proteins include mouse fetal liver cells (see example 1); human fetal 
spleen cells (see example 3); and human fetal liver (example 3). Some 
stromal fetal thymus cells contain flk-1 ligand (example 3). 
To clone the cDNA that encodes the ligand, a cDNA library is constructed 
from the isolated stromal cells in a suitable expression vector, 
preferably a phage such as CDMS, pSV Sport (BRL Gibco) or piH3, (Seed et 
al., Proc. Natl. Acad. Sci. U.S.A. 84, 3365-3369 (1987)). The library is 
transfected into suitable host cells, such as COS cells. Cells containing 
ligands on their surface are detected by known methods, see above. 
In one such method, transfected COS cells are distributed into single cell 
suspensions and incubated with the secreted alkaline phosphatase-flk 
receptor fusion protein, which is present in the medium from NIH/3T3 or 
COS cells prepared by the method described by Flanagan et al., see above. 
Alkaline phosphatase-receptor fusion proteins that are not bound to the 
cells are removed by centrifugation, and the cells are panned on plates 
coated with antibodies to alkaline phosphatase. Bound cells are isolated 
following several washes with a suitable wash reagent, such as 5% fetal 
bovine serum in PBS, and the DNA is extracted from the cells. Additional 
details of the panning method described above may be found in an article 
by Seed et al., Proc. Natl. Acad. Sci. U.S.A. 84, 3365-3369 (1987). 
In a second strategy, the putative extracellular ligand binding domains of 
the receptors are fused to the transmembrane and kinase domains of the 
human c-fms tyrosine kinase and introduced into 3T3 fibroblasts. The human 
c-fms kinase is necessary and sufficient to transduce proliferative 
signals in these cells after appropriate activation i.e. with the flk-1 or 
flk-2 ligand. The 3T3 cells expressing the chimeras are used to screen 
putative sources of ligand in a cell proliferation assay. 
An alternate approach for isolating ligands using the fusion 
receptor-expressing 3T3 cells and insertional activation is also possible. 
A retrovirus is introduced into random chromosomal positions in a large 
population of these cells. In a small fraction, the retrovirus is inserted 
in the vicinity of the ligand-encoding gene, thereby activating it. These 
cells proliferate due to autocrine stimulation of the receptor. The ligand 
gene is "tagged" by the retrovirus, thus facilitating its isolation. 
EXAMPLES 
Example 1 
Cells containing mouse flk-1 and flk-2 ligands. Murine stromal cell line 
2018. 
In order to establish stromal cell lines, fetal liver cells are 
disaggregated with collagen and grown in a mixture of Dulbecco's Modified 
Eagle's Medium (DMEM) and 10% heat-inactivated fetal calf serum at 
37.degree. C. The cells are immortalized by standard methods. A suitable 
method involves introducing DNA encoding a growth regulating- or 
oncogene-encoding sequence into the target host cell. The DNA may be 
introduced by means of transduction in a recombinant viral particle or 
transfection in a plasmid. See, for example, Hammerschmidt et al., Nature 
340, 393-397 (1989) and Abcouwer et al, Biotechnology 7, 939-946 (1989). 
Retroviruses are the preferred viral vectors, although SV40 and 
Epstein-Barr virus can also serve as donors of the growth-enhancing 
sequences. A suitable retrovirus is the ecotropic retrovirus containing a 
temperature sensitive SV40 T-antigen (tsA58) and a G418 resistance gene 
described by McKay in Cell 66, 713-729 (1991). After several days at 
37.degree. C., the temperature of the medium is lowered to 32.degree. C. 
Cells are selected with G418 (0.5 mg/ml). The selected cells are expanded 
and maintained. 
A mouse stromal cell line produced by this procedure is called 2018 and was 
deposited on Oct. 30, 1991 in the American Type Culture Collection, 
Rockville, Md., U.S.A. (ATCC); accession number CRL 10907. 
Example 2 
Cells containing human flk-1 and flk-2 ligands. 
Human fetal liver (18, 20, and 33 weeks after abortion), spleen (18 weeks 
after abortion), or thymus (20 weeks after abortion) is removed at the 
time of abortion and stored on ice in a balanced salt solution. After 
mincing into 1 mm fragments and forcing through a wire mesh, the tissue is 
washed one time in Hanks Balanced Salt Solution (HBSS). 
The disrupted tissue is centrifuged at 200.times.g for 15 minutes at room 
temperature. The resulting pellet is resuspended in 10-20 ml of a tissue 
culture grade trypsin-EDTA solution (Flow Laboratories). The resuspended 
tissue is transferred to a sterile flask and stirred with a stirring bar 
at room temperature for 10 minutes. One ml of heat-inactivated fetal 
bovine calf serum (Hyclone) is added to a final concentration of 10% in 
order to inhibit trypsin activity. Collagenase type IV (Sigma) is added 
from a stock solution (10 mg/ml in HBSS) to a final concentration of 100 
ug/ml in order to disrupt the stromal cells. The tissue is stirred at room 
temperature for an additional 2.5 hours; collected by centrifugation 
(400.times.g, 15 minutes); and resuspended in "stromal medium," which 
contains Iscove's modification of DMEM supplemented with 10% 
heat-inactivated fetal calf serum, 5% heat-inactivated human serum 
(Sigma), 4 mM L-glutamine, 1.times. sodium pyruvate, (stock of 100.times. 
Sigma), 1.times. non-essential amino acids (stock of 100.times., Flow), 
and a mixture of antibiotics kanomycin, neomycin, penicillin, 
streptomycin. Prior to resuspending the pellet in the stromal medium, the 
pellet is washed one time with HBSS. It is convenient to suspend the cells 
in 60 ml of medium. The number of cultures depends on the amount of 
tissue. 
Example 3 
Isolating Stromal cells 
Resuspended Cells (example 2) that are incubated at 37.degree. C. with 5% 
carbon dioxide begin to adhere to the plastic plate within 10-48 hours. 
Confluent monolayers may be observed within 7-10 days, depending upon the 
number of cells plated in the initial innoculum. Non-adherent and highly 
refractile cells adhering to the stromal cell layer as colonies are 
separately removed by pipetting and frozen. Non-adherent cells are likely 
sources of populations of self-renewing stem cells containing flk-2. The 
adherent stromal cell layers are frozen in aliquots for future studies or 
expanded for growth in culture. 
An unexpectedly high level of APtag-flk-2 fusion protein binding to the 
fetal spleen cells is observed. Two fetal spleen lines are grown in 
"stromal medium," which is described in example 2. 
Non-adherent fetal stem cells attach to the stromal cells and form colonies 
(colony forming unit--CFU). Stromal cells and CFU are isolated by means of 
sterile glass cylinders and expanded in culture. A clone, called Fsp 
62891, contains the flk-2 ligand. Fsp 62891 was deposited in the American 
Type Culture Collection, Rockville, Md., U.S.A on Nov. 21, 1991, accession 
number CRL 10935. 
Fetal liver and fetal thymus cells are prepared in a similar way. Both of 
these cell types produce ligands of flk-1 and, in the case of liver, some 
flk-2. One such fetal thymus cell line, called F.thy 62891, and one such 
fetal liver cell line, called FL 62891, were deposited in the American 
Type Culture Collection, Rockville, Md., U.S.A on Nov. 21, 1991 and Apr. 
2, 1992, respectively, accession numbers CRL 10936 and CRL 11005, 
respectively. 
Stable human cell lines are prepared from fetal cells with the same 
temperature sensitive immortalizing virus used to prepare the murine cell 
line described in example 1. 
Example 4 
Isolation of human stromal cell clone 
Highly refractile cells overgrow patches of stromal cells, presumably 
because the stromal cells produce factors that allow the formation of the 
CFU. To isolate stromal cell clones, sterile glass cylinders coated with 
vacuum grease are positioned over the CFU. A trypsin-EDTA solution (100 
ml) is added in order to detach the cells. The cells are added to 5 ml of 
stromal medium and each (clone) plated in a single well of 6-well plate. 
Example 5 
Plasmid (AP-tag) for expressing secretable alkaline phosphatase (SEAP) 
Plasmids that express secretable alkaline phosphatase are described by 
Flanagan and Leder in Cell 63, 185-194 (1990). The plasmids contain a 
promoter, such as the LTR promoter; a polylinker, including HindIII and 
BglII; DNA encoding SEAP; a poly-A signal; and ampicillin resistance gene; 
and replication site. 
Example 6 
Plasmid for expressing APtag-flk-2 and APtag-flk-1 fusion proteins 
Plasmids that express fusion proteins of SEAP and the extracellular portion 
of either flk-1 or flk-2 are prepared in accordance with the protocols of 
Flanagan and Leder in Cell 53, 185-194 (1990) and Berger et al., Gene 66, 
1-10 (1988). Briefly, a HindIII-Bam HI fragment containing the 
extracellular portion of flk-1 or flk-2 is prepared and inserted into the 
HindIII-BglII site of the plasmid described in example 5. 
Example 7 
Production Of APtaq-flk-1 Or -flk-2 Fusion Protein 
The plasmids from Example 6 are transfected into Cos-7 cells by 
DEAE-dextran (as described in Current Protocols in Molecular Biology, Unit 
16.13, "Transient Expression of Proteins Using Cos Cells," 1991); and 
cotransfected with a selectable marker, such as pSV7neo, into NIH/3T3 
cells by calcium precipitation. The NIH/3T3 cells are selected with 600 
.mu.g/ml G418 in 100 mm plates. Over 300 clones are screened for secretion 
of placental alkaline phosphatase activity. The assay is performed by 
heating a portion of the supernatant at 65.degree. C. for 10 minutes to 
inactivate background phosphatase activity, and measuring the OD.sub.405 
after incubating with 1M diethanolamine (pH 9.8), 0.5 mM MgCl.sub.2, 10 mM 
L-homoarginine (a phosphatase inhibitor), 0.5 mg/ml BSA, and 12 mM 
p-nitrophenyl phosphate. Human placental alkaline phosphatase is used to 
perform a standard curve. The APtaq-flk-1 clones (F-1AP21-4) produce up to 
10 .mu.g alkaline phosphatase activity/ml and the APtaq-flk-2 clones 
(F-2AP26-0) produce up to 0.5 .mu.g alkaline phosphatase activity/ml. 
Example 8 
Assay For APtaq-flk-1 Or APtaq-flk-2 Binding To Cells 
The binding of APtaq-flk-1 or APtag-flk-2 to cells containing the 
appropriate ligand is assayed by standard methods. See, for example, 
Flanagan and Leder, Cell 63:185-194, 1990). Cells (i.e., mouse stromal 
cells, human fetal liver, spleen or thymus, or various control cells) are 
grown to confluency in six-well plates and washed with HBHA (Hank's 
balanced salt solution with 0.5 mg/ml BSA, 0.02% NaN.sub.3, 20 mM HEPES, 
pH 7.0). Supernatants from transfected COS or NIH/3T3 cells containing 
either APtaq-flk-1 fusion protein, APtag-flk-2 fusion protein, or APtag 
without a receptor (as a control) are added to the cell monolayers and 
incubated for two hours at room temperature on a rotating platform. The 
concentration of the APtaq-flk-1 fusion protein, APtag-flk-2 fusion 
protein, or APtag without a receptor is 60 ng/ml of alkaline phosphatase 
as determined by the standard alkaline phosphatase curve (see above). The 
cells are then rinsed seven times with HBHA and lysed in 350 .mu.l of 1% 
Triton X-100, 10 mM Tris-HCl (pH 8.0). The lysates are transferred to a 
microfuge tube, along with a further 150 .mu.l rinse with the same 
solution. After vortexing vigorously, the samples are centrifuged for five 
minutes in a microfuge, heated at 65.degree. C. for 12 minutes to 
inactivate cellular phosphatases, and assayed for phosphatase activity as 
described previously. Results of experiments designed to show the time and 
dose responses of binding between stromal cells containing the ligands to 
flk-2 and flk-1 (2018) and APtag-flk-2, APtag-flk-1 and APtag without 
receptor (as a control) are shown in FIGS. 3 and 4, respectively. 
Example 8A 
Plasmids for expressing flk1/fms and flk2/fms fusion proteins 
Plasmids that express fusion proteins of the extracellular portion of 
either flk-1 or flk-2 and the intracellular portion of c-fms (also known 
as colony-stimulating factor-1 receptor) are prepared in a manner similar 
to that described under Example 6 (Plasmid for expressing APtag-flk-2 and 
APtag-flk-1 fusion proteins). Briefly, a Hind III-Bam HI fragment 
containing the extracellular portion of flk1 or flk2 is prepared and 
inserted into the Hind III-Bgl II site of a pLH expression vector 
containing the intracellular portion of c-fms. 
8B. Expression of flk1/fms or flk2/fms in 3T3 cells 
The plasmids from Example 11 are transfected into NIH/3T3 cells by calcium. 
The intracellular portion of c-fms is detected by Western blotting. 
Example 9 
Cloning and Expression of cDNA Coding For Mouse Ligand To flk-1 and flk-2 
Receptors 
cDNA expressing mouse ligand for flk-1 and flk-2 is prepared by known 
methods. See, for example, Seed, B., and Aruffo, A. PNAS 84:3365-3369, 
1987; Simmons, D. and Seed, B. J. Immunol. 141:2797-2800; and D'Andrea, A. 
D., Lodish, H. F. and Wong, G. G. Cell 57:277-285, 1989). 
The protocols are listed below in sequence: (a) RNA isolation; (b) poly A 
RNA preparation; (c) cDNA synthesis; (d) cDNA size fractionation; (e) 
propagation of plasmids (vector); (f) isolation of plasmid DNA; (g) 
preparation of vector pSV Sport (BRL Gibco) for cloning; (h) compilation 
of buffers for the above steps; (i) Transfection of cDNA encoding Ligands 
in Cos 7 Cells; (j) panning procedure; (k) Expression cloning of flk-1 or 
flk-2 ligand by establishment of an autocrine loop. 
9a. Guanidinium thiocyanate/LiCl Protocol for RNA Isolation 
For each ml of mix desired, 0.5 g guanidine thiocyanate (GuSCN) is 
dissolved in 0.55 ml of 25% LiCl (stock filtered through 0.45 micron 
filter). 20 .mu.l of mercaptoethanol is added. (The resulting solution is 
not good for more than about a week at room temperature.) 
The 2018 stromal cells are centrifuged, and 1 ml of the solution described 
above is added to up to 5.times.10.sup.7 cells. The cells are sheared by 
means of a polytron until the mixture is non-viscous. For small scale 
preparations (&lt;10.sup.8 cells), the sheared mixture is layered on 1.5 ml 
of 5.7M CsCl (RNase free; 1.26 g CsCl added to every ml 10 mM EDTA pH8), 
and overlaid with RNase-free water if needed. The mixture is spun in an 
SW55 rotor at 50 krpm for 2 hours. For large scale preparations, 25 ml of 
the mixture is layered on 12 ml CsCl in an SW28 tube, overlaid as above, 
and spun at 24 krpm for 8 hours. The contents of the tube are aspirated 
carefully with a sterile pasteur pipet connected to a vacuum flask. Once 
past the CsCl interface, a band around the tube is scratched with the 
pipet tip to prevent creeping of the layer on the wall down the tube. The 
remaining CsCl solution is aspirated. The resulting pellet is taken up in 
water, but not redissolved. 1/10 volume of sodium acetate and three 
volumes of ethanol are added to the mixture, and spun. The pellet is 
resuspended in water at 70.degree. C., if necessary. The concentration of 
the RNA is adjusted to 1 mg/ml and frozen. 
It should be noted that small RNA molecules (e.g., 5S) do not come down. 
For small amounts of cells, the volumes are scaled down, and the mixture 
is overlaid with GuSCN in RNase-free water on a gradient (precipitation is 
inefficient when RNA is dilute). 
9b. Poly A.sup.- RNA preparation 
(All buffers mentioned are compiled separately below) 
A disposable polypropylene column is prepared by washing with 5M NaOH and 
then rinsing with RNase-free water. For each milligram of total RNA, 
approximately 0.3 ml (final packed bed) of oligo dT cellulose is added. 
The oligo dT cellulose is prepared by resuspending approximately 0.5 ml of 
dry powder in 1 ml of 0.1M NaOH and transferring it into the column, or by 
percolating 0.1M NaOH through a previously used column. The column is 
washed with several column volumes of RNase-free water until the pH is 
neutral, and rinsed with 2-3 ml of loading buffer. The column bed is 
transferred to a sterile 15 ml tube using 4-6 ml of loading buffer. 
Total RNA from the 2018 cell line is heated to 70.degree. C. for 2-3 
minutes. LiCl from RNase-free stock is added to the mixture to a final 
concentration of 0.5M. The mixture is combined with oligo dT cellulose in 
the 15 ml tube, which is vortexed or agitated for 10 minutes. The mixture 
is poured into the column, and washed with 3 ml loading buffer, and then 
with 3 ml of middle wash buffer. The mRNA is eluted directly into an SW55 
tube with 1.5 ml of 2 mM EDTA and 0.1% SDS, discarding the first two or 
three drops. 
The eluted mRNA is precipitated by adding 1/10 volume of 3M sodium acetate 
and filling the tube with ethanol. The contents of the tube are mixed, 
chilled for 30 minutes at -20.degree. C., and spun at 50 krpm at 5.degree. 
C. for 30 minutes. After the ethanol is decanted, and the tube air dried, 
the mRNA pellet is resuspended in 50-100 .mu.l of RNase-free water. 5 
.mu.l of the resuspended mRNA is heated to 70.degree. C. in 
MOPS/EDTA/formaldehyde, and examined on an RNase-free 1% agarose gel. 
9c. cDNA Synthesis 
The protocol used is a variation of the method described by Gubler and 
Hoffman in Gene 25, 263-270 (1983) . 
1. First Strand. 4 .mu.g of mRNA is added to a microfuge tube, heated to 
approximately 100.degree. C. for 30 seconds, quenched on ice. The volume 
is adjusted to 70 .mu.l with RNAse-free water. 20 .mu.l of RT1 buffer, 2 
.mu.l of RNAse inhibitor (Boehringer 36 u/.mu.l), 1 .mu.l of 5 .mu.g/.mu.l 
of oligo dT (Collaborative Research), 2.5 .mu.l of 20 mM dXTP's 
(ultrapure--US Biochemicals), 1 .mu.l of 1M DTT and 4 .mu.l of RT-XL (Life 
Sciences, 24 u/.mu.l) are added. The mixture is incubated at 42.degree. C. 
for 40 minutes, and inactivated by heating at 70.degree. C. for 10 
minutes. 
2. Second Strand. 320 .mu.l of RNAse-free water, 80 .mu.l of RT2 buffer, 5 
.mu.l of DNA Polymerase I (Boehringer, 5 U/.mu.l), 2 .mu.l RNAse H (BRL 2 
u/.mu.l) are added to the solution containing the first strand. The 
solution is incubated at 15.degree. C. for one hour and at 22.degree. C. 
for an additional hour. After adding 20 .mu.l of 0.5M EDTA, pH 8.0, the 
solution is extracted with phenol and precipitated by adding NaCl to 0.5M 
linear polyacrylamide (carrier) to 20 .mu.g/ml, and filling the tube with 
EtOH. The tube is spun for 2-3 minutes in a microfuge, vortexed to 
dislodge precipitated material from the wall of the tube, and respun for 
one minute. 
3. Adaptors. Adaptors provide specific restriction sites to facilitate 
cloning, and are available from BRL Gibco, New England Biolabs, etc. Crude 
adaptors are resuspended at a concentration of 1 .mu.g/.mu.l. MgSO.sub.4 
is added to a final concentration of 10 mM, followed by five volumes of 
EtOH. The resulting precipitate is rinsed with 70% EtOH and resuspended in 
TE at a concentration of 1 .mu.g/.mu.l. To kinase, 25 .mu.l of resuspended 
adaptors is added to 3 .mu.l of 10.times. kinasing buffer and 20 units of 
kinase. The mixture is incubated at 37.degree. C. overnight. The 
precipitated cDNA is resuspended in 240 .mu.l of TE (10/1). After adding 
30 .mu.l of 10.times. low salt buffer, 30 .mu.l of 10.times. ligation 
buffer with 0.1 mM ATP, 3 .mu.l (2.4 .mu.g) of kinased 12-mer adaptor 
sequence, 2 .mu.l (1.6 .mu.g) of kinased 8-mer adaptor sequence, and 1 
.mu.l of T4 DNA ligase (BioLabs, 400 u/.mu.l, or Boehringer, 1 Weiss unit 
ml), the mixture is incubated at 15.degree. C. overnight. The cDNA is 
extracted with phenol and precipitated as above, except that the extra 
carrier is omitted, and resuspended in 100 .mu.l of TE. 
9d. cDNA Size Fractionation. 
A 20% KOAc, 2 mM EDTA, 1 .mu.g/ml ethidium bromide solution and a 5% KOAc, 
2 mM EDTA, 1 .mu.g/ml ethidium bromide solution are prepared. 2.6 ml of 
the 20% KOAc solution is added to the back chamber of a small gradient 
maker. Air bubbles are removed from the tube connecting the two chambers 
by allowing the 20% solution to flow into the front chamber and forcing 
the solution to return to the back chamber by tilting the gradient maker. 
The passage between the chambers is closed, and 2.5 ml of 5% solution is 
added to the front chamber. Any liquid in the tubing from a previous run 
is removed by allowing the 5% solution to flow to the end of the tubing, 
and then to return to its chamber. The apparatus is placed on a stirplate, 
and, with rapid stirring, the topcock connecting the two chambers and the 
front stopcock are opened. A polyallomer 5W55 tube is filled from the 
bottom with the KOAc solution. The gradient is overlaid with 100 .mu.l of 
cDNA solution, and spun for three hours at 50 k rpm at 22.degree. C. To 
collect fractions from the gradient, the SW55 tube is pierced close to the 
bottom of the tube with a butterfly infusion set (with the luer hub 
clipped off). Three 0.5 ml fractions and then six 0.25 ml fractions are 
collected in microfuge tubes (approximately 22 and 11 drops, 
respectively). The fractions are precipitated by adding linear 
polyacrylamide to 20 .mu.g/ml and filling the tube to the top with 
ethanol. The tubes are cooled, spun in a microfuge tube for three minutes, 
vortexed, and respun for one minute. The resulting pellets are rinsed with 
70% ethanol and respun, taking care not to permit the pellets to dry to 
completion. Each 0.25 ml fraction is resuspended in 10 .mu.l of TE, and 1 
.mu.l is run on a 1% agarose minigel. The first three fractions, and the 
last six which contain no material smaller than 1 kb are pooled. 
9e. Propagation of Plasmids 
SupF plasmids are selected in nonsuppressing bacterial hosts containing a 
second plasmid, p3, which contains amber mutated ampicillin and 
tetracycline drug resistance elements. See Seed, Nucleic Acids Res., 11, 
2427-2445 (1983). The p3 plasmid is derived from RP1, is 57 kb in length, 
and is a stably maintained, single copy episome. The ampicillin resistance 
of this plasmid reverts at a high rate so that amp.sup.r plasmids usually 
cannot be used in p3-containing strains. Selection for tetracycline 
resistance alone is almost as good as selection for 
ampicillin-tetracycline resistance. However, spontaneous appearance of 
chromosomal suppressor tRNA mutations presents an unavoidable background 
(frequency about 10.sup.-9) in this system. Colonies arising from 
spontaneous suppressor mutations are usually larger than colonies arising 
from plasmid transformation. Suppressor plasmids are selected in Luria 
broth (LB) medium containing ampicillin at 12.5 .mu.g/ml and tetracycline 
at 7.5 .mu.g/ml. For scaled-up plasmid preparations, M9 Casamino acids 
medium containing glycerol (0.8%) is employed as a carbon source. The 
bacteria are grown to saturation. 
Alternatively, pSV Sport (BRL, Gaithersberg, Md.) may be employed to 
provide SV40 derived sequences for replication, transcription initiation 
and termination in COS 7 cells, as well as those sequences necessary for 
replication and ampicillin resistance in E. coli. 
9f. Isolation of Vector DNA/Plasmid 
One liter of saturated bacterial cells are spun down in J6 bottles at 4.2 k 
rpm for 25 minutes. The cells are resuspended in 40 ml 10 mM EDTA, pH 8. 
80 ml 0.2M NaOH and 1% SDS are added, and the mixture is swirled until it 
is clear and viscous. 40 ml 5M KOAc, pH 4.7 (2.5M KOAc, 2.5M HOAc) is 
added, and the mixture is shaken semi-vigorously until the lumps are 
approximately 2-3 mm in size. The bottle is spun at 4.2 k rpm for 5 
minutes. The supernatant is poured through cheesecloth into a 250 ml 
bottle, which is then filled with isopropyl alcohol and centrifuged at 4.2 
k rpm for 5 minutes. The bottle is gently drained and rinsed with 70% 
ethanol, taking care not to fragment the pellet. After inverting the 
bottle and removing traces of ethanol, the mixture is resuspended in 3.5 
ml Tris base/EDTA (20 mM/10 mM). 3.75 ml of resuspended pellet and 0.75 ml 
10 mg/ml ethidium bromide are added to 4.5 g CsCl. VTi80 tubes are filled 
with solution, and centrifuged for at least 2.5 hours at 80 k rpm. Bands 
are extracted by visible light with 1 ml syringe and 20 gauge or lower 
needle. The top of the tube is cut off with scissors, and the needle is 
inserted upwards into the tube at an angle of about 30 degrees with 
respect to the tube at a position about 3 mm beneath the band, with the 
bevel of the needle up. After the band is removed, the contents of the 
tube are poured into bleach. The extracted band is deposited in a 13 ml 
Sarstedt tube, which is then filled to the top with n-butanol saturated 
with 1M NaCl extract. If the amount of DNA is large, the extraction 
procedure may be repeated. After aspirating the butanol into a trap 
containing 5M NaOH to destroy ethidium, an approximately equal volume of 
1M ammonium acetate and approximately two volumes of 95% ethanol are added 
to the DNA, which is then spun at 10 k rpm for 5 minutes. The pellet is 
rinsed carefully with 70% ethanol, and dried with a swab or lyophilizer. 
9g. Preparation of Vector for Cloning 
20 .mu.g of vector is cut in a 200 .mu.l reaction with 100 units of BstXI 
(New York Biolabs) at 50.degree. C. overnight in a well thermostated, 
circulating water bath. Potassium acetate solutions (5 and 20%) are 
prepared in 5W55 tubes as described above. 100 .mu.l of the digested 
vector is added to each tube and spun for three hours, 50 k rpm at 
22.degree. C. Under 300 nm UV light, the desired band is observed to 
migrate 2/3 of the length of the tube. Forward trailing of the band 
indicates that the gradient is overloaded. The band is removed with a 1 ml 
syringe fitted with a 20 gauge needle. After adding linear polyacrylamide 
and precipitating the plasmid by adding three volumes of ethanol, the 
plasmid is resuspended in 50 .mu.l of TE. Trial ligations are carried out 
with a constant amount of vector and increasing amounts of cDNA. Large 
scale ligation are carried out on the basis of these trial ligations. 
Usually the entire cDNA prep requires 1-2 .mu.g of cut vector. 
9h. Buffers 
______________________________________ 
Loading Buffer: 
.5M LiCl, 10 mM Tris pH 7.5, 1 mM 
EDTA .1% SDS. 
Middle Wash Buffer: 
.15M LiCl, 10 mM Tris pH 7.5, 1 mM 
EDTA .1% SDS. 
RT1 Buffer: .25M Tris pH 8.8 (8.2 at 42.sup.-), .25M 
KCl, 30 mM MgCl.sub.2. 
RT2 Buffer: .1M Tris pH 7.5, 25 mM MgCl2, .5M 
KCl, .25 mg/ml BSA, 50 mM 
dithiothreitol (DTT). 
10 X Low Salt: 
60 mM Tris pH 7.5, 60 mM MgCl.sub.2, 50 
mM NaCl, 2.5 mg/ml BSA 70 mM DME 
10 X Ligation 
1 mM ATP, 20 mM DTT, 1 mg/ml BSA 
Additions: 10 mM spermidine. 
10 X Kinasing Buffer: 
.5M Tris pH 7.5, 10 mM ATP, 20 mM 
DTT, 10 mM spermidine, 1 mg/ml BSA 
100 mM MgCl.sub.2 
______________________________________ 
9i. Transfection of cDNA encoding Ligands in Cos 7 Cells 
Cos 7 cells are split 1:5 into 100 mm plates in Dulbecco's modified Eagles 
medium (DME)/10% fetal calf serum (FCS), and allowed to grow overnight. 3 
ml Tris/DME (0.039M Tris, pH 7.4 in DME) containing 400 .mu.g/ml 
DEAE-dextran (Sigma, D-9885) is prepared for each 100 mm plate of Cos 7 
cells to be transfected. 10 ug of plasmid DNA preparation per plate is 
added. The medium is removed from the Cos-7 cells and the DNA/DEAE-dextran 
mixture is added. The cells are incubated for 4.5 hours. The medium is 
removed from the cells, and replaced with 3 ml of DME containing 2% fetal 
calf serum (FCS) and 0.1 mM chloroquine. The cells are incubated for one 
hour. After removing the chloroquine and replacing with 1.5 ml 20% 
glycerol in PBS, the cells are allowed to stand at room temperature for 
one minute. 3 ml Tris/DME is added, and the mixture is aspirated and 
washed two times with Tris/DME. 10 ml DME/10% FCS is added and the mixture 
is incubated overnight. The transfected Cos 7 cells are split 1:2 into 
fresh 100 mm plates with (DME)/10% FCS and allowed to grow. 
9j. Panning Procedure for Cos 7 cells Expressing Ligand 
1) Antibody-coated plates: 
Bacteriological 100 mm plates are coated for 1.5 hours with rabbit 
anti-human placental alkaline phosphatase (Dako, Calif.) diluted 1:500 in 
10 ml of 50 mM Tris.HCl, pH 9.5. The plates are washed three times with 
0.15M NaCl, and incubated with 3 mg BSA/ml PBS overnight. The blocking 
solution is aspirated, and the plates are utilized immediately or frozen 
for later use. 
2) Panning cells: 
The medium from transfected Cos 7 cells is aspirated, and 3 ml PBS/0.5 mM 
EDTA/0.02% sodium azide is added. The plates are incubated at 37.degree. 
C. for thirty minutes in order to detach the cells. The cells are 
triturated vigorously with a pasteur pipet and collected in a 15 ml 
centrifuge tube. The plate is washed with a further 2 ml PBS/EDTA/azide 
solution, which is then added to the centrifuge tube. After centrifuging 
at 200.times.g for five minutes, the cells are resuspended in 3 ml of 
APtaq-flk-1 (F-1AP21-4) or flk-2 (F-2AP26-0) supernatant from transfected 
NIH/3T3 cells (see Example 7.), and incubated for 1.5 hours on ice. The 
cells are centrifuged again at 200.times.g for five minutes. The 
supernatant is aspirated, and the cells are resuspended in 3 ml 
PBS/EDTA/azide solution. The cell suspension is layered carefully on 3 ml 
PBS/EDTA/azide/2% Ficoll, and centrifuged at 200.times.g for four minutes. 
The supernatant is aspirated, and the cells are resuspended in 0.5 ml 
PBS/EDTA/azide solution. The cells are added to the antibody-coated plates 
containing 4 ml PBS/EDTA/azide/5% FBS, and allowed to stand at room 
temperature one to three hours. Non-adhering cells are removed by washing 
gently two or three times with 3 ml PBS/5% FBS. 
3) Hirt Supernatant: 
0.4 ml 0.6% SDS and 10 mM EDTA are added to the panned plates, which are 
allowed to stand 20 minutes. The viscuous mixture is added by means of a 
pipet into a microfuge tube. 0.1 ml 5M NaCl is added to the tube, mixed, 
and chilled on ice for at least five hours. The tube is spun for four 
minutes, and the supernatant is removed carefully. The contents of the 
tube are extracted with phenol once, or, if the first interface is not 
clean, twice. Ten micrograms of linear polyacrylamide (or other carrier) 
is added, and the tube is filled to the top with ethanol. The resulting 
precipitate is resuspended in 0.1 ml water or TE. After adding 3 volumes 
of EtOH/NaOAc, the cells are reprecipitated and resuspended in 0.1 ml 
water or TE. The cDNA obtained is transfected into any suitable E. coli 
host by electroporation. Suitable hosts are described in various catalogs, 
and include MC1061/p3 or Electromax DH10B Cells of BRL Gibco. The cDNA is 
extracted by conventional methods. 
The above panning procedure is repeated until a pure E. coli clone bearing 
the cDNA as a unique plasmid recombinant capable of transfecting mammalian 
cells and yielding a positive panning assay is isolated. Normally, three 
repetitions are sufficient. 
9k. Expression cloning of flk1 or flk2 ligand by establishment of an 
autocrine loop 
Cells expressing flk1/fms or flk2/fms (Example 10) are transfected with 
20-30 ug of a cDNA library from either flk1 ligand or flk2 ligand 
expressing stromal cells, respectively. The cDNA library is prepared as 
described above (a-h). The cells are co-transfected with 1 ug pLTR neo 
cDNA. Following transfection the cells are passaged 1:2 and cultured in 
800 ug/ml of G418 in Dulbecco's medium (DME) supplemented with 10% CS. 
Approximately 12 days later the colonies of cells are passaged and plated 
onto dishes coated with poly-D-lysine (1 mg/ml) and human fibronectin (15 
ug/ml). The culture medium is defined serum-free medium which is a mixture 
(3:1) of DME and Ham's F12 medium. The medium supplements are 8 mM 
NaHCO.sub.3, 15 mM HEPES pH 7.4, 3 mM histidine, 4 uM MnCl.sub.2, 10 uM 
ethanolamine, 0.1 uM selenous acid, 2 uM hydrocortisone, 5 ug/ml 
transferrin, 500 ug/ml bovine serum albumin/linoleic acid complex, and 20 
ug/ml insulin (Ref. Zhan, X, et al. Oncogene 1: 369-376,1987). The 
cultures are refed the next day and every 3 days until the only cells 
capable of growing under the defined medium condition remain. The 
remaining colonies of cells are expanded and tested for the presence of 
the ligand by assaying for binding of APtag-flk1 or APtag-flk2 to the 
cells (as described in Example 8). The DNA would be rescued from cells 
demonstrating the presence of the flk1 or flk2 ligand and the sequence. 
Example 10 
Expression of Ligand cDNA 
The cDNA is sequenced, and expressed in a suitable host cell, such as a 
mammalian cell, preferably COS, CHO or NIH/3T3 cells. The presence of the 
ligand is confirmed by demonstrating binding of the ligand to APtag-flk2 
fusion protein (see above). 
Example 11 
Chemical Cross Linking of Receptor and Ligand 
Cross linking experiments are performed on intact cells using a 
modification of the procedure described by Blume-Jensen et al et al., EMBO 
J., 10, 4121-4128 (1991). Cells are cultured in 100 mm tissue culture 
plates to subconfluence and washed once with PBS-0.1% BSA. 
To examine chemical cross linking of soluble receptor to membrane-bound 
ligand, stromal cells from the 2018 stromal cell line are incubated with 
conditioned media (CM) from transfected 3T3 cells expressing the soluble 
receptor Flk2-APtag. Cross linking studies of soluble ligand to membrane 
bound receptor are performed by incubating conditioned media from 2018 
cells with transfected 3T3 cells expressing a Flk2-fms fusion construct. 
Binding is carried out for 2 hours either at room temperature with CM 
containing 0.02% sodium azide to prevent receptor internalization or at 
4.degree. C. with CM (and buffers) supplemented with sodium vanadate to 
prevent receptor dephosphorylation. Cells are washed twice with PBS-0.1% 
BSA and four times with PBS. 
Cross linking is performed in PBS containing 250 mM disuccinimidyl suberate 
(DSS; Pierce) for 30 minutes at room temperature. The reaction is quenched 
with Tris-HCL pH7.4 to a final concentration of 50 mM. 
Cells are solubilized in solubilization buffer: 0.5% Triton-X100, 0.5% 
deoxycholic acid, 20 mM Tris pH 7.4, 150 mM NaCl, 10 mM EDTA, 1 mM PMFS, 
50 mg/ml aprotinin, 2 mg/ml bestatin, 2 mg/ml pepstatin and 10 mg/ml 
leupeptin. Lysed cells are immediately transferred to 1.5 ml Nalgene tubes 
and solubilized by rolling end to end for 45 minutes at 4.degree. C. 
Lysates are then centrifuged in a microfuge at 14,000 g for 10 minutes. 
Solubilized cross linked receptor complexes are then retrieved from 
lysates by incubating supernatants with 10% (v/v) wheat germ 
lectin-Sepharose 6MB beads (Pharmacia) at 4.degree. C. for 2 hours or 
overnight. 
Beads are washed once with Tris-buffered saline (TBS) and resuspended in 
2.times. SDS-polyacrylamide nonreducing sample buffer. Bound complexes are 
eluted from the beads by heating at 95.degree. C. for 5 minutes. Samples 
are analyzed on 4-12% gradient gels (NOVEX) under nonreducing and reducing 
conditions (0.35M 2-mercaptoethanol) and then transferred to PVDF 
membranes for 2 hours using a Novex blotting apparatus. Blots are blocked 
in TBS-3% BSA for 1 hour at room temperature followed by incubation with 
appropriate antibody. 
Cross linked Flk2-APtag and Flk2-fms receptors are detected using rabbit 
polyclonal antibodies raised against human alkaline phosphatase and fms 
protein, respectively. The remainder of the procedure is carried out 
according to the instructions provided in the ABC Kit (Pierce). The kit is 
based on the use of a biotinylated secondary antibody and 
avidin-biotinylated horseradish peroxidase complex for detection. 
SUPPLEMENTAL ENABLEMENT 
The invention as claimed is enabled in accordance with the above 
specification and readily available references and starting materials. 
Nevertheless, Applicants have deposited with the American Type Culture 
Collection, Rockville, Md., U.S.A. (ATCC) the cell lines listed below: 
2018, ATCC accession no. CRL 10907, deposited Oct. 30, 1991. 
Fsp 62891, ATCC accession no. CRL 10935, deposited Nov. 21, 1991. 
F.thy 62891, ATCC accession no. CRL 10936, deposited Nov. 21, 1991. 
FL 62891, ATCC accession no. CRL 11005, deposited Apr. 2, 1992. 
These deposits were made under the provisions of the Budapest Treaty on the 
International Recognition of the Deposit of Microorganisms for the 
Purposes of Patent Procedure and the regulations thereunder (Budapest 
Treaty). This assures maintenance of a viable culture for 30 years from 
date of deposit. The organisms will be made available by ATCC under the 
terms of the Budapest Treaty, and subject to an agreement between 
Applicants and ATCC which assures unrestricted availability upon issuance 
of the pertinent U.S. patent. Availability of the deposited strains is not 
to be construed as a license to practice the invention in contravention of 
the rights granted under the authority of any government in accordance 
with its patent laws. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3453 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: N-terminal 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 31..3009 
(ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- peptide 
(B) LOCATION: 31..3006 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCGGCCTGGCTACCGCGCGCTCCGGAGGCCATGCGGGCGTTGGCGCAGCGCAGC54 
MetArgAlaLeuAlaGlnArgSer 
15 
GACCGGCGGCTGCTGCTGCTTGTTGTTTTGTCAGTAATGATTCTTGAG102 
AspArgArgLeuLeuLeuLeuValValLeuSerValMetIleLeuGlu 
101520 
ACCGTTACAAACCAAGACCTGCCTGTGATCAAGTGTGTTTTAATCAGT150 
ThrValThrAsnGlnAspLeuProValIleLysCysValLeuIleSer 
25303540 
CATGAGAACAATGGCTCATCAGCGGGAAAGCCATCATCGTACCGAATG198 
HisGluAsnAsnGlySerSerAlaGlyLysProSerSerTyrArgMet 
455055 
GTGCGAGGATCCCCAGAAGACCTCCAGTGTACCCCGAGGCGCCAGAGT246 
ValArgGlySerProGluAspLeuGlnCysThrProArgArgGlnSer 
606570 
GAAGGGACGGTATATGAAGCGGCCACCGTGGAGGTGGCCGAGTCTGGG294 
GluGlyThrValTyrGluAlaAlaThrValGluValAlaGluSerGly 
758085 
TCCATCACCCTGCAAGTGCAGCTCGCCACCCCAGGGGACCTTTCCTGC342 
SerIleThrLeuGlnValGlnLeuAlaThrProGlyAspLeuSerCys 
9095100 
CTCTGGGTCTTTAAGCACAGCTCCCTGGGCTGCCAGCCGCACTTTGAT390 
LeuTrpValPheLysHisSerSerLeuGlyCysGlnProHisPheAsp 
105110115120 
TTACAAAACAGAGGAATCGTTTCCATGGCCATCTTGAACGTGACAGAG438 
LeuGlnAsnArgGlyIleValSerMetAlaIleLeuAsnValThrGlu 
125130135 
ACCCAGGCAGGAGAATACCTACTCCATATTCAGAGCGAACGCGCCAAC486 
ThrGlnAlaGlyGluTyrLeuLeuHisIleGlnSerGluArgAlaAsn 
140145150 
TACACAGTACTGTTCACAGTGAATGTAAGAGATACACAGCTGTATGTG534 
TyrThrValLeuPheThrValAsnValArgAspThrGlnLeuTyrVal 
155160165 
CTAAGGAGACCTTACTTTAGGAAGATGGAAAACCAGGATGCACTGCTC582 
LeuArgArgProTyrPheArgLysMetGluAsnGlnAspAlaLeuLeu 
170175180 
TGCATCTCCGAGGGTGTTCCGGAGCCCACTGTGGAGTGGGTGCTCTGC630 
CysIleSerGluGlyValProGluProThrValGluTrpValLeuCys 
185190195200 
AGCTCCCACAGGGAAAGCTGTAAAGAAGAAGGCCCTGCTGTTGTCAGA678 
SerSerHisArgGluSerCysLysGluGluGlyProAlaValValArg 
205210215 
AAGGAGGAAAAGGTACTTCATGAGTTGTTCGGAACAGACATCAGATGC726 
LysGluGluLysValLeuHisGluLeuPheGlyThrAspIleArgCys 
220225230 
TGTGCTAGAAATGCACTGGGCCGCGAATGCACCAAGCTGTTCACCATA774 
CysAlaArgAsnAlaLeuGlyArgGluCysThrLysLeuPheThrIle 
235240245 
GATCTAAACCAGGCTCCTCAGAGCACACTGCCCCAGTTATTCCTGAAA822 
AspLeuAsnGlnAlaProGlnSerThrLeuProGlnLeuPheLeuLys 
250255260 
GTGGGGGAACCCTTGTGGATCAGGTGTAAGGCCATCCATGTGAACCAT870 
ValGlyGluProLeuTrpIleArgCysLysAlaIleHisValAsnHis 
265270275280 
GGATTCGGGCTCACCTGGGAGCTGGAAGACAAAGCCCTGGAGGAGGGC918 
GlyPheGlyLeuThrTrpGluLeuGluAspLysAlaLeuGluGluGly 
285290295 
AGCTACTTTGAGATGAGTACCTACTCCACAAACAGGACCATGATTCGG966 
SerTyrPheGluMetSerThrTyrSerThrAsnArgThrMetIleArg 
300305310 
ATTCTCTTGGCCTTTGTGTCTTCCGTGGGAAGGAACGACACCGGATAT1014 
IleLeuLeuAlaPheValSerSerValGlyArgAsnAspThrGlyTyr 
315320325 
TACACCTGCTCTTCCTCAAAGCACCCCAGCCAGTCAGCGTTGGTGACC1062 
TyrThrCysSerSerSerLysHisProSerGlnSerAlaLeuValThr 
330335340 
ATCCTAGAAAAAGGGTTTATAAACGCTACCAGCTCGCAAGAAGAGTAT1110 
IleLeuGluLysGlyPheIleAsnAlaThrSerSerGlnGluGluTyr 
345350355360 
GAAATTGACCCGTACGAAAAGTTCTGCTTCTCAGTCAGGTTTAAAGCG1158 
GluIleAspProTyrGluLysPheCysPheSerValArgPheLysAla 
365370375 
TACCCACGAATCCGATGCACGTGGATCTTCTCTCAAGCCTCATTTCCT1206 
TyrProArgIleArgCysThrTrpIlePheSerGlnAlaSerPhePro 
380385390 
TGTGAACAGAGAGGCCTGGAGGATGGGTACAGCATATCTAAATTTTGC1254 
CysGluGlnArgGlyLeuGluAspGlyTyrSerIleSerLysPheCys 
395400405 
GATCATAAGAACAAGCCAGGAGAGTACATATTCTATGCAGAAAATGAT1302 
AspHisLysAsnLysProGlyGluTyrIlePheTyrAlaGluAsnAsp 
410415420 
GACGCCCAGTTCACCAAAATGTTCACGCTGAATATAAGAAAGAAACCT1350 
AspAlaGlnPheThrLysMetPheThrLeuAsnIleArgLysLysPro 
425430435440 
CAAGTGCTAGCAAATGCCTCAGCCAGCCAGGCGTCCTGTTCCTCTGAT1398 
GlnValLeuAlaAsnAlaSerAlaSerGlnAlaSerCysSerSerAsp 
445450455 
GGCTACCCGCTACCCTCTTGGACCTGGAAGAAGTGTTCGGACAAATCT1446 
GlyTyrProLeuProSerTrpThrTrpLysLysCysSerAspLysSer 
460465470 
CCCAATTGCACGGAGGAAATCCCAGAAGGAGTTTGGAATAAAAAGGCT1494 
ProAsnCysThrGluGluIleProGluGlyValTrpAsnLysLysAla 
475480485 
AACAGAAAAGTGTTTGGCCAGTGGGTGTCGAGCAGTACTCTAAATATG1542 
AsnArgLysValPheGlyGlnTrpValSerSerSerThrLeuAsnMet 
490495500 
AGTGAGGCCGGGAAAGGGCTTCTGGTCAAATGCTGTGCGTACAATTCT1590 
SerGluAlaGlyLysGlyLeuLeuValLysCysCysAlaTyrAsnSer 
505510515520 
ATGGGCACGTCTTGCGAAACCATCTTTTTAAACTCACCAGGCCCCTTC1638 
MetGlyThrSerCysGluThrIlePheLeuAsnSerProGlyProPhe 
525530535 
CCTTTCATCCAAGACAACATCTCCTTCTATGCGACCATTGGGCTCTGT1686 
ProPheIleGlnAspAsnIleSerPheTyrAlaThrIleGlyLeuCys 
540545550 
CTCCCCTTCATTGTTGTTCTCATTGTGTTGATCTGCCACAAATACAAA1734 
LeuProPheIleValValLeuIleValLeuIleCysHisLysTyrLys 
555560565 
AAGCAATTTAGGTACGAGAGTCAGCTGCAGATGATCCAGGTGACTGGC1782 
LysGlnPheArgTyrGluSerGlnLeuGlnMetIleGlnValThrGly 
570575580 
CCCCTGGATAACGAGTACTTCTACGTTGACTTCAGGGACTATGAATAT1830 
ProLeuAspAsnGluTyrPheTyrValAspPheArgAspTyrGluTyr 
585590595600 
GACCTTAAGTGGGAGTTCCCGAGAGAGAACTTAGAGTTTGGGAAGGTC1878 
AspLeuLysTrpGluPheProArgGluAsnLeuGluPheGlyLysVal 
605610615 
CTGGGGTCTGGCGCTTTCGGGAGGGTGATGAACGCCACGGCCTATGGC1926 
LeuGlySerGlyAlaPheGlyArgValMetAsnAlaThrAlaTyrGly 
620625630 
ATTAGTAAAACGGGAGTCTCAATTCAGGTGGCGGTGAAGATGCTAAAA1974 
IleSerLysThrGlyValSerIleGlnValAlaValLysMetLeuLys 
635640645 
GAGAAAGCTGACAGCTGTGAAAAAGAAGCTCTCATGTCGGAGCTCAAA2022 
GluLysAlaAspSerCysGluLysGluAlaLeuMetSerGluLeuLys 
650655660 
ATGATGACCCACCTGGGACACCATGACAACATCGTGAATCTGCTGGGG2070 
MetMetThrHisLeuGlyHisHisAspAsnIleValAsnLeuLeuGly 
665670675680 
GCATGCACACTGTCAGGGCCAGTGTACTTGATTTTTGAATATTGTTGC2118 
AlaCysThrLeuSerGlyProValTyrLeuIlePheGluTyrCysCys 
685690695 
TATGGTGACCTCCTCAACTACCTAAGAAGTAAAAGAGAGAAGTTTCAC2166 
TyrGlyAspLeuLeuAsnTyrLeuArgSerLysArgGluLysPheHis 
700705710 
AGGACATGGACAGAGATTTTTAAGGAACATAATTTCAGTTCTTACCCT2214 
ArgThrTrpThrGluIlePheLysGluHisAsnPheSerSerTyrPro 
715720725 
ACTTTCCAGGCACATTCAAATTCCAGCATGCCTGGTTCACGAGAAGTT2262 
ThrPheGlnAlaHisSerAsnSerSerMetProGlySerArgGluVal 
730735740 
CAGTTACACCCGCCCTTGGATCAGCTCTCAGGGTTCAATGGGAATTCA2310 
GlnLeuHisProProLeuAspGlnLeuSerGlyPheAsnGlyAsnSer 
745750755760 
ATTCATTCTGAAGATGAGATTGAATATGAAAACCAGAAGAGGCTGGCA2358 
IleHisSerGluAspGluIleGluTyrGluAsnGlnLysArgLeuAla 
765770775 
GAAGAAGAGGAGGAAGATTTGAACGTGCTGACGTTTGAAGACCTCCTT2406 
GluGluGluGluGluAspLeuAsnValLeuThrPheGluAspLeuLeu 
780785790 
TGCTTTGCGTACCAAGTGGCCAAAGGCATGGAATTCCTGGAGTTCAAG2454 
CysPheAlaTyrGlnValAlaLysGlyMetGluPheLeuGluPheLys 
795800805 
TCGTGTGTCCACAGAGACCTGGCAGCCAGGAATGTGTTGGTCACCCAC2502 
SerCysValHisArgAspLeuAlaAlaArgAsnValLeuValThrHis 
810815820 
GGGAAGGTGGTGAAGATCTGTGACTTTGGACTGGCCCGAGACATCCTG2550 
GlyLysValValLysIleCysAspPheGlyLeuAlaArgAspIleLeu 
825830835840 
AGCGACTCCAGCTACGTCGTCAGGGGCAACGCACGGCTGCCGGTGAAG2598 
SerAspSerSerTyrValValArgGlyAsnAlaArgLeuProValLys 
845850855 
TGGATGGCACCCGAGAGCTTATTTGAAGGGATCTACACAATCAAGAGT2646 
TrpMetAlaProGluSerLeuPheGluGlyIleTyrThrIleLysSer 
860865870 
GACGTCTGGTCCTACGGCATCCTTCTCTGGGAGATATTTTCACTGGGT2694 
AspValTrpSerTyrGlyIleLeuLeuTrpGluIlePheSerLeuGly 
875880885 
GTGAACCCTTACCCTGGCATTCCTGTCGACGCTAACTTCTATAAACTG2742 
ValAsnProTyrProGlyIleProValAspAlaAsnPheTyrLysLeu 
890895900 
ATTCAGAGTGGATTTAAAATGGAGCAGCCATTCTATGCCACAGAAGGG2790 
IleGlnSerGlyPheLysMetGluGlnProPheTyrAlaThrGluGly 
905910915920 
ATATACTTTGTAATGCAATCCTGCTGGGCTTTTGACTCAAGGAAGCGG2838 
IleTyrPheValMetGlnSerCysTrpAlaPheAspSerArgLysArg 
925930935 
CCATCCTTCCCCAACCTGACTTCATTTTTAGGATGTCAGCTGGCAGAG2886 
ProSerPheProAsnLeuThrSerPheLeuGlyCysGlnLeuAlaGlu 
940945950 
GCAGAAGAAGCATGTATCAGAACATCCATCCATCTACCAAAACAGGCG2934 
AlaGluGluAlaCysIleArgThrSerIleHisLeuProLysGlnAla 
955960965 
GCCCCTCAGCAGAGAGGCGGGCTCAGAGCCCAGTCGCCACAGCGCCAG2982 
AlaProGlnGlnArgGlyGlyLeuArgAlaGlnSerProGlnArgGln 
970975980 
GTGAAGATTCACAGAGAAAGAAGTTAGCGAGGAGGCCTTGGACCCCGCCACCCT3036 
ValLysIleHisArgGluArgSer 
985990 
AGCAGGCTGTAGACCGCAGAGCCAAGATTAGCCTCGCCTCTGAGGAAGCGCCCTACAGCG3096 
CGTTGCTTCGCTGGACTTTTCTCTAGATGCTGTCTGCCATTACTCCAAAGTGACTTCTAT3156 
AAAATCAAACCTCTCCTCGCACAGGCGGGAGAGCCAATAATGAGACTTGTTGGTGAGCCC3216 
GCCTACCCTGGGGGCCTTTCCACGAGCTTGAGGGGAAAGCCATGTATCTGAAATATAGTA3276 
TATTCTTGTAAATACGTGAAACAAACCAAACCCGTTTTTTGCTAAGGGAAAGCTAAATAT3336 
GATTTTTAAAAATCTATGTTTTAAAATACTATGTAACTTTTTCATCTATTTAGTGATATA3396 
TTTTATGGATGGAAATAAACTTTCTACTGTAAAAAAAAAAAAAAAAAAAAAAAAAAA3453 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 992 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetArgAlaLeuAlaGlnArgSerAspArgArgLeuLeuLeuLeuVal 
151015 
ValLeuSerValMetIleLeuGluThrValThrAsnGlnAspLeuPro 
202530 
ValIleLysCysValLeuIleSerHisGluAsnAsnGlySerSerAla 
354045 
GlyLysProSerSerTyrArgMetValArgGlySerProGluAspLeu 
505560 
GlnCysThrProArgArgGlnSerGluGlyThrValTyrGluAlaAla 
65707580 
ThrValGluValAlaGluSerGlySerIleThrLeuGlnValGlnLeu 
859095 
AlaThrProGlyAspLeuSerCysLeuTrpValPheLysHisSerSer 
100105110 
LeuGlyCysGlnProHisPheAspLeuGlnAsnArgGlyIleValSer 
115120125 
MetAlaIleLeuAsnValThrGluThrGlnAlaGlyGluTyrLeuLeu 
130135140 
HisIleGlnSerGluArgAlaAsnTyrThrValLeuPheThrValAsn 
145150155160 
ValArgAspThrGlnLeuTyrValLeuArgArgProTyrPheArgLys 
165170175 
MetGluAsnGlnAspAlaLeuLeuCysIleSerGluGlyValProGlu 
180185190 
ProThrValGluTrpValLeuCysSerSerHisArgGluSerCysLys 
195200205 
GluGluGlyProAlaValValArgLysGluGluLysValLeuHisGlu 
210215220 
LeuPheGlyThrAspIleArgCysCysAlaArgAsnAlaLeuGlyArg 
225230235240 
GluCysThrLysLeuPheThrIleAspLeuAsnGlnAlaProGlnSer 
245250255 
ThrLeuProGlnLeuPheLeuLysValGlyGluProLeuTrpIleArg 
260265270 
CysLysAlaIleHisValAsnHisGlyPheGlyLeuThrTrpGluLeu 
275280285 
GluAspLysAlaLeuGluGluGlySerTyrPheGluMetSerThrTyr 
290295300 
SerThrAsnArgThrMetIleArgIleLeuLeuAlaPheValSerSer 
305310315320 
ValGlyArgAsnAspThrGlyTyrTyrThrCysSerSerSerLysHis 
325330335 
ProSerGlnSerAlaLeuValThrIleLeuGluLysGlyPheIleAsn 
340345350 
AlaThrSerSerGlnGluGluTyrGluIleAspProTyrGluLysPhe 
355360365 
CysPheSerValArgPheLysAlaTyrProArgIleArgCysThrTrp 
370375380 
IlePheSerGlnAlaSerPheProCysGluGlnArgGlyLeuGluAsp 
385390395400 
GlyTyrSerIleSerLysPheCysAspHisLysAsnLysProGlyGlu 
405410415 
TyrIlePheTyrAlaGluAsnAspAspAlaGlnPheThrLysMetPhe 
420425430 
ThrLeuAsnIleArgLysLysProGlnValLeuAlaAsnAlaSerAla 
435440445 
SerGlnAlaSerCysSerSerAspGlyTyrProLeuProSerTrpThr 
450455460 
TrpLysLysCysSerAspLysSerProAsnCysThrGluGluIlePro 
465470475480 
GluGlyValTrpAsnLysLysAlaAsnArgLysValPheGlyGlnTrp 
485490495 
ValSerSerSerThrLeuAsnMetSerGluAlaGlyLysGlyLeuLeu 
500505510 
ValLysCysCysAlaTyrAsnSerMetGlyThrSerCysGluThrIle 
515520525 
PheLeuAsnSerProGlyProPheProPheIleGlnAspAsnIleSer 
530535540 
PheTyrAlaThrIleGlyLeuCysLeuProPheIleValValLeuIle 
545550555560 
ValLeuIleCysHisLysTyrLysLysGlnPheArgTyrGluSerGln 
565570575 
LeuGlnMetIleGlnValThrGlyProLeuAspAsnGluTyrPheTyr 
580585590 
ValAspPheArgAspTyrGluTyrAspLeuLysTrpGluPheProArg 
595600605 
GluAsnLeuGluPheGlyLysValLeuGlySerGlyAlaPheGlyArg 
610615620 
ValMetAsnAlaThrAlaTyrGlyIleSerLysThrGlyValSerIle 
625630635640 
GlnValAlaValLysMetLeuLysGluLysAlaAspSerCysGluLys 
645650655 
GluAlaLeuMetSerGluLeuLysMetMetThrHisLeuGlyHisHis 
660665670 
AspAsnIleValAsnLeuLeuGlyAlaCysThrLeuSerGlyProVal 
675680685 
TyrLeuIlePheGluTyrCysCysTyrGlyAspLeuLeuAsnTyrLeu 
690695700 
ArgSerLysArgGluLysPheHisArgThrTrpThrGluIlePheLys 
705710715720 
GluHisAsnPheSerSerTyrProThrPheGlnAlaHisSerAsnSer 
725730735 
SerMetProGlySerArgGluValGlnLeuHisProProLeuAspGln 
740745750 
LeuSerGlyPheAsnGlyAsnSerIleHisSerGluAspGluIleGlu 
755760765 
TyrGluAsnGlnLysArgLeuAlaGluGluGluGluGluAspLeuAsn 
770775780 
ValLeuThrPheGluAspLeuLeuCysPheAlaTyrGlnValAlaLys 
785790795800 
GlyMetGluPheLeuGluPheLysSerCysValHisArgAspLeuAla 
805810815 
AlaArgAsnValLeuValThrHisGlyLysValValLysIleCysAsp 
820825830 
PheGlyLeuAlaArgAspIleLeuSerAspSerSerTyrValValArg 
835840845 
GlyAsnAlaArgLeuProValLysTrpMetAlaProGluSerLeuPhe 
850855860 
GluGlyIleTyrThrIleLysSerAspValTrpSerTyrGlyIleLeu 
865870875880 
LeuTrpGluIlePheSerLeuGlyValAsnProTyrProGlyIlePro 
885890895 
ValAspAlaAsnPheTyrLysLeuIleGlnSerGlyPheLysMetGlu 
900905910 
GlnProPheTyrAlaThrGluGlyIleTyrPheValMetGlnSerCys 
915920925 
TrpAlaPheAspSerArgLysArgProSerPheProAsnLeuThrSer 
930935940 
PheLeuGlyCysGlnLeuAlaGluAlaGluGluAlaCysIleArgThr 
945950955960 
SerIleHisLeuProLysGlnAlaAlaProGlnGlnArgGlyGlyLeu 
965970975 
ArgAlaGlnSerProGlnArgGlnValLysIleHisArgGluArgSer 
980985990 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3501 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 58..3039 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CGAGGCGGCATCCGAGGGCTGGGCCGGCGCCCTGGGGGACCCCGGGCTCCGGAGGCC57 
ATGCCGGCGTTGGCGCGCGACGCGGGCACCGTGCCGCTGCTCGTTGTT105 
MetProAlaLeuAlaArgAspAlaGlyThrValProLeuLeuValVal 
151015 
TTTTCTGCAATGATATTTGGGACTATTACAAATCAAGATCTGCCTGTG153 
PheSerAlaMetIlePheGlyThrIleThrAsnGlnAspLeuProVal 
202530 
ATCAAGTGTGTTTTAATCAATCATAAGAACAATGATTCATCAGTGGGG201 
IleLysCysValLeuIleAsnHisLysAsnAsnAspSerSerValGly 
354045 
AAGTCATCATCATATCCCATGGTATCAGAATCCCCGGAAGACCTCGGG249 
LysSerSerSerTyrProMetValSerGluSerProGluAspLeuGly 
505560 
TGTGCGTTGAGACCCCAGAGCTCAGGGACAGTGTACGAAGCTGCCGCT297 
CysAlaLeuArgProGlnSerSerGlyThrValTyrGluAlaAlaAla 
65707580 
GTGGAAGTGGATGTATCTGCTTCCATCACACTGCAAGTGCTGGTCGAT345 
ValGluValAspValSerAlaSerIleThrLeuGlnValLeuValAsp 
859095 
GCCCCAGGGAACATTTCCTGTCTCTGGGTCTTTAAGCACAGCTCCCTG393 
AlaProGlyAsnIleSerCysLeuTrpValPheLysHisSerSerLeu 
100105110 
AATTGCCAGCCACATTTTGATTTACAAAACAGAGGAGTTGTTTCCATG441 
AsnCysGlnProHisPheAspLeuGlnAsnArgGlyValValSerMet 
115120125 
GTCATTTTGAAAATGACAGAAACCCAAGCTGGAGAATACCTACTTTTT489 
ValIleLeuLysMetThrGluThrGlnAlaGlyGluTyrLeuLeuPhe 
130135140 
ATTCAGAGTGAAGCTACCAATTACACAATATTGTTTACAGTGAGTATA537 
IleGlnSerGluAlaThrAsnTyrThrIleLeuPheThrValSerIle 
145150155160 
AGAAATACCCTGCTTTACACATTAAGAAGACCTTACTTTAGAAAAATG585 
ArgAsnThrLeuLeuTyrThrLeuArgArgProTyrPheArgLysMet 
165170175 
GAAAACCAGGACGCCCTGGTCTGCATATCTGAGAGCGTTCCAGAGCCG633 
GluAsnGlnAspAlaLeuValCysIleSerGluSerValProGluPro 
180185190 
ATCGTGGAATGGGTGCTTTGCGATTCACAGGGGGAAAGCTGTAAAGAA681 
IleValGluTrpValLeuCysAspSerGlnGlyGluSerCysLysGlu 
195200205 
GAAAGTCCAGCTGTTGTTAAAAAGGAGGAAAAAGTGCTTCATGAATTA729 
GluSerProAlaValValLysLysGluGluLysValLeuHisGluLeu 
210215220 
TTTGGGACGGACATAAGGTGCTGTGCCAGAAATGAACTGGGCAGGGAA777 
PheGlyThrAspIleArgCysCysAlaArgAsnGluLeuGlyArgGlu 
225230235240 
TGCACCAGGCTGTTCACAATAGATCTAAATCAAACTCCTCAGACCACA825 
CysThrArgLeuPheThrIleAspLeuAsnGlnThrProGlnThrThr 
245250255 
TTGCCACAATTATTTCTTAAAGTAGGGGAACCCTTATGGATAAGGTGC873 
LeuProGlnLeuPheLeuLysValGlyGluProLeuTrpIleArgCys 
260265270 
AAAGCTGTTCATGTGAACCATGGATTCGGGCTCACCTGGGAATTAGAA921 
LysAlaValHisValAsnHisGlyPheGlyLeuThrTrpGluLeuGlu 
275280285 
AACAAAGCACTCGAGGAGGGCAACTACTTTGAGATGAGTACCTATTCA969 
AsnLysAlaLeuGluGluGlyAsnTyrPheGluMetSerThrTyrSer 
290295300 
ACAAACAGAACTATGATACGGATTCTGTTTGCTTTTGTATCATCAGTG1017 
ThrAsnArgThrMetIleArgIleLeuPheAlaPheValSerSerVal 
305310315320 
GCAAGAAACGACACCGGATACTACACTTGTTCCTCTTCAAAGCATCCC1065 
AlaArgAsnAspThrGlyTyrTyrThrCysSerSerSerLysHisPro 
325330335 
AGTCAATCAGCTTTGGTTACCATCGTAGGAAAGGGATTTATAAATGCT1113 
SerGlnSerAlaLeuValThrIleValGlyLysGlyPheIleAsnAla 
340345350 
ACCAATTCAAGTGAAGATTATGAAATTGACCAATATGAAGAGTTTTGT1161 
ThrAsnSerSerGluAspTyrGluIleAspGlnTyrGluGluPheCys 
355360365 
TTTTCTGTCAGGTTTAAAGCCTACCCACAAATCAGATGTACGTGGACC1209 
PheSerValArgPheLysAlaTyrProGlnIleArgCysThrTrpThr 
370375380 
TTCTCTCGAAAATCATTTCCTTGTGAGCAAAAGGGTCTTGATAACGGA1257 
PheSerArgLysSerPheProCysGluGlnLysGlyLeuAspAsnGly 
385390395400 
TACAGCATATCCAAGTTTTGCAATCATAAGCACCAGCCAGGAGAATAT1305 
TyrSerIleSerLysPheCysAsnHisLysHisGlnProGlyGluTyr 
405410415 
ATATTCCATGCAGAAAATGATGATGCCCAATTTACCAAAATGTTCACG1353 
IlePheHisAlaGluAsnAspAspAlaGlnPheThrLysMetPheThr 
420425430 
CTGAATATAAGAAGGAAACCTCAAGTGCTCGCAGAAGCATCGGCAAGT1401 
LeuAsnIleArgArgLysProGlnValLeuAlaGluAlaSerAlaSer 
435440445 
CAGGCGTCCTGTTTCTCGGATGGATACCCATTACCATCTTGGACCTGG1449 
GlnAlaSerCysPheSerAspGlyTyrProLeuProSerTrpThrTrp 
450455460 
AAGAAGTGTTCAGACAAGTCTCCCAACTGCACAGAAGAGATCACAGAA1497 
LysLysCysSerAspLysSerProAsnCysThrGluGluIleThrGlu 
465470475480 
GGAGTCTGGAATAGAAAGGCTAACAGAAAAGTGTTTGGACAGTGGGTG1545 
GlyValTrpAsnArgLysAlaAsnArgLysValPheGlyGlnTrpVal 
485490495 
TCGAGCAGTACTCTAAACATGAGTGAAGCCATAAAAGGGTTCCTGGTC1593 
SerSerSerThrLeuAsnMetSerGluAlaIleLysGlyPheLeuVal 
500505510 
AAGTGCTGTGCATACAATTCCCTTGGCACATCTTGTGAGACGATCCTT1641 
LysCysCysAlaTyrAsnSerLeuGlyThrSerCysGluThrIleLeu 
515520525 
TTAAACTCTCCAGGCCCCTTCCCTTTCATCCAAGACAACATCTCATTC1689 
LeuAsnSerProGlyProPheProPheIleGlnAspAsnIleSerPhe 
530535540 
TATGCAACAATTGGTGTTTGTCTCCTCTTCATTGTCGTTTTAACCCTG1737 
TyrAlaThrIleGlyValCysLeuLeuPheIleValValLeuThrLeu 
545550555560 
CTAATTTGTCACAAGTACAAAAAGCAATTTAGGTATGAAAGCCAGCTA1785 
LeuIleCysHisLysTyrLysLysGlnPheArgTyrGluSerGlnLeu 
565570575 
CAGATGGTACAGGTGACCGGCTCCTCAGATAATGAGTACTTCTACGTT1833 
GlnMetValGlnValThrGlySerSerAspAsnGluTyrPheTyrVal 
580585590 
GATTTCAGAGAATATGAATATGATCTCAAATGGGAGTTTCCAAGAGAA1881 
AspPheArgGluTyrGluTyrAspLeuLysTrpGluPheProArgGlu 
595600605 
AATTTAGAGTTTGGGAAGGTACTAGGATCAGGTGCTTTTGGAAAAGTG1929 
AsnLeuGluPheGlyLysValLeuGlySerGlyAlaPheGlyLysVal 
610615620 
ATGAACGCAACAGCTTATGGAATTAGCAAAACAGGAGTCTCAATCCAG1977 
MetAsnAlaThrAlaTyrGlyIleSerLysThrGlyValSerIleGln 
625630635640 
GTTGCCGTCAAAATGCTGAAAGAAAAAGCAGACAGCTCTGAAAGAGAG2025 
ValAlaValLysMetLeuLysGluLysAlaAspSerSerGluArgGlu 
645650655 
GCACTCATGTCAGAACTCAAGATGATGACCCAGCTGGGAAGCCACGAG2073 
AlaLeuMetSerGluLeuLysMetMetThrGlnLeuGlySerHisGlu 
660665670 
AATATTGTGAACCTGCTGGGGGCGTGCACACTGTCAGGACCAATTTAC2121 
AsnIleValAsnLeuLeuGlyAlaCysThrLeuSerGlyProIleTyr 
675680685 
TTGATTTTTGAATACTGTTGCTATGGTGATCTTCTCAACTATCTAAGA2169 
LeuIlePheGluTyrCysCysTyrGlyAspLeuLeuAsnTyrLeuArg 
690695700 
AGTAAAAGAGAAAAATTTCACAGGACTTGGACAGAGATTTTCAAGGAA2217 
SerLysArgGluLysPheHisArgThrTrpThrGluIlePheLysGlu 
705710715720 
CACAATTTCAGTTTTTACCCCACTTTCCAATCACATCCAAATTCCAGC2265 
HisAsnPheSerPheTyrProThrPheGlnSerHisProAsnSerSer 
725730735 
ATGCCTGGTTCAAGAGAAGTTCAGATACACCCGGACTCGGATCAAATC2313 
MetProGlySerArgGluValGlnIleHisProAspSerAspGlnIle 
740745750 
TCAGGGCTTCATGGGAATTCATTTCACTCTGAAGATGAAATTGAATAT2361 
SerGlyLeuHisGlyAsnSerPheHisSerGluAspGluIleGluTyr 
755760765 
GAAAACCAAAAAAGGCTGGAAGAAGAGGAGGACTTGAATGTGCTTACA2409 
GluAsnGlnLysArgLeuGluGluGluGluAspLeuAsnValLeuThr 
770775780 
TTTGAAGATCTTCTTTGCTTTGCATATCAAGTTGCCAAAGGAATGGAA2457 
PheGluAspLeuLeuCysPheAlaTyrGlnValAlaLysGlyMetGlu 
785790795800 
TTTCTGGAATTTAAGTCGTGTGTTCACAGAGACCTGGCCGCCAGGAAC2505 
PheLeuGluPheLysSerCysValHisArgAspLeuAlaAlaArgAsn 
805810815 
GTGCTTGTCACCCACGGGAAAGTGGTGAAGATATGTGACTTTGGATTG2553 
ValLeuValThrHisGlyLysValValLysIleCysAspPheGlyLeu 
820825830 
GCTCGAGATATCATGAGTGATTCCAACTATGTTGTCAGGGGCAATGCC2601 
AlaArgAspIleMetSerAspSerAsnTyrValValArgGlyAsnAla 
835840845 
CGTCTGCCTGTAAAATGGATGGCCCCCGAAAGCCTGTTTGAAGGCATC2649 
ArgLeuProValLysTrpMetAlaProGluSerLeuPheGluGlyIle 
850855860 
TACACCATTAAGAGTGATGTCTGGTCATATGGAATATTACTGTGGGAA2697 
TyrThrIleLysSerAspValTrpSerTyrGlyIleLeuLeuTrpGlu 
865870875880 
ATCTTCTCACTTGGTGTGAATCCTTACCCTGGCATTCCGGTTGATGCT2745 
IlePheSerLeuGlyValAsnProTyrProGlyIleProValAspAla 
885890895 
AACTTCTACAAACTGATTCAAAATGGATTTAAAATGGATCAGCCATTT2793 
AsnPheTyrLysLeuIleGlnAsnGlyPheLysMetAspGlnProPhe 
900905910 
TATGCTACAGAAGAAATATACATTATAATGCAATCCTGCTGGGCTTTT2841 
TyrAlaThrGluGluIleTyrIleIleMetGlnSerCysTrpAlaPhe 
915920925 
GACTCAAGGAAACGGCCATCCTTCCCTAATTTGACTTCGTTTTTAGGA2889 
AspSerArgLysArgProSerPheProAsnLeuThrSerPheLeuGly 
930935940 
TGTCAGCTGGCAGATGCAGAAGAAGCGATGTATCAGAATGTGGATGGC2937 
CysGlnLeuAlaAspAlaGluGluAlaMetTyrGlnAsnValAspGly 
945950955960 
CGTGTTTCGGAATGTCCTCACACCTACCAAAACAGGCGACCTTTCAGC2985 
ArgValSerGluCysProHisThrTyrGlnAsnArgArgProPheSer 
965970975 
AGAGAGATGGATTTGGGGCTACTCTCTCCGCAGGCTCAGGTCGAAGAT3033 
ArgGluMetAspLeuGlyLeuLeuSerProGlnAlaGlnValGluAsp 
980985990 
TCGTAGAGGAACAATTTAGTTTTAAGGACTTCATCCCTCCACCTATCCCTAAC3086 
Ser 
AGGCTGTAGATTACCAAAACAAGATTAATTTCATCACTAAAAGAAAATCTATTATCAACT3146 
GCTGCTTCACCAGACTTTTCTCTAGAAGCCGTCTGCGTTTACTCTTGTTTTCAAAGGGAC3206 
TTTTGTAAAATCAAATCATCCTGTCACAAGGCAGGAGGAGCTGATAATGAACTTTATTGG3266 
AGCATTGATCTGCATCCAAGGCCTTCTCAGGCCGGCTTGAGTGAATTGTGTACCTGAAGT3326 
ACAGTATATTCTTGTAAATACATAAAACAAAAGCATTTTGCTAAGGAGAAGCTAATATGA3386 
TTTTTTAAGTCTATGTTTTAAAATAATATGTAAATTTTTCAGCTATTTAGTGATATATTT3446 
TATGGGTGGGAATAAAATTTCTACTACAGAAAAAAAAAAAAAAAAAAAAAAAAAA3501 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 993 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetProAlaLeuAlaArgAspAlaGlyThrValProLeuLeuValVal 
151015 
PheSerAlaMetIlePheGlyThrIleThrAsnGlnAspLeuProVal 
202530 
IleLysCysValLeuIleAsnHisLysAsnAsnAspSerSerValGly 
354045 
LysSerSerSerTyrProMetValSerGluSerProGluAspLeuGly 
505560 
CysAlaLeuArgProGlnSerSerGlyThrValTyrGluAlaAlaAla 
65707580 
ValGluValAspValSerAlaSerIleThrLeuGlnValLeuValAsp 
859095 
AlaProGlyAsnIleSerCysLeuTrpValPheLysHisSerSerLeu 
100105110 
AsnCysGlnProHisPheAspLeuGlnAsnArgGlyValValSerMet 
115120125 
ValIleLeuLysMetThrGluThrGlnAlaGlyGluTyrLeuLeuPhe 
130135140 
IleGlnSerGluAlaThrAsnTyrThrIleLeuPheThrValSerIle 
145150155160 
ArgAsnThrLeuLeuTyrThrLeuArgArgProTyrPheArgLysMet 
165170175 
GluAsnGlnAspAlaLeuValCysIleSerGluSerValProGluPro 
180185190 
IleValGluTrpValLeuCysAspSerGlnGlyGluSerCysLysGlu 
195200205 
GluSerProAlaValValLysLysGluGluLysValLeuHisGluLeu 
210215220 
PheGlyThrAspIleArgCysCysAlaArgAsnGluLeuGlyArgGlu 
225230235240 
CysThrArgLeuPheThrIleAspLeuAsnGlnThrProGlnThrThr 
245250255 
LeuProGlnLeuPheLeuLysValGlyGluProLeuTrpIleArgCys 
260265270 
LysAlaValHisValAsnHisGlyPheGlyLeuThrTrpGluLeuGlu 
275280285 
AsnLysAlaLeuGluGluGlyAsnTyrPheGluMetSerThrTyrSer 
290295300 
ThrAsnArgThrMetIleArgIleLeuPheAlaPheValSerSerVal 
305310315320 
AlaArgAsnAspThrGlyTyrTyrThrCysSerSerSerLysHisPro 
325330335 
SerGlnSerAlaLeuValThrIleValGlyLysGlyPheIleAsnAla 
340345350 
ThrAsnSerSerGluAspTyrGluIleAspGlnTyrGluGluPheCys 
355360365 
PheSerValArgPheLysAlaTyrProGlnIleArgCysThrTrpThr 
370375380 
PheSerArgLysSerPheProCysGluGlnLysGlyLeuAspAsnGly 
385390395400 
TyrSerIleSerLysPheCysAsnHisLysHisGlnProGlyGluTyr 
405410415 
IlePheHisAlaGluAsnAspAspAlaGlnPheThrLysMetPheThr 
420425430 
LeuAsnIleArgArgLysProGlnValLeuAlaGluAlaSerAlaSer 
435440445 
GlnAlaSerCysPheSerAspGlyTyrProLeuProSerTrpThrTrp 
450455460 
LysLysCysSerAspLysSerProAsnCysThrGluGluIleThrGlu 
465470475480 
GlyValTrpAsnArgLysAlaAsnArgLysValPheGlyGlnTrpVal 
485490495 
SerSerSerThrLeuAsnMetSerGluAlaIleLysGlyPheLeuVal 
500505510 
LysCysCysAlaTyrAsnSerLeuGlyThrSerCysGluThrIleLeu 
515520525 
LeuAsnSerProGlyProPheProPheIleGlnAspAsnIleSerPhe 
530535540 
TyrAlaThrIleGlyValCysLeuLeuPheIleValValLeuThrLeu 
545550555560 
LeuIleCysHisLysTyrLysLysGlnPheArgTyrGluSerGlnLeu 
565570575 
GlnMetValGlnValThrGlySerSerAspAsnGluTyrPheTyrVal 
580585590 
AspPheArgGluTyrGluTyrAspLeuLysTrpGluPheProArgGlu 
595600605 
AsnLeuGluPheGlyLysValLeuGlySerGlyAlaPheGlyLysVal 
610615620 
MetAsnAlaThrAlaTyrGlyIleSerLysThrGlyValSerIleGln 
625630635640 
ValAlaValLysMetLeuLysGluLysAlaAspSerSerGluArgGlu 
645650655 
AlaLeuMetSerGluLeuLysMetMetThrGlnLeuGlySerHisGlu 
660665670 
AsnIleValAsnLeuLeuGlyAlaCysThrLeuSerGlyProIleTyr 
675680685 
LeuIlePheGluTyrCysCysTyrGlyAspLeuLeuAsnTyrLeuArg 
690695700 
SerLysArgGluLysPheHisArgThrTrpThrGluIlePheLysGlu 
705710715720 
HisAsnPheSerPheTyrProThrPheGlnSerHisProAsnSerSer 
725730735 
MetProGlySerArgGluValGlnIleHisProAspSerAspGlnIle 
740745750 
SerGlyLeuHisGlyAsnSerPheHisSerGluAspGluIleGluTyr 
755760765 
GluAsnGlnLysArgLeuGluGluGluGluAspLeuAsnValLeuThr 
770775780 
PheGluAspLeuLeuCysPheAlaTyrGlnValAlaLysGlyMetGlu 
785790795800 
PheLeuGluPheLysSerCysValHisArgAspLeuAlaAlaArgAsn 
805810815 
ValLeuValThrHisGlyLysValValLysIleCysAspPheGlyLeu 
820825830 
AlaArgAspIleMetSerAspSerAsnTyrValValArgGlyAsnAla 
835840845 
ArgLeuProValLysTrpMetAlaProGluSerLeuPheGluGlyIle 
850855860 
TyrThrIleLysSerAspValTrpSerTyrGlyIleLeuLeuTrpGlu 
865870875880 
IlePheSerLeuGlyValAsnProTyrProGlyIleProValAspAla 
885890895 
AsnPheTyrLysLeuIleGlnAsnGlyPheLysMetAspGlnProPhe 
900905910 
TyrAlaThrGluGluIleTyrIleIleMetGlnSerCysTrpAlaPhe 
915920925 
AspSerArgLysArgProSerPheProAsnLeuThrSerPheLeuGly 
930935940 
CysGlnLeuAlaAspAlaGluGluAlaMetTyrGlnAsnValAspGly 
945950955960 
ArgValSerGluCysProHisThrTyrGlnAsnArgArgProPheSer 
965970975 
ArgGluMetAspLeuGlyLeuLeuSerProGlnAlaGlnValGluAsp 
980985990 
Ser 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5406 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(v) FRAGMENT TYPE: N-terminal 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 208..4311 
(ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- peptide 
(B) LOCATION: 208..4308 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CTGTGTCCCGCAGCCGGATAACCTGGCTGACCCGATTCCGCGGACACCCGTGCAGCCGCG60 
GCTGGAGCCAGGGCGCCGGTGCCCGCGCTCTCCCCGGTCTTGCGCTGCGGGGGCCGATAC120 
CGCCTCTGTGACTTCTTTGCGGGCCAGGGACGGAGAAGGAGTCTGTGCCTGAGAAACTGG180 
GCTCTGTGCCCAGGCGCGAGGTGCAGGATGGAGAGCAAGGGCCTGCTAGCT231 
MetGluSerLysGlyLeuLeuAla 
15 
GTCGCTCTGTGGTTCTGCGTGGAGACCCGAGCCGCCTCTGTGGGTTTG279 
ValAlaLeuTrpPheCysValGluThrArgAlaAlaSerValGlyLeu 
101520 
CCTGGCGATTTTCTCCATCCCCCCAAGCTCAGCACACAGAAAGACATA327 
ProGlyAspPheLeuHisProProLysLeuSerThrGlnLysAspIle 
25303540 
CTGACAATTTTGGCAAATACAACCCTTCAGATTACTTGCAGGGGACAG375 
LeuThrIleLeuAlaAsnThrThrLeuGlnIleThrCysArgGlyGln 
455055 
CGGGACCTGGACTGGCTTTGGCCCAATGCTCAGCGTGATTCTGAGGAA423 
ArgAspLeuAspTrpLeuTrpProAsnAlaGlnArgAspSerGluGlu 
606570 
AGGGTATTGGTGACTGAATGCGGCGGTGGTGACAGTATCTTCTGCAAA471 
ArgValLeuValThrGluCysGlyGlyGlyAspSerIlePheCysLys 
758085 
ACACTCACCATTCCCAGGGTGGTTGGAAATGATACTGGAGCCTACAAG519 
ThrLeuThrIleProArgValValGlyAsnAspThrGlyAlaTyrLys 
9095100 
TGCTCGTACCGGGACGTCGACATAGCCTCCACTGTTTATGTCTATGTT567 
CysSerTyrArgAspValAspIleAlaSerThrValTyrValTyrVal 
105110115120 
CGAGATTACAGATCACCATTCATCGCCTCTGTCAGTGACCAGCATGGC615 
ArgAspTyrArgSerProPheIleAlaSerValSerAspGlnHisGly 
125130135 
ATCGTGTACATCACCGAGAACAAGAACAAAACTGTGGTGATCCCCTGC663 
IleValTyrIleThrGluAsnLysAsnLysThrValValIleProCys 
140145150 
CGAGGGTCGATTTCAAACCTCAATGTGTCTCTTTGCGCTAGGTATCCA711 
ArgGlySerIleSerAsnLeuAsnValSerLeuCysAlaArgTyrPro 
155160165 
GAAAAGAGATTTGTTCCGGATGGAAACAGAATTTCCTGGGACAGCGAG759 
GluLysArgPheValProAspGlyAsnArgIleSerTrpAspSerGlu 
170175180 
ATAGGCTTTACTCTCCCCAGTTACATGATCAGCTATGCCGGCATGGTC807 
IleGlyPheThrLeuProSerTyrMetIleSerTyrAlaGlyMetVal 
185190195200 
TTCTGTGAGGCAAAGATCAATGATGAAACCTATCAGTCTATCATGTAC855 
PheCysGluAlaLysIleAsnAspGluThrTyrGlnSerIleMetTyr 
205210215 
ATAGTTGTGGTTGTAGGATATAGGATTTATGATGTGATTCTGAGCCCC903 
IleValValValValGlyTyrArgIleTyrAspValIleLeuSerPro 
220225230 
CCGCATGAAATTGAGCTATCTGCCGGAGAAAAACTTGTCTTAAATTGT951 
ProHisGluIleGluLeuSerAlaGlyGluLysLeuValLeuAsnCys 
235240245 
ACAGCGAGAACAGAGCTCAATGTGGGGCTTGATTTCACCTGGCACTCT999 
ThrAlaArgThrGluLeuAsnValGlyLeuAspPheThrTrpHisSer 
250255260 
CCACCTTCAAAGTCTCATCATAAGAAGATTGTAAACCGGGATGTGAAA1047 
ProProSerLysSerHisHisLysLysIleValAsnArgAspValLys 
265270275280 
CCCTTTCCTGGGACTGTGGCGAAGATGTTTTTGAGCACCTTGACAATA1095 
ProPheProGlyThrValAlaLysMetPheLeuSerThrLeuThrIle 
285290295 
GAAAGTGTGACCAAGAGTGACCAAGGGGAATACACCTGTGTAGCGTCC1143 
GluSerValThrLysSerAspGlnGlyGluTyrThrCysValAlaSer 
300305310 
AGTGGACGGATGATCAAGAGAAATAGAACATTTGTCCGAGTTCACACA1191 
SerGlyArgMetIleLysArgAsnArgThrPheValArgValHisThr 
315320325 
AAGCCTTTTATTGCTTTCGGTAGTGGGATGAAATCTTTGGTGGAAGCC1239 
LysProPheIleAlaPheGlySerGlyMetLysSerLeuValGluAla 
330335340 
ACAGTGGGCAGTCAAGTCCGAATCCCTGTGAAGTATCTCAGTTACCCA1287 
ThrValGlySerGlnValArgIleProValLysTyrLeuSerTyrPro 
345350355360 
GCTCCTGATATCAAATGGTACAGAAATGGAAGGCCCATTGAGTCCAAC1335 
AlaProAspIleLysTrpTyrArgAsnGlyArgProIleGluSerAsn 
365370375 
TACACAATGATTGTTGGCGATGAACTCACCATCATGGAAGTGACTGAA1383 
TyrThrMetIleValGlyAspGluLeuThrIleMetGluValThrGlu 
380385390 
AGAGATGCAGGAAACTACACGGTCATCCTCACCAACCCCATTTCAATG1431 
ArgAspAlaGlyAsnTyrThrValIleLeuThrAsnProIleSerMet 
395400405 
GAGAAACAGAGCCACATGGTCTCTCTGGTTGTGAATGTCCCACCCCAG1479 
GluLysGlnSerHisMetValSerLeuValValAsnValProProGln 
410415420 
ATCGGTGAGAAAGCCTTGATCTCGCCTATGGATTCCTACCAGTATGGG1527 
IleGlyGluLysAlaLeuIleSerProMetAspSerTyrGlnTyrGly 
425430435440 
ACCATGCAGACATTGACATGCACAGTCTACGCCAACCCTCCCCTGCAC1575 
ThrMetGlnThrLeuThrCysThrValTyrAlaAsnProProLeuHis 
445450455 
CACATCCAGTGGTACTGGCAGCTAGAAGAAGCCTGCTCCTACAGACCC1623 
HisIleGlnTrpTyrTrpGlnLeuGluGluAlaCysSerTyrArgPro 
460465470 
GGCCAAACAAGCCCGTATGCTTGTAAAGAATGGAGACACGTGGAGGAT1671 
GlyGlnThrSerProTyrAlaCysLysGluTrpArgHisValGluAsp 
475480485 
TTCCAGGGGGGAAACAAGATCGAAGTCACCAAAAACCAATATGCCCTG1719 
PheGlnGlyGlyAsnLysIleGluValThrLysAsnGlnTyrAlaLeu 
490495500 
ATTGAAGGAAAAAACAAAACTGTAAGTACGCTGGTCATCCAAGCTGCC1767 
IleGluGlyLysAsnLysThrValSerThrLeuValIleGlnAlaAla 
505510515520 
AACGTGTCAGCGTTGTACAAATGTGAAGCCATCAACAAAGCGGGACGA1815 
AsnValSerAlaLeuTyrLysCysGluAlaIleAsnLysAlaGlyArg 
525530535 
GGAGAGAGGGTCATCTCCTTCCATGTGATCAGGGGTCCTGAAATTACT1863 
GlyGluArgValIleSerPheHisValIleArgGlyProGluIleThr 
540545550 
GTGCAACCTGCTGCCCAGCCAACTGAGCAGGAGAGTGTGTCCCTGTTG1911 
ValGlnProAlaAlaGlnProThrGluGlnGluSerValSerLeuLeu 
555560565 
TGCACTGCAGACAGAAATACGTTTGAGAACCTCACGTGGTACAAGCTT1959 
CysThrAlaAspArgAsnThrPheGluAsnLeuThrTrpTyrLysLeu 
570575580 
GGCTCACAGGCAACATCGGTCCACATGGGCGAATCACTCACACCAGTT2007 
GlySerGlnAlaThrSerValHisMetGlyGluSerLeuThrProVal 
585590595600 
TGCAAGAACTTGGATGCTCTTTGGAAACTGAATGGCACCATGTTTTCT2055 
CysLysAsnLeuAspAlaLeuTrpLysLeuAsnGlyThrMetPheSer 
605610615 
AACAGCACAAATGACATCTTGATTGTGGCATTTCAGAATGCCTCTCTG2103 
AsnSerThrAsnAspIleLeuIleValAlaPheGlnAsnAlaSerLeu 
620625630 
CAGGACCAAGGCGACTATGTTTGCTCTGCTCAAGATAAGAAGACCAAG2151 
GlnAspGlnGlyAspTyrValCysSerAlaGlnAspLysLysThrLys 
635640645 
AAAAGACATTGCCTGGTCAAACAGCTCATCATCCTAGAGCGCATGGCA2199 
LysArgHisCysLeuValLysGlnLeuIleIleLeuGluArgMetAla 
650655660 
CCCATGATCACCGGAAATCTGGAGAATCAGACAACAACCATTGGCGAG2247 
ProMetIleThrGlyAsnLeuGluAsnGlnThrThrThrIleGlyGlu 
665670675680 
ACCATTGAAGTGACTTGCCCAGCATCTGGAAATCCTACCCCACACATT2295 
ThrIleGluValThrCysProAlaSerGlyAsnProThrProHisIle 
685690695 
ACATGGTTCAAAGACAACGAGACCCTGGTAGAAGATTCAGGCATTGTA2343 
ThrTrpPheLysAspAsnGluThrLeuValGluAspSerGlyIleVal 
700705710 
CTGAGAGATGGGAACCGGAACCTGACTATCCGCAGGGTGAGGAAGGAG2391 
LeuArgAspGlyAsnArgAsnLeuThrIleArgArgValArgLysGlu 
715720725 
GATGGAGGCCTCTACACCTGCCAGGCCTGCAATGTCCTTGGCTGTGCA2439 
AspGlyGlyLeuTyrThrCysGlnAlaCysAsnValLeuGlyCysAla 
730735740 
AGAGCGGAGACGCTCTTCATAATAGAAGGTGCCCAGGAAAAGACCAAC2487 
ArgAlaGluThrLeuPheIleIleGluGlyAlaGlnGluLysThrAsn 
745750755760 
TTGGAAGTCATTATCCTCGTCGGCACTGCAGTGATTGCCATGTTCTTC2535 
LeuGluValIleIleLeuValGlyThrAlaValIleAlaMetPhePhe 
765770775 
TGGCTCCTTCTTGTCATTCTCGTACGGACCGTTAAGCGGGCCAATGAA2583 
TrpLeuLeuLeuValIleLeuValArgThrValLysArgAlaAsnGlu 
780785790 
GGGGAACTGAAGACAGGCTACTTGTCTATTGTCATGGATCCAGATGAA2631 
GlyGluLeuLysThrGlyTyrLeuSerIleValMetAspProAspGlu 
795800805 
TTGCCCTTGGATGAGCGCTGTGAACGCTTGCCTTATGATGCCAGCAAG2679 
LeuProLeuAspGluArgCysGluArgLeuProTyrAspAlaSerLys 
810815820 
TGGGAATTCCCCAGGGACCGGCTGAAACTAGGAAAACCTCTTGGCCGC2727 
TrpGluPheProArgAspArgLeuLysLeuGlyLysProLeuGlyArg 
825830835840 
GGTGCCTTCGGCCAAGTGATTGAGGCAGACGCTTTTGGAATTGACAAG2775 
GlyAlaPheGlyGlnValIleGluAlaAspAlaPheGlyIleAspLys 
845850855 
ACAGCGACTTGCAAAACAGTAGCCGTCAAGATGTTGAAAGAAGGAGCA2823 
ThrAlaThrCysLysThrValAlaValLysMetLeuLysGluGlyAla 
860865870 
ACACACAGCGAGCATCGAGCCCTCATGTCTGAACTCAAGATCCTCATC2871 
ThrHisSerGluHisArgAlaLeuMetSerGluLeuLysIleLeuIle 
875880885 
CACATTGGTCACCATCTCAATGTGGTGAACCTCCTAGGCGCCTGCACC2919 
HisIleGlyHisHisLeuAsnValValAsnLeuLeuGlyAlaCysThr 
890895900 
AAGCCGGGAGGGCCTCTCATGGTGATTGTGGAATTCTCGAAGTTTGGA2967 
LysProGlyGlyProLeuMetValIleValGluPheSerLysPheGly 
905910915920 
AACCTATCAACTTACTTACGGGGCAAGAGAAATGAATTTGTTCCCTAT3015 
AsnLeuSerThrTyrLeuArgGlyLysArgAsnGluPheValProTyr 
925930935 
AAGAGCAAAGGGGCACGCTTCCGCCAGGGCAAGGACTACGTTGGGGAG3063 
LysSerLysGlyAlaArgPheArgGlnGlyLysAspTyrValGlyGlu 
940945950 
CTCTCCGTGGATCTGAAAAGACGCTTGGACAGCATCACCAGCAGCCAG3111 
LeuSerValAspLeuLysArgArgLeuAspSerIleThrSerSerGln 
955960965 
AGCTCTGCCAGCTCAGGCTTTGTTGAGGAGAAATCGCTCAGTGATGTA3159 
SerSerAlaSerSerGlyPheValGluGluLysSerLeuSerAspVal 
970975980 
GAGGAAGAAGAAGCTTCTGAAGAACTGTACAAGGACTTCCTGACCTTG3207 
GluGluGluGluAlaSerGluGluLeuTyrLysAspPheLeuThrLeu 
9859909951000 
GAGCATCTCATCTGTTACAGCTTCCAAGTGGCTAAGGGCATGGAGTTC3255 
GluHisLeuIleCysTyrSerPheGlnValAlaLysGlyMetGluPhe 
100510101015 
TTGGCATCAAGGAAGTGTATCCACAGGGACCTGGCAGCACGAAACATT3303 
LeuAlaSerArgLysCysIleHisArgAspLeuAlaAlaArgAsnIle 
102010251030 
CTCCTATCGGAGAAGAATGTGGTTAAGATCTGTGACTTCGGCTTGGCC3351 
LeuLeuSerGluLysAsnValValLysIleCysAspPheGlyLeuAla 
103510401045 
CGGGACATTTATAAAGACCCGGATTATGTCAGAAAAGGAGATGCCCGA3399 
ArgAspIleTyrLysAspProAspTyrValArgLysGlyAspAlaArg 
105010551060 
CTCCCTTTGAAGTGGATGGCCCCGGAAACCATTTTTGACAGAGTATAC3447 
LeuProLeuLysTrpMetAlaProGluThrIlePheAspArgValTyr 
1065107010751080 
ACAATTCAGAGCGATGTGTGGTCTTTCGGTGTGTTGCTCTGGGAAATA3495 
ThrIleGlnSerAspValTrpSerPheGlyValLeuLeuTrpGluIle 
108510901095 
TTTTCCTTAGGTGCCTCCCCATACCCTGGGGTCAAGATTGATGAAGAA3543 
PheSerLeuGlyAlaSerProTyrProGlyValLysIleAspGluGlu 
110011051110 
TTTTGTAGGAGATTGAAAGAAGGAACTAGAATGCGGGCTCCTGACTAC3591 
PheCysArgArgLeuLysGluGlyThrArgMetArgAlaProAspTyr 
111511201125 
ACTACCCCAGAAATGTACCAGACCATGCTGGACTGCTGGCATGAGGAC3639 
ThrThrProGluMetTyrGlnThrMetLeuAspCysTrpHisGluAsp 
113011351140 
CCCAACCAGAGACCCTCGTTTTCAGAGTTGGTGGAGCATTTGGGAAAC3687 
ProAsnGlnArgProSerPheSerGluLeuValGluHisLeuGlyAsn 
1145115011551160 
CTCCTGCAAGCAAATGCGCAGCAGGATGGCAAAGACTATATTGTTCTT3735 
LeuLeuGlnAlaAsnAlaGlnGlnAspGlyLysAspTyrIleValLeu 
116511701175 
CCAATGTCAGAGACACTGAGCATGGAAGAGGATTCTGGACTCTCCCTG3783 
ProMetSerGluThrLeuSerMetGluGluAspSerGlyLeuSerLeu 
118011851190 
CCTACCTCACCTGTTTCCTGTATGGAGGAAGAGGAAGTGTGCGACCCC3831 
ProThrSerProValSerCysMetGluGluGluGluValCysAspPro 
119512001205 
AAATTCCATTATGACAACACAGCAGGAATCAGTCATTATCTCCAGAAC3879 
LysPheHisTyrAspAsnThrAlaGlyIleSerHisTyrLeuGlnAsn 
121012151220 
AGTAAGCGAAAGAGCCGGCCAGTGAGTGTAAAAACATTTGAAGATATC3927 
SerLysArgLysSerArgProValSerValLysThrPheGluAspIle 
1225123012351240 
CCATTGGAGGAACCAGAAGTAAAAGTGATCCCAGATGACAGCCAGACA3975 
ProLeuGluGluProGluValLysValIleProAspAspSerGlnThr 
124512501255 
GACAGTGGGATGGTCCTTGCATCAGAAGAGCTGAAAACTCTGGAAGAC4023 
AspSerGlyMetValLeuAlaSerGluGluLeuLysThrLeuGluAsp 
126012651270 
AGGAACAAATTATCTCCATCTTTTGGTGGAATGATGCCCAGTAAAAGC4071 
ArgAsnLysLeuSerProSerPheGlyGlyMetMetProSerLysSer 
127512801285 
AGGGAGTCTGTGGCCTCGGAAGGCTCCAACCAGACCAGTGGCTACCAG4119 
ArgGluSerValAlaSerGluGlySerAsnGlnThrSerGlyTyrGln 
129012951300 
TCTGGGTATCACTCAGATGACACAGACACCACCGTGTACTCCAGCGAC4167 
SerGlyTyrHisSerAspAspThrAspThrThrValTyrSerSerAsp 
1305131013151320 
GAGGCAGGACTTTTAAAGATGGTGGATGCTGCAGTTCACGCTGACTCA4215 
GluAlaGlyLeuLeuLysMetValAspAlaAlaValHisAlaAspSer 
132513301335 
GGGACCACACTGCAGCTCACCTCCTGTTTAAATGGAAGTGGTCCTGTC4263 
GlyThrThrLeuGlnLeuThrSerCysLeuAsnGlySerGlyProVal 
134013451350 
CCGGCTCCGCCCCCAACTCCTGGAAATCACGAGAGAGGTGCTGCTTAGATTTT4318 
ProAlaProProProThrProGlyAsnHisGluArgGlyAlaAla 
135513601365 
AGTGTTGTTCTTTCCACCACCCGGAAGTAGCCACATTTGATTTTCATTTTTGGAGGAGGG4378 
ACCTCAGACTGCAAGGAGCTTGTCCTCAGGGCATTTCCAGAGAAGATGCCCATGACCCAA4438 
GAATGTGTTGACTCTACTCTCTTTTCCATTCATTTAAAAGTCCTATATAATGTGCCCTGC4498 
TGTGGTCTCACTACCAGTTAAAGCAAAAGACTTTCAAACACGTGGACTCTGTCCTCCAAG4558 
AAGTGGCAACGGCACCTCTGTGAAACTGGATCGAATGGGCAATGCTTTGTGTGTTGAGGA4618 
TGGGTGAGATGTCCCAGGGCCGAGTCTGTCTACCTTGGAGGCTTTGTGGAGGATGCGGCT4678 
ATGAGCCAAGTGTTAAGTGTGGGATGTGGACTGGGAGGAAGGAAGGCGCAAGCCGTCCGG4738 
AGAGCGGTTGGAGCCTGCAGATGCATTGTGCTGGCTCTGGTGGAGGTGGGCTTGTGGCCT4798 
GTCAGGAAACGCAAAGGCGGCCGGCAGGGTTTGGTTTTGGAAGGTTTGCGTGCTCTTCAC4858 
AGTCGGGTTACAGGCGAGTTCCCTGTGGCGTTTCCTACTCCTAATGAGAGTTCCTTCCGG4918 
ACTCTTACGTGTCTCCTGGCCTGGCCCCAGGAAGGAAATGATGCAGCTTGCTCCTTCCTC4978 
ATCTCTCAGGCTGTGCCTTAATTCAGAACACCAAAAGAGAGGAACGTCGGCAGAGGCTCC5038 
TGACGGGGCCGAAGAATTGTGAGAACAGAACAGAAACTCAGGGTTTCTGCTGGGTGGAGA5098 
CCCACGTGGCGCCCTGGTGGCAGGTCTGAGGGTTCTCTGTCAAGTGGCGGTAAAGGCTCA5158 
GGCTGGTGTTCTTCCTCTATCTCCACTCCTGTCAGGCCCCCAAGTCCTCAGTATTTTAGC5218 
TTTGTGGCTTCCTGATGGCAGAAAAATCTTAATTGGTTGGTTTGCTCTCCAGATAATCAC5278 
TAGCCAGATTTCGAAATTACTTTTTAGCCGAGGTTATGATAACATCTACTGTATCCTTTA5338 
GAATTTTAACCTATAAAACTATGTCTACTGGTTTCTGCCTGTGTGCTTATGTTAAAAAAA5398 
AAAAAAAA5406 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1367 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
MetGluSerLysGlyLeuLeuAlaValAlaLeuTrpPheCysValGlu 
151015 
ThrArgAlaAlaSerValGlyLeuProGlyAspPheLeuHisProPro 
202530 
LysLeuSerThrGlnLysAspIleLeuThrIleLeuAlaAsnThrThr 
354045 
LeuGlnIleThrCysArgGlyGlnArgAspLeuAspTrpLeuTrpPro 
505560 
AsnAlaGlnArgAspSerGluGluArgValLeuValThrGluCysGly 
65707580 
GlyGlyAspSerIlePheCysLysThrLeuThrIleProArgValVal 
859095 
GlyAsnAspThrGlyAlaTyrLysCysSerTyrArgAspValAspIle 
100105110 
AlaSerThrValTyrValTyrValArgAspTyrArgSerProPheIle 
115120125 
AlaSerValSerAspGlnHisGlyIleValTyrIleThrGluAsnLys 
130135140 
AsnLysThrValValIleProCysArgGlySerIleSerAsnLeuAsn 
145150155160 
ValSerLeuCysAlaArgTyrProGluLysArgPheValProAspGly 
165170175 
AsnArgIleSerTrpAspSerGluIleGlyPheThrLeuProSerTyr 
180185190 
MetIleSerTyrAlaGlyMetValPheCysGluAlaLysIleAsnAsp 
195200205 
GluThrTyrGlnSerIleMetTyrIleValValValValGlyTyrArg 
210215220 
IleTyrAspValIleLeuSerProProHisGluIleGluLeuSerAla 
225230235240 
GlyGluLysLeuValLeuAsnCysThrAlaArgThrGluLeuAsnVal 
245250255 
GlyLeuAspPheThrTrpHisSerProProSerLysSerHisHisLys 
260265270 
LysIleValAsnArgAspValLysProPheProGlyThrValAlaLys 
275280285 
MetPheLeuSerThrLeuThrIleGluSerValThrLysSerAspGln 
290295300 
GlyGluTyrThrCysValAlaSerSerGlyArgMetIleLysArgAsn 
305310315320 
ArgThrPheValArgValHisThrLysProPheIleAlaPheGlySer 
325330335 
GlyMetLysSerLeuValGluAlaThrValGlySerGlnValArgIle 
340345350 
ProValLysTyrLeuSerTyrProAlaProAspIleLysTrpTyrArg 
355360365 
AsnGlyArgProIleGluSerAsnTyrThrMetIleValGlyAspGlu 
370375380 
LeuThrIleMetGluValThrGluArgAspAlaGlyAsnTyrThrVal 
385390395400 
IleLeuThrAsnProIleSerMetGluLysGlnSerHisMetValSer 
405410415 
LeuValValAsnValProProGlnIleGlyGluLysAlaLeuIleSer 
420425430 
ProMetAspSerTyrGlnTyrGlyThrMetGlnThrLeuThrCysThr 
435440445 
ValTyrAlaAsnProProLeuHisHisIleGlnTrpTyrTrpGlnLeu 
450455460 
GluGluAlaCysSerTyrArgProGlyGlnThrSerProTyrAlaCys 
465470475480 
LysGluTrpArgHisValGluAspPheGlnGlyGlyAsnLysIleGlu 
485490495 
ValThrLysAsnGlnTyrAlaLeuIleGluGlyLysAsnLysThrVal 
500505510 
SerThrLeuValIleGlnAlaAlaAsnValSerAlaLeuTyrLysCys 
515520525 
GluAlaIleAsnLysAlaGlyArgGlyGluArgValIleSerPheHis 
530535540 
ValIleArgGlyProGluIleThrValGlnProAlaAlaGlnProThr 
545550555560 
GluGlnGluSerValSerLeuLeuCysThrAlaAspArgAsnThrPhe 
565570575 
GluAsnLeuThrTrpTyrLysLeuGlySerGlnAlaThrSerValHis 
580585590 
MetGlyGluSerLeuThrProValCysLysAsnLeuAspAlaLeuTrp 
595600605 
LysLeuAsnGlyThrMetPheSerAsnSerThrAsnAspIleLeuIle 
610615620 
ValAlaPheGlnAsnAlaSerLeuGlnAspGlnGlyAspTyrValCys 
625630635640 
SerAlaGlnAspLysLysThrLysLysArgHisCysLeuValLysGln 
645650655 
LeuIleIleLeuGluArgMetAlaProMetIleThrGlyAsnLeuGlu 
660665670 
AsnGlnThrThrThrIleGlyGluThrIleGluValThrCysProAla 
675680685 
SerGlyAsnProThrProHisIleThrTrpPheLysAspAsnGluThr 
690695700 
LeuValGluAspSerGlyIleValLeuArgAspGlyAsnArgAsnLeu 
705710715720 
ThrIleArgArgValArgLysGluAspGlyGlyLeuTyrThrCysGln 
725730735 
AlaCysAsnValLeuGlyCysAlaArgAlaGluThrLeuPheIleIle 
740745750 
GluGlyAlaGlnGluLysThrAsnLeuGluValIleIleLeuValGly 
755760765 
ThrAlaValIleAlaMetPhePheTrpLeuLeuLeuValIleLeuVal 
770775780 
ArgThrValLysArgAlaAsnGluGlyGluLeuLysThrGlyTyrLeu 
785790795800 
SerIleValMetAspProAspGluLeuProLeuAspGluArgCysGlu 
805810815 
ArgLeuProTyrAspAlaSerLysTrpGluPheProArgAspArgLeu 
820825830 
LysLeuGlyLysProLeuGlyArgGlyAlaPheGlyGlnValIleGlu 
835840845 
AlaAspAlaPheGlyIleAspLysThrAlaThrCysLysThrValAla 
850855860 
ValLysMetLeuLysGluGlyAlaThrHisSerGluHisArgAlaLeu 
865870875880 
MetSerGluLeuLysIleLeuIleHisIleGlyHisHisLeuAsnVal 
885890895 
ValAsnLeuLeuGlyAlaCysThrLysProGlyGlyProLeuMetVal 
900905910 
IleValGluPheSerLysPheGlyAsnLeuSerThrTyrLeuArgGly 
915920925 
LysArgAsnGluPheValProTyrLysSerLysGlyAlaArgPheArg 
930935940 
GlnGlyLysAspTyrValGlyGluLeuSerValAspLeuLysArgArg 
945950955960 
LeuAspSerIleThrSerSerGlnSerSerAlaSerSerGlyPheVal 
965970975 
GluGluLysSerLeuSerAspValGluGluGluGluAlaSerGluGlu 
980985990 
LeuTyrLysAspPheLeuThrLeuGluHisLeuIleCysTyrSerPhe 
99510001005 
GlnValAlaLysGlyMetGluPheLeuAlaSerArgLysCysIleHis 
101010151020 
ArgAspLeuAlaAlaArgAsnIleLeuLeuSerGluLysAsnValVal 
1025103010351040 
LysIleCysAspPheGlyLeuAlaArgAspIleTyrLysAspProAsp 
104510501055 
TyrValArgLysGlyAspAlaArgLeuProLeuLysTrpMetAlaPro 
106010651070 
GluThrIlePheAspArgValTyrThrIleGlnSerAspValTrpSer 
107510801085 
PheGlyValLeuLeuTrpGluIlePheSerLeuGlyAlaSerProTyr 
109010951100 
ProGlyValLysIleAspGluGluPheCysArgArgLeuLysGluGly 
1105111011151120 
ThrArgMetArgAlaProAspTyrThrThrProGluMetTyrGlnThr 
112511301135 
MetLeuAspCysTrpHisGluAspProAsnGlnArgProSerPheSer 
114011451150 
GluLeuValGluHisLeuGlyAsnLeuLeuGlnAlaAsnAlaGlnGln 
115511601165 
AspGlyLysAspTyrIleValLeuProMetSerGluThrLeuSerMet 
117011751180 
GluGluAspSerGlyLeuSerLeuProThrSerProValSerCysMet 
1185119011951200 
GluGluGluGluValCysAspProLysPheHisTyrAspAsnThrAla 
120512101215 
GlyIleSerHisTyrLeuGlnAsnSerLysArgLysSerArgProVal 
122012251230 
SerValLysThrPheGluAspIleProLeuGluGluProGluValLys 
123512401245 
ValIleProAspAspSerGlnThrAspSerGlyMetValLeuAlaSer 
125012551260 
GluGluLeuLysThrLeuGluAspArgAsnLysLeuSerProSerPhe 
1265127012751280 
GlyGlyMetMetProSerLysSerArgGluSerValAlaSerGluGly 
128512901295 
SerAsnGlnThrSerGlyTyrGlnSerGlyTyrHisSerAspAspThr 
130013051310 
AspThrThrValTyrSerSerAspGluAlaGlyLeuLeuLysMetVal 
131513201325 
AspAlaAlaValHisAlaAspSerGlyThrThrLeuGlnLeuThrSer 
133013351340 
CysLeuAsnGlySerGlyProValProAlaProProProThrProGly 
1345135013551360 
AsnHisGluArgGlyAlaAla 
1365 
__________________________________________________________________________