DNA encoding a novel human .beta..sub.2 integrin .alpha. subunit polypeptide, designated .alpha..sub.d, is disclosed along with methods and materials for production of the same by recombinant procedures. Binding molecules specific for .alpha..sub.d are also disclosed as useful for modulating the biological activities of .alpha..sub.d. DNA from other species which show homology to human .alpha..sub.d encoding sequences are also disclosed.

FIELD OF THE INVENTION 
The present invention relates to the cloning and expression of nucleotide 
sequences encoding a novel human .beta..sub.2 integrin .alpha. subunit, 
designated .alpha..sub.d, which is structurally related to the known human 
.beta..sub.2 integrin .alpha. subunits, CD11a, CD11b and CD11c. The 
present invention also relates to nucleotide sequences isolated other 
species which show homology to human .alpha..sub.d encoding sequences. 
BACKGROUND OF THE INVENTION 
The integrins are a class of membrane-associated molecules which actively 
participate in cellular adhesion. Integrins are transmembrane heterodimers 
comprising an .alpha. subunit in noncovalent association with a .beta. 
subunit. To date, at least fourteen .alpha. subunits and eight .beta. 
subunits have been identified [reviewed in Springer, Nature 346:425-434 
(1990)]. The .beta. subunits are generally capable of association with 
more than one .alpha. subunit and the heterodimers sharing a common .beta. 
subunit have been classified as subfamilies within the integrin 
population. 
One class of human integrins, restricted to expression in white blood 
cells, is characterized by a common .beta..sub.2 subunit. As a result of 
this cell-specific expression, these integrins are commonly referred to as 
the leukocyte integrins, Leu-CAMs or leukointegrins. Because of the common 
.beta..sub.2 subunit, an alternative designation of this class is the 
.beta..sub.2 integrins. The .beta..sub.2 subunit (CD18) has previously 
been isolated in association with one of three distinct .alpha. subunits, 
CD11a, CD11b or CD11c. The isolation of a cDNA encoding human CD18 is 
described in Kishimoto, et al., Cell 48:681-690 (1987). In official WHO 
nomenclature, the heterodimeric proteins are referred to as CD11a/CD18, 
CD11b/CD18, and CD11c/CD18; in common nomenclature they are referred to as 
LFA-1, Mac-1 or Mol and p150,95 or LeuM5, respectively [Cobbold, et al., 
in Leukocyte Typing III, McMichael (ed), Oxford Press, p.788 (1987)]. The 
human .beta..sub.2 integrin .alpha. subunits CD11a, CD11b and CD11c have 
been demonstrated to migrate under reducing condition in electrophoresis 
with apparent molecular weights of approximately 180 kD, 155 kD and 150 
kD, respectively, and DNAs encoding these subunits have been cloned 
[CD11a, Larson, et al., J. Cell Biol. 108:703-712 (1989); CD11b, Corbi, et 
al., J. Biol. Chem. 263:12403-12411 (1988) and CD11c, Corbi, et al. EMBO 
J. 6:4023-4028 (1987)]. Putative homologs of the human .beta..sub.2 
integrin .alpha. and .beta. chains, defined by approximate similarity in 
molecular weight, have been variously identified in other species 
including monkeys and other primates [Letvin, et al., Blood 61:408-410 
(1983)], mice [Sanchez-Madrid, et al., J. Exp. Med. 154:1517 (1981)], and 
dogs [Moore, et al., Tissue Antigens 36:211-220 (1990)]. 
The absolute molecular weights of presumed homologs from other species have 
been shown to vary significantly [see, e.g., Danilenko et al., Tissue 
Antigens 40:13-21 (1992)], and in the absence of sequence information, a 
definitive correlation between human integrin subunits and those 
identified in other species has not been possible. Moreover, variation in 
the number of members in a protein family has been observed between 
different species. Consider, for example, that more IgA isotypes have been 
isolated in rabbits than in humans [Burnett, et al., EMBO J. 8:4041-4047 
(1989) and Schneiderman, et al., Proc. Natl. Acad. Sci.(USA) 86:7561-7565 
(1989)]. Similarly, in humans, at least six variants of the 
metallothionine protein have been previously identified [Karin and 
Richards, Nature 299:797-802 (1982) and Varshney, et al., Mol. Cell. Biol. 
6:26-37, (1986)], whereas in the mouse, only two such variants are in 
evidence [Searle, et al., Mol. Cell. Biol. 4:1221-1230 (1984)]. Therefore, 
existence of multiple members of a protein family in one species does not 
necessarily imply that corresponding family members exist in another 
species. 
In the specific context of .beta..sub.2 integrins, in dogs it has been 
observed that the presumed canine .beta..sub.2 counterpart to the human 
CD18 is capable of dimer formation with as many as four potentially 
distinct .alpha. subunits [Danilenko, et al., supra]. Antibodies generated 
by immunizing mice with canine splenocytes resulted in monoclonal 
antibodies which immunoprecipitated proteins tentatively designated as 
canine homologs to human CD18, CD11a, CD11b and CD11c based mainly on 
similar, but not identical, molecular weights. Another anti-canine 
splenocyte antibody, Ca11.8H2, recognized and immunoprecipitated a fourth 
.alpha.-like canine subunit also capable of association with the 
.beta..sub.2 subunit, but having a unique molecular weight and restricted 
in expression to a subset of differentiated tissue macrophages. Antibodies 
generated by immunization of hamsters with murine dendritic cells resulted 
in two anti-integrin antibodies [Metlay, et al., J. Exp. Med. 
171:1753-1771 (1990)]. One antibody, 2E6, immunoprecipitated a predominant 
heterodimer with subunits having approximate molecular weights of 180 kD 
and 90 kD in addition to minor bands in the molecular weight range of 
150-160 kD. The second antibody, N418, precipitated another apparent 
heterodimer with subunits having approximate molecular weights of 150 kD 
and 90 Kd. Based on cellular adhesion blocking studies, it was 
hypothesized that antibody 2E6 recognized a murine counterpart to human 
CD18. While the molecular weight of the N418 antigen suggested recognition 
of a murine homolog to human CD11c/CD18, further analysis indicated that 
the murine antigen exhibited a tissue distribution pattern which was 
inconsistent with that observed for human CD11c/CD18. 
The antigens recognized by the canine Ca11.8H2 antibody and the murine N418 
antibody could represent a variant species (e.g., a glycosylation or 
splice variant) of a previously identified canine or murine .alpha. 
subunit. Alternatively, these antigens may represent unique canine and 
murine integrin .alpha. subunits. In the absence of specific information 
regarding primary structure, these alternatives cannot be distinguished. 
In humans, CD11a/CD18 is expressed on all leukocytes. CD11b/CD18 and 
CD11c/CD18 are essentially restricted to expression on monocytes, 
granulocytes, macrophages and natural killer (NK) cells, but CD11c/CD18 is 
also detected on some B-cell types. In general, CD11a/CD18 predominates on 
lymphocytes, CD11b/CD18 on granulocytes and CD11c/CD18 on macrophages [see 
review, Arnaout, Blood 75:1037-1050 (1990)]. Expression of the .alpha. 
chains, however, is variable with regard to the state of activation and 
differentiation of the individual cell types [See review, Larson and 
Springer, Immunol. Rev. 114:181-217 (1990).] 
The involvement of the .beta..sub.2 integrins in human immune and 
inflammatory responses has been demonstrated using monoclonal antibodies 
which are capable of blocking .beta..sub.2 integrin-associated cell 
adhesion. For example, Cd11a/CD18, CD11b/CD18 and CD11c/CD18 actively 
participate in natural killer (NK) cell binding to lymphoma and 
adenocarcinoma cells [Patarroyo, et al., J. Immunol. Rev. 114:67-108 
(1990)], granulocyte accumulation [Nourshargh, et al., J. Immunol. 
142:3193-3198 (1989)], granulocyte-independent plasma leakage [Arfors, et 
al., Blood 69:338-340 (1987)], chemotactic response of stimulated 
leukocytes [Arfors, et al., supra] and leukocyte adhesion to vascular 
endothelium [Price, et al., J. Immunol. 139:4174-4177 (1987) and Smith, et 
al., J. Clin. Invest. 83:2008-2017 (1989)]. The fundamental role of 
.beta..sub.2 integrins in immune and inflammatory responses is made 
apparent in the clinical syndrome referred to as leukocyte adhesion 
deficiency (LAD), wherein clinical manifestations include recurrent and 
often life threatening bacterial infections. LAD results from 
heterogeneous mutations in the .beta..sub.2 subunit [Kishimoto, et al., 
Cell 50:193-202 (1987)] and the severity of the disease state is 
proportional to the degree of the deficiency in .beta..sub.2 subunit 
expression. Formation of the complete integrin heterodimer is impaired by 
the .beta..sub.2 mutation [Kishimoto, et al., supra]. 
Interestingly, at least one antibody specific for CD18 has been shown to 
inhibit human immunodeficiency virus type-1 (HIV-1) syncytia formation in 
vitro, albeit the exact mechanism of this inhibition is unclear [Hildreth 
and Orentas, Science 244:1075-1078 (1989)]. This observation is consistent 
with the discovery that a principal counterreceptor of CD11a/CD18, ICAM-1, 
is also a surface receptor for the major group of rhinovirus serotypes 
[Greve, et al., Cell 56:839 (1989)]. 
The significance of .beta..sub.2 integrin binding activity in human immune 
and inflammatory responses underscores the necessity to develop a more 
complete understanding of this class of surface proteins. Identification 
of yet unknown members of this subfamily, as well as their 
counterreceptors, and the generation of monoclonal antibodies or other 
soluble factors which can alter biological activity of the .beta..sub.2 
integrins will provide practical means for therapeutic intervention in 
.beta..sub.2 integrin-related immune and inflammatory responses. 
BRIEF DESCRIPTION OF THE INVENTION 
In one aspect, the present invention provides novel purified and isolated 
polynucleotides (e.g., DNA and RNA transcripts, both sense and antisense 
strands) encoding a novel human .beta..sub.2 integrin .alpha. subunit, 
.alpha..sub.d, and variants thereof (i.e., deletion, addition or 
substitution analogs) which possess binding and/or immunological 
properties inherent to .alpha..sub.d. Preferred DNA molecules of the 
invention include cDNA, genomic DNA and wholly or partially chemically 
synthesized DNA molecules. A presently preferred polynucleotide is the DNA 
as set forth in SEQ ID NO: 1, encoding the polypeptide of SEQ ID NO: 2. 
Also provided are recombinant plasmid and viral DNA constructions 
(expression constructs) which include .alpha..sub.d encoding sequences, 
wherein the .alpha..sub.d encoding sequence is operatively linked to a 
homologous or heterologous transcriptional regulatory element or elements. 
Also provided by the present invention are isolated and purified mouse and 
rat polynucleotides which exhibit homology to polynucleotides encoding 
human .alpha..sub.d. A preferred mouse polynucleotide is set forth in SEQ 
ID NO: 45; a preferred rat polynucleotide is set forth in SEQ ID NO: 36. 
As another aspect of the invention, prokaryotic or eukaryotic host cells 
transformed or transfected with DNA sequences of the invention are 
provided which express .alpha..sub.d polypeptide or variants thereof. Host 
cells of the invention are particularly useful for large scale production 
of .alpha..sub.d polypeptide, which can be isolated from either the host 
cell itself or from the medium in which the host cell is grown. Host cells 
which express .alpha..sub.d polypeptide on their extracellular membrane 
surface are also useful as immunogens in the production of .alpha..sub.d 
-specific antibodies. Preferably, host cells transfected with 
.alpha..sub.d will be cotransfected to express a .beta..sub.2 integrin 
subunit in order to allow surface expression of the heterodimer. 
Also provided by the present invention are purified and isolated 
.alpha..sub.d polypeptides, fragments and variants thereof. Preferred 
.alpha..sub.d polypeptides are as set forth in SEQ ID NO: 2. Novel 
.alpha..sub.d products of the invention may be obtained as isolates from 
natural sources, but, along with .alpha..sub.d variant products, are 
preferably produced by recombinant procedures involving host cells of the 
invention. Completely glycosylated, partially glycosylated and wholly 
deglycosylated forms of the .alpha..sub.d polypeptide may be generated by 
varying the host cell selected for recombinant production and/or 
post-isolation processing. Variant .alpha..sub.d polypeptides of the 
invention may comprise water soluble and insoluble .alpha..sub.d 
polypeptides including analogs wherein one or more of the amino acids are 
deleted or replaced: (1) without loss, and preferably with enhancement, of 
one or more biological activities or immunological characteristics 
specific for .alpha..sub.d ; or (2) with specific disablement of a 
particular ligand/receptor binding or signalling function. Fusion 
polypeptides are also provided, wherein .alpha..sub.d amino acid sequences 
are expressed contiguously with amino acid sequences from other 
polypeptides. Such fusion polypeptides may possess modified biological, 
biochemical, and/or immunological properties in comparison to wild-type 
.alpha..sub.d. Analog polypeptides including additional amino acid (e.g., 
lysine or cysteine) residues that facilitate multimer formation are 
contemplated. 
Also comprehended by the present invention are polypeptides and other 
non-peptide molecules which specifically bind to .alpha..sub.d. Preferred 
binding molecules include antibodies (e.g., monoclonal and polyclonal), 
counterreceptors (e.g., membrane-associated and soluble forms) and other 
ligands (e.g., naturally occurring or synthetic molecules), including 
those which competitively bind .alpha..sub.d in the presence of 
.alpha..sub.d monoclonal antibodies and/or specific counterreceptors. 
Binding molecules are useful for purification of .alpha..sub.d 
polypeptides and identifying cell types which express .alpha..sub.d. 
Binding molecules are also useful for modulating (i.e., inhibiting, 
blocking or stimulating) of in vivo binding and/or signal transduction 
activities of .alpha..sub.d. 
Assays to identify .alpha..sub.d binding molecules are also provided, 
including immobilized ligand binding assays, solution binding assays, 
scintillation proximity assays, di-hybrid screening assays, and the like. 
In vitro assays for identifying antibodies or other compounds that modulate 
the activity of .alpha..sub.d may involve, for example, immobilizing 
.alpha..sub.d or a natural ligand to which .alpha..sub.d binds, detectably 
labelling the nonimmobilized binding partner, incubating the binding 
partners together and determining the effect of a test compound on the 
amount of label bound wherein a reduction in the label bound in the 
presence of the test compound compared to the amount of label bound in the 
absence of the test compound indicates that the test agent is an inhibitor 
of .alpha..sub.d binding. 
Another type of assay for identifying compounds that modulate the 
interaction between .alpha..sub.d and a ligand involves immobilizing 
.alpha..sub.d or a fragment thereof on a solid support coated (or 
impregnated with) a fluorescent agent, labelling the ligand with a 
compound capable of exciting the fluorescent agent, contacting the 
immobilized .alpha..sub.d with the labelled ligand in the presence and 
absence of a putative modulator compound, detecting light emission by the 
fluorescent agent, and identifying modulating compounds as those compounds 
that affect the emission of light by the fluorescent agent in comparison 
to the emission of light by the fluorescent agent in the absence of a 
modulating compound. Alternatively, the .alpha..sub.d ligand may be 
immobilized and .alpha..sub.d may be labelled in the assay. 
Yet another method contemplated by the invention for identifying compounds 
that modulate the interaction between .alpha..sub.d and a ligand involves 
transforming or transfecting appropriate host cells with a DNA construct 
comprising a reporter gene under the control of a promoter regulated by a 
transcription factor having a DNA-binding domain and an activating domain, 
expressing in the host cells a first hybrid DNA sequence encoding a first 
fusion of part or all of .alpha..sub.d and either the DNA binding domain 
or the activating domain of the transcription factor, expressing in the 
host cells a second hybrid DNA sequence encoding part or all of the ligand 
and the DNA binding domain or activating domain of the transcription 
factor which is not incorporated in the first fusion, evaluating the 
effect of a putative modulating compound on the interaction between 
.alpha..sub.d and the ligand by detecting binding of the ligand to 
.alpha..sub.d in a particular host cell by measuring the production of 
reporter gene product in the host cell in the presence or absence of the 
putative modulator, and identifying modulating compounds as those 
compounds altering production of the reported gene product in comparison 
to production of the reporter gene product in the absence of the 
modulating compound. Presently preferred for use in the assay are the ADHI 
promoter, the lexA DNA binding domain, the GAL4 transactivation domain, 
the lacZ reporter gene, and a yeast host cell. 
A modified version of the foregoing assay may be used in isolating a 
polynucleotide encoding a protein that binds to .alpha..sub.d by 
transforming or transfecting appropriate host cells with a DNA construct 
comprising a reporter gene under the control of a promoter regulated by a 
transcription factor having a DNA-binding domain and an activating domain, 
expressing in the host cells a first hybrid DNA sequence encoding a first 
fusion of part or all of .alpha..sub.d and either the DNA binding domain 
or the activating domain of the transcription factor, expressing in the 
host cells a library of second hybrid DNA sequences encoding second 
fusions of part or all of putative .alpha..sub.d binding proteins and the 
DNA binding domain or activating domain of the transcription factor which 
is not incorporated in the first fusion, detecting binding of an 
.alpha..sub.d binding protein to .alpha..sub.d in a particular host cell 
by detecting the production of reporter gene product in the host cell, and 
isolating second hybrid DNA sequences encoding .alpha..sub.d binding 
protein from the particular host cell. 
Hybridoma cell lines which produce antibodies specific for .alpha..sub.d 
are also comprehended by the invention. Techniques for producing 
hybridomas which secrete monoclonal antibodies are well known in the art. 
Hybridoma cell lines may be generated after immunizing an animal with 
purified .alpha..sub.d, variants of .alpha..sub.d or cells which express 
.alpha..sub.d or a variant thereof on the extracellular membrane surface. 
Immunogen cell types include cells which express .alpha..sub.d in vivo, or 
transfected prokaryotic or eukaryotic cell lines which normally do not 
normally express .alpha..sub.d in vivo. 
The value of the information contributed through the disclosure of the DNA 
and amino acid sequences of .alpha..sub.d is manifest. In one series of 
examples, the disclosed .alpha..sub.d CDNA sequence makes possible the 
isolation of the human .alpha..sub.d genomic DNA sequence, including 
transcriptional control elements for the genomic sequence. Identification 
of .alpha..sub.d allelic variants and heterologous species (e.g., rat or 
mouse) DNAs is also comprehended. Isolation of the human .alpha..sub.d 
genomic DNA and heterologous species DNAs can be accomplished by standard 
DNA/DNA hybridization techniques, under appropriately stringent 
conditions, using all or part of the .alpha..sub.d CDNA sequence as a 
probe to screen an appropriate library. Alternatively, polymerase chain 
reaction (PCR) using oligonucleotide primers that are designed based on 
the known CDNA sequence can be used to amplify and identify genomic 
.alpha..sub.d DNA sequences. Synthetic DNAs encoding the .alpha..sub.d 
polypeptide, including fragments and other variants thereof, may be 
produced by conventional synthesis methods. 
DNA sequence information of the invention also makes possible the 
development, by homologous recombination or "knockout" strategies [see, 
e.g., Kapecchi, Science 244:1288-1292 (1989)], to produce rodents that 
fail to express a functional .alpha..sub.d polypeptide or that express a 
variant .alpha..sub.d polypeptide. Such rodents are useful as models for 
studying the activities of .alpha..sub.d and .alpha..sub.d modulators in 
vivo. 
DNA and amino acid sequences of the invention also make possible the 
analysis of .alpha..sub.d epitopes which actively participate in 
counterreceptor binding as well as epitopes which may regulate, rather 
than actively participate in, binding. Identification of epitopes which 
may participate in transmembrane signal transduction is also comprehended 
by the invention. 
DNA of the invention is also useful for the detection of cell types which 
express .alpha..sub.d polypeptide. Standard DNA/RNA hybridization 
techniques which utilize .alpha..sub.d DNA to detect .alpha..sub.d RNA may 
be used to determine the constitutive level of .alpha..sub.d transcription 
within a cell, as well as changes in the level of transcription in 
response to internal or external agents. Identification of agents which 
modify transcription and/or translation of .alpha..sub.d can, in turn, be 
assessed for potential therapeutic or prophylactic value. DNA of the 
invention also makes possible in situ hybridization of .alpha..sub.d DNA 
to cellular RNA to determine the cellular localization of .alpha..sub.d 
specific messages within complex cell populations and tissues. 
DNA of the invention is also useful for identification of non-human 
polynucleotide sequences which display homology to human .alpha..sub.d 
sequences. Possession of non-human .alpha..sub.d DNA sequences permits 
development of animal models (including, for example, transgenic models) 
of the human system. 
As another aspect of the invention, monoclonal or polyclonal antibodies 
specific for .alpha..sub.d may be employed in immunohistochemical analysis 
to localize .alpha..sub.d to subcellular compartments or individual cells 
within tissues. Immunohistochemical analyses of this type are particularly 
useful when used in combination with in situ hybridization to localize 
both .alpha..sub.d mRNA and polypeptide products of the .alpha..sub.d 
gene. 
Identification of cell types which express .alpha..sub.d may have 
significant ramifications for development of therapeutic and prophylactic 
agents. It is anticipated that the products of the invention related to 
.alpha..sub.d can be employed in the treatment of diseases wherein 
macrophages are an essential element of the disease process. 
For example, in BB rats which spontaneously develop type 1 or insulin 
dependent diabetes, macrophages have been documented as the predominant 
immune cell infiltrating the earliest detectable pancreatic lesions 
[Hanenberg, et al., Diabetologia 32:126-134 (1989)]. Non-specific removal 
of macrophages from the system prevents the onset of diabetes. It is 
therefore anticipated that .alpha..sub.d may play a significant role 
either in the initial sequestration of the macrophages at the lesion site 
and/or their subsequent destructive effector functions. 
Similarly, the genesis of atherosclerotic lesions is thought to involve the 
participation of specialized lipid laden macrophages termed foam cells, 
potentially both as triggering and sustaining elements of lesion 
progression. Since one ligand of .alpha..sub.d, ICAM-R, is known to be 
expressed on activated endothelial cells at neovascularizing sites as 
might occur in certain types of nascent atherosclerotic lesions, it is 
anticipated that interactions mediated by .alpha..sub.d on macrophages may 
serve both to facilitate the initial sequestration of the cells within the 
nascent lesion and potentially to promote their activation. 
Other diseases which involve activated macrophages as a central participant 
include, but are not limited to, multiple sclerosis, asthma, psoriasis, 
and rheumatoid arthritis. 
Pharmaceutical compositions for treatment of these and other disease states 
are provided by the invention. Pharmaceutical compositions are designed 
for the purpose of inhibiting interaction between .alpha..sub.d and its 
ligand(s) and include various soluble and membrane-associated forms of 
.alpha..sub.d (comprising the entire .alpha..sub.d polypeptide, or 
fragments thereof, which actively participate in .alpha..sub.d binding), 
soluble and membrane-associated forms of .alpha..sub.d binding proteins 
(including antibodies, ligands, and the like), intracellular or 
extracellular modulators of .alpha..sub.d binding activity, and/or 
modulators of .alpha..sub.d and/or .alpha..sub.d -ligand polypeptide 
expression, including modulators of transcription, translation, 
posttranslational processing and/or intracellular transport. The invention 
also comprehends methods for treatment of disease states in which 
.alpha..sub.d binding is implicated, wherein a patient suffering from said 
disease state is provided an amount of a pharmaceutical composition of the 
invention sufficient to modulate levels of .alpha..sub.d binding. The 
method of treatment of the invention is applicable to disease states such 
as, but not limited to, type I diabetes, atherosclerosis, multiple 
sclerosis, asthma, psoriasis, and rheumatoid arthritis.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is illustrated by the following examples relating to 
the isolation of a cDNA clone encoding .alpha..sub.d from a human spleen 
cDNA library. More particularly, Example 1 illustrates the use of 
anti-canine .alpha..sub.TM1 antibody in an attempt to detect a homologous 
human protein. Example 2 details purification of canine .alpha..sub.TM1 
and N-terminal sequencing of the polypeptide to design oligonucleotide 
primers for PCR amplification of the canine .alpha..sub.TM1 gene. Example 
3 addresses large scale purification of canine .alpha..sub.TM1 for 
internal sequencing in order to design additional PCR primers. Example 4 
describes use of the PCR and internal sequence primers to amplify a 
fragment of the canine .alpha..sub.TM1 gene. Example 5 addresses cloning 
of the human .alpha..sub.d -encoding cDNA sequence. Example 6 describes 
Northern blot hybridization analysis of human tissue and cells for 
expression of .alpha..sub.d mRNA. Example 7 details the construction of 
human .alpha..sub.d expression plasmids and transfection of COS cells with 
the resulting plasmids. Example 8 addresses ELISA analysis of 
.alpha..sub.d expression in transfected COS cells. Example 9 describes 
FACS analysis of COS cells transfected with human .alpha..sub.d expression 
plasmids. Example 10 addresses immunoprecipitation of CD18 in association 
with .alpha..sub.d in co-transfected COS cells. Example 11 relates to 
stable transfection of .alpha..sub.d expression constructs in Chinese 
hamster ovary cells. Example 12 addresses CD18-dependent binding of 
.alpha..sub.d to the intercellular adhesion molecule, ICAM-R. Example 13 
describes scintillation proximity screening assays to identify inhibitors 
of .alpha..sub.d ligand/anti-ligand binding interactions. Example 14 
addresses construction of expression plasmids which encode soluble forms 
of .alpha..sub.d. Example 15 relates to production of .alpha..sub.d 
-specific monoclonal antibodies. Example 16 describes isolation of rat 
cDNA sequences which show homology to human .alpha..sub.d gene sequences. 
Example 17 addresses isolation of mouse cDNA sequences which show homology 
to human .alpha..sub.d gene sequences. Example 18 relates to in situ 
hybridization analysis of various mouse tissues to determine tissues and 
cell specific expression of the putative mouse homolog to human 
.alpha..sub.d. Example 19 describes generation of expression constructs 
which encode the putative mouse homolog of human .alpha..sub.d . Example 
20 addresses design of a "knockout" mouse wherein the gene encoding the 
putative mouse homolog of human .alpha..sub.d is disrupted. 
EXAMPLE 1 
Attempt to Detect a Human Homolog of Canine .alpha..sub.TM1 
The monoclonal antibody Ca11.8H2 [Moore, et al., supra] specific for canine 
.alpha..sub.TM1 was tested for cross-reactivity on human peripheral blood 
leukocytes in an attempt to identify a human homolog of canine 
.alpha..sub.TM1. Cell preparations (typically 1.times.10.sup.6 cells) were 
incubated with undiluted hybridoma supernatant or a purified mouse 
IgG-negative control antibody (10 .mu.g/ml) on ice in the presence of 0.1% 
sodium azide. Monoclonal antibody binding was detected by subsequent 
incubation with FITC-conjugated horse anti-mouse IgG (Vector Laboratories, 
Burlingame, Calif.) at 6 .mu.g/ml. Stained cells were fixed with 2% w/v 
paraformaldehyde in phosphate buffered saline (PBS) and were analyzed with 
a Facstar Plus fluorescence-activated cell sorter (Becton Dickinson, 
Mountain View, Calif.). Typically, 10,000 cells were analyzed using 
logarithmic amplification for fluorescence intensity. 
The results indicated that Ca11.8H2 did not cross-react with surface 
proteins expressed on human peripheral blood leukocytes, while the control 
cells, neoplastic canine peripheral blood lymphocytes, were essentially 
all positive for .alpha..sub.TM1. 
Because the monoclonal antibody Ca11.8H2 specific for the canine .alpha. 
subunit did not cross react with a human homolog, isolation of canine 
.alpha..sub.TM1 DNA was deemed a necessary prerequisite to isolate a 
counterpart human gene if one existed. 
EXAMPLE 2 
Affinity Purification Of Canine .alpha..sub.TM1 For N-Terminal Sequencing 
Canine .alpha..sub.TM1 was affinity purified in order to determine 
N-terminal amino acid sequences for oligonucleotide probe/primer design. 
Briefly, anti-.alpha..sub.TM1 monoclonal antibody Ca11.8H2 was coupled to 
Affigel 10 chromatographic resin (BioRad, Hercules, Calif.) and protein 
was isolated by specific antibody-protein interaction. Antibody was 
conjugated to the resin, according to the BioRad suggested protocol, at a 
concentration of approximately 5 mg antibody per ml of resin. Following 
the conjugation reaction, excess antibody was removed and the resin 
blocked with three volumes of 0.1M ethanolamine. The resin was then washed 
with thirty column volumes of phosphate buffered saline (PBS). 
Twenty-five grams of a single dog spleen were homogenized in 250 ml of 
buffer containing 0.32M sucrose in 25 mM Tris-HCl, Ph 8.0, with protease 
inhibitors. Nuclei and cellular debris were pelleted with centrifugation 
at 1000 g for 15 minutes. Membranes were pelleted from the supernatant 
with centrifugation at 100,000 g for 30 minutes. The membrane pellet was 
resuspended in 200 ml lysis buffer (50 mM NaCl, 50 mM borate, pH 8.0, with 
2 % NP-40) and incubated for 1 hour on ice. Insoluble material was then 
pelleted by centrifugation at 100,000 g for 60 minutes. Ten milliliters of 
the cleared lysate were transferred to a 15 ml polypropylene tube with 0.5 
ml Call.8H2-conjugated Affigel 10 resin described above. The tube was 
incubated overnight at 4.degree. C. with rotation and the resin 
subsequently washed with 50 column volumes D-PBS. The resin was then 
transferred to a microfuge tube and boiled for ten minutes in 1 ml Laemmli 
(non-reducing) sample buffer containing 0.1M Tris-HCl, pH 6.8, 2% SDS, 20% 
glycerol and 0.002% bromophenol blue. The resin was pelleted by 
centrifugation and discarded; the supernatant was treated with 1/15 volume 
.beta.-mercaptoethanol (Sigma, St. Louis, Mo.) and run on a 7% 
polyacrylamide gel. The separated proteins were transferred to Immobilon 
PVDF membrane (Millipore, Bedford, Mass.) as follows. 
The gels were washed once in deionized, Millipore-filtered water and 
equilibrated for 15-45 minutes in 10 mM 
3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) transfer buffer, pH 
10.5, with 10% methanol. Immobilon membranes were moistened with methanol, 
rinsed with filtered water, and equilibrated for 15-30 minutes in CAPS 
transfer buffer. The initial transfer was carried out using a Biorad 
transfer apparatus at 70 volts for 3 hours. The Immobilon membrane was 
removed after transfer and stained in filtered 0.1% R250 Coomassie stain 
for 10 minutes. Membranes were destained in 50% methanol/10% acetic acid 
three times, ten minutes each time. After destaining, the membranes were 
washed in filtered water and air-dried. 
Protein bands of approximately 150 kD, 95 kD, 50 kD and 30 kD were 
detected. Presumably the 50 kD and 30 kD bands resulted from antibody 
contamination. N-terminal sequencing was then attempted on both the 150 kD 
and 95 kD bands, but the 95 kD protein was blocked, preventing sequencing. 
The protein band of 150 kD was excised from the membrane and directly 
sequenced with an Applied Biosystems (Foster City, Calif.) Model 473A 
protein sequencer according to the manufacturer's instructions. The 
resulting amino acid sequence is set in SEQ ID NO: 5 using single letter 
amino acid designations. 
EQU FNLDVEEPMVFQ (SEQ ID NO: 5) 
The identified sequence included the FNLD sequence characteristic of 
.alpha. subunits of the integrin family [Tamura, et al., J. Cell. Biol. 
111:1593-1604 (1990)]. 
Primer Design and Attempt to Amplify Canine .alpha..sub.TM1 Sequences 
From the N-terminal sequence information, three oligonucleotide probes were 
designed for hybridization: a) "Tommer," a fully degenerate 
oligonucleotide; b) "Patmer," a partially degenerate oligonucleotide; and 
c) "Guessmer," a nondegenerate oligonucleotide based on mammalian codon 
usage. These probes are set out below as SEQ ID NOS: 6, 7 and 8, 
respectively. Nucleic acid symbols are in accordance with 37 C.F.R. 
.sctn.1.882 for these and all other nucleotide sequences herein. 
EQU 5'-TRYAAYYTGGAYGTNGAROARCCNATGGTNTTYCA-3' (SEQ ID NO: 6) 
EQU 5'-TTCAACCTGGACGTGGAGGAGCCCATGGTGTTCCAA-3' (SEQ ID NO: 7) 
EQU 5'-TTCAACCTGGACGTNGAASANCCCATGGTCTTCCAA-3' (SEQ ID NO: 8) 
Based on sequencing dam, no relevant clones were detected using these 
oligonucleotides in several low stringency hybridizations to a canine 
spleen/peripheral blood macrophage cDNA library cloned into .lambda.ZAP 
(Stratagene, La Jolla, Calif.). 
Four other oligonucleotide primers, designated 5'Deg, 5'Spec, 3'Deg and 
3'Spec (as set out in SEQ ID NOS: 9, 10, 11 and 12, respectively, wherein 
Deg indicates degenerate and Spec indicates non-degenerate) were 
subsequently designed based on the deduced N-terminal sequence for 
attempts to amplify canine .alpha..sub.TM1 sequences by PCR from phage 
library DNA purified from plate lysates of the Stratagene library 
described above. 
EQU 5'-TTYAAYYTNGAYGTNGARGARCC-3' (SEQ ID NO: 9) 
EQU 5'-TTYAAYYTGGACGTNGAAGA-3' (SEQ ID NO: 10) 
EQU 5'-TGRAANACCATNGGYTC-3' (SEQ ID NO: 11) 
EQU 5'-TTGGAAGACCATNGGYTC-3' (SEQ ID NO: 12) 
The .alpha..sub.TM1 oligonucleotide primers were paired with T3 or T7 
vector primers, as set out in SEQ ID NOS: 13 and 14, respectively, which 
hybridize to sequences flanking the polylinker region in the Bluescript 
phagemid found in .lambda.ZAP. 
EQU 5'-ATTAACCCTCACTAAAG-3' (SEQ ID NO: 13) 
EQU 5'-AATACGACTCACTATAG-3' (SEQ ID NO: 14) 
The PCR amplification was carried out in Taq buffer (Boehringer Mannheim, 
Indianapolis, Ind.) containing magnesium with 150 ng of library DNA, 1 
.mu.g of each primer, 200 .mu.M dNTPs and 2.5 units Taq polymerase 
(Boehringer Mannheim) and the products were separated by electrophoresis 
on a 1% agarose gel in Tris-Acetate-EDTA (TAE) buffer with 0.25 .mu.g/ml 
ethidium bromide. DNA was transferred to a Hybond (Amersham, Arlington 
Heights, Ill.) membrane by wicking overnight in 10X SSPE. After transfer, 
the immobilized DNA was denatured with 0.5M NaOH with 0.6M NaCl, 
neutralized with 1.0M Tris-HCl, pH 8.0, in 1.5M NaCl, and washed with 2X 
SSPE before UV crosslinking with a Stratalinker (Stratagene) crosslinking 
apparatus. The membrane was incubated in prehybridization buffer (5X SSPE, 
4X Denhardts, 0.8% SDS, 30% formamide) for 2 hr at 50.degree. C. with 
agitation. 
Oligonucleotide probes 5'Deg, 5'Spec, 3'Deg and 3'Spec (SEQ ID NOS: 9, 10, 
11 and 12, respectively) were labeled using a Boehringer Mannheim kinase 
buffer with 100-300 .mu.Ci .gamma.P.sup.32 -dATP and 1-3 units of 
polynucleotide kinase for 1-3 hr at 37.degree. C. Unincorporated label was 
removed with Sephadex G-25 fine (Pharmacia, Piscataway, N.J.) 
chromatography using 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (TE) buffer and the 
flow-through added directly to the prehybridization solution. Membranes 
were probed for 16 hr at 42.degree. C. with agitation and washed 
repeatedly, with a final stringency wash of 1X SSPE/0.1% SDS at 50.degree. 
for 15 min. The blot was then exposed to Kodak X-Omat AR film for 1-4 
hours at -80.degree. C. 
The oligonucleotides 5'Deg, 5'Spec, 3'Deg and 3'Spec only hybridized to PCR 
products from the reactions in which they were used as primers and failed 
to hybridize as expected to PCR products from the reactions in which they 
were not used as primers. Thus, it was concluded that none of the PCR 
products were specific for .alpha..sub.TM1 because no product hybridized 
with all of the appropriate probes. 
EXAMPLE 3 
Large Scale Affinity Purification Of Canine .alpha..sub.TM1 For Internal 
Sequencing 
In order to provide additional amino acid sequence for primer design, 
canine .alpha.TM.sub.1 was purified for internal sequencing. Three 
sections of frozen spleen (approximately 50 g each) and frozen cells from 
two partial spleens from adult dogs were used to generate protein for 
internal sequencing. Fifty grams of spleen were homogenized in 200-300 ml 
borate buffer with a Waring blender. The homogenized material was diluted 
with 1 volume of buffer containing 4 % NP-40, and the mixture then gently 
agitated for at least one hour. The resulting lysate was cleared of large 
debris by centrifugation at 2000 g for 20 rain, and then filtered through 
either a Coming (Coming, N.Y.) prefilter or a Coming 0.8 micron filter. 
The lysate was further clarified by filtration through the Corning 0.4 
micron filter system. 
Splenic lysate and the antibody-conjugated Affigel 10 resin described in 
Example 2 were combined at a 150:1 volume ratio in 100 ml aliquots and 
incubated overnight at 4.degree. C. with rocking. The lysate was removed 
after centrifugation at 1000 g for 5 minutes, combined with more 
antibody-conjugated Affigel 10 resin and incubated overnight as above. The 
absorbed resin aliquots were then combined and washed with 50 volumes 
D-PBS/0.1% Tween 20 and the resin transferred to a 50 ml Biorad column. 
Adsorbed protein was eluted from the resin with 3-5 volumes of 0.1M 
glycine (pH 2.5); fractions of approximately 900 .mu.l were collected and 
neutralized with 100 .mu.l 1M Tris buffer, pH 8.0. Aliquots of 15 .mu.l 
were removed from each fraction and boiled in an equal volume of 2X 
Laemmli sample buffer with 1/15 volume 1M dithiothreitol (DTT). These 
samples were electrophoresed on 8 % Novex (San Diego, Calif.) 
polyacrylamide gels and visualized either by Coomassie stain or by silver 
stain using a Daiichi kit (Enprotech, Natick, Mass.) according to the 
manufacturer's suggested protocol. Fractions which contained the largest 
amounts of protein were combined and concentrated by vacuum. The remaining 
solution was diluted by 50% with reducing Laemmli sample buffer and run on 
1.5 mm 7% polyacrylamide gels in Tris-glycine/SDS buffer. Protein was 
transferred from the gels to Immobilon membrane by the procedure described 
in Example 2 using the Hoefer transfer apparatus. 
The protein bands corresponding to canine .alpha..sub.TM1 were excised from 
10 PVDF membranes and resulted in approximately 47 .mu.g total protein. 
The bands were destained in 4 ml 50% methanol for 5 minutes, air dried and 
cut into 1.times.2 mm pieces. The membrane pieces were submerged in 2 ml 
95% acetone at 4.degree. C. for 30 minutes with occasional vortexing and 
then air dried. 
Prior to proteolytic cleavage of the membrane bound protein, 3 mg of 
cyanogen bromide (CNBr) (Pierce, Rockford, Ill.) were dissolved in 1.25 ml 
70% formic acid. This solution was then added to a tube containing the 
PVDF membrane pieces and the tube incubated in the dark at room 
temperature for 24 hours. The supernatant (S1) was then removed to another 
tube and the membrane pieces washed with 0.25 ml 70% formic acid. This 
supernatant (S2) was removed and added to the previous supernatant (S1). 
Two milliliters of Milli Q water were added to the combined supernatants 
(S1 and S2) and the solution lyophilized. The PVDF membrane pieces were 
dried under nitrogen and extracted again with 1.25 ml 60% acetonitrile, 
0.1% tetrafluoroacetic acid (TFA) at 42.degree. C. for 17 hours. This 
supernatant (S3) was removed and the membrane pieces extracted again with 
1.0 ml 80% acetonitrile with 0.08% TFA at 42.degree. C. for 1 hour. This 
supernatant (S4) was combined with the previous supernatants (S1 , S2 and 
S3) and vacuum dried. 
The dried CNBr fragments were then dissolved in 63 .mu.l 8M urea, 0.4M 
NH.sub.4 HCO.sub.3. The fragments were reduced in 5 .mu.l 45 mM 
dithiothreitol (DTT) and subsequently incubated at 50.degree. C. for 15 
minutes. The solution was then cooled to room temperature and the 
fragments alkylated by adding 5 .mu.l 100 mM iodoacetamide (Sigma, St. 
Louis, Mo.). Following a 15 minute incubation at room temperature, the 
sample was diluted with 187 .mu.l Milli Q water to a final urea 
concentration of 2.0M. Trypsin (Worthington, Freehold, N.J.) was then 
added at a ratio of 1:25 (w:w) of enzyme to protein and the protein 
digested for 24 hours at 37.degree. C. Digestion was terminated with 
addition of 30 .mu.l TFA. 
The protein fragments were then separated with high performance liquid 
chromatography (HPLC) on a Waters 625 LC system (Millipore, Milford, 
Mass.) using a 2.1.times.250 mm, 5 micron Vydac C-18 column (Vydac, 
Hesperia, Calif.) equilibrated in 0.05% TFA and HPLC water (buffer A). The 
peptides were eluted with increasing concentration of 80% acetonitrile in 
0.04% TFA (buffer B) with a gradient of 38-75% buffer B for 65-95 minutes 
and 75-98% buffer B for 95-105 minutes. Peptides were fractionated at a 
flow rate of 0.2 ml/minute and detected at 210 nm. 
Following fractionation, the amino acid sequence of the peptides was 
analyzed by automated Edman degradation performed on an Applied Biosystems 
Model 437A protein sequencer using the manufacturer's standard cycles and 
the Model 610A Data Analysis software program, Version 1.2.1. All 
sequencing reagents were supplied by Applied Biosystems. The amino acid 
sequences of seven of the eight internal fragments are set out below 
wherein "X" indicates the identity of the amino acid was not certain. 
EQU VFQEXGAGFGQ (SEQ ID NO: 15) 
EQU LYDXVAATGLXQPI (SEQ ID NO: 16) 
EQU PLEYXDVIPQAE (SEQ ID NO: 17) 
EQU FQEGFSXVLX (SEQ ID NO: 18) 
EQU TSPTFIXMSQENVD (SEQ ID NO: 19) 
EQU LVVGAPLEVVAVXQTGR (SEQ ID NO: 20) 
EQU LDXKPXDTA (SEQ ID NO: 21) 
Primer Design 
One internal amino acid sequence (set out in SEQ ID NO: 22) obtained was 
then used to design a fully degenerate oligonucleotide primer, designated 
p4(R) as set out in SEQ ID NO: 23. 
EQU FGEQFSE (SEQ ID NO: 22) 
EQU 5'-RAANCCYTCYTGRAAACTYTC-3' (SEQ ID NO: 23) 
EXAMPLE 4 
PCR Cloning Of A Canine .alpha..sub.TM1 Fragment 
The 5' portion of the canine .alpha..sub.TM1 gene was amplified from 
double-stranded canine splenic cDNA by PCR. 
A. Generation of Double Stranded Canine Spleen cDNA 
One gram of frozen material from a juvenile dog spleen was ground in liquid 
nitrogen on dry ice and homogenized in 20 ml RNA-Stat 60 buffer (Tel-Test 
B, Inc, Friendswood, Tex.). Four ml chloroform were added, and the 
solution extracted by centrifugation at 12,000 g for 15 minutes. RNA was 
precipitated from the aqueous layer with 10 ml ethanol. Poly A.sup.+ RNA 
was then selected on Dynal Oligo dT Dynabeads (Dynal, Oslo, Norway). Five 
aliquots of 100 .mu.g total RNA were combined and diluted with an equal 
volume of 2X binding buffer (20 mM Tris-HCl, pH 7.5, 1.0M LiCl, 1 mM EDTA, 
0.1% SDS). RNA was then incubated 5 minutes with the Oligo dT Dynabeads 
(1.0 ml or 5 mg beads for all the samples). Beads were washed with buffer 
containing 10 mM Tris-HCl, pH 7.5, 0.15M LiCl, 1 mM EDTA and 0.1% SDS, 
according to the manufacturer's suggested protocol prior to elution of 
poly A.sup.+ mRNA with 2 mM EDTA, pH 7.5. Double-stranded cDNA was then 
generated using the eluted poly A.sup.+ mRNA and the Boehringer Mannheim 
cDNA Synthesis Kit according to the manufacturer's suggested protocol. 
B. Isolation of a Partial Canine .alpha..sub.TM1 cDNA 
Oligonucleotide primers 5'Deg (SEQ ID NO: 9) and p4(R) (SEQ ID NO: 23) were 
employed in a standard PCR reaction using 150 ng double-stranded cDNA, 500 
ng of each primer, 200 .mu.M dNTPs and 1.5 units Taq polymerase 
(Boehringer Mannheim) in Taq buffer (Boehringer Mannheim) with magnesium. 
The resulting products (1 .mu.l of the original reaction) were subjected 
to a second round of PCR with the same primers to increase product yield. 
This band was eluted from a 1% agarose gel onto Schleicher & Schuell 
(Keene, N.H.) NA45 paper in a buffer containing 10 mM Tris-HCl, pH 8, 1 mM 
EDTA, 1.5M NaCl at 65.degree. C., precipitated, and ligated into the 
pCR.TM.II vector (Invitrogen, San Diego, Calif.) using the TA cloning kit 
(Invitrogen) and the manufacturer's suggested protocol. The ligation 
mixture was transformed by electroporation into XL-1 Blue bacteria 
(Stratagene). One clone, 2.7, was determined to contain sequences 
corresponding to .alpha..sub.TM1 peptide sequences which were not utilized 
in design of the primers. 
Sequencing was performed with an Applied Biosystems 373A DNA sequencer 
(Foster City, Calif.) with a Dye-deoxy terminator cycle sequence kit (ABI) 
in which fluorescent-labeled dNTPs were incorporated in an asymmetric PCR 
reaction [McCabe, "Production of Single Stranded DNA by Asymmetric PCR," 
in PCR Protocols: A Guide to Methods and Applications, Innis, et al. 
(eds.) pp. 76-83 Academic Press: New York (1990)] as follows. Samples were 
held at 96.degree. C. for 4 minutes and subjected to 25 cycles of the step 
sequence: 96.degree. C., for 15 seconds; 50.degree. C. for 1 second; 
60.degree. C. for 4 minutes. Sequence data was automatically down-loaded 
into sample files on the computer that included chromatogram and text 
files. The sequence of the entire insert of clone 2.7 is set out in SEQ ID 
NO: 24. 
Attempts to isolate the full length canine .alpha..sub.TM1 cDNA from the 
Stratagene library (as described in Example 2) were unsuccessful. 
Approximately 1.times.10.sup.6 phage plaques were screened by 
hybridization under low stringency conditions using 30% formamide with 
clone 2.7 as a probe, but no positive clones resulted. Attempts to amplify 
relevant sequences downstream from those represented in clone 2.7 using 
specific oligonucleotides derived from clone 2.7 or degenerate primers 
based on amino acid sequence from other peptide fragments paired with a 
degenerate oligonucleotide based on the conserved .alpha. subunit amino 
acid motif GFFKR [Tamura, et al., supra] were also unsuccessful. 
EXAMPLE 5 
Cloning Of A Putative Human Homolog Of Canine .alpha..sub.TM1 
To attempt the isolation of a human sequence homologous to canine 
.alpha..sub.TM1 the approximately 1 kb canine .alpha..sub.TM1 fragment 
from clone 2.7 was used as a probe. The probe was generated by PCR under 
conditions described in Example 2 using NT2 (as set out in SEQ ID NO: 25) 
and p4(R) (SEQ ID NO: 23) primers. 
EQU 5'-GTNTTYCARGARGAYGG-3' (SEQ ID NO: 25) 
The PCR product was purified using the Qiagen (Chatsworth, Ga.) Quick Spin 
kit and the manufacturer's suggested protocol. The purified DNA (200 ng) 
was labeled with 200 .mu.Ci .alpha..sup.32 PdCTP using the Boehringer 
Mannheim Random Prime Labelling kit and the manufacturer's suggested 
protocol. Unincorporated isotope was removed with Sephadex G25 (fine) 
gravity chromatography. The probe was denatured with 0.2N NaOH and 
neutralized with 0.4M Tris-HCl, pH 8.0, before use. 
Colony lifts on Hybond filters (Amersham) of a human spleen cDNA library in 
pCDNA/Amp (Invitrogen, San Diego, Calif.) were prepared. The filters were 
initially denatured and neutralized as described in Example 2 and 
subsequently incubated in a prehybridization solution (8 ml/filter) with 
30% formamide at 50.degree. C. with gentle agitation for 2 hours. Labeled 
probe as described above was added to this solution and incubated with the 
filters for 14 hours at 42.degree. C. The filters were washed twice in 2X 
SSC/0.1% SDS at 37.degree. C. and twice in 2X SSC/0.1% SDS at 50.degree. 
C. Final stringency washes were 1X SSC/0.1% SDS, twice at 65.degree. C. 
(1X SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0). Filters were 
exposed to Kodak X-Omat AR film for six hours with an intensifying screen. 
Colonies giving signals on duplicate lifts were streaked on LB medium with 
magnesium (LBM)/carbenicillin plates and incubated overnight at 37.degree. 
C. Resulting streaked colonies were lifted with Hybond filters and these 
filters were treated as above. The filters were hybridized under more 
stringent conditions with the 1 kb probe from clone 2.7, labeled as 
previously described, in a 50% formamide hybridization solution at 
50.degree. C. for 3 hours. Probed filters were washed with a final 
stringency of 0.1 X SSC/0.1% SDS at 65 .degree. C. and exposed to Kodak 
X-Omar AR film for 2.5 hours at -80.degree. C. with an intensifying 
screen. Positive colonies were identified and cultured in 
LBM/carbenicillin medium overnight. DNA from the cultures was prepared 
using the Promega Wizard miniprep kit according to the manufacturer's 
suggested protocol and the resulting DNA was sequenced. 
The initial screening resulted in 18 positive clones, while the secondary 
screening under more stringent hybridization conditions produced one 
positive clone which was designated 19A2. The DNA and deduced amino acid 
sequences of the human .alpha..sub.d clone 19A2 are set out in SEQ ID NOS: 
1 and 2, respectively. 
Characteristics Of The Human .alpha..sub.d cDNA and Predicted Polypeptide 
Clone 19A2 encompasses the entire coding region for the mature protein, 
plus 48 bases (16 amino acid residues) of the 5' upstream signal sequence 
and 241 bases of 3' untranslated sequence which do not terminate in a 
polyadenylation sequence. The core molecular weight of the mature protein 
is predicted to be around 125 kD. The extracellular domain is predicted to 
encompass approximately amino acid residues 17 through 1108 of SEQ ID NO: 
2. This extracellular region is contiguous with about a 20 amino acid 
region homologous to the human CD11c transmembrane region (residues 1109 
through 1128 of SEQ ID NO: 2). The cytoplasmic domain comprises 
approximately 30 amino acids (about residues 1129 through 1161 of SEQ ID 
NO: 2). The protein also contains a region (around residues 150 through 
352) of approximately 202 amino acids homologous to the I (insertion) 
domain common to CD11a, CD11b and CD11c [Larson and Springer, supra], 
.alpha..sub.E [Shaw, et al., J. Biol. Chem. 269:6016-6025 (1994)] and in 
VLA-1 and VLA-2, [Tamura, et al., supra]. The I domain in other integrins 
has been shown to participate in ICAM binding [Landis, et al., J. Cell. 
Biol. 120:1519-1527 (1993); Diamond, et al., J. Cell. Biol. 120:1031-1043 
(1993)], suggesting that .alpha..sub.d may also bind members of the ICAM 
family of surface molecules. This region has not been demonstrated to 
exist in any other integrin subunits. 
The deduced amino acid sequence of .alpha..sub.d shows approximately 36% 
identity to that of CD11a, approximately 60% identity to CD11b and 
approximately 66% identity to CD11c. An alignment of amino acid sequences 
for (CD11b SEQ ID NO: 3), CD11c (SEQ ID NO: 4) and .alpha..sub.d (SEQ ID 
NO: 2) is presented in FIG. 1. 
The cytoplasmic domains of .alpha. subunits in .beta.2 integrins are 
typically distinct from one another within the same species, while 
individual .alpha. subunits show high degrees of homology across species 
boundaries. Consistent with these observations, the cytoplasmic region of 
.alpha..sub.d differs markedly from CD11a, CD11b, and CD11c except for a 
membrane proximal GFFKR amino acid sequence which has been shown to be 
conserved among all .alpha. integrins [Rojiani, et al., Biochemistry 
30:9859-9866 (1991)]. Since the cytoplasmic tail region of integrins has 
been implicated in "inside out" signaling and in avidity regulation 
[Landis et al., supra], it is possible that .alpha..sub.d interacts with 
cytosolic molecules distinct from those interacting with CD11a, CD11b, and 
CD11c, and, as a result, participates in signaling pathways distinct from 
those involving other .beta. integrins. 
The extracellular domain of .alpha..sub.d contains a conserved DGSGS amino 
acid sequence adjacent the I-domain; in CD11b, the DGSGS sequence is a 
metal-binding region required for ligand interaction [Michishita, et al. 
Cell 72:857-867 (1993)]. Three additional putative cation binding sites in 
CD11b and CD11c are conserved in the .alpha..sub.d sequence at amino acids 
465-474, 518-527, and 592-600 in clone 19A2 (SEQ ID NO: 1 ). The 
.alpha..sub.d I-domain is 36%, 62%, and 57% identical to the corresponding 
regions in CD11a, CD11b, and CD11c, respectively, and the relatively low 
sequence homology in this region suggests that .alpha..sub.d may interact 
with a set of extracellular proteins distinct from proteins with which 
other known .beta. integrins interact. Alternatively, the affinity of 
.alpha..sub.d for known .beta..sub.2 integrin ligands, for example, 
ICAM-1, ICAM-2 and/or ICAM-R, may be distinct from that demonstrated for 
the other .beta. integrin/ICAM interactions. [See Example 12.] 
EXAMPLE 6 
Northern Analysis of Human .alpha..sub.d Expression in Tissues 
In order to determine the relative level of expression and tissue 
specificity of .alpha..sub.d, Northern analysis was performed using 
fragments from clone 19A2 as probes. Approximately 10 .mu.g of total RNA 
from each of several human tissues or cultured cell lines were loaded on a 
formaldehyde agarose gel in the presence of 1 .mu.g of ethidium bromide. 
After electrophoresis at 100 V for 4 hr, the RNA was transferred to a 
nitrocellulose membrane (Schleicher & Schuell) by wicking in 10X SSC 
overnight. The membrane was baked 1.5 hr at 80.degree. C. under vacuum. 
Prehybridization solution containing 50% formamide in 
3-(N-morpholino)propane sulfonic acid (MOPS) buffer was used to block the 
membrane for 3 hr at 42.degree. C. Fragments of clone 19A2 were labeled 
with the Boehringer Mannheim Random Prime kit according to the 
manufacturer's instructions including both .alpha.P.sup.32 dCTP and 
.alpha.P.sup.32 dTTP. Unincorporated label was removed on a Sephadex G25 
column in TE buffer. The membrane was probed with 1.5.times.10.sup.6 
counts per ml of prehybridization buffer. The blot was then washed 
successively with 2X SSC/0.1% SDS at room temperature, 2X SSC/0.1% SDS at 
42.degree. C., 2X SSC/0.1% SDS at 50.degree. C., 1X SSC/0.1% SDS at 
50.degree. C., 0.5X SSC/0.1% SDS at 50.degree. C. and 0.1X SSC/0.1% SDS at 
50.degree. C. The blot was then exposed to film for 19 hr. 
Hybridization using a BstXI fragment from clone 19A2 (corresponding to 
nucleotides 2011 to 3388 in SEQ ID NO: 1) revealed a weak signal in the 
approximately 5 kb range in liver, placenta, thymus, and tonsil total RNA. 
No signal was detected in kidney, brain or heart samples. The amount of 
RNA present in the kidney lane was minimal, as determined with ethidium 
bromide staining. 
When using a second fragment of clone 19A2 (encompassing the region from 
bases 500 to 2100 in SEQ ID NO: 1), RNA transcripts of two different sizes 
were detected in a human multi-tissue Northern (MTN) blot using 
polyA.sup.+ RNA (Clontech). An approximately 6.5 kb band was observed in 
spleen and skeletal muscle, while a 4.5 kb band was detected in lung and 
peripheral blood leukocytes. The variation in sizes observed could be 
caused by tissue specific polyadenylation, cross reactivity of the probe 
with other integrin family members, or hybridization with alternatively 
spliced mRNAs. 
Northern analysis using a third fragment from clone 19A2, spanning 
nucleotides 2000 to 3100 in SEQ ID NO: 1, gave results consistent with 
those using the other clone 19A2 fragments. 
RNA from three myeloid lineage cell lines was also probed using the 
fragments corresponding to nucleotides 500 to 2100 and 2000 to 3100 in SEQ 
ID NO: 1. A THP-1 cell line, previously stimulated with PMA, gave a 
diffuse signal in the same size range (approximately 5.0 kb), with a 
slightly stronger intensity than the tissue signals. RNA from unstimulated 
and DMSO-stimulated HL-60 cells hybridized with the .alpha..sub.d probe at 
the same intensity as the tissue samples, however, PMA treatment seemed to 
increase the signal intensity. Since PMA and DMSO drive HL-60 cell 
differentiation toward monocyte/macrophage and granulocyte pathways, 
respectively, this result suggests enhanced .alpha..sub.d expression in 
monocyte/macrophage cell types. U937 cells expressed the .alpha..sub.d 
message and this signal did not increase with PMA stimulation. No band was 
detected in Molt, Daudi, H9, JY, or Jurkat cells. 
EXAMPLE 7 
Transient Expression of Human .alpha..sub.d Constructs 
A. Generation of expression constructs 
The human clone 19A2 lacks an initiating methionine codon and possibly some 
of the 5' signal sequence. Therefore, in order to generate a human 
expression plasmid containing 19A2 sequences, two different strategies 
were used. In the first, two plasmids were constructed in which signal 
peptide sequences derived from genes encoding either CD11b or CD11c were 
spliced into clone 19A2 to generate a chimeric .alpha..sub.d sequence. In 
the second approach, a third plasmid was constructed in which an adenosine 
base was added at position 0 in clone 19A2 to encode an initiating 
methionine. 
The three plasmids contained different regions which encoded the 5' portion 
of the .alpha..sub.d sequence or the chimeric .alpha..sub.d sequence. The 
.alpha..sub.d region was PCR amplified (see conditions in Example 2) with 
a specific 3' primer BamRev (set out below in SEQ ID NO: 26) and one of 
three 5' primers. The three 5' primers contained in sequence: (1) 
identical nonspecific bases at positions 1-6 allowing for digestion, an 
EcoRI site from positions 7-12 and a consensus Kozak sequence from 
positions 13-18; (2) a portion of the CD11b (primer ER1B) or CD11c (primer 
ER1C) signal sequence, or an adenosine (primer ER1D); and (3) an 
additional 15-17 bases specifically overlapping 5' sequences from clone 
19A2 to allow primer annealing. Primers ER1B, ER1C or ER1D are set out in 
SEQ ID NOS: 27, 28 or 29, respectively, where the initiating methionine 
codon is underlined and the EcoRI site is double underlined. 
EQU Primer BamRev (SEQ ID NO: 26) 
EQU 5 '-CCACTGTCAGGATGCCCGTG-3' 
EQU Primer ER1B (SEQ ID NO: 27) 
EQU 5'-A GTTACGAATTCGCCACCATGGCTCTACGGGTGCTTCTTCTG-3' 
EQU Primer ER1C (SEQ ID NO: 28) 
EQU 5'-AGTTACGAATTCGCCACCATGACTCGGACTGTGCTTCTTCTG-3' 
EQU Primer ER1D (SEQ ID NO: 29) 
EQU 5'-AGTTACGAATTCGCCACCATGACCTTCGGCACTGTG-3' 
The resulting PCR product was digested with EcoRI and BamHI. 
All three plasmids contained a common second .alpha..sub.d region (to be 
inserted immediately downstream from the 5' region described in the 
previous paragraph) including the 3' end of the .alpha..sub.d clone. The 
second .alpha..sub.d region, which extended from nucleotide 625 into the 
XbaI site in the vector 3' polylinker region of clone 19A2, was isolated 
by digestion of clone 19A2 with BamHI and XbaI. 
Three ligation reactions were prepared in which the 3' .alpha..sub.d 
BamHI/XbaI fragment was ligated to one of the three 5' .alpha..sub.d 
EcoRI/BamHI fragments using Boehringer Mannheim ligase buffer and T4 
ligase (1 unit per reaction). After a 4 hour incubation at 14.degree. C., 
an appropriate amount of vector pcDNA.3 (Invitrogen) digested with EcoRI 
and XbaI was added to each reaction with an additional unit of ligase. 
Reactions were allowed to continue for another 14 hours. One tenth of the 
reaction mixture was then transformed into competent XL-1 Blue cells. The 
resulting colonies were cultured and the DNA isolated as in Example 5. 
Digestion with EcoRI identified three clones which were positive for that 
restriction site, and thus, the engineered signal sequences. The clones 
were designated pATM.B1 (CD11b/.alpha..sub.d % from primer ER1B), pATM.C10 
(CD11c/.alpha..sub.d, from primer ER1C) and pATM.D12 
(adenosine/.alpha..sub.d from primer ER1d). The presence of the 
appropriate signal sequences in each clone was verified by nucleic acid 
sequencing. 
B. Transfection of COS Cells 
Expression from the .alpha..sub.d plasmids discussed above was effected by 
cotransfection of COS cells with the individual plasmids and a CD18 
expression plasmid, pRC.CD18. As a positive control, COS cells were also 
co-transfected with the plasmid pRC.CD18 and a CD11a expression plasmid, 
pDC.CD11A. 
Cells were passaged in culture medium (DMEM/10%FBS/penstrep) into 10 cm 
Corning tissue culture-treated petri dishes at 50% confluency 16 hours 
prior to transfection. Cells were removed from the plates with Versene 
buffer (0.5 mM NaEDTA in PBS) without trypsin for all procedures. Before 
transfection, the plates were washed once with serum-free DMEM. Fifteen 
micrograms of each plasmid were added to 5 ml transfection buffer (DMEM 
with 20 .mu.g/ml DEAE-Dextran and 0.5 mM chloroquine) on each plate. After 
1.5 hours incubation at 37.degree. C., the cells were shocked for 1 minute 
with 5 ml DMEM/10% DMSO. This DMSO solution was then replaced with 10 
ml/plate culture medium. 
Resulting transfectants were analyzed by ELISA, FACS , and 
immunoprecipitation as described in Examples 8, 9, and 10. 
EXAMPLE 8 
ELISA Analysis of COS Transfectants 
In order to determine if the COS cells co-transfected with CD18 expression 
plasmid pRC.CD 18 and an .alpha..sub.d plasmid expressed .alpha..sub.d on 
the cell surface in association with CD18, ELISAs were performed using 
primary antibodies raised against CD18 (e.g., TS1/18 purified from ATCC 
HB203). As a positive control, ELISAs were also performed on cells 
co-transfected with the CD18 expression plasmid and a CD11a expression 
plasmid, pDC.CD 11A. The primary antibodies in this control included CD18 
antibodies and anti-CD11a antibodies (e.g., TS1/22 purified from ATCC 
HB202). 
For ELISA, cells from each plate were removed with Versene buffer and 
transferred to a single 96-well flat-bottomed Corning tissue culture 
plate. Cells were allowed to incubate in culture media 2 days prior to 
assay. The plates were then washed twice with 150 .mu.l/well D-PBS/0.5% 
teleost skin gelatin (Sigma) solution. This buffer was used in all steps 
except during the development. All washes and incubations were performed 
at room temperature. The wells were blocked with gelatin solution for 1 
hour. Primary antibodies were diluted to 10 .mu.g/ml in gelatin solution 
and 50 .mu.l were then added to each well. Triplicate wells were set up 
for each primary antibody. After 1 hour incubation, plates were washed 3X 
with 150 .mu.l/well gelatin solution. Secondary antibody (goat anti-mouse 
Ig/HRP-Fc specific [Jackson, West Grove, Pa.]) at a 1:3500 dilution was 
added at 50 .mu.l/well and plates were incubated for 1 hour. After three 
washes, plates were developed for 20 minutes with 100 .mu.l/well 
o-phenyldiamine (OPD) (Sigma) solution (1 mg/ml OPD in citrate buffer) 
before addition of 50 .mu.l/well 15% sulfuric acid. 
Analysis of transfectants in the ELISA format with anti-CD18 specific 
antibodies revealed no significant expression above background in cells 
transfected only with the plasmid encoding CD18. Cells co-transfected with 
plasmid containing CD11a and CD18 showed an increase in expression over 
background when analyzed with CD18 specific antibodies or with reagents 
specific for CD11a. Further analysis of cells co-transfected with plasmids 
encoding CD18 and one of the .alpha..sub.d expression constructs (pATM.C10 
or pATM.D12) revealed that cell surface expression of CD18 was rescued by 
concomitant expression of .alpha..sub.d. The increase in detectable CD18 
expression in COS cells transfected with pATM.C10 or pATM.D12 was 
comparable to that observed in co-transfected CD11a/CD 18 positive control 
cells. 
EXAMPLE 9 
FACS Analysis of COS Transfectants 
For FACS analysis, cells in petri dishes were fed with fresh culture medium 
the day after transfection and allowed to incubate 2 days prior to the 
assay. Transfectant cells were removed from the plates with 3 ml Versene, 
washed once with 5 ml FACS buffer (DMEM/2% FBS/0.2% sodium azide) and 
diluted to 500,000 cells/sample in 0.1 ml FACS buffer. Ten microliters of 
either 1 mg/ml FITC-conjugated CD18, CD11a, or CD11b specific antibodies 
(Becton Dickinson) or 800 .mu.g/ml CFSE-conjugated murine 23F2G 
(anti-CD18) (ATCC HB11081 ) were added to each sample. Samples were then 
incubated on ice for 45 minutes, washed 3X with 5 ml/wash FACS buffer and 
resuspended in 0.2 ml FACS buffer. Samples were processed on a Becton 
Dickinson FACscan and the data analyzed using Lysys II software (Becton 
Dickinson). 
COS cells transfected with CD18 sequences only did not stain for CD18, 
CD11a or CD11b. When co-transfected with CD11a/CD18, about 15% of the 
cells stained with antibodies to CD11a or CD18. All cells transfected with 
CD18 and any .alpha..sub.d construct resulted in no detectable staining 
for CD11a and CD11b. The pATM.B1, pATM.C10 and pATM.D12 groups stained 4%, 
13% and 8% positive for CD 18, respectively. Fluorescence of the positive 
population in the CD11a/CD18 group was 4-fold higher than background. In 
comparison, the co-transfection of .alpha..sub.d constructs with the CD18 
construct produced a positive population that showed a 4- to 7-fold 
increase in fluorescence intensity over background. 
EXAMPLE 10 
Biotin-Labeled Immunoprecipitation of Human .alpha.d/CD18 Complexes from 
Co-transfected COS Cells 
Immunoprecipitation was attempted on cells co-transfected with CD18 and 
each of the .alpha..sub.d expression plasmids separately described in 
Example 7 in order to determine if .alpha..sub.d could be isolated as part 
of the .alpha..beta. heterodimer complex characteristic of integrins. 
Transfected cells (1-3.times.10.sup.8 cells/group) were removed from petri 
dishes with Versene buffer and washed 3 times in 50 ml/group D-PBS. Each 
sample was labeled with 2 mg Sulpho-NHS Biotin (Pierce, Rockford, Ill.) 
for 15 minutes at room temperature. The reaction was quenched by washing 3 
times in 50 ml/sample cold D-PBS. Washed cells were resuspended in 1 ml 
lysis buffer (1% NP40,50mM Tris-HCl, pH 8.0, 0.2 M NaCl, 2 mM Ca.sup.++, 2 
mM Mg.sup.++, and protease inhibitors) and incubated 15 minutes on ice. 
Insoluble material was pelleted by centrifugation at 10,000 g for 5 
minutes, and the supernatant removed to fresh tubes. In order to remove 
material non-specifically reactive with mouse immunoglobulin, a 
pre-clearance step was initially performed. Twenty-five micrograms of 
mouse immunoglobulin (Cappel, West Chester, Pa.) was incubated with 
supernatants at 4.degree. C. After 2.5 hr, 100 .mu.l (25 .mu.g) rabbit 
anti-mouse Ig conjugated Sepharose (prepared from Protein A Sepharose 4B 
and rabbit anti-mouse IgG, both from Zymed, San Francisco, Calif.) was 
added to each sample; incubation was continued at 4.degree. C. with 
rocking for 16 hours. Sepharose beads were removed from the supernatants 
by centrifugation. After pre-clearance, the supernatants were then treated 
with 20 .mu.g anti-CD18 antibody (TS1.18) for 2 hours at 4.degree. C. 
Antibody/antigen complexes were isolated from supernatants by incubation 
with 100 .mu.l/sample rabbit anti-mouse/Protein A-sepharose preparation 
described above. Beads were washed 4 times with 10 mM HEPES, 0.2M NaCl, 
and 1% Triton-X 100. Washed beads were pelleted and boiled for 10 minutes 
in 20 .mu.l 2X Laemmli sample buffer with 2% .beta.-mercaptoethanol. 
Samples were centrifuged and run on an 8% prepoured Novex polyacrylamide 
gel (Novex) at 100 V for 30 minutes. Protein was transferred to 
nitrocellulose membranes (Schleicher & Schuell) in TBS-T buffer at 200 
mAmps for 1 hour. Membranes were blocked for 2 hr with 3% BSA in TBS-T. 
Membranes were treated with 1:6000 dilution of Strep-avidin horse radish 
peroxidase (POD) (Boehringer Mannheim) for 1 hour, followed by 3 washes in 
TBS-T. The Amersham Enhanced Chemiluminescence kit was then used according 
to the manufacturer's instructions to develop the blot. The membrane was 
exposed to Hyperfilm MP (Amersham) for 0.5 to 2 minutes. 
Immunoprecipitation of CD18 complexes from cells transfected with pRC.CD18 
and either pATM.B1, pATM.C10 or pATM.D12 revealed surface expression of a 
heterodimeric species consisting of approximately 100 kD .beta. chain, 
consistent with the predicted size of CD18, and an .alpha. chain of 
approximately 150 kD, corresponding to .alpha..sub.d. 
EXAMPLE 11 
Stable Transfection of Human .alpha..sub.d in Chinese Hamster Ovary Cells 
To determine whether .alpha..sub.d is expressed on the cell surface as a 
heterodimer in association with CD18, cDNAs encoding each chain were both 
transiently and stably transfected into a cell line lacking both 
.alpha..sub.d and CD18. 
For these experiments, .alpha..sub.d cDNA was augmented with additional 
leader sequences and a Kozak consensus sequence, as described in Example 
7, and subcloned into expression vector pcDNA3. The final construct, 
designated pATM.D 12, was co-transfected with a modified commercial 
vector, pDC1.CD 18 encoding human CD18 into dihydrofolate reductase 
(DHFR).sup.- Chinese hamster ovary (CHO) cells. The plasmid pDC1.CD 18 
encodes a DHFR.sup.+ marker and transfectants can be selected using an 
appropriate nucleoside-deficient medium. The modifications which resulted 
in pDC1.CD18 are as follows. 
The plasmid pRC/CMV (Invitrogen) is a mammalian expression vector with a 
cytomegalovirus promoter and ampicillin resistance marker gene. A DHFR 
gene from the plasmid pSC1190-DHFR was inserted into pRC/CMV 5' of the 
SV40 origin of replication. In addition, a polylinker from the 5' region 
of the plasmid pHF2G-DHF was ligated into the pRC/CMV/DHFR construct, 3' 
to the DHFR gene. CD18 encoding sequences are subsequently cloned into the 
resulting plasmid between the 5' flanking polylinker region and the bovine 
growth hormone poly A encoding region. 
Surface expression of CD18 was analyzed by flow cytometry using the 
monoclonal antibody TS1/18. Heterodimer formation detected between 
.alpha..sub.d and CD18 in this cell line was consistent with the 
immunoprecipitation described in Example 10 with transient expression in 
COS cells. 
EXAMPLE 12 
Human .alpha..sub.d binds to ICAM-R in a CD 18-dependent fashion 
In view of reports that demonstrate interactions between the leukocyte 
integrins and intercellular adhesion molecules (ICAMs) which mediate 
cell-cell contact [Hynes, Cell 69:11-25 (1992)], the ability of CHO cells 
expressing .alpha..sub.d /CD18 to bind ICAM-1, ICAM-R, or VCAM-1 was 
assessed by two methods. 
In replicate assays, soluble ICAM-1, ICAM-R, or VCAM-1 IgG1 fusion proteins 
were immobilized on plastic and the ability of .alpha..sub.d /CD18 CHO 
transfected cells to bind the immobilized ligand was determined. 
Transfected cells were labeled internally with calcein, washed in binding 
buffer (RPMI with 1% BSA), and incubated in either buffer only (with or 
without 10 ng/ml PMA) or buffer with anti-CD18 monoclonal antibodies at 10 
.mu.g/ml. Transfected cells were added to 96-well Immulon 4 microtiter 
plates previously coated with soluble ICAM-1/IgG1, ICAM-R/IgG1 or 
VCAM-1/IgG1 fusion protein, or bovine serum albumin (BSA) as a negative 
control. Design of the soluble forms of these adhesion molecules is 
described and fully disclosed in co-pending and co-owned U.S. patent 
application Ser. No. 08/102,852, filed Aug. 5, 1993. Wells were blocked 
with 1% BSA in PBS prior to addition of labeled cells. After washing the 
plates by immersion in PBS with 0.1% BSA for 20 minutes, total 
fluorescence remaining in each well was measured using a Cytofluor 2300 
(Millipore, Milford, Mass.). 
In experiments with immobilized ICAMs, .alpha..sub.d /CD18 co-transfectants 
consistently showed a 3-5 fold increase in binding to ICAM-R/IgG1 wells 
over BSA coated wells. The specificity and CD18-dependence of this binding 
was demonstrated by the inhibitory effects of anti-CD18 antibody TS1/18. 
The binding of cells transfected with CD11a/CD18 to ICAM-1/IgG1 wells was 
comparable to the binding observed with BSA coated wells. CD11a/CD18 
transfected cells showed a 2-3 fold increase in binding to ICAM-1/IgG1 
wells only following pretreatment with PMA. PMA treatment of .alpha..sub.d 
/CD 18 transfectants did not affect binding to ICAM-1/IgG1 or ICAM-R/IgG1 
wells. No detectable binding of .alpha..sub.d /CD18 transfectants to 
VCAM-1/IgG1 wells was observed. 
Binding of .alpha..sub.d /CD18-transfected cells to soluble ICAM-1/IgG1, 
ICAM-R/IgG1, or VCAM-1/IgG1 fusion proteins was determined by flow 
cytometry. Approximately one million .alpha..sub.d /CD18-transfected CHO 
cells (grown in spinner flasks for higher expression) per measurement were 
suspended in 100 .mu.l binding buffer (RPMI and 1% BSA) with or without 10 
.mu.g/ml anti-CD18 antibody. After a 20 minute incubation at room 
temperature, the cells were washed in binding buffer and soluble 
ICAM-1/IgG1 or ICAM-R/IgG1 fusion protein was added to a final 
concentration of 5 .mu.g/ml. Binding was allowed to proceed for 30 minute 
at 37.degree. C., after which the cells were washed three times and 
resuspended in 100 .mu.l binding buffer containing FITC-conjugated sheep 
anti-human IgG1 at a 1:100 dilution. After a 30 minute incubation, samples 
were washed three times and suspended in 200 .mu.l binding buffer for 
analysis with a Becton Dickinson FACScan. 
Approximately 40-50% of the .alpha..sub.d /CD18 transfectants indicated 
binding to ICAM-R/IgG1, but no binding to ICAM-1/IgG1 or VCAM-1/IgG1 
proteins. Pretreatment of transfected cells with PMA has no effect on 
.alpha.d/CD18 binding to either ICAM-1/IgG1, ICAM-R/IgG1 or VCAM-1/IgG1, 
which was consistent with the immobilized adhesion assay. Binding by 
ICAM-R was reduced to background levels after treatment of .alpha..sub.d 
/CD18 transfectants with anti-CD18 antibody TS1/18. 
The collective data from these two binding assays illustrate that 
.alpha..sub.d/CD 18 binds to ICAM-R and does so preferentially as compared 
to ICAM-1 and VCAM-1. The .alpha.d/CD18 binding preference for ICAM-R over 
ICAM-1 is opposite that observed with CD11a/CD18 and CD11b/CD18. Thus 
modulation of .alpha..sub.d /CD18 binding may be expected to selectively 
affect normal and pathologic immune function where ICAM-R plays a 
prominent role. Moreover, results of similar assays, in which antibodies 
immunospecific for various extracellular domains of ICAM-R were tested for 
their ability to inhibit binding of ICAM-R to .alpha..sub.d /CD18 
transfectants, indicated that .alpha..sub.d /CD18 and CD11a/CD18 interact 
with different domains of ICAM-R. 
The failure of CD11a/CD18 to bind ICAM-1/IgG1 or ICAM-R/IgG1 in solution 
suggests that the affinity of binding between CD11a/CD18 and ICAM-1 or 
ICAM-R is too low to permit binding in solution. Detection of 
.alpha..sub.d /CD18 binding to ICAM-R/IgG1, however, suggests an unusually 
high binding affinity. 
EXAMPLE 13 
Screening by Scintillation Proximity Assay 
Specific inhibitors of binding between the .alpha..sub.d ligands of the 
present invention and their binding partners (.alpha..sub.d 
ligand/anti-ligand pair) may be determined by a variety of means, such as 
scintillation proximity assay techniques as generally described in U.S. 
Pat. No. 4,271,139, Hart and Greenwald, Mol. Immunol. 12:265-267 (1979), 
and Hart and Greenwald, J. Nuc. Med. 20:1062-1065 (1979), each of which is 
incorporated herein by reference. 
Briefly, one member of the .alpha..sub.d ligand/anti-ligand pair is bound 
to a solid support. A fluorescent agent is also bound to the support. 
Alternatively, the fluorescent agent may be integrated into the solid 
support as described in U.S. Pat. No. 4,568,649, incorporated herein by 
reference. The non-support bound member of the .alpha..sub.d 
ligand/anti-ligand pair is labeled with a radioactive compound that emits 
radiation capable of exciting the fluorescent agent. When the ligand binds 
the radiolabeled anti-ligand, the label is brought sufficiently close to 
the support-bound fluorescer to excite the fluorescer and cause emission 
of light. When not bound, the label is generally too distant from the 
solid support to excite the fluorescent agent, and light emissions are 
low. The emitted light is measured and correlated with binding between the 
ligand and the anti-ligand. Addition of a binding inhibitor to the sample 
will decrease the fluorescent emission by keeping the radioactive label 
from being captured in the proximity of the solid support. Therefore, 
binding inhibitors may be identified by their effect on fluorescent 
emissions from the samples. Potential anti-ligands to .alpha..sub.d may 
also be identified by similar means. 
EXAMPLE 14 
Soluble Human .alpha..sub.d Expression Constructs 
The expression of full-length, soluble human .alpha..sub.d /CD18 
heterodimeric protein provides easily purified material for immunization 
and binding assays. The advantage of generating soluble protein is that it 
can be purified from supernatants rather than from cell lysates (as with 
full-length membrane-bound .alpha..sub.d /CD18); recovery in therefore 
improved and impurities reduced. 
The soluble .alpha..sub.d expression plasmid was constructed as follows. A 
nucleotide fragment corresponding to the region from bases 0 to 3161 in 
SEQ ID NO: 1, cloned into plasmid pATM.D12, was isolated by digestion with 
HindIII and AatII. A PCR fragment corresponding to bases 3130 to 3390 in 
SEQ ID NO: 1, overlapping the HindIII/AatII fragment and containing an 
addition MluI restriction site at the 3' terminus, was amplified from 
pATM.D12 with primers sHAD.5 and sHAD.3 set out in SEQ ID NOS: 30 and 31, 
respectively. 
EQU TTGCTGACTGCCTGCAGTTC (SEQ ID NO: 30) 
EQU GTTCTGACGCGTAATGGCATTGTAGACCTCGTCTTC (SEQ ID NO: 31) 
The PCR amplification product was digested with AatII and MluI and ligated 
to the HindIII/AatII fragment. The resulting product was ligated into 
HindIII/MluI-digested plasmid PDC.1s. 
This construct is co-expressed with soluble CD18 in stably transfected CHO 
cells, and expression is detected by autoradiographic visualization of 
immunoprecipitated CD18 complexes derived from .sup.35 S-methionine 
labeled cells. 
Soluble Human .alpha..sub.d I Domain Expression Constructs 
It has previously been reported that the I domain in CD11a can be expressed 
as an independent structural unit that maintains ligand binding 
capabilities and antibody recognition [Randi and Hogg, J. Biol. Chem. 
269:12395-12398 (1994); Zhout, et al., J. Biol. Chem. 269:17075-17079 
(1994)]]. To generate a soluble fusion protein comprising the 
.alpha..sub.d I domain and human IgG4, the .alpha..sub.d I domain is 
amplified by PCR using primers designed to add flanking BamHI and XhoI 
restriction sites to facilitate subcloning. These primers are set out in 
SEQ ID NOS: 32 and 33 with restriction sites underlined. 
EQU ACGTATGCAGGATCCCATCAAGAGATGGACATCGCT (SEQ ID NO: 32) 
EQU ACTGCATGTCTCGAGGCTGAAGCCTTCTTGGGACATC (SEQ ID NO: 33) 
The C nucleotide immediately 3' to the BamHI site in SEQ ID NO: 32 
corresponds to nucleotide 435 in SEQ ID NO: 1; the G nucleotide 3' to the 
XhoI site in SEQ ID NO: 33 is complementary to nucleotide 1067 in SEQ ID 
NO: 1. The amplified I domain is digested with the appropriate enzymes, 
the purified fragment ligated into the mammalian expression vector pDCs 
and the prokaryotic expression vector pGEX-4T-3 (Pharmacia) and the I 
domain fragment sequenced. The fusion protein is then expressed in COS, 
CHO or E. coli cells transfected or transformed with an appropriate 
expression construct. 
Given the affinity of .alpha..sub.d for ICAM-R, expression of the 
.alpha..sub.d I domain may be of sufficient affinity to be a useful 
inhibitor of cell adhesion in which .alpha..sub.d participates. 
EXAMPLE 15 
Production of Human .alpha..sub.d Monoclonal Antibodies 
Transiently transfected cells from Example 7 were washed three times in 
Dulbecco's phosphate buffered saline (D-PBS) and injected at 
5.times.10.sup.6 cells/mouse into Balb/c mice with 50 .mu.g/mouse muramyl 
dipeptidase (Sigma) in PBS. Mice were injected two more times in the same 
fashion at two week intervals. The pre-bleed and immunized serum from the 
mice were screened by FACS analysis as outlined in Example 9 and the 
spleen from the mouse with the highest reactivity to cells transfected 
with .alpha..sub.d /CD18 was fused. Hybridoma culture supernatants were 
then screened separately for lack of reactivity against COS cells 
transfected with CD11a/CD18 and for reactivity with cells cotransfected 
with an .alpha..sub.d expression plasmid and CD18. 
This method resulted in no monoclonal antibodies. 
As an alternative, monoclonal antibodies are generated as follows. Affinity 
purified .alpha..sub.d /CD 18 heterodimeric protein from detergent lysates 
of stably transfected CHP cells is used with 50 .mu.g/ml muramyl 
dipeptidase to immunize Balb/c mice as described above. Mice receive three 
immunizations before serum reactivity against .alpha..sub.d /CD18 is 
determined by immunoprecipitation of biotinylated complexes in the CHO 
transfectants. Hybridomas from positive animals are established according 
to standard protocols, after which hybridoma cultures are selected by flow 
cytometry using .alpha..sub.d /CD18 transfectants. CD11a/CD18 
transfectants are utilized to control for CD18-only reactivity. 
As another alternative for production of monoclonal antibodies, soluble 
.alpha..sub.d I domain IgG4 fusion protein is affinity purified from 
supernatant of stably transfected CHO cells and used to immunized Balb/c 
mice as described above. Hybridomas are established and supernatant from 
these hybridomas are screened by ELISA for reactivity against 
.alpha..sub.d I domain fusion protein. Positive cultures are then analyzed 
for reactivity with full length .alpha..sub.d /CD18 complexes expressed on 
CHO transfectants. 
As another alternative for monoclonal antibody production, Balb/c mice 
undergo an immunization/immunosuppression protocol designed to reduce 
reactivity to CHO cell determinants on transfectants used for 
immunization. This protocol involves immunization with untransfected CHO 
cells and subsequent killing of CHO-reactive B-cell blasts with 
cyclophosphamide treatment. After three rounds of immunization and 
cyclophosphamide treatment are performed, the mice are immunized with 
.alpha..sub.d /CD18 CHO transfected cells as described above. 
EXAMPLE 16 
Isolation of Rat cDNA Clones 
In view of the existence of both canine and human .alpha..sub.d integrins, 
attempts were made to isolate homologous genes in other species, including 
rat (this example) and mouse (Example 17, infra). 
A partial sequence of a rat cDNA showing homology to the human 
.alpha..sub.d gene was obtained from a rat splenic .lambda.gt10 library 
(Clontech). The library was plated at 2.times.10.sup.4 pfu/plate onto 150 
mm LBM/agar plates. The library was lifted onto Hybond membranes 
(Amersham), denatured 3 minutes, neutralized 3 minutes and washed 5 
minutes with buffers as described in standard protocols [Sambrook, et al., 
Molecular Cloning: a laboratory manual, p.2.110]. The membranes were 
placed immediately into a Stratalinker (Stratagene) and the DNA 
crosslinked using the autocrosslinking setting. The membranes were 
prehybridized and hybridized in 30% or 50% formamide, for low and high 
stringency conditions, respectively. Membranes were initially screened 
with a .sup.32 P-labeled probe generated from the human .alpha..sub.d 
cDNA, corresponding to bases 500 to 2100 in clone 19A2 (SEQ ID NO: 1). The 
probe was labeled using Boehringer Mannheim's Random Prime Kit according 
to manufacturer's suggested protocol. Filters were washed with 2X SSC at 
55.degree. C. 
Two clones, designated 684.3 and 705.1, were identified which showed 
sequence homology to human .alpha..sub.d, human CD11b, and human CD11c. 
Both clones aligned to the human .alpha..sub.d gene in the 3' region of 
the gene, starting at base 1871 and extending to base 3012 for clone 
684.3, and bases 1551 to 3367 for clone 705.1. 
In order to isolate a more complete rat sequence which included the 5' 
region, the same library was rescreened using the same protocol as 
employed for the initial screening, but using a mouse probe generated from 
clone A1160 (See Example 17, infra). Single, isolated plaques were 
selected from the second screening and maintained as single clones on 
LBM/agar plates. Sequencing primers 434FL and 434FR (SEQ ID NOS: 34 and 
35, respectively) were used in a standard PCR protocol to generate DNA for 
sequencing. 
EQU 434FL (SEQ ID NO: 34) 
EQU TATAGACTGCTGGGTAGTCCCCAC 
EQU 434FR (SEQ ID NO: 35) 
EQU TGAAGATTGGGGGTAAATACAGA 
DNA from the PCR was purified using a Quick Spin Column (Qiagen) according 
to manufacturer's suggested protocol. 
Two clones, designated 741.4 and 741.11, were identified which overlapped 
clones 684.3 and 705.1; in the overlapping regions, clones 741.1 and 
741.11 were 100% homologous to clones 684.3 and 705.1. A composite rat 
cDNA having homology to the human .alpha..sub.d gene is set out in SEQ ID 
NO: 36; the predicted amino acid sequence is set forth in SEQ ID NO: 37. 
Characteristics of the Rat cDNA and Amino Acid Sequences 
Neither nucleic acid nor amino acid sequences have previously been reported 
for rat .alpha. subunits in .beta..sub.2 integrins. However sequence 
comparisons to reported human .beta..sub.2 integrin .alpha. subunits 
suggests that the isolated rat clone and its predicted amino acid sequence 
are most closely related to .alpha..sub.d nucleotide and amino acid 
sequences. 
At the nucleic acid level, the isolated rat cDNA clone shows 80% identity 
in comparison to the human .alpha..sub.d cDNA; 68% identity in comparison 
to human CD11b; 70% identity in comparison to human CD11c; and 65% 
identity in comparison to mouse CD11b. No significant identity is found in 
comparison to human CD11a and to mouse CD11a. 
At the amino acid level, the predicted rat polypeptide encoded by the 
isolated cDNA shows 70% identity in comparison to human .alpha..sub.d 
polypeptide; 28% identity in comparison to human CD11a; 58% identity in 
comparison to human CD11b; 61% identity in comparison to human CD11c; 28% 
identity in comparison to mouse CD11a; and 55% identity in comparison to 
mouse CD11b. 
EXAMPLE 17 
Isolation of Mouse cDNA Clones 
Isolation of a mouse cDNA exhibiting homology to human .alpha..sub.d by 
cross-species hybridization was attempted with two PCR-generated probes: a 
1.5 kb fragment corresponding to bases 522 to 2047 from human clone 19A2 
(SEQ ID NO: 1), and a 1.0 kb rat fragment which corresponds to bases 1900 
to 2900 in human clone 19A2 (SEQ ID NO: 1). The human probe was generated 
by PCR using primer pairs designated ATM-2 and 9-10.1 set out in SEQ ID 
NOS: 38 and 9, respectively; the rat probe was generated using primer 
pairs 434L and 434R, set out in SEQ ID NOS: 34 and 35, respectively. 
Samples were incubated at 4.degree. C. for 4 minutes and subjected to 30 
cycles of the temperature step sequence: 4.degree. C.; 50.degree. C. 2 
minutes; 72.degree. C., 4 minutes. 
EQU ATM-2 (SEQ ID NO: 38) 
EQU 5'-GTCCAAGCTGTCATGGGCCAG-3' 
EQU 9-10.1 (SEQ ID NO: 39) 
EQU 5'-GTCCAGCAGACTGAAGAGCACGG-3' 
The PCR products were purified using the Qiagen Quick Spin kit according to 
manufacturer's suggested protocol, and approximately 180 ng DNA was 
labeled with 200 .mu.Ci [.sup.32 P]-dCTP using a Boehringer Mannheim 
Random Primer Labeling kit according to manufacturer's suggested protocol. 
Unincorporated isotope was removed using a Centri-sep Spin Column 
(Princeton Separations, Adelphia, N.J.) according to manufacturer's 
suggested protocol. The probes were denatured with 0.2N NaOH and 
neutralized with 0.4M Tris-HCl, pH 8.0, before use. 
A mouse thymic oligo dT-primed cDNA library in lambda ZAP II (Stratagene) 
was plated at approximately 30,000 plaques per 15 cm plate. Plaque lifts 
on nitrocellulose filters (Schleicher & Schuell, Keene, N.H.) were 
incubated at 50.degree. C. with agitation for 1 hour in a prehybridization 
solution (8 ml/lift) containing 30% formamide. Labeled human and rat 
probes were added to the prehybridization solution and incubation 
continued overnight at 50.degree. C. Filters were washed twice in 2X 
SSC/0.1% at room temperature, once in 2X SSC/0.1% SDS at 37.degree. C., 
and once in 2X SSC/0.1% SDS at 42.degree. C. Filters were exposed on Kodak 
X-Omat AR film at -80.degree. C. for 27 hours with an intensifying screen. 
Four plaques giving positive signals on duplicate lifts were restreaked on 
LB medium with magnesium (LBM)/carbenicillin (100 mg/ml) plates and 
incubated overnight at 37.degree. C. The phage plaques were lifted with 
Hybond filters (Amersham), probed as in the initial screen, and exposed on 
Kodak X-Omat AR film for 24 hours at -80.degree. C. with an intensifying 
screen. 
Twelve plaques giving positive signals were transferred into low Mg.sup.++ 
phage diluent containing 10 mM Tris-HCl and 1 mM MgCl.sub.2. Insert size 
was determined by PCR amplification using T3 and T7 primers (SEQ ID NOS: 
13 and 14, respectively) and the following reaction conditions. Samples 
were incubated at 94.degree. C. for 4 minutes and subjected to 30 cycles 
of the temperature step sequence: 94.degree. C., for 15 seconds; 
50.degree. C., for 30 seconds; and 72.degree. C. for 1 minute. 
Six samples produced distinct bands that ranged in size from 300 bases to 1 
kb. Phagemids were released via co-infection with helper phage and 
recircularized to generate Bluescript SK.sup.- (Stratagene). The 
resulting colonies were cultured in LBM/carbenicillin (100 mg/ml) 
overnight. DNA was isolated with a Promega Wizard miniprep kit (Madison, 
Wis.) according to manufacturer's suggested protocol. EcoRI restriction 
analysis of purified DNA confirmed the molecular weights which were 
detected using PCR. Insert DNA was sequenced with M13 and M13 reverse. 1 
primers set out in SEQ ID NOS: 40 and 41, respectively. 
EQU 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO: 40) 
EQU 5'-GGAAACAGCTATGACCATG-3' (SEQ ID NO: 41) 
Sequencing was performed as described in Example 4. 
Of the six clones, only two, designated 10.3-1 and 10.5-2, provided 
sequence information and were identical 600 bp fragments. The 600 bp 
sequence was 68% identical to a corresponding region of human 
.alpha..sub.d, 40% identical to human CD11a, 58% identical to human CD11c, 
and 54% identical to mouse CD11b. This 600 bp fragment was then utilized 
to isolate a more complete cDNA encoding a putative mouse .alpha..sub.d 
homolog. 
A mouse splenic cDNA library (oligo dT.sup.- and random-primed) in lambda 
Zap II (Stratagene) was plated at 2.5.times.10.sup.4 phage/15 cm LBM 
plate. Plaques were lifted on Hybond nylon transfer membranes (Amersham), 
denatured with 0.5M NaOH/1.5M NaCl, neutralized with 0.5M Tris Base/1.5M 
NaCl/11.6 HCl, and washed in 2X SSC. The DNA was cross-linked to filters 
by ultraviolet irradiation. 
Approximately 500,000 plaques were screened using probes 10.3-1 and 10.5-2 
previously labeled as described supra. Probes were added to a 
prehybridization solution and incubated overnight at 50.degree. C. The 
filters were washed twice in 2X SSC/0.1% SDS at room temperature, once in 
2X SSC/0.1% SDS at 37.degree. C., and once in 2X SSC/0.1% SDS at 
42.degree. C. Filters were exposed on Kodak X-Omat AR film for 24 hours at 
-80.degree. C. with an intensifying screen. Fourteen plaques giving 
positive signals on duplicate lifts were subjected to a secondary screen 
identical to that for the initial screen except for additional final high 
stringency washes in 2X SSC/0.1% SDS at 50.degree. C., in 0.5X SSC/0.1% 
SDS at 50.degree. C., and at 55.degree. C. in 0.2X SSC/0.1% SDS. The 
filters were exposed on Kodak X-Omat AR film at -80.degree. C. for 13 
hours with an intensifying screen. 
Eighteen positive plaques were transferred into low Mg.sup.++ phage 
diluent and insert size determined by PCR amplification as described 
above. Seven of the samples gave single bands that ranged in size from 600 
bp to 4 kb. EcoRI restriction analysis of purified DNA confirmed the sizes 
observed from PCR and the DNA was sequenced with primers M13 and M13 
reverse. 1 (SEQ ID NOS: 40 and 41, respectively). 
One clone designated B3800 contained a 4 kb insert which corresponded to a 
region 200 bases downstream of the 5' end of the human .alpha..sub.d 19A2 
clone and includes 553 bases of a 3' untranslated region. Clone B3800 
showed 77% identity to a corresponding region of human .alpha..sub.d, 44% 
identity to a corresponding region of human CD11a, 59% identity to a 
corresponding region of human CD11c, and 51% identity to a corresponding 
region of mouse CD11b. The second clone A1160 was a 1.2 kb insert which 
aligned to the 5' end of the coding region of human .alpha..sub.d 
approximately 12 nucleic acids downstream of the initiating methionine. 
Clone A1160 showed 75% identity to a corresponding region of human 
.alpha..sub.d, 46% identity to a corresponding region of human CD11a, 2% 
identity to a corresponding region of human CD11c, and 66% identity to a 
corresponding region of mouse CD11b. 
Clone A1160, the fragment closer to the 5' end of human clone 19A2, is 1160 
bases in length, and shares a region of overlap with clone B3800 starting 
at base 205 and continuing to base 1134. Clone A1160 has a 110-base 
insertion (bases 704-814 of clone A 1160) not present in the overlapping 
region of clone B3800. This insertion occurs at a probable exon-intron 
boundary [Fleming, et al., J. Immunol. 150:480-490 (1993)] and was removed 
before subsequent ligation of clones A1160 and B3800. 
Rapid Amplification of 5' cDNA End of the Putative Mouse .alpha..sub.d 
Clone 
RACE PCR [Frohman, "RACE: Rapid Amplification of cDNA Ends," in PCR 
Protocols: A Guide to Methods and Applications, Innis, et al. (eds.) pp. 
28-38, Academic Press:New York (1990)] was used to obtain missing 5' 
sequences of the putative mouse .alpha..sub.d clone, including 5' 
untranslated sequence and initiating methionine. A mouse splenic 
RACE-Ready kit (Clontech, Palo Alto, Calif.) was used according to the 
manufacturer's suggested protocol. Two antisense, gene-specific primers 
(SEQ ID NOS: 42 and 43) were designed to perform primary and nested PCR. 
EQU A1160 RACE1-primary (SEQ ID NO: 42) 
EQU 5'-GGACATGTTCACTGCCTCTAGG-3' 
EQU A1160 RACE2-nested (SEQ ID NO: 43) 
EQU 5'-GGCGGACAGTCAGACGACTGTCCTG-3' 
The primers, SEQ ID NOS: 42 and 43, correspond to regions starting 302 and 
247 bases from the 5' end, respectively. PCR was performed as described, 
supra, using the 5' anchor primer (SEQ ID NO: 44) and mouse spleen cDNA 
supplied with the kit. 
EQU 5' anchor primer (SEQ ID NO: 44) 
EQU CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG 
Electrophoresis of the PCR product revealed a band approximately 280 bases 
in size, which was subcloned using a TA cloning kit (Invitrogen) according 
to manufacturer's suggested protocol. Ten resulting colonies were 
cultured, and the DNA isolated and sequenced. An additional 60 bases of 5' 
sequence were identified by this method, which correspond to bases 1 to 60 
in SEQ ID NO: 45. 
Characteristics of the Mouse cDNA and Predicted Amino Acid Sequence 
A composite sequence of the mouse cDNA encoding a putative homolog of human 
.alpha..sub.d is set out in SEQ ID NO: 45. Although homology between the 
external domains of the human and mouse clones is high, homology between 
the cytoplasmic domains is only 30%. The observed variation may indicate 
C-terminal functional differences between the human and mouse proteins. 
Alternatively, the variation in the cytoplasmic domains may result from 
splice variation, or may indicate the existence of an additional 
.beta..sub.2 integrin gene(s). 
At the amino acid level, the mouse cDNA predicts a protein (SEQ ID NO: 46) 
with 28% identity to mouse CD11a, 53% identity to mouse CD11b, 28% 
identity to human CD11a, 55% identity to human CD11b, 59% identity to 
human CD11c, and 70% identity to human .alpha..sub.d. Comparison of the 
amino acid sequences of the cytoplasmic domains of human .alpha..sub.d and 
the putative mouse homolog indicates regions of the same length, but 
having divergent primary structure. Similar sequence length in these 
regions suggests species variation rather than splice variant forms. In 
comparison to the predicted rat polypeptide, Example 16,supra, however, 
mouse and rat cytoplasmic domains show greater than 60% identity. 
EXAMPLE 18 
In situ hybridizations in Mouse 
A single stranded 200 bp mRNA probe was generated from a DNA template, 
corresponding to nucleotides 3460 to 3707 in the cytoplasmic tail region 
of the murine cDNA, by in vitro RNA transcription incorporating 35S-UTP 
(Amersham). 
Whole mouse embryos (harvested at days 11-18 after fertilization) and 
various mouse tissues, including spleen, kidney, liver, intestine, and 
thymus, were hybridized in situ with the radiolabeled single-stranded mRNA 
probe. 
Tissues were sectioned at 6 .mu.m thickness, adhered to Vectabond (Vector 
Laboratories, Inc., Burlingame, Calif.) coated slides, and stored at 
-70.degree. C. Prior to use, slides were removed from -70.degree. C. and 
placed at 50.degree. C. for approximately 5 minutes. Sections were fixed 
in 4% paraformaldehyde for 20 minutes at 4.degree. C., dehydrated with an 
increasing ethanol gradient (70-95-100%) for 1 minute at 4.degree. C. at 
each concentration, and air dried for 30 minutes at room temperature. 
Sections were denatured for 2 minutes at 70.degree. C. in 70% formamide/2X 
SSC, rinsed twice in 2X SSC, dehydrated with the ethanol gradient 
described supra and air dried for 30 minutes. Hybridization was carried 
out overnight (12-16 hours) at 55.degree. C. in a solution containing 
.sup.35 S-labeled riboprobes at 6.times.10.sup.5 cpm/section and 
diethylpyrocarbonate (DEPC)-treated water to give a final concentration of 
50% formamide, 0.3M NaCl, 20 mM Tris-HCl, pH 7.5, 10% dextran sulfate, 1X 
Denhardt's solution, 100 mM dithiothreitol (DTT) and 5 mM EDTA. After 
hybridization, sections were washed for 1 hour at room temperature in 4X 
SSC/10 mM DTT, 40 minutes at 60.degree. C. in 50% formamide/2X SSC/10 mM 
DTT, 30 minutes at room temperature in 2X SSC, and 30 minutes at room 
temperature in 0.1X SSC. The sections were dehydrated, air dried for 2 
hours, coated with Kodak NTB2 photographic emulsion, air dried for 2 
hours, developed (after storage at 4.degree. C. in complete darkness) and 
counter-stained with hematoxylin/eosin. 
Spleen tissue showed a strong signal primarily in the red pulp. This 
pattern is consistent with that of tissue macrophage distribution in the 
spleen, but does not exclude other cell types. 
EXAMPLE 19 
Generation of Mouse Expression Constructs 
In order to construct an expression plasmid including mouse cDNA sequences 
exhibiting homology to human .alpha..sub.d, inserts from clones A1160 and 
B3800 were ligated. Prior to this ligation, however, a 5' leader sequence, 
including an initiating methionine, was added to clone A1160. A primer 
designated "5' PCR leader" (SEQ ID NO: 47) was designed to contain: (1) 
identical nonspecific bases at positions 1-6 allowing for digestion; (2) a 
BamHI site (underlined in SEQ ID NO: 47) from positions 7-12 to facilitate 
subcloning into an expression vector; (3) a consensus Kozak sequence from 
positions 13-18, (4) a signal sequence including a codon for an initiating 
methionine (bold in SEQ ID NO: 47), and (5) an additional 31 bases of 
specifically overlapping 5' sequence from clone A1160 to allow primer 
annealing. A second primer designated "3' end frag" (SEQ ID NO: 48) was 
used with primer "5' PCR leader" to amplify the insert from clone A1160. 
EQU 5' PCR leader (SEQ ID NO: 47) 
EQU 5'-AGTTACGGATCCGGCACCATGACCTTCGGCACTGTGATCCTCCTGTGTG-3' 
EQU 3' end flag (SEQ ID NO: 48) 
EQU 5'-GCTGGACGATGGCATCCAC-3' 
The resulting PCR product did not digest with BamHI, suggesting that an 
insufficient number of bases preceded the restriction site, prohibiting 
recognition by the enzyme. The length of the "tail" sequence preceding the 
BamHI site in the 5' primer (SEQ ID NO: 47) was increased and PCR was 
repeated on the amplification product from the first PCR. A 5' primer, 
designated mAD.5'.2 (SEQ ID NO: 49), was designed with additional 
nonspecific bases at positions 1-4 and an additional 20 bases specifically 
overlapping the previously employed "5' PCR leader" primer sequences. 
EQU mAD.5'.2 (SEQ ID NO: 49) 
EQU 5'-GTAGAGTTACGGATCCGGCACCAT-3' 
Primers "mAD.5'. 2" and "3' end frag" were used together in PCR with the 
product from the first amplification as template. A resulting secondary 
PCR product was subcloned into plasmid pCRtmII (Invitrogen) according to 
manufacturer's suggested protocol and transformed into competent Oneshot 
cells (Invitrogen). One clone containing the PCR product was identified by 
restriction enzyme analysis using BamHI and EcoRI and sequenced. After the 
sequence was verified, the insert was isolated by digestion with BamHI and 
EcoRI and gel purified. 
The insert from clone B3800 was isolated by digestion with EcoRI and NotI, 
gel purified, and added to a ligation reaction which included the 
augmented A1160 BarnHI/EcoRI fragment. Ligation was allowed to proceed for 
14 hours at 14.degree. C. Vector pcDNA.3 (Invitrogen), digested with BamHi 
and NotI, was added to the ligation reaction with additional ligase and 
the reaction was continued for another 12 hours. An aliquot of the 
reaction mixture was transformed into competent E. coli cells, the 
resulting colonies cultured, and one positive clone identified by PCR 
analysis with the primers 11.b-1/2FOR1 and 11.b-1/2REV11 (SEQ ID NOS: 50 
and 51, respectively). 
EQU 5'-GCAGCCAGCTTCGGACAGAC-3' (SEQ ID NO: 50) 
EQU 5'-CCATGTCCACAGAACAGAGAG-3' (SEQ ID NO: 51) 
These primers bridge the A1160 and B3800 fragments, therefore detection of 
an amplification product indicates the two fragments were ligated. The 
sequence of the positive clone was verified with the primers set out in 
SEQ ID NOS: 50 and 51, which amplify from base 100 to 1405 after the 
initiating methionine. 
EXAMPLE 20 
Construction of a Knock-out Mouse 
In order to more accurately assess the immunological role of the protein 
encoded by the putative mouse .alpha..sub.d cDNA, a "knock-out" mouse is 
designed wherein the genomic DNA sequence encoding the putative 
.alpha..sub.d homolog is disrupted by homologous recombination. The 
significance of the protein encoded by the disrupted gene is thereby 
assessed by the absence of the encoded protein. 
Design of such a mouse begins with construction of a plasmid containing 
sequences to be "knocked out" by homologous recombination events. A 750 
base pair fragment of the mouse cDNA (corresponding to nucleotides 1985 to 
2733 in SEQ ID NO: 45)was used to identify a mouse genomic sequence 
encoding the putative mouse .alpha..sub.d homolog from a .lambda.FIX 
library. Primary screening resulted in 14 positive plaques, seven of which 
were confirmed by secondary screening. Liquid lysates were obtained from 
two of the plaques giving the strongest signal and the .alpha. DNA was 
isolated by conventional methods. Restriction mapping and Southern 
analysis confirmed the authenticity of one clone, designated 14-1, and the 
insert DNA was isolated by digestion with NotI. This fragment was cloned 
into Bluescript SKII.sup.+. 
In order to identify a restriction fragment of approximately 9 to 14 kb, a 
length reported to optimize the probability of homologous recombination 
events, Southern hybridization was performed with the 750 bp cDNA probe. 
Prior to hybridization, a restriction map was constructed for clone 14-1. 
A 12 kb fragment was identified as a possible candidate and this fragment 
was subcloned into pBluescript SKII.sup.+ in a position wherein the mouse 
DNA is flanked by thymidine kinase encoding cassettes. 
A neomycin resistance (neo.sup.r) gene is then inserted into the resulting 
plasmid in a manner that interrupts the protein coding sequence of the 
genomic mouse DNA. The resulting plasmid therefore contains aneo.sup.r 
gene within the mouse genomic DNA sequences, all of which are positioned 
within a thymidine kinase encoding region. Plasmid construction in this 
manner is required to favor homologous recombination over random 
recombination [Chisaka, et al., Nature 355:516-520 (1992)]. 
Numerous modifications and variations in the invention as set forth in the 
above illustrative examples are expected to occur to those skilled in the 
art. Consequently only such limitations as appear in the appended claims 
should be placed on the invention. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 51 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3726 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 3..3485 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
TGACCTTCGGCACTGTGCTTCTTCTGAGTGTCCTGGCTTCTTATCAT47 
ThrPheGlyThrValLeuLeuLeuSerValLeuAlaSerTyrHis 
1 51015 
GGATTCAACCTGGATGTGGAGGAGCCTACGATCTTCCAGGAGGATGCA95 
GlyPheAsnLeuAspValGluGluProThrIlePheGlnGluAspAla 
202530 
GGCGGCTTTGGGCAGAGCGTGGTGCAGTTCGGTGGATCTCGACTCGTG143 
GlyGlyPheGlyGlnSerValValGlnPheGlyGlySerArgLeuVal 
354045 
GTGGGAGCACCCCTGGAGGTGGTGGCGGCCAACCAGACGGGACGGCTG191 
ValGlyAlaProLeuGluValValAlaAlaAsnGlnThrGlyArgLeu 
505560 
TATGACTGCGCAGCTGCCACCGGCATGTGCCAGCCCATCCCGCTGCAC239 
TyrAspCysAlaAlaAlaThrGlyMetCysGlnProIleProLeuHis 
657075 
ATCCGCCCTGAGGCCGTGAACATGTCCTTGGGCCTGACCCTGGCAGCC287 
IleArgProGluAlaValAsnMetSerLeuGlyLeuThrLeuAlaAla 
80 859095 
TCCACCAACGGCTCCCGGCTCCTGGCCTGTGGCCCGACCCTGCACAGA335 
SerThrAsnGlySerArgLeuLeuAlaCysGlyProThrLeuHisArg 
100105110 
GTCTGTGGGGAGAACTCATACTCAAAGGGTTCCTGCCTCCTGCTGGGC383 
ValCysGlyGluAsnSerTyrSerLysGlySerCysLeuLeuLeuGly 
115120125 
TCGCGCTGGGAGATCATCCAGACAGTCCCCGACGCCACGCCAGAGTGT431 
SerArgTrpGluIleIleGlnThrValProAspAlaThrProGluCys 
130135140 
CCACATCAAGAGATGGACATCGTCTTCCTGATTGACGGCTCTGGAAGC479 
ProHisGlnGluMetAspIleValPheLeuIleAspGlySerGlySer 
145150155 
ATTGACCAAAATGACTTTAACCAGATGAAGGGCTTTGTCCAAGCTGTC527 
IleAspGlnAsnAspPheAsnGlnMetLysGlyPheValGlnAlaVal 
160 165170175 
ATGGGCCAGTTTGAGGGCACTGACACCCTGTTTGCACTGATGCAGTAC575 
MetGlyGlnPheGluGlyThrAspThrLeuPheAlaLeuMetGlnTyr 
180185190 
TCAAACCTCCTGAAGATCCACTTCACCTTCACCCAATTCCGGACCAGC623 
SerAsnLeuLeuLysIleHisPheThrPheThrGlnPheArgThrSer 
195200205 
CCGAGCCAGCAGAGCCTGGTGGATCCCATCGTCCAACTGAAAGGCCTG671 
ProSerGlnGlnSerLeuValAspProIleValGlnLeuLysGlyLeu 
210215220 
ACGTTCACGGCCACGGGCATCCTGACAGTGGTGACACAGCTATTTCAT719 
ThrPheThrAlaThrGlyIleLeuThrValValThrGlnLeuPheHis 
225230235 
CATAAGAATGGGGCCCGAAAAAGTGCCAAGAAGATCCTCATTGTCATC767 
HisLysAsnGlyAlaArgLysSerAlaLysLysIleLeuIleValIle 
240 245250255 
ACAGATGGGCAGAAGTACAAAGACCCCCTGGAATACAGTGATGTCATC815 
ThrAspGlyGlnLysTyrLysAspProLeuGluTyrSerAspValIle 
260265270 
CCCCAGGCAGAGAAGGCTGGCATCATCCGCTACGCTATCGGGGTGGGA863 
ProGlnAlaGluLysAlaGlyIleIleArgTyrAlaIleGlyValGly 
275280285 
CACGCTTTCCAGGGACCCACTGCCAGGCAGGAGCTGAATACCATCAGC911 
HisAlaPheGlnGlyProThrAlaArgGlnGluLeuAsnThrIleSer 
290295300 
TCAGCGCCTCCGCAGGACCACGTGTTCAAGGTGGACAACTTTGCAGCC959 
SerAlaProProGlnAspHisValPheLysValAspAsnPheAlaAla 
305310315 
CTTGGCAGCATCCAGAAGCAGCTGCAGGAGAAGATCTATGCAGTTGAG1007 
LeuGlySerIleGlnLysGlnLeuGlnGluLysIleTyrAlaValGlu 
320 325330335 
GGAACCCAGTCCAGGGCAAGCAGCTCCTTCCAGCACGAGATGTCCCAA1055 
GlyThrGlnSerArgAlaSerSerSerPheGlnHisGluMetSerGln 
340345350 
GAAGGCTTCAGCACAGCCCTCACAATGGATGGCCTCTTCCTGGGGGCT1103 
GluGlyPheSerThrAlaLeuThrMetAspGlyLeuPheLeuGlyAla 
355360365 
GTGGGGAGCTTTAGCTGGTCTGGAGGTGCCTTCCTGTATCCCCCAAAT1151 
ValGlySerPheSerTrpSerGlyGlyAlaPheLeuTyrProProAsn 
370375380 
ATGAGCCCCACCTTCATCAACATGTCTCAGGAGAATGTGGACATGAGG1199 
MetSerProThrPheIleAsnMetSerGlnGluAsnValAspMetArg 
385390395 
GACTCTTACCTGGGTTACTCCACCGAGCTAGCCCTGTGGAAGGGGGTA1247 
AspSerTyrLeuGlyTyrSerThrGluLeuAlaLeuTrpLysGlyVal 
400 405410415 
CAGAACCTGGTCCTGGGGGCCCCCCGCTACCAGCATACCGGGAAGGCT1295 
GlnAsnLeuValLeuGlyAlaProArgTyrGlnHisThrGlyLysAla 
420425430 
GTCATCTTCACCCAGGTGTCCAGGCAATGGAGGAAGAAGGCCGAAGTC1343 
ValIlePheThrGlnValSerArgGlnTrpArgLysLysAlaGluVal 
435440445 
ACAGGGACGCAGATCGGCTCCTACTTCGGGGCCTCCCTCTGCTCCGTG1391 
ThrGlyThrGlnIleGlySerTyrPheGlyAlaSerLeuCysSerVal 
450455460 
GATGTGGACAGCGATGGCAGCACCGACCTGATCCTCATTGGGGCCCCC1439 
AspValAspSerAspGlySerThrAspLeuIleLeuIleGlyAlaPro 
465470475 
CATTACTATGAGCAGACCCGAGGGGGCCAGGTGTCCGTGTGTCCCTTG1487 
HisTyrTyrGluGlnThrArgGlyGlyGlnValSerValCysProLeu 
480 485490495 
CCTAGGGGGCAGAGGGTGCAGTGGCAGTGTGACGCTGTTCTCCGTGGT1535 
ProArgGlyGlnArgValGlnTrpGlnCysAspAlaValLeuArgGly 
500505510 
GAGCAGGGCCACCCCTGGGGCCGCTTTGGGGCAGCCCTGACAGTGTTG1583 
GluGlnGlyHisProTrpGlyArgPheGlyAlaAlaLeuThrValLeu 
515520525 
GGGGATGTGAATGAGGACAAGCTGATAGACGTGGCCATTGGGGCCCCG1631 
GlyAspValAsnGluAspLysLeuIleAspValAlaIleGlyAlaPro 
530535540 
GGAGAGCAGGAGAACCGGGGTGCTGTCTACCTGTTTCACGGAGCCTCA1679 
GlyGluGlnGluAsnArgGlyAlaValTyrLeuPheHisGlyAlaSer 
545550555 
GAATCCGGCATCAGCCCCTCCCACAGCCAGCGGATTGCCAGCTCCCAG1727 
GluSerGlyIleSerProSerHisSerGlnArgIleAlaSerSerGln 
560 565570575 
CTCTCCCCCAGGCTGCAGTATTTTGGGCAGGCGCTGAGTGGGGGTCAG1775 
LeuSerProArgLeuGlnTyrPheGlyGlnAlaLeuSerGlyGlyGln 
580585590 
GACCTCACCCAGGATGGACTGATGGACCTGGCCGTGGGGGCCCGGGGC1823 
AspLeuThrGlnAspGlyLeuMetAspLeuAlaValGlyAlaArgGly 
595600605 
CAGGTGCTCCTGCTCAGGAGTCTGCCGGTGCTGAAAGTGGGGGTGGCC1871 
GlnValLeuLeuLeuArgSerLeuProValLeuLysValGlyValAla 
610615620 
ATGAGATTCAGCCCTGTGGAGGTGGCCAAGGCTGTGTACCGGTGCTGG1919 
MetArgPheSerProValGluValAlaLysAlaValTyrArgCysTrp 
625630635 
GAAGAGAAGCCCAGTGCCCTGGAAGCTGGGGACGCCACCGTCTGTCTC1967 
GluGluLysProSerAlaLeuGluAlaGlyAspAlaThrValCysLeu 
640 645650655 
ACCATCCAGAAAAGCTCACTGGACCAGCTAGGTGACATCCAAAGCTCT2015 
ThrIleGlnLysSerSerLeuAspGlnLeuGlyAspIleGlnSerSer 
660665670 
GTCAGGTTTGATCTGGCACTGGACCCAGGTCGTCTGACTTCTCGTGCC2063 
ValArgPheAspLeuAlaLeuAspProGlyArgLeuThrSerArgAla 
675680685 
ATTTTCAATGAAACCAAGAACCCCACTTTGACTCGAAGAAAAACCCTG2111 
IlePheAsnGluThrLysAsnProThrLeuThrArgArgLysThrLeu 
690695700 
GGACTGGGGATTCACTGTGAAACCCTGAAGCTGCTTTTGCCAGATTGT2159 
GlyLeuGlyIleHisCysGluThrLeuLysLeuLeuLeuProAspCys 
705710715 
GTGGAGGATGTGGTGAGCCCCATCATTCTGCACCTCAACTTCTCACTG2207 
ValGluAspValValSerProIleIleLeuHisLeuAsnPheSerLeu 
720 725730735 
GTGAGAGAGCCCATCCCCTCCCCCCAGAACCTGCGTCCTGTGCTGGCC2255 
ValArgGluProIleProSerProGlnAsnLeuArgProValLeuAla 
740745750 
GTGGGCTCACAAGACCTCTTCACTGCTTCTCTCCCCTTCGAGAAGAAC2303 
ValGlySerGlnAspLeuPheThrAlaSerLeuProPheGluLysAsn 
755760765 
TGTGGGCAAGATGGCCTCTGTGAAGGGGACCTGGGTGTCACCCTCAGC2351 
CysGlyGlnAspGlyLeuCysGluGlyAspLeuGlyValThrLeuSer 
770775780 
TTCTCAGGCCTGCAGACCCTGACCGTGGGGAGCTCCCTGGAGCTCAAC2399 
PheSerGlyLeuGlnThrLeuThrValGlySerSerLeuGluLeuAsn 
785790795 
GTGATTGTGACTGTGTGGAACGCAGGTGAGGATTCCTACGGAACCGTG2447 
ValIleValThrValTrpAsnAlaGlyGluAspSerTyrGlyThrVal 
800 805810815 
GTCAGCCTCTACTATCCAGCAGGGCTGTCGCACCGACGGGTGTCAGGA2495 
ValSerLeuTyrTyrProAlaGlyLeuSerHisArgArgValSerGly 
820825830 
GCCCAGAAGCAGCCCCATCAGAGTGCCCTGCGCCTGGCATGTGAGACA2543 
AlaGlnLysGlnProHisGlnSerAlaLeuArgLeuAlaCysGluThr 
835840845 
GTGCCCACTGAGGATGAGGGCCTAAGAAGCAGCCGCTGCAGTGTCAAC2591 
ValProThrGluAspGluGlyLeuArgSerSerArgCysSerValAsn 
850855860 
CACCCCATCTTCCATGAGGGCTCTAACGGCACCTTCATAGTCACATTC2639 
HisProIlePheHisGluGlySerAsnGlyThrPheIleValThrPhe 
865870875 
GATGTCTCCTACAAGGCCACCCTGGGAGACAGGATGCTTATGAGGGCC2687 
AspValSerTyrLysAlaThrLeuGlyAspArgMetLeuMetArgAla 
880 885890895 
AGTGCAAGCAGTGAGAACAATAAGGCTTCAAGCAGCAAGGCCACCTTC2735 
SerAlaSerSerGluAsnAsnLysAlaSerSerSerLysAlaThrPhe 
900905910 
CAGCTGGAGCTCCCGGTGAAGTATGCAGTCTACACCATGATCAGCAGG2783 
GlnLeuGluLeuProValLysTyrAlaValTyrThrMetIleSerArg 
915920925 
CAGGAAGAATCCACCAAGTACTTCAACTTTGCAACCTCCGATGAGAAG2831 
GlnGluGluSerThrLysTyrPheAsnPheAlaThrSerAspGluLys 
930935940 
AAAATGAAAGAGGCTGAGCATCGATACCGTGTGAATAACCTCAGCCAG2879 
LysMetLysGluAlaGluHisArgTyrArgValAsnAsnLeuSerGln 
945950955 
CGAGATCTGGCCATCAGCATTAACTTCTGGGTTCCTGTCCTGCTGAAC2927 
ArgAspLeuAlaIleSerIleAsnPheTrpValProValLeuLeuAsn 
960 965970975 
GGGGTGGCTGTGTGGGATGTGGTCATGGAGGCCCCATCTCAGAGTCTC2975 
GlyValAlaValTrpAspValValMetGluAlaProSerGlnSerLeu 
980985990 
CCCTGTGTTTCAGAGAGAAAACCTCCCCAGCATTCTGACTTCCTGACC3023 
ProCysValSerGluArgLysProProGlnHisSerAspPheLeuThr 
99510001005 
CAGATTTCAAGAAGTCCCATGCTGGACTGCTCCATTGCTGACTGCCTG3071 
GlnIleSerArgSerProMetLeuAspCysSerIleAlaAspCysLe u 
101010151020 
CAGTTCCGCTGTGACGTCCCCTCCTTCAGCGTCCAGGAGGAGCTGGAT3119 
GlnPheArgCysAspValProSerPheSerValGlnGluGluLeuAsp 
102510301035 
TTCACCCTGAAGGGCAATCTCAGTTTCGGCTGGGTCCGCGAGACATTG3167 
PheThrLeuLysGlyAsnLeuSerPheGlyTrpValArgGluThrLeu 
1040 104510501055 
CAGAAGAAGGTGTTGGTCGTGAGTGTGGCTGAAATTACGTTCGACACA3215 
GlnLysLysValLeuValValSerValAlaGluIleThrPheAspThr 
106010651070 
TCCGTGTACTCCCAGCTTCCAGGACAGGAGGCATTTATGAGAGCTCAG3263 
SerValTyrSerGlnLeuProGlyGlnGluAlaPheMetArgAl aGln 
107510801085 
ATGGAGATGGTGCTAGAAGAAGACGAGGTCTACAATGCCATTCCCATC3311 
MetGluMetValLeuGluGluAspGluValTyrAsnAlaIleP roIle 
109010951100 
ATCATGGGCAGCTCTGTGGGGGCTCTGCTACTGCTGGCGCTCATCACA3359 
IleMetGlySerSerValGlyAlaLeuLeuLeuLeuAlaLeuIle Thr 
110511101115 
GCCACACTGTACAAGCTTGGCTTCTTCAAACGCCACTACAAGGAAATG3407 
AlaThrLeuTyrLysLeuGlyPhePheLysArgHisTyrLysGluMet 
1 120112511301135 
CTGGAGGACAAGCCTGAAGACACTGCCACATTCAGTGGGGACGATTTC3455 
LeuGluAspLysProGluAspThrAlaThrPheSerGlyAspAs pPhe 
114011451150 
AGCTGTGTGGCCCCAAATGTGCCTTTGTCCTAATAATCCACTTTCCTGTT3505 
SerCysValAlaProAsnValProLeuSer 
11551160 
TATCTCTACCACTGTGGGCTGGACTTGCTTGCAACCATAAATCAACTTACATGGAAACAA3565 
CTTCTGCATAGATCTGCACTGGCCTAAGCAACCTACCAGGTGCTAAGCACCTTCTCGGAG3625 
AGATAGAGATTGTAATGT TTTTACATATCTGTCCATCTTTTTCAGCAATGACCCACTTTT3685 
TACAGAAGCAGGCATGGTGCCAGCATAAATTTTCATATGCT3726 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1161 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ThrPheGlyThrValLeuLeuLeuSerValLeuAlaSerTyrHisGly 
151015 
PheAsnLeuAspValGl uGluProThrIlePheGlnGluAspAlaGly 
202530 
GlyPheGlyGlnSerValValGlnPheGlyGlySerArgLeuValVal 
3540 45 
GlyAlaProLeuGluValValAlaAlaAsnGlnThrGlyArgLeuTyr 
505560 
AspCysAlaAlaAlaThrGlyMetCysGlnProIleProLeuHis Ile 
65707580 
ArgProGluAlaValAsnMetSerLeuGlyLeuThrLeuAlaAlaSer 
859095 
ThrAsnGlySerArgLeuLeuAlaCysGlyProThrLeuHisArgVal 
100105110 
CysGlyGluAsnSerTyrSerLysGlySerCysLeuLeuLeuGlySer 
115120125 
ArgTrpGluIleIleGlnThrValProAspAlaThrProGluCysPro 
130135140 
HisGlnGluMetAspIleValPheLe uIleAspGlySerGlySerIle 
145150155160 
AspGlnAsnAspPheAsnGlnMetLysGlyPheValGlnAlaValMet 
165 170175 
GlyGlnPheGluGlyThrAspThrLeuPheAlaLeuMetGlnTyrSer 
180185190 
AsnLeuLeuLysIleHisPheThrPheThrGlnPhe ArgThrSerPro 
195200205 
SerGlnGlnSerLeuValAspProIleValGlnLeuLysGlyLeuThr 
210215220 
PheThrA laThrGlyIleLeuThrValValThrGlnLeuPheHisHis 
225230235240 
LysAsnGlyAlaArgLysSerAlaLysLysIleLeuIleValIleThr 
245250255 
AspGlyGlnLysTyrLysAspProLeuGluTyrSerAspValIlePro 
260265270 
GlnAlaGluLysAlaGl yIleIleArgTyrAlaIleGlyValGlyHis 
275280285 
AlaPheGlnGlyProThrAlaArgGlnGluLeuAsnThrIleSerSer 
290295 300 
AlaProProGlnAspHisValPheLysValAspAsnPheAlaAlaLeu 
305310315320 
GlySerIleGlnLysGlnLeuGlnGluLysIleTyrAla ValGluGly 
325330335 
ThrGlnSerArgAlaSerSerSerPheGlnHisGluMetSerGlnGlu 
340345350 
GlyPheSerThrAlaLeuThrMetAspGlyLeuPheLeuGlyAlaVal 
355360365 
GlySerPheSerTrpSerGlyGlyAlaPheLeuTyrProProAsnMet 
370 375380 
SerProThrPheIleAsnMetSerGlnGluAsnValAspMetArgAsp 
385390395400 
SerTyrLeuGlyTyrSerTh rGluLeuAlaLeuTrpLysGlyValGln 
405410415 
AsnLeuValLeuGlyAlaProArgTyrGlnHisThrGlyLysAlaVal 
420 425430 
IlePheThrGlnValSerArgGlnTrpArgLysLysAlaGluValThr 
435440445 
GlyThrGlnIleGlySerTyrPheGlyAlaSerLeuCys SerValAsp 
450455460 
ValAspSerAspGlySerThrAspLeuIleLeuIleGlyAlaProHis 
465470475480 
T yrTyrGluGlnThrArgGlyGlyGlnValSerValCysProLeuPro 
485490495 
ArgGlyGlnArgValGlnTrpGlnCysAspAlaValLeuArgGlyGlu 
500505510 
GlnGlyHisProTrpGlyArgPheGlyAlaAlaLeuThrValLeuGly 
515520525 
AspValAsnGluAspLysLe uIleAspValAlaIleGlyAlaProGly 
530535540 
GluGlnGluAsnArgGlyAlaValTyrLeuPheHisGlyAlaSerGlu 
545550555 560 
SerGlyIleSerProSerHisSerGlnArgIleAlaSerSerGlnLeu 
565570575 
SerProArgLeuGlnTyrPheGlyGlnAlaLeuSer GlyGlyGlnAsp 
580585590 
LeuThrGlnAspGlyLeuMetAspLeuAlaValGlyAlaArgGlyGln 
595600605 
V alLeuLeuLeuArgSerLeuProValLeuLysValGlyValAlaMet 
610615620 
ArgPheSerProValGluValAlaLysAlaValTyrArgCysTrpGlu 
625 630635640 
GluLysProSerAlaLeuGluAlaGlyAspAlaThrValCysLeuThr 
645650655 
IleGlnLysSerSerLe uAspGlnLeuGlyAspIleGlnSerSerVal 
660665670 
ArgPheAspLeuAlaLeuAspProGlyArgLeuThrSerArgAlaIle 
675680 685 
PheAsnGluThrLysAsnProThrLeuThrArgArgLysThrLeuGly 
690695700 
LeuGlyIleHisCysGluThrLeuLysLeuLeuLeuProAspCys Val 
705710715720 
GluAspValValSerProIleIleLeuHisLeuAsnPheSerLeuVal 
725730735 
ArgGluProIleProSerProGlnAsnLeuArgProValLeuAlaVal 
740745750 
GlySerGlnAspLeuPheThrAlaSerLeuProPheGluLysAsnCys 
755760765 
GlyGlnAspGlyLeuCysGluGlyAspLeuGlyValThrLeuSerPhe 
770775780 
SerGlyLeuGlnThrLeuThrValGl ySerSerLeuGluLeuAsnVal 
785790795800 
IleValThrValTrpAsnAlaGlyGluAspSerTyrGlyThrValVal 
805 810815 
SerLeuTyrTyrProAlaGlyLeuSerHisArgArgValSerGlyAla 
820825830 
GlnLysGlnProHisGlnSerAlaLeuArgLeuAla CysGluThrVal 
835840845 
ProThrGluAspGluGlyLeuArgSerSerArgCysSerValAsnHis 
850855860 
ProIleP heHisGluGlySerAsnGlyThrPheIleValThrPheAsp 
865870875880 
ValSerTyrLysAlaThrLeuGlyAspArgMetLeuMetArgAlaSer 
885890895 
AlaSerSerGluAsnAsnLysAlaSerSerSerLysAlaThrPheGln 
900905910 
LeuGluLeuProValLy sTyrAlaValTyrThrMetIleSerArgGln 
915920925 
GluGluSerThrLysTyrPheAsnPheAlaThrSerAspGluLysLys 
930935 940 
MetLysGluAlaGluHisArgTyrArgValAsnAsnLeuSerGlnArg 
945950955960 
AspLeuAlaIleSerIleAsnPheTrpValProValLeu LeuAsnGly 
965970975 
ValAlaValTrpAspValValMetGluAlaProSerGlnSerLeuPro 
980985990 
CysValSerGluArgLysProProGlnHisSerAspPheLeuThrGln 
99510001005 
IleSerArgSerProMetLeuAspCysSerIleAlaAspCysLeuGln 
1010 10151020 
PheArgCysAspValProSerPheSerValGlnGluGluLeuAspPhe 
1025103010351040 
ThrLeuLysGlyAsnLeu SerPheGlyTrpValArgGluThrLeuGln 
104510501055 
LysLysValLeuValValSerValAlaGluIleThrPheAspThrSer 
1060 10651070 
ValTyrSerGlnLeuProGlyGlnGluAlaPheMetArgAlaGlnMet 
107510801085 
GluMetValLeuGluGluAspGluValTyrAsnA laIleProIleIle 
109010951100 
MetGlySerSerValGlyAlaLeuLeuLeuLeuAlaLeuIleThrAla 
1105111011151 120 
ThrLeuTyrLysLeuGlyPhePheLysArgHisTyrLysGluMetLeu 
112511301135 
GluAspLysProGluAspThrAlaThrPheSerGlyAspAspPheSer 
114011451150 
CysValAlaProAsnValProLysSer 
11551160 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1153 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetAlaLeuArgValLeuLeuLeuThrAlaLeuThrLeuCysHisGly 
1510 15 
PheAsnLeuAspThrGluAsnAlaMetThrPheGlnGluAsnAlaArg 
202530 
GlyPheGlyGlnSerValValGlnLeuGlnGlySerArg ValValVal 
354045 
GlyAlaProGlnGluIleValAlaAlaAsnGlnArgGlySerLeuTyr 
505560 
GlnCysAspTyrSerThrGlySerCysGluProIleArgLeuGlnVal 
65707580 
ProValGluAlaValAsnMetSerLeuGlyLeuSerLeuAl aAlaThr 
859095 
ThrSerProProGlnLeuLeuAlaCysGlyProThrValHisGlnThr 
100105 110 
CysSerGluAsnThrTyrValLysGlyLeuCysPheLeuPheGlySer 
115120125 
AsnLeuArgGlnGlnProGlnLysPheProGluAlaLe uArgGlyCys 
130135140 
ProGlnGluAspSerAspIleAlaPheLeuIleAspGlySerGlySer 
145150155 160 
IleIleProHisAspPheArgArgMetLysGluPheValSerThrVal 
165170175 
MetGluGlnLeuLysLysSerLysThrLeuP heSerLeuMetGlnTyr 
180185190 
SerGluGluPheArgIleHisPheThrPheLysGluPheGlnAsnAsn 
195200 205 
ProAsnProArgSerLeuValLysProIleThrGlnLeuLeuGlyArg 
210215220 
ThrHisThrAlaThrGlyIleArgLysValValArg GluLeuPheAsn 
225230235240 
IleThrAsnGlyAlaArgLysAsnAlaPheLysIleLeuValValIle 
245 250255 
ThrAspGlyGluLysPheGlyAspProLeuGlyTyrGluAspValIle 
260265270 
ProGluAlaAspArgGluGlyVal IleArgTyrValIleGlyValGly 
275280285 
AspAlaPheArgSerGluLysSerArgGlnGluLeuAsnThrIleAla 
290295 300 
SerLysProProArgAspHisValPheGlnValAsnAsnPheGluAla 
305310315320 
LeuLysThrIleGlnAsnGlnLe uArgGluLysIlePheAlaIleGlu 
325330335 
GlyThrGlnThrGlySerSerSerSerPheGluHisGluMetSerGln 
340 345350 
GluGlyPheSerAlaAlaIleThrSerAsnGlyProLeuLeuSerThr 
355360365 
ValGlySerTyrAspTrpA laGlyGlyValPheLeuTyrThrSerLys 
370375380 
GluLysSerThrPheIleAsnMetThrArgValAspSerAspMetAsn 
385390 395400 
AspAlaTyrLeuGlyTyrAlaAlaAlaIleIleLeuArgAsnArgVal 
405410415 
GlnSerLeuVal LeuGlyAlaProArgTyrGlnHisIleGlyLeuVal 
420425430 
AlaMetPheArgGlnAsnThrGlyMetTrpGluSerAsnAlaAsnVal 
43 5440445 
LysGlyThrGlnIleGlyAlaTyrPheGlyAlaSerLeuCysSerVal 
450455460 
AspValAspSerAsnGly SerThrAspLeuValLeuIleGlyAlaPro 
465470475480 
HisTyrTyrGluGlnThrArgGlyGlyGlnValSerValCysProLeu 
485490495 
ProArgGlyGlnArgAlaArgTrpGlnCysAspAlaValLeuTyrGly 
500505510 
GluGl nGlyGlnProTrpGlyArgPheGlyAlaAlaLeuThrValLeu 
515520525 
GlyAspValAsnGlyAspLysLeuThrAspValAlaIleGlyAlaPro 
530535540 
GlyGluGluAspAsnArgGlyAlaValTyrLeuPheHisGlyThrSer 
545550555560 
GlyS erGlyIleSerProSerHisSerGlnArgIleAlaGlySerLys 
565570575 
LeuSerProArgLeuGlnTyrPheGlyGlnSerLeuSerGlyGlyGln 
580585590 
AspLeuThrMetAspGlyLeuValAspLeuThrValGlyAlaGlnGly 
595600605 
HisValLeuLeuLeuArgSerGlnProValLeuArgValLysAlaIle 
610615620 
MetGluPheAsnProArgGluValAlaArgAsnValPheGluCysAsn 
62 5630635640 
AspGlnValValLysGlyLysGluAlaGlyGluValArgValCysLeu 
645650655 
HisValGlnLysSerThrArgAspArgLeuArgGluGlyGlnIleGln 
660665670 
SerValValThrTyrAspLeuAlaLeuAspSerGlyArgPro HisSer 
675680685 
ArgAlaValPheAsnGluThrLysAsnSerThrArgArgGlnThrGln 
690695700 
ValLeuGlyLeuThrGlnThrCysGluThrLeuLysLeuGlnLeuPro 
705710715720 
AsnCysIleGluAspProValSerProIleValLeuArgLeu AsnPhe 
725730735 
SerLeuValGlyThrProLeuSerAlaPheGlyAsnLeuArgProVal 
740745 750 
LeuAlaGluAspAlaGlnArgLeuPheThrAlaLeuPheProPheGlu 
755760765 
LysAsnCysGlyAsnAspAsnIleCysGlnAspAspLe uSerIleThr 
770775780 
PheSerPheMetSerLeuAspCysLeuValValGlyGlyProArgGlu 
785790795 800 
PheAsnValThrValThrValArgAsnAspGlyGluAspSerTyrArg 
805810815 
ThrGlnValThrPhePhePheProLeuAspL euSerTyrArgLysVal 
820825830 
SerThrLeuGlnAsnGlnArgSerGlnArgSerTrpArgLeuAlaCys 
835840 845 
GluSerAlaSerSerThrGluValSerGlyAlaLeuLysSerThrSer 
850855860 
CysSerIleAsnHisProIlePheProGluAsnSer GluValThrPhe 
865870875880 
AsnIleThrPheAspValAspSerLysAlaSerLeuGlyAsnLysLeu 
885 890895 
LeuLeuLysAlaAsnValThrSerGluAsnAsnMetProArgThrAsn 
900905910 
LysThrGluPheGlnLeuGluLeu ProValLysTyrAlaValTyrMet 
915920925 
ValValThrSerHisGlyValSerThrLysTyrLeuAsnPheThrAla 
930935 940 
SerGluAsnThrSerArgValMetGlnHisGlnTyrGlnValSerAsn 
945950955960 
LeuGlyGlnArgSerLeuProIl eSerLeuValPheLeuValProVal 
965970975 
ArgLeuAsnGlnThrValIleTrpAspArgProGlnValThrPheSer 
980 985990 
GluAsnLeuSerSerThrCysHisThrLysGluArgLeuProSerHis 
99510001005 
SerAspPheLeuAlaGlu LeuArgLysAlaProValValAsnCysSer 
101010151020 
IleAlaValCysGlnArgIleGlnCysAspIleProPhePheGlyIle 
10251030 10351040 
GlnGluGluPheAsnAlaThrLeuLysGlyAsnLeuSerPheAspTrp 
104510501055 
TyrIleLys ThrSerHisAsnHisLeuLeuIleValSerThrAlaGlu 
106010651070 
IleLeuPheAsnAspSerValPheThrLeuLeuProGlyGlnGlyAla 
107510801085 
PheValArgSerGlnThrGluThrLysValGluProPheGluValPro 
109010951100 
AsnProLeuPro LeuIleValGlySerSerValGlyGlyLeuLeuLeu 
1105111011151120 
LeuAlaLeuIleThrAlaAlaLeuTyrLysLeuGlyPhePheLysArg 
112511301135 
GlnTyrLysAspMetMetSerGluGlyGlyProProGlyAlaGluPro 
114011451150 
Gln 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1163 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetThrArgThrArgAlaAlaLeuLeuLeuPheThrAl aLeuAlaThr 
151015 
SerLeuGlyPheAsnLeuAspThrGluGluLeuThrAlaPheArgVal 
2025 30 
AspSerAlaGlyPheGlyAspSerValValGlnTyrAlaAsnSerTrp 
354045 
ValValValGlyAlaProGlnLysIleIleAlaAla AsnGlnIleGly 
505560 
GlyLeuTyrGlnCysGlyTyrSerThrGlyAlaCysGluProIleGly 
657075 80 
LeuGlnValProProGluAlaValAsnMetSerLeuGlyLeuSerLeu 
859095 
AlaSerThrThrSerProSerGlnLeuLeuAl aCysGlyProThrVal 
100105110 
HisHisGluCysGlyArgAsnMetTyrLeuThrGlyLeuCysPheLeu 
115120 125 
LeuGlyProThrGlnLeuThrGlnArgLeuProValSerArgGlnGlu 
130135140 
CysProArgGlnGluGlnAspIleValPheLeuIleA spGlySerGly 
145150155160 
SerIleSerSerArgAsnPheAlaThrMetMetAsnPheValArgAla 
165 170175 
ValIleSerGlnPheGlnArgProSerThrGlnPheSerLeuMetGln 
180185190 
PheSerAsnLysPheGlnThrHis PheThrPheGluGluPheArgArg 
195200205 
ThrSerAsnProLeuSerLeuLeuAlaSerValHisGlnLeuGlnGly 
210215 220 
PheThrTyrThrAlaThrAlaIleGlnAsnValValHisArgLeuPhe 
225230235240 
HisAlaSerTyrGlyAlaArgArg AspAlaIleLysIleLeuIleVal 
245250255 
IleThrAspGlyLysLysGluGlyAspSerLeuAspTyrLysAspVal 
260 265270 
IleProMetAlaAspAlaAlaGlyIleIleArgTyrAlaIleGlyVal 
275280285 
GlyLeuAlaPheGlnAsnAr gAsnSerTrpLysGluLeuAsnAspIle 
290295300 
AlaSerLysProSerGlnGluHisIlePheLysValGluAspPheAsp 
305310 315320 
AlaLeuLysAspIleGlnAsnGlnLeuLysGluLysIlePheAlaIle 
325330335 
GluGlyThrGluT hrIleSerSerSerSerPheGluLeuGluMetAla 
340345350 
GlnGluGlyPheSerAlaValPheThrProAspGlyProValLeuGly 
355 360365 
AlaValGlySerPheThrTrpSerGlyGlyAlaPheLeuTyrProPro 
370375380 
AsnMetSerProThrPhe IleAsnMetSerGlnGluAsnValAspMet 
385390395400 
ArgAspSerTyrLeuGlyTyrSerThrGluLeuAlaLeuTrpLysGly 
405410415 
ValGlnSerLeuValLeuGlyAlaProArgTyrGlnHisIleGlyLys 
420425430 
AlaVal IlePheIleGlnValSerArgGlnTrpArgMetLysAlaGlu 
435440445 
ValIleGlyThrGlnIleGlySerTyrPheGlyAlaSerLeuCysSer 
4 50455460 
ValAspValAspThrAspGlySerThrAspLeuValLeuIleGlyAla 
465470475480 
ProHi sTyrTyrGluGlnThrArgGlyGlyGlnValSerValCysPro 
485490495 
LeuProArgGlyTrpArgArgTrpTrpCysAspAlaValLeuTyrGly 
500505510 
GluGlnGlyHisProTrpGlyArgPheGlyAlaAlaLeuThrValLeu 
515520525 
G lyAspValAsnGlyAspLysLeuThrAspValValIleGlyAlaPro 
530535540 
GlyGluGluGluAsnArgGlyAlaValTyrLeuPheHisGlyValLeu 
545 550555560 
GlyProSerIleSerProSerHisSerGlnArgIleAlaGlySerGln 
565570575 
LeuSerSerArgLeuGlnTyrPheGlyGlnAlaLeuSerGlyGlyGln 
580585590 
AspLeuThrGlnAspGlyLeuValAspLeuAlaValGlyAlaA rgGly 
595600605 
GlnValLeuLeuLeuArgThrArgProValLeuTrpValGlyValSer 
610615620 
MetGlnPheIleProAlaGluIleProArgSerAlaPheGluCysArg 
625630635640 
GluGlnValValSerGluGlnThrLeuValGlnSerAsnIle CysLeu 
645650655 
TyrIleAspLysArgSerLysAsnLeuLeuGlySerArgAspLeuGln 
660665 670 
SerSerValThrLeuAspLeuAlaLeuAlaProGlyArgLeuSerPro 
675680685 
ArgAlaIlePheGlnGluThrLysAsnArgSerLeuSer ArgValArg 
690695700 
ValLeuGlyLeuLysAlaHisCysGluAsnPheAsnLeuLeuLeuPro 
705710715 720 
SerCysValGluAspSerValIleProIleIleLeuArgLeuAsnPhe 
725730735 
ThrLeuValGlyLysProLeuLeuAlaPheAr gAsnLeuArgProMet 
740745750 
LeuAlaAlaLeuAlaGlnArgTyrPheThrAlaSerLeuProPheGlu 
755760 765 
LysAsnCysGlyAlaAspHisIleCysGlnAspAsnLeuGlyIleSer 
770775780 
PheSerPheProGlyLeuLysSerLeuLeuValGlyS erAsnLeuGlu 
785790795800 
LeuAsnAlaGluValMetValTrpAsnAspGlyGluAspSerTyrGly 
805 810815 
ThrThrIleThrPheSerHisProAlaGlyLeuSerTyrArgTyrVal 
820825830 
AlaGluGlyGlnLysGlnGlyGln LeuArgSerLeuHisLeuThrCys 
835840845 
CysSerAlaProValGlySerGlnGlyThrTrpSerThrSerCysArg 
850855 860 
IleAsnHisLeuIlePheArgGlyGlyAlaGlnIleThrPheLeuAla 
865870875880 
ThrPheAspValSerProLysAla ValGlyLeuAspArgLeuLeuLeu 
885890895 
IleAlaAsnValSerSerGluAsnAsnIleProArgThrSerLysThr 
900 905910 
IlePheGlnLeuGluLeuProValLysTyrAlaValTyrIleValVal 
915920925 
SerSerHisGluGlnPheTh rLysTyrLeuAsnPheSerGluSerGlu 
930935940 
GluLysGluSerHisValAlaMetHisArgTyrGlnValAsnAsnLeu 
945950 955960 
GlyGlnArgAspLeuProValSerIleAsnPheTrpValProValGlu 
965970975 
LeuAsnGlnGluA laValTrpMetAspValGluValSerHisProGln 
980985990 
AsnProSerLeuArgCysSerSerGluLysIleAlaProProAlaSer 
995 10001005 
AspPheLeuAlaHisIleGlnLysAsnProValLeuAspCysSerIle 
101010151020 
AlaGlyCysLeuArgPh eArgCysAspValProSerPheSerValGln 
1025103010351040 
GluGluLeuAspPheThrLeuLysGlyAsnLeuSerPheGlyTrpVal 
104510501055 
ArgGlnIleLeuGlnLysLysValSerValValSerValAlaGluIle 
106010651070 
Il ePheAspThrSerValTyrSerGlnLeuProGlyGlnGluAlaPhe 
107510801085 
MetArgAlaGlnThrIleThrValLeuGluLysTyrLysValHisAsn 
109010951100 
ProIleProLeuIleValGlySerSerIleGlyGlyLeuLeuLeuLeu 
1105111011151120 
AlaLeuIleThrAlaValLeuTyrLysValGlyPhePheLysArgGln 
112511301135 
TyrLysGluMetMetGluGluAlaAsnGlyGlnIleAlaProGl uAsn 
114011451150 
GlyThrGlnThrProSerProProSerGluLys 
11551160 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
PheAsnLeuAspValGluGluProMetValPheGln 
1510 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TTYAAYYTGGAYGTNGARGARCCNATGGTNTTYCA35 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
TTCAACCTGGACGTGGAGGAGCCCATGGTGTTCCAA36 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TTCAACCTGGACGTNGAASANCCCATGGTCTTCCAA 36 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TTYAAYYTNGAYGTNGARGARCC 23 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TTYAAYYTGGACGTNGAAGA 20 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
TGRAANACCATNGGYTC 17 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
TTGGAAGACCATNGGYTC 18 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
ATTAACCCTCAC TAAAG17 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AAT ACGACTCACTATAG17 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
ValPh eGlnGluXaaGlyAlaGlyPheGlyGln 
1510 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
LeuTyrAspXaaValAlaAlaThrGlyLeuXaaGlnProIle 
1510 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
ProLeuGluTyrXaaAspValIleProGlnAlaGlu 
1510 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
PheGlnGluGlyPheSerXaaValLeuXaa 
1510 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
ThrSerProThrPheIleXaaMetSerGlnGluAsnValAsp 
1510 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
LeuValValGlyAlaProLeuGluValValAlaValXaaGlnThrGly 
1510 15 
Arg 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
LeuAspXaaLysProXaaAspThrAla 
1 5 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
PheGlyGluGlnPheSerGlu 
1 5 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
RAANCCYTCYTGRAAACTYTC 21 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1006 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
TTCAACCTGGACGTGGAGGAGCCCATGGTGTTCAAGAGGA TGGAGCTGGCTTTGGACAGA60 
GCGTGGCCCAGCTTGGCGGATCTAGACTCGTGGTGGGAGCCCCCCTGGAGGTGGTGGCGG120 
TCAACCAAACAGGAAGGTTGTATGACTGTGTGGCTGCCACTGGCCTTGTCAACCCATACC180 
CCTGCACACACCCC CAGATGCTGTGAACATGTCCCTGGGTCTGTCCCTGTCAGCCGCCGC240 
CAGTCGCCCCTGGCTGCTGGCCTGTGGCCCAACCATGCACAGAGCCTGTGGGGAGAATAT300 
GTATGCAGAAGGCTTTTGCCTCCTGTTGGACTCCCATCTGCAGACCATTTGGACAGTA CC360 
TGCTGCCCTACCAGAGTGTCCAAGTCAAGAGATGGACATTGTCTTCCTGATTGATGGTTC420 
TGGCAGTATGAGCAAAGTGACTTTAAACAAATGAAGGATTTGTGAGAGCTGTGATGGGAC480 
AGTTTGAGGGCACCCAAACCCTGTTCTCACTG ATACAGTATCCCACCTCCCTGAAGATCC540 
ACTTCACCTTCACGCAATTCCAGAGCAGCTGGAACCCTCTGAGCCTGGTGGATCCCATTG600 
TCCAACTGGACGGCCTGACATATACAGCCACGGGCATCCGGAAAGTGGTGGAGGAACTGT660 
TTCATAG TAAGAATGGGGCCCGTAAAAGTGCCAAGAAGATCCTCATTGTCATCACAGATG720 
GCAAAAATACAAAGACCCCCTGGAGTACGAGGACGTATCCCCAGGCAGAGAGAGCGGATC780 
ATCCGCTATGCCATTGGGGTGGGAGATGCTTTCTGGAAACCCAGTGCCAA GCAGGAGCTG840 
GACAACATTGGCTCAGAGCCGGCTCAGGACCATGTGTTCAGGGTGGACAACTTTGCAGCA900 
CTCAGCAGCATCCAGGAGCAGCTGCAGGAGAAGATCTTTGCACTCGAAGGAACCCAGTCG960 
ACGACAAGTAGCTCTTTCCAACATG AGATGTTCCAAGAAGGGTTCA1006 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
GTNTTYCARGARGAYG G17 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
CCACTGT CAGGATGCCCGTG20 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
AGTTACGAATTCGCCACCATGGCTCTACGGGTGCTTCTTCTG42 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
AGTTACGAATTCGCCACCATGACTCGGACTGTGCTTCTTCTG42 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
AGTTACGAATTCGCCACCATGACCTTCGGCACTGTG36 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
TTGCTGACTGCCTGCAGTTC20 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
GTTCTGACGCGTAATGGCATTGTAGACCTCGTCTTC36 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
ACGTATGCAGGATCCCATCAAGAGATGGACATCGCT36 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 37 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
ACTGCATGTCTCGAGGCTGAAGCCTTCTTGGGACATC37 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
TATAGACTGCTGGGTAGTCCCCAC24 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
TGAAGATTGGGGGTAAATAACAGA24 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3528 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..3456 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
GGCTGGGCCCTGGCTTCCTGTCATGGGTCTAACCTGGATGTGGA GGAA48 
GlyTrpAlaLeuAlaSerCysHisGlySerAsnLeuAspValGluGlu 
151015 
CCCATCGTGTTCAGAGAGGATGCAGCCAGCTTTGGACAGAC TGTGGTG96 
ProIleValPheArgGluAspAlaAlaSerPheGlyGlnThrValVal 
202530 
CAGTTTGGTGGATCTCGACTCGTGGTGGGAGCCCCTCTGGA GGCGGTG144 
GlnPheGlyGlySerArgLeuValValGlyAlaProLeuGluAlaVal 
354045 
GCAGTCAACCAAACAGGACGGTTGTATGACTGTGCACCTGCCAC TGGC192 
AlaValAsnGlnThrGlyArgLeuTyrAspCysAlaProAlaThrGly 
505560 
ATGTGCCAGCCCATCGTACTGCGCAGTCCCCTAGAGGCAGTGAACATG 240 
MetCysGlnProIleValLeuArgSerProLeuGluAlaValAsnMet 
65707580 
TCCCTGGGCCTGTCTCTGGTGACTGCCACCAATAACGCCCAGTT GCTG288 
SerLeuGlyLeuSerLeuValThrAlaThrAsnAsnAlaGlnLeuLeu 
859095 
GCTTGTGGTCCAACTGCACAGAGAGCTTGTGTGAAGAACAT GTATGCG336 
AlaCysGlyProThrAlaGlnArgAlaCysValLysAsnMetTyrAla 
100105110 
AAAGGTTCCTGCCTCCTTCTCGGCTCCAGCTTGCAGTTCAT CCAGGCA384 
LysGlySerCysLeuLeuLeuGlySerSerLeuGlnPheIleGlnAla 
115120125 
GTCCCTGCCTCCATGCCAGAGTGTCCAAGACAAGAGATGGACAT TGCT432 
ValProAlaSerMetProGluCysProArgGlnGluMetAspIleAla 
130135140 
TTCCTGATTGATGGTTCTGGCAGCATTAACCAAAGGGACTTTGCCCAG 480 
PheLeuIleAspGlySerGlySerIleAsnGlnArgAspPheAlaGln 
145150155160 
ATGAAGGACTTTGTCAAAGCTTTGATGGGAGAGTTTGCGAGCAC CAGC528 
MetLysAspPheValLysAlaLeuMetGlyGluPheAlaSerThrSer 
165170175 
ACCTTGTTCTCCCTGATGCAATACTCGAACATCCTGAAGAC CCATTTT576 
ThrLeuPheSerLeuMetGlnTyrSerAsnIleLeuLysThrHisPhe 
180185190 
ACCTTCACTGAATTCAAGAACATCCTGGACCCTCAGAGCCT GGTGGAT624 
ThrPheThrGluPheLysAsnIleLeuAspProGlnSerLeuValAsp 
195200205 
CCCATTGTCCAGCTGCAAGGCCTGACCTACACAGCCACAGGCAT CCGG672 
ProIleValGlnLeuGlnGlyLeuThrTyrThrAlaThrGlyIleArg 
210215220 
ACAGTGATGGAAGAGCTATTTCATAGCAAGAATGGGTCCCGTAAAAGT 720 
ThrValMetGluGluLeuPheHisSerLysAsnGlySerArgLysSer 
225230235240 
GCCAAGAAGATCCTCCTTGTCATCACAGATGGGCAGAAATACAG AGAC768 
AlaLysLysIleLeuLeuValIleThrAspGlyGlnLysTyrArgAsp 
245250255 
CCCCTGGAGTATAGTGATGTCATTCCCGCCGCAGACAAAGC TGGCATC816 
ProLeuGluTyrSerAspValIleProAlaAlaAspLysAlaGlyIle 
260265270 
ATTCGTTATGCTATTGGGGTGGGAGATGCCTTCCAGGAGCC CACTGCC864 
IleArgTyrAlaIleGlyValGlyAspAlaPheGlnGluProThrAla 
275280285 
CTGAAGGAGCTGAACACCATTGGCTCAGCTCCCCCACAGGACCA CGTG912 
LeuLysGluLeuAsnThrIleGlySerAlaProProGlnAspHisVal 
290295300 
TTCAAGGTAGGCAACTTTGCAGCACTTCGCAGCATCCAGAGGCAACTT 960 
PheLysValGlyAsnPheAlaAlaLeuArgSerIleGlnArgGlnLeu 
305310315320 
CAGGAGAAAATCTTCGCCATTGAGGGAACTCAATCAAGGTCAAG TAGT1008 
GlnGluLysIlePheAlaIleGluGlyThrGlnSerArgSerSerSer 
325330335 
TCCTTTCAGCACGAGATGTCACAAGAAGGTTTCAGTTCAGC TCTCACA1056 
SerPheGlnHisGluMetSerGlnGluGlyPheSerSerAlaLeuThr 
340345350 
TCGGATGGACCCGTTCTGGGGGCCGYGGGAAGCTTCAGCTG GTCCGGA1104 
SerAspGlyProValLeuGlyAlaXaaGlySerPheSerTrpSerGly 
355360365 
GGTGCCTTCTTATATCCCCCAAATACGAGACCCACCTTTATCAA CATG1152 
GlyAlaPheLeuTyrProProAsnThrArgProThrPheIleAsnMet 
370375380 
TCTCAGGAGAATGTGGACATGAGAGACTCCTACCTGGGTTACTCCACC 1200 
SerGlnGluAsnValAspMetArgAspSerTyrLeuGlyTyrSerThr 
385390395400 
GCAGTGGCCTTTTGGAAGGGGGTTCACAGCCTGATCCTGGGGGC CCCG1248 
AlaValAlaPheTrpLysGlyValHisSerLeuIleLeuGlyAlaPro 
405410415 
CGTCACCAGCACACGGGGAAGGTTGTCATCTTTACCCAGGA AGCCAGG1296 
ArgHisGlnHisThrGlyLysValValIlePheThrGlnGluAlaArg 
420425430 
CATTGGAGGCCCAAGTCTGAAGTCAGAGGGACACAGATCGG CTCCTAC1344 
HisTrpArgProLysSerGluValArgGlyThrGlnIleGlySerTyr 
435440445 
TTCGGGGCCTCTCTCTGTTCTGTGGACGTGGATAGAGATGGCAG CACY1392 
PheGlyAlaSerLeuCysSerValAspValAspArgAspGlySerXaa 
450455460 
GACCTGGTCCTGATCGGAGCCCCCCATTACTATGAGCAGACCCGAGGG 1440 
AspLeuValLeuIleGlyAlaProHisTyrTyrGluGlnThrArgGly 
465470475480 
GGGCAGGTCTCAGTGTKCCCCGTGCCCGGTGTGAGGGGCAGGTG GCAG1488 
GlyGlnValSerValXaaProValProGlyValArgGlyArgTrpGln 
485490495 
TGTGAGGCCACCCTCCACGGGGAGCAGGRCCATCCTTGGGG CCGCTTT1536 
CysGluAlaThrLeuHisGlyGluGlnXaaHisProTrpGlyArgPhe 
500505510 
GGGGTGGCTCTGACAGTGCTGGGGGACGTAAACGGGGACAA TCTGGCA1584 
GlyValAlaLeuThrValLeuGlyAspValAsnGlyAspAsnLeuAla 
515520525 
GACGTGGCTATTGGTGCCCCTGGAGAGGAGGAGAGCAGAGGTGC TGTC1632 
AspValAlaIleGlyAlaProGlyGluGluGluSerArgGlyAlaVal 
530535540 
TACATATTTCATGGAGCCTCGAGACTGGAGATCATGCCCTCACCCAGC 1680 
TyrIlePheHisGlyAlaSerArgLeuGluIleMetProSerProSer 
545550555560 
CAGCGGGTCACTGGCTCCCAGCTCTCCCTGAGACTGCAGTATTT TGGG1728 
GlnArgValThrGlySerGlnLeuSerLeuArgLeuGlnTyrPheGly 
565570575 
CAGTCATTGAGTGGGGGTCAGGACCTTACACAGGATGGCCT GGTGGAC1776 
GlnSerLeuSerGlyGlyGlnAspLeuThrGlnAspGlyLeuValAsp 
580585590 
CTGGCCGTGGGAGCCCAGGGGCACGTACTGCTGCTCAGGAG TCTGCCT1824 
LeuAlaValGlyAlaGlnGlyHisValLeuLeuLeuArgSerLeuPro 
595600605 
CTGCTGAAAGTGGAGCTCTCCATAAGATTCGCCCCCATGGAGGT GGCA1872 
LeuLeuLysValGluLeuSerIleArgPheAlaProMetGluValAla 
610615620 
AAGGCTGTGTACCAGTGCTGGGAAAGGACTCCCACTGTCCTCGAAGCT 1920 
LysAlaValTyrGlnCysTrpGluArgThrProThrValLeuGluAla 
625630635640 
GGAGAGGCCACTGTCTGTCTCACTGTCCACAAAGGCTCACCTGA CCTG1968 
GlyGluAlaThrValCysLeuThrValHisLysGlySerProAspLeu 
645650655 
TTAGGTAATGTCCAAGGCTCTGTCAGGTATGATCTGGCGTT AGATCCG2016 
LeuGlyAsnValGlnGlySerValArgTyrAspLeuAlaLeuAspPro 
660665670 
GGCCGCCTGATTTCTCGTGCCATTTTTGATGAGACTAAGAA CTGCACT2064 
GlyArgLeuIleSerArgAlaIlePheAspGluThrLysAsnCysThr 
675680685 
TTGACGGGAAGGAAGACTCTGGGGCTTGGTGATCACTGCGAAAC AGTG2112 
LeuThrGlyArgLysThrLeuGlyLeuGlyAspHisCysGluThrVal 
690695700 
AAGCTGCTTTTGCCGGACTGTGTGGAAGATGCAGTGAGCCCTATCATC 2160 
LysLeuLeuLeuProAspCysValGluAspAlaValSerProIleIle 
705710715720 
CTGCGCCTCAACTTTTCCCTGGTGAGAGACTCTGCTTCACCCAG GAAC2208 
LeuArgLeuAsnPheSerLeuValArgAspSerAlaSerProArgAsn 
725730735 
CTGCATCCTGTGCTGGCTGTGGGCTCACAAGACCACATAAC TGCTTCT2256 
LeuHisProValLeuAlaValGlySerGlnAspHisIleThrAlaSer 
740745750 
CTGCCGTTTGAGAAGAACTGTAAGCAAGAACTCCTGTGTGA GGGGGAC2304 
LeuProPheGluLysAsnCysLysGlnGluLeuLeuCysGluGlyAsp 
755760765 
CTGGGCATCAGCTTTAACTTCTCAGGCCTGCAGGTCTTGGTGGT GGGA2352 
LeuGlyIleSerPheAsnPheSerGlyLeuGlnValLeuValValGly 
770775780 
GGCTCCCCAGAGCTCACTGTGACAGTCACTGTGTGGAATGAGGGTGAG 2400 
GlySerProGluLeuThrValThrValThrValTrpAsnGluGlyGlu 
785790795800 
GACAGCTATGGAACTTTAGTCAAGTTCTACTACCCAGCAGGGCT ATCT2448 
AspSerTyrGlyThrLeuValLysPheTyrTyrProAlaGlyLeuSer 
805810815 
TACCGACGGGTAACAGGGACTCAGCAACCTCATCAGTACCC ACTACGC2496 
TyrArgArgValThrGlyThrGlnGlnProHisGlnTyrProLeuArg 
820825830 
TTGGCCTGTGAGGCTGAGCCCGCTGCCCAGGAGGACCTGAG GAGCAGC2544 
LeuAlaCysGluAlaGluProAlaAlaGlnGluAspLeuArgSerSer 
835840845 
AGCTGTAGCATTAATCACCCCATCTTCCGAGAAGGTGCAAAGAC CACC2592 
SerCysSerIleAsnHisProIlePheArgGluGlyAlaLysThrThr 
850855860 
TTCATGATCACATTCGATGTCTCCTACAAGGCCTTCCTAGGAGACAGG 2640 
PheMetIleThrPheAspValSerTyrLysAlaPheLeuGlyAspArg 
865870875880 
TTGCTTCTGAGGGCCAAAGCCAGCAGTGAGAATAATAAGCCTGA TACC2688 
LeuLeuLeuArgAlaLysAlaSerSerGluAsnAsnLysProAspThr 
885890895 
AACAAGACTGCCTTCCAGCTGGAGCTCCCAGTGAAGTACAC CGTCTAT2736 
AsnLysThrAlaPheGlnLeuGluLeuProValLysTyrThrValTyr 
900905910 
ACCCTGATCAGTAGGCAAGAAGATTCCACCAACCATGTCAA CTTTTCA2784 
ThrLeuIleSerArgGlnGluAspSerThrAsnHisValAsnPheSer 
915920925 
TCTTCCCACGGGGGGAGAAGGCAAGAAGCCGCACATCGCTATCG TGTG2832 
SerSerHisGlyGlyArgArgGlnGluAlaAlaHisArgTyrArgVal 
930935940 
AATAACCTGAGTCCACTGAAGCTGGCCGTCAGAGTTAACTTCTGGGTC 2880 
AsnAsnLeuSerProLeuLysLeuAlaValArgValAsnPheTrpVal 
945950955960 
CCTGTCCTTCTGAACGGTGTGGCTGTGTGGGACGTGACTCTGAG CAGC2928 
ProValLeuLeuAsnGlyValAlaValTrpAspValThrLeuSerSer 
965970975 
CCAGCACAGGGTGTCTCCTGCGTGTCCCAGATGAAACCTCC TCAGAAT2976 
ProAlaGlnGlyValSerCysValSerGlnMetLysProProGlnAsn 
980985990 
CCCGACTTTCTGACCCAGATTCAGAGACGTTCTGTGCTGGA CTGCTCC3024 
ProAspPheLeuThrGlnIleGlnArgArgSerValLeuAspCysSer 
99510001005 
ATTGCTGACTGCCTGCACTCCCGCTGTGACATCCCCTCCTTGG ACATC3072 
IleAlaAspCysLeuHisSerArgCysAspIleProSerLeuAspIle 
101010151020 
CAGGATGAACTTGACTTCATTCTGAGGGGCAACCTCAGCTTCGGCTGG 3120 
GlnAspGluLeuAspPheIleLeuArgGlyAsnLeuSerPheGlyTrp 
1025103010351040 
GTCAGTCAGACATTGCAGGAAAAGGTGTTGCTTGTGAGTGAG GCTGAA3168 
ValSerGlnThrLeuGlnGluLysValLeuLeuValSerGluAlaGlu 
104510501055 
ATCACTTTCGACACATCTGTGTACTCCCAGCTGCCAGG ACAGGAGGCA3216 
IleThrPheAspThrSerValTyrSerGlnLeuProGlyGlnGluAla 
106010651070 
TTTCTGAGAGCCCAGGTGGAGACAACGTTAGAAGAAT ACGTGGTCTAT3264 
PheLeuArgAlaGlnValGluThrThrLeuGluGluTyrValValTyr 
107510801085 
GAGCCCATCTTCCTCGTGGCGGGCAGCTCGGTGGGAGGT CTGCTGTTA3312 
GluProIlePheLeuValAlaGlySerSerValGlyGlyLeuLeuLeu 
109010951100 
CTGGCTCTCATCACAGTGGTACTGTACAAGCTTGGCTYCTYCAAA CGT3360 
LeuAlaLeuIleThrValValLeuTyrLysLeuGlyXaaXaaLysArg 
1105111011151120 
CAGTACAAAGAAATGCTGGACGGCAAGGCTGCAGATCC TGTCACAGCC3408 
GlnTyrLysGluMetLeuAspGlyLysAlaAlaAspProValThrAla 
112511301135 
GGCCAGGCAGATTTCGGCTGTGAGACTCCTCCAT ATCTCGTGAGCTAGGAATC3463 
GlyGlnAlaAspPheGlyCysGluThrProProTyrLeuValSer 
114011451150 
CTCTCCTGCCTATCTCTGNAATGAAGATTGGTCCTGCCTATGAG TCTACTGGCATGGGAA3523 
CGAGT3528 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1151 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
GlyTrpAlaLeuAlaSerCysHisGlySerAsnLeuAspValGluGlu 
151015 
ProIleValPheArgGluAspAlaAlaSerPheGlyGln ThrValVal 
202530 
GlnPheGlyGlySerArgLeuValValGlyAlaProLeuGluAlaVal 
354045 
AlaV alAsnGlnThrGlyArgLeuTyrAspCysAlaProAlaThrGly 
505560 
MetCysGlnProIleValLeuArgSerProLeuGluAlaValAsnMet 
657 07580 
SerLeuGlyLeuSerLeuValThrAlaThrAsnAsnAlaGlnLeuLeu 
859095 
AlaCysGlyProThrAlaGl nArgAlaCysValLysAsnMetTyrAla 
100105110 
LysGlySerCysLeuLeuLeuGlySerSerLeuGlnPheIleGlnAla 
115120 125 
ValProAlaSerMetProGluCysProArgGlnGluMetAspIleAla 
130135140 
PheLeuIleAspGlySerGlySerIleAsnGlnArgAspPheAlaGln 
145150155160 
MetLysAspPheValLysAlaLeuMetGlyGluPheAlaSerThrSer 
165170175 
T hrLeuPheSerLeuMetGlnTyrSerAsnIleLeuLysThrHisPhe 
180185190 
ThrPheThrGluPheLysAsnIleLeuAspProGlnSerLeuValAsp 
19 5200205 
ProIleValGlnLeuGlnGlyLeuThrTyrThrAlaThrGlyIleArg 
210215220 
ThrValMetGluGluLeuPheHisSerLy sAsnGlySerArgLysSer 
225230235240 
AlaLysLysIleLeuLeuValIleThrAspGlyGlnLysTyrArgAsp 
245250 255 
ProLeuGluTyrSerAspValIleProAlaAlaAspLysAlaGlyIle 
260265270 
IleArgTyrAlaIleGlyValGlyAspAlaPheGlnGlu ProThrAla 
275280285 
LeuLysGluLeuAsnThrIleGlySerAlaProProGlnAspHisVal 
290295300 
PheLysValG lyAsnPheAlaAlaLeuArgSerIleGlnArgGlnLeu 
305310315320 
GlnGluLysIlePheAlaIleGluGlyThrGlnSerArgSerSerSer 
325330335 
SerPheGlnHisGluMetSerGlnGluGlyPheSerSerAlaLeuThr 
340345350 
SerAspGlyProValLeuGl yAlaXaaGlySerPheSerTrpSerGly 
355360365 
GlyAlaPheLeuTyrProProAsnThrArgProThrPheIleAsnMet 
370375 380 
SerGlnGluAsnValAspMetArgAspSerTyrLeuGlyTyrSerThr 
385390395400 
AlaValAlaPheTrpLysGlyValHisSerLeuIleLeuGly AlaPro 
405410415 
ArgHisGlnHisThrGlyLysValValIlePheThrGlnGluAlaArg 
420425430 
H isTrpArgProLysSerGluValArgGlyThrGlnIleGlySerTyr 
435440445 
PheGlyAlaSerLeuCysSerValAspValAspArgAspGlySerXaa 
450 455460 
AspLeuValLeuIleGlyAlaProHisTyrTyrGluGlnThrArgGly 
465470475480 
GlyGlnValSerValXaaProVa lProGlyValArgGlyArgTrpGln 
485490495 
CysGluAlaThrLeuHisGlyGluGlnXaaHisProTrpGlyArgPhe 
500505 510 
GlyValAlaLeuThrValLeuGlyAspValAsnGlyAspAsnLeuAla 
515520525 
AspValAlaIleGlyAlaProGlyGluGluGluSerArgGly AlaVal 
530535540 
TyrIlePheHisGlyAlaSerArgLeuGluIleMetProSerProSer 
545550555560 
GlnA rgValThrGlySerGlnLeuSerLeuArgLeuGlnTyrPheGly 
565570575 
GlnSerLeuSerGlyGlyGlnAspLeuThrGlnAspGlyLeuValAsp 
580585590 
LeuAlaValGlyAlaGlnGlyHisValLeuLeuLeuArgSerLeuPro 
595600605 
LeuLeuLysValGluLeuSerIl eArgPheAlaProMetGluValAla 
610615620 
LysAlaValTyrGlnCysTrpGluArgThrProThrValLeuGluAla 
625630635 640 
GlyGluAlaThrValCysLeuThrValHisLysGlySerProAspLeu 
645650655 
LeuGlyAsnValGlnGlySerValArgTyrAspLeuAla LeuAspPro 
660665670 
GlyArgLeuIleSerArgAlaIlePheAspGluThrLysAsnCysThr 
675680685 
LeuT hrGlyArgLysThrLeuGlyLeuGlyAspHisCysGluThrVal 
690695700 
LysLeuLeuLeuProAspCysValGluAspAlaValSerProIleIle 
70571 0715720 
LeuArgLeuAsnPheSerLeuValArgAspSerAlaSerProArgAsn 
725730735 
LeuHisProValLeuAlaVa lGlySerGlnAspHisIleThrAlaSer 
740745750 
LeuProPheGluLysAsnCysLysGlnGluLeuLeuCysGluGlyAsp 
755760 765 
LeuGlyIleSerPheAsnPheSerGlyLeuGlnValLeuValValGly 
770775780 
GlySerProGluLeuThrValThrValThrValTrpAsnGluGlyGlu 
785790795800 
AspSerTyrGlyThrLeuValLysPheTyrTyrProAlaGlyLeuSer 
805810815 
T yrArgArgValThrGlyThrGlnGlnProHisGlnTyrProLeuArg 
820825830 
LeuAlaCysGluAlaGluProAlaAlaGlnGluAspLeuArgSerSer 
83 5840845 
SerCysSerIleAsnHisProIlePheArgGluGlyAlaLysThrThr 
850855860 
PheMetIleThrPheAspValSerTyrLy sAlaPheLeuGlyAspArg 
865870875880 
LeuLeuLeuArgAlaLysAlaSerSerGluAsnAsnLysProAspThr 
885890 895 
AsnLysThrAlaPheGlnLeuGluLeuProValLysTyrThrValTyr 
900905910 
ThrLeuIleSerArgGlnGluAspSerThrAsnHisVal AsnPheSer 
915920925 
SerSerHisGlyGlyArgArgGlnGluAlaAlaHisArgTyrArgVal 
930935940 
AsnAsnLeuS erProLeuLysLeuAlaValArgValAsnPheTrpVal 
945950955960 
ProValLeuLeuAsnGlyValAlaValTrpAspValThrLeuSerSer 
965970975 
ProAlaGlnGlyValSerCysValSerGlnMetLysProProGlnAsn 
980985990 
ProAspPheLeuThrGlnIl eGlnArgArgSerValLeuAspCysSer 
99510001005 
IleAlaAspCysLeuHisSerArgCysAspIleProSerLeuAspIle 
10101015 1020 
GlnAspGluLeuAspPheIleLeuArgGlyAsnLeuSerPheGlyTrp 
1025103010351040 
ValSerGlnThrLeuGlnGluLysValLeuLeuValSer GluAlaGlu 
104510501055 
IleThrPheAspThrSerValTyrSerGlnLeuProGlyGlnGluAla 
106010651070 
PheLeuArgAlaGlnValGluThrThrLeuGluGluTyrValValTyr 
107510801085 
GluProIlePheLeuValAlaGlySerSerValGlyGlyLeuLeuLeu 
1090 10951100 
LeuAlaLeuIleThrValValLeuTyrLysLeuGlyXaaXaaLysArg 
1105111011151120 
GlnTyrLysGluMetLe uAspGlyLysAlaAlaAspProValThrAla 
112511301135 
GlyGlnAlaAspPheGlyCysGluThrProProTyrLeuValSer 
1140 11451150 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
GTCCAAGCTGTCATGGGCCAG 21 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
GTCCAGCAGACTGAAGAGCACGG 23 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
TGTAAAACGACGGCC AGT18 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
GGAAAC AGCTATGACCATG19 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
GGACATGTTCACTGCCTCTAGG22 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
GGCGGACAGTCAGACGACTGTCCTG25 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG38 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3519 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 52..3519 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
GCTTTCTGAAGGTTCCAGAATCGATAGTGAATTCGTGGGCACTGCTCAGATATGGTC57 
MetVal 
CGTGGAGTTGTGATCCTCCTGTGTGGCTGGGCCCTGGCTTCCTGTCAT105 
ArgGlyValValIleLeuLeuCysGly TrpAlaLeuAlaSerCysHis 
51015 
GGGTCTAACCTGGATGTGGAGAAGCCCGTCGTGTTCAAAGAGGATGCA153 
GlySerAsnLeuAspValGluLysProVal ValPheLysGluAspAla 
202530 
GCCAGCTTCGGACAGACTGTGGTGCAGTTTGGTGGATCTCGACTCGTG201 
AlaSerPheGlyGlnThrValValGlnPheGlyGly SerArgLeuVal 
35404550 
GTGGGAGCCCCTCTGGAGGCGGTGGCAGTCAACCAAACAGGACAGTCG249 
ValGlyAlaProLeuGluAlaValAlaVal AsnGlnThrGlyGlnSer 
556065 
TCTGACTGTCCGCCTGCCACTGGCGTGTGCCAGCCCATCTTACTGCAC297 
SerAspCysProProAlaThrGlyVal CysGlnProIleLeuLeuHis 
707580 
ATTCCCCTAGAGGCAGTGAACATGTCCCTGGGCCTGTCTCTGGTGGCT345 
IleProLeuGluAlaValAsnMetSer LeuGlyLeuSerLeuValAla 
859095 
GACACCAATAACTCCCAGTTGCTGGCTTGTGGTCCAACTGCACAGAGA393 
AspThrAsnAsnSerGlnLeuLeuAlaCys GlyProThrAlaGlnArg 
100105110 
GCTTGTGCAAAGAACATGTATGCAAAAGGTTCCTGCCTCCTTCTGGGC441 
AlaCysAlaLysAsnMetTyrAlaLysGlySerCys LeuLeuLeuGly 
115120125130 
TCCAGCTTGCAGTTCATCCAGGCAATCCCTGCTACCATGCCAGAGTGT489 
SerSerLeuGlnPheIleGlnAlaIlePro AlaThrMetProGluCys 
135140145 
CCAGGACAAGAGATGGACATTGCTTTCCTGATTGATGGCTCCGGCAGC537 
ProGlyGlnGluMetAspIleAlaPhe LeuIleAspGlySerGlySer 
150155160 
ATTGATCAAAGTGACTTTACCCAGATGAAGGACTTCGTCAAAGCTTTG585 
IleAspGlnSerAspPheThrGlnMet LysAspPheValLysAlaLeu 
165170175 
ATGGGCCAGTTGGCGAGCACCAGCACCTCGTTCTCCCTGATGCAATAC633 
MetGlyGlnLeuAlaSerThrSerThrSer PheSerLeuMetGlnTyr 
180185190 
TCAAACATCCTGAAGACTCATTTTACCTTCACGGAATTCAAGAGCAGC681 
SerAsnIleLeuLysThrHisPheThrPheThrGlu PheLysSerSer 
195200205210 
CTGAGCCCTCAGAGCCTGGTGGATGCCATCGTCCAGCTCCAAGGCCTG729 
LeuSerProGlnSerLeuValAspAlaIle ValGlnLeuGlnGlyLeu 
215220225 
ACGTACACAGCCTCGGGCATCCAGAAAGTGGTGAAAGAGCTATTTCAT777 
ThrTyrThrAlaSerGlyIleGlnLys ValValLysGluLeuPheHis 
230235240 
AGCAAGAATGGGGCCCGAAAAAGTGCCAAGAAGATACTAATTGTCATC825 
SerLysAsnGlyAlaArgLysSerAla LysLysIleLeuIleValIle 
245250255 
ACAGATGGGCAGAAATTCAGAGACCCCCTGGAGTATAGACATGTCATC873 
ThrAspGlyGlnLysPheArgAspProLeu GluTyrArgHisValIle 
260265270 
CCTGAAGCAGAGAAAGCTGGGATCATTCGCTATGCTATAGGGGTGGGA921 
ProGluAlaGluLysAlaGlyIleIleArgTyrAla IleGlyValGly 
275280285290 
GATGCCTTCCGGGAACCCACTGCCCTACAGGAGCTGAACACCATTGGC969 
AspAlaPheArgGluProThrAlaLeuGln GluLeuAsnThrIleGly 
295300305 
TCAGCTCCCTCGCAGGACCACGTGTTCAAGGTGGGCAATTTTGTAGCA1017 
SerAlaProSerGlnAspHisValPhe LysValGlyAsnPheValAla 
310315320 
CTTCGCAGCATCCAGCGGCAAATTCAGGAGAAAATCTTTGCCATTGAA1065 
LeuArgSerIleGlnArgGlnIleGln GluLysIlePheAlaIleGlu 
325330335 
GGAACCGAATCAAGGTCAAGTAGTTCCTTTCAGCACGAGATGTCACAA1113 
GlyThrGluSerArgSerSerSerSerPhe GlnHisGluMetSerGln 
340345350 
GAAGGTTTCAGCTCAGCTCTCTCAATGGATGGACCAGTTCTGGGGGCT1161 
GluGlyPheSerSerAlaLeuSerMetAspGlyPro ValLeuGlyAla 
355360365370 
GTGGGAGGCTTCAGCTGGTCTGGAGGTGCCTTCTTGTACCCCTCAAAT1209 
ValGlyGlyPheSerTrpSerGlyGlyAla PheLeuTyrProSerAsn 
375380385 
ATGAGATCCACCTTCATCAACATGTCTCAGGAGAACGAGGATATGAGG1257 
MetArgSerThrPheIleAsnMetSer GlnGluAsnGluAspMetArg 
390395400 
GACGCTTACCTGGGTTACTCCACCGCACTGGCCTTTTGGAAGGGGGTC1305 
AspAlaTyrLeuGlyTyrSerThrAla LeuAlaPheTrpLysGlyVal 
405410415 
CACAGCCTGATCCTGGGGGCCCCTCGCCACCAGCACACGGGGAAGGTT1353 
HisSerLeuIleLeuGlyAlaProArgHis GlnHisThrGlyLysVal 
420425430 
GTCATCTTTACCCAGGAATCCAGGCACTGGAGGCCCAAGTCTGAAGTC1401 
ValIlePheThrGlnGluSerArgHisTrpArgPro LysSerGluVal 
435440445450 
AGAGGGACACAGATCGGCTCCTACTTTGGGGCATCTCTCTGTTCTGTG1449 
ArgGlyThrGlnIleGlySerTyrPheGly AlaSerLeuCysSerVal 
455460465 
GACATGGATAGAGATGGCAGCACTGACCTGGTCCTGATTGGAGTCCCC1497 
AspMetAspArgAspGlySerThrAsp LeuValLeuIleGlyValPro 
470475480 
CATTACTATGAGCACACCCGAGGGGGGCAGGTGTCGGTGTGCCCCATG1545 
HisTyrTyrGluHisThrArgGlyGly GlnValSerValCysProMet 
485490495 
CCTGGTGTGAGGAGCAGGTGGCATTGTGGGACCACCCTCCATGGGGAG1593 
ProGlyValArgSerArgTrpHisCysGly ThrThrLeuHisGlyGlu 
500505510 
CAGGGCCATCCTTGGGGCCGCTTTGGGGCGGCTCTGACAGTGCTAGGG1641 
GlnGlyHisProTrpGlyArgPheGlyAlaAlaLeu ThrValLeuGly 
515520525530 
GACGTGAATGGGGACAGTCTGGCGGATGTGGCTATTGGTGCACCCGGA1689 
AspValAsnGlyAspSerLeuAlaAspVal AlaIleGlyAlaProGly 
535540545 
GAGGAGGAGAACAGAGGTGCTGTCTACATATTTCATGGAGCCTCGAGA1737 
GluGluGluAsnArgGlyAlaValTyr IlePheHisGlyAlaSerArg 
550555560 
CAGGACATCGCTCCCTCGCCTAGCCAGCGGGTCACTGGCTCCCAGCTC1785 
GlnAspIleAlaProSerProSerGln ArgValThrGlySerGlnLeu 
565570575 
TTCCTGAGGCTCCAATATTTTGGGCAGTCATTAAGTGGGGGTCAGGAC1833 
PheLeuArgLeuGlnTyrPheGlyGlnSer LeuSerGlyGlyGlnAsp 
580585590 
CTTACACAGGATGGCCTGGTGGACCTGGCCGTGGGAGCCCAGGGGCAC1881 
LeuThrGlnAspGlyLeuValAspLeuAlaValGly AlaGlnGlyHis 
595600605610 
GTGCTGCTGCTTAGGAGTCTGCCTTTGCTGAAAGTGGGGATCTCCATT1929 
ValLeuLeuLeuArgSerLeuProLeuLeu LysValGlyIleSerIle 
615620625 
AGATTTGCCCCCTCAGAGGTGGCAAAGACTGTGTACCAGTGCTGGGGA1977 
ArgPheAlaProSerGluValAlaLys ThrValTyrGlnCysTrpGly 
630635640 
AGGACTCCCACTGTCCTCGAAGCTGGAGAGGCCACCGTCTGTCTCACT2025 
ArgThrProThrValLeuGluAlaGly GluAlaThrValCysLeuThr 
645650655 
GTCCGCAAAGGTTCACCTGACCTGTTAGGTGATGTCCAAAGCTCTGTC2073 
ValArgLysGlySerProAspLeuLeuGly AspValGlnSerSerVal 
660665670 
AGGTATGATCTGGCGTTGGATCCGGGCCGTCTGATTTCTCGTGCCATT2121 
ArgTyrAspLeuAlaLeuAspProGlyArgLeuIle SerArgAlaIle 
675680685690 
TTTGATGAGACGAAGAACTGCACTTTGACCCGAAGGAAGACTCTGGGG2169 
PheAspGluThrLysAsnCysThrLeuThr ArgArgLysThrLeuGly 
695700705 
CTTGGTGATCACTGCGAAACAATGAAGCTGCTTTTGCCAGACTGTGTG2217 
LeuGlyAspHisCysGluThrMetLys LeuLeuLeuProAspCysVal 
710715720 
GAGGATGCAGTGACCCCTATCATCCTGCGCCTTAACTTATCCCTGGCA2265 
GluAspAlaValThrProIleIleLeu ArgLeuAsnLeuSerLeuAla 
725730735 
GGGGACTCTGCTCCATCCAGGAACCTTCGTCCTGTGCTGGCTGTGGGC2313 
GlyAspSerAlaProSerArgAsnLeuArg ProValLeuAlaValGly 
740745750 
TCACAAGACCATGTAACAGCTTCTTTCCCGTTTGAGAAGAACTGTGAG2361 
SerGlnAspHisValThrAlaSerPheProPheGlu LysAsnCysGlu 
755760765770 
GGGAACCTGGGCGTCAGCTTCAACTTCTCAGGCCTGCAGGTCTTGGAG2409 
GlyAsnLeuGlyValSerPheAsnPheSer GlyLeuGlnValLeuGlu 
775780785 
GTAGGAAGCTCCCCAGAGCTCACTGTGACAGTAACAGTTTGGAATGAG2457 
ValGlySerSerProGluLeuThrVal ThrValThrValTrpAsnGlu 
790795800 
GGTGAGGACAGCTATGGAACCTTAATCAAGTTCTACTACCCAGCAGAG2505 
GlyGluAspSerTyrGlyThrLeuIle LysPheTyrTyrProAlaGlu 
805810815 
CTATCTTACCGACGGGTGACAAGAGCCCAGCAACCTCATCCGTACCCA2553 
LeuSerTyrArgArgValThrArgAlaGln GlnProHisProTyrPro 
820825830 
CTACGCCTGGCATGTGAGGCTGAGCCCACGGGCCAGGAGAGCCTGAGG2601 
LeuArgLeuAlaCysGluAlaGluProThrGlyGln GluSerLeuArg 
835840845850 
AGCAGCAGCTGTAGCATCAATCACCCCATCTTCCGAGAAGGTGCCAAG2649 
SerSerSerCysSerIleAsnHisProIle PheArgGluGlyAlaLys 
855860865 
GCCACCTTCATGATCACATTTGATGTCTCCTACAAGGCCTTCCTGGGA2697 
AlaThrPheMetIleThrPheAspVal SerTyrLysAlaPheLeuGly 
870875880 
GACAGGTTGCTTCTGAGGGCCAGCGCAAGCAGTGAGAATAATAAGCCT2745 
AspArgLeuLeuLeuArgAlaSerAla SerSerGluAsnAsnLysPro 
885890895 
GAAACCAGCAAGACTGCCTTCCAGCTGGAGCTTCCGGTGAAGTACACG2793 
GluThrSerLysThrAlaPheGlnLeuGlu LeuProValLysTyrThr 
900905910 
GTCTATACCGTGATCAGTAGGCAGGAAGATTCTACCAAGCATTTCAAC2841 
ValTyrThrValIleSerArgGlnGluAspSerThr LysHisPheAsn 
915920925930 
TTCTCATCTTCCCACGGGGAGAGACAGAAAGAGGCCGAACATCGATAT2889 
PheSerSerSerHisGlyGluArgGlnLys GluAlaGluHisArgTyr 
935940945 
CGTGTGAATAACCTGAGTCCATTGACGCTGGCCATCAGCGTTAACTTC2937 
ArgValAsnAsnLeuSerProLeuThr LeuAlaIleSerValAsnPhe 
950955960 
TGGGTCCCCATCCTTCTGAATGGTGTGGCCGTGTGGGATGTGACTCTG2985 
TrpValProIleLeuLeuAsnGlyVal AlaValTrpAspValThrLeu 
965970975 
AGGAGCCCAGCACAGGGTGTCTCCTGTGTGTCACAGAGGGAACCTCCT3033 
ArgSerProAlaGlnGlyValSerCysVal SerGlnArgGluProPro 
980985990 
CAACATTCCGACCTTCTGACCCAGATCCAAGGACGCTCTGTGCTGGAC3081 
GlnHisSerAspLeuLeuThrGlnIleGlnGlyArg SerValLeuAsp 
995100010051010 
TGCGCCATCGCCGACTGCCTGCACCTCCGCTGTGACATCCCCTCCTTG3129 
CysAlaIleAlaAspCysLeuHisLeuArg CysAspIleProSerLeu 
101510201025 
GGCACCCTGGATGAGCTTGACTTCATTCTGAAGGGCAACCTCAGCTTC3177 
GlyThrLeuAspGluLeuAspPheIl eLeuLysGlyAsnLeuSerPhe 
103010351040 
GGCTGGATCAGTCAGACATTGCAGAAAAAGGTGTTGCTCCTGAGTGAG3225 
GlyTrpIleSerGlnThrLeuGlnL ysLysValLeuLeuLeuSerGlu 
104510501055 
GCTGAAATCACATTCAACACATCTGTGTATTCCCAGCTGCCGGGACAG3273 
AlaGluIleThrPheAsnThrSerVal TyrSerGlnLeuProGlyGln 
106010651070 
GAGGCATTTCTGAGAGCCCAGGTGTCAACGATGCTAGAAGAATACGTG3321 
GluAlaPheLeuArgAlaGlnValSerThrMet LeuGluGluTyrVal 
1075108010851090 
GTCTATGAGCCCGTCTTCCTCATGGTGTTCAGCTCAGTGGGAGGTCTG3369 
ValTyrGluProValPheLeuMetVa lPheSerSerValGlyGlyLeu 
109511001105 
CTGTTACTGGCTCTCATCACTGTGGCGCTGTACAAGCTTGGCTTCTTC3417 
LeuLeuLeuAlaLeuIleThrV alAlaLeuTyrLysLeuGlyPhePhe 
111011151120 
AAACGTCAGTATAAAGAGATGCTGGATCTACCATCTGCAGATCCTGAC3465 
LysArgGlnTyrLysGluMet LeuAspLeuProSerAlaAspProAsp 
112511301135 
CCAGCCGGCCAGGCAGATTCCAACCATGAGACTCCTCCACATCTCACG3513 
ProAlaGlyGlnAlaAspSerAsn HisGluThrProProHisLeuThr 
114011451150 
TCCTAG3519 
Ser 
1155 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1155 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
MetValArgGlyValValIleLeuLeuCysGlyTrpAlaLeuAlaSer 
1510 15 
CysHisGlySerAsnLeuAspValGluLysProValValPheLysGlu 
202530 
AspAlaAlaSerPheGlyGlnThrValValGlnPheGlyGl ySerArg 
354045 
LeuValValGlyAlaProLeuGluAlaValAlaValAsnGlnThrGly 
505560 
GlnSerSerAsp CysProProAlaThrGlyValCysGlnProIleLeu 
65707580 
LeuHisIleProLeuGluAlaValAsnMetSerLeuGlyLeuSerLeu 
859095 
ValAlaAspThrAsnAsnSerGlnLeuLeuAlaCysGlyProThrAla 
100105110 
GlnArgAlaCysAlaLysAsnM etTyrAlaLysGlySerCysLeuLeu 
115120125 
LeuGlySerSerLeuGlnPheIleGlnAlaIleProAlaThrMetPro 
130135 140 
GluCysProGlyGlnGluMetAspIleAlaPheLeuIleAspGlySer 
145150155160 
GlySerIleAspGlnSerAspPheThrGlnMetLysAspPheVa lLys 
165170175 
AlaLeuMetGlyGlnLeuAlaSerThrSerThrSerPheSerLeuMet 
180185190 
Gln TyrSerAsnIleLeuLysThrHisPheThrPheThrGluPheLys 
195200205 
SerSerLeuSerProGlnSerLeuValAspAlaIleValGlnLeuGln 
210 215220 
GlyLeuThrTyrThrAlaSerGlyIleGlnLysValValLysGluLeu 
225230235240 
PheHisSerLysAsnGlyAlaArgL ysSerAlaLysLysIleLeuIle 
245250255 
ValIleThrAspGlyGlnLysPheArgAspProLeuGluTyrArgHis 
260265 270 
ValIleProGluAlaGluLysAlaGlyIleIleArgTyrAlaIleGly 
275280285 
ValGlyAspAlaPheArgGluProThrAlaLeuGlnGluLeuAs nThr 
290295300 
IleGlySerAlaProSerGlnAspHisValPheLysValGlyAsnPhe 
305310315320 
ValAla LeuArgSerIleGlnArgGlnIleGlnGluLysIlePheAla 
325330335 
IleGluGlyThrGluSerArgSerSerSerSerPheGlnHisGluMet 
3 40345350 
SerGlnGluGlyPheSerSerAlaLeuSerMetAspGlyProValLeu 
355360365 
GlyAlaValGlyGlyPheSerTrpS erGlyGlyAlaPheLeuTyrPro 
370375380 
SerAsnMetArgSerThrPheIleAsnMetSerGlnGluAsnGluAsp 
385390395 400 
MetArgAspAlaTyrLeuGlyTyrSerThrAlaLeuAlaPheTrpLys 
405410415 
GlyValHisSerLeuIleLeuGlyAlaProArgHisGlnHi sThrGly 
420425430 
LysValValIlePheThrGlnGluSerArgHisTrpArgProLysSer 
435440445 
GluVal ArgGlyThrGlnIleGlySerTyrPheGlyAlaSerLeuCys 
450455460 
SerValAspMetAspArgAspGlySerThrAspLeuValLeuIleGly 
465470 475480 
ValProHisTyrTyrGluHisThrArgGlyGlyGlnValSerValCys 
485490495 
ProMetProGlyValArgSerA rgTrpHisCysGlyThrThrLeuHis 
500505510 
GlyGluGlnGlyHisProTrpGlyArgPheGlyAlaAlaLeuThrVal 
515520 525 
LeuGlyAspValAsnGlyAspSerLeuAlaAspValAlaIleGlyAla 
530535540 
ProGlyGluGluGluAsnArgGlyAlaValTyrIlePheHisGlyAla 
545550555560 
SerArgGlnAspIleAlaProSerProSerGlnArgValThrGlySer 
565570575 
Gln LeuPheLeuArgLeuGlnTyrPheGlyGlnSerLeuSerGlyGly 
580585590 
GlnAspLeuThrGlnAspGlyLeuValAspLeuAlaValGlyAlaGln 
595 600605 
GlyHisValLeuLeuLeuArgSerLeuProLeuLeuLysValGlyIle 
610615620 
SerIleArgPheAlaProSerGluValAlaL ysThrValTyrGlnCys 
625630635640 
TrpGlyArgThrProThrValLeuGluAlaGlyGluAlaThrValCys 
645650 655 
LeuThrValArgLysGlySerProAspLeuLeuGlyAspValGlnSer 
660665670 
SerValArgTyrAspLeuAlaLeuAspProGlyArgLeuIl eSerArg 
675680685 
AlaIlePheAspGluThrLysAsnCysThrLeuThrArgArgLysThr 
690695700 
LeuGlyLeuGly AspHisCysGluThrMetLysLeuLeuLeuProAsp 
705710715720 
CysValGluAspAlaValThrProIleIleLeuArgLeuAsnLeuSer 
7 25730735 
LeuAlaGlyAspSerAlaProSerArgAsnLeuArgProValLeuAla 
740745750 
ValGlySerGlnAspHisValT hrAlaSerPheProPheGluLysAsn 
755760765 
CysGluGlyAsnLeuGlyValSerPheAsnPheSerGlyLeuGlnVal 
770775 780 
LeuGluValGlySerSerProGluLeuThrValThrValThrValTrp 
785790795800 
AsnGluGlyGluAspSerTyrGlyThrLeuIleLysPheTyrTy rPro 
805810815 
AlaGluLeuSerTyrArgArgValThrArgAlaGlnGlnProHisPro 
820825830 
Tyr ProLeuArgLeuAlaCysGluAlaGluProThrGlyGlnGluSer 
835840845 
LeuArgSerSerSerCysSerIleAsnHisProIlePheArgGluGly 
850 855860 
AlaLysAlaThrPheMetIleThrPheAspValSerTyrLysAlaPhe 
865870875880 
LeuGlyAspArgLeuLeuLeuArgA laSerAlaSerSerGluAsnAsn 
885890895 
LysProGluThrSerLysThrAlaPheGlnLeuGluLeuProValLys 
900905 910 
TyrThrValTyrThrValIleSerArgGlnGluAspSerThrLysHis 
915920925 
PheAsnPheSerSerSerHisGlyGluArgGlnLysGluAlaGl uHis 
930935940 
ArgTyrArgValAsnAsnLeuSerProLeuThrLeuAlaIleSerVal 
945950955960 
AsnPhe TrpValProIleLeuLeuAsnGlyValAlaValTrpAspVal 
965970975 
ThrLeuArgSerProAlaGlnGlyValSerCysValSerGlnArgGlu 
9 80985990 
ProProGlnHisSerAspLeuLeuThrGlnIleGlnGlyArgSerVal 
99510001005 
LeuAspCysAlaIleAlaAspCys LeuHisLeuArgCysAspIlePro 
101010151020 
SerLeuGlyThrLeuAspGluLeuAspPheIleLeuLysGlyAsnLeu 
102510301035 1040 
SerPheGlyTrpIleSerGlnThrLeuGlnLysLysValLeuLeuLeu 
104510501055 
SerGluAlaGluIleThrPheAsnThrSerValTyrSe rGlnLeuPro 
106010651070 
GlyGlnGluAlaPheLeuArgAlaGlnValSerThrMetLeuGluGlu 
107510801085 
Ty rValValTyrGluProValPheLeuMetValPheSerSerValGly 
109010951100 
GlyLeuLeuLeuLeuAlaLeuIleThrValAlaLeuTyrLysLeuGly 
1105 111011151120 
PhePheLysArgGlnTyrLysGluMetLeuAspLeuProSerAlaAsp 
112511301135 
ProAspProAlaGly GlnAlaAspSerAsnHisGluThrProProHis 
114011451150 
LeuThrSer 
1155 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 49 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
AGTTACGGATCCGGCACCATGACCTTCGGCACTGTGATCCTCCTGTGTG49 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
GCTGGACGATGGCATCCAC19 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
GTAGAGTTACGGATCCGGCACCAT24 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
GCAGCCAGCTTCGGACAGAC20 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
CCATGTCCACAGAACAGAGAG21