Human phospholipase inhibitor

The present invention provides a polynucleotide (gipl) the partial sequence for which was initially isolated from a THP-1 cDNA library and which identifies and encodes a novel human phospholipase inhibitor (GIPL). The invention provides for genetically engineered expression vectors and host cells comprising the nucleic acid sequence encoding GIPL.

FIELD OF THE INVENTION 
The present invention relates to nucleic acid and amino acid sequences of a 
novel human phospholipase inhibitor and to the use of these sequences in 
the diagnosis, study, prevention and treatment of disease. 
BACKGROUND OF THE INVENTION 
Phospholipase enzymes catalyze the removal of fatty acid residues from 
phosphoglycerides. Specifically, phospholipase A2 (PLA2) cleaves the ester 
bond at the 2 position of the glycerol moiety of membrane phospholipids 
giving rise to equimolar amounts of arachidonic acid and 
lysophospholipids. Although PLA2 preferentially cleaves arachidonic acid 
from phospholipids, arachidonic acid is generated secondarily from 
intermediates of the S1, phospholipase C- and phospholipase D-activated 
pathways. 
Although the known PLA2s were originally divided into groups by source 
organism, shown in parentheses, and their primary amino acid sequences, 
they are now characterized by a growing list of other attributes. Group 1 
includes the 80-90 kD PLA2s (mammalian) which are active at pH 6-8 and 
dependent on the presence of calcium ion (Ca.sup.++). Group II is a 
mixture of approximately 14 kD, secreted PLA2s (snake) which bind heparin 
and are inhibited by dexamethadone, dithiothreitol and deoxycholate. Group 
III is the cytosolic, 100 kD PLA2s (honey bee venom). The prokaryotic 
versions of PLA2 are produced by bacteria such as Streptomyces 
violaceoruber. 
Several recent scientific studies reveal pertinent facts towards the 
characterization of PLA2s and their lipolytic activity. Van den Berg B et 
al. (1995; EMBO J 14:4123-31) reported that PLA2 appears to be more active 
in the degradation of high molecular weight aggregates than of monomers. 
In vitro experiments by de Carvalho M G et al. (1995; J Biol Chem 
270:20439-46) showed that with a bilayer substrate, PLA2 preferentially 
and sequentially deacylates sn-2 and then sn-1 acyl groups. Ross et al. 
(1995; J Neurochem 64:2213-21) reported that in the temporal cortex of the 
human brain, PLA2 activity was higher in membrane fraction than in the 
cytosolic fraction. 
Arachidonic acid, the product of PLA2 activity, is processed into bioactive 
lipid mediators such as lyso-platelet-activating factor (lyso-PAF) or 
shuttled into pathways for the synthesis of eicosanoids. In fact the 
release of arachidonic acid from membrane phospholipids is the 
rate-limiting step in the biosynthesis of the four major classes of 
eicosanoids (prostaglandins, prostacyclins, thrombosanes and leukotrienes) 
involved in pain, fever, and inflammation. Furthermore, leukotriene-B4 
(LKB4) is known to function in a feedback loop which induces further 
increased PLA2 activity. 
PLA2 has many other known activators which include tumor necrosis factor 
(Jaattela M et al. (1995) Oncogene 10:2297-305); the protein phosphatase 
inhibitor, okadaic acid (Gewert K and R Sundler (1995) Biochem J 
307:499-504); the neuroleptics, fluphenazine and thioridazine (Trzeciak H 
I et al (1995) Eur Arch Psychiatry Clin Neurosci 245:179-182); the 
mammalian phospholipase A2-activating protein (PLAP; Yamada H and Bitar KN 
(1995) Biochem Biophys Res Commun 217:203-10); and the eicosanoids, LKB4, 
5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (Wijkander J 
et al (1995) J Biol Chem 270:26543-9). Epidermal growth factor 
specifically induces serine phosphorylation-dependent and 
calcium-dependent activation of cytosolic PLA2 (Schalkwijk C G et al. 
(1995) Eur J Biochem 231:593-601). 
Inhibitors of PLA2 are useful in the regulation of the signaling cascades 
that result from or correlate with PLA2 activity. One pronounced example 
of this signaling is seen in sepsis where the increase in PLA2 was found 
to be more than 12,000-fold normal and PLA2 was associated with complement 
C3-derived anaphylatoxin. PLA2 inhibitors include chemical molecules such 
as p-bromophenacyl bromide and biological molecules such as the specific 
inhibitor, thielocin A1 beta, produced by a fungus (Tanaka et al. (1995) 
Eur J Pharmacol 279:143-8) and nonspecific inhibitors such as 
glucocorticoids. 
Fortes-Dias C L et al. (1994; J Biol Chem 269:15646-51) have isolated and 
characterized a PLA2 inhibitor from the plasma of a South American 
rattlesnake, Crotalus durissus terrificus. This 20-24 kD protein, 
designated Crotalus neutralizing factor (CNF), appears to self-associate 
as a 6-8 oligomeric aggregate. The crotoxin molecule which CNF neutralizes 
is active only as a dimer and consists of an acidic molecule (CA) 
associated with one of two basic isoforms of PLA2 (CB.sub.1 and CB.sub.2). 
CNF actually displaces CA to form a stable association with one of the CB 
molecules. This displacement inactivates the neurotoxic, cardiotoxic, 
myotoxic, anticoagulent and platelet-activating activities of crotoxin. 
The full length 840 bp cDNA of CNF was cloned from Crotalus liver tissue. 
The nucleotide sequence encodes a 19 residue signal peptide and a 181 
residue mature protein with 16 cysteines, a pl of 5.45, and a possible 
glycosylation site at N.sub.157. Fortes-Dias states that the cDNA contains 
noncoding sequence and lacks a putative polyadenylation site. In 
inhibitory assays, the acidic CNF molecule also inhibits the activity of 
bee venom, and in 100-fold excess in plasma, porcine pancreatic PLA2. 
SUMMARY 
The present invention relates to a novel phospholipase inhibitor initially 
identified among the partial cDNAs from a THP-1 cell library and to the 
use of the nucleic acid and amino acid sequences in the study, diagnosis, 
prevention and treatment of disease. 
The phospholipase inhibitor of the present invention was first identified 
within Incyte Clone 156817 through a computer generated search for amino 
acid sequence alignments. The nucleic acid sequence, SEQ ID NO: 1, 
disclosed herein and designated in lower case, gipl, encodes the amino 
acid sequence, SEQ ID NO: 2, designated in upper case, GIPL. The present 
invention is based, in part, on the chemical and structural homology 
between GIPL and Crotalus phospholipase A2 inhibitor, CNF (GenBank GI 
501050; Fortes-Dias C L et al. 1994; J Biol Chem 269:15646-51). 
GIPL has 23% identity to the mature CNF molecule, and lacks homology to any 
other nucleotide or protein sequence in GenBank. GIPL is 204 amino acids 
long and lacks potential N-linked glycosylation sites. It contains 19 
cysteine residues, 16 of which align with the cysteine residues of the 
mature CNF. The nucleic acid sequence, oligonucleotides, fragments, 
portions or antisense molecules thereof, may be used in diagnostic assays 
of body fluids or biopsied tissues to detect the expression level of gipl. 
For example, gipl sequences designed from the consensus sequence or the 
contiguous sequences found in Incyte Clones 8491, 10033, 10644, 10774, 
72861, 74452, 75814, 155045, 156817, 619856, 683480 and 1291208 (SEQ ID 
NOs: 4-15) can be used to detect the presence of the mRNA transcripts in a 
patient or to monitor the decrease in transcripts during treatment. 
The nucleic acid sequence also provides for the design of antisense 
molecules useful in diminishing or eliminating expression of the genomic 
nucleotide sequence in individuals in which normal or increased 
phospholipase activity would ameliorate disease. The present invention 
also relates, in part, to the inclusion of the polynucleotide in an 
expression vector which can be used to transform host cells or organisms. 
Such transgenic hosts are useful for production of GIPL. 
The invention further provides diagnostic kits for the detection of 
naturally occurring GIPL. It provides for the use of purified GIPL to 
produce antibodies or to use in the identification of agonists which 
induce GIPL or antagonists or inhibitors which bind GIPL. Such agonists, 
antagonists or inhibitors can be delivered into the vascular system, lymph 
or cerebrospinal fluid to interact with GIPL and alter the activity of 
phospholipases. Anti-GIPL antibodies are useful in inhibition of GIPL and 
to monitor GIPL activity in biopsied tissues where GIPL is expressed. 
The invention comprises pharmaceutical compositions comprising the protein, 
antisense molecules capable of disrupting expression of the native gene, 
and agonists, antibodies, antagonists or inhibitors of the disclosed 
protein. These compositions are useful for the prevention or treatment of 
conditions such as viral, bacterial or fungal infections including septic 
and toxic shock and gangrene; autoimmune responses encompassing but not 
limited to anemias, asthma, systemic lupus, and myasthenia gravis; 
hereditary or cancerous conditions such as Alzheimer's, breast carcinoma, 
diabetes mellitus, osteoporosis, and schizophrenia; glomerulonephritis; 
pregnancy; rheumatoid and osteoarthritis; scleroderma; and insect or snake 
bites or stings in which phospholipases are a component of the injected 
venom.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a novel phospholipase inhibitor, the 
partial nucleic acid sequence for which was initially identified in Incyte 
Clone 156817 from a cDNA library (THP1PLB02) prepared from phorbol and 
lipopolysaccharide stimulated THP-1 cells and to the use of the 
polynucleotide (lower case, gipl; SEQ ID NO: 1) and polypeptide (upper 
case, GIPL; SEQ ID NO: 2) shown in FIG. 1 in the study, diagnosis, 
prevention and treatment of disease. The present invention is based, in 
part, on the chemical and structural homology between the mature proteins 
of GIPL and Crotalus phospholipase A2 inhibitor, CNF (GenBank GI 501050; 
Fortes-Dias C L et al. 1994; J Biol Chem 269:15646-51). 
GIPL has 23% identity to the known mature CNF molecule, and lacks 
statistically significant homology to any other nucleotide or protein 
sequence in GenBank. GIPL is 204 amino acids long and lacks potential 
N-linked glycosylation sites. However, it contains 19 cysteine residues, 
16 of which align with the cysteine residues of CNF. 
The nucleic acid sequence, oligonucleotides, fragments, portions or 
antisense molecules thereof, may be used in diagnostic assays of body 
fluids or biopsied tissues to detect the expression level of gipl. For 
example, gipl sequences designed from the consensus sequence SEQ ID NO: 1 
or the related sequences found in Incyte Clones 8491 (SEQ ID NO: 4, from 
cDNA library HMC1NOT01), 10033 (SEQ ID NO: 5, from cDNA library 
THP1PLB01), 10644 (SEQ ID NO: 6, from cDNA library THP1PLB01), 10774 (SEQ 
ID NO: 7, from cDNA library THP1PLB01), 72861 (SEQ ID NO: 8, from cDNA 
library THP1PEB01), 74452 (SEQ ID NO: 9, from cDNA library THP1PEB01), 
75814 ((SEQ ID NO: 10, from cDNA library THP1PEB01), 155045 (SEQ ID NO: 
11, from cDNA library THP1PIB02), 156187 (SEQ ID NO: 12, from cDNA library 
THP1PIB02), 619856 (SEQ ID NO: 13 from cDNA library PGANNOT01), 683480 
(SEQ ID NO: 14 from cDNA library UTRSNOT02) and 1291208 (SEQ ID: 15 from 
cDNA library BRAINOT11) can be used to detect the presence of the mRNA 
transcripts or to monitor the changes in the number of transcripts in a 
patient's cells or tissues during treatment. 
The nucleic acid sequence also provides for the design of antisense 
molecules useful in diminishing or eliminating expression of the 
inhibitory genomic nucleotide sequence. In individuals with Alzheimer's, 
the maintenance of normal phospholipase activity could slow down 
progression of the disease. The present invention also relates, in part, 
to the inclusion of the polynucleotide in an expression vector which can 
be used to transform host cells or organisms. Such transgenic hosts are 
useful for production of GIPL. 
The invention further provides diagnostic kits for the detection of 
naturally occurring GIPL. It provides for the use of purified GIPL to 
produce antibodies or to use in the identification of agonists which 
induce GIPL or antagonists or inhibitors which bind GIPL. Such agonists, 
antagonists or inhibitors can be delivered into the vascular system, lymph 
or cerebrospinal fluid to bind with GIPL and influence the activity of 
phospholipases. Anti-GIPL antibodies are useful in inhibition of GIPL and 
to monitor GIPL activity in biopsied tissues where GIPL is expressed. 
The invention comprises pharmaceutical compositions comprising the protein, 
antisense molecules capable of disrupting expression of the genomic 
sequence, and agonists, antibodies, antagonists or inhibitors of the 
disclosed protein. These compositions are useful for the prevention or 
treatment of conditions such as viral, bacterial or fungal infections 
including septic and toxic shock and gangrene; autoimmune responses 
encompassing but not limited to anemias, asthma, systemic lupus, and 
myasthenia gravis; hereditary or cancerous conditions such as Alzheimer's, 
breast carcinoma, diabetes mellitus, osteoporosis, and schizophrenia; 
glomerulonephritis; pregnancy; rheumatoid and osteoarthritis; scleroderma; 
and insect or snake bites or stings in which phospholipases are a 
component of the injected venom. 
The nucleotide sequences encoding GIPL (or its complement) have numerous 
applications in techniques known to those skilled in the art of molecular 
biology. These techniques include use as hybridization probes, use as 
oligomers for PCR, use for chromosome and gene mapping, use in the 
recombinant production of GIPL, and use in generation of antisense DNA or 
RNA, their chemical analogs and the like. Furthermore, the nucleotide 
sequences disclosed herein may be used in molecular biology techniques 
that have not yet been developed, provided the new techniques rely on 
properties of nucleotide sequences that are currently known such as the 
triplet genetic code, specific base pair interactions, and the like. 
It will be appreciated by those skilled in the art that as a result of the 
degeneracy of the genetic code, a multitude of GIPL-encoding nucleotide 
sequences, some bearing minimal homology to the nucleotide sequences of 
any known and naturally occurring gene may be produced. The invention has 
specifically contemplated each and every possible variation of nucleotide 
sequence that could be made by selecting combinations based on possible 
codon choices. These combinations are made in accordance with the standard 
triplet genetic code as applied to the nucleotide sequence of naturally 
occurring GIPL, and all such variations are to be considered as being 
specifically disclosed. 
Although nucleotide sequences which encode GIPL and its variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring gipl under appropriately selected conditions of 
stringency, it may be advantageous to produce nucleotide sequences 
encoding GIPL or its derivatives possessing a substantially different 
codon usage. Codons may be selected to increase the rate at which 
expression of the peptide occurs in a particular prokaryotic or eukaryotic 
expression host in accordance with the frequency with which particular 
codons are utilized by the host. Other reasons for substantially altering 
the nucleotide sequence encoding GIPL and its derivatives without altering 
the encoded amino acid sequences include the production of RNA transcripts 
having more desirable properties, such as a greater half-life, than 
transcripts produced from the naturally occurring sequence. 
The nucleotide sequences encoding GIPL may be joined to a variety of other 
nucleotide sequences by means of well established recombinant DNA 
techniques (cf Sambrook J et al. (1989) Molecular Cloning: A Laboratory 
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.). Useful 
nucleotide sequences for joining to gipl include an assortment of cloning 
vectors, e.g., plasmids, cosraids, lambda phage derivatives, phagemids, 
and the like, that are well known in the art. Vectors of interest include 
expression vectors, replication vectors, probe generation vectors, and 
sequencing vectors. In general, vectors of interest will contain an origin 
of replication functional in at least one organism, convenient restriction 
endonuclease sensitive sites, and selectable markers for the host cell. 
Another aspect of the subject invention is to provide for gipl specific 
nucleic acid hybridization probes capable of hybridizing with naturally 
occurring nucleotide sequences encoding GIPL. Such probes may also be used 
for the detection of related inhibitor encoding sequences and should 
preferably contain at least 50% of the nucleotides from any of these GIPL 
encoding sequences. The hybridization probes of the subject invention may 
be derived from the nucleotide sequence of the SEQ ID NOs: 1 and 5-12 or 
from genomic sequence including promoter, enhancer elements and introns of 
the naturally occurring gipl. Hybridization probes may be labeled by a 
variety of reporter groups, including radionuclides such as .sup.32 P or 
.sup.35 S, or enzymatic labels such as alkaline phosphatase coupled to the 
probe via avidin/biotin coupling systems, and the like. 
PCR as described U.S. Pat. Nos 4,683,195 and 4,965,188 provide additional 
uses for oligonucleotides based upon the nucleotide sequences which encode 
GIPL. Such probes used in PCR may be of recombinant origin, may be 
chemically synthesized, or a mixture of both. The probe will comprise a 
discrete nucleotide sequence for the detection of identical sequences or a 
degenerate pool of possible sequences for identification of closely 
related genomic sequences. 
Other means for producing specific hybridization probes for gipl DNAs 
include the cloning of nucleic acid sequences encoding GIPL or GIPL 
derivatives into vectors for the production of mRNA probes. Such vectors 
are known in the art and are commercially available and may be used to 
synthesize RNA probes in vitro by means of the addition of the appropriate 
RNA polymerase as T7 or SP6 RNA polymerase and the appropriate 
radioactively labeled nucleotides. 
It is now possible to produce a DNA sequence, or portions thereof, encoding 
a GIPL and its derivatives entirely by synthetic chemistry, after which 
the synthetic gene may be inserted into any of the many available DNA 
vectors and cell systems using reagents that are well known in the art at 
the time of the filing of this application. Moreover, synthetic chemistry 
may be used to introduce mutations into a gipl sequence or any portion 
thereof. 
The nucleotide sequences may be used to construct an assay to detect 
activation or induction of gipl due to inflammation or disease. The 
nucleotide sequence may be labeled by methods known in the art and added 
to a fluid or tissue sample from a patient under hybridizing conditions. 
After an incubation period, the sample is washed with a compatible fluid 
which optionally contains a dye (or other label requiring a developer) if 
the nucleotide has been labeled with an enzyme. After the compatible fluid 
is rinsed off, the dye is quantitated and compared with a standard. If the 
amount of dye in the biopsied or extracted sample is significantly 
elevated over that of a comparable control sample, the nucleotide sequence 
has hybridized with the sample, and the assay indicates the presence of 
the inducing inflammation and/or disease. 
The nucleotide sequences for gipl may be used to construct hybridization 
probes for mapping their respective genomic sequences. The nucleotide 
sequence provided herein may be mapped to a chromosome or specific regions 
of a chromosome using well known genetic and/or chromosomal mapping 
techniques. These techniques include in situ hybridization, linkage 
analysis against known chromosomal markers, hybridization screening with 
libraries or flow-sorted chromosomal preparations specific to known 
chromosomes, and the like. The technique of fluorescent in situ 
hybridization of chromosome spreads has been described, among other 
places, in Verma et al (1988) Human Chromosomes: A Manual of Basic 
Techniques, Pergamon Press, New York N.Y. 
Fluorescent in situ hybridization of chromosomal preparations and other 
physical chromosome mapping techniques may be correlated with additional 
genetic map data. Examples of genetic map data can be found in the 1994 
Genome Issue of Science (265:1981f). Correlation between the location of a 
gipl on a physical chromosomal map and a specific disease (or 
predisposition to a specific disease) may help delimit the region of DNA 
associated with that genetic disease. The nucleotide sequences of the 
subject invention may be used to detect differences in gene sequences 
between normal, carrier or affected individuals. 
The nucleotide sequence encoding GIPL may be used to produce purified GIPL 
using well known methods of recombinant DNA technology. Among the many 
publications that teach methods for the expression of genes after they 
have been isolated is Goeddel (1990) Gene Expression Technology, Methods 
and Enzymology, Vol 185, Academic Press, San Diego. GIPL may be expressed 
in a variety of host cells, either prokaryotic or eukaryotic. Host cells 
may be from the same species from which a particular gipl nucleotide 
sequence was isolated or from a different species. Advantages of producing 
GIPL by recombinant DNA technology include obtaining adequate amounts of 
the protein for purification and the availability of simplified 
purification procedures. 
Cells transformed with DNA encoding GIPL may be cultured under conditions 
suitable for the expression of GIPLs and recovery of the protein. GIPL 
produced by a recombinant cell may be secreted, contained intracellularly, 
or inserted into a membrane depending on the particular genetic 
construction used. In general, it is more convenient to prepare 
recombinant proteins in secreted form. Purification steps vary with the 
production process, the host organism and the particular protein produced. 
In addition to recombinant production, fragments of GIPL may be produced by 
direct peptide synthesis using solid-phase techniques (cf Stewart et al 
(1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; 
Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitro protein 
synthesis may be performed using manual techniques or by automation. 
Automated synthesis may be achieved, for example, using Applied Biosystems 
431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance 
with the instructions provided by the manufacturer. Various fragments of 
GIPL may be chemically synthesized separately and combined using chemical 
methods to produce the full length molecule. 
GIPL for antibody induction does not require biological activity; however, 
the protein fragment, or oligopeptide must be immunogenic. Peptides used 
to induce specific antibodies may have an amino acid sequence consisting 
of at least five amino acids, preferably at least 10 amino acids. They 
should mimic a portion of the amino acid sequence of the natural protein 
and may contain the entire amino acid sequence of a small, naturally 
occurring molecule. Short stretches of GIPL amino acids may be fused with 
those of another protein such as keyhole limpet hemocyanin and antibody 
produced against the chimeric molecule. 
Antibodies specific for GIPL may be produced by inoculation of an 
appropriate animal with the polypeptide or an antigenic fragment. An 
antibody is specific for the particular GIPL if it is produced against an 
epitope of the polypeptide and binds to at least part of the natural or 
recombinant protein. Antibody production includes not only the stimulation 
of an immune response by injection into animals, but also analogous steps 
in the production of synthetic antibodies or other specific-binding 
molecules such as the screening of recombinant immunoglobulin libraries 
(cf Orlandi R et al (1989) PNAS 86:3833-3837, or Huse W D et al (1989) 
Science 256:1275-1281) or the in vitro stimulation of lymphocyte 
populations. Current technology (Winter G and Milstein C (1991) Nature 
349:293-299) provides for a number of highly specific binding reagents 
based on the principles of antibody formation. These techniques may be 
adapted to produce molecules specifically binding GIPL. 
An additional embodiment of the subject invention is the use of GIPL 
specific antibodies, as bioactive agents to treat conditions associated 
with excessive phospholipase activity. 
Bioactive compositions comprising agonists or antagonists of GIPL may be 
administered in a suitable therapeutic dose determined by any of several 
methodologies including clinical studies on mammalian species to determine 
maximum tolerable dose and on normal human subjects to determine safe 
dosage. Additionally, the bioactive agent may be complexed with a variety 
of well established compounds or compositions which enhance stability or 
pharmacological properties such as half-life. It is contemplated that a 
therapeutic, bioactive composition may be delivered by intravenous 
infusion into the bloodstream or any other effective means which could be 
used for treatment. 
"Nucleic acid sequence" as used herein refers to an oligonucleotide, 
nucleotide or polynucleotide, and fragments or portions thereof, and to 
DNA or RNA of genomic or synthetic origin which may be single- or 
double-stranded, and represent the sense or antisense strand. Similarly, 
amine acid sequence as used herein refers to an oligopeptide, peptide, 
polypeptide or protein sequence. 
"Peptide nucleic acid" as used herein refers to a molecule which comprises 
an oligomer to which an amine acid residue, such as lysine, and an amine 
group have been added. These small molecules, also designated anti-gene 
agents, stop transcript elongation by binding to their complementary 
(template) strand of nucleic acid (Nielsen P E et al (1993) Anticancer 
Drug Des 8:53-63). 
As used herein, GIPL refers to the amine acid sequence of GIPL from any 
species, particularly mammalian, including bovine, ovine, porcine, murine, 
equine, and preferably human, in a naturally occurring form or from any 
source whether natural, synthetic, semi-synthetic or recombinant. As used 
herein, "naturally occurring" refers to an amine acid sequence which is 
found in nature. 
The present invention also encompasses GIPL variants. A preferred GIPL 
variant is one having at least 80% amine acid sequence similarity, a more 
preferred GIPL variant is one having at least 90% amine acid sequence 
similarity and a most preferred GIPL variant is one having at least 95% 
amine acid sequence similarity to the GIPL amine acid sequence (SEQ ID NO: 
2). A "variant" of GIPL may have an amine acid sequence that is different 
by one or more amine acid "substitutions". The variant may have 
"conservative" changes, wherein a substituted amine acid has similar 
structural or chemical properties, eg, replacement of leucine with 
isoleucine. More rarely, a variant may have "nonconservative" changes, eg, 
replacement of a glycine with a tryptophan. Similar minor variations may 
also include amine acid deletions or insertions, or both. Guidance in 
determining which and how many amine acid residues may be substituted, 
inserted or deleted without abolishing biological or immunological 
activity may be found using computer programs well known in the art, for 
example, DNAStar software. 
The term "biologically active" refers to a GIPL having structural, 
regulatory or biochemical functions of the naturally occurring GIPL. 
Likewise, "immunelogically active" defines the capability of the natural, 
recombinant or synthetic GIPL, or any oligopeptide thereof, to induce a 
specific immune response in appropriate animals or cells and to bind with 
specific antibodies. 
The term "derivative" as used herein refers to the chemical modification of 
a gipl or the encoded GIPL. Illustrative of such modifications would be 
replacement of hydrogen by an alkyl, acyl, or amino group. A GIPL 
derivative would encode a polypeptide which retains essential biological 
characteristics of natural GIPL. 
As used herein, the term "purified" refers to molecules, either nucleic or 
amino acid sequences, that are removed from their natural environment and 
isolated or separated from at least one other component with which they 
are naturally associated. 
The GIPL Coding Sequences 
The nucleic acid and deduced amino acid sequences of GIPL are shown in FIG. 
1. In accordance with the invention, any nucleic acid sequence which 
encodes the amino acid sequence of GIPL can be used to generate 
recombinant molecules which express GIPL. In a specific embodiment 
described herein, the sequence for gipl was first isolated as Incyte Clone 
156817 from a THP-1 cDNA library (THP1PBL02), patent application Ser. No. 
08/438,571, entitled "Polynucleotides Derived from THP-1 Cells" by 
Delegeane et al. and filed May 10, 1995, the disclosure of which is 
incorporated herein by reference. 
Methods for DNA sequencing are well known in the art and employ such 
enzymes as the Klenow fragment of DNA polymerase I, Sequenase.RTM. (US 
Biochemical Corp, Cleveland Ohio)), Taq polymerase (Perkin Elmer, Norwalk 
Conn.), thermostable T7 polymerass (Amersham, Chicago Ill.), or 
combinations of recombinant polymerases and proofreading exonucleases such 
as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg 
Md.). 
Methods to extend the DNA from an oligonucleotide primer annealed to the 
DNA template of interest have been developed for both single-stranded and 
double-stranded templates. Chain termination reaction products were 
separated using electrophoresis and detected via their incorporated, 
labeled precursors. Recent improvements in mechanized reaction 
preparation, sequencing and analysis have permitted expansion in the 
number of sequences that can be determined per day. Preferably, the 
process is automated with machines such as the Hamilton Micro Lab 2200 
(Hamilton, Reno Nev.), Peltier Thermal Cycler (PTC200; MJ Research, 
Watertown Mass.) and the ABI 377 DNA sequencers (Perkin Elmer). 
The quality of any particular cDNA library may be determined by performing 
a pilot scale analysis of the cDNAs and checking for percentages of clones 
containing vector, lambda or E. coli DNA, mitochondrial or repetitive DNA, 
and clones with exact or homologous matches to sequences in public 
databases. 
Extending the Polynucleotide Sequence 
The polynucleotide sequence of gipl may be extended utilizing partial 
nucleotide sequence and various methods known in the art to detect 
upstream sequences such as promoters and regulatory elements. Gobinda et 
al (1993; PCR Methods Applic 2:318-22) disclose "restriction-site" 
polymerase chain reaction (PCR) as a direct method which uses universal 
primers to retrieve unknown sequence adjacent to a known locus. First, 
genomic DNA is amplified in the presence of primer to a linker sequence 
and a primer specific to the known region. The amplified sequences are 
subjected to a second round of PCR with the same linker primer and another 
specific primer internal to the first one. Products of each round of PCR 
are transcribed with an appropriate RNA polymerase and sequenced using 
reverse transcriptase. 
Inverse PCR can be used to amplify or extend sequences using divergent 
primers based on a known region (Triglia T et al (1988) Nucleic Acids Res 
16:8186). The primers may be designed using OLIGO.RTM.4.06 Primer Analysis 
Software (1992; National Biosciences Inc, Plymouth Minn.), or another 
appropriate program, to be 22-30 nucleotides in length, to have a GC 
content of 50% or more, and to anneal to the target sequence at 
temperatures about 68.degree.-72.degree. C. The method uses several 
restriction enzymes to generate a suitable fragment in the known region of 
a gene. The fragment is then circularized by intramolecular ligation and 
used as a PCR template. 
Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic 1:111-19) is a 
method for PCR amplification of DNA fragments adjacent to a known sequence 
in human and yeast artificial chromosome DNA. Capture PCR also requires 
multiple restriction enzyme digestions and ligations to place an 
engineered double-stranded sequence into an unknown portion of the DNA 
molecule before PCR. 
Parker J D et al (1991; Nucleic Acids Res 19:3055-60), teach walking PCR, a 
method for targeted gene walking which permits retrieval of unknown 
sequence. PromoterFinder.TM. a new kit available from Clontech (Palo Alto 
Calif.) uses PCR, nested primers and PromoterFinder libraries to walk in 
genomic DNA. This process avoids the need to screen libraries and is 
useful in finding intron/exon junctions. 
Another PCR method, "Improved Method for Obtaining Full Length cDNA 
Sequences" by Guegler et al, patent application Ser. No. 08/487,112, filed 
Jun. 7, 1995 and hereby incorporated by reference, employs XL-PCR.TM. 
(Perkin Elmer) to amplify and extend nucleotide sequences. 
Preferred libraries for screening for full length cDNAs are ones that have 
been size-selected to include larger cDNAs. Also, random primed libraries 
are preferred in that they will contain more sequences which contain the 
5' and upstream regions of genes. A randomly primed library may be 
particularly useful if an oligo d(T) library does not yield a full-length 
cDNA. Genomic libraries are useful for extension into the 5' nontranslated 
regulatory region. 
A new method for analyzing either the size or confirming the nucleotide 
sequence of sequencing or PCR products is capillary electrophoresis. 
Systems for rapid sequencing are available from Perkin Elmer, Beckman 
Instruments (Fullerton Calif.), and other companies. Capillary sequencing 
employs flowable polymers for electrophoretic separation, four different 
fluorescent dyes (one for each nucleotide) which are laser activated, and 
detection of the emitted wavelengths by a charge coupled devise camera. 
Output/light intensity is converted to electrical signal using appropriate 
software (eg. Genotyper.TM. and Sequence Navigator.TM. from Perkin Elmer) 
and the entire process from loading of samples to computer analysis and 
electronic data display is computer controlled. Capillary electrophoresis 
is particularly suited to the sequencing of small pieces of DNA which 
might be present in limited amounts in a particular sample. The 
reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min has 
been reported (Ruiz-Martinez M C et al (1993) Anal Chem 65:2851-8). 
Expression of the Nucleotide Sequence 
In accordance with the present invention, polynucleotide sequences which 
encode GIPL, fragments of the polypeptide, fusion proteins or functional 
equivalents thereof may be used to generate recombinant DNA molecules that 
direct the expression of GIPL in appropriate host cells. Due to the 
inherent degeneracy of the genetic code, other DNA sequences which encode 
substantially the same or a functionally equivalent amino acid sequence, 
may be used to clone and express GIPL. As will be understood by those of 
skill in the art, it may be advantageous to produce GIPL-encoding 
nucleotide sequences possessing non-naturally occurring codons. Codons 
preferred by a particular prokaryotic or eukaryotic host (Murray E et al 
(1989) Nuc Acids Res 17:477-508) can be selected, for example, to increase 
the rate of GIPL expression or to produce recombinant RNA transcripts 
having desirable properties, such as a longer half-life, than transcripts 
produced from naturally occurring sequence. 
Also included within the scope of the present invention are polynucleotide 
sequences that are capable of hybridizing to the nucleotide sequence of 
Figure I under various conditions of stringency. Hybridization conditions 
are based on the melting temperature (Tm) of the nucleic acid binding 
complex or probe, as taught in Berger and Kimmel (1987, Guide to Molecular 
Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San 
Diego Calif.) incorporated herein by reference, and confer may be used at 
a defined "stringency" as explained below. 
"Maximum stringency" typically occurs at about Tm-5.degree. C. (5.degree. 
C. below the Tm of the probe); "high stringency" at about 5.degree. C. to 
10.degree. C. below Tm; "intermediate stringency" at about 10.degree. C. 
to 20.degree. C. below Tm; and "low stringency" at about 20.degree. C. to 
25.degree. C. below Tm. As will be understood by those of skill in the 
art, a maximum stringency hybridization can be used to identify or detect 
identical polynucleotide sequences while an intermediate (or low) 
stringency hybridization can be used to identify or detect similar or 
related polynucleotide sequences. 
The term "hybridization" as used herein shall include "any process by which 
a strand of nucleic acid joins with a complementary strand through base 
pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New 
York N.Y.). Then by definition, hybridization includes the process of 
amplification as carried out in the polymerase chain reaction technologies 
described in Dieffenbach C W and G S Dveksler (1995, PCR Primer, a 
Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.) and 
incorporated herein by reference. 
A "deletion" is defined as a change in either nucleotide or amino acid 
sequence in which one or more nucleotides or amino acid residues, 
respectively, are absent. 
An "insertion" or "addition" is that change in a nucleotide or amino acid 
sequence which has resulted in the addition of one or more nucleotides or 
amino acid residues, respectively, as compared to the naturally occurring 
gipl. 
A "substitution" results from the replacement of one or more nucleotides or 
amino acids by different nucleotides or amino acids, respectively. 
Altered gipl nucleic acid sequences which may be used in accordance with 
the invention include deletions, insertions or substitutions of different 
nucleotides resulting in a polynucleotide that encodes the same or a 
functionally equivalent GIPL. The protein may also show deletions, 
insertions or substitutions of amino acid residues which produce a silent 
change and result in a functionally equivalent GIPL. Deliberate amino acid 
substitutions may be made on the basis of similarity in polarity, charge, 
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature 
of the residues as long as the biological activity of GIPL is retained. 
For example, negatively charged amino acids include aspartic acid and 
glutamic acid; positively charged amino acids include lysine and arginine; 
and amino acids with uncharged polar head groups having similar 
hydrophilicity values include leucine, isoleucine, valine; gtycine, 
alanine; asparagine, glutamine; serine, threonine phenylalanine, and 
tyrosine. 
Included within the scope of the present invention are alleles of gipl. As 
used herein, an "allele" or "allelic sequence" is an alternative form of 
gipl. Alleles result from a mutation, ie, a change in the nucleic acid 
sequence, and generally produce altered mRNAs or polypeptides whose 
structure or function may or may not be altered. Any given gene may have 
none, one or many allelic forms. Common mutational changes which give rise 
to alleles are generally ascribed to natural deletions, additions or 
substitutions of amino acids. Each of these types of changes may occur 
alone, or in combination with the others, one or more times in a given 
sequence. 
The nucleotide sequences of the present invention can be engineered in 
order to alter a gipl coding sequence for a variety of reasons, including 
but not limited to, alterations which modify the cloning, processing 
and/or expression of the gens product. For example, mutations may be 
introduced using techniques which are well known in the art, eg, 
site-directed mutagenesis to insert new restriction sites, to alter 
glycosylation patterns, to change codon preference, to produce splice 
variants, etc. 
In another embodiment of the invention, a natural, modified or recombinant 
gipl sequence may be ligated to a heterologous sequence to encode a fusion 
protein. For example, for screening of peptide libraries for inhibitors of 
GIPL activity, it may be useful to encode a chimeric GIPL protein that is 
recognized by a commercially available antibody. A fusion protein may also 
be engineered to contain a cleavage site located between a GIPL sequence 
and the heterologous protein sequence, so that the GIPL may be cleaved and 
purified away from the heterologous moiety. 
In an alternate embodiment of the invention, the coding sequence of gipl 
could be synthesized, whole or in part, using chemical methods well known 
in the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser 215-23, 
Horn T et al (1980) Nuc Acids Res Symp Ser 225-32, etc). Alternatively, 
the protein itself could be produced using chemical methods to synthesize 
a GIPL amino acid sequence, whole or in part. For example, peptide 
synthesis can be performed using various solid-phase techniques (Roberge J 
Y et al (1995) Science 269:202-204) and automated synthesis may be 
achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin 
Elmer) in accordance with the instructions provided by the manufacturer. 
The newly synthesized peptide can be purified by preparative high 
performance liquid chromatography (eg, Creighton (1983) Proteins, 
Structures and Molecular Principles, WH Freeman and Co, New York N.Y.). 
The composition of the synthetic peptides may be confirmed by amino acid 
analysis or sequencing (eg, the Edman degradation procedure; Creighton, 
supra). Additionally the amino acid sequence of GIPL, or any part thereof, 
may be altered during direct synthesis and/or combined using chemical 
methods with sequences from other proteins, or any part thereof, to 
produce a variant polypeptide. 
Expression Systems 
In order to express a biologically active GIPL, the nucleotide sequence 
encoding GIPL or its functional equivalent, is inserted into an 
appropriate expression vector, ie, a vector which contains the necessary 
elements for the transcription and translation of the inserted coding 
sequence. 
Methods which are well known to those skilled in the art can be used to 
construct expression vectors containing a GIPL coding sequence and 
appropriate transcriptional or translational controls. These methods 
include in vitro recombinant DNA techniques, synthetic techniques and in 
vivo recombination or genetic recombination. Such techniques are described 
in Maniatis et al (1989) Molecular Cloning, A Laboratory Manual, Cold 
Spring Harbor Press, Plainview N.Y. and Ausubel F M et al. (1989) Current 
Protocols in Molecular Biology, John Wiley & Sons, New York N.Y. 
A variety of expression vector/host systems may be utilized to contain and 
express a gipl coding sequence. These include but are not limited to 
microorganisms such as bacteria transformed with recombinant 
bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed 
with yeast expression vectors; insect cell systems infected with virus 
expression vectors (eg, baculovirus); plant cell systems transfected with 
virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco 
mosaic virus, TMV) or transformed with bacterial expression vectors (eg, 
Ti or pBR322 plasmid); or animal cell systems. 
The "control elements" or "regulatory sequences" of these systems vary in 
their strength and specificities and are those nontranslated regions of 
the vector, enhancers, promoters, and 3' untranslated regions, which 
interact with host cellular proteins to carry out transcription and 
translation. Depending on the vector system and host utilized, any number 
of suitable transcription and translation elements, including constitutive 
and inducible promoters, may be used. For example, when cloning in 
bacterial systems, inducible promoters such as the hybrid lacZ promoter of 
the Bluescript.RTM. phagemid (Stratagene, LaJolla Calif.) or pSport1 
(Gibco BRL) and ptrp-lac hybrids and the like may be used. The baculovirus 
polyhedrin promoter may be used in insect cells. Promoters or enhancers 
derived from the genomes of plant cells (eg, heat shock, RUBISCO; and 
storage protein genes) or from plant viruses (eg, viral promoters or 
leader sequences) may be cloned into the vector. In mammalian cell 
systems, promoters from the mammalian genes or from mammalian viruses are 
most appropriate. If it is necessary to generate a cell line that contains 
multiple copies of gipl, vectors based on SV40 or EBV may be used with an 
appropriate selectable marker. 
In bacterial systems, a number of expression vectors may be selected 
depending upon the use intended for GIPL. For example, when large 
quantities of GIPL are needed for the induction of antibodies, vectors 
which direct high level expression of fusion proteins that are readily 
purified may be desirable. Such vectors include, but are not limited to, 
the multifunctional E. coli cloning and expression vectors such as 
Bluescript.RTM. (Stratagene), in which the gipl coding sequence may be 
ligated into the vector in frame with sequences for the amino-terminal Met 
and the subsequent 7 residues of .beta.-galactosidase so that a hybrid 
protein is produced; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 
264:5503-5509); and the like. pGEX vectors (Promega, Madison Wis.) may 
also be used to express foreign polypeptides as fusion proteins with 
glutathione S-transferase (GST). In general, such fusion proteins are 
soluble and can easily be purified from lysed cells by adsorption to 
glutathione-agarose beads followed by elution in the presence of free 
glutathione. Proteins made in such systems are designed to include 
heparin, thrombin or factor XA protease cleavage sites so that the cloned 
polypeptide of interest can be released from the GST moiety at will. 
In the yeast, Saccharomyces cerevisiae, a number of vectors containing 
constitutive or inducible promoters such as alpha factor, alcohol oxidase 
and PGH may be used. For reviews, see Ausubel et al (supra) and Grant et 
al (1987) Methods in Enzymology 153:516-544. 
In cases where plant expression vectors are used, the expression of a 
sequence encoding GIPL may be driven by any of a number of promoters. For 
example, viral promoters such as the 35S and 19S promoters of CaMV 
(Brisson et al (1984) Nature 310:511-514) may be used alone or in 
combination with the omega leader sequence from TMV (Takamatsu et al 
(1987) EMBO J 6:307-311). Alternatively, plant promoters such as the small 
subunit of RUBISCO (Coruzzi et al (1984) EMBO J 3:1671:1680; Broglie et al 
(1984) Science 224:838-843); or heat shock promoters (Winter J and 
Sinibaldi RM (1991) Results Probl Cell Differ 17:85-105) may be used. 
These constructs can be introduced into plant cells by direct DNA 
transformation or pathogen-mediated transfection. For reviews of such 
techniques, see Hobbs S or Murry L E in McGraw Yearbook of Science and 
Technology (1992) McGraw Hill New York N.Y., pp 191-196 or Weissbach and 
Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New 
York N.Y., pp 421-463. 
An alternative expression system which could be used to express gipl is an 
insect system. In one such system, Autographa californica nuclear 
polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in 
Spodoptera frugiperda cells or in Trichoplusia larvae. The gipl coding 
sequence may be cloned into a nonessential region of the virus, such as 
the polyhedrin gene, and placed under control of the polyhedrin promoter. 
Successful insertion of gipl will render the polyhedrin gene inactive and 
produce recombinant virus lacking coat protein coat. The recombinant 
viruses are then used to infect S. frugiperda cells or Trichoplusia larvae 
in which GIPL is expressed (Smith et al (1983) J Virol 46:584; Engelhard E 
K et al (1994) Proc Nat Acad Sci 91:3224-7). 
In mammalian host cells, a number of viral-based expression systems may be 
utilized. In cases where an adenovirus is used as an expression vector, a 
gipl coding sequence may be ligated into an adenovirus 
transcription/translation complex consisting of the late promoter and 
tripartite leader sequence. Insertion in a nonessential E1 or E3 region of 
the viral genome will result in a viable virus capable of expressing GIPL 
in infected host cells. (Logan and Shenk (1984) Proc Natl Acad Sci 
81:3655-59). In addition, transcription enhancers, such as the rous 
sarcoma virus (RSV) enhancer, may be used to increase expression in 
mammalian host cells. 
Specific initiation signals may also be required for efficient translation 
of a gipl sequence. These signals include the ATG initiation codon and 
adjacent sequences. In cases where gipl, its initiation codon and upstream 
sequences are inserted into the appropriate expression vector, no 
additional translational control signals may be needed. However, in cases 
where only coding sequence, or a portion thereof, is inserted, exogenous 
transcriptional control signals including the ATG initiation codon must be 
provided. Furthermore, the initiation codon must be in the correct reading 
frame to ensure transcription of the entire insert. Exogenous 
transcriptional elements and initiation codohs can be of various origins, 
both natural and synthetic. The efficiency of expression may be enhanced 
by the inclusion of enhancers appropriate to the cell system in use 
(Scharf D et al (1994) Results Probl Cell Differ 20:125-62; Bittner et al 
(1987) Methods in Enzymol 153:516-544). 
In addition, a host cell strain may be chosen for its ability to modulate 
the expression of the inserted sequences or to process the expressed 
protein in the desired fashion. Such modifications of the polypeptide 
include, but are not limited to, acetylation, carboxylation, 
glycosylation, phosphorylation, lipidation and acylation. 
Post-translational processing which cleaves a "prepro" form of the protein 
may also be important for correct insertion, folding and/or function. 
Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc have specific 
cellular machinery and characteristic mechanisms for such 
post-translational activities and may be chosen to ensure the correct 
modification and processing of the introduced, foreign protein. 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express gipl 
may be transformed using expression vectors which contain viral origins of 
replication or endogenous expression elements and a selectable marker 
gene. Following the introduction of the vector, cells may be allowed to 
grow for 1-2 days in an enriched media before they are switched to 
selective media. The purpose of the selectable marker is to confer 
resistance to selection, and its presence allows growth and recovery of 
cells which successfully express the introduced sequences. Resistant 
clumps of stably transformed cells can be proliferated using tissue 
culture techniques appropriate to the cell type. 
Any number of selection systems may be used to recover transformed cell 
lines. These include, but are not limited to, the herpes simplex virus 
thymidine kinase (Wigler M et al (1977) Cell 11:223-32) and adenine 
phosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) genes which 
can be employed in tk.sup.- or aprt.sup.- cells, respectively. Also, 
antimetabolite, antibiotic or herbicide resistance can be used as the 
basis for selection; for example, dhfr which confers resistance to 
methotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt, 
which confers resistance to the aminoglycosides neomycin and G-418 
(Colbere-Garapin F et al (1981) J Mol Biol 150:1-14) and als or pat, which 
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, 
respectively (Murry, supra). Additional selectable genes have been 
described, for example, trpB, which allows cells to utilize indole in 
place of tryptophan, or hisD, which allows cells to utilize histinol in 
place of histidine (Hartman SC and RC Mulligan (1988) Proc Natl Acad Sci 
85:8047-51). Recently, the use of visible markers has gained popularity 
with such markers as anthocyanins, .beta. glucuronidase and its substrate, 
GUS, and luciferase and its substrate, luciferin, being widely used not 
only to identify transformants, but also to quantify the amount of 
transient or stable protein expression attributable to a specific vector 
system (Rhodes C A et al (1995) Methods Mol Biol 55:121-131). 
Identification of Transformants Containing the Polynucleotide Sequence 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, its presence and expression should be 
confirmed. For example, if the gipl is inserted within a marker gene 
sequence, recombinant cells containing gipl can be identified by the 
absence of marker gene function. Alternatively, a marker gene can be 
placed in tandem with a GIPL sequence under the control of a single 
promoter. Expression of the marker gene in response to induction or 
selection usually indicates expression of the tandem gipl as well. 
Alternatively, host cells which contain the coding sequence for gipl and 
express GIPL may be identified by a variety of procedures known to those 
of skill in the art. These procedures include, but are not limited to, 
DNA--DNA or DNA-RNA hybridization and protein bioassay or immunoassay 
techniques which include membrane, solution, or chip based technologies 
for the detection and/or quantification of the nucleic acid or protein. 
The presence of the gipl polynucleotide sequence can be detected by 
DNA--DNA or DNA-RNA hybridization or amplification using probes, portions 
or fragments of gipl. Nucleic acid amplification based assays involve the 
use of oligonucleotides or oligomers based on the gipl sequence to detect 
transformants containing gipl DNA or RNA. As used herein 
"oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at 
least about 10 nucleotides and as many as about 60 nucleotides, preferably 
about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides 
which can be used as a probe or amplimer. 
A variety of protocols for detecting and measuring the expression of GIPL, 
using either polyclonal or monoclonal antibodies specific for the protein 
are known in the art. Examples include enzyme-linked immunosorbent assay 
(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting 
(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal 
antibodies reactive to two non-interfering epitopes on GIPL is preferred, 
but a competitive binding assay may be employed. These and other assays 
are described, among other places, in Hampton R et al (1990, Serological 
Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox D E et 
al (1983, J Exp Med 158:1211). 
A wide variety of labels and conjugation techniques are known by those 
skilled in the art and can be used in various nucleic acid and amino acid 
assays. Means for producing labeled hybridization or PCR probes for 
detecting sequences related to gipl include oligolabeling, nick 
translation, end-labeling or PCR amplification using a labeled nucleotide. 
Alternatively, the gipl sequence, or any portion of it, may be cloned into 
a vector for the production of an mRNA probe. Such vectors are known in 
the art, are commercially available, and may be used to synthesize RNA 
probes in vitro by addition of an appropriate RNA polymerase such as T7, 
T3 or SP6 and labeled nucleotides. 
A number of companies such as Pharmacia Biotech (Piscataway N.J.), Promega 
(Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supply commercial 
kits and protocols for these procedures. Suitable reporter molecules or 
labels include those radionuclides, enzymes, fluorescent, 
chemiluminescent, or chromogenic agents as well as substrates, cofactors, 
inhibitors, magnetic particles and the like. Patents teaching the use of 
such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 
3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant 
immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567 
incorporated herein by reference. 
Purification of GIPL 
Host cells transformed with a gipl nucleotide sequence may be cultured 
under conditions suitable for the expression and recovery of the encoded 
protein from cell culture. The protein produced by a recombinant cell may 
be secreted or contained intracellularly depending on the sequence and/or 
the vector used. As will be understood by those of skill in the art, 
expression vectors containing gipl can be designed with signal sequences 
which direct secretion of GIPL through a prokaryotic or eukaryotic cell 
membrane. Other recombinant constructions may join gipl to nucleotide 
sequence encoding a polypeptide domain which will facilitate purification 
of soluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53; cf 
discussion of vectors infra containing fusion proteins). 
GIPL may also be expressed as a recombinant protein with one or more 
additional polypeptide domains added to facilitate protein purification. 
Such purification facilitating domains include, but are not limited to, 
metal chelating peptides such as histidine-tryptophan modules that allow 
purification on immobilized metals, protein A domains that allow 
purification on immobilized immunoglobulin, and the domain utilized in the 
FLAGS extension/affinity purification system (Immunex Corp, Seattle 
Wash.). The inclusion of a clearable linker sequences such as Factor XA or 
enterokinase (Invitrogen, San Diego Calif.) between the purification 
domain and GIPL is useful to facilitate purification. 
Uses of GIPL 
The rationale for diagnostic and therapeutic uses of the nucleotide and 
peptide sequences disclosed herein is based on the disclosed nucleic acid 
and amino acid sequences, the information from comparisons between GIPL 
and CNF as shown in FIGS. 3-7, and the presence of the gipl transcript in 
the mast cell (HMC1NOT1), THP-1, neuronal (PGANNOT01) and uterus 
(UTRSNOT01) cDNA libraries. It must be noted that these libraries were 
made from activated cells or the cells or tissues removed from patients or 
victims of accidental death. 
The nucleic acid sequence (SEQ ID NO: 1), its complement, fragments or 
oligomers, and anti-GIPL antibodies may be used as diagnostic compositions 
to assay bodily fluids or extracts of biological samples for expression of 
gipl. Purified polynucleotides and polypeptides can be used as positive 
controls in their respective nucleic acid or protein based assays to 
validate and quantitate the expression of gipl either during preliminary 
diagnosis or during the course of therapeutic treatment for a particular 
condition or disease. In some cases, the mere presence of gipl or GIPL 
(expression vs. absence of expression) will connote disease, while in 
other cases, gipl expression will be abnormal because gipl or GIPL 
deviates from a predetermined normal level. 
Some of the conditions in which the moderation of phospholipase expression 
may be important include viral, bacterial or fungal infections including 
septic and toxic shock and gangrene; autoimmune responses encompassing but 
not limited to anemias, asthma, systemic lupus, and myasthenia gravis; 
hereditary or cancerous conditions such as Alzheimer's, breast carcinoma, 
diabetes mellitus, osteoporosis, and schizophrenia; glomerulonephritis; 
pregnancy; rheumatoid and osteoarthritis; scleroderma; and insect or snake 
bites or stings in which phospholipases are a component of the injected 
venom. 
The use of GIPL, and of the nucleic acid sequences which encode it, is 
based on the amino acid sequence and structural homology based on cysteine 
distribution between GIPL and CNF. The timing of and amount of expression 
of phospholipases and GIPL are implicated in the conditions previously 
recited. In each of the next three situations, the level of phospholipase 
expression precedes or exceeds the expression of GIPL. 
Phospholipases play a role in membrane turnover in normal as well as 
cancerous tissues. Supplying purified GIPL to the cancerous tissues would 
interfere with metastasis and growth of neoplastic cells. Similarly, 
supplying purified GIPL to individuals with developing tumors would 
interfere with anglogenesis, the vascularization of the tumor which 
supports prolific growth. 
In reproductive studies, Reponen P et al. (1995; Dev Dyn 202:388-96) has 
discussed the enzymes which are active in remodeling during implantation 
of the embryo. The phospholipases are involved in membrane reconstruction 
during this process. Therefore, the timely inhibition of these 
phospholipases, post coitus, by supplying women with recombinant GIPL 
would inhibit the membrane remodeling process, in effect, preventing 
implantation and pregnancy. 
In diseases such as osteoarthritis, rheumatoid arthritis, pulmonary 
emphysema, periodontal disease, systemic lupus, and osteoporosis or with 
infectious conditions such as septic or toxic shock or gangrene, the 
excess activity of the phospholipases producing arachidonic acid or 
diacylglycerol and contributing to the formation or eicosanoids causes 
inflammation, tissue destruction, impaired function or death. Appropriate 
delivery of GIPL would inhibit the activity of the phospholipases and 
their continued induction by eicosanoids (such as the LKB4) significantly 
reducing inflammation and damage. Similarly, the delivery of a 
GIPL-specific agonist would prolong the effects of GIPL. 
Purified GIPL may also be administered as an antivenom in those cases of 
snake or insect bite where phospholipases are an active component of the 
venom. Supplying a pharmaceutical composition containing GIPL inactivates 
the neurotoxic, cardiotoxic, myotoxic, anticoagulent and 
platelet-activating activities of venom. 
GIPL Antibodies 
Procedures well known in the art can be used for the production of 
antibodies to GIPL. Such antibodies include, but are not limited to, 
polyclonal, monoclonal, chimeric, single chain, Fab fragments and 
fragments produced by a Fab expression library. Neutralizing antibodies, 
ie, those which inhibit dimer formation, are especially preferred for 
diagnostics and therapeutics. 
For the production of antibodies, various hosts including goats, rabbits, 
rats, mice, etc. may be immunized by injection with GIPL or any portion, 
fragment or oligopeptide which retains immunogenic properties. Depending 
on the host species, various adjuvants may be used to increase 
immunological response. Such adjuvants include but are not limited to 
Freund's, mineral gels such as aluminum hydroxide, and surface active 
substances such as lysolecithin, pluronic polyols, polyanions, peptides, 
oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli 
Calmette-Guerin) and Corynebacterium parvum are potentially useful human 
adjuvants. 
Monoclonal antibodies to GIPL may be prepared using any technique which 
provides for the production of antibody molecules by continuous cell lines 
in culture. These include but are not limited to the hybridoma technique 
originally described by Koehler and Milstein (1975 Nature 256:495-497), 
the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 
4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the 
EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and 
Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96). 
In addition, techniques developed for the production of "chimeric 
antibodies", the splicing of mouse antibody genes to human antibody genes 
to obtain a molecule with appropriate antigen specificity and biological 
activity can be used (Morrison et al (1984) Proc Natl Acad Sci 
81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al 
(1985) Nature 314:452-454). Alternatively, techniques described for the 
production of single chain antibodies (U.S. Pat. No. 4,946,778) can be 
adapted to produce GIPL-specific single chain antibodies. 
Antibodies may also be produced by inducing in vivo production in the 
lymphocyte population or by screening recombinant immunoglobulin libraries 
or panels of highly specific binding reagents as disclosed in Orlandi et 
al (1989, Proc Natl Acad Sci 86:3833-3837), and Winter G and Milstein C 
(1991; Nature 349:293-299). 
Antibody fragments which contain specific binding sites for GIPL may also 
be generated. For example, such fragments include, but are not limited to, 
the F(ab').sub.2 fragments which can be produced by pepsin digestion of 
the antibody molecule and the Fab fragments which can be generated by 
reducing the disulfide bridges of the F(ab').sub.2 fragments. 
Alternatively, Fab expression libraries may be constructed to allow rapid 
and easy identification of monoclonal Fab fragments with the desired 
specificity (Huse W D et al (1989) Science 256:1275-1281). 
GIPL-specific antibodies are useful for the diagnosis of conditions and 
diseases associated with expression of GIPL. A variety of protocols for 
competitive binding or immunoradiometric assays using either polyclonal or 
monoclonal antibodies with established specificities are well known in the 
art. Such immunoassays typically involve the formation of complexes 
between GIPL and its specific antibody and the measurement of complex 
formation. A two-site, monoclonal-based immunoassay utilizing monoclonal 
antibodies reactive to two noninterfering epitopes on a specific GIPL 
protein is preferred, but a competitive binding assay may also be 
employed. These assays are described in Maddox D E et al (1983, J Exp Med 
158:1211). 
Diagnostic Assays Using GIPL Specific Antibodies 
Particular GIPL antibodies are useful for the diagnosis of conditions or 
diseases characterized by expression of GIPL or in assays to monitor 
patients being treated with GIPL, agonists or inhibitors. Diagnostic 
assays for GIPL include methods utilizing the antibody and a label to 
detect GIPL in human body fluids or extracts of cells or tissues. The 
polypeptides and antibodies of the present invention may be used with or 
without modification. Frequently, the polypeptides and antibodies will be 
labeled by joining them, either covalently or noncovalently, with a 
reporter molecule. A wide variety of reporter molecules are known, several 
of which were described above, 
A variety of protocols for measuring GIPL, using either polyclonal or 
monoclonal antibodies specific for the respective protein are known in the 
art. Examples include enzyme-linked immunosorbent assay (ELISA), 
radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A 
two-site, monoclonal-based immunoassay utilizing monoclonal antibodies 
reactive to two non-interfering epitopes on GIPL is preferred, but a 
competitive binding assay may be employed. These assays are described, 
among other places, in Maddox, D E et al (1983, J Exp Med 158:1211). 
In order to provide a basis for diagnosis, normal or standard values for 
GIPL expression must be established. This is accomplished by combining 
body fluids or cell extracts taken from normal subjects, either animal or 
human, with antibody to GIPL under conditions suitable for complex 
formation which are well known in the art. The amount of standard complex 
formation may be quantified by comparing various artificial membranes 
containing known quantities of GIPL with both control and disease samples 
from biopsied tissues. Then, standard values obtained from normal samples 
may be compared with values obtained from samples from subjects 
potentially affected by disease. Deviation between standard and subject 
values establishes the presence of disease state. 
Drug Screening 
GIPL, its catalytic or immunogenic fragments or oligopeptides, can be used 
for screening therapeutic compounds in any of a variety of drug screening 
techniques. The fragment employed in such a test may be free in solution, 
affixed to a solid support, borne on a cell surface, or located 
intracellularly. The formation of binding complexes, between GIPL and the 
agent being tested, may be measured. 
Another technique for drug screening which provides for high throughput 
screening of compounds having suitable binding affinity to the GIPL is 
described in detail in "Determination of Amino Acid Sequence Antigenicity" 
by Geysen H N, WO application Ser. No. 84/03564, published on Sep. 13, 
1984, and incorporated herein by reference. In summary, large numbers of 
different small peptide test compounds are synthesized on a solid 
substrate, such as plastic pins or some other surface. The peptide test 
compounds are reacted with fragments of GIPL and washed. Bound GIPL is 
then detected by methods well known in the art. Purified GIPL can also be 
coated directly onto plates for use in the aforementioned drug screening 
techniques. Alternatively, non-neutralizing antibodies can be used to 
capture the peptide and immobilize it on a solid support. 
This invention also contemplates the use of competitive drug screening 
assays in which neutralizing antibodies capable of binding GIPL 
specifically compete with a test compound for binding GIPL. In this 
manner, the antibodies can be used to detect the presence of any peptide 
which shares one or more antigenic determinants with GIPL. 
Uses of the Polynucleotide Encoding GIPL 
A polynucleotide, gipl, or any part thereof, may be used for diagnostic 
and/or therapeutic purposes. For diagnostic purposes, the gipl of this 
invention may be used to detect and quantitate gene expression in biopsied 
tissues in which expression of GIPL may be implicated. The diagnostic 
assay is useful to distinguish between absence, presence, and excess 
expression of gipl and to monitor regulation of gipl levels during 
therapeutic intervention. Included in the scope of the invention are 
oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs. 
Another aspect of the subject invention is to provide for hybridization or 
PCR probes which are capable of detecting polynucleotide sequences, 
including genomic sequences, encoding GIPL or closely related molecules. 
The specificity of the probe, whether it is made from a highly specific 
region, eg, 10 unique nucleotides in the 5' regulatory region, or a less 
specific region, eg, especially in the 3' region, and the stringency of 
the hybridization or amplification (maximal, high, intermediate or low) 
will determine whether the probe identifies only naturally occurring gipl, 
alleles or related sequences. 
Diagnostics 
Polynucleotide sequences encoding GIPL may be used for the diagnosis of 
conditions or diseases with which the expression of GIPL is associated. 
For example, polynucleotide sequences encoding GIPL may be used in 
hybridization or PCR assays of fluids or tissues from biopsies to detect 
gipl expression. The form of such qualitative or quantitative methods may 
include Southern or northern analysis, dot blot or other membrane-based 
technologies; PCR technologies; dip stick, pin, chip and ELISA 
technologies. All of these techniques are well known in the art and are 
the basis of many commercially available diagnostic kits. 
Such assays may also be used to evaluate the efficacy of a particular 
therapeutic treatment regime in animal studies, in clinical trials, or in 
monitoring the treatment of an individual patient. In order to provide a 
basis for the diagnosis of disease, a normal or standard profile for gipl 
expression must be established. This is accomplished by combining body 
fluids or cell extracts taken from normal subjects, either animal or 
human, with gipl, or a portion thereof, under conditions suitable for 
hybridization or amplification. Standard hybridization may be quantified 
by comparing the values obtained for normal subjects with a dilution 
series of gipl run in the same experiment where a known amount of purified 
gipl is used. Standard values obtained from normal samples may be compared 
with values obtained from samples from patients affected by 
gipl-associated diseases. Deviation between standard and subject values 
establishes the presence of disease. 
Once disease is established, a therapeutic agent is administered; and a 
treatment profile is generated. Such assays may be repeated on a regular 
basis to evaluate whether the values in the profile progress toward or 
return to the normal or standard pattern. Successive treatment profiles 
may be used to show the efficacy of treatment over a period of several 
days or several months. 
PCR as described in U.S. Pat. Nos. 4,683,195 and 4,965,188 provides 
additional uses for oligonucleotides based upon the gipl sequence. Such 
oligomers are generally chemically synthesized, but they may be generated 
enzymatically or produced from a recombinant source. Oligomers generally 
comprise two nucleotide sequences, one with sense orientation (5'-&gt;3') and 
one with antisense (3'&lt;-5'), employed under optimized conditions for 
identification of a specific gene or condition. The same two oligomers, 
nested sets of oligomers, or even a degenerate pool of oligomers may be 
employed under less stringent conditions for detection and/or quantitation 
of closely related DNA or RNA sequences. 
Additionally, methods to quantirate the expression of a particular molecule 
include radiolabeling (Melby P C et al 1993 J Immunol Methods 159:235-44) 
or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, 
coamplification of a control nucleic acid, and standard curves onto which 
the experimental results are interpolated. Quantitation of multiple 
samples may be speeded up by running the assay in an ELISA format where 
the oligomer of interest is presented in various dilutions and a 
spectrophotometric or colorimetric response gives rapid quantitation. For 
example, the presence of gipl in extracts of biopsied tissues may indicate 
the onset of cancer. A definitive diagnosis of this type may allow health 
professionals to begin aggressive treatment and prevent further worsening 
of the condition. Similarly, further assays can be used to monitor the 
progress of a patient during treatment. 
Therapeutics 
The polynucleotide disclosed herein may be useful in the treatment of 
various inherited conditions such as autoimmune, hereditary and cancerous 
conditions, pregnancy, and infections or bites and stings resulting in 
shock or anaphylaxis. For example, administration of a vector containing 
and expressing gipl provides a means to moderate the phospholipase 
activity which leads to rapid membrane remodeling in schizophrenia. By 
introducing the antisense molecules (anti-gipl) into the cerebrospinal 
fluid, gene therapy via expression of GIPL can be used to reduce or 
eliminate phospholipase activity. 
Expression vectors derived from retroviruses, adenovirus, herpes or 
vaccinia viruses, or from various bacterial plasmids, may be used for 
delivery of nucleotide sequences to the targeted organ, tissue or cell 
population. Methods which are well known to those skilled in the art can 
be used to construct recombinant vectors which will express anti-gipl. 
See, for example, the techniques described in Maniatis et al (supra) and 
Ausubel et al (supra). 
The polynucleotides comprising full length cDNA sequence and/or its 
regulatory elements enable researchers to use gipl as an investigative 
tool in sense (Youssoufian H and H F Lodish 1993 Mol Cell Biol 13:98-104) 
or antisense (Eguchi et al (1991) Annu Rev Biochem 60:631-652) regulation 
of gene function. Such technology is now well known in the art, and sense 
or antisense oligomers, or larger fragments, can be designed from various 
locations along the coding or control regions. 
Genes encoding GIPL can be turned off by transfecting a cell or tissue with 
expression vectors which express high levels of the desired fragment. Such 
constructs can flood cells with untranslatable sense or antisense 
sequences. Even in the absence of integration into the DNA, such vectors 
may continue to transcribe RNA molecules until all copies are disabled by 
endogenous nucleases. Transient expression may last for a month or more 
with a non-replicating vector (Mettier I, personal communication) and even 
longer if appropriate replication elements are part of the vector system. 
As mentioned above, modifications of gene expression can be obtained by 
designing antisense molecules, DNA, RNA or PNA, to the control regions of 
gipl, ie, the promoters, enhancers, and introns. Oligonucleotides derived 
from the transcription initiation site, eg, between -10 and +10 regions of 
the leader sequence, are preferred. The antisense molecules may also be 
designed to block translation of mRNA by preventing the transcript from 
binding to ribosomes. Similarly, inhibition can be achieved using "triple 
helix" base-pairing methodology. Triple helix pairing compromises the 
ability of the double helix to open sufficiently for the binding of 
polymerases, transcription factors, or regulatory molecules. Recent 
therapeutic advances using triplex DNA were reviewed by Gee J E et al. 
(In: Huber B E and B I Carr (1994) Molecular and Immunologic Approaches, 
Futura Publishing Co, Mt Kisco N.Y.). 
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific 
cleavage of RNA. The mechanism of ribozyme action involves 
sequence-specific hybridization of the ribozyme molecule to complementary 
target RNA, followed by endonucleolytic cleavage. Within the scope of the 
invention are engineered hammerhead motif ribozyme molecules that can 
specifically and efficiently catalyze endonucleolytic cleavage of gipl. 
Specific ribozyme cleavage sites within any potential RNA target are 
initially identified by scanning the target molecule for ribozyme cleavage 
sites which include the following sequences, GUA, GUU and GUC. Once 
identified, short RNA sequences of between 15 and 20 ribonucleotides 
corresponding to the region of the target gene containing the cleavage 
site may be evaluated for secondary structural features which may render 
the oligonucleotide inoperable. The suitability of candidate targets may 
also be evaluated by testing accessibility to hybridization with 
complementary oligonucleotides using ribonuclease protection assays. 
Antisense molecules and ribozymes of the invention may be prepared by any 
method known in the art for the synthesis of RNA molecules. These include 
techniques for chemically synthesizing oligonucleotides such as solid 
phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may 
be generated by in Vitro and in Viv0 transcription of DNA sequences 
encoding GIPL. Such DNA sequences may be incorporated into a wide variety 
of vectors with suitable RNA polymerase promoters such as T7 or SP6. 
Alternatively, antisense cDNA constructs that synthesize antisense RNA 
constitutively or inducibly can be introduced into cell lines, cells or 
tissues. 
RNA molecules may be modified to increase intracellular stability and 
half-life. Possible modifications include, but are not limited to, the 
addition of flanking sequences at the 5' and/or 3' ends of the molecule or 
the use of phosphorothioate or 2'O-methyl rather than phosphodiesterase 
linkages within the backbone of the molecule. This concept is inherent in 
the production of PNAs and can be extended in all of these molecules by 
the inclusion of nontraditional bases such as inosine, queosine and 
wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms 
of adenine, cytidine, guanine, thymine, and uridine which are not as 
easily recognized by endogenous endonucleases. 
Methods for introducing vectors into cells or tissues include those methods 
discussed infra and which are equally suitable for in vivo, in vitro and 
ex vivo therapy. For ex vivo therapy, vectors are introduced into stem 
cells taken from the patient and clonally propagated for autologous 
transplant back into that same patient is presented as U.S. Pat. Nos. 
5,399,493 and 5,437,994, disclosed herein by reference. Delivery by 
transfection and by liposome are quite well known in the art. 
Furthermore, the nucleotide sequences for gipl disclosed herein may be used 
in molecular biology techniques that have not yet been developed, provided 
the new techniques rely on properties of nucleotide sequences that are 
currently known, including but not limited to such properties as the 
triplet genetic code and specific base pair interactions. 
Detection and Mapping of Related Polynucleotide Sequences 
The nucleic acid sequence for gipl can also be used to generate 
hybridization probes for mapping the naturally occurring genomic sequence. 
The sequence may be mapped to a particular chromosome or to a specific 
region of the chromosome using well known techniques. These include in 
situ hybridization to chromosomal spreads, flow-sorted chromosomal 
preparations, or artificial chromosome constructions such as yeast 
artificial chromosomes, bacterial artificial chromosomes, bacterial P1 
constructions or single chromosome cDNA libraries as reviewed in Price C M 
(1993; Blood Rev 7:127-34) and Trask B J (1991; Trends Genet 7:149-54). 
In situ hybridization of chromosomal preparations and physical mapping 
techniques such as linkage analysis using established chromosomal markers 
are invaluable in extending genetic maps. A recent example of an STS based 
map of the human genome was recently published by the Whitehead-MIT Center 
for Genomic Research (Hudson T J et al. (1995) Science 270:1945-1954). 
Often the placement of agene on the chromosome of another mammalian 
species such as mouse (Whitehead Institute/MIT Center for Genome Research, 
Genetic Map of the Mouse, Database Release 10, Apr. 28, 1995) may reveal 
associated markers even if the number or arm of a particular human 
chromosome is not known. New sequences can be assigned to chromosomal 
arms, or parts thereof, by physical mapping. This provides valuable 
information to investigators searching for disease genes using positional 
cloning or other gene discovery techniques. Once a disease or syndrome, 
such as ataxia telangiectasia (AT), has been crudely localized by genetic 
linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti 
et al (1988) Nature 336:577-580), any sequences mapping to that area may 
represent associated or regulatory genes for further investigation. The 
nucleotide sequence of the subject invention may also be used to detect 
differences in the chromosomal location due to translocation, inversion, 
etc. among normal, carrier or affected individuals. 
Pharmaceutical Compositions 
The present invention comprises pharmaceutical compositions which may 
comprise nucleotides, proteins, antibodies, antagonists, or inhibitors, 
alone or in combination with at least one other agent, such as stabilizing 
compound, which may be administered in any sterile, biocompatible 
pharmaceutical carrier, including, but not limited to, saline, buffered 
saline, dextrose, and water. Any of these molecules can be administered to 
a patient alone, or in combination with other agents, drugs or hormones, 
in pharmaceutical compositions where it is mixed with excipient(s) or 
pharmaceutically acceptable carriers. In one embodiment of the present 
invention, the pharmaceutically acceptable carrier is pharmaceutically 
inert. 
Administration of Pharmaceutical Compositions 
Administration of pharmaceutical compositions is accomplished orally or 
parenterally, Methods of parenteral delivery include topical, 
intra-arterial (directly to the tumor), intramuscular, subcutaneous, 
intramedullary, intrathecal, intraventricular, intravenous, 
intraperitoneal, or intranasal administration. In addition to the active 
ingredients, these pharmaceutical compositions may contain suitable 
pharmaceutically acceptable carriers comprising excipients and auxiliaries 
which facilitate processing of the active compounds into preparations 
which can be used pharmaceutically. Further details on techniques for 
formulation and administration may be found in the latest edition of 
"Remington's Pharmaceutical Sciences" (Maack Publishing Co, Easton 
Penna.). 
Pharmaceutical compositions for oral administration can be formulated using 
pharmaceutically acceptable carriers well known in the art in dosages 
suitable for oral administration. Such carriers enable the pharmaceutical 
compositions to be formulated as tablets, pills, dragees, capsules, 
liquids, gels, syrups, slurries, suspensions and the like, for ingestion 
by the patient. 
Pharmaceutical preparations for oral use can be obtained through 
combination of active compounds with solid excipient, optionally grinding 
a resulting mixture, and processing the mixture of granules, after adding 
suitable auxiliaries, if desired, to obtain tablets or dragee cores. 
Suitable excipients are carbohydrate or protein fillers such as sugars, 
including lactose, sucrose, mannitol, or sorbitol; starch from corn, 
wheat, rice, potato, or other plants; cellulose such as methyl cellulose, 
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums 
including arabic and tragacanth; and proteins such as gelatin and 
collagen. If desired, disintegrating or solubilizing agents may be added, 
such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a 
salt thereof, such as sodium alginate. 
Dragee cores are provided with suitable coatings such as concentrated sugar 
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, 
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer 
solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or 
pigments may be added to the tablets or dragee coatings for product 
identification or to characterize the quantity of active compound, ie, 
dosage. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a coating such as glycerol or sorbitol. Push-fit capsules can contain 
active ingredients mixed with a filler or binders such as lactose or 
starches, lubricants such as talc or magnesium stearate, and, optionally, 
stabilizers. In soft capsules, the active compounds may be dissolved or 
suspended in suitable liquids, such as fatty oils, liquid paraffin, or 
liquid polyethylene glycol with or without stabilizers. 
Pharmaceutical formulations for parenteral administration include aqueous 
solutions of active compounds. For injection, the pharmaceutical 
compositions of the invention may be formulated in aqueous solutions, 
preferably in physiologically compatible buffers such as Hanks's solution, 
Ringer's solution, or physiologically buffered saline. Aqueous injection 
suspensions may contain substances which increase the viscosity of the 
suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. 
Additionally, suspensions of the active compounds may be prepared as 
appropriate oily injection suspensions. Suitable lipophilic solvents or 
vehicles include fatty oils such as sesame oil, or synthetic fatty acid 
esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, 
the suspension may also contain suitable stabilizers or agents which 
increase the solubility of the compounds to allow for the preparation of 
highly concentrated solutions. 
For topical or nasal administration, penerrants appropriate to the 
particular barrier to be permeated are used in the formulation. Such 
penetrants are generally known in the art. 
Manufacture and Storage 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that known in the art, eg, by means of 
conventional mixing, dissolving, granulating, dragee-making, levigating, 
emulsifying, encapsulating, entrapping or lyophilizing processes. 
The pharmaceutical composition may be provided as a salt and can be formed 
with many acids, including but not limited to hydrochloric, sulfuric, 
acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more 
soluble in aqueous or other protonic solvents that are the corresponding 
free base forms. In other cases, the preferred preparation may be a 
lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% 
mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to 
use. 
After pharmaceutical compositions comprising a compound of the invention 
formulated in a acceptable carrier have been prepared, they can be placed 
in an appropriate container and labeled for treatment of an indicated 
condition. For administration of GIPL, such labeling would include amount, 
frequency and method of administration. 
Therapeutically Effective Dose 
Pharmaceutical compositions suitable for use in the present invention 
include compositions wherein the active ingredients are contained in an 
effective amount to achieve the intended purpose. The determination of an 
effective dose is well within the capability of those skilled in the art. 
For any compound, the therapeutically effective dose can be estimated 
initially either in cell culture assays, eg, of neoplastic cells, or in 
animal models, usually mice, rabbits, dogs, or pigs. The animal model is 
also used to achieve a desirable concentration range and route of 
administration. Such information can then be used to determine useful 
doses and routes for administration in humans. 
A therapeutically effective dose refers to that amount of protein or its 
antibodies, antagonists, or inhibitors which ameliorate the symptoms or 
condition. Therapeutic efficacy and toxicity of such compounds can be 
determined by standard pharmaceutical procedures in cell cultures or 
experimental animals, eg, ED50 (the dose therapeutically effective in 50% 
of the population) and LD50 (the dose lethal to 50% of the population). 
The dose ratio between therapeutic and toxic effects is the therapeutic 
index, and it can be expressed as the ratio, ED50/LD50. Pharmaceutical 
compositions which exhibit large therapeutic indices are preferred. The 
data obtained from cell culture assays and animal studies is used in 
formulating a range of dosage for human use. The dosage of such compounds 
lies preferably within a range of circulating concentrations that include 
the ED50 with little or no toxicity. The dosage varies within this range 
depending upon the dosage form employed, sensitivity of the patient, and 
the route of administration. 
The exact dosage is chosen by the individual physician in view of the 
patient to be treated. Dosage and administration are adjusted to provide 
sufficient levels of the active moiety or to maintain the desired effect. 
Additional factors which may be taken into account include the severity of 
the disease state, eg, tumor size and location; age, weight and gender of 
the patient; diet, time and frequency of administration, drug 
combination(s), reaction sensitivities, and tolerance/response to therapy. 
Long acting pharmaceutical compositions might be administered every 3 to 4 
days, every week, or once every two weeks depending on half-life and 
clearance rate of the particular formulation. 
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a 
total dose of about 1 g, depending upon the route of administration. 
Guidance as to particular dosages and methods of delivery is provided in 
the literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. 
Those skilled in the art will employ different formulations for 
nucleotides than for proteins or their inhibitors. Similarly, delivery of 
polynucleotides or polypeptides will be specific to particular cells, 
conditions, locations, etc. 
It is contemplated, for example, that GIPL can be delivered in a suitable 
formulation to block the accelerated turnover of membranes associated with 
phospholipase activity in schizophrenia (Gattaz W F et al. (1995) 
Schizophr Res 16:1-6). In a clinical setting, the monitoring of the 
patient's mental and emotional condition as well as arachidonic acid, 
diacylglycerol, or eicosanoid levels will allow the attending physician to 
adjust dosage of the pharmaceutical composition. Similarly, administration 
of identified agonists should accelerate or extend native GIPL activity 
accomplishing a similar amelioration of the disease state. 
The examples below are provided to illustrate the subject invention and are 
not included for the purpose of limiting the invention. 
EXAMPLES 
I THP1PLB02 cDNA Library Construction 
THP-1 is a human leukemic cell line with distinct monocytic 
characteristics. This cell line was derived from the blood of a 1-year-old 
boy with acute monocytic leukemia (Tsuchiya S et al (1980) Int J Cancer 
26:171-176). Cells were cultured for 48 hr with 100 nm phorbol in DMSO and 
for 4 hr with 1 .mu.g/ml LPS. The PMA+LPS-stimulated cells represent 
activated macrophages. The cDNA libraries was custom constructed by 
Stratagene (La Jolla Calif.) essentially as described below. 
Stratagene prepared the cDNA library using oligo d(T) priming. Synthetic 
adapter oligonucleotides were ligated onto the cDNA molecules enabling 
them to be inserted into the Uni-ZAP.TM. vector system (Stratagene). The 
quality of the cDNA library was screened using DNA probes, and then, the 
pBluescript.RTM. phagemid (Stratagene) was excised. The library phage 
particles were infected into E. coil host strain XL1-Blue.RTM. 
(Stratagene). Alternative unidirectional vectors include, but are not 
limited to, pcDNAI (Invitrogen, San Diego Calif.) and pSHIox-1 (Novagen, 
Madison Wis.). 
II Isolation of cDNA Clones 
The phagemid forms of individual cDNA clones were obtained by the in vivo 
excision process, in which the host bacterial strain was co-infected with 
both the library phage and an f1 helper phage. Polypeptides or enzymes 
derived from both the library-containing phage and the helper phage nicked 
the DNA, initiated new DNA synthesis from defined sequences on the target 
DNA, and created a smaller, single stranded circular phagemid DNA molecule 
that included all DNA sequences of the pBiuescript phagemid and the cDNA 
insert. The phagemid DNA was released from the cells, purified, and used 
to reinfect fresh SOLR.TM. host cells (Stratagene) The newly transformed 
bacteria were selected on medium containing ampicillin and produced 
double-stranded phagemid DNA. 
An alternate method for purifying phagemid utilizes the Miniprep Kit 
(Catalog No. 77468, available from Advanced Genetic Technologies 
Corporation, Gaithersburg Md.). This kit has a 96-well format and provides 
reagents for 960 purifications. Each kit is provided with a recommended 
protocol, which has been employed except for the following changes. First, 
the 96 wells are each filled with only 1 ml of sterile terrific broth with 
carbenicillin at 25 mg/L and glycerol at 0.4%. After the wells are 
inoculated, the bacteria are cultured for 24 hours and lysed with 60 .mu.l 
of lysis buffer. A centrifugation step (2900 rpm for 5 minutes) is 
performed before the contents of the block are added to the primary filter 
plate. The optional step of adding isopropanol to TRIS buffer is not 
routinely performed. After the last step in the protocol, samples are 
transferred to a Beckman 96-well block for storage. 
Phagemid DNA may also be purified using the QIAWELL-8 Plasmid Purification 
System from QIAGEN Inc (Chatsworth Calif.). This high throughput method 
provides lysis of the bacterial cells and isolation of highly purified 
phagemid DNA using QIAGEN.RTM. anion-exchange resin particles with 
EMPORE.TM. membrane technology (3M, Minneapolis Minn.) in a multiwell 
format. The DNA was eluted from the purification resin and prepared for 
DNA sequencing and other analytical manipulations. 
The cDNAs were sequenced by the method of Sanger F and A R Coulson (1975; J 
Mol Biol 94:441f), using a either Applied Biosystems Catalyst 800 or a 
Hamilton Micro Lab 2200 (Hamilton, Reno Nev.) in combination with four 
Peltier Thermal Cyclers (PTC200 from MJ Research, Watertown Mass.) and 
Applied Biosystems 377 or 373 DNA Sequencing Systems; and the reading 
frame was determined. 
III Homology Searching of cDNA Clones and their Deduced Proteins 
Each cDNA was compared to sequences in GenBank using a search algorithm 
developed by Applied Biosystems and incorporated into the INHERIT.TM. 670 
Sequence Analysis System. In this algorithm, Pattern Specification 
Language (TRW Inc, Los Angeles Calif.) was used to determine regions of 
homology. The three parameters that determine how the sequence comparisons 
run were window size, window offset, and error tolerance. Using a 
combination of these three parameters, the DNA database was searched for 
sequences containing regions of homology to the query sequence, and the 
appropriate sequences were scored with an initial value. Subsequently, 
these homologous regions were examined using dot matrix homology plots to 
distinguish regions of homology from chance matches. Smith-Waterman 
alignments were used to display the results of the homology search. 
Peptide and protein sequence homologies were ascertained using the 
INHERIT.TM. 670 Sequence Analysis System in a way similar to that used in 
DNA sequence homologies. Pattern Specification Language and parameter 
windows were used to search protein databases for sequences containing 
regions of homology which were scored with an initial value. Dot-matrix 
homology plots were examined to distinguish regions of significant 
homology from chance matches. 
BLAST, which stands for Basic Local Alignment Search Tool (Altschul SF 
(1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol 
215:403-10), was used to search for local sequence alignments. BLAST 
produces alignments of both nucleotide and amino acid sequences to 
determine sequence similarity. Because of the local nature of the 
alignments, BLAST is especially useful in determining exact matches or in 
identifying homologs. BLAST is useful for matches which do not contain 
gaps. The fundamental unit of BLAST algorithm output is the High-scoring 
Segment Pair (HSP). 
An HSP consists of two sequence fragments of arbitrary but equal lengths 
whose alignment is locally maximal and for which the alignment score meets 
or exceeds a threshold or cutoff score set by the user. The BLAST approach 
is to look for HSPs between a query sequence and a database sequence, to 
evaluate the statistical significance of any matches found, and to report 
only those matches which satisfy the user-selected threshold of 
significance. The parameter E establishes the statistically significant 
threshold for reporting database sequence matches. E is interpreted as the 
upper bound of the expected frequency of chance occurrence of an HSP (or 
set of HSPs) within the context of the entire database search. Any 
database sequence whose match satisfies E is reported in the program 
output. 
IV Extension of GIPL to Full Length or to Recover Regulatory Elements 
The nucleic acid sequence of full length GIPL (SEQ ID NO: 1) may be used to 
design oligonucleotide primers for extending a partial nucleotide sequence 
to full length or for obtaining 5' sequences from genomic libraries. One 
primer is synthesized to initiate extension in the antisense direction 
(XLR) and the other is synthesized to extend sequence in the sense 
direction (XLF). Primers allow the extension of the known GIPL sequence 
"outward" generating ampiicons containing new, unknown nucleotide sequence 
for the region of interest. The initial primers may be designed from the 
cDNA using OLIGO.RTM. 4.06 Primer Analysis Software (National 
Biosciences), or another appropriate program, to be 22-30 nucleotides in 
length, to have a GC content of 50% or more, and to anneal to the target 
sequence, at temperatures about 68.degree.-72.degree. C. Any stretch of 
nucleotides which would result in hairpin structures and primer-primer 
dimerizations is avoided. 
The original cDNA library may be used to extend the sequence or a human 
genomic library is used to extend and amplify 5' upstream regions. If more 
extension is necessary or desired, additional sets of primers are designed 
to further extend the known region. 
By following the instructions for the XL-PCR kit (Perkin Elmer) and 
thoroughly mixing the enzyme and reaction mix, high fidelity amplification 
is obtained. Beginning with 40 pmol of each primer and the recommended 
concentrations of all other components of the kit, PCR is performed using 
the Peltier Thermal Cycler (PTC200; MJ Research, Watertown Mass.) and the 
following parameters: 
Step 1 94.degree. C. for 1 min (initial denaturation) 
Step 2 65.degree. C. for 1 min 
Step 3 68.degree. C. for 6 min 
Step 4 94.degree. C. for 15 sec 
Step 5 65.degree. C. for 1 min 
Step 6 68.degree. C. for 7 min 
Step 7 Repeat step 4-6 for 15 additional cycles 
Step 8 94.degree. C. for 15 sec 
Step 9 65.degree. C. for 1 min 
Step 10 68.degree. C. for 7:15 min 
Step 11 Repeat step 8-10 for 12 cycles 
Step 12 72.degree. C. for 8 min 
Step 13 4.degree. C. (and holding) 
A 5-10 .mu.l aliquot of the reaction mixture is analyzed by electrophoresis 
on a low concentration (about 0.6-0.8%) agarose mini-gel to determine 
which reactions were successful in extending the sequence. The largest 
products or bands were selected and cut out of the gel. Further 
purification involves using a commercial gel extraction method such as 
QIAQuick.TM. (QIAGEN Inc). After recovery of the DNA, Klenow enzyme was 
used to trim single-stranded, nucleotide overhangs creating blunt ends 
which facilitate religation and cloning. 
After ethanol precipitation, the products are redissolved in 13 .mu.l of 
ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1.mu.l T4 
polynucleotide kinase are added, and the mixture is incubated at room 
temperature for 2-3 hours or overnight at 16.degree. C. Competent E. coli 
cells (in 40 .mu.l of appropriate media) are transformed with 3 .mu.l of 
ligation mixture and cultured in 80 .mu.l of SOC medium (Sambrook J et al, 
supra). After incubation for one hour at 37.degree. C., the whole 
transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J et 
al, supra) containing 2.times.Carb. The following day, several colonies 
are randomly picked from each plate and cultured in 150 .mu.l of liquid 
LB/2.times.Carb medium placed in an individual well of an appropriate, 
commercially-available, sterile 96-well microtiter plate. The following 
day, 5 .mu.l of each overnight culture is transferred into a non-sterile 
96-well plate and after dilution 1:10 with water, 5 .mu.l of each sample 
is transferred into a PCR array. 
For PCR amplification, 18 .mu.l of concentrated PCR reaction mix 
(3.3.times.) containing 4 units of rTth DNA polymerase, a vector primer 
and one or both of the gene specific primers used for the extension 
reaction are added to each well. Amplification is performed using the 
following conditions: 
Step 1 94.degree. C. for 60 sec 
Step 2 94.degree. C. for 20 sec 
Step 3 55.degree. C. for 30 sec 
Step 4 72.degree. C. for 90 sec 
Step 5 Repeat steps 2-4 for an additional 29 cycles 
Step 6 72.degree. C. for 180 sec 
Step 7 4.degree. C. (and holding) 
Aliquots of the PCR reactions are run on agarose gels together with 
molecular weight markers. The sizes of the PCR products are compared to 
the original partial cDNAs, and appropriate clones are selected, ligated 
into plasmid and sequenced. 
V Labeling of Hybridization Probes 
Hybridization probes derived from SEQ ID NO: 1 may be employed to screen 
cDNAs, genomic DNAs or mRNAs. Although the labeling of oligonucleotides, 
consisting of about 20 base-pairs, is specifically described, essentially 
the same procedure may be used with larger cDNA fragments. 
Oligonucleotides are designed using state-of-the-art software such as 
OLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of each 
oligomer and 250 mCi of [.gamma.-.sup.32 P] adenosine triphosphate 
(Amersham, Chicago Ill.) and T4 polynucleotide kinase (DuPont NEN.RTM., 
Boston Mass.). The labeled oligonucleotides are purified with Sephadex 
G-25 super fine resin column (Pharmacia). A portion containing 10.sup.7 
counts per minute of each of the sense and antisense oligonucleotides is 
used in a typical membrane based hybridization analysis of human genomic 
DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco 
Rl, Pst I, Xba 1, or Pvu II; DuPont NEN.RTM.). 
The DNA from each digest is fractionated on a 0.7 percent agarose gel and 
transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham 
N.H.). Hybridization is carried out for 16 hours at 40.degree. C. To 
remove nonspecific signals, blots are sequentially washed at room 
temperature under increasingly stringent conditions up to 0.1.times. 
saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR.TM. 
film (Kodak, Rochester N.Y.) is exposed to the blots in a Phosphoimager 
cassette (Molecular Dynamics, Sunnyvale Calif.) for several hours, 
hybridization patterns are compared visually. 
VI Antisense Molecules 
The gipl sequence, or any part thereof, may be used to inhibit in vivo or 
in vitro expression of native gipl. Although use of antisense 
oligonucleotides, comprising about 20 base-pairs, is specifically 
described, essentially the same procedure may be used with larger cDNA 
fragments. An oligonucleotide based on the coding sequence of GIPL as 
shown in FIG. 1 may be used to inhibit expression of native GIPL. The 
complementary oligonucleotide can be designed from the most unique 5' 
sequence as shown in FIG. 2 and used either to inhibit transcription by 
preventing promoter binding to the upstream nontranslated sequence or 
translation of a gipl transcript by preventing the ribosome from binding. 
Using an appropriate portion of the leader and 5' sequence of SEQ ID NO: 
1, an effective antisense oligonucleotide would include approximately 66 
codons spanning the region which translates into the first 22 residues of 
the signal and coding sequence of the polypeptide as shown in FIG. 1. 
VII Expression of GIPL 
Expression of the GIPL may be accomplished by subcloning the cDNAs into 
appropriate vectors and transfecting the vectors into host cells. In this 
case, the cloning vector, pSport, previously used for the generation of 
the cDNA library is used to express GIPL in E. coil. Upstream of the 
cloning site, this vector contains a promoter for .beta.-galactosidase, 
followed by sequence containing the amino-terminal Met and the subsequent 
7 residues of .beta.-galactosidase. Immediately following these eight 
residues is a bacteriophage promoter useful for transcription and a linker 
containing a number of unique restriction sites. 
Induction of an isolated, transfected bacterial strain with IPTG using 
standard methods produces a fusion protein which consists of the first 
seven residues of .beta.-galactosidase, about 5 to 15 residues of linker, 
and the full length GIPL. The signal sequence directs the secretion of 
GIPL into the bacterial growth media which can be used directly in the 
following assay for activity. 
VIII GIPL Activity 
An erthrocyte membrane assay may be used to quantitate GIPL activity. 
Erthrocytes and various phospholipases are placed in eppendorf tubes in 
appropriate media and under appropriate conditions for the digestion of 
membrane phospholipids. After approximately one hour, samples are removed 
and analyzed for arachidonic acid and diacyglycerol using high performance 
liquid chromatography (HPLC). The HPLC results represent the control set 
against which a GIPL-containing experimental set is compared. In the 
experimental set, erythrocytes are incubated with phospholipases and GIPL. 
The effective inhibition of the phospholipases by the GIPL molecule 
disclosed herein is reflected in a lower amount of (or lack of) 
arachidonic acid or diacylglycerol in the media from the experimental 
tubes. 
IX Production of GIPL Specific Antibodies 
Although GIPL purified using PAGE electrophoresis (Maniatis, supra) can be 
used to immunize rabbits using standard protocols, a monoclonal approach 
is more commonly employed. The amino acid sequence translated from GIPL is 
analyzed using DNAStar software (DNAStar Inc) to determine regions of high 
immunogenicity and a corresponding oligopolypeptide is synthesized and 
used to raise antibodies by means known to those of skill in the art. 
Analysis to select appropriate epitopes, such as those near the C-terminus 
or in hydrophilic regions is described by Ausubel F M et al (supra) and 
shown in FIG. 4. 
Typically, the oligopeptides are 15 residues in length, synthesized using 
an Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry, 
and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with 
M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel F M et al, 
supra). Rabbits are immunized with the oligopeptide-KLH complex in 
complete Freund's adjuvant. The resulting antisera are tested for 
antipeptide activity, for example, by binding the peptide to plastic, 
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting 
with radioiodinated, goat anti-rabbit IgG. 
X Purification of Native GIPL Using Specific Antibodies 
Native or recombinant GIPL can be purified by immunoaffinity chromatography 
using antibodies specific for GIPL. An immunoaffinity column is 
constructed by covalently coupling GIPL antibody to an activated 
chromatographic resin such as CnBr-activated Sepharose (Pharmacia 
Biotech). After the coupling, the resin is blocked and washed according to 
the manufacturer's instructions. 
Media containing GIPL is passed over the immunoaffinity column, and the 
column is washed under conditions that allow the preferential absorbance 
of GIPL (eg, high ionic strength buffers in the presence of detergent). 
The column is eluted under conditions that disrupt antibody/GIPL binding 
(eg, a buffer of pH 2-3 or a high concentration of a chaotrope such as 
urea or thiocyanate ion), and GIPL is collected. 
XI Identification of Molecules Which Interact with GIPL 
GIPL, or biologically active fragments thereof, are labelled with .sup.125 
I Bolton-Hunter reagent (Bolton, A E and Hunter, W M (1973) Biochem J 
133:529). Candidate molecules previously arrayed in the wells of a 96 well 
plate are incubated with the labelled GIPL, washed and any wells with 
labelled GIPL complex are assayed. Data obtained using different 
concentrations of GIPL are used to calculate values for the number, 
affinity, and association of GIPL with the candidate molecules. 
All publications and patents mentioned in the above specification are 
herein incorporated by reference. Various modifications and variations of 
the described method and system of the invention will be apparent to those 
skilled in the art without departing from the scope and spirit of the 
invention. Although the invention has been described in connection with 
specific preferred embodiments, it should be understood that the invention 
as claimed should not be unduly limited to such specific embodiments. 
Indeed, various modifications of the described modes for carrying out the 
invention which are obvious to those skilled in molecular biology or 
related fields are intended to be within the scope of the following 
claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 15 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 839 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1LPB02 
(B) CLONE: CONSENSUS 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
NCAATGGGCCGGCCGTGGGAAGGGTGAATGTGGGTCCAGACCCGCCCCTCCTCAGCTTCC60 
TATAAAAGCTGGGGACCAGGTACTGCTGATACACACACCATGAGGCTCTCCAGGAGACCA120 
GAGACCTTTCTGCTGGCCTTTGTGTTGCTCTGCACCCTCCTGGGTCTTGGGTGCCCACTA180 
CACTGCGAAATATGTACGGCGGCGGGGAGCAGGTGCCATGGCCAAATGAAGACCTGCAGC240 
AGTGACAAGGACACATGTGTGCTCCTGGTCGGGAAGGCTACTTCAAAGGGCAAGGAGTTG300 
GTGCACACCTACAAGGGCTGCATCAGGTCCCAGGACTGCTACTCCGGCGTTATATCCACC360 
ACCATGGGCCCCAAGGACCACATGGTAACCAGCTCCTTCTGCTGCCAGAGCGACGGCTGC420 
AACAGTGCCTTTTTGTCTGTTCCCTTGACCAATCTTACTGAGAATGGCCTGATGTGCCCC480 
GCCTGCACTGCGAGCTTCAGGGACAAATGCATGGGGCCCATGACCCACTGTACTGGAAAG540 
GAAAACCACTGCGTCTCCTTATCTGGACACGTGCAGGCTGGTATTTTCAAACCCAGATTT600 
GCTATGCGGGGCTGTGCTACAGAGAGTATGTGCTTTACCAAGCCTGGTGCTGAAGTACCC660 
ACAGGCACCAATGTCCTCTTCCTCCATCATATAGAGTGCACTCACTCCCCCTGAAAAGCT720 
ATCTGAACAGAGGAAGATAATGTAGTGTGAAGTCCCCATTTGTCCTCAGCCTGTAACTTC780 
CCCGTGTGCCTATAAAGAAGTTAATAGAGCAAAAAAAAAAAAAAAAAAAAAAACTCGAG839 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 204 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1LPB02 
(B) CLONE: CONSENSUS 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetArgLeuSerArgArgProGluThrPheLeuLeuAlaPheValLeu 
151015 
LeuCysThrLeuLeuGlyLeuGlyCysProLeuHisCysGluIleCys 
202530 
ThrAlaAlaGlySerArgCysHisGlyGlnMetLysThrCysSerSer 
354045 
AspLysAspThrCysValLeuLeuValGlyLysAlaThrSerLysGly 
505560 
LysGluLeuValHisThrTyrLysGlyCysIleArgSerGlnAspCys 
65707580 
TyrSerGlyValIleSerThrThrMetGlyProLysAspHisMetVal 
859095 
ThrSerSerPheCysCysGlnSerAspGlyCysAsnSerAlaPheLeu 
100105110 
SerValProLeuThrAsnLeuThrGluAsnGlyLeuMetCysProAla 
115120125 
CysThrAlaSerPheArgAspLysCysMetGlyProMetThrHisCys 
130135140 
ThrGlyLysGluAsnHisCysValSerLeuSerGlyHisValGlnAla 
145150155160 
GlyIlePheLysProArgPheAlaMetArgGlyCysAlaThrGluSer 
165170175 
MetCysPheThrLysProGlyAlaGluValProThrGlyThrAsnVal 
180185190 
LeuPheLeuHisHisIleGluCysThrHisSerPro 
195200 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 200 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: GI 501050 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetLysTyrLeuHisThrIleCysLeuLeuPheIlePheValAlaArg 
151015 
GlyAsnSerArgSerCysAspPheCysHisAsnIleGlyLysAspCys 
202530 
AspGlyTyrGluGluGluCysSerSerProGluAspValCysGlyLys 
354045 
ValLeuLeuGluIleSerSerAlaSerLeuSerValArgThrValHis 
505560 
LysAsnCysPheSerSerSerIleCysLysLeuGlyGlnPheAspVal 
65707580 
AsnIleGlyHisHisSerTyrIleArgGlyArgIleAsnCysCysGlu 
859095 
LysGluLeuCysGluAspGlnProPheProGlyLeuProLeuSerLys 
100105110 
ProAsnGlyTyrTyrCysProGlyAlaIleGlyLeuPheThrLysAsp 
115120125 
SerThrGluTyrGluAlaIleCysLysGlyThrGluThrLysCysIle 
130135140 
AsnIleValGlyHisArgTyrGluGlnPheProGlyAspIleSerTyr 
145150155160 
AsnLeuLysGlyCysValSerSerCysProLeuLeuSerLeuSerAsn 
165170175 
AlaThrPheGluGlnAsnArgAsnTyrLeuGluLysValGluCysLys 
180185190 
AspAlaIleArgLeuAlaSerLeu 
195200 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 327 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: HMC1N0T01 
(B) CLONE: 8941 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GGAGGCCTAGGGTTAGGCAAGACCTTGAGGCAGGGGTTGAAGCCAGGGAGTGGTCAGCCA60 
GCACTGTCCCTGCCTGTCCCCATCCCACAGAGGGCAAGGAGTTGGTGCACACCTACAAGG120 
GCTGCATCAGGTCCCAGGACTGCTACTCCGGCGTTATATCNACCACCATGGGCCCCAAGG180 
ACCACATGGTAACCAGCTCCTTCTGNTGCCAGAGCGACGGCTGCAACAGTGCCTTTTTGT240 
CTGTTCCCTTGACCAATCTTACTGAGAATGGCCTGATGTGCCCNGCTGCACTGCGAGTTT300 
NAGGGNCAAAATNCATGGGGGCCCATT327 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 324 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1LPB01 
(B) CLONE: 10033 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GTTGCTCTNCACCCTCCTGGGTCTTGGGTGCCCACTACACTGCGAAATATGTACGGCGGC60 
GGGGAGCAGGTGCCATGGCCAAATGAAGACCTGCAGCAGTGACAAGGACACATGTGTGCT120 
CCTGGTCGGGAAGGCTACTTCAAAGGGCAAGGAGTTGGTGCACACCTACAAGGGCTGCAT180 
CAGGTCCCAGGACTGCTACTCCGGCGTTATATCCACCACCATGGGCCCCAAGGACCACAT240 
GGTAACCAGCTCCTTCTGCTGCAGAGCGACGGCTGCAACAGTGCCTTTTTGTCTGTTCCC300 
TTGACCAATCTTACTGAGAATGGT324 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 262 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1LPB01 
(B) CLONE: 10644 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GACACATGTGTNCTCCTGGTCGGGAAGGCTACTTCAAAGGGCAAGGAGTTGGTGCACACC60 
TACAAGGGCTGCATCAGGTNCCAGGACTGCTACTCCGGNGTTATATCCACCACCATGGGC120 
CCCAAGGACCACATGGTAACCAGCTCCTTCTGCTGCCAGAGCGACGGCTGCAACAGTGCC180 
TTTTTGTCTGTTCCCTTGACCAATNTTACTGAGAATNGNCTGATGTGCCCCGNCTGCACT240 
GNGAGCTTCAGGGACAAATGCT262 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 310 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1LPB01 
(B) CLONE: 10774 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GACACATGTGTGCTCCTGGTCGGGAAGGCTACTTCAAAGGGCAAGGAGTTGGTGCACACC60 
TACAAGGGCTGCATCAGGTCCCAGGACTGCTACTCCGGNGTTATATCCACCACCATGGGC120 
CCCAAGGACCACATGGTAACCAGCTCCTTCTGCTGCCAGAGCGACGGCTGCAACAGTGCC180 
TTTTTGTCTGTTCCCTTANCCAATCTTACTGAGAATGGCCTGATGTGCCCCGNCTGAACT240 
NCGAGCTTCAGGGACAAATNCATGGGNCNATGACCCACTGTACTGGNAAGNNAAACCACT300 
GNGTGTCCTT310 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 185 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1PEB01 
(B) CLONE: 71854 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
CTCCGGCGTTATATCCACCACCATGGGCCCCAAGGACCACATGGTAACCAGCTCCTTCTG60 
CTGCCAGAGCGACGGCTGCAACANTGCCTTTTTNTNTGTTCCCTTGACCAATCTTACTGA120 
GAATGGCCTGATGTGCCCCGCCTGCACTGCGAGCTTCAGGGACAAATGCATGGGGCCCAT180 
GACCC185 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 151 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1PEB01 
(B) CLONE: 72861 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
TTTGGTGCACACCTACAAGGGCTGCATCAGGTCCCAGGACTTCTACTCCGGNGTTATATC60 
CACCACCATGGGCCCCAAGGACCACATGGTAACCAGCTCCTTNTGCTGCCAGAGCGACGG120 
CTGCAACATTGCCTTTTTNTNTGTNCCCTTG151 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 144 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1PEB01 
(B) CLONE: 74452 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
CAGGACTGCTACTCCGGNGTTATATCCACCACCATGGGCCCCAAGGACCACATGGTAACC60 
AGCTCCTTCTGCTGCCAGAGCNACGGCTGCAACANTGCCTTTNTGTCTGTNCCCTTGACC120 
AATCTNACTGAGAATNGCCTGATT144 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 174 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1LPB02 
(B) CLONE: 155045 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CNTGGCCTTAGTTTTNCNCTCACCCTCCNGGGTCTNGGGTGCCCACNACACTGCGAANTA60 
TGTACGGCGGCGGGTAGCAGGTTCCATGNCCAAATNAAGANCTTCANCNGTGACAAGGAC120 
ACATGTNTGCTCCTGGTCGGNAAGNCTACTTCAAAGGGCAAGGAGTTGGTGCAC174 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 194 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: THP1LPB02 
(B) CLONE: 156817 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
CTGGCCTTTGTGTTGCTCTGCACCCTCCTGGGTCTTGGGTGCCCACTACACTGCGAAATA60 
TGTACGGCGGCGGGGAGCAGGTGCCATGGCCAAATGAAGACCTGCAGCAGTGACAAGGAC120 
ACATGTGTGCTCCTGGTCGGGAAGGCTACTTCAAAGGGCAAGGAGTTNGTGCACACCTAC180 
AAGGGCTGCATCAT194 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 224 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PGANNOT01 
(B) CLONE: 619856 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
AGAAAGAGACCATNCCAGGAAGTTGTGGGGTTGGGGAGGCCTAGGGTTAGGCAAGACCTT60 
GAGGCAGGGGTTGAAGCCAGGGAGTGGTCAGCCAGCACTGTCCCTGCCTGTCCCCATCCC120 
ACAGAGGGCAAGGAGTTGGTGCACAACTACAAGGGCTGCATCAGGTCCCAGGACTGCTAC180 
TNCGGNGTTATATCCACCACCATGGGCCCCAAGGACCACATGGT224 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 252 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: UTRSN0T02 
(B) CLONE: 683480 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GTTGGTGCACACCTACAAGGGCTGCATCAGGTCCCAGGACTGCTACTCCGGCGTTATATN60 
CACCACCATGGGNCCCAAGGACCACATGGTAACCAGCTCCTTNTGCTGCCAGAGCGACGN120 
CTGCAACAGTGCCTTTTTGTCTGTTCCCTTGACCAATCTTACTGAGAATGGCCTGATGTG180 
CCCCGNCTGCACTGCGAGCTTNAGGGACAAATGCATGGGGCCCATGACCCACTGTACTGG240 
AGAGGAAAACCA252 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 250 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: BRAINOT11 
(B) CLONE: 1291208 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GGGAATCCCAGTTCTTGCAGCCACTGGGAATCAAGAGGCCCAACTCCGTCTTGGTCTTNN60 
NNNNNNNNNNNNNNNNNCAATGGGCCGGCCGTGGGAAGGGTGAATGTGGGTCCAGACCCG120 
CCCCTCCTCAGCTTCCTATAAAAGCTGGGGACCAGGTACTGCTGATACACACACCATGAG180 
GCTCTCCAGGAGACCAGAGACCTTTCTGCTGGCCTTTGTGTTGCTCTGCACCCTCCTGGG240 
TCTTGGGTGC250 
__________________________________________________________________________