Molecular clone of a P58 receptor protein and uses thereof

The present invention provides a purified and isolated nucleic acid molecule encoding the p58 receptor protein. The present invention also provides vectors encoding this nucleic acid molecule, a host cell stably transformed or transfected with the vector, as well as the p58 receptor protein produced by this host cell. The present invention further provides methods for detecting nucleic acid encoding the p58 receptor as well as the p58 receptor protein in biological samples, methods for identifying a ligand capable of binding to the p58 receptor protein, and methods for screening for drugs capable of acting as agonists or antagonists to the p58 receptor protein. The present invention also provides the agonists and antagonists identified by the screening methods, as well as their use. Lastly, the present invention provides chimeric protein comprising the cytoplasmic region of a p58 receptor protein.

Natural killer (NK) cells are important in the immune defense against 
tumors, viruses and intracellular parasites. NK cells also may be involved 
in the rejection of bone marrow transplants, and other transplants. The 
ability to intervene with the immune function of the NK cells, by either 
boosting NK cell responses to pathogens, or by lowering or stopping 
unwanted NK cell responses, is a long recognized need. 
The p58 receptor of NK cells is a family of cell surface molecules with a 
molecular mass of about 58,000 which is involved in the recognition of 
target cells by NK cells. The p58 receptor contributes to the specificity 
of NK cells in their recognition of major histocompatibility complex (MHC) 
class I molecules on target cells. The specific recognition of MHC 
molecules mediated by the p58 receptor protects the target cell from lysis 
by the NK cell. Conversely, absence of the appropriate MHC class I 
molecule on target cells leads to NK-mediated lysis. 
Prior to the present invention, however, no members of the p58 family have 
been purified sufficiently to permit their isolation and characterization. 
This is because it is impossible to grow large enough populations of the 
NK cells to permit isolation of sufficient amounts of p58 receptor protein 
for sequencing. Furthermore, the known NK cell lines express high levels 
of Fc receptor which could interfere with immunoaffinity purifications 
utilizing monoclonal antibodies directed to the p58 receptor protein, 
since these antibodies also bind to the Fc receptor. 
In particular, Moretta, et al. (1990) described the use of a monoclonal 
antibody designated GL183 to immunoprecipitate either a 58 Kd band or a 
broad 55-58 Kd band from NK clones which had been labeled with .sup.125 I 
(Moretta, et al. J. Exp. Med. 171: 695-714 (1990); Moretta, et al. 
Advances in Immunology 55: 341-380 (1994)). However, Moretta, et al. did 
not sufficiently purify the alleged p58 receptor protein to permit its 
isolation and characterization, and could not have done so utilizing the 
particular NK cells described therein, or known NK cells. 
This is because NK cells described in Moretta, et al., as most NK cells, 
cannot be grown to large enough populations to permit isolation of 
sufficient amounts of p58 receptor protein for sequencing. Specifically, 
about 1.times.10.sup.10 NK cells would have to be obtained in order to 
purify an amount of pure p58 protein sufficient for sequencing. However, 
NK cells cannot be expanded much beyond 1.times.10.sup.7 cells since they 
do not grow well in culture. 
Moreover, the NK cell lines described in Moretta, et al. express high 
levels of Fc receptor which could interfere with immunoaffinity 
purifications utilizing the GL183 monoclonal antibody, since this antibody 
also binds to the Fc receptor. Furthermore, the inventors of the present 
invention found that the p58 and Fc receptors (CD16) have very similar 
isoelectric points and relative masses, which could make their separation 
difficult. 
The inventors of the present invention overcame the potential problems in 
the art by employing a long term NK cell line designated NK 3.3 which, 
unlike previously identified NK clones, grows well under long-term culture 
conditions, can be readily expanded to sufficient numbers, and expresses 
very low levels of the Fc receptor. Accordingly, the inventors of the 
present invention were the first to purify a member of the p58 receptor 
family, characterize it, and subsequently clone its DNA. 
SUMMARY OF THE INVENTION 
The present invention provides a purified and isolated nucleic acid 
molecule encoding the p58 receptor protein. The present invention also 
provides vectors encoding this nucleic acid molecule, a host cell stably 
transformed or transfected with the vector, as well as the p58 receptor 
protein produced by this host cell. 
The present invention also provides a method for detecting nucleic acid 
encoding the p58 receptor in a biological sample which comprises the steps 
of: (a) contacting nucleic acid from the biological sample with the probe 
made from nucleic acid encoding the p58 receptor protein under conditions 
permitting a complex to be formed between the probe and nucleic acid 
presence in the sample; and (b) detecting the formation of the complex. 
The present invention further provides a method for detecting nucleic acid 
encoding the p58 receptor in a biological sample which comprises the steps 
of: (a) contacting nucleic acid from the biological sample with a sense 
and an antisense primer prepared from nucleic acid encoding the p58 
receptor protein under conditions permitting PCR amplification to occur; 
and (b) detecting amplification of the nucleic acid from the biological 
sample. 
Moreover, the present invention provides a method for detecting a p58 
receptor protein in a biological sample which comprises the steps of: (a) 
contacting the biological sample with a reagent which specifically reacts 
with a p58 receptor protein; and (b) detecting the formation of a complex 
between the protein and the reagent. 
The present invention also provides a method for identifying a ligand 
capable of binding to the p58 receptor protein which comprises the steps 
of: (a) contacting a p58 receptor protein or cells which express the p58 
receptor protein with a candidate ligand; and (b) detecting the formation 
of a complex between the p58 receptor protein and the ligand. 
In addition, the present invention provides a method for identifying a 
ligand capable of binding to the p58 receptor protein which comprises the 
steps of: (a) contacting a p58 receptor protein with cells transfected 
with cDNA encoding a candidate ligand; and (b) detecting the formation of 
a complex between the p58 receptor protein and the ligand expressed by the 
cells. 
Also provided by the present invention is a method for screening for drugs 
capable of acting as agonists or antagonists to the p58 receptor protein 
which comprises the steps of: (a) contacting cells which express the p58 
receptor protein with candidate drugs; and (b) evaluating the biological 
activity mediated by said contact. 
The present invention also provides the agonists and antagonists identified 
by the method above as well as uses of these agonists and antagonists. 
In particular, the present invention provides a method for preventing 
immunosuppressive rejection of a transplant in a subject receiving the 
transplant which comprises administering to the subject an amount of an 
agonist to a p58 receptor protein effective to prevent immunosuppressive 
rejection of the transplant. 
The present invention also provides a method for treating a neoplastic cell 
growth in a subject in need of such treatment which comprises 
administering an amount of an antagonist to a p58 receptor protein 
effective to treat the neoplastic cell growth. 
Lastly, the present invention provides a chimeric protein comprising (a) 
the cytoplasmic region of a p58 receptor protein which transmits a 
dominant negative signal to a cell expressing the p58 receptor protein; 
and (b) a transmembrane domain and an extracellular domain of a known 
receptor.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides a purified and isolated nucleic acid 
molecule encoding the p58 receptor protein. The nucleic acid molecule 
includes all nucleic acid sequences which encode for the p58 receptor 
including all degenerate forms. The term "nucleic acid" includes but is 
not limited to DNA, RNA or cDNA. 
In the preferred embodiment, the p58 receptor protein has the nucleotide 
sequence contained in SEQ ID NO:1, or a sequence which is substantially 
homologous thereto. "Substantially homologous" as used herein refers to 
substantial correspondence between the nucleic acid sequences of various 
members of the p58 receptor protein family. "Substantially homologous" 
means at least 50% identical, preferably about 65% identical, and most 
preferably, about 70-90% identical. The nucleic acid is mammalian, and is 
preferably human or murine. 
The nucleic acid sequences for the additional members of the p58 receptor 
protein family may be isolated by screening libraries of cDNA clones 
derived from NK cell RNA with a probe corresponding to the sequence 
contained in SEQ ID NO:2 using standard DNA hybridization techniques. It 
is expected that the DNA sequences encoding the different members of the 
p58 receptor protein family will exhibit a level of similarity that 
clearly exceeds the 55% identity required for cross-hybridization. This is 
because analogous families of related genes encoding other receptors 
usually display similarities with about 70 to 90% identity among different 
members (e.g. HLA class I genes, HLA class II genes, genes encoding 
different T cell receptors, genes encoding different immunoglobulin 
molecules). 
Furthermore, genomic clones encoding the p58 receptor family also may be 
isolated by screening human genomic libraries with a probe corresponding 
to the sequence contained in SEQ ID NO:2 using standard DNA hybridization 
techniques. 
In addition, the corresponding gene in mice may be isolated by 
cross-hybridization techniques as long as some portion of the probe above 
has at least 55% nucleotide sequence identity with the mouse genes. If 
cross-hybridization is not successful, oligonucleotide primers based on 
the same sequence may be used to isolate the mouse genes using PCR. The 
mouse genes may be used to generate "knock-out" mice with a defect in one 
or several of the genes encoding the mouse counterpart of the human p58 
receptor protein. Such knock-out mice would be extremely valuable tools to 
study the role of p58 receptors in immune responses. 
The present invention also provides a vector comprising the nucleic acid 
molecule encoding the p58 receptor protein, as described above. In the 
preferred embodiment, the vector is an expression vector which comprises 
at least one expression control element operationally linked to the 
nucleic acid sequence encoding the p58 receptor. The expression control 
elements are inserted in the vector to control and regulate the expression 
of the nucleic acid sequence, and are known in the art. The vectors of the 
present invention may be constructed by known techniques (see U.S. Pat. 
Nos. 4,704,362, 4,366,246, 4,425,437, 4,356,270, and 4,571,421), using 
commercially available plasmids. 
The present invention also provides a host cell stably transformed or 
transfected with the vector above. The host cells may be prokaryotic such 
as E. coli, or eukaryotic such as animal, plant, insect and yeast cells. 
The host cells are stably transformed or transfected by known procedures 
(see U.S. Pat. Nos. 4,704,362, 4,366,246, 4,425,437, 4,356,270, and 
4,571,421). In the particularly preferred embodiment, amplification 
protocols (e.g. methotrexate selection with dhfr vectors) may be used to 
obtain cells that produce high levels of recombinant proteins. Other 
expression systems based on virus vectors may be used for extremely high 
level expression, but only transiently (e.g. baculovirus, Sindbis virus). 
The host cells may be screened for clones which produce the recombinant 
p58 receptor protein by known procedures such as Coomassie blue staining 
and Western blotting. 
It is also within the confines of the present invention that modified forms 
of the p58 receptor protein may be engineered and produced. Truncated or 
mutated forms can be produced to define functional domains of the p58 
receptor protein. Secreted forms can be engineered for the production of 
soluble receptors. 
The present invention also provides the p58 receptor protein produced by 
the host cell above. The recombinant protein expressed by the host cells 
may be obtained as a crude lysate or can be purified by standard protein 
purification procedures known in the art such as differential 
precipitation, molecular sieve chromatography, ion-exchange 
chromatography, isoelectric focusing, gel electrophoresis, affinity 
chromatography, immunoaffinity chromatography, and the like. 
Preferably, the p58 receptor protein has the amino acid sequence contained 
in SEQ ID NO:2, or is a biologically active analogue thereof. The phrase 
"biologically active analogue thereof" refers to proteins having 
substantially homologous sequences to the sequence contained in SEQ ID 
NO:2, and possess similar activity as that protein. The isolated and 
purified p58 receptor protein of the present invention includes all 
members of the p58 receptor protein family. 
The present invention also provides a method for detecting nucleic acid 
encoding the p58 receptor protein in a biological sample utilizing as 
probes the nucleic acid above, or portions thereof, labeled with 
detectable markers. In particular, the method comprises the steps of: (a) 
contacting the probe with a biological sample under conditions permitting 
a complex to be formed between the probe and nucleic acid present in the 
sample; and (b) detecting the formation of the complex. 
The biological samples include mammalian tissue or cell samples. The 
nucleic acid may be extracted from the biological samples by known 
procedures. Specifically, the cells may be lysed using an enzyme such as 
proteinase K, in the presence of detergents such as sodium dodecyl sulfate 
(SDS), NP40, or Tween 20. If the nucleic acid is genomic DNA, it may be 
extracted using known techniques such as phenol/chloroform extraction, or 
other procedures (see U.S. Pat. Nos. 4,900,677 and 5,047,345). 
Alternatively, the DNA may be isolated using one of the commercially 
available kits such as the Oncor Genomic DNA isolation kit. RNA may be 
extracted using various known procedures such as guanidinium thiocyanate 
followed by centrifugation in cesium chloride (Sambrook, J., Fritsch, E. 
F., and Maniatis, T. "Molecular Cloning, A Laboratory Manual," second 
edition, Cold Spring Harbor Laboratory Press, pp. 7.0-7.25 (1989)). 
The formation of the complex may be detected using various conventional 
techniques known in the art including but not limited to Northern 
blotting, Southern blotting, dot and slot hybridization, S1 nuclease 
assay, ribonuclease protection assay, and filter hybridization (Southern, 
E. M. J. Mol. Biol. 98:503 (1975); Chirgwin, J. M., et al. Biochemistry 
18: 5294-5299 (1979); Kafatos, et al. Nuc. Acids. Res. 7: 1541 (1979); 
Thomas, P. S. Proc. Natl. Acad. Sci. 77: 5201 (1980); White, B. A. and F. 
C. Bancroft J. Bio. Chem. 257: 8569 (1982); Berk, A. J. and P. A. Sharp 
Cell 12: 721 (1977); Casey, J. and N. Davidson Nuc. Acids Res. 4:1539 
(1977); Sambrook, J., Fritsch, E. F., and Maniatis, T. "Molecular Cloning, 
A Laboratory Manual," second edition, Cold Spring Harbor Laboratory Press, 
pp. 7.30-7.87 and 9.31-9.62 (1989)). 
In the method above, the probe is labeled with a detectable marker which 
includes but is not limited to fluorescence, enzyme or radiolabeled 
markers such as .sup.32 P and biotin. In the preferred embodiment, the 
marker is .sup.32 P. The probe is labeled by known procedures such as 
phosphorylation with bacteriophage T4 polynucleotide kinase (Sambrook, J., 
Fritsch, E. F., and Maniatis, T. "Molecular Cloning, A Laboratory Manual," 
second edition, Cold Spring Harbor Laboratory Press, pp. 11.31-11.32 
(1989)). 
It is within the confines of the present invention that probes may be 
designed to detect all members of the p58 family, or may be unique in 
detecting only one or more specific members of the p58 family. 
The present invention also provides a method for detecting nucleic acid 
encoding the p58 receptor in a biological sample using standard PCR 
procedures. In this regard, the method comprises the steps of: (a) 
contacting nucleic acid from the biological sample with a sense and an 
antisense primer prepared from the sequence of the present invention under 
conditions permitting PCR amplification to occur; and (b) detecting 
amplification of the nucleic acid from the biological sample. 
Polymerase chain reaction is performed by methods and conditions disclosed 
in U.S. Pat. Nos. 4,683,202 and 4,683,195 and in Perkin Elmer Cetus PCR 
kit protocols. The DNA polymerase, deoxyribonucleotide triphosphates 
(dNTPS) (e.g. dATP, dCTP, dTTP, and dGTP), and amplification buffer (e.g. 
glycerol, tris-hydrochloric acid, potassium chloride, Tween 20, and 
magnesium chloride) are readily commercially available (Perkin Elmer 
Cetus). The polymerase chain reaction may be performed as many cycles as 
desired. Reverse transcription (RT) of mRNA and RT-PCR are performed by 
methods described in commercially available kits such as the RT and RT-PCR 
kits (Perkin Elmer Cetus). 
The sense and antisense primers for use in PCR and RT-PCR may be prepared 
from the nucleic acid sequence above using automated instruments sold by a 
variety of manufacturers or may be commercially synthesized. 
It is within the confines of the present invention that primers may be 
designed to detect all members of the p58 family, or may be unique in 
detecting only one or more specific members of the p58 family. 
The present invention also provides a method for detecting a p58 receptor 
protein in a biological sample using particular reagents which 
specifically react with the p58 receptor protein. 
The term "reagent" includes monoclonal or polyclonal antibodies. Exemplary 
antibody molecules for use in the detection methods of the present 
invention are intact immunoglobulin molecules, substantially intact 
immunoglobulin molecules, or those portions of an immunoglobulin molecule 
that contain the antigen binding site, including those portions known in 
the art as F(ab), F(ab'), F(ab').sub.2, and F(v). In the preferred 
embodiment, antibodies specific to each different member of the p58 
receptor protein family, or portions thereof, may be used to detect 
expression of their corresponding p58 receptor protein family member. The 
complete protein or unique portions thereof of the particular members of 
the p58 family may be utilized as an immunogen, with or without a carrier 
molecule, to produce these antibodies. Examples of carrier molecules 
includes human albumin, bovine albumin, keyhole limpet hemo-cyanin, and 
the like. 
Polyclonal and monoclonal antibodies, or their fragments, may be produced 
by methods known in the art, or by genetic engineering (Kohler and 
Milstein (1975) Nature 256: 495-497; Campbell "Monoclonal Antibody 
Technology," the "Production and Characterization of Rodent and Human 
Hybridomas" in Burdon, et al. (eds.) (1985) "Laboratory Techniques in 
Biochemistry and Molecular Biology," Volume 13, Elsevier Science 
Publishers, Amsterdam). 
The antibodies are used in immunoassays to detect the p58 receptor protein 
in biological samples. In particular, the method comprises the steps of: 
(a) contacting the biological sample with an antibody; and (b) detecting 
the formation of a complex between the protein and the antibody. In the 
particularly preferred embodiment, the immunoassays are used to detect 
different members of the p58 receptor protein family in the biological 
sample using their respective antibodies. 
Suitable immunoassays include but are not limited to radioimmunoassay, 
Western blot assay, immunofluorescent assay, enzyme immunoassay, 
chemiluminescent assay, and immunohistochemical assay. These assays may be 
performed using procedures well known in the art. (In "Principles and 
Practice of Immunoassay" (1991) Christopher P. Price and David J. Neoman 
(eds), Stockton Press, New York, N.Y.; Ausubel, et al. (eds) (1991) in 
"Current Protocols in Molecular Biology" John Wiley and Sons, New York, 
N.Y.). 
Target cells that do not express a cell surface ligand able to interact 
with the p58 receptor expressed by a particular NK cell are likely to be 
lysed by that NK cell. This deadly fate results from the fact that the p58 
receptor on NK cells serves to provide a negative signal to the NK cell 
and thus stop the lytic machinery of the NK cell. Rejection of bone marrow 
transplants, and possibly autoimmune reactions, are mediated by NK cells 
when the negative signal fails to be delivered to the NK cells. It is 
therefore very important, and potentially very useful clinically, to 
determine exactly which molecules on target cells serve as ligands for the 
p58 receptor proteins and as protective elements on target cells that 
would otherwise be killed. 
Therefore, the present invention also provides a method for identifying a 
ligand capable of binding to the p58 receptor protein. In one embodiment, 
the method comprises the steps of: (a) contacting a p58 receptor protein 
or cells which express the p58 receptor protein with a candidate ligand; 
and (b) detecting the formation of a complex between the p58 receptor 
protein and the ligand. In another embodiment, the method comprises the 
steps of: (a) contacting a p58 receptor protein with cells transfected 
with cDNA encoding a candidate ligand; and (b) detecting the formation of 
a complex between the p58 receptor protein and the ligand expressed by the 
cells. 
The term "ligand" refers to any protein or proteins that may interact with 
the p58 receptor protein. Said ligand may be soluble or membrane bound. 
The ligand may be a naturally occurring protein, or synthetically or 
recombinantly produced. The ligand may also be a nonprotein molecule that 
acts as a ligand when it interacts with the p58 receptor. In the preferred 
embodiment, the ligands are HLA class I molecules and their isotypes. 
Interactions between the ligand and receptor include but are not limited 
to covalent or non-covalent interactions. The receptor binding domain is 
any region of the receptor that interacts directly or indirectly with the 
ligand. 
The p58 receptor protein is preferably recombinant p58 receptor protein, 
and most preferably is soluble. It is within the confines of the present 
invention that the soluble recombinant molecules may be produced as fusion 
proteins with a secreted form of immunoglobulin. This leads to efficient 
expression of a secreted soluble form and also to detection using 
well-established antibodies specific for the immunoglobulin molecules. The 
soluble p58-immunoglobulin fusion proteins may be used as reagents for 
analysis by flow cytometry to test for the presence of ligands on 
different cells. 
Cells stably transfected with a complete cDNA encoding a p58 receptor 
protein may be used to test binding of soluble ligands or binding of 
immobilized ligands. To increase valency, ligands may be attached to a 
support such as a latex bead. These assays are known in the art as rosette 
formation assays. 
The present invention also provides a method for screening for drugs 
capable of acting as agonists or antagonists to the p58 receptor protein 
which comprises the steps of: (a) contacting cells which express the p58 
receptor protein with candidate drugs; and (b) evaluating the biological 
activity mediated by said contact. 
The term "drugs" includes but is not limited to proteins, peptides, agents 
purified from conditioned cell medium, organic molecules, inorganic 
molecules, antibodies oligonucleotides, or analogs of the ligand described 
above. The drug may be naturally occurring or synthetically or 
recombinantly produced. Specific agonists of the p58 receptor protein 
would be extremely useful in preventing unwanted NK-mediated lysis of 
target cells (e.g. in bone marrow transplantation). Specific antagonists 
would be useful for boosting NK cell responses to unwanted pathogens such 
as cancer cells. 
The term "biological activity" as used herein means the triggering of the 
p58 receptor protein to transmit a negative signal to the cell which 
expresses it. This is evaluated by tranfecting the cDNA encoding the p58 
receptor protein with known reporter gene constructs, and measuring the 
triggering effect of specific drugs on the p58 receptor protein using 
known calorimetric assays (e.g. beta-galactosidase) or luminometry (e.g. 
luciferase). 
The present invention also provides an agonist or antagonist to a p58 
receptor protein identified using the methods above, as well as uses 
thereof. 
In particular, the present invention provides a method for preventing 
immunosuppressive rejection of a transplant in a subject receiving the 
transplant which comprises administering to the subject an amount of an 
agonist to a p58 receptor protein effective to prevent immunosuppressive 
rejection of the transplant. 
As used herein, the term "transplant" refers to any cell, tissue or organ 
which may be suspectable to immunosuppressive rejection when transplanted 
into a patient. In the preferred embodiment, the transplant is bone 
marrow. 
The present invention also provides a method for treating a neoplastic cell 
growth in a subject in need of such treatment which comprises 
administering an amount of an antagonist to a p58 receptor protein 
effective to treat the neoplastic cell growth. 
The term "neoplastic cell growth" includes any of the known cancers such as 
cancers of the breast, lung, brain, groin, and the like. The term 
"treatment" includes the partial or total inhibition of neoplastic cell 
growth, as well as the partial or total destruction of the neoplastic 
cells. The term "subject" includes a human or animal subject diagnosed as 
having cancer. It is within the confines of the present invention that the 
antagonist may be administered in conjunction with an antibody which 
specifically recognizes the tumor. 
The administration for both methods above may be affected by means known to 
those skilled in the art such as oral, rectal, topical intravenous, 
subcutaneous, intramuscular, or intraperitoneal routes of administration. 
The dosage form and amount for each method can be readily established by 
reference to known immunosupressive treatments and antineoplastic 
treatments, respectively. The actual dose will depend upon the route of 
administration, the pharmacokinetic properties of the individual treated, 
as well as the results desired. 
Studies of chimeric receptor proteins consisting of extracellular domain of 
one receptor and the intracellular domain of a different receptor indicate 
that a common mechanism of signal transduction through the cell membrane 
is shared by different growth factor receptors (Riedel, et al. Nature 324: 
68-70 (1986); Riedel, et al. EMBO J. 8: 2943-2954 (1989)). The specificity 
of the signal transmitted by the receptors resides in the intracellular 
domain. Chimeras between the extracellular domain of a known ligand and 
intracellular domain of an orphan receptor can be activated by the known 
ligand, yet the biological function elicited is similar to the receptor 
with the intracellular domain. Numerous chimeric receptors including 
insulin receptor (IR)/epidermal growth factor receptor (EGFR) (Riedel, et 
al. Nature 324: 68-70 (1986), EGFR/IR (Riedel, et al. EMBO J. 8: 2943-2954 
(1989)), platelet-derived growth factor receptor beta 
(PDGFR-beta)/fibroblast growth factor receptor-1 (FGFR-1) (Mares, et al. 
Growth Factors 6:93-101 (1992)) have been constructed and elicited proper 
responses. 
Accordingly, the present invention also provides a chimeric protein 
comprising (a) the cytoplasmic region of a p58 receptor protein which 
transmits a dominant negative signal to a cell expressing the p58 receptor 
protein; and (b) a transmembrane domain and an extracellular domain of a 
known receptor. This chimeric protein may be constructed as described in 
the publications above. The chimeric protein is useful for studying the 
recognition of target molecules mediated by the p58 receptor protein 
family. 
The present invention is described in the following Experimental Details 
section, which sets forth specific examples to aid in an understanding of 
the invention, and should not be construed to limit in any way the 
invention as defined in the claims which follow thereafter. 
EXPERIMENTAL DETAILS SECTION 
A. Purification of p58 Receptor Protein 
1. NK3.3 Cells Express 183 Ag 
The mAb GL183 recognizes a surface antigen (183 antigen, 183 Ag) expressed 
on a subset of freshly isolated human NK cells and on some NK clones 
(Moretta, et al. J. Exp. Med. 171: 695-714 (1990); Moretta, et al. 
Advances in Immunology 55: 341-380 (1994)). The difficulty of generating 
and growing human NK clones has precluded the isolation of large amounts 
of 183 antigen from these cells necessary for sequencing the 183 antigen. 
Purification also has been hindered because most known NK cells express 
the Fc receptor (CD16), which also has a similar isoelectric point and 
molecular mass as the 183 antigen. 
The inventors of the present invention have found by FACS analysis using 
GL183 that a human NK cell line NK3.3 obtained from Jackie Kornbluth 
(Kornbluth, J., et al. J. Immunol. 129:2831 (1982)) expresses the 183 
antigen at the cell surface, and also express low levels of the Fc 
receptor (CD16). Unlike previously identified clones, the NK3.3 cell line 
also grows well under long term conditions, and can be readily expanded. 
Analytical experiments were performed to biochemically characterize the 183 
Ag expressed on NK3.3, in order to compare it to the Ag expressed on NK 
clones. NK3.3 and NK clones were surface iodinated and lysed in lysis 
buffer containing Triton and protease inhibitors. Immunoprecipitation 
using GL183 and control antibodies, and analysis of the precipitates on 
SDS-PAGE indicated that the 183 Ag derived from the two sources were very 
similar. 
2. Immunoprecipitation 
GL183 was purified from ascites and coupled to Sepharose CL-2B. These 
GL183-Sepharose beads were used to immunoprecipitate 183 Ag from detergent 
lysates of surface iodinated NK3.3. The immuno-precipitates were then 
incubated in an elution buffer at pH 11.2 to test whether the 183 Ag could 
be eluted from the beads under those conditions. The eluted material was 
analyzed on SDS gels followed by autoradiography. 
It was found that incubation at pH 11.2 released a I-125 labelled protein 
of the expected size (MW). However, these experiments also showed that 
control beads, either unconjugated or conjugated to control Abs, 
immunoprecipitated large amounts of I-125 labelled proteins, which were 
also eluted from the beads under these conditions, indicating that 
extensive preclearing of the detergent lysates would be necessary. 
The lysate was therefore passed over four sequential columns containing the 
following beads: Unconjugated Sepharose CL-2B, Mouse IgG-agarose, 
L243-Sepharose-CL-2B and finally GL183-Sepharose-CL-2B. The first two 
columns were strictly preclearing columns. The third, containing beads 
conjugated to L243, a mAb specific for HLA-DR, served as both a 
preclearing column and a positive control, since the NK3.3 cells also 
express HLA-DR. To monitor the purification, small amounts of 
radio-iodinated NK3.3 was used as a tracer. 
A lysate prepared from 2.times.10.sup.9 NK3.3 cells and spiked with a 
second lysate prepared from 6.times.10.sup.7 surface-iodinated NK3.3 was 
passed over the 4 columns described above connected to each other in 
series. The columns were disconnected and the L243 and the GL183 columns 
were washed with wash buffer in parallel. The bound proteins were eluted 
with the elution buffer at pH 11.2 and 50 drops/fraction (about 500 
.mu.l/fraction) were collected. Each fraction was immediately neutralized 
by addition of dilute acetic acid. 2 .mu.l of each fraction was counted in 
a gamma counter to identify fractions containing peak radioactivity. The 
material in those fractions containing the most radioactivity was further 
analyzed. 
3. SDS-PAGE and Other Purification 
60 .mu.l of each fraction was run in a SDS-PAGE gel which was stained with 
Coomassie Blue to visualize total protein and then exposed to 
autoradiography film to detect radiolabeled proteins. A single 
radiolabeled protein migrating at 58 kD was eluted from the GL183 column 
and not from the L243 column. Although the autoradiography indicated that 
the GL183 had been purified to apparent homogeneity, Coomassie-staining 
revealed the presence of several additional non-radioactive proteins, 
mostly of lower molecular weight. These non-radioactive contaminants made 
it necessary to further purify the 183 Ag before subjecting it to 
sequencing. 
Several fractions of affinity-column eluate were pooled, EtOH-precipitated 
and resuspended in sample buffer. The material was run in a preparative 
SDS-PAGE gel. The proteins were electrophoretically transferred to a 
ProBlot PVDF membrane which was stained with Coomassie blue. The band at 
about 58 kD was cut out and subjected to automated Edman degradation. This 
yielded an N-terminal sequence of 19 amino acids. 
Computer searches of the translated Genbank and Swissprot databases showed 
that the sequence was unique. However, there was no evidence that the 
sequence was derived from the 183 Ag, rather than from a contaminant 
protein with the same electrophoretic mobility. 
Other methods were evaluated for further purification. Reverse-phase HPLC 
was found to resolve the 183 Ag too well, in that it separated the 
material into several fractions. This fractionation procedure revealed 
that a contaminant with the same electrophoretic mobility was present in 
the immunoaffinity-purified 183 Ag. This molecule was not detected in the 
cell surface-iodinated sample and probably represents an intracellular 
protein. 
Anion-exchange HPLC was also attempted but did not separate the 183 Ag from 
the contaminants. The 183 Ag also became refractive to N-terminal amino 
acid sequencing during these procedures. 
Accordingly, different wash buffers were evaluated during the 
affinity-purification step to see if conditions could be found where these 
contaminants would be removed during the column washes without removing 
the 183 Ag. The following wash procedure was adopted: After washing 
overnight with 0,1% Triton X-100, 10 mM Tris-HCL, pH 7.5, 150 mM NaCl, 
0,02% NaN.sub.3 the columns were washed with 0.1% C12E9, 10 mM Tris-HCL pH 
9.0, 500 mM NaCl followed by a final wash with 0.1% C.sub.12 E.sub.9, 10 
mM Tris-HCL, pH 7.5, 150 mM NaCl. The wash at pH 9.0 was found to remove 
significant amounts of contaminants with only small decreases in yield of 
183 Ag. 
Affinity-purified material from about 4.times.10.sup.9 cells was subjected 
to SDS-gel separation and transfer to Problot as described above. After 
cutting out the region of the membrane containing 183 Ag, it was subjected 
to digestion with trypsin. The digest was separated by reverse-phase HPLC 
using a vydac C-18 column. Aliquots of material from the prominent peaks 
were analyzed by Matrix-Assisted Laser Desorption Mass Spectrometry and 
fractions containing one main peak were subjected to Edman degradation. 
The amino acid sequences of eight tryptic peptides were obtained. Computer 
searches revealed that these sequences were previously unknown. 
B. Cloning cDNA of the p58 Receptor Protein 
1. PCR Amplification 
Based on the amino acid sequences of the N-terminus and internal tryptic 
peptides of 183, various oligonucleotide primers for PCR amplification 
were designed. The use of degenerate primers produced too much 
non-specific amplifications to be practical. Primers were therefore made 
with inosine substitutions at positions of maximum degeneracy (183N-FOR32; 
183P20-BACK32). However, these inosine-containing products could not be 
cloned into bacterial plasmids for further analysis. Primers without 
inosines were therefore used in the re-amplification of the initial PCR 
product (183P20-BACK32B; 183N-FOR32-B). The best primers used are as 
follows: 
183N-FOR32: 5' GGI CCI (C/T)TI GTN AA(A/G) TCN GAA GAG AC 3' (SEQ ID NO:3) 
183P20-BACK32: 5' IC(G/T) (A/G)CT IA(A/G) (A/G)TG (A/G)TA CAT (A/G)TC ATA 
3' (SEQ ID NO:4) 
183P20-BACK32B: 5' NC(G/T) (A/G)CT NA(A/G) (A/G)TG (A/G)TA CAT (A/G)TC ATA 
3' (SEQ ID NO:5) 
183N-FOR32-B: 5' GGN CCN (C/T)TN GTN AA(A/G) TCN GAA GAG AC 3' (SEQ ID 
NO:6) 
2. Construction and Screening of NK3.3 cDNA Library 
Total RNA was isolated from NK3.3 by a scaled-up version of the method of 
Chomczynksi and Sacchi (Analytical Biochemistry 162:156-159 (1987)). 
Poly(A)+ selection was carried out by two passages of the total RNA over 
an Oligo(dT) column. 
The cDNA library was constructed using a kit from Gibco BRL and was size 
selected as follows. The cDNA library was amplified, and purified 
double-stranded plasmid was linearized by digestion with NotI and 
separated by agarose gel electrophoresis. The gel was cut in 21 sections, 
each containing DNA of a different size, i.e. with inserts of different 
length. The DNA was purified from the agarose and an aliquot of each 
fraction was run on an agarose gel. It was transferred to Genescreen, and 
hybridization was done using the .sup.32 P-labelled 183 PCR amplification 
product as probe. This identified the fraction containing inserts 
corresponding to the PCR amplification product. 
This fraction was re-circularized with DNA ligase and transformed into E. 
coli. The bacteria were plated on LB plates and after lifting onto 
Genescreen discs they were screened with the probed described above. 
Several positive clones were isolated, and 6 were further characterized by 
sequencing. The 6 clones turned out to be identical in their sequence. 
C. Expression of cDNA 
The insert from one of the clones was subcloned into the eukaryotic 
expression vector RSV.5(gpt). This construct was transfected into the 
human T cell line Jurkat and into a human B-LCL by electroporation. Stable 
transfectants were selected by culture in medium containing Mycophenolic 
acid and Xanthine and were screened for surface expression of 183 Ag by 
FACS analysis using the GL183 as primary antibody followed by 
FITC-conjugated rabbit anti-mouse IgG (see FIGS. 1B-1C). The results show 
that the p58 cDNA directs the surface expression of the 183 Ag in 
transfected cells. 
All publications mentioned hereinabove are hereby incorporated by reference 
in their entirety. 
While the foregoing invention has been described in some detail for 
purposes of clarity and understanding, it will be appreciated by one 
skilled in the art from a reading of the disclosure that various changes 
in form and detail can be made without departing from the true scope of 
the invention in the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1594 BASE PAIRS 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: UNKNOWN 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: cDNA to mRNA 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: HUMAN 
(C) INDIVIDUAL ISOLATE: NK 3.3 CELL 
(xi) SEQUENCE DESCRIPTION:SEQ ID NO:1: 
CCTGTCTGCACAGACAGCACCATGTCGCTCATGGTCGTCAGCATG45 
MetSerLeuMetValValSerMet 
15 
GTGTGTGTTGGGTTCTTCTTGCTGCAGGGGGCCTGGCCACAT87 
ValCysValGlyPhePheLeuLeuGlnGlyAlaTrpProHis 
101520 
GAGGGAGTCCACAGAAAACCTTCCCTCCTGGCCCACCCAGGT129 
GluGlyValHisArgLysProSerLeuLeuAlaHisProGly 
253035 
CCCCTGGTGAAATCAGAAGAGACAGTCATCCTGCAATGTTGG171 
ProLeuValLysSerGluGluThrValIleLeuGlnCysTrp 
404550 
TCAGATGTCAGGTTTCAGCACTTCCTTCTGCACAGAGAAGGG213 
SerAspValArgPheGlnHisPheLeuLeuHisArgGluGly 
5560 
AAGTTTAAGGACACTTTGCACCTCATTGGAGAGCACCATGAT255 
LysPheLysAspThrLeuHisLeuIleGlyGluHisHisAsp 
657075 
GGGGTCTCCAAGGCCAACTTCTCCATCGGTCCCATGATGCAA297 
GlyValSerLysAlaAsnPheSerIleGlyProMetMetGln 
808590 
GACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCAC339 
AspLeuAlaGlyThrTyrArgCysTyrGlySerValThrHis 
95100105 
TCCCCCTATCAGTTGTCAGCTCCCAGTGACCCTCTGGACATC381 
SerProTyrGlnLeuSerAlaProSerAspProLeuAspIle 
110115120 
GTCATCACAGGTCTATATGAGAAACCTTCTCTCTCACGCCAG423 
ValIleThrGlyLeuTyrGluLysProSerLeuSerArgGln 
125130 
CCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCC465 
ProGlyProThrValLeuAlaGlyGluSerValThrLeuSer 
135140145 
TGCAGCTCCCGGAGCTCCTATGACATGTACCATCTATCCAGG507 
CysSerSerArgSerSerTyrAspMetTyrHisLeuSerArg 
150155160 
GAGGGGGAGGCCCATGAACGTAGGTTCTCTGCAGGGCCCAAG549 
GluGlyGluAlaHisGluArgArgPheSerAlaGlyProLys 
165170175 
GTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCC591 
ValAsnGlyThrPheGlnAlaAspPheProLeuGlyProAla 
180185190 
ACCCACGGAGGAACCTACAGATGCTTCGGCTCTTTCCGTGAC633 
ThrHisGlyGlyThrTyrArgCysPheGlySerPheArgAsp 
195200 
TCTCCATACGAGTGGTCAAACTCGAGTGACCCACTGCTTGTT675 
SerProTyrGluTrpSerAsnSerSerAspProLeuLeuVal 
205210215 
TCTGTCACAGGAAACCCTTCAAATAGTTGGCCTTCACCCACT717 
SerValThrGlyAsnProSerAsnSerTrpProSerProThr 
220225230 
GAACCAAGCTCCGAAACCGGTAACCCCAGACACCTGCATGTT759 
GluProSerSerGluThrGlyAsnProArgHisLeuHisVal 
235240245 
CTGATTGGGACCTCAGTGGTCATCATCCTCTTCATCCTCCTC801 
LeuIleGlyThrSerValValIleIleLeuPheIleLeuLeu 
250255260 
CTCTTCTTTCTCCTTCATCGCTGGTGCTGCAACAAAAAAAAT843 
LeuPhePheLeuLeuHisArgTrpCysCysAsnLysLysAsn 
265270 
GCTGTTGTAATGGACCAAGAGCCTGCAGGGAACAGAACAGTG885 
AlaValValMetAspGlnGluProAlaGlyAsnArgThrVal 
275280285 
AACAGGGAGGACTCTGATGAACAAGACCCTCAGGAGGTGACA927 
AsnArgGluAspSerAspGluGlnAspProGlnGluValThr 
290295300 
TATGCACAGTTGAATCACTGCGTTTTCACACAGAGAAAAATC969 
TyrAlaGlnLeuAsnHisCysValPheThrGlnArgLysIle 
305310315 
ACTCGCCCTTCTCAGAGGCCCAAGACACCCCCAACAGATATC1011 
ThrArgProSerGlnArgProLysThrProProThrAspIle 
320325330 
ATCGTGTACACGGAACTTCCAAATGCTGAGCCCTGATCC1050 
IleValTyrThrGluLeuProAsnAlaGluPro 
335340 
AAAGTTGTCTCCTGCCCATGAGCACCACAGTCAGGCCTTGAGGGGATCTT1100 
CTAGGGAGACAACAGCCCTGTCTCAAAACTGGGTTGCCAGCTCCAATGTA1150 
CCAGCAGCTGGAATCTGAAGGCGTGAGTCTGCATCTTAGGGCATCGCTCT1200 
TCCTCACACCACAAATCTGAACGTGCCTCTCCCTTGCTTACAAATGTCTA1250 
AGGTCCCCACTGCCTGCTGGAGAGAAAACACACTCCTTTGCTTAGCCCAC1300 
AATTCTCCATTTCACTTGACCCCTGCCCACCTCTCCAACCTAACTGGCTT1350 
ACTTCCTAGTCTACTTGAGGCTGCAATCACACTGAGGAACTCACAATTCC1400 
AAACATACAAGAGGCTCCCTCTTAACACGGCACTTAGACACGTGCTGTTC1450 
CACCTTCCCTCATGCTGTTCCACCTCCCCTCAGACTAGCTTTCAGCCTTC1500 
TGTCAGCAGTAAAACTTATATATTTTTTAAAATAATTTCAATGTAGTTTT1550 
CCCTCCTTCAAATAAACATGTCTGCCCTCAAAAAAAAAAAAAAA1594 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 341 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(C) STRANDEDNESS: UNKNOWN 
(D) TOPOLOGY: UNKNOWN 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: PROTEIN 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: HUMAN 
(C) INDIVIDUAL ISOLATE: NK 3.3 CELL 
(xi) SEQUENCE DESCRIPTION:SEQ ID NO:2: 
MetSerLeuMetValValSerMetValCysValGlyPhePhe 
1510 
LeuLeuGlnGlyAlaTrpProHisGluGlyValHisArgLys 
152025 
ProSerLeuLeuAlaHisProGlyProLeuValLysSerGlu 
303540 
GluThrValIleLeuGlnCysTrpSerAspValArgPheGln 
455055 
HisPheLeuLeuHisArgGluGlyLysPheLysAspThrLeu 
606570 
HisLeuIleGlyGluHisHisAspGlyValSerLysAlaAsn 
7580 
PheSerIleGlyProMetMetGlnAspLeuAlaGlyThrTyr 
859095 
ArgCysTyrGlySerValThrHisSerProTyrGlnLeuSer 
100105110 
AlaProSerAspProLeuAspIleValIleThrGlyLeuTyr 
115120125 
GluLysProSerLeuSerArgGlnProGlyProThrValLeu 
130135140 
AlaGlyGluSerValThrLeuSerCysSerSerArgSerSer 
145150 
TyrAspMetTyrHisLeuSerArgGluGlyGluAlaHisGlu 
155160165 
ArgArgPheSerAlaGlyProLysValAsnGlyThrPheGln 
170175180 
AlaAspPheProLeuGlyProAlaThrHisGlyGlyThrTyr 
185190195 
ArgCysPheGlySerPheArgAspSerProTyrGluTrpSer 
200205210 
AsnSerSerAspProLeuLeuValSerValThrGlyAsnPro 
215220 
SerAsnSerTrpProSerProThrGluProSerSerGluThr 
225230235 
GlyAsnProArgHisLeuHisValLeuIleGlyThrSerVal 
240245250 
ValIleIleLeuPheIleLeuLeuLeuPhePheLeuLeuHis 
255260265 
ArgTrpCysCysAsnLysLysAsnAlaValValMetAspGln 
270275280 
GluProAlaGlyAsnArgThrValAsnArgGluAspSerAsp 
285290 
GluGlnAspProGlnGluValThrTyrAlaGlnLeuAsnHis 
295300305 
CysValPheThrGlnArgLysIleThrArgProSerGlnArg 
310315320 
ProLysThrProProThrAspIleIleValTyrThrGluLeu 
325330335 
ProAsnAlaGluPro 
340 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 BASE PAIRS 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: UNKNOWN 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: OLIGONUCLEOTIDE 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: HUMAN 
(C) INDIVIDUAL ISOLATE: NK 3.3 CELL 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(C) IDENTIFICATION METHOD: 
(D) OTHER INFORMATION: The N at nucleotides 3, 
6, and 9 is inosine (I); the N at nucleotide 
7 is C or T; the N at nucleotides 12 and 18 
is C, T, A or G; and the N at nucleotide 15 
is A or G. 
(xi) SEQUENCE DESCRIPTION:SEQ ID NO:3: 
GGNCCNNTNGTNAANTCNGAAGAGAC26 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 BASE PAIRS 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: UNKNOWN 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: OLIGONUCLEOTIDE 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: HUMAN 
(C) INDIVIDUAL ISOLATE: NK 3.3 CELL 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(C) IDENTIFICATION METHOD: 
(D) OTHER INFORMATION: The N at nucleotides 
1 and 7 is inosine (I); the N at 
nucleotide 3 is G or T; and the N at 
nucleotides 4, 9, 10, 13, and 19 is A or 
G. 
(xi) SEQUENCE DESCRIPTION:SEQ ID NO:4: 
NCNNCTNANNTGNTACATNTCATA24 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 BASE PAIRS 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: UNKNOWN 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: OLIGONUCLEOTIDE 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: HUMAN 
(C) INDIVIDUAL ISOLATE: NK 3.3 CELL 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(C) IDENTIFICATION METHOD: 
(D) OTHER INFORMATION: The N at nucleotides 
1 and 7 is C, T, A, or G; the N at 
nucleotide 3 is G or T; and the N at 
nucleotides 4, 9, 10, 13, and 19 is A or 
G. 
(xi) SEQUENCE DESCRIPTION:SEQ ID NO:5: 
NCNNCTNANNTGNTACATNTCATA24 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 BASE PAIRS 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: UNKNOWN 
(ii) MOLECULE TYPE: 
(A) DESCRIPTION: OLIGONUCLEOTIDE 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: HUMAN 
(C) INDIVIDUAL ISOLATE: NK 3.3 CELL 
(ix) FEATURE: 
(A) NAME/KEY: 
(B) LOCATION: 
(C) IDENTIFICATION METHOD: 
(D) OTHER INFORMATION: The N at nucleotides 
3, 6, 9, 11, and 18 is C, T, A, or G; 
the N at nucleotide 7 is C or T; and the 
N at nucleotide 15 is A or G. 
(xi) SEQUENCE DESCRIPTION:SEQ ID NO:6: 
GGNCCNNTNGTNAANTCNGAAGAGAC26 
__________________________________________________________________________