Modified receptors that continuously signal

Engineered cell surface receptors are described that are constitutively active in the absence of the cytokine, hormone or molecule that normally activates the receptor. Receptors that constitutively signal are generally created by engineering the receptor to form multimers at the cell surface. Disclosed are various DNA, protein and cellular compositions and methods of making and using such constitutively active receptors. Particular examples are fusion proteins in which the stem, transmembrane domain and cytoplasmic domain are derived from a TNF receptor, and the extracellular, multimerizing domain is derived from an erythropoietin receptor. Transfection of such fusion protein constructs into cells is shown to result in a strong cytotoxic effect.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to the fields of cell surface 
receptors and their functions. More particularly, it concerns the 
generation of engineered receptor molecules that are constitutively active 
in the absence of the cytokine, hormone or molecule that normally 
activates the receptor. Disclosed are various DNA, protein and cellular 
compositions and methods of making and using constitutively active 
receptors, particularly, receptors engineered to form multimers. 
2. Description of the Related Art 
Cytokines are a group of peptide hormones that interact with cell surface 
receptors to signal specific biological effects. The tyrosine kinase 
receptors have a consensus tyrosine kinase sequence in the cytoplasmic 
domain that is involved in the signal transduction mechanism. Other types 
of receptors signal by mechanisms that are less well understood, although 
there is evidence for tyrosine phosphorylation in some receptors. 
The erythropoietin receptor (EPOR) recognizes a glycoprotein hormone 
ligand. This hormone (erythropoietin) is required for the survival, 
proliferation and differentiation of committed erythroid progenitors. 
Other members of the cytokine family to which this receptor belongs 
include the receptors for hematopoietic growth factors such as 
interleukins, colony-stimulating factors and growth hormones. Unlike the 
tyrosine kinase receptors, the mode of signalling activity of these 
cytokine receptors is not known. However, it has been suggested that 
dimerization or oligomerization of these receptors plays an important role 
in the signal mechanism (Oyashi et al., 1994). 
Previous studies have also shown that a mutation in the EPOR which converts 
an arginine to cysteine at position 129 confers constitutive expression 
and induces stable receptor homooligomers (Oyashi et al., 1994; Longmore 
and Lodish, 1991). Chimeric receptors have also been expressed by the same 
group in which the cytoplasmic and extracellular domains of the EPOR and 
the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, 
have been fused to form chimeric receptors that respond to the 
extracellular domain ligand by the response mechanism of the cytoplasmic 
domain. 
Tumor necrosis factor (TNF) is a cytokine mediator which initially showed 
much promise as an antineoplastic agent, since the protein specifically 
destroys transformed cells in vitro, and causes the hemorrhagic necrosis 
of transplantable tumors in vivo without killing normal cells. 
Subsequently, however, it was demonstrated that TNF has many toxic effects 
in living animals. Specifically, it appears to be a central mediator of 
endotoxic shock. Unfortunately, this has limited the therapeutic 
application of TNF. No human tumor has ever been successfully treated with 
the protein. 
All of the biological effects of TNF are mediated by two types of cell 
membrane receptor. The larger of these, a 75 kD glycoprotein, transduces 
the proliferative effect of TNF, although some cytotoxic activity may be 
generated through this molecule as well. The smaller receptor, a 55 kD 
cell surface glycoprotein, bears some homology to the larger TNF receptor 
in the region of the extracellular domain, but has an entirely different 
cytoplasmic domain. It therefore generates a different signal when 
activated by ligand binding. It is the 55 kD cell surface receptor that 
appears to be chiefly responsible for the induction of TNF-mediated 
cytotoxicity. 
TNF is a trimeric molecule. The TNF receptors exist as a mixed population 
of monomers on the surface of virtually all somatic cells. TNF initiates 
signals through both of the two types of receptor by cross-linking three 
identical monomeric subunits (either three 75 kD subunits or three 55 kD 
subunits) on the cell surface. The juxtaposition of the monomers leads to 
generation of a signal through a process that is not completely 
understood. It is clear, however, that the cross-linking of monomers, and 
not the engagement of TNF per se, is the important event in signal 
transduction. Therefore, anti-receptor antibodies can substitute for TNF, 
generating an agonist signal. There still exists a need however, for a 
mechanism of inducing the cytotoxic TNF response without inducing the side 
effects that are caused by administration of TNF. 
SUMMARY OF THE INVENTION 
The present invention, in a general and overall sense, concerns engineered 
receptor molecules capable of constitutively signalling, i.e., receptors 
that exert or induce a biological function or reaction even in the absence 
of the ligand that normally induces the receptor to signal a positive 
response. The invention arises from the surprising discovery that the 
formation of multimeric receptor complexes on the extracellular surface is 
sufficient for transmitting a positive signal to the cytoplasmic domain of 
the receptors even in the absence of the cognate ligand or in the presence 
of a ligand that has entirely different activities. For clinical use, a 
ligand with minimal extraneous effects would be selected. 
The receptors used in the practice of the invention are preferably cytokine 
receptors and have three basic parts, the extracellular domain, the 
cytoplasmic domain and the transmembrane domain. The extracellular domain 
functions to recognize and bind the ligand. Binding of the ligand induces 
the receptors to form dimers, trimers or higher order multimers. The 
presence of the multimeric receptors then transmits a signal through the 
transmembrane domain to the cytoplasmic domain which mediates a biological 
response. 
The present inventor(s) has(ve) discovered that a chimeric receptor which 
comprises an extracellular domain that is capable of forming multimeric 
complexes may be joined to a cytoplasmic domain from a different receptor, 
and that the signal is transmitted and results in a biological response. 
In the examples disclosed herein, the transmembrane domain is derived from 
the same receptor as the cytoplasmic domain, however, it is contemplated 
that any of several receptor transmembrane domains would function in the 
chimeric receptors and would be included within the scope of the present 
invention. For example, the transmembrane domain may be derived from the 
same receptor as the extracellular domain, or it may even be derived from 
a different receptor than either the extracellular or cytoplasmic domain. 
Alternatively, the transmembrane domain may be encoded by a chemically 
synthesized DNA fragment based on consensus transmembrane domain 
sequences. 
In a preferred embodiment, the present invention is a polypeptide 
comprising a cytokine receptor cytoplasmic domain functionally connected 
to an extracellular domain. The extracellular domain is capable of forming 
multimers and stimulating constitutive signal activity in the cytoplasmic 
domain. One aspect of the invention is an extracellular domain that forms 
multimers in the absence of any signal such as the binding of a ligand. 
Examples of this type of domain are the tumor necrosis factor receptor 
extracellular domain directly linked to the tumor necrosis factor ligand, 
or the mouse erythropoietin extracellular receptor which contains an 
arginine to cysteine mutation at position 129. It is understood that any 
such extracellular domain covalently complexed to the cognate ligand, or 
an extracellular receptor domain/antibody complex, or any extracellular 
domain that contains a mutation that confers the spontaneous formation of 
multimers would be encompassed by the present claimed invention. Other 
examples include, but are not limited to receptors for growth hormones and 
the interferons. Moreover, the extracellular domain responsible for 
multimer formation need not be derived from a receptor. Portions of the 
immunoglobulin heavy chain, influenza hemagglutinin and other proteins 
that multimerize, either in the extracellular compartment or within the 
cytosol are contemplated to be useful in the design of molecules that 
exhibit constitutive signalling activity. 
The receptor cytoplasmic domain of the present invention determines the 
biological response to the signal. Any type of receptor domain that 
responds to the formation of multimers is an acceptable embodiment of the 
present invention. A preferred domain is the cytoplasmic domain of the 
tumor necrosis factor receptor, and more particularly, the cytoplasmic 
domain from the 55 kDa or the 75 kDa tumor necrosis factor receptors. 
Other receptors that confer different biological activities are also 
contemplated to be encompassed by the present claimed invention. Examples 
of such receptors include, but are not limited to the Fas antigen, growth 
hormone receptor, insulin receptor and erythropoietin receptor cytoplasmic 
domains. 
In certain embodiments, the present invention is an expression vector 
construct comprising a DNA sequence encoding the amino acid sequence in 
accordance with SEQ ID NO:7. By an expression vector is meant a segment of 
DNA that comprises one or more structural genes, functionally connected to 
the necessary promoter/enhancer regions, ribosome binding sites and 
polyadenylation sites necessary for the production of polypeptide products 
in a cell, and preferably in a eukaryotic cell. The expression vector 
further comprises an origin of replication and other DNA segments 
necessary for its own replication in a cell. 
The expression vector construct may further include a DNA segment encoding 
a polypeptide comprising a cytokine receptor cytoplasmic domain 
functionally connected to an extracellular domain as the structural gene. 
In this embodiment, the vector directs the expression of a chimeric 
receptor construct that is capable of forming multimers and conferring 
constitutive signal activity. 
In a further embodiment, the invention is a method of inducing a cytotoxic 
effect in a cell. The method comprises obtaining an expression vector 
construct comprising a cytokine receptor with constitutive signal activity 
and expressing the cytokine receptor in a cell. The signal confers a 
cytotoxic reaction. A preferred signal activity is the tumor necrosis 
factor receptor activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
It was an objective of the disclosed studies to identify multimerizing 
groups that might be used to create constitutive signalling activity. 
Multimeric and constitutively active forms of 55 kDa and 75 kDa forms of 
TNF as well as TNF itself and the extracellular domain of influenza 
hemagglutinin were tested. Both TNF and the influenza hemagglutinin are 
homotrimeric proteins in their native state. Additionally, the 
erythropoietin receptor extracellular domain was employed as a 
multimerizing group, with the expectation that this protein should 
dimerize in the presence of erythropoietin, in view of a previously 
described mutant dimeric form of the receptor. 
A soluble form of TNF was observed, rather than the multimerizing forms 
expected. Surprisingly, malignant transformation of NIH 3T3 cells 
transfected with an expression construct coding for a secreted, soluble 
variant of TNF was observed. 
The use of influenza hemagglutinin as a multimerizing group did not yield 
detectable cell surface expression of protein when the cytoplasmic domain 
of the TNF receptors was attached. This was surprising since truncated 
variants of the influenza hemagglutinin molecule, lacking any cytoplasmic 
domain were expressed at the cell surface. 
High levels of chimeric TNF/EpoR protein were expressed at the cell surface 
and constitutive signal transduction was detected through the TNF 
cytoplasmic domain. Erythropoietin receptor extracellular domain was 
successfully used not only as a multimerizing group, but also the chimeric 
fusion proteins were active even in the absence of added ligand. 
The following examples are included to demonstrate preferred embodiments of 
the invention. It should be appreciated by those of skill in the art that 
the techniques disclosed in the examples which follow represent techniques 
discovered by the inventor to function well in the practice of the 
invention, and thus can be considered to constitute preferred modes for 
its practice. However, those of skill in the art should, in light of the 
present disclosure, appreciate that many changes can be made in the 
specific embodiments which are disclosed and still obtain a like or 
similar result without departing from the spirit and scope of the 
invention. 
EXAMPLE I 
TNF-RECEPTOR:TNF LIGAND GENE CONSTRUCTION 
This example describes the generation of a genetic construct that directs 
the expression of a protein in which the TNF ligand is coupled to the 
extracellular domain of the TNF receptor, but which does not contain a 
cytoplasmic domain. 
From genomic DNA, the entire TNF coding sequence and introns (excluding the 
TNF 5'-UTR and 3'-UTR sequences) was amplified by polymerase 
chain-reaction (PCR). The stop-codon of the TNF gene was omitted, and the 
terminal codon (for leucine) was spliced to a sequence encoding the 
extracellular domain of the TNF 55 kD receptor beginning just above the 
plasma membrane in the protein sequence, and continuing through the 
C-terminus. An aspartic acid residue was interposed to assure the 
formation of a salt bridge with Lysine-11 of each subunit, permitting 
stable trimer formation. A cDNA clone was used for this part of the 
amplification. 
The construct produced by recombinant PCR coded for a molecule that is 
exteriorized under the influence of the TNF secretory signal peptide. The 
molecule formed a homotrimer, based on the association of TNF monomers. 
The extracellular "stem" acts as a spacer, to give enough flexibility to 
avoid tension on the TNF trimer. 
The same type of construction was applied to produce a constitutive form of 
the 75 kD TNF receptor. As before, the TNF gene, and the receptor cDNA, 
were used to produce the recombinant molecule, in which an aspartic acid 
residue follows the C-terminal residue of the TNF monomer. These 
constructions were made using mouse sequences; however, the same 
methodology may be straightforwardly applied to the human genes and their 
products. The mouse provides a useful in vivo model. 
After synthesis of this construct, a frame-shift mutation was detected 
within the expression unit. This was expected to cause termination of the 
protein prior to synthesis of the cytoplasmic domain; however, as shown in 
Example II, biological activity was detected. 
EXAMPLE II 
CHARACTERIZATION OF TNF-RECEPTOR:TNF CONSTRUCT 
The gene described in Example I, expressing a chimeric protein without a 
cytoplasmic domain, was transfected into cells in vitro. The gene was 
transcribed, leading to the production of high levels of chimeric 
messenger RNA in the cytoplasm of transfected cells. 
The protein was not detected using immunostaining techniques, in which 
rabbit anti-mouse TNF antibody was applied to the transfected cells, 
followed by a washing step and application of a secondary antibody of 
caprine origin (goat anti-rabbit IgG) coupled to fluorescein. However, 
secreted TNF activity was detected in the culture medium of transfected 
cells employing the L-929 bioassay system. Briefly, culture medium was 
harvested and added at various dilutions to monolayers of L-929 cells 
grown at a density of 70,000 cells per well of a 96-well plate. 
Cycloheximide was added to the assay system at a concentration of 0.1 
mg/mL. The cells were allowed to incubate for 16 hours at 37.degree. C. in 
a humidified CO.sub.2 incubator, after which time residual cells adherent 
to the plastic were stained with crystal violet and quantified by 
densitometry. Moreover, there was evidence that the chimeric molecule 
produced had biological activity, despite the frame-shift mutation. The 
engineered molecule, though lacking a cytoplasmic domain, caused rapid 
destruction of transfected NIH 3T3 fibroblasts when cycloheximide was 
added to the culture (FIG. 1A, FIG. 1B, FIG. 1C, and FIG. 1D). As such, 
the molecule still mimicked the effect of TNF itself, providing evidence 
that the expressed mutant protein has certain functional activities. 
The presence of soluble TNF activity in the culture medium suggested that 
the truncated protein was a secreted product, rather than a 
membrane-anchored product. 
EXAMPLE III 
TNF-RECEPTOR:TNF CONSTRUCT ONCOGENIC ACTIVITY IN VIVO 
A surprising new activity of the chimeric molecule was discovered when 
cells expressing the construct were administered to mice. These studies 
showed that the modified partial receptor:ligand molecule was highly 
oncogenic. 
Even when very low levels of the chimeric messenger RNA of Example I were 
expressed in stably transfected NIH 3T3 cells, the cells formed large 
tumors in nude mice in a matter of days (FIG. 2). This phenomenon was 
observed in 8 out of 8 mice, inoculated with four separate clones 
expressing the recombinant proteins. None of the control animals 
(receiving cells transfected with empty vector) developed tumors. Thus, 
the signal generated by the recombinant protein is capable of transforming 
cells. 
However, owing to the fact that this engineered molecule may be a secreted 
product, rather than a membrane-anchored product, it was not established 
whether the oncogenic effect is transduced by the 75 kD receptor, the 55 
kD receptor, or both receptors acting in conjunction with one another. 
The inventor contemplates that it is possible that certain cancers are 
caused by this type of rearrangement, involving the TNF receptor or other 
cytokine receptors, in vivo. This example provides a model for studying 
the formation of tumors. 
EXAMPLE IV 
CHIMERIC TNF RECEPTOR:HEMAGGLUTININ CONSTRUCTS 
The inventor has investigated the use of a "multimerizing group" other than 
TNF to maintain the cytoplasmic domains in close proximity. 
The influenza hemagglutinin (a trimeric protein) was first employed as the 
extracellular domain, on the assumption that it would maintain three 
cytoplasmic domains of the TNF receptors in close proximity with one 
another. The relevant constructs were made and expressed within COS cells. 
The native influenza hemagglutinin construct, as well as a construct 
containing only a few amino acids on the cytoplasmic side, were well 
expressed at the cell surface, as assessed by the ability of transfected 
cells to form rosettes with erythropoietin. On the other hand, the fall 
length construct apparently was not expressed at high levels in a native 
form at the cell surface, apparently because of failure to fold properly. 
Furthermore, no cytotoxic effect was observed on transfection of the 
hemagglutinin chimeras. 
EXAMPLE V 
CHIMERIC TNF RECEPTOR:ERYTHROPOIETIN RECEPTOR CONSTRUCTS 
The next extracellular moiety investigated was the erythropoietin receptor. 
Fusion proteins were constructed using the erythropoietin receptor as the 
extracellular domain, together with the cytoplasmic domains of the two TNF 
receptors. 
In these studies, four types of recombinant molecule were produced. The 
extracellular domain was derived either from the wild-type mouse 
erythropoietin receptor (EpoR), or from a mutant receptor in which an 
arginine to cysteine substitution at residue 129 results in spontaneous 
dimerization (Li et al., 1990). The "stem," transmembrane domain, and 
cytoplasmic domain of each recombinant was derived either from the 55 kD 
or 75 kD TNF receptor. The approach was to amplify and clone each 
component separately, sequence the fragments to ensure that no mutation 
was introduced, and then splice the fragments together using a restriction 
sites built into the primers used for amplification. 
1. Amplifications 
For the EpoR signal peptide and extracellular domain, the 5' EpoR primer 
used had the sequence of SEQ ID NO:1 (Kpn-I site underlined). This primer 
hybridizes with the 5'-untranslated region of the EpoR cDNA. The 3' EpoR 
primer for the EpoR signal peptide and extracellular domain had the 
sequence of SEQ ID NO:2 (Hind-III site underlined). 
CGT GGT ACC TGA GCT TCC TGA AGC GGC (SEQ ID NO:1) 
GGA CCT AAG CTT CAGG GTC CAG CTC GCT AGC GGT (SEQ 10 NO:2) 
These primers were used for amplifying extracellular domains from both wild 
type and mutant EpoR clones (obtained from Dr. S. Watowich, Massachusetts 
Institute of Technology, Boston, Mass.). The mutant EpoR clone s had a 
mutation of arginine to cysteine at residue 129. The entire extracellular 
domain was amplified, encompassing the initiator methionine and continuing 
up to the last extracellular residue (no. 249). The amplification product 
encodes 24 amino acids of propeptide, plus 225 amino acids of the mature 
protein. The human erythropoietin receptor DNA sequence is found in 
Genbank, accession number J04843, SEQ ID NO:9 incorporated herein by 
reference. 
The TNF receptor sequences are available in Genbank, as accession number 
M59378 (SEQ ID NO:11) for the 75 k and accession number M59377 SEQ ID 
NO:10) for the 55 k DNA segments, herein incorporated by reference. For 
the 55 kD TNF receptor "stem," transmembrane domain, and cytoplasmic 
domain, the 5' primer used had the sequence of SEQ ID NO:3 (Hind-III site 
underlined); and the 3' primer had the sequence of SEQ ID NO:4 (Xba-I site 
underlined; stop codon in bold). 
GGA CCT AAG CTT CCT CCG CTT GCA AAT GTC ACA (SEQ ID NO:3) 
GCT CTA GAG CTT ATC GCG GGA GGC GGG TCG TGG A (SEQ ID NO:4) 
These primers were used for amplifying the 55 kD TNF receptor stem, 
transmembrane domain and cytoplasmic domain from nucleotide 687 to the 
stop codon at nucleotide 1460, of SEQ ID NO:10 incorporating 15 
extracellular domain residues in the stem, 23 transmembrane domain 
residues and 219 cytoplasmic domain residues, for a total of 257 residues. 
For the 75 kD TNF receptor "stem," transmembrane domain, and cytoplasmic 
domain, the 5' primer used had the sequence of SEQ ID NO:5 (Hind-III site 
underlined); and the 3' primer used had the sequence of SEQ ID NO:6 (Xba-I 
site underlined; stop codon in bold). 
GGA CCT AAG CTT CCA AGC ATC CTT ACA TCG TTG (SEQ ID NO:5) 
GCT CTA GAT CAG GCC ACT TTG ACT GCA AT (SEQ ID NO:6) 
These primers were used for amplifying the 75 kD TNF receptor stem, 
transmembrane domain and cytoplasmic domain from nucleotide 757 to stop 
codon at nucleotide 1467, of SEQ ID 11 incorporating 20 extracellular 
domain resides in the "stem", 29 transmembrane domain residues, and 187 
cytoplasmic domain residues for a total of 236 residues. 
2. Cloning 
All components were cloned into Bluescript-KS, and sequenced independently 
using the Sanger dideoxynucleotide method. The fragments were then cloned 
Kpn-I to Hind-III (for the EpoR coding regions) and Hind-III to Xba-I (for 
the TNFR coding regions) into the vector pCMV4 for expression. The 
full-length EpoR construct was also cloned into pCMVr for expression as a 
control in some studies. 
For constructs containing either the wild-type or mutant EpoR domain, the 
predicted junctional amino acid sequences are as follows: 
55 kD: . . . L-D-P-(F-E-L)-P-P- . . . (SEQ ID NO:7) 
75 kD: . . . L-D-P-(F-E)-P-S-I . . . (SEQ ID NO:8) 
The underlined amino acids are derived from the Epo receptor. The amino 
acids in parentheses are artificially introduced. The remaining amino 
acids represent the start of the TNF receptor moiety. 
EXAMPLE VI 
CHARACTERIZATION OF CHIMERIC TNF:ERYTHROPOIETIN RECEPTORS 
1. Detection of Expression of Chimeric Proteins on the Plasma Membrane by 
Measurement of Epo Binding 
Radiolabeled (.sup.1251) Epo was obtained from NEN. 2.times.10.sup.6 COS 
cells were plated in a 10 cm plate, transfected according to a CaPO.sub.4 
method (Chen & Okayama, 1987), using 20 .mu.g of total DNA purified by the 
Quiagen technique. Transfections were performed in duplicate plates, from 
which the cells were pooled after 12 hours. The cells were then split into 
triplicate wells of a six-well plate at a density of 1.times.10.sup.6 per 
well. After five hours allowed for adherence to the plastic, cells were 
incubated on ice in 1 ml of medium containing 2% serum and 2% antibiotic 
mixture (pen/strep/GIBCO), 25 mM HEPES buffer, and 0.525 pM iodinated Epo 
(415 nCi/ml). Incubation was allowed to continue for 2 hours with 
occasional gentle shaking by hand (on ice). 3 ml of ice-cold PBS 
containing 1 mM EDTA was then added to each well. The cells were harvested 
by trituration, pelleted, transferred to Eppendorf tube, and washed once 
more with PBS/EDTA solution (all procedures were carried out in the cold). 
The cell pellets were then counted for bound radioactivity. 
As illustrated in FIG. 3, transfection with the chimeric or wild-type Epo 
receptors led to greatly augmented Epo binding. The introduction of 
competing unlabeled Epo essentially abolished binding, demonstrating the 
specificity of the interaction. The fact that labeled Epo is capable of 
binding tightly and specifically to intact transfected cells indicates 
that the recombinant molecules are expressed on the plasma membrane of the 
cells. The fact that far more Epo is bound by recombinant forms of the 
receptor, compared with the native form of the Epo receptor, suggests that 
the recombinant molecules may exhibit higher affinity for the ligand, or a 
faster on-rate, than the native molecule. This is particularly likely in 
view of the fact that similar quantities of the native and recombinant 
receptors were expressed, as shown by immunoprecipitation studies. 
2. Immunoprecipitation Studies 
To further characterize the molecules that were expressed, 
immunoprecipitation analysis of .sup.35 S-labeled proteins was performed. 
2.times.10.sup.6 COS cells were transfected in two 10 cm plates as 
described above. Cells were pooled and replated as described above after 
12 hours. After adherence, cells were washed in methionine/cysteine-free 
medium and allowed to incubate in 3 ml of the same medium for one hour. 
Fresh methionine/cysteine-free medium (1 ml per well), supplemented with 
100 .mu.Ci of .sup.35 S translabel (ICN), was then added to each 
monolayer. Cells were allowed to incubate in the presence of the label for 
3 hours. They were then collected by trituration in PBS/EDTA solution. 
The cells were then pelleted, and resuspended in an Eppendorf tube in 250 
.mu.l of lysis buffer (0.15 M NaCl, 50 mM tris/HCl, pH 7.4, 1 mM EDTA, and 
1.0% triton X-100). Nuclei were removed by centrifugation, and the 
supernatants were transferred to a tube containing an equal volume of the 
same lysis buffer, to which 1.0% sodium deoxycholate and 0.2% SDS had been 
added. 
Rabbit antiserum raised against the NH.sub.2 terminus of the EpoR (5-10) 
was added at a dilution of 1:250. A non-immune serum was used as a 
control. Antibody was allowed to incubate overnight at 4.degree. C. with 
gentle agitation. Pansorbin was then added to each tube, and allowed to 
react for 2 hours under the same conditions. The pansorbin was washed five 
times with lysis buffer, with one final wash in PBS alone (all washes on 
ice). The pansorbin was then resuspended in reducing SDS sample buffer, 
boiled, and the supernatant subjected to electrophoresis in a 10%-15% 
polyacrylamide gradient gel under denaturing conditions. Results are shown 
in FIG. 4 using .sup.35 S-labeled proteins from transfected COS cells 
immunoprecipitated with anti-EpoR antisera. 
EXAMPLE VII 
ACTIVITY OF CHIMERIC TNF:ERYTHROPOIETIN RECEPTORS 
Convincing evidence of the biological activity of the chimeric TNF:EPOR 
proteins was also obtained from expression in mammalian cells. Cytotoxic 
activity was demonstrated in two ways. 
First, PAM 212 cells (transformed mouse keratinocytes) were cotransfected 
with two vectors: pCMV5 encoding the fusion protein; and pCH110 encoding 
.beta.-galactosidase at a ratio of 10:1 respectively. It was assumed that 
using this ratio all, or nearly all, cells transfected with the 
.beta.-galactosidase vector would also be cotransfected with the 
expression vector encoding the fusion protein. Control cells were 
cotransfected with an empty pCMV5 expression vector instead of vector 
containing the fusion protein construct. 
Cells thus cotransfected were stained for detection of .beta.-galactosidase 
after 24 hrs and after 72 hrs, according to the method of Kolls, et al. 
(1994). As shown in FIG. 5A, FIG. 5B and FIG. 5C, control cultures 
contained abundant quantities of large, blue-staining cells at 24 and 72 
hrs post transfection. Cultures cotransfected with either the 55 kDa or 75 
kDa TNF receptor EPOR fusion constructs showed fewer blue cells; about 1/3 
as many at 24 hrs and about 120 as many at 72 hrs. The blue-staining cells 
were very small, and appeared to be undergoing condensation, as typically 
occurs prior to TNF-induced apoptosis. 
Second, NIH 3T3 cells were cotransfected with two vectors, one encoding 
either of the two fusion proteins; one in which the Epo receptor 
extracellular domain was attached to the 55 kDa or 75 kDa TNF receptor 
cytoplasmic domain, and the other encoding neomycin phosphotransferase 
(pcDNAneo) which confers resistance to the antibiotic G418. Transfection 
was accomplished using a ratio of ten parts fusion construct to one part 
pcDNAneo resistance vector. Cells were then selected for growth in the 
presence of G418. Cotransfection with either of the two vectors encoding 
fusion proteins led to a greater-than-ten-fold decrease in the number of 
G418 resistant colonies that formed over a two-week period of time. 
Some of the surviving colonies, transfected with each of the two types of 
fusion construct, exhibited morphology in vitro which was suggestive of 
malignant transformation. 
In these studies, erythropoietin was added to the cultures continuously at 
a concentration of 0.5 nM; however, substantially similar results were 
seen in the absence of added erythropoietin. The inventor had originally 
considered that the fusion proteins created with an erythropoietin 
receptor extracellular domain would be biologically inactive under normal 
conditions in culture, but activated by erythropoietin. Alternatively, an 
unusual conformation of the fusion protein may lead to dimerization in the 
absence of added ligand. Regardless of the mechanism, the inventor has 
shown that the recombinant erythropoietin receptor is active. 
Surprisingly, a considerable amount of cell killing was observed following 
transfection with the chimeric constructs. To confirm the effect of the 
recombinant vector, cotransfection studies were established to determine 
whether fewer cells would express a reporter construct cotransfected with 
the chimeric expression construct than those cotransfected with an empty 
vector. 
Transfections were performed in 293 cells, using a ratio of one part pCH110 
(a .beta.-galactosidase-encoding vector) to nine parts of chimeric EpoR 
encoding plasmid, or empty vector as a control. After 48 hours, cells were 
stained for .beta.-galactosidase activity as described by Kolls et al., 
(1994). 
Using the wild-type EpoR extracellular domains, fewer than 10% of the 
number of blue staining cells, as compared with the number in control 
plates, were consistently observed when cotransfected with the empty 
vector. 
EXAMPLE VIII 
FURTHER USES OF CONSTITUTIVELY ACTIVE RECEPTORS 
When expressed as the products of transgenes driven by tissue specific 
enhancer/silencer combinations, the constitutively active receptors are 
contemplated for use in effectively mimicking the tissue-specific 
expression of TNF. At present, no system for the actual expression of TNF 
can achieve this, since TNF rapidly emigrates from the site of production. 
The present invention also provides for the selective expression of 
constitutively active modified receptor conjugates in specific tissues 
within an animal. To achieve this, the modified receptor DNA construct is 
placed under the control of a cell- or tissue-specific promoter or 
enhancer/silencer combination, and then administered to an animal. 
Virtually any means of gene delivery may be employed, such as, for 
example, an adenoviral or retroviral vector delivery system. Whether or 
not many cell types receive the construct, the use of a specific genetic 
control element will result in only specific cells or tissues expressing 
the constitutively active receptor. 
It is envisioned that the delivery of an engineered receptor construct, 
such as the one described in Example I, or Example V, to tumor cells in 
vivo may be used to elicit a strongly destructive effect on the tumor 
cells. Using cell or tissue-specific genetic control elements would mean 
that the lack of expression of the recombinant receptors in normal cell 
types would lead to low toxicity to the host. Therefore, in certain 
aspects the engineered receptors may be employed as effective 
chemotherapeutic agents for of cancer treatment. It is recognized that 
fusion proteins may by themselves have transforming potential. 
Additionally, the inventor has shown that TNF itself may have transforming 
activity. The selective cytotoxic action of TNF on tumor cells suggests 
that it might serve as an effective therapeutic agent if not for its 
simultaneous ability to activate neutrophils and endothelial cells, which 
may cause shock. It is possible that this dose-limiting toxicity might be 
overcome by directing the activity of TNF to the tumor cell population, to 
the exclusion of neutrophils, endothelial cells and other cells that 
actively promote an inflammatory response. 
As one example, melanoma cells are often sensitive to the cytolytic effect 
of TNF. Melanoma cells, along with melanocytes and certain neurons, are 
also unique in that they express sequences placed under the control of a 
tyrosinase promoter. As such, the inventor contemplates creating an 
expression vector comprising a constitutively active TNF receptor or 
TNFR:EpoR receptor under the influence of the tyrosinase promoter. The 
administration of such a construct into an animal through the use of a 
viral vector, e.g., adenoviral or other vector, would result in many cells 
taking up the construct. However, only melanoma cells would succumb to the 
lethal effect of gene expression, with the melanocytes and neurons would 
not be expected to react to TNF-mimetic signals because no non-transformed 
cells are known to be killed by the direct action of TNF, even at 
extremely high concentrations. One might employ the insulin promoter in 
the therapy of insulinomas or other tumors capable of utilizing this 
promoter. The insulin promoter is expressed in beta-islet cells and their 
transformed derivatives. 
All of the compositions and methods disclosed and claimed herein can be 
made and executed without undue experimentation in light of the present 
disclosure. While the compositions and methods of this invention have been 
described in terms of preferred embodiments, it will be apparent to those 
of skill in the art that variations may be applied to the composition, 
methods and in the steps or in the sequence of steps of the method 
described herein without departing from the concept, spirit and scope of 
the invention. More specifically, it will be apparent that certain agents 
which are both chemically and physiologically related may be substituted 
for the agents described herein while the same or similar results would be 
achieved. All such similar substitutes and modifications apparent to those 
skilled in the art are deemed to be within the spirit, scope and concept 
of the invention as defined by the appended claims. 
REFERENCES 
The following references, to the extent that they provide exemplary 
procedural or other details supplementary to those set forth herein, are 
specifically incorporated herein by reference. 
Chen, C. and H. Okayama. 1987. High-efficiency transformation of mammalian 
cells by plasmid DNA. Mol. Cell. Biol. 7:2745-2752. 
Kolls, J., K. Peppel, M. Silva, and B. Beutler. 1994. Prolonged and 
effective blockade of TNF activity through adenovirus-mediated gene 
transfer. Proc. Natl. Acad. Sci. 91:215-219. 
Li, J. P., A. D. D'Andrea, H. F. Lodish, and D. Baltimore. 1990. Activation 
of cell growth by binding of Friend spleen focus-forming virus gp55 
glycoprotein to the erythropoietin receptor. Nature 343:762-764. 
Longmore, G. D. and H. F. Lodish. 1991. An activating mutation in the 
murine erythropoietin receptor induces erythroleukemia in mice: a cytokine 
receptor superfamily oncogene. Cell 67:1089-1102. 
Oyashi, J., Maruyama, K., Liu, Y.-C. and A. Yoshimura. 1994. Ligand-induced 
activation of chimeric receptors between the erythropoietin receptor and 
receptor tyrosine kinases. Proc. Natl. Acad. Sci. USA 91:158-162. 
Schall, T. J., M. Lewis, K. J. Koller, A. Lee, G. C. Rice, G. H. W. Wong, 
T. Gatanaga, G. A. Granger, R. Lentz, H. Raab, W. J. Kohr, and D. V. 
Goeddel. 1990. Molecular cloning and expression of a receptor for human 
tumor necrosis factor. Cell 61:361-370. 
Smith, C. A., T. Davis, D. Anderson, L. Solam, M. P. Beckmann, R. Jerzy, S. 
K. Dower, D. Cosman, and R. G. Goodwin. 1990. A receptor for tumor 
necrosis factor defines an unusual family of cellular and viral proteins. 
Science 248:1019-1023. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 11 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 30 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
# 30 CCTG AAGCTAGGGC 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 33 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 33 TCCA GGTCGCTAGC GGT 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 33 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
# 33 CGCT TGCAAATGTC ACA 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 34 CGGG AGGCGGGTCG TGGA 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 33 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 33 GCAT CCTTACATCG TTG 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 29 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 29 CTTT GACTGCAAT 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 8 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- Leu Glu Pro Phe Glu Leu Pro Pro 
# 5 1 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 8 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- Leu Glu Pro Phe Glu Pro Pro Ile 
# 5 1 
- (2) INFORMATION FOR SEQ ID NO:9: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 774 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- TGAGCTTCCT GAAGCTAGGG CTGCATCATG GACAAACTCA GGGTGCCCCT CT - #GGCCTCGG 
60 
- GTAGGCCCCC TCTGTCTCCT ACTTGCTGGG GCAGCCTGGG CACCTTCACC CA - #GCCTCCCG 
120 
- GACCCCAAGT TTGAGAGCAA AGCGGCCCTG CTGGCATCCC GGGGCTCCGA AG - #AACTTCTG 
180 
- TGCTTCACCC AACGCTTGGA AGACTTGGTG TGTTTCTGGG AGGAAGCGGC GA - #GCTCCGGG 
240 
- ATGGACTTCA ACTACAGCTT CTCATACCAG CTCGAGGGTG AGTCACGAAA GT - #CATGTAGC 
300 
- CTGCACCAGG CTCCCACCGT CCGCGGCTCC GTGCGTTTCT GGTGTTCACT GC - #CAACAGCG 
360 
- GACACATCGA GTTTTGTGCC GCTGGAGCTG CAGGTGACGG AGGCGTCCGG TT - #CTCCTCGC 
420 
- TATCACCGCA TCATCCATAT CAATGAAGTA GTGCTCCTGG ACGCCCCCGC GG - #GGCTGCTG 
480 
- GCGCGCCGGG CAGAAGAGGG CAGCCACGTG GTGCTGCGCT GGCTGCCACC TC - #CTGGAGCA 
540 
- CCTATGACCA CCCACATCCG ATATGAAGTG GACGTGTCGG CAGGCAACCG GG - #CAGGAGGG 
600 
- ACACAAAGGG TGGAGGTCCT GGAAGGCCGC ACTGAGTGTG TTCTGAGCAA CC - #TGCGGGGC 
660 
- GGGACGCGCT ACACCTTCGC TGTTCGAGCG CGCATGGCCG AGCCGAGCTT CA - #GCGGATTC 
720 
- TGGAGTGCCT GGTCTGAGCC CGCGTCACTA CTGACCGCTA GCGACCTGGA CC - #CT 
774 
- (2) INFORMATION FOR SEQ ID NO:10: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1956 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- CCTGGTCCGA TCATCTTACT TCATTCACGA GCGTTGTCAA TTGCTGCCCT GT - #CCCCAGCC 
60 
- CCAATGGGGG AGTGAGAGGC CACTGCCGGC CGGACATGGG TCTCCCCACC GT - #GCCTGGCC 
120 
- TGCTGCTGTC ACTGGTGCTC CTGGCTCTGC TGATGGGGAT ACATCCATCA GG - #GGTCACTG 
180 
- GACTAGTCCC TTCTCTTGGT GACCGGGAGA AGAGGGATAG CTTGTGTCCC CA - #AGGAAAGT 
240 
- ATGTCCATTC TAAGAACAAT TCCATCTGCT GCACCAAGTG CCACAAAGGA AC - #CTACTTGG 
300 
- TGAGTGACTG TCCGAGCCCA GGGCGGGATA CAGTCTGCAG GGAGTGTGAA AA - #GGGCACCT 
360 
- TTACGGCTTC CCAGAATTAC CTCAGGCAGT GTCTCAGTTG CAAGACATGT CG - #GAAAGAAA 
420 
- TGTCCCAGGT GGAGATCTCT CCTTGCCAAG CTGACAAGGA CACGGTGTGT GG - #CTGTAAGG 
480 
- AGAACCAGTT CCAACGCTAC CTGAGTGAGA CACACTTCCA GTGCGTGGAC TG - #CAGCCCCT 
540 
- GCTTCAACGG CACCGTGACA ATCCCCTGTA AGGAGACTCA GAACACCGTG TG - #TAACTGCC 
600 
- ATGCAGGGTT CTTTCTGAGA GAAAGTGAGT GCGTCCCTTG CAGCCACTGC AA - #GAAAAATG 
660 
- AGGAGTGTAT GAAGTTGTGC CTACCTCCTC CGCTTGCAAA TGTCACAAAC CC - #CCAGGACT 
720 
- CAGGTACTGC GGTGCTGTTG CCCCTGGTTA TCTTGCTAGG TCTTTGCCTT CT - #ATCCTTTA 
780 
- TCTTCATCAG TTTAATGTGC CGATATCCCC GGTGGAGGCC CGAAGTCTAC TC - #CATCATTT 
840 
- GTAGGGATCC CGTGCCTGTC AAAGAGGAGA AGGCTGGAAA GCCCCTAACT CC - #AGCCCCCT 
900 
- CCCCAGCCTT CAGCCCCACC TCCGGCTTCA ACCCCACTCT GGGCTTCAGC AC - #CCCAGGCT 
960 
- TTAGTTCTCC TGTCTCCAGT ACCCCCATCA GCCCCATCTT CGGTCCTAGT AA - #CTGGCACT 
1020 
- TCATGCCACC TGTCAGTGAG GTAGTCCCAA CCCAGGGAGC TGACCCTCTG CT - #CTACGAAT 
1080 
- CACTCTGCTC CGTGCCAGCC CCCACCTCTG TTCAGAAATG GGAAGACTCC GC - #CCACCCGC 
1140 
- AACGTCCTGA CAATGCAGAC CTTGCGATTC TGTATGCTGT GGTGGATGGC GT - #GCCTCCAG 
1200 
- CGCGCTGGAA GGAGTTCATG CGTTTCATGG GGCTGAGCGA GCACGAGATC GA - #GAGGCTGG 
1260 
- AGATGCAGAA CGGGCGCTGC CTGCGCGAGG CTCAGTACAG CATGCTGGAA GC - #CTGGCGGC 
1320 
- GCCGCACGCC GCGCCACGAG GACACGCTGG AAGTAGTGGG CCTCGTGCTT TC - #CAAGATGA 
1380 
- ACCTGGCTGG GTGCCTGGAG AATATCCTCG AGGCTCTGAG AAATCCCGCC CC - #CTCGTCCA 
1440 
- CGACCCGCCT CCCGCGATAA AGCCACACCC ACAACCTTAG GAAGAGGGAC TT - #GAACTTCA 
1500 
- AGGACCATTC TGCTAGATGC CCTACTCCCT GTGGGTGAAA AGTGGGCAAA GG - #TCTCTAAG 
1560 
- GGGAAGGCTC GAGCTGGTAG CCACTTCCTT GGTGCTACCA ACTTGGTGTA CA - #TAGCTTTT 
1620 
- CTCAGCCGCC GAGGACTGCC TGAGCCAGCC ACTTGTGAGT GGCAGGGAGA TG - #TACCATCA 
1680 
- GCTCCTGGCC AGCTGAGGGT GCCAAAGACA GGATTGTAGA GGAAAGGCAC AA - #TGTATCTG 
1740 
- GTGCCCACTT GGGATGCACA GGGCCCAAGC CAAGCTTCTC AGGGCCTCCT CA - #GTGGGTTT 
1800 
- CTGGGCCTTT TTCACTTTTG ATAAGCAATC TTTGTATCAA TTATATCACA CT - #AATGGATG 
1860 
- AACTGTGTAA GGTAAGGACA AGCATAGAAA GGCGGGGTCT CCAGCTGGAG CC - #CTCGACTC 
1920 
# 1956 CGTC TAAAAATGAA AAAAAA 
- (2) INFORMATION FOR SEQ ID NO:11: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 3796 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- ACTAGAGCTC CAGGCACAAG GGCGGGAGCC ACCGCTGCCC CTATGGCGCC CG - #CCGCCCTC 
60 
- TGGGTCGCGC TGGTCTTCGA ACTGCAGCTG TGGGCCACCG GGCACACAGT GC - #CCGCCCAG 
120 
- GTTGTCTTGA CACCCTACAA ACCGGAACCT GGGTACGAGT GCCAGATCTC AC - #AGGAATAC 
180 
- TATGACAGGA AGGCTCAGAT GTGCTGTGCT AAGTGTCCTC CTGGCCAATA TG - #TGAAACAT 
240 
- TTCTGCAACA AGACCTCGGA CACCGTGTGT GCGGACTGTG AGGCAAGCAT GT - #ATACCCAG 
300 
- GTCTGGAACC AGTTTCGTAC ATGTTTGAGC TGCAGTTCTT CCTGTACCAC TG - #ACCAGGTG 
360 
- GAGATCCGCG CCTGCACTAA ACAGCAGAAC CGAGTGTGTG CTTGCGAAGC TG - #GCAGGTAC 
420 
- TGCGCCTTGA AAACCCATTC TGGCAGCTGT CGACAGTGCA TGAGGCTGAG CA - #AGTGCGGC 
480 
- CCTGGCTTCG GAGTGGCCAG TTCAAGAGCC CCAAATGGAA ATGTGCTATG CA - #AGGCCTGT 
540 
- GCCCCAGGGA CGTTCTCTGA CACCACATCA TCCACTGATG TGTGCAGGCC CC - #ACCGCATC 
600 
- TGTAGCATCC TGGCTATTCC CGGAAATGCA AGCACAGATG CAGTCTGTGC GC - #CCGAGTCC 
660 
- CCAACTCTAA GTGCCATCCC AAGGACACTC TACGTATCTC AGCCAGAGCC CA - #CAAGATCC 
720 
- CAACCCCTGG ATCAAGAGCC AGGGCCCAGC CAAACTCCAA GCATCCTTAC AT - #CGTTGGGT 
780 
- TCAACCCCCA TTATTGAACA AAGTACCAAG GGTGGCATCT CTCTTCCAAT TG - #GTCTGATT 
840 
- GTTGGAGTGA CATCACTGGG TCTGCTGATG TTAGGACTGG TGAACTGCAT CA - #TCCTGGTG 
900 
- CAGAGGAAAA AGAAGCCCTC CTGCCTACAA AGAGATGCCA AGGTGCCTCA TG - #TGCCTGAT 
960 
- GAGAAATCCC AGGATGCAGT AGGCCTTGAG CAGCAGCACC TGTTGACCAC AG - #CACCCAGT 
1020 
- TCCAGCAGCA GCTCCCTAGA GAGCTCAGCC AGCGCTGGGG ACCGAAGGGC GC - #CCCCTGGG 
1080 
- GGCCATCCCC AAGCAAGAGT CATGGCGGAG GCCCAAGGGT TTCAGGAGGC CC - #GTGCCAGC 
1140 
- TCCAGGATTT CAGATTCTTC CCACGGAAGC CACGGGACCC ACGTCAACGT CA - #CCTGCATC 
1200 
- GTGAACGTCT GTAGCAGCTC TGACCACAGT TCTCAGTGCT CTTCCCAAGC CA - #GCGCCACA 
1260 
- GTGGGAGACC CAGATGCCAA GCCCTCAGCG TCCCCAAAGG ATGAGCAGGT CC - #CCTTCTCT 
1320 
- CAGGAGGAGT GTCCGTCTCA GTCCCCGTGT GAGACTACAG AGACACTGCA GA - #GCCATGAG 
1380 
- AAGCCCTTGC CCCTTGGTGT GCCGGATATG GGCATGAAGC CCAGCCAAGC TG - #GCTGGTTT 
1440 
- GATCAGATTG CAGTCAAAGT GGCCTGACCC CTGACAGGGG TAACACCCTG CA - #AAGGGACC 
1500 
- CCCGAGACCC TGAACCCATG GAACTTCATG ACTTTTGCTG GATCCATTTC CC - #TTAGTGGC 
1560 
- TTCCAGAGCC CCAGTTGCAG GTCAAGTGAG GGCTGAGACA GCTAGAGTGG TC - #AAAAACTG 
1620 
- CCATGGTGTT TTATGGGGGC AGTCCCAGGA AGTTGTTGCT CTTCCATGAC CC - #CTCTGGAT 
1680 
- CTCCTGGGCT CTTGCCTGAT TCTTGCTTCT GAGAGGCCCC AGTATTTTTT CC - #TTCTAAGG 
1740 
- AGCTAACATC CTCTTCCATG AATAGCACAG CTCTTCAGCC TGAATGCTGA CA - #CTGCAGGG 
1800 
- CGGTTCCAGC AAGTAGGAGC AAGTGGTGGC CTGGTAGGGC ACAGAGGCCC TT - #CAGGTTAG 
1860 
- TGCTAAACTC TTAGGAAGTA CCCTCTCCAA GCCCACCGAA ATTCTTTTGA TG - #CAAGAATC 
1920 
- AGAGGCCCCA TCAGGCAGAG TTGCTCTGTT ATAGGATGGT AGGGCTGTAA CT - #CAGTGGTC 
1980 
- CAGTGTGCTT TTAGCATGCC CTGGGTTTGA TCCTCAGCAA CACATGCAAA AC - #GTAAGTAG 
2040 
- ACAGCAGACA GCAGACAGCA CAGCCAGCCC CCTGTGTGGT TTGCAGCCTC TG - #CCTTTGAC 
2100 
- TTTTACTCTG GTGGGCACAC AGAGGGCTGG AGCTCCTCCT CCTGACCTTC TA - #ATGAGCCC 
2160 
- TTCCAAGGCC ACGCCTTCCT TCAGGGAATC TCAGGGACTG TAGAGTTCCC AG - #GCCCCTGC 
2220 
- AGCCACCTGT CTCTTCCTAC CTCAGCCTGG AGCACTCCCT CTAACTCCCC AA - #CGGCTTGG 
2280 
- TACTGTACTT GCTGTGACCC CAAGTGCATT GTCCGGGTTA GGCACTGTGA GT - #TGGAACAG 
2340 
- CTGATGACAT CGGTTGAAAG GCCCACCCGG AAACAGCTGA AGCCAGCTCT TT - #TGCCAAAG 
2400 
- GATTCATGCC GGTTTTCTAA TCAACCTGCT CCCCTAGCAT GCCTGGAAGG AA - #AGGGTTCA 
2460 
- GGAGACTCCT CAAGAAGCAA GTTCAGTCTC AGGTGCTTGG ATGCCATGCT CA - #CCGATTCC 
2520 
- ACTGGATATG AACTTGGCAG AGGAGCCTAG TTGTTGCCAT GGAGACTTAA AG - #AGCTCAGC 
2580 
- ACTCTGGAAT CAAGATACTG GACACTTGGG GCCGACTTGT TAAGGCTCTG CA - #GCATCAGA 
2640 
- CTGTAGAGGG GAAGGAACAC GTCTGCCCCC TGGTGGCCCG TCCTGGGATG AC - #CTCGGGCC 
2700 
- TCCTAGGCAA CAAAAGAATG AATTGGAAAG GACTGTTCCT GGGTGTGGCC TA - #GCTCCTGT 
2760 
- GCTTGTGTGG ATCCCTAAAG GGTGTGCTAA GGAGCAATTG CACTGTGTGC TG - #GACAGAAT 
2820 
- TCCTGCTTAT AAATGCTTTT TGTTGTTGTT TTGTACACTG AGCCCTGGCT GA - #GCCACCCC 
2880 
- ACCCCACCTC CCATCCCACC TTTACAGCCA CTCTTGCAGA GAACCTGGCT GT - #CTCCCACT 
2940 
- TGTAGCCTGT GGATGCTGAG GAAACACCCA GCCAAGTAGA CTCCAGGCTT GC - #CCCTATCT 
3000 
- CCTGCTCTGA GTCTGGCCTC CTCATTGTGT TGTGGGAAGG AGACGGGTTC TG - #TCATCTCG 
3060 
- GAAGCCCACA CCGTGGATGT GAACAATGGC TGTACTAGCT TAGACCAGCT TA - #GGGCTCTG 
3120 
- CAATCAGAGG AGGGGGAGCA GGGAACAATT TGAGTGCTGA CCTATAACAC AT - #TCCTAAAG 
3180 
- GATGGGCAGT CCAGAATCTC CCTCCTTCAG TGTGTGTGTG TGTGTGTGTG TG - #TGTGTGTG 
3240 
- TGTGTGTGTG TGTCCATGTT TGCATGTATG TGTGTGCCAG TGTGTGGAGG CC - #CGAGGTTG 
3300 
- GCTTTGGGTG TGTTTGATCA CTCTCCAGTT ACTGAGGCGG GCTCTCATCT GT - #ACCCAGAG 
3360 
- CTTGCACATT TTCTAGTCTA ACTTGCTTCA GGGATCTCTG TCTGCCTATG GA - #GTGCTCAG 
3420 
- GTTACAGGCA GGCTGCCATA CCTGCCCGAC ATTTACATGA ATACTAGAGA TC - #TGAATTCT 
3480 
- GGTCCTCACA CTTGTATACC TGCATTTTAT CCACTAAGAC ATCTCTCCAA GG - #GCTCCCCC 
3540 
- TTCCTATTTA ATAAGTTAGT TTTGAACTGG CAAGATGGCT CAGTGGGTAA GG - #CAGTTTGC 
3600 
- GGACAAACCT GATGACCTGA GTTGGATCCC TGACCATAAG GTAGAAGAGA CC - #TGATTCCT 
3660 
- GCAAGTTGTC CTCTGACCAC CACCCCATAC ATGCTTCTGC ATATGTGCAC AC - #ATCACATT 
3720 
- CTTGCACACA CACTCACATA CCATAAATGT AATAAATTTT TTTAAATAAA TT - #GATTTTAT 
3780 
# 3796 
__________________________________________________________________________