Bombesin receptor subtype-3sb polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing Bombesin receptor subtype-3sb polypeptides and polynucleotides in therapy, and diagnostic assays for such.

FIELD OF THE INVENTION 
This invention relates to newly identified polypeptides and polynucleotides 
encoding such polypeptides, to their use in therapy and in identifying 
compounds which may be agonists, antagonists and/or inhibitors which are 
potentially useful in therapy, and to production of such polypeptides and 
polynucleotides. 
BACKGROUND OF THE INVENTION 
The drug discovery process is currently undergoing a fundamental revolution 
as it embraces `functional genomics`, that is, high throughput genome- or 
gene-based biology. This approach is rapidly superceding earlier 
approaches based on `positional cloning`. A phenotype, that is a 
biological function or genetic disease, would be identified and this would 
then be tracked back to the responsible gene, based on its generic map 
position. 
Functional genomics relies heavily on the various tools of bioinformatics 
to identify gene sequences of potential interest from the many molecular 
biology databases now available. There is a continuing need to identify 
and characterise further genes and their related polypeptides/proteins, as 
targets for drug discovery. 
It is well established that many medically significant biological processes 
are mediated by proteins participating in signal transduction pathways 
that involve G-proteins and/or second messengers, e.g., cAMP (Lefkowitz, 
Nature, 1991, 351:353-354). Herein these proteins are referred to as 
proteins participating in pathways with G-proteins or PPG proteins. Some 
examples of these proteins include the GPC receptors, such as those for 
adrenergic agents and dopamine (Kobilka, B. K., et al., Proc. Natl Acad. 
Sci., USA, 1987, 84:46-50; Kobilka, B. K., et al., Science, 1987, 
238:650-656; Bunzow, J. R., et al., Nature, 1988, 336:783-787). G-proteins 
themselves, effector proteins, e.g., phospholipase C, adenyl cyclase, and 
phosphodiesterase, and actuator proteins, e.g., protein kinase A and 
protein kinase C (Simon, M. I., et al., Science, 1991, 252:802-8). 
For example, in one form of signal transduction, the effect of hormone 
binding is activation of the enzyme, adenylate cyclase, inside the cell. 
Enzyme activation by hormones is dependent on the presence of the 
nucleotide, GTP. GTP also influences hormone binding. A G-protein connects 
the hormone receptor to adenilate cyclase. G-protein was shown to exchange 
GTP for bound GDP when activated by a hormone receptor. The GTP-carrying 
form then binds to activated adenylate cyclase. Hydrolysis of GTP to GDP, 
catalyzed by the G-protein itself, returns the G-protein to its basal, 
inactive form. Thus, the G-protein serves a dual role, as an intermediate 
that relays the signal from receptor to effector, and as a clock that 
controls the duration of the signal. 
The membrane protein gene superfamily of G-protein coupled receptors has 
been characterized as having seven putative transmembrane domains. The 
domains are believed to represent transmembrane .alpha.-helices connected 
by extracellular or cytoplasmic loops. G-protein coupled receptors include 
a wide range of biologically active receptors, such as hormone, viral, 
growth factor and neuroreceptors. 
G-protein coupled receptors (otherwise known as 7TM receptors) have been 
characterized as including these seven conserved hydrophobic stretches of 
about 20 to 30 amino acids, connecting at least eight divergent 
hydrophilic loops. The G-protein family of coupled receptors includes 
dopamine receptors which bind to neuroleptic drugs used for treating 
psychotic and neurological disorders. Other examples of members of this 
family include, but are not limited to, calcitonin, adrenergic, 
endothelin, cAMP, adenosine, muscarinic, acetylcholine, serotonin, 
histamine, thrombin, kinin, follicle stimulating hormone, opsins, 
endothelial differentiation gene-1, rhodopsins, odorant, and 
cytomegalovirus receptors. 
Most G-protein coupled receptors have single conserved cysteine residues in 
each of the first two extracellular loops which form disulfide bonds that 
are believed to stabilize functional protein structure. The 7 
transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and 
TM7. TM3 has been implicated in signal transduction. 
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine 
residues can influence signal transduction of some G-protein coupled 
receptors. Most G-protein coupled receptors contain potential 
phosphorylation sites within the third cytoplasmic loop and/or the carboxy 
terminus. For several G-protein coupled receptors, such as the 
.beta.-adrenoreceptor, phosphorylation by protein kinase A and/or specific 
receptor kinases mediates receptor desensitization. 
For some receptors, the ligand binding sites of G-protein coupled receptors 
are believed to comprise hydrophilic sockets formed by several G-protein 
coupled receptor transmembrane domains, said sockets being; surrounded by 
hydrophobic residues of the G-protein coupled receptors. The hydrophilic 
side of each G-protein coupled receptor transmembrane helix is postulated 
to face inward and form a polar ligand binding site. TM3 has been 
implicated in several G-protein coupled receptors as having a ligand 
binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 
asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated 
in ligand binding. 
G-protein coupled receptors can be intracellularly coupled by 
heterotrimeric G-proteins to various intracellular enzymes, ion channels 
and transporters (see, Johnson et al., Endoc. Rev., 1989, 10:317-331). 
Different G-protein .alpha.-subunits preferentially stimulate particular 
effectors to modulate various biological functions in a cell. 
Phosphorylation of cytoplasmic residues of G-protein coupled receptors has 
been identified as an important mechanism for the regulation of G-protein 
coupling of some G-protein coupled receptors. G-protein coupled receptors 
are found in numerous sites within a mammalian host. 
Over the past 15 years, nearly 350 therapeutic agents targeting 7 
transmembrane (7 TM) receptors have been successfully introduced onto the 
market. 
SUMMARY OF THE INVENTION 
The present invention relates to Bombesin receptor subtype-3sb, in 
particular Bombesin receptor subtype-3sb polypeptides and Bombesin 
receptor subtype-3sb polynucleotides, recombinant materials and methods 
for their production. In another aspect, the invention relates to methods 
for using such polypeptides and polynucleotides, including the treatment 
of infections such as bacterial, fungal, protozoan and viral infections, 
particularly infections caused by HIV-1 or HIV-2; pain; cancers; diabetes, 
obesity; anorexia; bulimia; asthma, Parkinson's disease; acute heart 
failure; hypotension; hypertension; urinary retention; osteoporosis; 
angina pectoris; myocardial infarction; stroke; ulcers; asthma; allergies; 
benign prostatic hypertrophy; migraine; vomiting; psychotic and 
neurological disorders, including anxiety, schizophrenia, manic 
depression, depression, delirium, dementia, and severe mental retardation; 
and dyskinesias, such as Huntngton's disease or Gilles dela Tourett's 
syndrome, hereinafter referred to as "the Diseases", amongst others. In a 
further aspect, the invention relates to methods for identifying agonists 
and antagonists/inhibitors using the materials provided by the invention, 
and treating conditions associated with Bombesin receptor subtype-3sb 
imbalance with the identified compounds. In a still further aspect, the 
invention relates to diagnostic assays for detecting diseases associated 
with inappropriate Bombesin receptor subtype-3sb activity or levels. 
DESCRIPTION OF THE INVENTION 
In a first aspect, the present invention relates to Bombesin receptor 
subtype-3sb polypeptides. Such peptides include isolated polypeptides 
comprising an amino acid sequence which has at least 70% identity, 
preferably at least 80% identity, more preferably at least 90% identity, 
yet more preferably at least 95% identity, most preferably at least 97-99% 
identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2. 
Such polypeptides include those comprising the amino acid of SEQ ID NO:2. 
Further peptides of the present invention include isolated polypeptides in 
which the amino acid sequence has at least 70% identity, preferably at 
least 80% identity, more preferably at least 90% identity, yet more 
preferably at least 95% identity, most preferably at least 97-99% 
identity, to the amino acid sequence of SEQ ID NO:2 over the entire length 
of SEQ ID NO:2. Such polypeptides include the polypeptide of SEQ ID NO:2. 
Further peptides of the present invention include isolated polypeptides 
encoded by a polynucleotide comprising the sequence contained in SEQ ID 
NO:1. 
Polypeptides of the present invention are believed to be members of the 
G-protien coupled 7 transmembrane receptor gene family of polypeptides. 
They are therefore of interest because G-protein coupled receptors, more 
than any other gene family, have been the targets of pharmaceutical 
intervention. These properties are hereinafter referred to as "Bombesin 
receptor subtype-3sb activity" or "Bombesin receptor subtype-3sb 
polypeptide activity" or "biological activity of Bombesin receptor 
subtype-3sb". Also included amongst these activities are antigenic and 
immunogenic activities of said Bombesin receptor subtype-3sb polypeptides, 
in particular the antigenic and immunogenic activities of the polypeptide 
of SEQ ID NO:2. Preferably, a polypeptide of the present invention 
exhibits at least one biological activity of Bombesin receptor 
subtype-3sb. 
The polypeptides of the present invention may be in the form of the 
"mature" protein or may be a part of a larger protein such as a fusion 
protein. It is often advantageous to include an additional amino acid 
sequence which contains secretory or leader sequences, pro-sequences, 
sequences which aid in purification such as multiple histidine residues, 
or an additional sequence for stability during recombinant production. 
The present invention also includes include variants of the aforementioned 
polypeptides, that is polypeptides that vary from the referents by 
conservative amino acid substitutions, whereby a residue is substituted by 
another with like characteristics. Typical such substitutions are among 
Ala, Val, Leu and Ile; among Ser and Thr; among the acidic residues Asp 
and Glu; among Asn and Gln; and among the basic residues Lys and Arg; or 
aromatic residues Phe and Tyr. Particularly preferred are variants in 
which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, 
deleted, or added in any combination. 
Polypeptides of the present invention can be prepared in any suitable 
manner. Such polypeptides include isolated naturally occurring 
polypeptides, recombinantly produced polypeptides, synthetically produced 
polypeptides, or polypeptides produced by a combination of these methods. 
Means for preparing such polypeptides are well understood in the art. 
In a further aspect, the present invention relates to Bombesin receptor 
subtype-3sb polynucleotides. Such polynucleotides include isolated 
polynucleotides comprising a nucleotide sequence encoding a polypeptide 
which has at least 70% identity, preferably at least 80% identity, more 
preferably at least 90% identity, yet more preferably at least 95% 
identity, to the amino acid sequence of SEQ ID NO:2, over the entire 
length of SEQ ID NO:2. In this regard, polypeptides which have at least 
97% identity are highly preferred, whilst those with at least 98-99% 
identity are more highly preferred, and those with at least 99% identity 
are most highly preferred. Such polynucleotides include a polynucleotide 
comprising the nucleotide sequence contained in SEQ ID NO:1 encoding the 
polypeptide of SEQ ID NO:2. 
Further polynucleotides of the present invention include isolated 
polynucleotides comprising a nucleotide sequence that has at least 70% 
identity, preferably at least 80% identity, more preferably at least 90% 
identity, yet more preferably at least 95% identity, to a nucleotide 
sequence encoding a polypeptide of SEQ ID NO:2, over the entire coding 
region. In this regard, polynucleotides which have at least 97% identity 
are highly preferred, whilst those with at least 98-99% identity are more 
highly preferred, and those with at least 99% identity are most highly 
preferred. 
Further polynucleotides of the present invention include isolated 
polynucleotides comprising a nucleotide sequence which has at least 70% 
identity, preferably at least 80% identity, more preferably at least 90% 
identity, yet more preferably at least 95% identity, to SEQ ID NO:1 over 
the entire length of SEQ ID NO:1. In this regard, polynucleotides which 
have at least 97% identity are highly preferred, whilst those with at 
least 98-99% identity are more highly preferred, and those with at least 
99% identity are most highly preferred. Such polynucleotides include a 
polynucleotide comprising the polynucleotide of SEQ ID NO:1 as well as the 
polynucleotide of SEQ ID NO:1. 
The invention also provides polynucleotides which are complementary to all 
the above described polynucleotides. 
The nucleotide sequence of SEQ ID NO:1 shows homology with Human bombesin 
receptor subtype-3. (Fathi, Z., J. biol. Chem. 268, 5979-5984 (1993)). The 
nucleotide sequence of SEQ ID NO:1 is a cDNA sequence and comprises a 
polypeptide encoding sequence (nucleotide 3 to 1202) encoding a 
polypeptide of 399 amino acids, the polypeptide of SEQ ID NO:2. The 
nucleotide sequence encoding the polylieptide of SEQ ID NO:2 may be 
identical to the polypeptide encoding sequence contained in SEQ ID NO:1 or 
it may be a sequence other than the one contained in SEQ ID NO:1, which, 
as a result of the redundancy (degeneracy) of the genetic code, also 
encodes the polypeptide of SEQ ID NO:2. The polypeptide of SEQ ID NO:2 is 
structurally related to other proteins of the G-protein coupled (7 
transmembrane) receptor gene family, having homology and/or structural 
similarity with Bombesin receptor subtype-3. 
Preferred polypeptides and polynucleotides of the present invention are 
expected to have, inter alia, similar biological functions/properties to 
their homologous polypeptides and polynucleotides. Furthermore, preferred 
polypeptides and polynucleotides of the present invention have at least 
one Bombesin receptor subtype-3sb activity. 
Polynucleotides of the present invention may be obtained, using standard 
cloning and screening techniques, from a cDNA library derived from mRNA in 
cells of human placenta, using the expressed sequence tag (EST) analysis 
(Adams, M. D., et al. Science (1991) 252:1651-1656; Adams, M. D. et al., 
Nature, (1992) 355:632-634; Adams, M. D., et al., Nature (1995) 377 
Supp:3-174). Polynucleotides of the invention can also be obtained from 
natural sources such as genomic DNA libraries or can be synthesized using 
well known and commercially available techniques. 
When polynucleotides of the present invention are used for the recombinant 
production of polypeptides of the present invention, the polynucleotide 
may include the coding sequence for the mature polypeptide, by itself, or 
the coding sequence for the mature polypeptide in reading frame with other 
coding sequences, such as those encoding a leader or secretory sequence, a 
pre-, or pro- or prepro-protein sequence, or other fusion peptide 
portions. For example, a marker sequence which facilitates purification of 
the fused polypeptide can be encoded. In certain preferred embodiments of 
this aspect of the invention, the marker sequence is a hexa-histidine 
peptide, as provided in the pQE vector (Qiagen, Inc.) and described in 
Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag. 
The polynucleotide may also contain non-coding 5' and 3' sequences, such 
as transcribed, non-translated sequences, splicing and polyadenylation 
signals, ribosome binding sites and sequences that stabilize mRNA. 
Further embodiments of the present invention include polynucleotides 
encoding polypeptide variants which comprise the amino acid sequence of 
SEQ ID NO:2 and in which several, for instance from 5 to 10, 1 to 5, 1 to 
3, 1 to 2 or 1, amino acid residues are substituted, deleted or added, in 
any combination. 
Polynucleotides which are identical or sufficiently identical to a 
nucleotide sequence contained in SEQ ID NO:1, may be used as hybridization 
probes for cDNA and genomic DNA or as primers for a nucleic acid 
amplification (PCR) reaction, to isolate full-length cDNAs and genomic 
clones encoding polypeptides of the present invention and to isolate cDNA 
and genomic clones of other genes (including genes encoding homologs and 
orthologs from species other than human) that have a high sequence 
similarity to SEQ ID NO:1. Typically these nucleotide sequences are 70% 
identical, preferably 80% identical, more preferably 90% identical, most 
preferably 95% identical to that of the referent. The probes or primers 
will generally comprise at least 15 nucleotides, preferably, at least 30 
nucleotides and may have at least 50 nucleotides. Particularly preferred 
probes will have between 30 and 50 nucleotides. 
A polynucleotide encoding a polypeptide of the present invention, including 
homologs and orthologs from species other than human, may be obtained by a 
process which comprises the steps of screening an appropriate library 
under stringent hybridization conditions with a labeled probe having the 
sequence of SEQ ID NO:1 or a fragment thereof; and isolating fill-length 
cDNA and genomic clones containing said polynucleotide sequence. Such 
hybridization techniques are well known to the skilled artisan. Preferred 
stringent hybridization conditions include overnight incubation at 
42.degree. C. in a solution comprising: 50% formamide, 5.times.SSC (150 mM 
NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5.times. 
Denbardt's solution, 10% dextran sulfate, and 20 microgram/ml denatured, 
sheared salmon sperm DNA; followed by washing the filters in 0.1.times.SSC 
at about 65.degree. C. Thus the present invention also includes 
polynucleotides obtainable by screening an appropriate library under 
stringent hybridization conditions with a labeled probe having the 
sequence of SEQ ID NO:1 or a fragment thereof. 
The skilled artisan will appreciate that, in many cases, an isolated cDNA 
sequence will be incomplete, in that the region coding for the polypeptide 
is cut short at the 5' end of the cDNA. This is a consequence of reverse 
transcriptase, an enzyme with inherently low `processivity` (a measure of 
the ability of the enzyme to remain attached to the template during the 
polymerisation reaction), failing to complete a DNA copy of the mRNA 
template during 1st strand cDNA synthesis. 
There are several methods available and well known to those skilled in the 
art to obtain full-length cDNAs, or extend short cDNAs, for example those 
based on the method of Rapid Amplification of cDNA ends (RACE) (see, for 
example, Frohman et al., PNAS USA 85, 8998-9002, 1988). Recent 
modifications of the technique, exemplified by the Marathon.TM. technology 
(Clontech Laboratories Inc.) for example, have significantly simplified 
the search for longer cDNAs. In the Marathon.TM. technology, cDNAs have 
been prepared from mRNA extracted from a chosen tissue and an `adaptor` 
sequence ligated onto each end. Nucleic acid amplification (PCR) is then 
carried out to amplify the `missing` 5' end of the cDNA using a 
combination of gene specific and adaptor specific oligonucleotide primers. 
The PCR reaction is then repeated using `nested` primers, that is, primers 
designed to anneal within the amplified product (typically an adaptor 
specific primer that anneals further 3' in the adaptor sequence and a gene 
specific primer that anneals further 5' in the known gene sequence). The 
products of this reaction can then be analysed by DNA sequencing and a 
full-length cDNA constructed either by joining the product directly to the 
existing cDNA to give a complete sequence, or carrying out a separate 
full-length PCR using the new sequence information for the design of the 
5' primer. 
Recombinant polypeptides of the present invention may be prepared by 
processes well known in the art from genetically engineered host cells 
comprising expression systems. Accordingly, in a further aspect, the 
present invention relates to expression systems which comprise a 
polynucleotide or polynucleotides of the present invention, to host cells 
which are genetically engineered with such expression systems and to the 
production of polypeptides of the invention by recombinant techniques. 
Cell-free translation systems can also be employed to produce such 
proteins using RNAs derived from the DNA constructs of the present 
invention. 
For recombinant production, host cells can be genetically engineered to 
incorporate expression systems or portions thereof for polynucleotides of 
the present invention. Introduction of polynucleotides into host cells can 
be effected by methods described in many standard laboratory manuals, such 
as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et 
al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, N.Y. (1989). Preferred such methods 
include, for instance, calcium phosphate transfection, DEAE-dextran 
mediated transfection, transfection, microinjection, cationic 
lipid-mediated transfection, electroporation, transduction, scrape 
loading, ballistic introduction or infection. 
Representative examples of appropriate hosts include bacterial cells, such 
as streptococci, staphylococci, E. coli, Streptomyces and Bacillus 
subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; 
insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells 
such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; 
and plant cells. 
A great variety of expression systems can be used, for instance, 
chromosomal, episomal and virus-derived systems, e.g., vectors derived 
from bacterial plasmids, from bacteriophage, from transposons, from yeast 
episomes, from insertion elements, from yeast chromosomal elements, from 
viruses such as baculoviruses, papova viruses, such as SV40, vaccinia 
viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and 
retroviruses, and vectors derived from combinations thereof, such as those 
derived from plasmid and bacteriophage genetic elements, such as cosmids 
and phagemids. The expression systems may contain control regions that 
regulate as well as engender expression. Generally, any system or vector 
which is able to maintain, propagate or express a polynucleotide to 
produce a polypeptide in a host may be used. The appropriate nucleotide 
sequence may be inserted into an expression system by any of a variety of 
well-known and routine techniques, such as, for example, those set forth 
in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra). 
Appropriate secretion signals may be incorporated into the desired 
polypeptide to allow secretion of the translated protein into the lumen of 
the endoplasmic reticulum, the periplasmic space or the extracellular 
environment. These signals may be endogenous to the polypeptide or they 
may be heterologous signals. 
If a polypeptide of the present invention is to be expressed for use in 
screening assays, it is generally preferred that the polypeptide be 
produced at the surface of the cell. In this event, the cells may be 
harvested prior to use in the screening assay. If the polypeptide is 
secreted into the medium, the medium can be recovered in order to recover 
and purify the polypeptide. If produced intracellularly, the cells must 
first be lysed before the polypeptide is recovered. 
Polypeptides of the present invention can be recovered and purified from 
recombinant cell cultures by well-known methods including ammonium sulfate 
or ethanol precipitation, acid extraction, anion or cation exchange 
chromatography, phosphocellulose chromatography, hydrophobic interaction 
chromatography, affinity chromatography, hydroxylapatite chromatography 
and lectin chromatography. Most preferably, high performance liquid 
chromatography is employed for purification. Well known techniques for 
refolding proteins may be employed to regenerate active conformation when 
the polypeptide is denatured during isolation and or purification. 
This invention also relates to the use of polynucleotides of the present 
invention as diagnostic reagents. Detection of a mutated form of the gene 
characterised by the polynucleotide of SEQ ID NO:1 which is associated 
with a dysfunction will provide a diagnostic tool that can add to, or 
define, a diagnosis of a disease, or susceptibility to a disease, which 
results from under-expression, over-expression or altered expression of 
the gene. Individuals carrying mutations in the gene may be detected at 
the DNA level by a variety of techniques. 
Nucleic acids for diagnosis may be obtained from a subject's cells, such as 
from blood, urine, saliva, tissue biopsy or autopsy material. The genomic 
DNA may be used directly for detection or may be amplified enzymatically 
by using PCR or other amplification techniques prior to analysis. RNA or 
cDNA may also be used in similar fashion. Deletions and insertions can be 
detected by a change in size of the amplified product in comparison to the 
normal genotype. Point mutations can be identified by hybridizing 
amplified DNA to labeled Bombesin receptor subtype-3sb nucleotide 
sequences. Perfectly matched sequences can be distinguished from 
mismatched duplexes by RNase digestion or by differences in melting 
temperatures. DNA sequence differences may also be detected by alterations 
in electrophoretic mobility of DNA fragments in gels;, with or without 
denaturing agents, or by direct DNA sequencing (ee, e.g., Myers et at, 
Science (1985)230:1242). Sequence changes at specific locations may also 
be revealed by nuclease protection assays, such as RNase and S1 protection 
or the chemical cleavage method (see Cotton et al., Proc Natl Acad Sci USA 
(1985) 85: 4397-4401). In another embodiment, an array of oligonucleotides 
probes comprising Bombesin receptor subtype-3sb nucleotide sequence or 
fragments thereof can be constructed to conduct efficient screening of 
e.g., genetic mutations. Array technology methods are well known and have 
general applicability and can be used to address a variety of questions in 
molecular genetics including gene expression, genetic linkage, and genetic 
variability (see for example: M. Chee et al., Science, Vol 274, pp 610-613 
(1996)). 
The diagnostic assays offer a process for diagnosing or determining a 
susceptibility to the Diseases through detection of mutation in the 
Bombesin receptor subtype-3sb gene by the methods described. In addition, 
such diseases may be diagnosed by methods comprising determining from a 
sample derived from a subject an abnormally decreased or increased level 
of polypeptide or mRNA. Decreased or increased expression can be measured 
at the RNA level using any of the methods well known in the art for the 
quantitation of polynucleotides, such as, for example, nucleic acid 
amplification, for instance PCR, RT-PCR, RNase protection, Northern 
blotting and other hybridization methods. Assay techniques that can be 
used to determine levels of a protein, such as a polypeptide of the 
present invention, in a sample derived from a host are well-known to those 
of skill in the art. Such assay methods include radioimmunoassays, 
competitive-binding assays, Western Blot analysis and ELISA assays. 
Thus in another aspect, the present invention relates to a diagonostic kit 
which comprises: 
(a) a polynucleotide of the present invention, preferably the nucleotide 
sequence of SEQ ID NO:1, or a fragment thereof; 
(b) a nucleotide sequence complementary to that of (a); 
(c) a polypeptide of the present invention, preferably the polypeptide of 
SEQ ID NO:2 or a fragment thereof; or 
(d) an antibody to a polypeptide of the present invention, preferably to 
the polypeptide of SEQ ID NO:2. 
It will be appreciated that in any such kit, (a), (b), (c) or (d) may 
comprise a substantial component. Such a kit will be of use in diagnosing 
a disease or suspectability to a disease, particularly infections such as 
bacterial, fungal, protozoan and viral infections, particularly infections 
caused by HIV-1 or HIV-2; pain; cancers; obesity; anorexia; bulimia; 
asthma; Parkinson's disease; acute heart failure; hypotension; 
hypertension; urinary retention; osteoporosis; angina pectoris; myocardial 
infarction; stroke; ulcers; asthma; allergies; benign prostatic 
hypertrophy; migraine; vomiting; psychotic and neurological disorders, 
including anxiety, schizophrenia, manic depression, depression, delirium, 
dementia, and severe mental retardation; and dyskinesias, such as 
Huntington's disease or Gilles dela Tourett's syndrome, amongst others. 
The nucleotide sequences of the present invention are also valuable for 
chromosome identification. The sequence is specifically targeted to, and 
can hybridize with, a particular location on an individual human 
chromosome. The mapping of relevant sequences to chromosomes according to 
the present invention is an important first step in correlating those 
sequences with gene associated disease. Once a sequence has been mapped to 
a precise chromosomal location, the physical position of the sequence on 
the chromosome can be correlated with genetic map data. Such data are 
found in, for example, V. McKusick, Mendelian Inheritance in Man 
(available on-line through Johns Hopkins University Welch Medical 
Library). The relationship between genes and diseases that have been 
mapped to the same chromosomal region are then identified through linkage 
analysis (coinheritance of physically adjacent genes). 
The differences in the cDNA or genomic sequence between affected and 
unaffected individuals can also be determined. If a mutation is observed 
in some or all of the affected individuals but not in any normal 
individuals, then the mutation is likely to be the causative agent of the 
disease. 
The polypeptides of the invention or their fragments or analogs thereof, or 
cells expressing them, can also be used as immunogens to produce 
antibodies immunospecific for polypeptides of the present invention. The 
term "immunospecific" means that the antibodies have substantially greater 
affinity for the polypeptides of the invention than their affinity for 
other related polypeptides in the prior art. 
Antibodies generated against polypeptides of the present invention may be 
obtained by administering the polypeptides or epitope-bearing fragments, 
analogs or cells to an animal, preferably a non-human animal, using 
routine protocols. For preparation of monoclonal antibodies, any technique 
which provides antibodies produced by continuous cell line cultures can be 
used. Examples include the hybridoma technique (Kohler, G. and Milstein, 
C., Nature (1975) 256:495-497), the trioma technique, the human B-cell 
hybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) and the 
EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER 
THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985). 
Techniques for the production of single chain antibodies, such as those 
described in U.S. Pat. No. 4,946,778, can also be adapted to produce 
single chain antibodies to polypeptides of this invention. Also, 
transgenic mice, or other organisms, including other mammals, may be used 
to express humanized antibodies. 
The above-described antibodies may be employed to isolate or to identify 
clones expressing the polypeptide or to purify the polypeptides by 
affinity chromatography. 
Antibodies against polypeptides of the present invention may also be 
employed to treat the Diseases, amongst others. 
In a further aspect, the present invention relates to genetically 
engineered soluble fusion proteins comprising a polypeptide of the present 
invention, or a fragment thereof, and various portions of the constant 
regions of heavy or light chains of immunoglobulins of various subclasses 
(IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part 
of the heavy chain of human IgG, particularly IgG1, where fusion takes 
place at the hinge region. In a particular embodiment, the Fc part can be 
removed simply by incorporation of a cleavage sequence which can be 
cleaved with blood clotting factor Xa. Furthermore, this invention relates 
to processes for the preparation of these fusion proteins by genetic 
engineering, and to the use thereof for drug screening, diagnosis and 
therapy. A further aspect of the invention also relates to polynucleotides 
encoding such fusion proteins. Examples of fusion protein technology can 
be found in International Patent Application Nos. WO94/29458 and 
WO94/22914. 
Another aspect of the invention relates to a method for inducing an 
immunological response in a mammal which comprises inoculating the mammal 
with a polypeptide of the present invention, adequate to produce antibody 
and/or T cell immune response to protect said animal from the Diseases 
hereinbefore mentioned, amongst others. Yet another aspect of the 
invention relates to a method of inducing immunological response in a 
mammal which comprises, delivering a polypeptide of the present invention 
via a vector directing expression of the polynucleotide and coding for the 
polypeptide in vivo in order to induce such an immunological response to 
produce antibody to protect said animal from diseases. 
A further aspect of the invention relates to an immunological/vaccine 
formulation (composition) which, when introduced into a mammalian host, 
induces an immunological response in that mammal to a polypeptide of the 
present invention wherein the composition comprises a polypeptide or 
polynucleotide of the present invention. The vaccine formulation may 
further comprise a suitable carrier. Since a polypeptide may be broken 
down in the stomach, it is preferably administered parenterally (for 
instance, subcutaneous, intramuscular, intravenous, or intradermal 
injection). Formulations suitable for parenteral administration include 
aqueous and non-aqueous sterile injection solutions which may contain 
anti-oxidants, buffers, bacteriostats and solutes which render the 
formulation instonic with the blood of the recipient; and aqueous and 
non-aqueous sterile suspensions which may include suspending agents or 
thickening agents. The formulations may be presented in unit-dose or 
multi-dose containers, for example, sealed ampoules and vials and may be 
stored in a freeze-dried condition requiring only the addition of the 
sterile liquid carrier immediately prior to use. The vaccine formulation 
may also include adjuvant systems for enhancing the immunogenicity of the 
formulation, such as oil-in water systems and other systems known in the 
art. The dosage will depend on the specific activity of the vaccine and 
can be readily determined by routine experimentation. 
Polypeptides of the present invention are responsible for many biological 
functions, including many disease states, in particular the Diseases 
hereinbefore mentioned. It is therefore desirous to devise screening 
methods to identify compounds which stimulate or which inhibit the 
function of the polypeptide. Accordingly, in a further aspect the present 
invention provides for a method of screening compounds to identify those 
which stimulate or which inhibit the function of the polypeptide. In 
general, agonists or antagonists may be employed for therapeutic and 
prophylactic purposes for such Diseases as hereinbefore mentioned. 
Compounds may be identified from a variety of sources, for example, cells, 
cell-free preparations, chemical libraries, and natural product mixtures. 
Such agonists, antagonists or inhibitors so-identified may be natural or 
modified substrates, ligands, receptors, enzymes, etc., as the case may 
be, of the polypeptide; or may be structural or functional mimetics 
thereof (see Coligan et al., Current Protocols in Immunology 1(2):Chapter 
5 (1991)). 
The screening method may simply measure the binding of a candidate compound 
to the polypeptide, or to cells or membranes bearing the polypeptide, or a 
fusion protein thereof by means of a label directly or indirectly 
associated with the candidate compound. Alternatively, the screening 
method may involve competition with a labeled competitor. Further, these 
screening methods may test whether the candidate compound results in a 
signal generated by activation or inhibition of the polypeptide, using 
detection systems appropriate to the cells bearing the polypeptide. 
Inhibitors of activation are generally assayed in the presence of a known 
agonist and the effect on activation by the agonist by the presence of the 
candidate compound is observed. Constitutively active polpypeptides may be 
employed in screening methods for inverse agonists or inhibitors, in the 
absence of an agonist or inhibitor, by testing whether the candidate 
compound results in inhibition of activation of the polypeptide. Further, 
the screening methods may simply comprise the steps of mixing a candidate 
compound with a solution containing a polypeptide of the present 
invention, to form a mixture, measuring Bombesin receptor subtype-3sb 
activity in the mixture, and comparing the Bombesin receptor subtype-3sb 
activity of the mixture to a standard. Fusion proteins, such as those made 
from Fc portion and Bombesin receptor subtype-3sb polypeptide, as 
hereinbefore described, can also be used for high-throughput screening 
assays to identify antagonists for the polypeptide of the present 
invention (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and 
K. Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)). 
The polynucleotides, polypeptides and antibodies to the polypeptide of the 
present invention may also be used to configure screening methods for 
detecting the effect of added compounds on the production of mRNA and 
polypeptide in cells. For example, an ELISA assay may be constructed for 
measuring secreted or cell associated levels of polypeptide using 
monoclonal and polyclonal antibodies by standard methods known in the art. 
This can be used to discover agents which may inhibit or enhance the 
production of polypeptide (also called antagonist or agonist, 
respectively) from suitably manipulated cells or tissues. 
The polypeptide may be used to identify membrane bound or soluble 
receptors, if any, through standard receptor binding techniques known in 
the art. These include, but are not limited to, ligand binding and 
crosslinking assays in which the polypeptide is labeled with a radioactive 
isotope (for instance, .sup.125 I), chemically modified (for instance, 
biotinylated), or fused to a peptide sequence suitable for detection or 
purification, and incubated with a source of the putative receptor (cells, 
cell membranes, cell supernatants, tissue extracts, bodily fluids). Other 
methods include biophysical techniques such as surface plasmon resonance 
and spectroscopy. These screening methods may also be used to identify 
agonists and antagonists of the polypeptide which compete with the binding 
of the polypeptide to its receptors, if any. Standard methods for 
conducting such assays are well understood in the art. 
Examples of potential polypeptide antagonists include antibodies or, in 
some cases, oligonucleotides or proteins which are closely related to the 
ligands, substrates, receptors, enzymes, etc., as the case may be, of the 
polypeptide, e.g., a fragment of the ligands, substrates, receptors, 
enzymes, etc.; or small molecules which bind to the polypeptide of the 
present invention but do not elicit a response, so that the activity of 
the polypeptide is prevented. 
Thus, in another aspect, the present invention relates to a screening kit 
for identifying agonists, antagonists, ligands, receptors, substrates, 
enzymes, etc. for polypeptides of the present invention; or compounds 
which decrease or enhance the production of such polypeptides, which 
comprises: 
(a) a polypeptide of the present invention; 
(b) a recombinant cell expressing a polypeptide of the present invention; 
(c) a cell membrane expressing a polypeptide of the present invention; or 
(d) antibody to a polypeptide of the present invention; which polypeptide 
is preferably that of SEQ ID NO:2. 
It will be appreciated that in any such kit, (a), (b), (c) or (d) may 
comprise a substantial component. 
It will be readily appreciated by the skilled artisan that a polypeptide of 
the present invention may also be used in a method for the structure-based 
design of an agonist, antagonist or inhibitor of the polypeptide, by: 
(a) determining in the first instance the three dimensional structure of 
the polypeptide; 
(b) deducing the three-dimensional structure for the likely reactive or 
binding site(s) of an agonist, antagonist or inhibitor; 
(c) synthesing candidate compounds that are predicted to bind to or react 
with the deduced binding or reactive site; and 
(d) testing whether the candidate compounds are indeed agonists, 
antagonists or inhibitors. 
It will be further appreciated that this will normally be an interative 
process. 
In a further aspect, the present invention provides methods of treating 
abnormal conditions such as, for instance, infections such as bacterial, 
fungal, protozoan and viral infections, particularly infections caused by 
HIV-1 or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; 
asthma; Parkion's disease; acute heart failure; hypotension; hypertension; 
urinary retention; osteoporosis; angina pectoris; myocardial infarction; 
stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; 
vomiting; psychotic and neurological disorders, including anxiety, 
schizophrenia, manic depression, depression, delirium, dementia, and 
severe mental retardation; and dyskinesias, such as Huntington's disease 
or Gilles dela Tourett's syndrome, related to either an excess of, or an 
under-expression of, Bombesin receptor subtype-3sb polypeptide activity. 
If the activity of the polypeptide is in excess, several approaches are 
available. One approach comprises administering to a subject in need 
thereof an inhibitor compound (antagonist) as hereinabove described, 
optionally in combination with a pharmaceutically acceptable carrier, in 
an amount effective to inhibit the function of the polypeptide, such as, 
for example, by blocking the binding of ligands, substrates, receptors, 
enzymes, etc., or by inhibiting a second signal, and thereby alleviating 
the abnormal condition. In another approach, soluble forms of the 
polypeptides still capable of binding the ligand, substrate, enzymes, 
receptors, etc. in competition with endogenous polypeptide may be 
administered. Typical examples of such competitors include fragments of 
the Bombesin receptor subtype-3sb polypeptide. 
In still another approach, expression of the gene encoding endogenous 
Bombesin receptor subtype-3sb polypeptide can be inhibited using 
expression blocking techniques. Known such techniques involve the use of 
antisense sequences, either internally generated or separately 
administered (see, for example, O'Connor, J Neurochem (1991) 56:560 in 
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC 
Press, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides which 
form triple helices with the gene can be supplied (see, for example, Lee 
et al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988) 
241:456; Dervan et al., Science (1991) 251:1360). These oligomers can be 
administered per se or the relevant oligomers can be expressed in vivo. 
For treating abnormal conditions related to an under-expression of Bombesin 
receptor subtype-3sb and its activity, several approaches are also 
available. One approach comprises administering to a subject a 
therapeutically effective amount of a compound which activates a 
polypeptide of the present invention, i.e., an agonist as described above, 
in combination with a pharmaceutically acceptable carrier, to thereby 
alleviate the abnormal condition. Alternatively, gene therapy may be 
employed to effect the endogenous production of Bombesin receptor 
subtype-3sb by the relevant cells in the subject. For example, a 
polynucleotide of the invention may be engineered for expression in a 
replication defective retroviral vector, as discussed above. The 
retroviral expression construct may then be isolated and introduced into a 
packaging cell transduced with a retroviral plasmid vector containing RNA 
encoding a polypeptide of the present invention such that the packaging 
cell now produces infectious viral particles containing the gene of 
interest. These producer cells may be administered to a subject for 
engineering cells in vivo and expression of the polypeptide in vivo. For 
an overview of gene therapy, see Chapter 20, Gene Therapy and other 
Molecular Genetic-based Therapeutic Approaches, (and references cited 
therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS 
Scientific Publishers Ltd (1996). Another approach is to administer a 
therapeutic amount of a polypeptide of the present invention in 
combination with a suitable pharmaceutical carrier. 
In a further aspect, the present invention provides for pharmaceutical 
compositions comprising a therapeutically effective amount of a 
polypeptide, such as the soluble form of a polypeptide of the present 
invention, agonist/antagonist peptide or small molecule compound, in 
combination with a pharmaceutically acceptable carrier or excipient. Such 
carriers include, but are not limed to, saline, buffered saline, dextrose, 
water, glycerol, ethanol, and combinations thereof The invention further 
relates to pharmaceutical packs and kits comprising one or more containers 
filled with one or more of the ingredients of the aforementioned 
compositions of the invention. Polypeptides and other compounds of the 
present invention may be employed alone or in conjunction with other 
compounds, such as therapeutic compounds. 
The composition will be adapted to the route of administration, for 
instance by a systemic or an oral route. Preferred forms of systemic 
administration include injection, typically by intravenous injection. 
Other injection routes, such as subcutaneous, intramuscular, or 
intraperitoneal, can be used. Alternative means for systemic 
administration include transmucosal and transdermal administration using 
penetrants such as bile salts or fusidic acids or other detergents. In 
addition, if a polypeptide or other compounds of the present invention can 
be formulated in an enteric or an encapsulated formulation, oral 
administration may also be possible. Administration of these compounds may 
also be topical and/or localized, in the form of salves, pastes, gels, and 
the like. 
The dosage range required depends on the choice of peptide or other 
compounds of the present invention, the route of administration, the 
nature of the formulation, the nature of the subject's condition, and the 
judgment of the attending practitioner. Suitable dosages, however, are in 
the range of 0.1-100 .mu.g/kg of subject. Wide variations in the needed 
dosage, however, are to be expected in view of the variety of compounds 
available and the differing efficiencies of various routes of 
administration. For example, oral administration would be expected to 
require higher dosages than administration by intravenous injection. 
Variations in these dosage levels can be adjusted using standard empirical 
routines for optimization, as is well understood in the art. 
Polypeptides used in treatment can also be generated endogenously in the 
subject, in treatment modalities often referred to as "gene therapy" as 
described above. Thus, for example, cells from a subject may be engineered 
with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex 
vivo, and for example, by the use of a retroviral plasmid vector. The 
cells are then introduced into the subject. 
Polynucleotide and polypeptide sequences form a valuable information 
resource with which to identify further sequences of similar homology. 
This is most easily facilitated by storing the sequence in a computer 
readable medium and then using the stored data to search a sequence 
database using well known searching tools, such as GCC. Accordingly, in a 
further aspect, the present invention provides for a computer readable 
medium having stored thereon a polynucleotide comprising the sequence of 
SEQ ID NO:1 and/or a polypeptide sequence encoded thereby. 
The following definitions are provided to facilitate understanding of 
certain terms used frequently hereinbefore. 
"Antibodies" as used herein includes polyclonal and monoclonal antibodies, 
chimeric, single chain, and humanized antibodies, as well as Fab 
fragments, including the products of an Fab or other immunoglobulin 
expression library. 
"Isolated" means altered "by the hand of man" from the natural state. If an 
"isolated" composition or substance occurs in nature, it has been changed 
or removed from its original environment, or both. For example, a 
polynucleotide or a polypeptide naturally present in a living animal is 
not "isolated," but the same polynucleotide or polypeptide separated from 
the coexisting materials of its natural state is "isolated", as the term 
is employed herein. 
"Polynucleotide" generally refers to any polyribonucleotide or 
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA 
or DNA. "Polynucleotides" include, without limitation, single- and 
double-stranded DNA, DNA that is a mixture of single- and double-stranded 
regions, single- and double-stranded RNA, and RNA that is mixture of 
single- and double-stranded regions, hybrid molecules comprising DNA and 
RNA that may be single-stranded or, more typically, double-stranded or a 
mixture of single- and double-stranded regions. In addition, 
"polynucleotide" refers to triple-stranded regions comprising RNA or DNA 
or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs 
containing one or more modified bases and DNAs or RNAs with backbones 
modified for stability or for other reasons. "Modified" bases include, for 
example, tritylated bases and unusual bases such as inosine. A variety of 
modifications may be made to DNA and RNA; thus, "polynucleotide" embraces 
chemically, enzymatically or metabolically modified forms of 
polynucleotides as typically found in nature, as well as the chemical 
forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" 
also embraces relatively short polynucleotides, often referred to as 
oligonucleotides. 
"Polypeptide" refers to any peptide or protein comprising two or more amino 
acids joined to each other by peptide bonds or modified peptide bonds, 
i.e., peptide isosteres. "Polypeptide" refers to both short chains, 
commonly referred to as peptides, oligopeptides or oligomers, and to 
longer chains, generally referred to as proteins. Polypeptides may contain 
amino acids other than the 20 gene-encoded amino acids. "Polypeptides" 
include amino acid sequences modified either by natural processes, such as 
post-translational processing, or by chemical modification techniques 
which are well known in the art. Such modifications are well described in 
basic texts and in more detailed monographs, as well as in a voluminous 
research literature. Modifications may occur anywhere in a polypeptide, 
including the peptide backbone, the amino acid side-chains and the amino 
or carboxyl termini. It will be appreciated that the same type of 
modification may be present to the same or varying degrees at several 
sites in a given polypeptide. Also, a given polypeptide may contain many 
types of modifications. Polypeptides may be branched as a result of 
ubiquitination, and they may be cyclic, with or without branching. Cyclic, 
branched and branched cyclic polypeptides may result from post-translation 
natural processes or may be made by synthetic methods. Modifications 
include acetylation, acylation, ADP-ribosylation, amidation, covalent 
attachment of flavin, covalent attachment of a heme moiety, covalent 
attachment of a nucleotide or nucleotide derivative, covalent attachment 
of a lipid or lipid derivative, covalent attachment of 
phosphotidylinositol, cross-linking, cyclization, disulfide bond 
formation, demethylation, formation of covalent cross-links, formation of 
cystine, formation of pyroglutamate, formylation, gamma-carboxylation, 
glycosylation, GPI anchor formation, hydroxylation, iodination, 
methylation, myristoylation, oxidation, proteolytic processing, 
phosphorylation, prenylation, racemization, selenoylation, sulfation, 
transfer-RNA mediated addition of amino acids to proteins such as 
arginylation, and ubiquitination (see, for instance, PROTEINS--STRUCTURE 
AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and 
Company, New York, 1993; Wold, F., Post-translational Protein 
Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL 
COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New 
York, 1983; Seifter et al., "Analysis for protein modifications and 
nonprotein cofactors", Meth Enzymol (1990) 182:626-646 and Rattan et al., 
"Protein Synthesis: Post-translational Modifications and Aging", Ann NY 
Acad Sci (1992) 663:48-62). 
"Variant" refers to a polynucleotide or polypeptide that differs from a 
reference polynucleotide or polypeptide, but retains essential properties. 
A typical variant of a polynucleotide differs in nucleotide sequence from 
another, reference polynucleotide. Changes in the nucleotide sequence of 
the variant may or may not alter the amino acid sequence of a polypeptide 
encoded by the reference polynucleotide. Nucleotide changes may result in 
amino acid substitutions, additions, deletions, fusions and truncations in 
the polypeptide encoded by the reference sequence, as discussed below. A 
typical variant of a polypeptide differs in amino acid sequence from 
another, reference polypeptide. Generally, differences are limited so that 
the sequences of the reference polypeptide and the variant are closely 
similar overall and, in many regions, identical. A variant and reference 
polypeptide may differ in amino acid sequence by one or more 
substitutions, additions, deletions in any combination. A substituted or 
inserted amino acid residue may or may not be one encoded by the genetic 
code. A variant of a polynucleotide or polypeptide may be a naturally 
occurring such as an allelic variant, or it may be a variant that is not 
known to occur naturally. Non-naturally occurring variants of 
polynucleotides and polypeptides may be made by mutagenesis techniques or 
by direct synthesis. 
"Identity," as known in the art, is a relationship between two or more 
polypeptide sequences or two or more polynucleotide sequences, as the case 
may be, as determined by comparing the sequences. In the art, "identity" 
also means the degree of sequence relatedness between polypeptide or 
polynucleotide sequences, as the case may be, as determined by the match 
between strings of such sequences. "Identity" can be readily calculated by 
known methods, including but not limited to those described in 
(Computational Molecular Biology, Lesk, A. M., ed., Oxford University 
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, 
Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of 
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana 
Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von 
Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, 
M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, 
H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to 
determine identity are designed to give the largest match between the 
sequences tested. Moreover, methods to determine identity are codified in 
publicly available computer programs. Computer program methods to 
determine identity between two sequences include, but are not limited to, 
the GCG program package (Devereux, J., et al., Nucleic Acids Research 
12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. 
Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly 
available from NCBI and other sources (BLAST Manual, Altschul, S., et al., 
NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 
403-410 (1990). The well known Smith Waterman algorithm may also be used 
to determine identity. 
Parameters for polypeptide sequence comparison include the following: 
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443453 (1970) 
Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. 
Acad. Sci. USA. 89:11)915-10919 (1992) 
Gap Penalty: 12 
Gap Length Penalty: 4 
A program useful with these parameters is publicly available as the "gap" 
program from Genetics Computer Group, Madison Wis. The aforementioned 
parameters are the default parameters for peptide comparisons (along with 
no penalty for end gaps). 
Parameters for polynucleotide comparison include the following: 
1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) 
Comparison matrix: matches=+10, mismatch=0 
Gap Penalty: 50 
Gap Length Penalty: 3 
Available as: The "gap" program from Genetics Computer Group, Madison WI. 
These are the default parameters for nucleic acid comparisons. 
A preferred meaning for "identity" for polynucleotides and polypeptides, as 
the case may be, are provided in (1) and (2) below. 
(1) Polynucleotide embodiments further include an isolated polynucleotide 
comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 
90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO:1, 
wherein said polynucleotide sequence may be identical to the reference 
sequence of SEQ ID NO:1 or may include up to a certain integer number of 
nucleotide alterations as compared to the reference sequence, wherein said 
alterations are selected from the group consisting of at least one 
nucleotide deletion, substitution, including transition and transversion, 
or insertion, and wherein said alterations may occur at the 5' or 3' 
terminal positions of the reference nucleotide sequence or anywhere 
between those terminal positions, interspersed either individually among 
the nucleotides in the reference sequence or in one or more contiguous 
groups within the reference sequence, and wherein said number of 
nucleotide alterations is determined by multiplying the total number of 
nucleotides in SEQ ID NO:1 by the integer defining the percent identity 
divided by 100 and then subtracting that product from said total number of 
nucleotides in SEQ ID NO:1, or: 
EQU n.sub.n .ltoreq.x.sub.n -(x.sub.n .multidot.y), 
wherein n.sub.n is the number of nucleotide alterations, x.sub.n is the 
total number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for 
60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 
0.97 for 97% or 1.00 for 100%, and .multidot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.n 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.n. Alterations of a polynucleotide sequence encoding the polypeptide 
of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in 
this coding sequence and thereby alter the polypeptide encoded by the 
polynucleotide following such alterations. 
By way of example, a polynucleotide sequence of the present invention may 
be identical to the reference sequence of SEQ ID NO:2, that is it may be 
100% identical, or it may include up to a certain integer number of amino 
acid alterations as compared to the reference sequence such that the 
percent identity is less than 100% identity. Such alterations are selected 
from the group consisting of at least one nucleic acid deletion, 
substitution, including transition and transversion, or insertion, and 
wherein said alterations may occur at the 5' or 3' terminal positions of 
the reference polynucleotide sequence or anywhere between those terminal 
positions, interspersed either individually among the nucleic acids in the 
reference sequence or in one or more contiguous groups within the 
reference sequence. The number of nucleic acid alterations for a given 
percent identity is determined by multiplying the total number of amino 
acids in SEQ ID NO:2 by the integer defining the percent identity divided 
by 100 and then subtracting that product from said total number of amino 
acids in SEQ ID NO:2, or: 
EQU n.sub.n .ltoreq.x.sub.n -(x.sub.n .multidot.y), 
wherein n.sub.n is the number of amino acid alterations, x.sub.n is the 
total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for 
70%, 0.80 for 80%, 0.85 for 85% etc., .multidot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.n 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.n. 
(2) Polypeptide embodiments further include an isolated polypeptide 
comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 
or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, 
wherein said polypeptide sequence may be identical to the reference 
sequence of SEQ ID NO:2 or may include up to a certain integer number of 
amino acid alterations as compared to the reference sequence, wherein said 
alterations are selected from the group consisting of at least one amino 
acid deletion, substitution, including conservative and non-conservative 
substitution, or insertion, and wherein said alterations may occur at the 
amino- or carboxy-terminal positions of the reference polypeptide sequence 
or anywhere between those terminal positions, interspersed either 
individually among the amino acids in the reference sequence or in one or 
more contiguous groups within the reference sequence, and wherein said 
number of amino acid alterations is determined by multiplying the total 
number of amino acids in SEQ ID NO:2 by the integer defining the percent 
identity divided by 100 and then subtracting that product from said total 
number of amino acids in SEQ ID NO:2, or: 
EQU n.sub.a .ltoreq.x.sub.a -(x.sub.a .multidot.y), 
wherein n.sub.a is the number of amino acid alterations, x.sub.a is the 
total number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for 
60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 
0.97 for 97% or 1.00 for 100%, and .multidot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.a 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.a. 
By way of example, a polypeptide sequence of the present invention may be 
identical to the reference sequence of SEQ ID NO:2, that is it may be 100% 
identical, or it may include up to a certain integer number of amino acid 
alterations as compared to the reference sequence such that the percent 
identity is less than 100% identity. Such alterations are selected from 
the group consisting of at least one amino acid deletion, substitution, 
including conservative and non-conservative substitution, or insertion, 
and wherein said alterations may occur at the amino- or carboxy-terminal 
positions of the reference polypeptide sequence or anywhere between those 
terminal positions, interspersed either individually among the amino acids 
in the reference sequence or in one or more contiguous groups within the 
reference sequence. The number of amino acid alterations for a given % 
identity is determined by multiplying the total number of amino acids in 
SEQ ID NO:2 by the integer defining the percent identity divided by 100 
and then subtracting that product from said total number of amino acids in 
SEQ ID NO:2, or: 
EQU n.sub.a .ltoreq.x.sub.a -(x.sub.a .multidot.y), 
wherein n.sub.a is the number of amino acid alterations, x.sub.a is the 
total number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for 
70%, 0.80 for 80%, 0.85 for 85% etc., and .multidot. is the symbol for the 
multiplication operator, and wherein any non-integer product of x.sub.a 
and y is rounded down to the nearest integer prior to subtracting it from 
x.sub.a. 
"Fusion protein" refers to a protein encoded by two, often unrelated, fused 
genes or fragments thereof. In one example, EP-A-0 464 discloses fusion 
proteins comprising various portions of constant region of immunoglobulin 
molecules together with another human protein or part thereof. In many 
cases, employing an immunoglobulin Fc region as a part of a fusion protein 
is advantageous for use in therapy and diagnosis resulting in, for 
example, improved pharmacokinetic properties [see, e.g., EP-A 0232 262]. 
On the other hand, for some uses it would be desirable to be able to 
delete the Fc part after the fusion protein has been expressed, detected 
and purified. 
All publications, including but not limited to patents and patent 
applications, cited in this specification are herein incorporated by 
reference as if each individual publication were specifically and 
individually indicated to be incorporated by reference herein as though 
fully set forth.