Genes encoding art, an agouti-related transcript

Disclosed is a novel gene termed ART which is expressed primarily in selected regions of the brain, as well as adrenal and lung tissues. Polypeptides encoded by ART are also disclosed, as are methods for preparing ART DNA and amino acid sequences.

This application claims the benefit of U.S. Provisional application Ser. 
No. 60/017,505 filed 10 May 1996. 
BACKGROUND 
1. Field of the Invention 
This invention relates to novel human gene sequences and proteins encoded 
by the gene sequences. More specifically, the invention concerns a novel 
gene, termed "ART" (Agouti Related Transcript), that is expressed in 
selected tissues, and increases food uptake. 
2. Description of Related Art 
1. Agouti Gene 
The agouti gene is present in most mammals, although its function in 
mammals other than rodents is unclear. The agouti gene product regulates 
the relative production of black or yellow pigment in the hair of many 
animals, including mice, squirrels, and wolves (A. G Searle, Comparative 
Genetics of Coat Color in Mammals, Academic Press, New York, N.Y. 1968!). 
The mouse agouti gene has been cloned and sequenced (Bultman et al., Cell, 
71:1195-1204 1992!), and it encodes a 131 amino acid protein that is 
secreted. The agouti protein appears to act as an antagonist to the 
melanocortin-1 receptor ("MC1r") which is expressed on melanocytes (see 
Takeuchi, J. Invest. Dermatol., 92:239S-242S 1989!; Jackson, Nature, 
362:587-588 1993!). MC1r, when occupied by melanocyte stimulating hormone 
(a-MSH), causes the melanocyte to synthesize black pigment (see Jackson, 
supra), and therefore, it appears that agouti blocks the action of a-MSH, 
thereby resulting in hairs with yellow pigment (Lu et al., Nature, 
371:799-802 1994!). 
Similarly, Willard et al. (Biochemistry, 34:12341-12346 1995!) have shown 
that partially purified mouse agouti protein acts as a potent antagonist 
of a-MSH at the MC1 receptor in B16F10 mouse melanoma cell cultures. 
Proteolytic cleavage of agouti protein at amino acid 83 generates a 
C-terminal fragment that is comparable in activity to full length agouti 
protein, suggesting that the active domain of agouti protein lies within 
its C-terminus (Willard et al., supra). This C-terminal fragment has 10 
cysteines (the full length molecule has 11 cysteines). 
In humans, the agouti gene is expressed in skin, heart, testes, ovary, and 
adipose tissue. This diverse tissue expression suggests that agouti may be 
involved in physiological processes other than pigmentation production 
(Wilson et al., Human Mol. Gen., 4:233-230 1995!; Kwon et al., Proc. 
Natl. Acad. Sci USA, 91:9760-9764 1994!). 
Several dominant phenotypes that result from agouti over-expression in 
transgenic mice have been identified. These include, for example, obesity, 
hyperinsulinemia, diabetes, and increased tumor susceptibility (see Manne 
et al., Proc. Natl. Acad. Sci USA, 92:4721-4724 1995!). The degree and 
time of onset of obesity and hyperinsulinemia appear to be related to the 
level of agouti gene expression (Manne et al., supra). Further, these 
phenotypes do not seem to be related to the excess production of yellow 
pigment, since mice which have an inactive MC1 receptor show the same 
phenotype. 
Mutant mice that over-express the agouti gene product have increased levels 
of intracellular calcium in the skeletal muscle (Zemel et al., Proc. Natl. 
Acad. Sci USA, 92:4728-4732 1995!). Although the mechanism by which 
agouti produces this effect is not known, it does not appear to result 
either from release of intracellular stores of calcium or from a decreased 
efflux rate of calcium. Since skeletal muscle is important in the uptake 
of insulin, and this process is regulated at least in part by calcium 
levels, this increased intracellular calcium may explain in part the 
hyperinsulinemia observed in agouti mutant mice. 
Interestingly, mouse agouti shares some amino acid sequence homology with 
certain spider and snail toxins that target specific neurotransmitter 
receptors or ion channels (Manne et al., supra; Ichida et al., Neurochem. 
Res., 18:1137-1144 1993!; Figueiredo et al., Toxicon, 33:83-93 1995!). 
This homology is primarily confined to the C-terminus of the agouti 
protein, where the toxins and agouti share 8 cysteine residues. In the 
toxins, these cysteine residues form 4 disulfide bonds that are critical 
for toxin activity. Structural activity relationships using 3-dimensional 
NMR predicts that the disulfide bonds are required to form the tertiary 
structure needed to block calcium channels (Kim et al., J. Mol. Biol., 
250:659-671 1995!). 
In view of the amino acid sequence homologies of agouti with the spider and 
snail toxins, and the results obtained from mutant mice that over-express 
agouti, it has been suggested that agouti may be a member of a new class 
of molecules that regulate the activity of melanocortin receptors or 
certain types of calcium channel proteins (Manne et al., supra). 
2. Melanocortin Receptors 
In humans, there are currently five known melanocortin receptors and they 
are known as MC1r-MC5r. Two of these, MC1r and MC2r, show relative 
specificity for the ligands a-MSH and ACTH, respectively. MC1r and MC2r 
are expressed in melanocytes and the adrenal gland, respectively (Mountjoy 
et al., Science, 257:1248-1251 1992!). MC3r is expressed in specific 
brain regions, while MC4r is expressed more widely throughout the brain, 
and MC5r is expressed in numerous peripheral tissues (Roselli-Reyfuss et 
al., Proc. Natl. Acad. Sci. USA, 90:8856-8860 1993!; Mountjoy et al., 
Science, supra; Labbe et al., Biochemistry, 33:4543-4549 1994!). The 
ligands and biological functions of MC3r, MC4r, and MC5r are presently 
unknown. 
A role for melanocortin receptors in the central control of obesity has 
recently been suggested by the observation that injection of melanin 
concentrating hormone (MCH) into the brain of rats stimulates a feeding 
response (Qu et al., Nature, 380:243-247 1996!). Although MCH does not 
have amino acid sequence homology with agouti, antibodies against MCH also 
recognize epitopes on agouti, and MCH also displays antagonistic activity 
at the MC1 receptor. 
In view of the variety of physiological disorders and diseases (obesity, 
insulinemia, diabetes) that agouti and MCH have been implicated in, and in 
view of the fact that agouti and MCH antagonize MC receptors, there is a 
need in the art to identify and analyze related genes and proteins that 
may be involved in these same disorders. 
Accordingly, it is an object to provide a compound that can modulate, 
either directly or indirectly, melanocortin receptor signaling, 
intra-cellular calcium levels, and/or body fat composition (such as 
adipose tissue level and/or distribution, circulating glucose levels, 
and/or insulin levels). 
It is a further object to provide a compound that can increase food uptake. 
These and other objectives will readily be apparent to one of ordinary 
skill in the art. 
SUMMARY OF THE INVENTION 
In one embodiment, the invention provides a nucleic acid molecule encoding 
a polypeptide selected from the group consisting of: a nucleic acid 
molecule encoding a polypeptide selected from the group consisting of: 
(a) the nucleic acid molecule of SEQ ID NO:4; 
(b) the nucleic acid molecule of SEQ ID NO:5; 
(c) the nucleic acid molecule of SEQ ID NO:6; 
(d) the nucleic acid molecule of SEQ ID NO:9 
(e) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:8; 
(f) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:10; 
(g) a nucleic acid molecule encoding the polypeptide of SEQ ID NO:11; 
(h) a nucleic acid molecule that encodes a polypeptide that is at least 70 
percent identical to the polypeptides of SEQ ID NO:7, SEQ ID NO:8, SEQ ID 
NO:10, SEQ ID NO:11; and 
(i) a nucleic acid molecule that is the complement of any of (a)-(h) above. 
In another embodiment, the invention provides a vector comprising a nucleic 
acid molecule selected from the group set forth above, and a host cell 
comprising the vector. 
In yet another embodiment, the invention provides a process for producing 
an ART polypeptide comprising the steps of: 
(a) expressing a polypeptide encoded by a nucleic acid selected from the 
group set forth above, wherein the nucleic acid has been inserted into a 
suitable host; and 
(b) isolating the polypeptide. 
The invention further provides an ART polypeptide selected from the group 
consisting of: 
(a) the polypeptide of SEQ ID NO:7; 
(b) the polypeptide of SEQ ID NO:8; 
(c) the polypeptide of SEQ ID NO:10; 
(d) the polypeptide of SEQ ID NO:11; and 
(e) a polypeptide that is 70 percent homologous with the polypeptide of (a) 
or (b), wherein the ART polypeptide may or may not possess an amino 
terminal methionine. 
In one further embodiment, the invention provides a method of increasing 
food uptake in a mammal comprising administering an ART polypeptide to the 
mammal.

DETAILED DESCRIPTION OF THE INVENTION 
As used herein, the term "ART" when used to describe a nucleic acid 
molecule refers to a nucleic acid molecule or fragment thereof that (a) 
has the nucleotide sequence as set forth in SEQ ID NO: 4, SEQ ID NO:5, or 
SEQ ID NO:6; (b) has a nucleic acid sequence encoding a polypeptide that 
is at least 70 percent identical, preferably at least 80 percent 
identical, and more preferably at least 90 percent identical to the 
polypeptide encoded by any of SEQ ID NOS:4, 5, or 6; (c) is a naturally 
occurring allelic variant of (a) or (b); (d) is a nucleic acid variant of 
(a)-(c) produced as provided for herein; and/or (e) is complementary to 
(a)-(d). 
Percent sequence identity can be determined by standard methods that are 
commonly used to compare the similarity in position of the amino acids of 
two polypeptides. Using a computer program such as BLAST or FASTA, two 
polypeptides are aligned for optimal matching of their respective amino 
acids (either along the full length of one or both sequences, or along a 
predetermined portion of one or both sequences). The programs provide a 
"default" opening penalty and a "default" gap penalty, and a scoring 
matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: 
Atlas of Protein Sequence and Structure, vol. 5, supp.3 1978!) can be 
used in conjunction with the computer program. The percent identity can 
then be calculated as: 
##EQU1## 
Polypeptides that are at least 70 percent identical will typically have 
one or more amino acid substitutions, deletions, and/or insertions. 
Usually, the substitutions will be conservative so as to have little or no 
effect on the overall net charge, polarity, or hydrophobicity of the 
protein. Conservative substitutions are set forth in Table I below. 
TABLE I 
______________________________________ 
Conservative amino acid substitutions 
______________________________________ 
Basic: arginine 
lysine 
histidine 
Acidic: glutamic acid 
aspartic acid 
Polar: glutamine 
asparagine 
Hydrophobic: leucine 
isoleucine 
valine 
Aromatic: phenylalanine 
tryptophan 
tyrosine 
Small: glycine 
alanine 
serine 
threonine 
methionine 
______________________________________ 
The term "stringent conditions" refers to hybridization and washing under 
conditions that permit only binding of a nucleic acid molecule such as an 
oligonucleotide or cDNA molecule probe to highly homologous sequences. One 
stringent wash solution is 0.015M NaCl, 0.005M NaCitrate, and 0.1 percent 
SDS used at a temperature of 55.degree. C.-65.degree. C. Another stringent 
wash solution is 0.2.times.SSC and 0.1 percent SDS used at a temperature 
of between 50.degree. C.-65.degree. C. Where oligonucleotide probes are 
used to screen cDNA or genomic libraries, the following stringent washing 
conditions may be used. One protocol uses 6.times.SSC with 0.05 percent 
sodium pyrophosphate at a temperature of 35.degree. C.-62.degree. C., 
depending on the length of the oligonucleotide probe. For example, 14 base 
pair probes are washed at 35.degree.-40.degree. C., 17 base pair probes 
are washed at 45.degree.-50.degree. C., 20 base pair probes are washed at 
52.degree.-57.degree. C., and 23 base pair probes are washed at 
57.degree.-63.degree. C. The temperature can be increased 
2.degree.-3.degree. C. where the background non-specific binding appears 
high. A second protocol utilizes tetramethylammonium chloride (TMAC) for 
washing oligonucleotide probes. One stringent washing solution is 3M TMAC, 
50 mM Tris-HCl, pH 8.0, and 0.2 percent SDS. The washing temperature using 
this solution is a function of the length of the probe. For example, a 17 
base pair probe is washed at about 45.degree.-50.degree. C. 
The term "ART protein" or "ART polypeptide" as used herein refers to any 
protein or polypeptide having the properties described herein for ART. The 
ART polypeptide may or may not have an amino terminal methionine, 
depending on the manner in which it is prepared. By way of illustration, 
ART protein or ART polypeptide includes, an amino acid sequence encoded by 
the nucleic acid molecule set forth in any of items (a)-(e) above and 
peptide or polypeptide fragments derived therefrom, to the amino acid 
sequence set forth in SEQ ID NOs:7 or 8, and/or to chemically modified 
derivatives as well as nucleic acid and or amino acid sequence variants 
thereof as provided for herein. 
As used herein, the term "ART fragment" refers to a peptide or polypeptide 
that is less than the full length amino acid sequence of naturally 
occurring ART protein but has substantially the same biological activity 
as ART polypeptide or ART protein described above. Such a fragment may be 
truncated at the amino terminus, the carboxy terminus, and/or internally, 
and may be chemically modified. Preferably, the ART fragment will be a 
carboxy terminal fragment which retains at least all 10 C-terminal 
cysteine residues. Such ART fragments may be prepared with or without an 
amino terminal methionine. A preferred ART fragment is set forth in SEQ ID 
NO:8. 
As used herein, the term "ART derivative" or "ART variant" refers to a ART 
polypeptide or ART protein that has 1) been chemically modified, as for 
example, by addition of polyethylene glycol or other compound, and/or 2) 
contains one or more nucleic acid or amino acid sequence substitutions, 
deletions, and/or insertions. 
As used herein, the terms "biologically active polypeptide" and 
"biologically active fragment" refer to a peptide or polypeptide that has 
ART activity (i.e., is capable of modulating the signaling activity of a 
melanocortin receptor, is capable of modulating intracellular calcium 
levels, and/or is capable of modulating lipid metabolism). 
As used herein, the terms "effective amount" and "therapeutically effective 
amount" refer to the amount of ART necessary to support one or more 
biological activities of ART as set forth above. 
The ART polypeptides that have use in practicing the present invention may 
be naturally occurring full length polypeptides, or truncated polypeptides 
or peptides (i.e, "fragments"). The polypeptides or fragments may be 
chemically modified, i.e., glycosylated, phosphorylated, and/or linked to 
a polymer, as described below, and they may have an amino terminal 
methionine, depending on how they are prepared. In addition, the 
polypeptides or fragments may be variants of the naturally occurring ART 
polypeptide (i.e., may contain one or more amino acid deletions, 
insertions, and/or substitutions as compared with naturally occurring 
ART). 
The full length ART polypeptide or fragment thereof can be prepared using 
well known recombinant DNA technology methods such as those set forth in 
Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989!) and/or Ausubel 
et al., eds, (Current Protocols in Molecular Biology, Green Publishers 
Inc. and Wiley and Sons, NY 1994!). A gene or cDNA encoding the ART 
protein or fragment thereof may be obtained for example by screening a 
genomic or cDNA library, or by PCR amplification. Alternatively, a gene 
encoding the ART polypeptide or fragment may be prepared by chemical 
synthesis using methods well known to the skilled artisan such as those 
described by Engels et al.(Angew. Chem. Intl. Ed., 28:716-734 1989!). 
These methods include, inter alia, the phosphotriester, phosphoramidite, 
and H-phosphonate methods for nucleic acid synthesis. A preferred method 
for such chemical synthesis is polymer-supported synthesis using standard 
phosphoramidite chemistry. Typically, the DNA encoding the ART polypeptide 
will be several hundred nucleotides in length. Nucleic acids larger than 
about 100 nucleotides can be synthesized as several fragments using these 
methods. The fragments can then be ligated together to form the full 
length ART polypeptide. Usually, the DNA fragment encoding the amino 
terminus of the polypeptide will have an ATG, which encodes a methionine 
residue. This methionine may or may not be present on the mature form of 
the ART polypeptide, depending on whether the polypeptide produced in the 
host cell is secreted from that cell. 
In some cases, it may be desirable to prepare nucleic acid and/or amino 
acid variants of naturally occurring ART. Nucleic acid variants (wherein 
one or more nucleotides are designed to differ from the wild-type or 
naturally occurring ART) may be produced using site directed mutagenesis 
or PCR amplification where the primer(s) have the desired point mutations 
(see Sambrook et al., supra, and Ausubel et al., supra, for descriptions 
of mutagenesis techniques). Chemical synthesis using methods described by 
Engels et al., supra, may also be used to prepare such variants. Other 
methods known to the skilled artisan may be used as well. Preferred 
nucleic acid variants are those containing nucleotide substitutions 
accounting for codon preference in the host cell that is to be used to 
produce ART. Other preferred variants are those encoding conservative 
amino acid changes (e.g., wherein the charge or polarity of the naturally 
occurring amino acid side chain is not altered substantially by 
substitution with a different amino acid) as compared to wild type, and/or 
those designed to either generate a novel glycosylation and/or 
phosphorylation site(s) on ART, or those designed to delete an existing 
glycosylation and/or phosphorylation site(s) on ART. 
The ART gene or cDNA can be inserted into an appropriate expression vector 
for expression in a host cell. The vector is selected to be functional in 
the particular host cell employed (i.e., the vector is compatible with the 
host cell machinery such that amplification of the ART gene and/or 
expression of the gene can occur). The ART polypeptide or fragment thereof 
may be amplified/expressed in prokaryotic, yeast, insect (baculovirus 
systems) and/or eukaryotic host cells. Selection of the host cell will 
depend at least in part on whether the ART polypeptide or fragment thereof 
is to be glycosylated. If so, yeast, insect, or mammalian host cells are 
preferable; yeast cells will glycosylate the polypeptide, and insect and 
mammalian cells can glycosylate and/or phosphorylate the polypeptide as it 
naturally occurs on the ART polypeptide (i.e., "native" glycosylation 
and/or phosphorylation). 
Typically, the vectors used in any of the host cells will contain 5' 
flanking sequence (also referred to as a "promoter") and other regulatory 
elements as well such as an enhancer(s), an origin of replication element, 
a transcriptional termination element, a complete intron sequence 
containing a donor and acceptor splice site, a signal peptide sequence, a 
ribosome binding site element, a polyadenylation sequence, a polylinker 
region for inserting the nucleic acid encoding the polypeptide to be 
expressed, and a selectable marker element. Each of these elements is 
discussed below. Optionally, the vector may contain a "tag" sequence, 
i.e., an oligonucleotide sequence located at the 5' or 3' end of the ART 
coding sequence that encodes polyHis (such as hexaHis) or another small 
immunogenic sequence. This tag will be expressed along with the protein, 
and can serve as an affinity tag for purification of the ART polypeptide 
from the host cell. Optionally, the tag can subsequently be removed from 
the purified ART polypeptide by various means such as using a selected 
peptidase for example. 
The 5' flanking sequence may be homologous (i.e., from the same species 
and/or strain as the host cell), heterologous (i.e., from a species other 
than the host cell species or strain), hybrid (i.e., a combination of 5' 
flanking sequences from more than one source), synthetic, or it may be the 
native ART 5' flanking sequence. As such, the source of the 5' flanking 
sequence may be any unicellular prokaryotic or eukaryotic organism, any 
vertebrate or invertebrate organism, or any plant, provided that the 5' 
flanking sequence is functional in, and can be activated by, the host cell 
machinery. 
The 5' flanking sequences useful in the vectors of this invention may be 
obtained by any of several methods well known in the art. Typically, 5' 
flanking sequences useful herein other than the ART 5' flanking sequence 
will have been previously identified by mapping and/or by restriction 
endonuclease digestion and can thus be isolated from the proper tissue 
source using the appropriate restriction endonucleases. In some cases, the 
full nucleotide sequence of the 5' flanking sequence may be known. Here, 
the 5' flanking sequence may be synthesized using the methods described 
above for nucleic acid synthesis or cloning. 
Where all or only a portion of the 5' flanking sequence is known, it may be 
obtained using PCR and/or by screening a genomic library with suitable 
oligonucleotide and/or 5' flanking sequence fragments from the same or 
another species. 
Where the 5' flanking sequence is not known, a fragment of DNA containing a 
5' flanking sequence may be isolated from a larger piece of DNA that may 
contain, for example, a coding sequence or even another gene or genes. 
Isolation may be accomplished by restriction endonuclease digestion using 
one or more carefully selected enzymes to isolate the proper DNA fragment. 
After digestion, the desired fragment may be isolated by agarose gel 
purification, Qiagen.RTM. column or other methods known to the skilled 
artisan. Selection of suitable enzymes to accomplish this purpose will be 
readily apparent to one of ordinary skill in the art. 
The origin of replication element is typically a part of prokaryotic 
expression vectors purchased commercially, and aids in the amplification 
of the vector in a host cell. Amplification of the vector to a certain 
copy number can, in some cases, be important for optimal expression of the 
ART polypeptide. If the vector of choice does not contain an origin of 
replication site, one may be chemically synthesized based on a known 
sequence, and ligated into the vector. 
The transcription termination element is typically located 3' to the end of 
the ART polypeptide coding sequence and serves to terminate transcription 
of the ART polypeptide. Usually, the transcription termination element in 
prokaryotic cells is a G-C rich fragment followed by a poly T sequence. 
While the element is easily cloned from a library or even purchased 
commercially as part of a vector, it can also be readily synthesized using 
methods for nucleic acid synthesis such as those described above. 
A selectable marker gene element encodes a protein necessary for the 
survival and growth of a host cell grown in a selective culture medium. 
Typical selection marker genes encode proteins that (a) confer resistance 
to antibiotics or other toxins, e.g., ampicillin, tetracycline, or 
kanamycin for prokaryotic host cells, (b) complement auxotrophic 
deficiencies of the cell; or (c) supply critical nutrients not available 
from complex media. Preferred selectable markers are the kanamycin 
resistance gene, the ampicillin resistance gene, and the tetracycline 
resistance gene. 
The ribosome binding element, commonly called the Shine-Dalgarno sequence 
(prokaryotes) or the Kozak sequence (eukaryotes), is necessary for 
translation initiation of mRNA. The element is typically located 3' to the 
promoter and 5' to the coding sequence of the ART polypeptide to be 
synthesized. The Shine-Dalgarno sequence is varied but is typically a 
polypurine (i.e., having a high A-G content). Many Shine-Dalgarno 
sequences have been identified, each of which can be readily synthesized 
using methods set forth above and used in a prokaryotic vector. 
In those cases where it is desirable for ART to be secreted from the host 
cell, a signal sequence may be used to direct the ART polypeptide out of 
the host cell where it is synthesized. Typically, the signal sequence is 
positioned in the coding region of ART nucleic acid sequence, or directly 
at the 5' end of the ART coding region. Many signal sequences have been 
identified, and any of them that are functional in the selected host cell 
may be used in conjunction with the ART gene. Therefore, the signal 
sequence may be homologous or heterologous to the ART polypeptide, and may 
be homologous or heterologous to the ART polypeptide. Additionally, the 
signal sequence may be chemically synthesized using methods set forth 
above. In most cases, secretion of the polypeptide from the host cell via 
the presence of a signal peptide will result in the removal of the amino 
terminal methionine from the polypeptide. 
In many cases, transcription of the ART polypeptide is increased by the 
presence of one or more introns on the vector; this is particularly true 
for eukaryotic host cells, especially mammalian host cells. The intron may 
be naturally occurring within the ART nucleic acid sequence, especially 
where the ART sequence used is a full length genomic sequence or a 
fragment thereof. Where the intron is not naturally occurring within the 
ART DNA sequence (as for most cDNAs), the intron(s) may be obtained from 
another source. The position of the intron with respect to the 5' flanking 
sequence and the ART coding sequence is important, as the intron must be 
transcribed to be effective. As such, where the ART nucleic acid sequence 
is a cDNA sequence, the preferred position for the intron is 3' to the 
transcription start site, and 5' to the polyA transcription termination 
sequence. Preferably for ART cDNAs, the intron will be located on one side 
or the other (i.e., 5' or 3') of the ART coding sequence such that it does 
not interrupt the this coding sequence. Any intron from any source, 
including any viral, prokaryotic and eukaryotic (plant or animal) 
organisms, may be used to practice this invention, provided that it is 
compatible with the host cell(s) into which it is inserted. Also included 
herein are synthetic introns. Optionally, more than one intron may be used 
in the vector. 
Where one or more of the elements set forth above are not already present 
in the vector to be used, they may be individually obtained and ligated 
into the vector. Methods used for obtaining each of the elements are well 
known to the skilled artisan and are comparable to the methods set forth 
above (i.e., synthesis of the DNA, library screening, and the like). 
The final vectors used to practice this invention are typically constructed 
from a starting vectors such as a commercially available vector. Such 
vectors may or may not contain some of the elements to be included in the 
completed vector. If none of the desired elements are present in the 
starting vector, each element may be individually ligated into the vector 
by cutting the vector with the appropriate restriction endonuclease(s) 
such that the ends of the element to be ligated in and the ends of the 
vector are compatible for ligation. In some cases, it may be necessary to 
"blunt" the ends to be ligated together in order to obtain a satisfactory 
ligation. Blunting is accomplished by first filling in "sticky ends" using 
Klenow DNA polymerase or T4 DNA polymerase in the presence of all four 
nucleotides. This procedure is well known in the art and is described for 
example in Sambrook et al., supra. 
Alternatively, two or more of the elements to be inserted into the vector 
may first be ligated together (if they are to be positioned adjacent to 
each other) and then ligated into the vector. 
One other method for constructing the vector to conduct all ligations of 
the various elements simultaneously in one reaction mixture. Here, many 
nonsense or nonfunctional vectors will be generated due to improper 
ligation or insertion of the elements, however the functional vector may 
be identified and selected by restriction endonuclease digestion. 
Preferred vectors for practicing this invention are those which are 
compatible with bacterial, insect, and mammalian host cells. Such vectors 
include, inter alia, pCRII (Invitrogen Company, San Diego, Calif.), pBSII 
(Stratagene Company, LaJolla, Calif.), and pETL (BlueBacII; Invitrogen). 
After the vector has been constructed and a ART nucleic acid has been 
inserted into the proper site of the vector, the completed vector may be 
inserted into a suitable host cell for amplification and/or ART 
polypeptide expression. 
Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic 
host cells (such as a yeast cell, an insect cell, or a vertebrate cell). 
The host cell, when cultured under appropriate conditions, can synthesize 
ART protein which can subsequently be collected from the culture medium 
(if the host cell secretes it into the medium) or directly from the host 
cell producing it (if it is not secreted). After collection, the ART 
protein can be purified using methods such as molecular sieve 
chromatography, affinity chromatography, and the like. 
Selection of the host cell will depend in part on whether the ART protein 
is to be glycosylated or phosphorylated (in which case eukaryotic host 
cells are preferred), and the manner in which the host cell is able to 
"fold" the protein into its native tertiary structure (e.g., proper 
orientation of disulfide bridges, etc.) such that biologically active 
protein is prepared by the cell. However, where the host cell does not 
synthesize biologically active ART, the ART may be "folded" after 
synthesis using appropriate chemical conditions as discussed below. 
Suitable cells or cell lines may be mammalian cells, such as Chinese 
hamster ovary cells (CHO) or 3T3 cells. The selection of suitable 
mammalian host cells and methods for transformation, culture, 
amplification, screening and product production and purification are known 
in the art. Other suitable mammalian cell lines, are the monkey COS-1 and 
COS-7 cell lines, and the CV-1 cell line. Further exemplary mammalian host 
cells include primate cell lines and rodent cell lines, including 
transformed cell lines. Normal diploid cells, cell strains derived from in 
vitro culture of primary tissue, as well as primary explants, are also 
suitable. Candidate cells may be genotypically deficient in the selection 
gene, or may contain a dominantly acting selection gene. Other suitable 
mammalian cell lines include but are not limited to, HeLa, mouse L-929 
cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK 
hamster cell lines. 
Similarly useful as host cells suitable for the present invention are 
bacterial cells. For example, the various strains of E. coli (e.g., HB101, 
DH5.alpha.,DH10, and MC1061) are well-known as host cells in the field of 
biotechnology. Various strains of B. subtilis, Pseudomonas spp., other 
Bacillus spp., Streptomyces spp., and the like may also be employed in 
this method. 
Many strains of yeast cells known to those skilled in the art are also 
available as host cells for expression of the polypeptides of the present 
invention. Additionally, where desired, insect cells may be utilized as 
host cells in the method of the present invention (Miller et al., Genetic 
Engineering 8:277-298 1986!). 
Insertion (also referred to as "transformation" or "transfection") of the 
vector into the selected host cell may be accomplished using such methods 
as calcium chloride, electroporation, microinjection, lipofection or the 
DEAE-dextran method. The method selected will in part be a function of the 
type of host cell to be used. These methods and other suitable methods are 
well known to the skilled artisan, and are set forth, for example, in 
Sambrook et al., supra. 
The host cells containing the vector (i.e., transformed or transfected) may 
be cultured using standard media well known to the skilled artisan. The 
media will usually contain all nutrients necessary for the growth and 
survival of the cells. Suitable media for culturing E. coli cells are for 
example, Luria Broth (LB) and/or Terrific Broth (TB). Suitable media for 
culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may be 
supplemented with serum and/or growth factors as required by the 
particular cell line being cultured. A suitable medium for insect cultures 
is Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, 
and/or fetal calf serum as necessary. 
Typically, an antibiotic or other compound useful for selective growth of 
the transformed cells only is added as a supplement to the media. The 
compound to be used will be dictated by the selectable marker element 
present on the plasmid with which the host cell was transformed. For 
example, where the selectable marker element is kanamycin resistance, the 
compound added to the culture medium will be kanamycin. 
The amount of ART polypeptide produced in the host cell can be evaluated 
using standard methods known in the art. Such methods include, without 
limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, 
non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, 
and/or activity assays such as DNA binding gel shift assays. 
If the ART polypeptide has been designed to be secreted from the host 
cells, the majority of polypeptide will likely be found in the cell 
culture medium. Polypeptides prepared in this way will typically not 
possess an amino terminal methionine, as it is removed during secretion 
from the cell. If however, the ART polypeptide is not secreted from the 
host cells, it will be present in the cytoplasm (for eukaryotic, gram 
positive bacteria, and insect host cells) or in the periplasm (for gram 
negative bacteria host cells) and may have an amino terminal methionine. 
For intracellular ART protein, the host cells are typically first disrupted 
mechanically or osmotically to release the cytoplasmic contents into a 
buffered solution. ART polypeptide can then be isolated from this 
solution. 
Purification of ART polypeptide from solution can be accomplished using a 
variety of techniques. If the polypeptide has been synthesized such that 
it contains a tag such as Hexahistidine (ART/hexaHis) or other small 
peptide at either its carboxyl or amino terminus, it may essentially be 
purified in a one-step process by passing the solution through an affinity 
column where the column matrix has a high affinity for the tag or for the 
polypeptide directly (i.e., a monoclonal antibody specifically recognizing 
ART). For example, polyhistidine binds with great affinity and specificity 
to nickel, thus an affinity column of nickel (such as the Qiagen nickel 
columns) can be used for purification of ART/polyHis. (See for example, 
Ausubel et al., eds., Current Protocols in Molecular Biology, Section 
10.11.8, John Wiley & Sons, New York 1993!). 
Where the ART polypeptide has no tag and no antibodies are available, other 
well known procedures for purification can be used. Such procedures 
include, without limitation, ion exchange chromatography, molecular sieve 
chromatography, HPLC, native gel electrophoresis in combination with gel 
elution, and preparative isoelectric focusing ("Isoprime" 
machine/technique, Hoefer Scientific). In some cases, two or more of these 
techniques may be combined to achieve increased purity. Preferred methods 
for purification include polyHistidine tagging and ion exchange 
chromatography in combination with preparative isoelectric focusing. 
If it is anticipated that the ART polypeptide will be found primarily in 
the periplasmic space of the bacteria or the cytoplasm of eukaryotic 
cells, the contents of the periplasm or cytoplasm, including inclusion 
bodies (e.g., gram-negative bacteria) if the processed polypeptide has 
formed such complexes, can be extracted from the host cell using any 
standard technique known to the skilled artisan. For example, the host 
cells can be lysed to release the contents of the periplasm by French 
press, homogenization, and/or sonication. The homogenate can then be 
centrifuged. 
If the ART polypeptide has formed inclusion bodies in the periplasm, the 
inclusion bodies can often bind to the inner and/or outer cellular 
membranes and thus will be found primarily in the pellet material after 
centrifugation. The pellet material can then be treated with a chaotropic 
agent such as guanidine or urea to release, break apart, and solubilize 
the inclusion bodies. The ART polypeptide in its now soluble form can then 
be analyzed using gel electrophoresis, immunoprecipitation or the like. If 
it is desired to isolate the ART polypeptide, isolation may be 
accomplished using standard methods such as those set forth below and in 
Marston et al. (Meth. Enz., 182:264-275 1990!). 
If ART polypeptide inclusion bodies are not formed to a significant degree 
in the periplasm of the host cell, the ART polypeptide will be found 
primarily in the supernatant after centrifugation of the cell homogenate, 
and the ART polypeptide can be isolated from the supernatant using methods 
such as those set forth below. 
In those situations where it is preferable to partially or completely 
isolate the ART polypeptide, purification can be accomplished using 
standard methods well known to the skilled artisan. Such methods include, 
without limitation, separation by electrophoresis followed by 
electroelution, various types of chromatography (immunoaffinity, molecular 
sieve, and/or ion exchange), and/or high pressure liquid chromatography. 
In some cases, it may be preferable to use more than one of these methods 
for complete purification. 
In addition to preparing and purifying ART polypeptide using recombinant 
DNA techniques, the ART polypeptides, fragments, and/or derivatives 
thereof may be prepared by chemical synthesis methods (such as solid phase 
peptide synthesis) using methods known in the art such as those set forth 
by Merrifield et al., (J. Am. Chem. Soc., 85:2149 1964!), Houghten et al. 
(Proc Natl Acad. Sci. USA, 82:5132 1985!), and Stewart and Young (Solid 
Phase Peptide Synthesis, Pierce Chem Co, Rockford, Ill. 1984!). Such 
polypeptides may be synthesized with or without a methionine on the amino 
terminus. Chemically synthesized ART polypeptides or fragments may be 
oxidized using methods set forth in these references to form disulfide 
bridges. The ART polypeptides or fragments may be employed as biologically 
active or immunological substitutes for natural, purified ART polypeptides 
in therapeutic and immunological processes. 
Chemically modified ART compositions (i.e., "derivatives") where the ART 
polypeptide is linked to a polymer ("ART-polymers") are included within 
the scope of the present invention. The polymer selected is typically 
water soluble so that the protein to which it is attached does not 
precipitate in an aqueous environment, such as a physiological 
environment. The polymer selected is usually modified to have a single 
reactive group, such as an active ester for acylation or an aldehyde for 
alkylation, so that the degree of polymerization may be controlled as 
provided for in the present methods. A preferred reactive aldehyde is 
polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 
alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714). The 
polymer may be branched or unbranched. Included within the scope of 
ART-polymers is a mixture of polymers. Preferably, for therapeutic use of 
the end-product preparation, the polymer will be pharmaceutically 
acceptable. The water soluble polymer or mixture thereof may be selected 
from the group consisting of, for example, polyethylene glycol (PEG), 
monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate 
based polymers, poly- (N-vinyl pyrrolidone) polyethylene glycol, propylene 
glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, 
polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol. For the 
acylation reactions, the polymer(s) selected should have a single reactive 
ester group. For reductive alkylation, the polymer(s) selected should have 
a single reactive aldehyde group. The polymer may be of any molecular 
weight, and may be branched or unbranched. 
Pegylation of ART may be carried out by any of the pegylation reactions 
known in the art, as described for example in the following references: 
Focus on Growth Factors 3 (2): 4-10 (1992); EP 0 154 316; and EP 0 401 
384. Preferably, the pegylation is carried out via an acylation reaction 
or an alkylation reaction with a reactive polyethylene glycol molecule (or 
an analogous reactive water-soluble polymer) as described below. 
Pegylation by acylation generally involves reacting an active ester 
derivative of polyethylene glycol (PEG) with an ART protein. Any known or 
subsequently discovered reactive PEG molecule may be used to carry out the 
pegylation of ART. A preferred activated PEG ester is PEG esterified to 
N-hydroxysuccinimide ("NHS"). As used herein, "acylation" is contemplated 
to include without limitation the following types of linkages between ART 
and a water soluble polymer such as PEG: amide, carbamate, urethane, and 
the like, as described in Bioconjugate Chem. 5:133-140 (1994). Reaction 
conditions may be selected from any of those known in the pegylation art 
or those subsequently developed, provided that conditions such as 
temperature, solvent, and pH that would inactivate the ART species to be 
modified are avoided. 
Pegylation by acylation usually results in a poly-pegylated ART product, 
wherein the lysine .epsilon.-amino groups are pegylated via an acyl 
linking group. Preferably, the connecting linkage will be an amide. Also 
preferably, the resulting product will be at least about 95 percent mono, 
di- or tri- pegylated. However, some species with higher degrees of 
pegylation (up to the maximum number of lysine .epsilon.-amino acid groups 
of ART plus one .alpha.-amino group at the amino terminus of ART) will 
normally be formed in amounts depending on the specific reaction 
conditions used. If desired, more purified pegylated species may be 
separated from the mixture, particularly unreacted species, by standard 
purification techniques, including, among others, dialysis, salting-out, 
ultrafiltration, ion-exchange chromatography, gel filtration 
chromatography and electrophoresis. 
Pegylation by alkylation generally involves reacting a terminal aldehyde 
derivative of PEG with a protein such as ART in the presence of a reducing 
agent. Regardless of the degree of pegylation, the PEG groups are 
preferably attached to the protein via a --CH.sub.2 --NH-- group. With 
particular reference to the --CH.sub.2 -- group, this type of linkage is 
referred to herein as an "alkyl" linkage. 
Derivatization via reductive alkylation to produce a monopegylated product 
exploits the differential reactivity of different types of primary amino 
groups (lysine versus the N-terminal) available for derivatization in ART. 
Typically, the reaction is performed at a pH (see below) which allows one 
to take advantage of the pK.sub.a differences between the .epsilon.-amino 
groups of the lysine residues and that of the .alpha.-amino group of the 
N-terminal residue of the protein. By such selective derivatization, 
attachment of a water soluble polymer that contains a reactive group such 
as an aldehyde, to a protein is controlled: the conjugation with the 
polymer occurs predominantly at the N-terminus of the protein without 
significant modification of other reactive groups such as the lysine side 
chain amino groups. The present invention provides for a substantially 
homogeneous preparation of ART-monopolymer protein conjugate molecules 
(meaning ART protein to which a polymer molecule has been attached 
substantially only (i.e., at least about 95%) in a single location on the 
ART protein. More specifically, if polyethylene glycol is used, the 
present invention also provides for pegylated ART protein lacking possibly 
antigenic linking groups, and having the polyethylene glycol molecule 
directly coupled to the ART protein. 
A particularly preferred water-soluble polymer for use herein is 
polyethylene glycol, abbreviated PEG. As used herein, polyethylene glycol 
is meant to encompass any of the forms of PEG that have been used to 
derivatize other proteins, such as mono-(C1-C10) alkoxy- or 
aryloxy-polyethylene glycol. 
In general, chemical derivatization may be performed under any suitable 
conditions used to react a biologically active substance with an activated 
polymer molecule. Methods for preparing pegylated ART will generally 
comprise the steps of (a) reacting an ART polypeptide with polyethylene 
glycol (such as a reactive ester or aldehyde derivative of PEG) under 
conditions whereby ART becomes attached to one or more PEG groups, and (b) 
obtaining the reaction product(s). In general, the optimal reaction 
conditions for the acylation reactions will be determined based on known 
parameters and the desired result. For example, the larger the ratio of 
PEG: protein, the greater the percentage of poly-pegylated product. 
Reductive alkylation to produce a substantially homogeneous population of 
mono-polymer/ART protein conjugate molecule will generally comprise the 
steps of: (a) reacting a ART protein with a reactive PEG molecule under 
reductive alkylation conditions, at a pH suitable to permit selective 
modification of the .alpha.-amino group at the amino terminus of said ART 
protein; and (b) obtaining the reaction product(s). 
For a substantially homogeneous population of mono-polymer/ART protein 
conjugate molecules, the reductive alkylation reaction conditions are 
those which permit the selective attachment of the water soluble polymer 
moiety to the N-terminus of ART. Such reaction conditions generally 
provide for pKa differences between the lysine amino groups and the 
.alpha.-amino group at the N-terminus (the pKa being the pH at which 50% 
of the amino groups are protonated and 50% are not). The pH also affects 
the ratio of polymer to protein to be used. In general, if the pH is 
lower, a larger excess of polymer to protein will be desired (i.e., the 
less reactive the N-terminal .alpha.-amino group, the more polymer needed 
to achieve optimal conditions). If the pH is higher, the polymer:protein 
ratio need not be as large (i.e., more reactive groups are available, so 
fewer polymer molecules are needed). For purposes of the present 
invention, the pH will generally fall within the range of 3-9, preferably 
3-6. 
Another important consideration is the molecular weight of the polymer. In 
general, the higher the molecular weight of the polymer, the fewer number 
of polymer molecules which may be attached to the protein. Similarly, 
branching of the polymer should be taken into account when optimizing 
these parameters. Generally, the higher the molecular weight (or the more 
branches) the higher the polymer:protein ratio. In general, for the 
pegylation reactions contemplated herein, the preferred average molecular 
weight is about 2 kDa to about 100 kDa (the term "about" indicating .+-.30 
kDa). The preferred average molecular weight is about 5 kDa to about 50 
kDa, particularly preferably about 12 kDa to about 25 kDa. The ratio of 
water-soluble polymer to ART protein will generally range from 1:1 to 
100:1, preferably (for polypegylation) 1:1 to 20:1 and (for 
monopegylation) 1:1 to 5:1. 
Using the conditions indicated above, reductive alkylation will provide for 
selective attachment of the polymer to any ART protein having an 
.alpha.-amino group at the amino terminus, and provide for a substantially 
homogenous preparation of monopolymer/ART protein conjugate. The term 
"monopolymer/ART protein conjugate" is used here to mean a composition 
comprised of a single polymer molecule attached to an ART protein 
molecule. The monopolymer/ART protein conjugate preferably will have a 
polymer molecule located at the N-terminus, but not on lysine amino side 
groups. The preparation will preferably be greater than 90% 
monopolymer/ART protein conjugate, and more preferably greater than 95% 
monopolymer ART protein conjugate, with the remainder of observable 
molecules being unreacted (i.e., protein lacking the polymer moiety). The 
examples below provide for a preparation which is at least about 90% 
monopolymer/protein conjugate, and about 10% unreacted protein. The 
monopolymer/protein conjugate has biological activity. 
For the present reductive alkylation, the reducing agent should be stable 
in aqueous solution and preferably be able to reduce only the Schiff base 
formed in the initial process of reductive alkylation. Preferred reducing 
agents may be selected from the group consisting of sodium borohydride, 
sodium cyanoborohydride, dimethylamine borane, trimethylamine borane and 
pyridine borane. A particularly preferred reducing agent is sodium 
cyanoborohydride. 
Other reaction parameters, such as solvent, reaction times, temperatures, 
etc., and means of purification of products, can be determined based on 
the published information relating to derivatization of proteins with 
water soluble polymers. 
A mixture of polymer-ART protein conjugate molecules may be prepared by 
acylation and/or alkylation methods, as described above,and one may select 
the proportion of monopolymer/protein conjugate to include in the mixture. 
Thus, where desired, a mixture of various protein with various numbers of 
polymer molecules attached (i.e., di-, tri-, tetra-, etc.) may be prepared 
and combined with the monopolymer/ART protein conjugate material prepared 
using the present methods. 
Generally, conditions which may be alleviated or modulated by 
administration of the present polymer/ART include those described herein 
for ART molecules in general. However, the polymer/ART molecules disclosed 
herein may have additional activities, enhanced or reduced activities, or 
other characteristics, as compared to the non-derivatized molecules. 
ART nucleic acid molecules, fragments, and/or derivatives that do not 
themselves encode polypeptides that are active in activity assays may be 
useful as hybridization probes in diagnostic assays to test, either 
qualitatively or quantitatively, for the presence of ART DNA or RNA in 
mammalian tissue or bodily fluid samples. 
ART polypeptide fragments and/or derivatives that are not themselves active 
in activity assays may be useful as modulators (e.g., inhibitors or 
stimulants) of the ART receptors in vitro or in vivo, or to prepare 
antibodies to ART polypeptides. 
The ART polypeptides and fragments thereof, whether or not chemically 
modified, may be employed alone, or in combination with other 
pharmaceutical compositions such as, for example, neurotrophic factors, 
cytokines, interferons, interleukins, growth factors, antibiotics, 
anti-inflammatories, neurotransmitter receptor agonists or antagonists 
and/or antibodies, in the treatment of endocrine system disorders. 
The ART polypeptides and/or fragments thereof may be used to prepare 
antibodies generated by standard methods. Thus, antibodies that react with 
the ART polypeptides, as well as reactive fragments of such antibodies, 
are also contemplated as within the scope of the present invention. The 
antibodies may be polyclonal, monoclonal, recombinant, chimeric, 
single-chain and/or bispecific, etc. The antibody fragments may be any 
fragment that is reactive with the ART of the present invention, such as, 
F.sub.ab, F.sub.ab', etc. Also provided by this invention are the 
hybridomas generated by presenting ART or a fragment thereof as an antigen 
to a selected mammal, followed by fusing cells (e.g., spleen cells) of the 
animal with certain cancer cells to create immortalized cell lines by 
known techniques. The methods employed to generate such cell lines and 
antibodies directed against all or portions of a human ART polypeptide of 
the present invention are also encompassed by this invention. 
The antibodies may be used therapeutically, such as to inhibit binding of 
the ART to its receptor. The antibodies may further be used for in vivo 
and in vitro diagnostic purposes, such as in labeled form to detect the 
presence of the ART in a body fluid. 
Therapeutic Compositions and Administration 
Therapeutic compositions for treating various endocrine and/or 
neuro-endocrine system disorders such as glucocorticoid resistance, 
Cushing's syndrome (either genetic or caued by ectopic ACTH production due 
to pituitary tumors, small lung carcinomas, or adrenal tumors), congenital 
adrenal hyperplasia, other disorders of the hypothalamic-pitutary axis 
(HPA), and/or obesity are within the scope of the present invention. Such 
compositions may comprise a therapeutically effective amount of a ART 
polypeptide or fragment thereof (either of which may be chemically 
modified) in admixture with a pharmaceutically acceptable carrier. The 
carrier material may be water for injection, preferably supplemented with 
other materials common in solutions for administration to mammals. 
Typically, a ART therapeutic compound will be administered in the form of 
a composition comprising purified protein (which may be chemically 
modified) in conjunction with one or more physiologically acceptable 
carriers, excipients, or diluents. Neutral buffered saline or saline mixed 
with serum albumin are exemplary appropriate carriers. Preferably, the 
product is formulated as a lyophilizate using appropriate excipients 
(e.g., sucrose). Other standard carriers, diluents, and excipients may be 
included as desired. Other exemplary compositions comprise Tris buffer of 
about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further 
include sorbitol or a suitable substitute therefor. 
The ART compositions can be systemically administered parenterally. 
Alternatively, the compositions may be administered intravenously or 
subcutaneously. When systemically administered, the therapeutic 
compositions for use in this invention may be in the form of a 
pyrogen-free, parenterally acceptable aqueous solution. The preparation of 
such pharmaceutically acceptable protein solutions, with due regard to pH, 
isotonicity, stability and the like, is within the skill of the art. 
Therapeutic formulations of ART compositions useful for practicing the 
present invention may be prepared for storage by mixing the selected 
composition having the desired degree of purity with optional 
physiologically acceptable carriers, excipients, or stabilizers 
(Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, ed., 
Mack Publishing Company 1990!) in the form of a lyophilized cake or an 
aqueous solution. Acceptable carriers, excipients or stabilizers are 
nontoxic to recipients and are preferably inert at the dosages and 
concentrations employed, and include buffers such as phosphate, citrate, 
or other organic acids; antioxidants such as ascorbic acid; low molecular 
weight polypeptides; proteins, such as serum albumin, gelatin, or 
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino 
acids such as glycine, glutamine, asparagine, arginine or lysine; 
monosaccharides, disaccharides, and other carbohydrates including glucose, 
mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such 
as mannitol or sorbitol; salt-forming counterions such as sodium; and/or 
nonionic surfactants such as Tween, Pluronics or polyethylene glycol 
(PEG). 
The ART composition to be used for in vivo administration must be sterile. 
This is readily accomplished by filtration through sterile filtration 
membranes. Where the ART composition is lyophilized, sterilization using 
these methods may be conducted either prior to, or following, 
lyophilization and reconstitution. The composition for parenteral 
administration ordinarily will be stored in lyophilized form or in 
solution. 
Therapeutic compositions generally are placed into a container having a 
sterile access port, for example, an intravenous solution bag or vial 
having a stopper pierceable by a hypodermic injection needle. 
The route of administration of the composition is in accord with known 
methods, e.g. oral, injection or infusion by intravenous, intraperitoneal, 
intracerebral (intraparenchymal), intracerebroventricular, intramuscular, 
intraocular, intraarterial, or intralesional routes, or by sustained 
release systems or implantation device which may optionally involve the 
use of a catheter. Where desired, the compositions may be administered 
continuously by infusion, bolus injection or by implantation device. 
Alternatively or additionally, ART may be administered locally via 
implantation into the affected area of a membrane, sponge, or other 
appropriate material on to which ART polypeptide has been absorbed. 
Where an implantation device is used, the device may be implanted any 
suitable tissue or organ, such as, for example, into a cerebral ventricle 
or into brain parenchyma, and delivery of ART may be directly through the 
device via bolus or continuous administration, or via a catheter using 
continuous infusion. 
ART polypeptide may be administered in a sustained release formulation or 
preparation. Suitable examples of sustained-release preparations include 
semipermeable polymer matrices in the form of shaped articles, e.g. films, 
or microcapsules. Sustained release matrices include polyesters, 
hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers 
of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 
22:547-556 1983!), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. 
Biomed. Mater. Res., 15:167-277 1981! and Langer, Chem. Tech., 12:98-105 
1982!), ethylene vinyl acetate (Langer et al., supra) or 
poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release 
compositions also may include liposomes, which can be prepared by any of 
several methods known in the art (e.g., DE 3,218,121; Epstein et al., 
Proc. Natl. Acad. Sci. USA, 82:3688-3692 1985!; Hwang et al., Proc. Natl. 
Acad. Sci. USA, 77:4030-4034 1980!; EP 52,322; EP 36,676; EP 88,046; EP 
143,949). 
In some cases, it may be desirable to use ART compositions in an ex vivo 
manner, i.e., to treat cells or tissues that have been removed from the 
patient and are then subsequently implanted back into the patient. 
In other cases, ART may be delivered through implanting into patients 
certain cells that have been genetically engineered (using methods 
described above) to express and secrete ART polypeptide. Such cells may be 
human cells, and may be derived from the patient's own tissue or from 
another source, either human or non-human. Optionally, the cells may be 
immortalized. The cells may be implanted into the brain, adrenal gland or 
into other body tissues or organs. 
In certain situations, it may be desirable to use gene therapy methods for 
administration of ART to patients suffering from certain endocrine and/or 
neuro-endocrine system disorders or diseases such as glucocorticoid 
resistance, Cushing's syndrome (either genetic or caued by ectopic ACTH 
production due to pituitary tumors, small lung carcinomas, or adrenal 
tumors), congenital adrenal hyperplasia, other disorders of the 
hypothalamic-pitutary axis (HPA), and/or obesity. In these situations, 
genomic DNA, cDNA, and/or synthetic DNA encoding ART or a fragment or 
variant thereof may be operably linked to a constitutive or inducible 
promoter that is active in the tissue into which the composition will be 
injected. This ART DNA construct can be injected directly into brain or 
other neuronal tissue to be treated. 
Alternatively, the ART DNA construct may be injected into muscle tissue 
where it can be taken up into the cells and expressed in the cells, 
provided that the ART DNA is operably linked to a promoter that is active 
in muscle tissue such as cytomegalovirus (CMV) promoter, Rous sarcoma 
virus (RSV) promoter, or muscle creatine kinase promoter. Typically, the 
DNA construct may include (in addition to the ART DNA and a promoter), 
vector sequence obtained from vectors such as adenovirus vector, 
adeno-associated virus vector, a retroviral vector, and/or a herpes virus 
vector. The vector/DNA construct may be admixed with a pharmaceutically 
acceptable carrier(s) for injection. 
An effective amount of the ART composition(s) to be employed 
therapeutically will depend, for example, upon the therapeutic objectives 
such as the indication for which ART is being used, the route of 
administration, and the condition of the patient. Accordingly, it will be 
necessary for the therapist to titer the dosage and modify the route of 
administration as required to obtain the optimal therapeutic effect. A 
typical daily dosage may range from about 0.1 .mu.g/kg to up to 100 mg/kg 
or more, depending on the factors mentioned above. Typically, a clinician 
will administer the ART composition until a dosage is reached that 
achieves the desired effect. The ART composition may therefore be 
administered as a single dose, or as two or more doses (which may or may 
not contain the same amount of ART) over time, or as a continuous infusion 
via implantation device or catheter. 
As further studies are conducted, information will emerge regarding 
appropriate dosage levels for treatment of various conditions in various 
patients, and the ordinary skilled worker, considering the therapeutic 
context, the type of disorder under treatment, the age and general health 
of the recipient, will be able to ascertain proper dosing. Generally, the 
dosage will be between 0.01 .mu.g/kg body weight (calculating the mass of 
the protein alone, without chemical modification) and 300 .mu.g/kg (based 
on the same). 
The ART proteins, fragments and/or derivatives thereof may be utilized to 
treat diseases and disorders of the endocrine system which may be 
associated with alterations in the pattern of ART expression or which may 
benefit from exposure to ART or anti-ART antibodies. 
ART protein, and/or fragments or derivatives thereof, may be used to treat 
patients in whom various cells of the endocrine and/or nervous system have 
degenerated and/or have been damaged by congenital disease, trauma, 
surgery, stroke, ischemia, infection, metabolic disease, nutritional 
deficiency, malignancy, and/or toxic agents. 
In other embodiments of the invention, ART protein and/or fragments or 
derivatives thereof can be used to treat endocrine and/or neuro-endocrine 
system disorders or diseases such as glucocorticoid resistance, Cushing's 
syndrome (either genetic or caued by ectopic ACTH production due to 
pituitary tumors, small lung carcinomas, or adrenal tumors), congenital 
adrenal hyperplasia, other disorders of the hypothalamic-pitutary axis 
(HPA), and/or obesity. In addition, ART compositions may be useful in 
modulating intra-cellular calcium levels. 
In addition, ART protein or peptide fragments or derivatives thereof can be 
used in conjunction with surgical implantation of tissue in the treatment 
of diseases in which tissue implantation is indicated. 
The following examples are intended for illustration purposes only, and 
should not be construed as limiting the scope of the invention in any way. 
EXAMPLES 
Example I: 
Identification of Human ART cDNA 
The publicly available Washington University/Merck DNA sequence database 
referred to as the EST (Expressed Sequence Tag) database was searched with 
a sequence profile (Gribskov et al., Proc. Natl. Acad. Sci, USA, 84:4355 
1987! and Luethy et al., Protein Science, 3:139-146 1994!) using a 
sequence alignment of the human and mouse agouti genes (starting at amino 
acid 22 of both mouse and human agouti), along with the PAM250 amino acid 
substitution table (Dayhoff et al., in: Atlas of Protein Sequence and 
Structure, vol 5, supp. 3 1978!). 
In order to search the database for homologous amino acid sequences, each 
entry in the EST database was first translated by computer from DNA to 
amino acid sequence prior to searching. One EST database submission cDNA 
clone, H63735, was found to have homology to this profile sequence. The 
submission containing the sequence of the opposite end of this cDNA clone, 
H63298, was examined but did not show any homology to the profile 
sequence. 
The E. coli stock containing the cDNA clone corresponding to H63735 and 
H63298 (stock number 208641) was obtained from Genome Systems Inc., St. 
Louis, Mo. The DNA from this clone was prepared using standard miniprep 
methods (Sambrook et al., Molecular Cloning: A Laboratoy Manual, Cold 
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989!). The DNA 
was purified by passage through a Qiagen column (Qiagen, Chatsworth, 
Calif.) and following the manufacturer's protocol. After purification, the 
DNA was sequenced using the standard dideoxy chain termination method. 
When this purified DNA was digested with the restriction endonucleases 
EcoRI and HindIII, two fragments of about 1.2 and 0.3 kb were obtained, 
indicating the clone contained an insert of approximately 1.5 kb. Sequence 
from the T3 and T7 primers of the sequencing vector yielded sequence which 
was nearly identical to the submitted sequences, indicating that clone 
208641 contained the DNA used to generate submissions H63735 and H63298. 
Analysis of the full cDNA sequence of clone 208641 confirmed the presence 
of homology with the agouti gene in the cysteine-rich carboxy terminus. 
Comparison of the cDNA sequence of clone 208641 with the original sequence 
submitted in the database (H63735) revealed an error in the submitted 
sequence. Specifically, an extra guanine nucleotide was present at 
position 164 of H63735, resulting in a frameshift mutation and a premature 
in-frame termination codon when H63735 was translated. This error, when 
corrected, revealed increased homology between the profile sequence and 
H63735. Correction of this error resulted in additional sequence homology 
between clone 208641 and the agouti gene as well. However, even with the 
correction of this frame shift, the predicted protein sequence of 208641 
from the open reading frame resulted in a protein of 94 amino acids, 
compared to 132 amino acids for human agouti. In addition, the predicted 
protein homology decreased dramatically towards the amino terminus. This 
suggested that 208641 was actually not a genuine cDNA, but rather a 
partially spliced genomic intron DNA-cDNA hybrid, and this was confirmed 
when the sequence for the human genomic clone (SEQ ID NO:4) was obtained, 
as described below. 
To assess the gene expression pattern of the clone, nylon Northern blots 
containing about 2 .mu.g per lane of polyA RNA from various human tissues 
(Clontech Labs, Palo Alto, Calif.) were screened for the presence of ART 
by probing the blots with an approximately 600 base pair probe (obtained 
by digesting clone 208641 with NcoI and NotI and isolating the 600 base 
pair fragment using the Qiagen Gel Purification Kit Qiagen, Chatsworth, 
Calif.!) and following the manufacturer's protocol. This isolated 600 bp 
fragment was radioactively labelled with a-.sup.32 P-dCTP using standard 
methods (RediVue, Amersham, Arlington Heights, Ill.) in a random primed 
reaction (RediPrime, Amersham). Unincorporated radioactivity was excluded 
by size exclusion chromatography (QuickSpin columns, Boehringer-Mannheim). 
The Northern filters were hybridized overnight at about 42.degree. C. in 
buffer containing 50% formamide, 2% SDS, 10.times.Denhardts, 100 mg/ml 
salmon sperm DNA, and 5.times.SSPE. The filters were then washed in 
2.times.SSC, 0.05% SDS at room temperature for about 40 minutes with three 
changes of wash solution, followed by 30 minutes at about 50.degree. C. in 
0.1.times.SSC, 0.1%SDS. Hybridization signals were detected by placing the 
filters in a phosphoimager cassette overnight. 
Hybridization of the Northern filters with the 600 bp NcoI-NotI probe 
revealed a striking and relatively specific pattern of expression of ART, 
as is shown in FIG. 6. The most abundant site of expression was the 
adrenal cortex, followed by the adrenal medulla, hypothalamus, subthalamic 
nucleus, and testis. A weak hybridization signal was detected in lung. 
When the relative intensities of the hybridization signals were 
quantitated on a phosphoimager and expressed relative to adrenal cortex, 
the following values were obtained; adrenal cortex, 100; adrenal medulla, 
46; hypothalamus, 23; testis, 15; subthalamic nucleus, 11; and lung, 3.6. 
The filters were then probed with a beta-actin probe to verify equal 
loading of RNA and accurate placement of RNA size markers. 
Examination of the Northern blot with reference to the size markers 
revealed an interesting difference in transcript length of ART between 
brain and peripheral tissues, which could be due to alternative exon 
splicing. The transcript size was approximately 0.8 kb for the brain 
tissues, while the peripheral tissues had a smaller transcript of 
approximately 0.5 kb. To resolve whether this represented the alternative 
splicing of coding and/or untranslated exons, the cDNA from both 
subthalamic nucleus and adrenal gland was cloned as described below. 
Initial attempts to clone the full length cDNA using standard phage 
libraries were unsuccessful, which was most likely due to the small 
transcript size being excluded during the preparation of such libraries. 
Accordingly, a more sophisticated and technically challenging cloning 
method utilizing PCR was attempted. To obtain the full-length human cDNA 
clone corresponding to clone 208641, human polyA RNA from adrenal gland, 
subthalamic nucleus, and lung (Clontech, Palo Alto, Calif.; catalog 
numbers 6571-1, 6581-1, and 6524-1, respectively) was reverse transcribed, 
second strand cDNA was synthesized, and ligated to adaptor primers using 
the Marathon cDNA amplification kit (Clontech, Palo Alto, Calif.), 
following the manufacturer's protocol. The final cDNA products were 
purified from unligated adaptor primers (PCR Clean-up kit, Qiagen, 
Chatsworth, Calif.), and used as templates for subsequent RACE reactions 
using PCR. PCR was performed for each cDNA using the following primers: 
EQU CCATCCTAATACGACTCACTATAGGGC (SEQ ID NO:1) 
EQU TAGCCCCGACCCTGACGTTGGC (SEQ ID NO:2) 
and using the Advantage PCR kit components (Clontech, Palo Alto, Calif.). 
Following an initial denaturation step (94.degree. C. for 3 minutes), the 
reactions were cycled 5 times at 94.degree. C. for 15 seconds and then 
72.degree. C. for 2 minutes; 5 times at 94.degree. C. for 15 seconds and 
then 70.degree. C. for 2 minutes; and 25 cycles at 94.degree. C. for 15 
seconds and then 68.degree. C. for 2 minutes. All reactions were conducted 
on a Perkin Elmer 2400 PCR machine. 
An aliquot of each PCR reaction mix was electrophoresed on an agarose gel, 
and the bands migrating at approximately 600 base pairs were excised and 
purified (Gel Extraction kit, Qiagen, Chatsworth, Calif.) and used as a 
template for subsequent PCR using the primer SEQ ID NO:2 and the primer: 
EQU ACTCACTATAGGGCTCGAGCGGC (SEQ ID NO:3) 
The PCR conditions were the same as described above. 
An aliquot of this second PCR reaction was electrophoresed on agarose, and 
the bands migrating at approximately 600 base pairs were excised, 
purified, and cloned into a plasmid (TA Cloning kit, Invitrogen, San 
Diego, Calif.). Bacterial host cells were then transformed with the 
plasmid, and grown overnight for DNA purification. The plasmid DNA was 
then isolated from the bacteria host cells using the Qiagen miniprep 
protocol, digested with EcoRI, and electrophoresed to confirm the presence 
and size of the inserts. Clones containing a variety of insert sizes were 
sequenced using various T7 and M13 primers. Sequencing of the clones 
indicated a polymorphism in the second position of codon 135 corresponding 
to the predicted amino acids Leu (CTG) or Pro (CCG; see FIGS. 4 and 9). 
The sequences obtained were used to determine which clones had inserts 
that contained ART cDNA, and to design oligonucleotide primers to the 5' 
portion of the ART cDNA. When a number of these inserts were sequenced, 
only the larger insert sizes of 700 bp and 500 bp for the subthalamic 
nucleus and adrenal gland, respectively, contained ART transcripts. Both 
of these inserts contained the same open reading frame (ORF), but differed 
in the amount of 5' untranslated region. This ORF matched the seqence from 
208641 in the 3' region. 
For the 3' RACE reaction, an oligonucleotide on the forward strand that 
overlapped the 5' RACE product by about 180 bp was used with SEQ ID NO:1. 
This resulted in the same sized amplicon (about 300 bp) from all three 
tissues. The sequence from this amplicon was the same from all three 
tissues, and also matched the sequence from clone 208641. The sequence of 
the adrenal gland and lung ("peripheral tissues") ART cDNA is shown in 
FIG. 3 (SEQ ID NO:6). 
The combined sequence from the subthalamic nucleus RACE reactions is shown 
in FIG. 2 (SEQ ID NO:5). As mentioned above, the sequence from the adrenal 
gland and lung was identical to this sequence except for the length of the 
5' untranslated region. This cDNA sequence contains in-frame termination 
codons from the presumed translation start site, a polyadenylation signal, 
and a polyA tail. The protein predicted from this ORF contains 132 amino 
acids, a signal peptide sequence and 11 cysteines, and this sequence is 
shown in FIG. 4 (SEQ ID NO:7). The signal peptide consist of the first 20 
amino acids, and the mature polypeptide starts at amino acid 21 (Ala). 
Example II 
Identification of Human ART Genomic DNA 
High density filters spotted with DNA from human genomic DNA (obtained from 
Genome Systems Inc., St. Louis, Mo.) were hybridized with the 600 bp 
a-.sup.32 P-dCTP labelled NcoI-NotI cDNA probe (see Example I) in RapidHyb 
buffer (Amersham, Arlington Heights, Ill.; catalog number RPN 1636) at 
about 65.degree. C. for about 4 hours. The filters were then washed in 
2.times.SSC containing 0.2% SDS at room temperature for 30 minutes, and 
then in 0.2.times.SSC containing 0.2% SDS at 65.degree. C. for 30 minutes. 
The filters were placed into autoradiography cassettes with Hyperfilm 
(Amersham) and placed at -80.degree. C. overnight. The film was then 
developed, and the coordinates of P1 clones which hybridized to the probe 
were recorded. Bacterial stocks containing these positive P1 clones were 
obtained from Genome Systems Inc. and the DNA from these stocks was 
isolated (Qiagen Miniprep System, Qiagen, Chatsworth, Calif.). 
An aliquot of DNA was digested with EcoRI, electrophoresed on a 0.9% 
agarose gel, and the bands migrating at approximately 2-3 kb were excised, 
purified, and subcloned into a plasmid (Bluescript-KSII, Stratagene) 
previously digested with EcoRI. DNA was isolated from bacteria containing 
inserts (Qiagen Miniprep System), digested with EcoRI, electrophoresed, 
transferred to nylon filters (Turboblotter, S&S, Keene, NH), and UV 
cross-linked (Stratagene, La Jolla, Calif.). These filters were then 
hybridized with the NcoI-NotI probe as described above to identify clones 
which contained ART sequences. A clone containing an approximately 2.3 kb 
EcoRI fragment was found to hybridize to the ART probe. DNA from this 
clone was then sequenced, and the nucleic acid sequence of this ART 
genomic DNA is shown in FIG. 1 (SEQ ID NO:4). When the sequence from this 
genomic clone was compared to the cDNA sequence obtained from adrenal 
gland and brain, the ART coding sequence was found to be divided into 3 
exons. Furthermore, the 5' untranslated sequence present in the brain cDNA 
was found to be a separate exon, located 5' to these 3 coding exons. 
Therefore, the ART gene appears to be composed of three coding exons and a 
variably spliced untranslated exon. 
It is possible that the smaller ART transcripts that were identified in 
Northern blots of peripheral tissues are due to the absence of this 
non-coding exon. Interestingly, mouse agouti is known to use 
alternatively-spliced non-coding exons during different phases of the 
hair-growth cycle. 
Example III 
Preparation of ART Peptides 
A synthetic peptide containing amino acids 79-132 of ART was prepared using 
standard solid phase FMOC protection chemistry. The sequence of this 
peptide is set forth in FIG. 5 (SEQ ID NO:8). To refold the ART peptide, 
about 5.0 mg of lyophilized powder was dissolved in 25 ml of 20 mM 
Tris-HCl and 4M urea (pH 7.0). This mixture was stirred slowly overnight 
at room temperature. After stirring, the sample was concentrated in an 
Amicon (Beverly, Mass.) stirred cell using a cutoff membrane of 3 kDa. The 
final volume after concentration was about 1 ml. This sample was then 
diluted with about 15 ml of sterile 1.times.D-PBS (Gibco/BRL, Grand 
Island, N.Y.) , and was then reconcentrated to a final volume of about 1 
ml. The stirred cell was rinsed twice with about 2 ml of D-PBS, and this 4 
ml of solution was added to the sample. This sample solution, now about 5 
ml, was concentrated further in an Amicon Centricon 3 device to a final 
volume of about 0.5 ml (equivalent to about 10 mg/ml). The sample was then 
sterile filtered in a Costar (Cambridge, Mass.) 0.22 .mu.m Spinex filter 
device and stored at 4.degree. C. 
This peptide sample was administered to rats as described in Example V 
below. 
Example IV 
Cloning of Mouse ART Genomic DNA 
A mouse liver tissue genomic library (Stratagene, La Jolla, Calif.) was 
screened for the mouse ART genomic DNA using the 600 bp a-.sup.32 P-dCTP 
labelled NcoI-NotI cDNA probe (see Example I) in RapidHyb buffer 
(Amersham, Arlington Heights, Ill.; catalog number RPN 1636) at about 
65.degree. C. for about 4 hours. The filters were then washed in 
2.times.SSC containing 0.2% SDS at room temperature for 30 minutes, and 
then in 0.2.times.SSC containing 0.2% SDS at 65.degree. C. for 30 minutes. 
The filters were placed into autoradiography cassettes with Hyperfilm 
(Amersham, Arlington Heights, Ill.) and placed at -80.degree. C. 
overnight. The film was then developed, and one clone was identified as 
binding to the probe. 
This clone, termed m-ARTg, was plaque purified using standard methods, the 
bacteria were lysed, and the DNA was then isolated using a Qiagen 
(Chatsworth, Calif.) Maxiprep column. The purified DNA was digested with 
XbaI, and an approximaately 2.8 kb fragment was found to hybridize with 
the human ART cDNA probe. This 2.8 kb fragment was subcloned into the 
vector pBlueScript (Stratagene, La Jolla, Calif.) and sequenced. The 
coding region of this sequence is set forth in FIG. 7. The splice 
donor/acceptor sites in this gene were found to be comparable to those in 
the human ART genomic DNA, indicating that the mouse ART gene also has 
three coding exons (2, 3, and 4) and one non-coding exon (exon 1). The 
predicted amino acid sequence of mouse ART is shown in FIG. 8. This 
sequence is about 81 percent identical to the human ART polypeptide 
sequence. 
Example V 
Feeding Behavior of Rats Treated With ART 
Long-Evans male rats weighing 300-500 grams were chronically implanted in 
the brain with a 22 gauge cannula aimed at the lateral ventricle. The 
steriotaxic coordinates for the canulas were approximately: 0.8 mm 
anterior/posterior; 1.4 mm medial/lateral; and 3.5 mm dorsal/ventral. A 
3.5 mm, 28 gauge stylet remained inside the implanted cannula until the 
animal was ready for an injection. ART peptide or control solutions were 
administered with a 28 gauge injector that extended about 1 mm beyond the 
tip of the cannula. 
ART peptide was dissolved in PBS (pH about 7.0) and injected into the 
lateral ventricle at doses ranging from about 0.075 nmol to about 7.5 nmol 
in a volume of about 2 .mu.l. Controls were PBS and an unfolded version of 
ART at about 7.5 nmol. Feeding measurements were taken from pre-weighed 
dishes containing a mixture of ground rodent chow, sugar, and condensed 
milk. (45%:28%:27%). Rats were offered this mixture along with their 
regular chow about 24 hours prior to injection. About one and one half 
hours prior to infusion, the regular chow was removed, but the rats were 
allowed to continue feeding on the sweetened mixture. Injections were done 
at 8:30 am or at 8:30 pm. Food intake was assesed by weighing the dishes 
over time at 90 minutes, 4 hours, 8 hours, 12 hours, and 24 hours after 
injection. 10-12 rats were used per group. 
The results are shown in FIG. 10. As can be seen, those rats receiving 
folded ART increased their food intake as compared to controls. Further, 
there is a correlation between the amount of ART injected and the amount 
of food eaten by the rats. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 11 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Oligonucleotide" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCATCCTAATACGACTCACTATAGGGC27 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Oligonucleotide" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
TAGCCCCGACCCTGACGTTGGC22 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: other nucleic acid 
(A) DESCRIPTION: /desc = "Oligonucleotide" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ACTCACTATAGGGCTCGAGCGGC23 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2371 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GAATTCTTGGAAGCACAGGAAACAACATGCCACATAGGGGTTGAGTAAGCATCTCTGGGG60 
CCACAAATTAAATTAAGCTTTCAGGGCCGCCTGCCTTGTTATTGCTAATGGTTCTAGCCC120 
TGCTCAGCTCCTAGGTCCCTGTCCTGTGGAAATTTGTGGACCCTGGGCACCCTCTCTTGC180 
TCCCAAATTTTAATCGGCTCCTGGAAACCTCACCCCAAATTGGAGATAGGCACTCCTCTT240 
GTAGAACAAAAGGCTCAGGTTCAGGGAGTGAGGGCCTGAACTGTGCCCCCACCCTCCAGG300 
AAGGGTCCTTCACGGCCTGGCTGCAGGGATCAGTCACGTGTGGCCCTTCATTAGGCCCTG360 
CCATATAAGCCAAGGGCACGGGGTGGCCGGGAACTCTCTAGGCAAGAATCCCGGAGGCAG420 
AGGTGAGTCCTCAGGTTGGGCAGGGACTCCTCCTCTCTGTGGGGTCTCTATCTGGGCACC480 
TAGAGGGGACTCCAAGGATAAGGAGGGACTAAGTGGTACATCTTCCTGCTGAGCCAGGCC540 
ATGCTGACCGCAGCGGTGCTGAGCTGTGCCCTGCTGCTGGCACTGCCTGCCACGCGAGGA600 
GCCCAGATGGGCTTGGCCCCCATGGAGGGCATCAGAAGGCCTGACCAGGCCCTGCTCCCA660 
GAGCTCCCAGGTCAGTGTGAGCAAGGGTGGGACTGGGCGGGGCCTGAATACCCTCTGGCC720 
ACAAATAGTCTCCCCTGGCATAAACCCTCTTTCTCCCTTCCCAAACCCTCCCCTGGGAGG780 
TGGGTGCTTTGTGCATGGGGGTTCCTGCCCTCACATCCTCTGCCCCAGGCCTGGGCCTGC840 
GGGCCCCACTGAAGAAGACAACTGCAGAACAGGCAGAAGAGGATCTGTTGCAGGAGGCTC900 
AGGCCTTGGCAGAGGTAACTGCTCAGGGAAAAGGGTAAGGTGGTGGCCCTTGGGAGGGGG960 
CATTGGGTATTAGCTCCTCTCCCCAGCTCCAAACTCCCTCACCAGCGACGACACTACCGA1020 
CCACCCCTTCCCATGCTCCACTGCCATCCTGCACAGGTTGGGACAGGTAAGATCCCTGGA1080 
TCTGTCTTTAGAGGCCTGTGCTGGTTCCCCACCCCTGCAGGTACTAGACCTGCAGGACCG1140 
CGAGCCCCGCTCCTCACGTCGCTGCGTAAGGCTGCATGAGTCCTGCCTGGGACAGCAGGT1200 
GCCTTGCTGTGACCCATGTGCCACGTGCTACTGCCGCTTCTTCAATGCCTTCTGCTACTG1260 
CCGCAAGCTGGGTACTGCCATGAATCCCTGCAGCCGCACCTAGCTGGCCAACGTCAGGGT1320 
CGGGGCTAGGGTAGGGGCAAGGAAACTCGAATAAAGGATGGGACCAACCCCAAGGCTGTG1380 
GTTATTTCAAACGTGGCCGTCAAAGGAGGGAGGGTTCATGGAGGGGGTGGGAGTGTCACC1440 
AAGCCAAGAAACCACACATACTCTTATCCCAGGGCCTGGGCTACCCTATCATAGGAGGCA1500 
CATACACGGGCGCTTTTAGGGGTCCTGGTGCCCCTGGGAAAAATAGAGAAGAGCCGCACT1560 
CCAGCTTTCGAAAATCTTGTACAGCAAGTGCGGGGAACGCAGGACGCAGCGTGGCACAGG1620 
GGCTATCACTCCTGGCTAACAAATAAGCCTTAGGCTCCAGGGCTTGCTGCTACTTCCACG1680 
CAAAGCCTGCCCCTCATCCTGTTACCAGAGGGAAGGCCAGGAGTGTGCGTTGTTCAGGTC1740 
CTTAGCGTTTCGAACAAAGAATTGAACAAAACCCAGAAAGTAACAAACGAATGACACACA1800 
GGAAGGAAGCAGACAGCTGGGATTTGTTAAAGCGAGAAAGCACTACGCAGGGTGGGAGTG1860 
GGCCTGAGCAAGAGGCTGAAGGGGCTCAGTTACAAAGTTTTCCGGGTTTTAAGTACTCCT1920 
TTTGCGGTCCCTGTCCGTTACCCCTTATCTGGATGAAGGGTTTGGTCCATGGCTAATTAA1980 
TCCATTTATGCCTGAGGTTGCAATCTTTTTGAATTTTTGCAATCAGACCTTGGCCATGAC2040 
CTTGAGCAGTAGGATATAAATAACTCCCATATGCTTAGCGTTCCAATAATGGAACACAAG2100 
GCATAAATGGGGCTAAGGTGAATTGGCGCCCTATGCAGATGAAGGGATGGCCCGTGCTTG2160 
GCCCGCAGCCAATCCAAGGCACTCTCCCTTTCAACTGAGACGTGGTGGAAGGGGGAGGGT2220 
TGTGGGGACAGTGGCCTTTGATCCTTTGTTACTTGGACATGGGGAGATGGGGTTTTTCTT2280 
TTTGGTTTAGCTTTAGTAAGCTCGCCTTAGTTGGCCTCCGGTTCCCTGCCCCCAGACCTT2340 
GGTGTTTTCCCTTGATTCAGCTTCAGAATTC2371 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 830 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGGC60 
TTGGTCCCTGTCCTGTGGAAATTTGTGGACCCTGGGCACCCTCTCTTGCTCCCAAATTTT120 
AATCGGCTCCTGGAAACCTCACCCCAAATTGGAGATAGGCACTCCTCTTGTAGAACAAAA180 
GGCTCAGGTTCAGGGAGTGAGGGCCTGAACTGTGCCCCCACCCTCCAGGAAGGGTCCTTC240 
ACGGCCTGGCTGCAGGGATCAGTCACGTGTGGCCCTTCATTAGGCCCTGCCATATAAGCC300 
AAAGGCACGGGGTGGCCGGGAACTCTCTAGGCAAGAATCCCGGAGGCAGAGGCCATGCTG360 
ACCGCAGCGGTGCTGAGCTGTGCCCTGCTGCTGGCACTGCCTGCCACGCGAGGAGCCCAG420 
ATGGGCTTGGCCCCCATGGAGGGCATCAGAAGGCCTGACCAGGCCCTGCTCCCAGAGCTC480 
CCAGGCCTGGGCCTGCGGGCCCCACTGAAGAAGACAACTGCAGAACAGGCAGAAGAGGAT540 
CTGTTGCAGGAGGCTCAGGCCTTGGCAGAGGTACTAGACCTGCAGGACCGCGAGCCCCGC600 
TCCTCACGTCGCTGCGTAAGGCTGCATGAGTCCTGCCTGGGACAGCAGGTGCCTTGCTGT660 
GACCCATGTGCCACGTGCTACTGCCGCTTCTTCAATGCCTTCTGCTACTGCCGCAAGCTG720 
GGTACTGCCATGAATCCCTGCAGCCGCACCTAGCTGGCCAACGTCAGGGTCGGGGCTAGG780 
GTAGGGGCAAGGAAACTCGAATAAAGGATGGGACCAACAAAAAAAAAAAA830 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 479 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GCCATGCTGACCGCAGCGGTGCTGAGCTGTGCCCTGCTGCTGGCACTGCCTGCCACGCGA60 
GGAGCCCAGATGGGCTTGGCCCCCATGGAGGGCATCAGAAGGCCTGACCAGGCCCTGCTC120 
CCAGAGCTCCCAGGCCTGGGCCTGCGGGCCCCACTGAAGAAGACAACTGCAGAACAGGCA180 
GAAGAGGATCTGTTGCAGGAGGCTCAGGCCTTGGCAGAGGTACTAGACCTGCAGGACCGC240 
GAGCCCCGCTCCTCACGTCGCTGCGTAAGGCTGCATGAGTCCTGCCTGGGACAGCAGGTG300 
CCTTGCTGTGACCCATGTGCCACGTGCTACTGCCGCTTCTTCAATGCCTTCTGCTACTGC360 
CGCAAGCTGGGTACTGCCATGAATCCCTGCAGCCGCACCTAGCTGGCCAACGTCAGGGTC420 
GGGGCTAGGGTAGGGGCAAGGAAACTCGAATAAAGGATGGGACCAACAAAAAAAAAAAA479 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 132 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
MetLeuThrAlaAlaValLeuSerCysAlaLeuLeuLeuAlaLeuPro 
151015 
AlaThrArgGlyAlaGlnMetGlyLeuAlaProMetGluGlyIleArg 
202530 
ArgProAspGlnAlaLeuLeuProGluLeuProGlyLeuGlyLeuArg 
354045 
AlaProLeuLysLysThrThrAlaGluGlnAlaGluGluAspLeuLeu 
505560 
GlnGluAlaGlnAlaLeuAlaGluValLeuAspLeuGlnAspArgGlu 
65707580 
ProArgSerSerArgArgCysValArgLeuHisGluSerCysLeuGly 
859095 
GlnGlnValProCysCysAspProCysAlaThrCysTyrCysArgPhe 
100105110 
PheAsnAlaPheCysTyrCysArgLysLeuGlyThrAlaMetAsnPro 
115120125 
CysSerArgThr 
130 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 54 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
ArgGluProArgSerSerArgArgCysValArgLeuHisGluSerCys 
151015 
LeuGlyGlnGlnValProCysCysAspProCysAlaThrCysTyrCys 
202530 
ArgPhePheAsnAlaPheCysTyrCysArgLysLeuGlyThrAlaMet 
354045 
AsnProCysSerArgThr 
50 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 734 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ATGCTGACTGCAATGTTGCTGAGTTGTGTTCTGCTGTTGGCACTGCCTCCCACACTGGGG60 
GTCCAGATGGGCGTGGCTCCACTGAAGGGCATCAGAAGGCCTGACCAGGCTCTGTTCCCA120 
GAGTTCCCAGGTGAGTATGGTCAGGTTGGGGATATGTGGGGCAACGACCATTGCTGGCCA180 
CAGACCTGCCCGCCCAGGCTTAGACCTCCTTCCCCAATCCCAATCCCAACCTAGGGAGGT240 
GGGTACTTGGTGCATGGTGGGTGTGGCCCTCACATCTTCTGCCCCAGGTCTAAGTCTGAA300 
TGGCCTCAAGAAGACAACTGCAGACCGAGCAGAAGAAGTTCTGCTGCAGAAGGCAGAAGC360 
TTTGGCGGAGGTAACTCATTAGGGAAAGGGATAAAGTAGAAGGTAGGGCGCATCAGATAC420 
CATCATCTCTCCCCACTTCCGGATTACCCAACCTGGGCAGAACTGCAGCCCCTCCCTGAC480 
CTCAGTCCACTGCCACCCTACTGGGGTCGGGGTTTGAGAGTTTCCTGAACCTTATTCCCC540 
TACGAATGCAGGTGCTAGATCCACAGAACCGCGAGTCTCGTTCTCCGCGTCGCTGTGTAA600 
GGCTGCACGAGTCCTGCTTGGGACAGCAGGTACCTTGCTGCGACCCGTGCGCTACGTGCT660 
ACTGCCGCTTCTTCAATGCCTTTTGCTACTGCCGCAAGCTGGGTACGGCCACGAACCTCT720 
GTAGTCGCACCTAG734 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 131 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
MetLeuThrAlaMetLeuLeuSerCysValLeuLeuLeuAlaLeuPro 
151015 
ProThrLeuGlyValGlnMetGlyValAlaProLeuLysGlyIleArg 
202530 
ArgProAspGlnAlaLeuPheProGluPheProGlyLeuSerLeuAsn 
354045 
GlyLeuLysLysThrThrAlaAspArgAlaGluGluValLeuLeuGln 
505560 
LysAlaGluAlaLeuAlaGluValLeuAspProGlnAsnArgGluSer 
65707580 
ArgSerProArgArgCysValArgLeuHisGluSerCysLeuGlyGln 
859095 
GlnValProCysCysAspProCysAlaThrCysTyrCysArgPhePhe 
100105110 
AsnAlaPheCysTyrCysArgLysLeuGlyThrAlaThrAsnLeuCys 
115120125 
SerArgThr 
130 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 132 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
MetLeuThrAlaAlaValLeuSerCysAlaLeuLeuLeuAlaLeuPro 
151015 
AlaThrArgGlyAlaGlnMetGlyLeuAlaProMetGluGlyIleArg 
202530 
ArgProAspGlnAlaLeuLeuProGluLeuProGlyProGlyLeuArg 
354045 
AlaProLeuLysLysThrThrAlaGluGlnAlaGluGluAspLeuLeu 
505560 
GlnGluAlaGlnAlaLeuAlaGluValLeuAspLeuGlnAspArgGlu 
65707580 
ProArgSerSerArgArgCysValArgLeuHisGluSerCysLeuGly 
859095 
GlnGlnValProCysCysAspProCysAlaThrCysTyrCysArgPhe 
100105110 
PheAsnAlaPheCysTyrCysArgLysLeuGlyThrAlaMetAsnPro 
115120125 
CysSerArgThr 
130 
__________________________________________________________________________