Human urotensin II

Human Urotensin II polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing Human Urotensin II 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 superseding 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 genetic 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. 
SUMMARY OF THE INVENTION 
The present invention relates to Human Urotensin II, in particular Human 
Urotensin II polypeptides and Human Urotensin II 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 acute heart failure; 
hypotension; hypertension; angina pectoris; myocardial infarction; ulcers; 
psychotic and neurological disorders, including anxiety, schizophrenia, 
manic depression, delirium, dementia, severe mental retardation and 
dyskinesias, 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 Human Urotensin II 
imbalance with the identified compounds. In a still further aspect, the 
invention relates to diagnostic assays for detecting diseases associated 
with inappropriate Human Urotensin II activity or levels.

DESCRIPTION OF THE INVENTION 
In a first aspect, the present invention relates to Human Urotensin II 
polypeptides. Such peptides include isolated polypetides 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 
Urotensin II family of polypeptides. They are therefore of interest 
because Urotensin II has been reported from fish and frog sources but the 
human gene has not been described. Urotensin II has been reported to both 
contract rat aorta strips (Itoh, et al., Br J. Phamacol 104: 847-852 
(1991)), and to produce potent endothelium-dependent relaxations and 
endothelium-independent contractions of rat aorta (Br-J-Pharmacol. May; 
91(1): 205-12 (1987)). Urotensin II has a hypertensive effect in trout 
(Le-Mevel, et al., Amer J. Physiol. 271: 1335-43 (1996)) and dogfish 
(Hazon et al., Amer J. Physiol. 265: 573-576 (1993)), and a spasmogenic 
effect on amphibian and teleost smooth muscle (Yano, et al., General 
Comparative Endocrin. 97: 103-110 (1995)). Urotensin II is described as a 
neurohormone which is important in the freshwater adaptation of teleosts 
by increasing the net chloride absorption and decreasing chloride backflux 
in intestinal epithelium (Loretz-CA, J-Exp-Zool-Suppl. 4: 31-6 (1990); and 
Baldisserotto, et al., Braz-J-Med-Biol-Res. 30: 35-39 (1997)). 
Furthermore, fish Urotensin II has recently been discovered to be the 
ligand for GPR14 receptor (U.S. patent Ser. No. 08/789,354) now U.S. Pat. 
No. 5,851,798. Here, we report a potential urotensin II homologue 
geneThese properties are hereinafter referred to as "Human Urotensin II 
activity" or "Human Urotensin II polypeptide activity" or "biological 
activity of Human Urotensin II". Also included amongst these activities 
are antigenic and immunogenic activities of said Human Urotensin II 
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 Human Urotensin II. 
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 
polypetides, 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. 
Based on the processing of fish urotensin II, it is predicted that the 
Human urotensin II precursor protein will be processed to an active 
peptide having an amino acid sequence consisting of ETPDCFWKYCV (SEQ ID 
NO: 5). Thus, the Human Urotensin II polypeptides of the present invention 
also relates to mature Human Urotensin II of SEQ ID NO: 5. 
In a further aspect, the present invention relates to Human Urotensin II 
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 both an EST 
(Accession number: AA535545) and with a genomic clone (Accession number: 
Z98884). Furthermore, the nucleotide sequence of SEQ ID NO 1 shows 
homology with Carp Urotensin II-alpha (J. Neuroscience 6, 2730-2735, 
1986). The nucleotide sequence of SEQ ID NO:1 is a cDNA sequence and 
comprises a polypeptide encoding sequence (nucleotide 118 to 534) encoding 
a polypeptide of 139 amino acids, the polypeptide of SEQ ID NO:2. The 
nucleotide sequence encoding the polypeptide 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 Urotensin II family, having 
homology and/or structural similarity with Goby Urotensin II (PNAS USA 
77(8), 5021-5024, 1980). 
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 Human Urotensin II activity. 
The present invention also relates to partial or other polynucleotide and 
polypeptide sequences which were first identified prior to the 
determination of the corresponding fill length sequences of SEQ ID NO:1 
and SEQ ID NO:2. 
Accordingly, in a further aspect, the present invention provides for an 
isolated polynucleotide comprising: 
(a) 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, even more preferably at least 97-99% 
identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3; 
(b) 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, even more preferably at least 97-99% 
identity, to SEQ ID NO:1 over the entire length of SEQ ID NO:3; 
(c) the polynucleotide of SEQ ID NO:3; or 
(d) 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, even more preferably 
at least 97-99% identity, to the amino acid sequence of SEQ ID NO:4, over 
the entire length of SEQ ID NO:4; 
as well as the polynucleotide of SEQ ID NO:3. 
The present invention further provides for a polypeptide which: 
(a) comprises 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:4; 
(b) has an amino acid sequence which is 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:4; 
(c) comprises the amino acid of SEQ ID NO:4; and 
(d) is the polypeptide of SEQ ID NO:4; 
as well as polypeptides encoded by a polynucleotide comprising the sequence 
contained in SEQ ID NO:3. 
The nucleotide sequence of SEQ ID NO:3 and the peptide sequence encoded 
thereby are derived from EST (Expressed Sequence Tag) sequences. It is 
recognised by those skilled in the art that there will inevitably be some 
nucleotide sequence reading errors in EST sequences (see Adams, M. D. et 
al, Nature 377 (supp) 3, 1995). Accordingly, the nucleotide sequence of 
SEQ ID NO:3 and the peptide sequence encoded therefrom are therefore 
subjec to the same inherent limitations in sequence accuracy. Furthermore, 
the peptide sequence encoded by SEQ ID NO:3 comprises a region of identity 
or close homology and/or close structural similarity (for example a 
conservative amino acid difference) with the closest homologous or 
structurally similar protein. 
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 colon tumors, placenta, and Burkitt's Lymphoma, 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 preproprotein 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 full-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.Denhardt'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 stingent 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 sytems 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, transvection, 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, IHEK 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 Human Urotensin II 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 al., 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 Human Urotensin II 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 Human 
Urotensin II 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 diagnosin a 
disease or suspectability to a disease, particularly acute heart failure, 
hypotension; hypertension, angina pectoris; myocardial infarction; ulcers; 
psychotic and neurological disorders, including anxiety, schizophrenia, 
manic depression, delirium, dementia, severe mental retardation and 
dyskinesias, 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 Hopkin 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 gene of the present invention is reported to map to human 
chromosome 1. 
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 Human Urotensin II activity in the 
mixture, and comparing the Human Urotensin II activity of the mixture to a 
standard. Fusion proteins, such as those made from Fc portion and Human 
Urotensin II 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 polypetide 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, acute heart failure; 
hypotension; hypertension; angina pectoris; myocardial infarction; ulcers; 
psychotic and neurological disorders, including anxiety, schizophrenia, 
manic depression, delirium, dementia, severe mental retardation and 
dyskinesias, related to either an excess of or an under-expression of, 
Human Urotensin II 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 Human Urotensin II polypeptide. 
In still another approach, expression of the gene encoding endogenous Human 
Urotensin II 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 Human 
Urotensin II 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 Human Urotensin II 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 limited 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" is a measure of the identity of nucleotide sequences or amino 
acid sequences. In general, the sequences are aligned so that the highest 
order match is obtained. "Identity" per se has an art-recognized meaning 
and can be calculated using published techniques (see, e.g.: 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, T 
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). While there exist a number of 
methods to measure identity between two polynucleotide or polypeptide 
sequences, the term "identity" is well known to skilled artisans (Carillo, 
H., and Lipton, D., SIAM J Applied Math (1988) 48:1073). Methods commonly 
employed to determine identity or similarity between two sequences 
include, but are not limited to, those disclosed in Guide to Huge 
Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and 
Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073. Methods 
to determine identity and similarity are codified in computer programs. 
Preferred computer program methods to determine identity and similarity 
between two sequences include, but are not limited to, GCG program package 
(Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, 
BLASTN, and FASTA (Atschul, S. F. et al., J Molec Biol (1990) 215:403). 
By way of example, a polynucleotide sequence of the present invention may 
be identical to the reference sequence of SEQ ID NO:1, that is be 100% 
identical, or it may include up to a certain integer number of nucleotide 
alterations as compared to the reference sequence. Such 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. The number of nucleotide alterations is determined by 
multiplying the total number of nucleotides in SEQ ID NO:1 by the 
numerical percent of the respective percent identity(divided by 100) and 
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, and 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 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. 
Similarly, a polypeptide sequence of the present invention may be identical 
to the reference sequence of SEQ ID NO:2, that is 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 % identity is less than 
100%. 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 numerical percent of the respective 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, and y is, for instance 0.70 
for 70%, 0.80 for 80%, 0.85 for 85% etc., 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. 
SEQUENCE INFORMATION 
- TGCCGGTGCCTTCATGGGCAGGACTA 
GGCCGAAAACTCCCCAGATTGTCATTCTTC 
AGGGSEQ ID NO:1 
- ATGGCAGCCCTAAACACAGCATGGCAACTCATCTACTCACTCATGAAAGATTAAAAAATG 
- GAAACCAACGTATTTCATCTTATGCT 
CTGCGTCACTTCTGCTCGGACTCATAAATC 
CACG 
- TCTCTTTGCTTTGGCCACTTCAACTCATATCCAAGCCTTCCTTTAATTCATGATTTATTG 
- CTGGAAATATCCTTTCAACTCTCAGC 
ACCTCATGAAGACGCGCGCTTAACTCCGGA 
GGAG 
- CTAGAAAGAGCTTCCCTTCTACAGATACTGCCAGAGATGCTGGGTGCAGAAAGAGGGGAT 
- ATTCTCAGGAAAGCAGACTCAAGTAC 
CAACATTTTTAACCCAAGAGGAAATTTGAG 
AAAG 
- TTTCAGGATTTCTCTGGACAAGATCCTAACATTTTACTGAGTCATCTTTTGGCCAGAATC 
- TGGAAACCATACAAGAAACGTGAGAC 
TCCTGATTGCTTCTGGAAATACTGTGTCTG 
AAGT 
- GAAATAAGCATCTGTTAGTCAGCTCAGAAACACCCATCTTAGAATATGAAAAATAACACA 
- ATGCTTGATTTGAAAACAGTGTGGAG 
AAAAACTAGGCAAACTACACCCTGTTCATT 
GTTA 
- CCTGGAAAATAAATCCTCTATGTTTTGC 
- METNVFHLMLCVTSARTHKSTSLCFGHFNSYPSLPLIHDLLLEISFQLSAPHEDARLTPESEQ ID 
NO:2 
- ELERASLLQILPEMLGAERGDILRKADSSTNIFNPRGNLRKFQDFSGQDPNILLSHLLAR 
- IWKPYKKRETPDCFWKYCV 
- TGAAGGATGAACCCAATTCTAAGTAT 
CTGTTTTTACTCTGTAGCACCTCATGAAGA 
CGCGSEQ ID NO:3 
- CGCTTAACTCCGGAGGAGCTAGAAAGAGCTTCCCTTCTACAGATACTGCCAGAGATGCTG 
- GGTGCAGAAAGAGGGGATATTCTCAG 
GAAAGCAGACTCAAGTACCAACATTTTTAA 
CCCA 
- AGAGGAAATTTGAGAAAGTTTCAGGATTTCTCTGGACAAGATCCTAACATTTTACTGAGT 
- CATCTTTTGGCCAGAATCTGGAAACC 
ATACAAGAAACGTGAGACTCCTGATTGCTT 
CTGG 
- AAATACTGTGTCTGA 
- RMNPILSICFYSVAPHEDARLTPEELERASLLQILPEMLGAERGDILRKADSSTNIFNPSEQ ID 
NO:4 
- RGNLRKFQDFSGQDPNILLSHLLARIWKPYKKRETPDCFWKYCV 
- ETPDCFWKYCVSEQ ID NO:5 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 5 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 688 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - TGCCGGTGCC TTCATGGGCA GGACTAGGCC GAAAACTCCC CAGATTGTCA - #TTCTTCAG 
GG 60 
- - ATGGCAGCCC TAAACACAGC ATGGCAACTC ATCTACTCAC TCATGAAAGA - #TTAAAAAAT 
G 120 
- - GAAACCAACG TATTTCATCT TATGCTCTGC GTCACTTCTG CTCGGACTCA - #TAAATCCAC 
G 180 
- - TCTCTTTGCT TTGGCCACTT CAACTCATAT CCAAGCCTTC CTTTAATTCA - #TGATTTATT 
G 240 
- - CTGGAAATAT CCTTTCAACT CTCAGCACCT CATGAAGACG CGCGCTTAAC - #TCCGGAGGA 
G 300 
- - CTAGAAAGAG CTTCCCTTCT ACAGATACTG CCAGAGATGC TGGGTGCAGA - #AAGAGGGGA 
T 360 
- - ATTCTCAGGA AAGCAGACTC AAGTACCAAC ATTTTTAACC CAAGAGGAAA - #TTTGAGAAA 
G 420 
- - TTTCAGGATT TCTCTGGACA AGATCCTAAC ATTTTACTGA GTCATCTTTT - #GGCCAGAAT 
C 480 
- - TGGAAACCAT ACAAGAAACG TGAGACTCCT GATTGCTTCT GGAAATACTG - #TGTCTGAAG 
T 540 
- - GAAATAAGCA TCTGTTAGTC AGCTCAGAAA CACCCATCTT AGAATATGAA - #AAATAACAC 
A 600 
- - ATGCTTGATT TGAAAACAGT GTGGAGAAAA ACTAGGCAAA CTACACCCTG - #TTCATTGTT 
A 660 
- - CCTGGAAAAT AAATCCTCTA TGTTTTGC - # - # 
688 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 139 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Glu Thr Asn Val Phe His Leu Met Leu - #Cys Val Thr Ser Ala Arg 
1 5 - # 10 - # 15 
- - Thr His Lys Ser Thr Ser Leu Cys Phe Gly - #His Phe Asn Ser Tyr Pro 
20 - # 25 - # 30 
- - Ser Leu Pro Leu Ile His Asp Leu Leu Leu - #Glu Ile Ser Phe Gln Leu 
35 - # 40 - # 45 
- - Ser Ala Pro His Glu Asp Ala Arg Leu Thr - #Pro Glu Glu Leu Glu Arg 
50 - # 55 - # 60 
- - Ala Ser Leu Leu Gln Ile Leu Pro Glu Met - #Leu Gly Ala Glu Arg Gly 
65 - # 70 - # 75 - # 80 
- - Asp Ile Leu Arg Lys Ala Asp Ser Ser Thr - #Asn Ile Phe Asn Pro Arg 
85 - # 90 - # 95 
- - Gly Asn Leu Arg Lys Phe Gln Asp Phe Ser - #Gly Gln Asp Pro Asn Ile 
100 - # 105 - # 110 
- - Leu Leu Ser His Leu Leu Ala Arg Ile Trp - #Lys Pro Tyr Lys Lys Arg 
115 - # 120 - # 125 
- - Glu Thr Pro Asp Cys Phe Trp Lys Tyr Cys - #Val 
130 - # 135 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 315 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: cDNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - TGAAGGATGA ACCCAATTCT AAGTATCTGT TTTTACTCTG TAGCACCTCA - #TGAAGACGC 
G 60 
- - CGCTTAACTC CGGAGGAGCT AGAAAGAGCT TCCCTTCTAC AGATACTGCC - #AGAGATGCT 
G 120 
- - GGTGCAGAAA GAGGGGATAT TCTCAGGAAA GCAGACTCAA GTACCAACAT - #TTTTAACCC 
A 180 
- - AGAGGAAATT TGAGAAAGTT TCAGGATTTC TCTGGACAAG ATCCTAACAT - #TTTACTGAG 
T 240 
- - CATCTTTTGG CCAGAATCTG GAAACCATAC AAGAAACGTG AGACTCCTGA - #TTGCTTCTG 
G 300 
- - AAATACTGTG TCTGA - # - # 
- # 315 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 103 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Arg Met Asn Pro Ile Leu Ser Ile Cys Phe - #Tyr Ser Val Ala Pro His 
1 5 - # 10 - # 15 
- - Glu Asp Ala Arg Leu Thr Pro Glu Glu Leu - #Glu Arg Ala Ser Leu Leu 
20 - # 25 - # 30 
- - Gln Ile Leu Pro Glu Met Leu Gly Ala Glu - #Arg Gly Asp Ile Leu Arg 
35 - # 40 - # 45 
- - Lys Ala Asp Ser Ser Thr Asn Ile Phe Asn - #Pro Arg Gly Asn Leu Arg 
50 - # 55 - # 60 
- - Lys Phe Gln Asp Phe Ser Gly Gln Asp Pro - #Asn Ile Leu Leu Ser His 
65 - # 70 - # 75 - # 80 
- - Leu Leu Ala Arg Ile Trp Lys Pro Tyr Lys - #Lys Arg Glu Thr Pro Asp 
85 - # 90 - # 95 
- - Cys Phe Trp Lys Tyr Cys Val 
100 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - Glu Thr Pro Asp Cys Phe Trp Lys Tyr Cys - #Val 
1 5 - # 10 
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