Human preprotachykinin B

The invention provides a human preprotachykinin B (PPT-B) and polynucleotides which identify and encode PPT-B. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of PPT-B.

FIELD OF THE INVENTION 
This invention relates to nucleic acid and amino acid sequences of a human 
preprotachykinin B and to the use of these sequences in the diagnosis, 
prevention, and treatment of neurological and neoplastic disorders. 
BACKGROUND OF THE INVENTION 
Biologically active peptides function as hormones, paracrine regulators, or 
chemical neurotransmitters. These peptides are usually released by 
proteolysis of a larger, often inactive, precursor molecule. Neuropeptides 
perform many functions in the central nervous system (CNS) as 
neurotransmitters, neuromodulators, and neurotrophic factors (Stewart, J. 
M. and Hall, M. E. (1993) Agents Actions Suppl. 42:211-226). Neuropeptides 
are also important regulators of amine neurotransmitter release and can be 
identified as playing important roles in several pathological states 
(Stewart and Hall (1993) supra). 
The tachykinins comprise a family of closely related peptides that 
participate in the regulation of diverse biological processes. They are 
characterized by the amino acid residue 
sequence-Phe-X-Gly-Leu-Met-NH.sub.2 at the C-terminus in which X 
represents an aromatic (Phe, Tyr) or branched aliphatic (Val, Ile) amino 
acid. The tachykinin peptides substance P (SP), neurokinin A (NKA), 
NKA(3-10), neuropeptide K, and neuropeptide .gamma. are produced from a 
single preprotachykinin gene, preprotachykinin A (PPT-A). Differential 
splicing of PPT-A mRNA yields .alpha.PPT-A, .beta.PPT-A, .gamma.PPT-A, and 
.delta.PPT-A mRNA species. Postranslational processing of the 
preproprotein gives rise to multiple active products both within a single 
cell and in different cells expressing the gene (c.f. Helke, C. J. (1990) 
FASEB J. 4:1606-1615; Nakanishi, S. (1987) Physiol. Rev. 67:1117-1142). 
N-terminally extended forms of SP and NKA, but not the mature peptides, 
are detected in measurable amounts in human cerebrospinal fluid (CSF) 
using high performance liquid chromatography (HPLC). It has been suggested 
that their levels in CSF can be used as markers of the activity in central 
SP and NKA neurons (Toresson, G. et al. (1993) Regul. Pept. 46:357-359). 
In mammals, SP and NKA cause vasodilation in the circulatory system, are 
involved in inflammation and immune processes, and play a role in the 
pathogenesis of allergic diseases (Otsuka, M. and Yoshioka, K. (1993) 
Physiol. Rev. 73:229-308). 
A second preprotachykinin gene, PPT-B, encodes proneurokinin B (Kotani, H. 
et al. (1986) Proc. Natl. Acad. Sci. USA 83:7074-7078). Proteolytic 
cleavage of rat proneurokinin B yields neurokinin B (NKB) and a 30-residue 
peptide (Lang, S. and Sperk, G. (1995) Regul. Pept. 57:183-192). PPT-B 
mRNA and peptide products are differentially distributed throughout the 
CNS (particularly in the hypothalamus) and peripheral tissue (Helke, C. J. 
(1990) supra). PPT-B mRNAs have been isolated from bovine and rat brain, 
and the proteins share 75% amino acid residue identity (Kotani, H. et al. 
(1986) supra; Bonner, T. I. et al. (1987) Brain Res. 388:243-249). 
NKB has been shown to be involved in a broad range of biological functions. 
Foe example, acute treatment of rats with synthetic rat NKB prevented a 
decline in cortical choline acetyltransferase activity associated with 
injection of N-methyl-D-aspartate into the nucleus basalis 
magnocellularis. NKB also attenuated impaired behavioral performance 
produced by entorhinalcortex lesions (Wenk, G. L. et al. (1997) Behav. 
Brain Res. 83:129-133). Levels of rat NKB mRNA and NKB-immunoreactivity in 
the granule cells of the rat hippocampus were enhanced following limbic 
epileptogenesis. It has been suggested that these changes may have 
profound effects on synaptic transmission and contribute to modulate 
hippocampal excitability (Schwarzer, C. et al. (1996) Brain Res. Brain 
Res. Rev. 22:27-50). Large amounts of NKB, but not of SP, or NKA, were 
found in tumors of the peripheral nervous system and it has been suggested 
that PPT-B gene expression predominates over that of PPT-A and may be used 
as a tumor marker of nervous tissue (McGregor, G. P. et al. (1990) FEBS 
Lett. 277:83-87). NKB induced airway mucus secretion in the rat, and an 
NKB agonist, [MePhe7]-NKB, caused contraction of guinea pig lung 
parenchymal strips in vitro (Wagner, U. et al. (1995) Life Sci. 
57:283-289; Killingsworth, C. R. and Shore, S. A. (1995) Regul. Pept. 
57:149-161). NKB modulates cellular biochemistry by interacting with NK-3, 
a G-protein coupled receptor, inducing second messenger pathways activated 
by Ca.sup.2+, inositoltrisphosphate, and diacylglycerol (Helke, C. J. et 
al. (1995) supra). The properties of the 30 residue peptide 
(preprotachykinin B(50-79)) are unknown. 
The discovery of a new human preprotachykinin B and the polynucleotides 
encoding it satisfies a need in the art by providing new compositions 
which are useful in the diagnosis, prevention and treatment of 
neurological and neoplastic disorders. 
SUMMARY OF THE INVENTION 
The invention features a substantially purified polypeptide, human 
preprotachykinin B (PPT-B), having the amino acid sequence shown in SEQ ID 
NO:1, or fragments thereof. 
The invention further provides an isolated and substantially purified 
polynucleotide sequence encoding the polypeptide comprising the amino acid 
sequence of SEQ ID NO:1 or fragments thereof and a composition comprising 
said polynucleotide sequence. The invention also provides a polynucleotide 
sequence which hybridizes under stringent conditions to the polynucleotide 
sequence encoding the amino acid sequence SEQ ID NO:1, or fragments of 
said polynucleotide sequence. The invention further provides a 
polynucleotide sequence comprising the complement of the polynucleotide 
sequence encoding the amino acid sequence of SEQ ID NO:1, or fragments or 
variants of said polynucleotide sequence. 
The invention also provides an isolated and purified sequence comprising 
SEQ ID NO.2 or variants thereof. In addition, the invention provides a 
polynucleotide sequence which hybridizes under stringent conditions to the 
polynucleotide sequence of SEQ ID NO:2. 
In another aspect the invention provides a composition comprising an 
isolated and purified polynucleotide sequence comprising the complement of 
SEQ ID NO:2, or fragments or variants thereof. The invention also provides 
a polynucleotide sequence comprising the complement of SEQ ID NO:2. 
The present invention further provides an expression vector containing at 
least a fragment of any of the claimed polynucleotide sequences. In yet 
another aspect, the expression vector containing the polynucleotide 
sequence is contained within a host cell. 
The invention also provides a method for producing a polypeptide comprising 
the amino acid sequence of SEQ ID NO:1 or a fragment thereof, the method 
comprising the steps of: a) culturing the host cell containing an 
expression vector containing at least a fragment of the polynucleotide 
sequence encoding PPT-B under conditions suitable for the expression of 
the polypeptide; and b) recovering the polypeptide from the host cell 
culture. 
The invention also provides a pharmaceutical composition comprising 
substantially purified PPT-B having the amino acid sequence of SEQ ID NO:1 
in conjunction with a suitable pharmaceutical carrier. 
The invention also provides a purified antagonist of a polypeptide of SEQ 
ID NO:1. In one aspect the invention provides a purified antibody which 
binds to a polypeptide comprising at least a fragment of the amino acid 
sequence of SEQ ID NO:1. 
Still further, the invention provides a purified agonist which modulates 
the activity of the polypeptide of SEQ ID NO:1. 
The invention also provides a method for treating or preventing a 
neurological disorder comprising administering to a subject in need of 
such treatment an effective amount of a pharmaceutical composition 
containing PPT-B. 
The invention also provides a method for treating or preventing a 
neoplastic disorder comprising administering to a subject in need of such 
treatment an effective amount of an antagonist to PPT-B. 
The invention also provides a method for detecting a polynucleotide which 
encodes PPT-B in a biological sample comprising the steps of: a) 
hybridizing the complement of the polynucleotide sequence encoding PPT-B 
to nucleic acid material of a biological sample, thereby forming a 
hybridization complex; and b) detecting the hybridization complex, wherein 
the presence of the complex correlates with the presence of a 
polynucleotide encoding PPT-B in the biological sample. In one aspect, 
prior to hybridization, the nucleic acid material of the biological sample 
is amplified by the polymerase chain reaction.

DESCRIPTION OF THE INVENTION 
Before the present proteins, nucleotide sequences, and methods are 
described, it is understood that this invention is not limited to the 
particular methodology, protocols, cell lines, vectors, and reagents 
described, as these may vary. It is also to be understood that the 
terminology used herein is for the purpose of describing particular 
embodiments only, and is not intended to limit the scope of the present 
invention which will be limited only by the appended claims. 
It must be noted that as used herein and in the appended claims, the 
singular forms "a", "an", and "the" include plural reference unless the 
context clearly dictates otherwise. Thus, for example, reference to "a 
host cell" includes a plurality of such host cells, reference to the 
"antibody" is a reference to one or more antibodies and equivalents 
thereof known to those skilled in the art, and so forth. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meanings as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods, 
devices, and materials are now described. All publications mentioned 
herein are incorporated herein by reference for the purpose of describing 
and disclosing the cell lines, vectors, and methodologies which are 
reported in the publications which might be used in connection with the 
invention. Nothing herein is to be construed as an admission that the 
invention is not entitled to antedate such disclosure by virtue of prior 
invention. 
DEFINITIONS 
PPT-B, as used herein, refers to the amino acid sequences of substantially 
purified PPT-B obtained from any species, particularly mammalian, 
including bovine, ovine, porcine, murine, equine, and preferably human, 
from any source whether natural, synthetic, semi-synthetic, or 
recombinant. 
The term "agonist", as used herein, refers to a molecule which, when bound 
to PPT-B, increases or prolongs the duration of the effect of PPT-B. 
Agonists may include proteins, nucleic acids, carbohydrates, or any other 
molecules which bind to and modulate the effect of PPT-B. 
An "allele" or "allelic sequence", as used herein, is an alternative form 
of the gene encoding PPT-B. Alleles may result from at least one mutation 
in the nucleic acid sequence and may result in altered mRNAs or 
polypeptides whose structure or function may or may not be altered. Any 
given natural or recombinant 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 nucleotides. 
Each of these types of changes may occur alone, or in combination with the 
others, one or more times in a given sequence. 
"Altered" nucleic acid sequences encoding PPT-B as used herein include 
those with deletions, insertions, or substitutions of different 
nucleotides resulting in a polynucleotide that encodes the same or a 
functionally equivalent PPT-B. Included within this definition are 
polymorphisms which may or may not be readily detectable using a 
particular oligonucleotide probe of the polynucleotide encoding PPT-B, and 
improper or unexpected hybridization to alleles, with a locus other than 
the normal chromosomal locus for the polynucleotide sequence encoding 
PPT-B. The encoded protein may also be "altered" and contain deletions, 
insertions, or substitutions of amino acid residues which produce a silent 
change and result in a functionally equivalent PPT-B. 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 or immunological activity 
of PPT-B is retained. For example, negatively charged amino acids may 
include aspartic acid and glutamic acid; positively charged amino acids 
may include lysine and arginine; and amino acids with uncharged polar head 
groups having similar hydrophilicity values may include leucine, 
isoleucine, and valine, glycine and alanine, asparagine and glutamine, 
serine and threonine, and phenylalanine and tyrosine. 
"Amino acid sequence" as used herein refers to an oligopeptide, peptide, 
polypeptide, or protein sequence, and fragment thereof, and to naturally 
occurring or synthetic molecules. Fragments of PPT-B are preferably about 
5 to about 15 amino acids in length and retain the biological activity or 
the immunological activity of PPT-B. Where "amino acid sequence" is 
recited herein to refer to an amino acid sequence of a naturally occurring 
protein molecule, amino acid sequence, and like terms, are not meant to 
limit the amino acid sequence to the complete, native amino acid sequence 
associated with the recited protein molecule. 
"Amplification" as used herein refers to the production of additional 
copies of a nucleic acid sequence and is generally carried out using 
polymerase chain reaction (PCR) technologies well known in the art 
(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory 
Manual, Cold Spring Harbor Press, Plainview, N.Y.). 
The term "antagonist" as used herein, refers to a molecule which, when 
bound to PPT-B, decreases the amount or the duration of the effect of the 
biological or immunological activity of PPT-B. Antagonists may include 
antibodies, proteins, nucleic acids, carbohydrates, or any other molecules 
which decrease the effect of PPT-B. 
As used herein, the term "antibody" refers to intact molecules as well as 
fragments thereof, such as Fa, F(ab').sub.2, and Fv, which are capable of 
binding the epitopic determinant. Antibodies that bind PPT-B polypeptides 
can be prepared using intact polypeptides or fragments containing small 
peptides of interest as the immunizing antigen. The polypeptide or 
oligopeptide used to immunize an animal can be derived from the 
translation of RNA or synthesized chemically and can be conjugated to a 
carrier protein, if desired. Commonly used carriers that are chemically 
coupled to peptides include bovine serum albumin and thyroglobulin, 
keyhole limpet hemocyanin. The coupled peptide is then used to immunize 
the animal (e.g., a mouse, a rat, or a rabbit). 
The term "antigenic determinant", as used herein, refers to that fragment 
of a molecule (i.e., an epitope) that makes contact with a particular 
antibody. When a protein or fragment of a protein is used to immunize a 
host animal, numerous regions of the protein may induce the production of 
antibodies which bind specifically to a given region or three-dimensional 
structure on the protein; these regions or structures are referred to as 
antigenic determinants. An antigenic determinant may compete with the 
intact antigen (i.e., the immunogen used to elicit the immune response) 
for binding to an antibody. 
The term "antisense", as used herein, refers to any composition containing 
nucleotide sequences which are complementary to a specific DNA or RNA 
sequence. The term "antisense strand" is used in reference to a nucleic 
acid strand that is complementary to the "sense" strand. Antisense 
molecules include peptide nucleic acids and may be produced by any method 
including synthesis or transcription. Once introduced into a cell, the 
complementary nucleotides combine with natural sequences produced by the 
cell to form duplexes and block either transcription or translation. The 
designation "negative" is sometimes used in reference to the antisense 
strand, and "positive" is sometimes used in reference to the sense strand. 
The term "biologically active", as used herein, refers to a protein having 
structural, regulatory, or biochemical functions of a naturally occurring 
molecule. Likewise, "immunologically active" refers to the capability of 
the natural, recombinant, or synthetic PPT-B, or any oligopeptide thereof, 
to induce a specific immune response in appropriate animals or cells and 
to bind with specific antibodies. 
The terms "complementary" or "complementarity", as used herein, refer to 
the natural binding of polynucleotides under permissive salt and 
temperature conditions by base-pairing. For example, the sequence "A-G-T" 
binds to the complementary sequence "T-C-A". Complementarity between two 
single-stranded molecules may be "partial", in which only some of the 
nucleic acids bind, or it may be complete when total complementarity 
exists between the single stranded molecules. The degree of 
complementarity between nucleic acid strands has significant effects on 
the efficiency and strength of hybridization between nucleic acid strands. 
This is of particular importance in amplification reactions, which depend 
upon binding between nucleic acids strands and in the design and use of 
PNA molecules. 
A "composition comprising a given polynucleotide sequence" as used herein 
refers broadly to any composition containing the given polynucleotide 
sequence. The composition may comprise a dry formulation or an aqueous 
solution. Compositions comprising polynucleotide sequences encoding PPT-B 
(SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 and fragments 
thereof) may be employed as hybridization probes. The probes may be stored 
in freeze-dried form and may be associated with a stabilizing agent such 
as a carbohydrate. In hybridizations, the probe may be deployed in an 
aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS) and 
other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, 
etc.). 
"Consensus", as used herein, refers to a nucleic acid sequence which has 
been resequenced to resolve uncalled bases, has been extended using 
XL-PCR.TM. (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3' 
direction and resequenced, or has been assembled from the overlapping 
sequences of more than one Incyte Clone using a computer program for 
fragment assembly (e.g., GELVIEW.TM. Fragment Assembly system, GCG, 
Madison, Wis.). Some sequences have been both extended and assembled to 
produce the consensus sequence. 
The term "correlates with expression of a polynucleotide", as used herein, 
indicates that the detection of the presence of ribonucleic acid that is 
similar to SEQ ID NO:2 by northern analysis is indicative of the presence 
of mRNA encoding PPT-B in a sample and thereby correlates with expression 
of the transcript from the polynucleotide encoding the protein. 
A "deletion", as used herein, refers to a change in the amino acid or 
nucleotide sequence and results in the absence of one or more amino acid 
residues or nucleotides. 
The term "derivative", as used herein, refers to the chemical modification 
of a nucleic acid encoding or complementary to PPT-B or the encoded PPT-B. 
Such modifications include, for example, replacement of hydrogen by an 
alkyl, acyl, or amino group. A nucleic acid derivative encodes a 
polypeptide which retains the biological or immunological function of the 
natural molecule. A derivative polypeptide is one which is modified by 
glycosylation, pegylation, or any similar process which retains the 
biological or immunological function of the polypeptide from which it was 
derived. 
The term "homology", as used herein, refers to a degree of complementarity. 
There may be partial homology or complete homology (i.e., identity). A 
partially complementary sequence that at least partially inhibits an 
identical sequence from hybridizing to a target nucleic acid is referred 
to using the functional term "substantially homologous." The inhibition of 
hybridization of the completely complementary sequence to the target 
sequence may be examined using a hybridization assay (Southern or northern 
blot, solution hybridization and the like) under conditions of low 
stringency. A substantially homologous sequence or hybridization probe 
will compete for and inhibit the binding of a completely homologous 
sequence to the target sequence under conditions of low stringency. This 
is not to say that conditions of low stringency are such that non-specific 
binding is permitted; low stringency conditions require that the binding 
of two sequences to one another be a specific (i.e., selective) 
interaction. The absence of non-specific binding may be tested by the use 
of a second target sequence which lacks even a partial degree of 
complementarity (e.g., less than about 30% identity). In the absence of 
non-specific binding, the probe will not hybridize to the second 
non-complementary target sequence. 
Human artificial chromosomes (HACs) are linear microchromosomes which may 
contain DNA sequences of 10K to 10M in size and contain all of the 
elements required for stable mitotic chromosome segregation and 
maintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355). 
The term "humanized antibody", as used herein, refers to antibody molecules 
in which amino acids have been replaced in the non-antigen binding regions 
in order to more closely resemble a human antibody, while still retaining 
the original binding ability. 
The term "hybridization", as used herein, refers to any process by which a 
strand of nucleic acid binds with a complementary strand through base 
pairing. 
The term "hybridization complex", as used herein, refers to a complex 
formed between two nucleic acid sequences by virtue of the formation of 
hydrogen bonds between complementary G and C bases and between 
complementary A and T bases; these hydrogen bonds may be further 
stabilized by base stacking interactions. The two complementary nucleic 
acid sequences hydrogen bond in an antiparallel configuration. A 
hybridization complex may be formed in solution (e.g., C.sub.0 t or 
R.sub.0 t analysis) or between one nucleic acid sequence present in 
solution and another nucleic acid sequence immobilized on a solid support 
(e.g., paper, membranes, filters, chips, pins or glass slides, or any 
other appropriate substrate to which cells or their nucleic acids have 
been fixed). 
An "insertion" or "addition", as used herein, refers to a change in an 
amino acid or nucleotide sequence resulting in the addition of one or more 
amino acid residues or nucleotides, respectively, as compared to the 
naturally occurring molecule. 
"Microarray" refers to an array of distinct polynucleotides or 
oligonucleotides synthesized on a substrate, such as paper, nylon or other 
type of membrane, filter, chip, glass slide, or any other suitable solid 
support. 
The term "modulate", as used herein, refers to a change in the activity of 
PPT-B. For example, modulation may cause an increase or a decrease in 
protein activity, binding characteristics, or any other biological, 
functional or immunological properties of PPT-B. 
"Nucleic acid sequence" as used herein refers to an oligonucleotide, 
nucleotide, or polynucleotide, and fragments 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. "Fragments" are those nucleic 
acid sequences which are greater than 60 nucleotides than in length, and 
most preferably includes fragments that are at least 100 nucleotides or at 
least 1000 nucleotides, and at least 10,000 nucleotides in length. 
The term "oligonucleotide" refers to a nucleic acid sequence of at least 
about 6 nucleotides to about 60 nucleotides, preferably about 15 to 30 
nucleotides, and more preferably about 20 to 25 nucleotides, which can be 
used in PCR amplification, or hybridization assays, or microassays. As 
used herein, oligonucleotide is substantially equivalent to the terms 
"amplimers", "primers", "oligomers", and "probes", as commonly defined in 
the art. 
"Peptide nucleic acid", PNA as used herein, refers to an antisense molecule 
or anti-gene agent which comprises an oligonucleotide of at least five 
nucleotides in length linked to a peptide backbone of amino acid residues 
which ends in lysine. The terminal lysine confers solubility to the 
composition. PNAs may be pegylated to extend their life-span in the cell 
where they preferentially bind complementary single stranded DNA and RNA 
and stop transcript elongation (Nielsen, P. E. et al. (1993) Anticancer 
Drug Des. 8:53-63). 
The term "portion", as used herein, with regard to a protein (as in "a 
portion of a given protein") refers to fragments of that protein. The 
fragments may range in size from five amino acid residues to the entire 
amino acid sequence minus one amino acid. Thus, a protein "comprising at 
least a portion of the amino acid sequence of SEQ ID NO:1" encompasses the 
full-length PPT-B and fragments thereof. 
The term "sample", as used herein, is used in its broadest sense. A 
biological sample suspected of containing nucleic acid encoding PPT-B, or 
fragments thereof, or PPT-B itself may comprise a bodily fluid, extract 
from a cell, chromosome, organelle, or membrane isolated from a cell, a 
cell, genomic DNA, RNA, or cDNA (in solution or bound to a solid support, 
a tissue, a tissue print, and the like. 
The terms "specific binding" or "specifically binding", as used herein, 
refers to that interaction between a protein or peptide and an agonist, an 
antibody and an antagonist. The interaction is dependent upon the presence 
of a particular structure (i.e., the antigenic determinant or epitope) of 
the protein recognized by the binding molecule. For example, if an 
antibody is specific for epitope "A", the presence of a protein containing 
epitope A (or free, unlabeled A) in a reaction containing labeled "A" and 
the antibody will reduce the amount of labeled A bound to the antibody. 
The terms "stringent conditions" or "stringency", as used herein, refer to 
the conditions for hybridization as defined by the nucleic acid, salt, and 
temperature. These conditions are well known in the art and may be altered 
in order to identify or detect identical or related polynucleotide 
sequences. Numerous equivalent conditions comprising either low or high 
stringency depend on factors such as the length and nature of the sequence 
(DNA, RNA, base composition), nature of the target (DNA, RNA, base 
composition), milieu (in solution or immobilized on a solid substrate), 
concentration of salts and other components (e.g., formamide, dextran 
sulfate and/or polyethylene glycol), and temperature of the reactions 
(within a range from about 5.degree. C. below the melting temperature of 
the probe to about 20.degree. C. to 25.degree. C. below the melting 
temperature). One or more factors be may be varied to generate conditions 
of either low or high stringency different from, but equivalent to, the 
above listed conditions. 
The term "substantially purified", as used herein, refers to nucleic or 
amino acid sequences that are removed from their natural environment, 
isolated or separated, and are at least 60% free, preferably 75% free, and 
most preferably 90% free from other components with which they are 
naturally associated. 
A "substitution", as used herein, refers to the replacement of one or more 
amino acids or nucleotides by different amino acids or nucleotides, 
respectively. 
"Transformation", as defined herein, describes a process by which exogenous 
DNA enters and changes a recipient cell. It may occur under natural or 
artificial conditions using various methods well known in the art. 
Transformation may rely on any known method for the insertion of foreign 
nucleic acid sequences into a prokaryotic or eukaryotic host cell. The 
method is selected based on the type of host cell being transformed and 
may include, but is not limited to, viral infection, electroporation, heat 
shock, lipofection, and particle bombardment. Such "transformed" cells 
include stably transformed cells in which the inserted DNA is capable of 
replication either as an autonomously replicating plasmid or as part of 
the host chromosome. They also include cells which transiently express the 
inserted DNA or RNA for limited periods of time. 
A "variant" of PPT-B, as used herein, refers to an amino acid sequence that 
is altered by one or more amino acids. The variant may have "conservative" 
changes, wherein a substituted amino acid has similar structural or 
chemical properties, e.g., replacement of leucine with isoleucine. More 
rarely, a variant may have "nonconservative" changes, e.g., replacement of 
a glycine with a tryptophan. Analogous minor variations may also include 
amino acid deletions or insertions, or both. Guidance in determining which 
amino 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 INVENTION 
The invention is based on the discovery of a new human preprotachykinin B 
(hereinafter referred to as "PPT-B"), the polynucleotides encoding PPT-B, 
and the use of these compositions for the diagnosis, prevention, or 
treatment of neurological disorders and neoplastic disorders. 
Nucleic acids encoding the PPT-B of the present invention were first 
identified in Incyte Clone 2109906 from the brain tumor tissue cDNA 
library (BRAITUT03) using a computer search for amino acid sequence 
alignments. A consensus sequence, SEQ ID NO:2, was derived from the 
following overlapping and/or extended nucleic acid sequences: Incyte 
Clones 866703, 866445, 2109906, 2106440 (BRAITUT03), 489306 (HNT2AGT01), 
2256223 (OVARTUT01), 2207643 (SINTFET03), 1500257, 1498317 (SINTBST01), 
1384531 and 1380957 (BRAITUT08). 
In one embodiment, the invention encompasses a polypeptide comprising the 
amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A and 1B. Human 
PPT-B is 122 amino acids in length and has a potential amidation site at 
residue M90; four potential casein kinase II phosphorylation sites at 
residues S37, S55, T75, S99; one potential protein kinase C 
phosphorylation site at residues S37; one potential tyrosine protein 
kinase phosphorylation site at residues Y36; and one potential leucine 
zipper pattern between residues L43 and L64. The hydrophobic amino acid 
residues M1 to G20 of human PPT-B are typical of those of a signal peptide 
of a secreted molecule. Human PPT-B has three potential sites for 
proteolytic cleavage at K38-R39, K79-R80, and K92-R93 to yield a 
38-residue peptide homologous to rat preprotachykinin B(50-79) and a 
peptide with 100% sequence identity to NKB (Helke, C. J. (1990) supra). As 
shown in FIG. 2, human PPT-B has chemical and structural homology with 
bovine PPT-B (GI 163590; SEQ ID NO:3), and rat PPT-B (GI 205724; SEQ ID 
NO:4). In particular, human PPT-B and bovine PPT-B share 66% identity, 
human PPT-B and rat PPT-B share 68% identity. As illustrated by FIGS. 3A 
and 3B, human PPT-B and bovine PPT-B have rather similar hydrophobicity 
plots. Northern analysis shows the expression of this sequence in various 
libraries, at least 50% of which are immortalized or cancerous, in 
particular tumors of the ovary, brain, and adrenal gland; and at least 25% 
of which involve the sympathetic nervous system. 
The invention also encompasses PPT-B variants. A preferred PPT-B variant is 
one having at least 80%, and more preferably 90%, amino acid sequence 
identity to the PPT-B amino acid sequence (SEQ ID NO:1). A most preferred 
PPT-B variant is one having at least 95% amino acid sequence identity to 
SEQ ID NO:1. 
The invention also encompasses polynucleotides which encode PPT-B. 
Accordingly, any nucleic acid sequence which encodes the amino acid 
sequence of PPT-B can be used to produce recombinant molecules which 
express PPT-B. In a particular embodiment, the invention encompasses the 
polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 as 
shown in FIGS. 1A and 1B. 
It will be appreciated by those skilled in the art that as a result of the 
degeneracy of the genetic code, a multitude of nucleotide sequences 
encoding PPT-B, some bearing minimal homology to the nucleotide sequences 
of any known and naturally occurring gene, may be produced. Thus, the 
invention contemplates 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 PPT-B, and all such variations are to be considered as being 
specifically disclosed. 
Although nucleotide sequences which encode PPT-B and its variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring PPT-B under appropriately selected conditions of 
stringency, it may be advantageous to produce nucleotide sequences 
encoding PPT-B 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 
host in accordance with the frequency with which particular codons are 
utilized by the host. Other reasons for substantially altering the 
nucleotide sequence encoding PPT-B 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 invention also encompasses production of DNA sequences, or fragments 
thereof, which encode PPT-B and its derivatives, entirely by synthetic 
chemistry. After production, the synthetic sequence may be inserted into 
any of the many available expression vectors and cell systems using 
reagents that are well known in the art. Moreover, synthetic chemistry may 
be used to introduce mutations into a sequence encoding PPT-B or any 
fragment thereof. 
Also encompassed by the invention are polynucleotide sequences that are 
capable of hybridizing to the claimed nucleotide sequences, and in 
particular, those shown in SEQ ID NO:2, under various conditions of 
stringency as taught in Wahl, G. M. and S. L. Berger (1987; Methods 
Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol. 
152:507-511). 
Methods for DNA sequencing which are well known and generally available in 
the art and may be used to practice any of the embodiments of the 
invention. The methods may employ such enzymes as the Klenow fragment of 
DNA polymerase I, Sequenase.RTM. (U.S. Biochemical Corp, Cleveland, Ohio), 
Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, 
Chicago, Ill.), or combinations of polymerases and proofreading 
exonucleases such as those found in the ELONGASE Amplification System 
marketed by Gibco/BRL (Gaithersburg, Md.). 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 Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer). 
The nucleic acid sequences encoding PPT-B may be extended utilizing a 
partial nucleotide sequence and employing various methods known in the art 
to detect upstream sequences such as promoters and regulatory elements. 
For example, one method which may be employed, "restriction-site" PCR, 
uses universal primers to retrieve unknown sequence adjacent to a known 
locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). In particular, 
genomic DNA is first amplified in the presence of primer to a linker 
sequence and a primer specific to the known region. The amplified 
sequences are then 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 may also 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 commercially available 
software such as OLIGO 4.06 Primer Analysis software (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. 
Another method which may be used is capture PCR which involves PCR 
amplification of DNA fragments adjacent to a known sequence in human and 
yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods 
Applic. 1:111-119). In this method, multiple restriction enzyme digestions 
and ligations may also be used to place an engineered double-stranded 
sequence into an unknown fragment of the DNA molecule before performing 
PCR. 
Another method which may be used to retrieve unknown sequences is that of 
Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060). 
Additionally, one may use PCR, nested primers, and PromoterFinder.TM. 
libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). This process 
avoids the need to screen libraries and is useful in finding intron/exon 
junctions. 
When screening for full-length cDNAs, it is preferable to use libraries 
that have been size-selected to include larger cDNAs. Also, random-primed 
libraries are preferable, in that they will contain more sequences which 
contain the 5' regions of genes. Use of a randomly primed library may be 
especially preferable for situations in which an oligo d(T) library does 
not yield a full-length cDNA. Genomic libraries may be useful for 
extension of sequence into 5' non-transcribed regulatory regions. 
Capillary electrophoresis systems which are commercially available may be 
used to analyze the size or confirm the nucleotide sequence of sequencing 
or PCR products. In particular, capillary sequencing may employ 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 may be converted to electrical signal using appropriate software 
(e.g. Genotyper.TM. and Sequence Navigator.TM., Perkin Elmer) and the 
entire process from loading of samples to computer analysis and electronic 
data display may be computer controlled. Capillary electrophoresis is 
especially preferable for the sequencing of small pieces of DNA which 
might be present in limited amounts in a particular sample. 
In another embodiment of the invention, polynucleotide sequences or 
fragments thereof which encode PPT-B may be used in recombinant DNA 
molecules to direct expression of PPT-B, fragments or functional 
equivalents thereof, 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 produced, and these sequences may be used to clone and express 
PPT-B. 
As will be understood by those of skill in the art, it may be advantageous 
to produce PPT-B-encoding nucleotide sequences possessing non-naturally 
occurring codons. For example, codons preferred by a particular 
prokaryotic or eukaryotic host can be selected to increase the rate of 
protein expression or to produce an RNA transcript having desirable 
properties, such as a half-life which is longer than that of a transcript 
generated from the naturally occurring sequence. 
The nucleotide sequences of the present invention can be engineered using 
methods generally known in the art in order to alter PPT-B encoding 
sequences for a variety of reasons, including but not limited to, 
alterations which modify the cloning, processing, and/or expression of the 
gene product. DNA shuffling by random fragmentation and PCR reassembly of 
gene fragments and synthetic oligonucleotides may be used to engineer the 
nucleotide sequences. For example, site-directed mutagenesis may be used 
to insert new restriction sites, alter glycosylation patterns, change 
codon preference, produce splice variants, introduce mutations, and so 
forth. 
In another embodiment of the invention, natural, modified, or recombinant 
nucleic acid sequences encoding PPT-B may be ligated to a heterologous 
sequence to encode a fusion protein. For example, to screen peptide 
libraries for inhibitors of PPT-B activity, it may be useful to encode a 
chimeric PPT-B protein that can be recognized by a commercially available 
antibody. A fusion protein may also be engineered to contain a cleavage 
site located between the PPT-B encoding sequence and the heterologous 
protein sequence, so that PPT-B may be cleaved and purified away from the 
heterologous moiety. 
In another embodiment, sequences encoding PPT-B may be synthesized, in 
whole or in part, using chemical methods well known in the art (see 
Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, 
T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the 
protein itself may be produced using chemical methods to synthesize the 
amino acid sequence of PPT-B, or a fragment thereof. 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). 
The newly synthesized peptide may be substantially purified by preparative 
high performance liquid chromatography (e.g., Creighton, T. (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 (e.g., the Edman degradation procedure; 
Creighton, supra). Additionally, the amino acid sequence of PPT-B, 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. 
In order to express a biologically active PPT-B, the nucleotide sequences 
encoding PPT-B or functional equivalents, may be inserted into appropriate 
expression vector, i.e., 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 may be used to 
construct expression vectors containing sequences encoding PPT-B and 
appropriate transcriptional and translational control elements. These 
methods include in vitro recombinant DNA techniques, synthetic techniques, 
and in vivo genetic recombination. Such techniques are described in 
Sambrook, J. 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 sequences encoding PPT-B. 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 (e.g., baculovirus); plant cell systems 
transformed with virus expression vectors (e.g., cauliflower mosaic virus, 
CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors 
(e.g., Ti or pBR322 plasmids); or animal cell systems. The invention is 
not limited by the host cell employed. 
The "control elements" or "regulatory sequences" are those non-translated 
regions of the vector--enhancers, promoters, 5' and 3' untranslated 
regions--which interact with host cellular proteins to carry out 
transcription and translation. Such elements may vary in their strength 
and specificity. 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.TM. plasmid (Gibco BRL) 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 (e.g., heat shock, RUBISCO; and 
storage protein genes) or from plant viruses (e.g., viral promoters or 
leader sequences) may be cloned into the vector. In mammalian cell 
systems, promoters from mammalian genes or from mammalian viruses are 
preferable. If it is necessary to generate a cell line that contains 
multiple copies of the sequence encoding PPT-B, 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 PPT-B. For example, when large 
quantities of PPT-B are needed for the induction of antibodies, vectors 
which direct high level expression of fusion proteins that are readily 
purified may be used. Such vectors include, but are not limited to, the 
multi-functional E. coli cloning and expression vectors such as 
Bluescript.RTM. (Stratagene), in which the sequence encoding PPT-B 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, G. and S. M. 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 may be 
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 Enzymol. 153:516-544. 
In cases where plant expression vectors are used, the expression of 
sequences encoding PPT-B may be driven by any of a number of promoters. 
For example, viral promoters such as the 35S and 19S promoters of CaMV may 
be used alone or in combination with the omega leader sequence from TMV 
(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters 
such as the small subunit of RUBISCO or heat shock promoters may be used 
(Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) 
Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell 
Differ. 17:85-105). These constructs can be introduced into plant cells by 
direct DNA transformation or pathogen-mediated transfection. Such 
techniques are described in a number of generally available reviews (see, 
for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science 
and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196. 
An insect system may also be used to express PPT-B. For example, in one 
such system, Autographa califomica nuclear polyhedrosis virus (AcNPV) is 
used as a vector to express foreign genes in Spodoptera frugiperda cells 
or in Trichoplusia larvae. The sequences encoding PPT-B may be cloned into 
a non-essential region of the virus, such as the polyhedrin gene, and 
placed under control of the polyhedrin promoter. Successful insertion of 
PPT-B will render the polyhedrin gene inactive and produce recombinant 
virus lacking coat protein. The recombinant viruses may then be used to 
infect, for example, S. frugiperda cells or Trichoplusia larvae in which 
PPT-B may be expressed (Engelhard, E. K. et al. (1994) Proc. Nat. Acad. 
Sci. 91:3224-3227). 
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, 
sequences encoding PPT-B may be ligated into an adenovirus 
transcription/translation complex consisting of the late promoter and 
tripartite leader sequence. Insertion in a non-essential E1 or E3 region 
of the viral genome may be used to obtain a viable virus which is capable 
of expressing PPT-B in infected host cells (Logan, J. and Shenk, T. (1984) 
Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcription 
enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to 
increase expression in mammalian host cells. 
Human artificial chromosomes (HACs) may also be employed to deliver larger 
fragments of DNA than can be contained and expressed in a plasmid. HACs of 
6 to 10M are constructed and delivered via conventional delivery methods 
(liposomes, polycationic amino polymers, or vesicles) for therapeutic 
purposes. 
Specific initiation signals may also be used to achieve more efficient 
translation of sequences encoding PPT-B. Such signals include the ATG 
initiation codon and adjacent sequences. In cases where sequences encoding 
PPT-B, its initiation codon, and upstream sequences are inserted into the 
appropriate expression vector, no additional transcriptional or 
translational control signals may be needed. However, in cases where only 
coding sequence, or a fragment thereof, is inserted, exogenous 
translational control signals including the ATG initiation codon should be 
provided. Furthermore, the initiation codon should be in the correct 
reading frame to ensure translation of the entire insert. Exogenous 
translational elements and initiation codons may be of various origins, 
both natural and synthetic. The efficiency of expression may be enhanced 
by the inclusion of enhancers which are appropriate for the particular 
cell system which is used, such as those described in the literature 
(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162). 
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 used to facilitate correct insertion, folding and/or function. 
Different host cells which have specific cellular machinery and 
characteristic mechanisms for post-translational activities (e.g., CHO, 
HeLa, MDCK, HEK293, and W138), are available from the American Type 
Culture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure the 
correct modification and processing of the foreign protein. 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express 
PPT-B may be transformed using expression vectors which may contain viral 
origins of replication and/or endogenous expression elements and a 
selectable marker gene on the same or on a separate vector. 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 clones of stably transformed cells may 
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, S. C. and R. C. 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). 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, its presence and expression may need to 
be confirmed. For example, if the sequence encoding PPT-B is inserted 
within a marker gene sequence, transformed cells containing sequences 
encoding PPT-B can be identified by the absence of marker gene function. 
Alternatively, a marker gene can be placed in tandem with a sequence 
encoding PPT-B under the control of a single promoter. Expression of the 
marker gene in response to induction or selection usually indicates 
expression of the tandem gene as well. 
Alternatively, host cells which contain the nucleic acid sequence encoding 
PPT-B and express PPT-B 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 hybridizations and protein bioassay or 
immunoassay techniques which include membrane, solution, or chip based 
technologies for the detection and/or quantification of nucleic acid or 
protein. 
The presence of polynucleotide sequences encoding PPT-B can be detected by 
DNA-DNA or DNA-RNA hybridization or amplification using probes or 
fragments or fragments of polynucleotides encoding PPT-B. Nucleic acid 
amplification based assays involve the use of oligonucleotides or 
oligomers based on the sequences encoding PPT-B to detect transformants 
containing DNA or RNA encoding PPT-B. 
A variety of protocols for detecting and measuring the expression of PPT-B, 
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 fluorescence activated cell sorting 
(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal 
antibodies reactive to two non-interfering epitopes on PPT-B 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. 158:1211-1216). 
A wide variety of labels and conjugation techniques are known by those 
skilled in the art and may be used in various nucleic acid and amino acid 
assays. Means for producing labeled hybridization or PCR probes for 
detecting sequences related to polynucleotides encoding PPT-B include 
oligolabeling, nick translation, end-labeling or PCR amplification using a 
labeled nucleotide. Alternatively, the sequences encoding PPT-B, or any 
fragments thereof 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. 
These procedures may be conducted using a variety of commercially 
available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison, 
Wis.); and U.S. Biochemical Corp., Cleveland, Ohio). Suitable reporter 
molecules or labels, which may be used for ease of detection, include 
radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic 
agents as well as substrates, cofactors, inhibitors, magnetic particles, 
and the like. 
Host cells transformed with nucleotide sequences encoding PPT-B may be 
cultured under conditions suitable for the expression and recovery of the 
protein from cell culture. The protein produced by a transformed 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 polynucleotides which encode PPT-B may be 
designed to contain signal sequences which direct secretion of PPT-B 
through a prokaryotic or eukaryotic cell membrane. Other constructions may 
be used to join sequences encoding PPT-B to nucleotide sequence encoding a 
polypeptide domain which will facilitate purification of soluble proteins. 
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 cleavable linker sequences such as those specific 
for Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the 
purification domain and PPT-B may be used to facilitate purification. One 
such expression vector provides for expression of a fusion protein 
containing PPT-B and a nucleic acid encoding 6 histidine residues 
preceding a thioredoxin or an enterokinase cleavage site. The histidine 
residues facilitate purification on IMAC (immobilized metal ion affinity 
chromatography as described in Porath, J. et al. (1992, Prot. Exp. Purif. 
3:263-281) while the enterokinase cleavage site provides a means for 
purifying PPT-B from the fusion protein. A discussion of vectors which 
contain fusion proteins is provided in Kroll, D. J. et al. (1993; DNA Cell 
Biol. 12:441-453). 
In addition to recombinant production, fragments of PPT-B may be produced 
by direct peptide synthesis using solid-phase techniques Merrifield J. 
(1963) J. Am. Chem. Soc. 85:2149-2154). 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). Various fragments of PPT-B may be chemically synthesized 
separately and combined using chemical methods to produce the full length 
molecule. 
THERAPEUTICS 
Chemical and structural homology exits among human PPT-B, bovine PPT-B (GI 
163590) and rat PPT-B (GI 205725). In addition, PPT-B is expressed in 
neural tissue, gastrointestinal tissue, adrenal tissue, proliferating 
tissue, and rapidly dividing cells. Therefore, PPT-B appears to play a 
role in neurological disorders and neoplastic disorders, particularly 
disorders in which PPT-B is overexpressed, or associated with regulation 
by the sympathetic nervous system. 
Therefore, in one embodiment, PPT-B or a fragment or derivative thereof may 
be administered to a subject to treat a neurological disorder. Such 
disorders include, but are not limited to, akathesia, Alzheimer's disease, 
amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, 
cerebral neoplasms, dementia, depression, Down's syndrome, tardive 
dyskinesia, dystonias, epilepsy, Huntington's disease, multiple sclerosis, 
neurofibromatosis, Parkinson's disease, paranoid psychoses, schizophrenia, 
and Tourette's disorder; angina, anaphylactic shock, arrhythmias, asthma, 
cardiovascular shock, Cushing's syndrome, hypertension, hypoglycemia, 
myocardial infarction, migraine, and pheochromocytoma. 
In another embodiment, a vector capable of expressing PPT-B, or a fragment 
or a derivative thereof, may also be administered to a subject to treat a 
neurological disorder, including, but not limited to, those listed above. 
In still another embodiment, an agonist of PPT-B may also be administered 
to a subject to treat a neurological disorder, including, but not limited 
to, those listed above. 
In one embodiment, an antagonist of PPT-B may be administered to a subject 
to prevent or treat a neoplastic disorder. Such disorders may include, but 
are not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, 
sarcoma, and teratocarcinoma, and particularly cancers of the adrenal 
gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, 
ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, 
ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, 
spleen, testis, thymus, thyroid, and uterus. In one aspect, antibodies 
which specifically bind PPT-B may be used directly as an antagonist or 
indirectly as a targeting or delivery mechanism for bringing a 
pharmaceutical agent to cells or tissue which express PPT-B. 
In another embodiment, a vector expressing the complement of the 
polynucleotide encoding PPT-B may be administered to a subject to treat or 
prevent a neoplastic disorder including, but not limited to, those listed 
above. 
In other embodiments, any of the proteins, antagonists, antibodies, 
agonists, complementary sequences or vectors of the invention may be 
administered in combination with other appropriate therapeutic agents. 
Selection of the appropriate agents for use in combination therapy may be 
made by one of ordinary skill in the art, according to conventional 
pharmaceutical principles. The combination of therapeutic agents may act 
synergistically to effect the treatment or prevention of the various 
disorders described above. Using this approach, one may be able to achieve 
therapeutic efficacy with lower dosages of each agent, thus reducing the 
potential for adverse side effects. 
Antagonists or inhibitors of PPT-B may be produced using methods which are 
generally known in the art. In particular, purified PPT-B may be used to 
produce antibodies or to screen libraries of pharmaceutical agents to 
identify those which specifically bind PPT-B. 
Antibodies to PPT-B may be generated using methods that are well known in 
the art. Such antibodies may include, but are not limited to, polyclonal, 
monoclonal, chimeric, single chain, Fab fragments, and fragments produced 
by a Fab expression library. Neutralizing antibodies, (i.e., those which 
inhibit dimer formation) are especially preferred for therapeutic use. 
For the production of antibodies, various hosts including goats, rabbits, 
rats, mice, humans, and others, may be immunized by injection with PPT-B 
or any fragment or oligopeptide thereof which has 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. Among 
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and 
Corynebacterium parvum are especially preferable. 
It is preferred that the oligopeptides, peptides, or fragments used to 
induce antibodies to PPT-B have an amino acid sequence consisting of at 
least five amino acids and more preferably at least 10 amino acids. It is 
also preferable that they are identical to a portion of the amino acid 
sequence of the natural protein, and they may contain the entire amino 
acid sequence of a small, naturally occurring molecule. Short stretches of 
PPT-B amino acids may be fused with those of another protein such as 
keyhole limpet hemocyanin and antibody produced against the chimeric 
molecule. 
Monoclonal antibodies to PPT-B 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, the human B-cell hybridoma technique, and the EBV-hybridoma 
technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. 
(1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. 
Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 
62:109-120). 
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, S. L. et al. (1984) Proc. Natl. Acad. Sci. 
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; Takeda, 
S. et al. (1985) Nature 314:452-454). Alternatively, techniques described 
for the production of single chain antibodies may be adapted, using 
methods known in the art, to produce PPT-B-specific single chain 
antibodies. Antibodies with related specificity, but of distinct idiotypic 
composition, may be generated by chain shuffling from random combinatorial 
immunoglobin libraries (Burton D. R. (1991) Proc. Natl. Acad. Sci. 
88:11120-3). 
Antibodies may also be produced by inducing in vivo production in the 
lymphocyte population or by screening immunoglobulin libraries or panels 
of highly specific binding reagents as disclosed in the literature 
(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, 
G. et al. (1991) Nature 349:293-299). 
Antibody fragments which contain specific binding sites for PPT-B may also 
be generated. For example, such fragments include, but are not limited to, 
the F(ab')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')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 254:1275-1281). 
Various immunoassays may be used for screening to identify antibodies 
having the desired specificity. Numerous 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 measurement of complex formation 
between PPT-B and its specific antibody. A two-site, monoclonal-based 
immunoassay utilizing monoclonal antibodies reactive to two 
non-interfering PPT-B epitopes is preferred, but a competitive binding 
assay may also be employed (Maddox, supra). 
In another embodiment of the invention, the polynucleotides encoding PPT-B, 
or any fragment or complement thereof, may be used for therapeutic 
purposes. In one aspect, the complement of the polynucleotide encoding 
PPT-B may be used in situations in which it would be desirable to block 
the transcription of the mRNA. In particular, cells may be transformed 
with sequences complementary to polynucleotides encoding PPT-B. Thus, 
complementary molecules or fragments may be used to modulate PPT-B 
activity, or to achieve regulation of gene function. Such technology is 
now well known in the art, and sense or antisense oligonucleotides or 
larger fragments, can be designed from various locations along the coding 
or control regions of sequences encoding PPT-B. 
Expression vectors derived from retro viruses, 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 vectors which will express nucleic acid sequence 
which is complementary to the polynucleotides of the gene encoding PPT-B. 
These techniques are described both in Sambrook et al. (supra) and in 
Ausubel et al. (supra). 
Genes encoding PPT-B can be turned off by transforming a cell or tissue 
with expression vectors which express high levels of a polynucleotide or 
fragment thereof which encodes PPT-B. Such constructs may be used to 
introduce untranslatable sense or antisense sequences into a cell. Even in 
the absence of integration into the DNA, such vectors may continue to 
transcribe RNA molecules until they are disabled by endogenous nucleases. 
Transient expression may last for a month or more with a non-replicating 
vector 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 complementary sequences or antisense molecules (DNA, RNA, or 
PNA) to the control, 5' or regulatory regions of the gene encoding PPT-B 
(signal sequence, promoters, enhancers, and introns). Oligonucleotides 
derived from the transcription initiation site, e.g., between positions 
-10 and +10 from the start site, are preferred. Similarly, inhibition can 
be achieved using "triple helix" base-pairing methodology. Triple helix 
pairing is useful because it causes inhibition of 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 have been described in the literature (Gee, J. 
E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular and 
Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). The 
complementary sequence or antisense molecule may also be designed to block 
translation of mRNA by preventing the transcript from binding to 
ribosomes. 
Ribozymes, enzymatic RNA molecules, may also be used to catalyze 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. Examples which may be 
used include engineered hammerhead motif ribozyme molecules that can 
specifically and efficiently catalyze endonucleolytic cleavage of 
sequences encoding PPT-B. 
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. 
Complementary ribonucleic acid molecules and ribozymes of the invention may 
be prepared by any method known in the art for the synthesis of nucleic 
acid 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 vivo 
transcription of DNA sequences encoding PPT-B. Such DNA sequences may be 
incorporated into a wide variety of vectors with suitable RNA polymerase 
promoters such as T7 or SP6. Alternatively, these cDNA constructs that 
synthesize complementary 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. 
Many methods for introducing vectors into cells or tissues are available 
and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo 
therapy, vectors may be introduced into stem cells taken from the patient 
and clonally propagated for autologous transplant back into that same 
patient. Delivery by transfection, by liposome injections or polycationic 
amino polymers (Goldman, C. K. et al. (1997) Nature Biotechnology 
15:462-66; incorporated herein by reference) may be achieved using methods 
which are well known in the art. 
Any of the therapeutic methods described above may be applied to any 
subject in need of such therapy, including, for example, mammals such as 
dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. 
An additional embodiment of the invention relates to the administration of 
a pharmaceutical composition, in conjunction with a pharmaceutically 
acceptable carrier, for any of the therapeutic effects discussed above. 
Such pharmaceutical compositions may consist of PPT-B, antibodies to 
PPT-B, mimetics, agonists, antagonists, or inhibitors of PPT-B. The 
compositions may be administered 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. The compositions 
may be administered to a patient alone, or in combination with other 
agents, drugs or hormones. 
The pharmaceutical compositions utilized in this invention may be 
administered by any number of routes including, but not limited to, oral, 
intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, 
intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, 
enteral, topical, sublingual, or rectal means. 
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, Pa.). 
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; 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 may be used in conjunction 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, i.e., 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, or liquid 
polyethylene glycol with or without stabilizers. 
Pharmaceutical formulations suitable for parenteral administration 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. Non-lipid polycationic amino polymers may 
also be used for delivery. 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, penetrants appropriate to the 
particular barrier to be permeated are used in the formulation. Such 
penetrants are generally known in the art. 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that is known in the art, e.g., 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 than are the corresponding 
free base forms. In other cases, the preferred preparation may be a 
lyophilized powder which may contain any or all of the following: 1-50 mM 
histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 
5.5, that is combined with buffer prior to use. 
After pharmaceutical compositions have been prepared, they can be placed in 
an appropriate container and labeled for treatment of an indicated 
condition. For administration of PPT-B, such labeling would include 
amount, frequency, and method of administration. 
Pharmaceutical compositions suitable for use in the 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, e.g., of neoplastic cells, or in 
animal models, usually mice, rabbits, dogs, or pigs. The animal model may 
also be used to determine the appropriate 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 active 
ingredient, for example PPT-B or fragments thereof, antibodies of PPT-B, 
agonists, antagonists or inhibitors of PPT-B, which ameliorates the 
symptoms or condition. Therapeutic efficacy and toxicity may be determined 
by standard pharmaceutical procedures in cell cultures or experimental 
animals, e.g., 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, LD50/ED50. 
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 
contained in such compositions is 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 will be determined by the practitioner, in light of 
factors related to the subject that requires treatment. Dosage and 
administration are adjusted to provide sufficient levels of the active 
moiety or to maintain the desired effect. Factors which may be taken into 
account include the severity of the disease state, general health of the 
subject, age, weight, and gender of the subject, diet, time and frequency 
of administration, drug combination(s), reaction sensitivities, and 
tolerance/response to therapy. Long-acting pharmaceutical compositions may 
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 and generally available to practitioners in the art. 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. 
DIAGNOSTICS 
In another embodiment, antibodies which specifically bind PPT-B may be used 
for the diagnosis of conditions or diseases characterized by expression of 
PPT-B, or in assays to monitor patients being treated with PPT-B, 
agonists, antagonists or inhibitors. The antibodies useful for diagnostic 
purposes may be prepared in the same manner as those described above for 
therapeutics. Diagnostic assays for PPT-B include methods which utilize 
the antibody and a label to detect PPT-B in human body fluids or extracts 
of cells or tissues. The antibodies may be used with or without 
modification, and may be labeled by joining them, either covalently or 
non-covalently, with a reporter molecule. A wide variety of reporter 
molecules which are known in the art may be used, several of which are 
described above. 
A variety of protocols including ELISA, RIA, and FACS for measuring PPT-B 
are known in the art and provide a basis for diagnosing altered or 
abnormal levels of PPT-B expression. Normal or standard values for PPT-B 
expression are established by combining body fluids or cell extracts taken 
from normal mammalian subjects, preferably human, with antibody to PPT-B 
under conditions suitable for complex formation The amount of standard 
complex formation may be quantified by various methods, but preferably by 
photometric, means. Quantities of PPT-B expressed in subject, control and 
disease, samples from biopsied tissues are compared with the standard 
values. Deviation between standard and subject values establishes the 
parameters for diagnosing disease. 
In another embodiment of the invention, the polynucleotides encoding PPT-B 
may be used for diagnostic purposes. The polynucleotides which may be used 
include oligonucleotide sequences, complementary RNA and DNA molecules, 
and PNAs. The polynucleotides may be used to detect and quantitate gene 
expression in biopsied tissues in which expression of PPT-B may be 
correlated with disease. The diagnostic assay may be used to distinguish 
between absence, presence, and excess expression of PPT-B, and to monitor 
regulation of PPT-B levels during therapeutic intervention. 
In one aspect, hybridization with PCR probes which are capable of detecting 
polynucleotide sequences, including genomic sequences, encoding PPT-B or 
closely related molecules, may be used to identify nucleic acid sequences 
which encode PPT-B. The specificity of the probe, whether it is made from 
a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory 
region, or a less specific region, e.g., especially in the 3' coding 
region, and the stringency of the hybridization or amplification (maximal, 
high, intermediate, or low) will determine whether the probe identifies 
only naturally occurring sequences encoding PPT-B, alleles, or related 
sequences. 
Probes may also be used for the detection of related sequences, and should 
preferably contain at least 50% of the nucleotides from any of the PPT-B 
encoding sequences. The hybridization probes of the subject invention may 
be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO:2 or 
from genomic sequence including promoter, enhancer elements, and introns 
of the naturally occurring PPT-B. 
Means for producing specific hybridization probes for DNAs encoding PPT-B 
include the cloning of nucleic acid sequences encoding PPT-B or PPT-B 
derivatives into vectors for the production of mRNA probes. Such vectors 
are known in the art, commercially available, and may be used to 
synthesize RNA probes in vitro by means of the addition of the appropriate 
RNA polymerases and the appropriate labeled nucleotides. Hybridization 
probes may be labeled by a variety of reporter groups, for example, 
radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline 
phosphatase coupled to the probe via avidin/biotin coupling systems, and 
the like. 
Polynucleotide sequences encoding PPT-B may be used for the diagnosis of 
conditions, disorders, or diseases which are associated with expression of 
PPT-B. Examples of such conditions or diseases include, but are not 
limited to, akathesia, Alzheimer's disease, amnesia, amyotrophic lateral 
sclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia, 
depression, Down's syndrome, tardive dyskinesia, dystonias, epilepsy, 
Huntington's disease, multiple sclerosis, neurofibromatosis, Parkinson's 
disease, paranoid psychoses, schizophrenia, and Tourette's disorder; 
angina, anaphylactic shock, arrhythmias, asthma, cardiovascular shock, 
Cushing's syndrome, hypertension, hypoglycemia, myocardial infarction, 
migraine, and pheochromocytoma; adenocarcinoma, leukemia, lymphoma, 
melanoma, myeloma, sarcoma, and teratocarcinoma, and particularly cancers 
of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, 
gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, 
muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, 
skin, spleen, testis, thymus, thyroid, and uterus. The polynucleotide 
sequences encoding PPT-B may be used in Southern or northern analysis, dot 
blot, or other membrane-based technologies; in PCR technologies; or in 
dipstick, pin, ELISA assays or microarrays utilizing fluids or tissues 
from patient biopsies to detect altered PPT-B expression. Such qualitative 
or quantitative methods are well known in the art. 
In a particular aspect, the nucleotide sequences encoding PPT-B may be 
useful in assays that detect activation or induction of various cancers, 
particularly those mentioned above. The nucleotide sequences encoding 
PPT-B may be labeled by standard methods, and added to a fluid or tissue 
sample from a patient under conditions suitable for the formation of 
hybridization complexes. After a suitable incubation period, the sample is 
washed and the signal is quantitated and compared with a standard value. 
If the amount of signal in the biopsied or extracted sample is 
significantly altered from that of a comparable control sample, the 
nucleotide sequences have hybridized with nucleotide sequences in the 
sample, and the presence of altered levels of nucleotide sequences 
encoding PPT-B in the sample indicates the presence of the associated 
disease. Such assays may also be used to evaluate the efficacy of a 
particular therapeutic treatment regimen 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 associated with 
expression of PPT-B, a normal or standard profile for expression is 
established. This may be accomplished by combining body fluids or cell 
extracts taken from normal subjects, either animal or human, with a 
sequence, or a fragment thereof, which encodes PPT-B, under conditions 
suitable for hybridization or amplification. Standard hybridization may be 
quantified by comparing the values obtained from normal subjects with 
those from an experiment where a known amount of a substantially purified 
polynucleotide is used. Standard values obtained from normal samples may 
be compared with values obtained from samples from patients who are 
symptomatic for disease. Deviation between standard and subject values is 
used to establish the presence of disease. 
Once disease is established and a treatment protocol is initiated, 
hybridization assays may be repeated on a regular basis to evaluate 
whether the level of expression in the patient begins to approximate that 
which is observed in the normal patient. The results obtained from 
successive assays may be used to show the efficacy of treatment over a 
period ranging from several days to months. 
With respect to cancer, the presence of a relatively high amount of 
transcript in biopsied tissue from an individual may indicate a 
predisposition for the development of the disease, or may provide a means 
for detecting the disease prior to the appearance of actual clinical 
symptoms. A more definitive diagnosis of this type may allow health 
professionals to employ preventative measures or aggressive treatment 
earlier thereby preventing the development or further progression of the 
cancer. 
Additional diagnostic uses for oligonucleotides designed from the sequences 
encoding PPT-B may involve the use of PCR. Such oligomers may be 
chemically synthesized, generated enzymatically, or produced in vitro. 
Oligomers will preferably consist of two nucleotide sequences, one with 
sense orientation (5'-&gt;3') and another 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. 
Methods which may also be used to quantitate the expression of PPT-B 
include radiolabeling or biotinylating nucleotides, coamplification of a 
control nucleic acid, and standard curves onto which the experimental 
results are interpolated (Melby, P. C. et al. (1993) J. Immunol. Methods, 
159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). The speed 
of quantitation of multiple samples may be accelerated by running the 
assay in an ELISA format where the oligomer of interest is presented in 
various dilutions and a spectrophotometric or calorimetric response gives 
rapid quantitation. 
In further embodiments, oligonucleotides derived from any of the 
polynucleotide sequences described herein may be used as targets in 
microarrays. The microarrays can be used to monitor the expression level 
of large numbers of genes simultaneously (to produce a transcript image), 
and to identify genetic variants, mutations and polymorphisms. This 
information will be useful in determining gene function, understanding the 
genetic basis of disease, diagnosing disease, and in developing and 
monitoring the activity of therapeutic agents. 
In one embodiment, the microarray is prepared and used according to the 
methods described in PCT application WO95/11995 (Chee et al.), Lockhart, 
D. J. et al. (1996; Nat. Biotech. 14:1675-1680) and Schena, M. et al. 
(1996; Proc. Natl. Acad. Sci. 93:10614-10619), all of which are 
incorporated herein in their entirety by reference. 
The microarray is preferably composed of a large number of unique, 
single-stranded nucleic acid sequences, usually either synthetic antisense 
oligonucleotides or fragments of cDNAs fixed to a solid support. 
Microarrays may contain oligonucleotides which cover the known 5', or 3', 
sequence, or contain sequential oligonucleotides which cover the full 
length sequence; or unique oligonucleotides selected from particular areas 
along the length of the sequence. Polynucleotides used in the microarray 
may be oligonucleotides that are specific to a gene or genes of interest 
in which at least a fragment of the sequence is known or that are specific 
to one or more unidentified cDNAs which are common to a particular cell 
type, developmental or disease state. 
In order to produce oligonucleotides to a known sequence for a microarray, 
the gene of interest is examined using a computer algorithm which starts 
at the 5' or more preferably at the 3' end of the nucleotide sequence. The 
algorithm identifies oligomers of defined length that are unique to the 
gene, have a GC content within a range suitable for hybridization, and 
lack predicted secondary structure that may interfere with hybridization. 
The oligomers are synthesized at designated areas on a substrate using a 
light-directed chemical process. The substrate may be paper, nylon or 
other type of membrane, filter, chip, glass slide or any other suitable 
solid support. 
In another aspect, the oligonucleotides may be synthesized on the surface 
of the substrate by using a chemical coupling procedure and an ink jet 
application apparatus, as described in PCT application WO95/251116 
(Baldeschweiler et al.) which is incorporated herein in its entirety by 
reference. In another aspect, a "gridded" array analogous to a dot (or 
slot) blot may be used to arrange and link cDNA fragments or 
oligonucleotides to the surface of a substrate using a vacuum system, 
thermal, UV, mechanical or chemical bonding procedures. An array may be 
produced by hand or using available devises (slot blot or dot blot 
apparatus) materials and machines (including robotic instruments) and 
contain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 
dots, or any other multiple which lends itself to the efficient use of 
commercially available instrumentation. 
In order to conduct sample analysis using the microarrays, the RNA or DNA 
from a biological sample is made into hybridization probes. The mRNA is 
isolated, and cDNA is produced and used as a template to make antisense 
RNA (aRNA). The aRNA is amplified in the presence of fluorescent 
nucleotides, and labeled probes are incubated with the microarray so that 
the probe sequences hybridize to complementary oligonucleotides of the 
microarray. Incubation conditions are adjusted so that hybridization 
occurs with precise complementary matches or with various degrees of less 
complementarity. After removal of nonhybridized probes, a scanner is used 
to determine the levels and patterns of fluorescence. The scanned images 
are examined to determine degree of complementarity and the relative 
abundance of each oligonucleotide sequence on the microarray. The 
biological samples may be obtained from any bodily fluids (such as blood, 
urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or 
other tissue preparations. A detection system may be used to measure the 
absence, presence, and amount of hybridization for all of the distinct 
sequences simultaneously. This data may be used for large scale 
correlation studies on the sequences, mutations, variants, or 
polymorphisms among samples. 
In another embodiment of the invention, the nucleic acid sequences which 
encode PPT-B may also be used to generate hybridization probes which are 
useful for mapping the naturally occurring genomic sequence. The sequences 
may be mapped to a particular chromosome, to a specific region of a 
chromosome or to artificial chromosome constructions, such as human 
artificial chromosomes (HACs), yeast artificial chromosomes (YACs), 
bacterial artificial chromosomes (BACs), bacterial P1 constructions or 
single chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood 
Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154. 
Fluorescent in situ hybridization (FISH as described in Verma et al. (1988) 
Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, 
N.Y.) may be correlated with other physical chromosome mapping techniques 
and genetic map data. Examples of genetic map data can be found in various 
scientific journals or at Online Mendelian Inheritance in Man (OMIM). 
Correlation between the location of the gene encoding PPT-B 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. 
In situ hybridization of chromosomal preparations and physical mapping 
techniques such as linkage analysis using established chromosomal markers 
may be used for extending genetic maps. Often the placement of a gene on 
the chromosome of another mammalian species, such as mouse, 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 the disease or syndrome 
has been crudely localized by genetic linkage to a particular genomic 
region, for example, AT to 11q22-23 (Gatti, R. A. 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. 
In another embodiment of the invention, PPT-B, its catalytic or immunogenic 
fragments or oligopeptides thereof, can be used for screening libraries of 
compounds in any of a variety of drug screening techniques. The fragment 
employed in such screening may be free in solution, affixed to a solid 
support, borne on a cell surface, or located intracellularly. The 
formation of binding complexes, between PPT-B and the agent being tested, 
may be measured. 
Another technique for drug screening which may be used provides for high 
throughput screening of compounds having suitable binding affinity to the 
protein of interest as described in published PCT application WO84/03564. 
In this method, as applied to PPT-B large numbers of different small test 
compounds are synthesized on a solid substrate, such as plastic pins or 
some other surface. The test compounds are reacted with PPT-B, or 
fragments thereof, and washed. Bound PPT-B is then detected by methods 
well known in the art. Purified PPT-B 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. 
In another embodiment, one may use competitive drug screening assays in 
which neutralizing antibodies capable of binding PPT-B specifically 
compete with a test compound for binding PPT-B. In this manner, the 
antibodies can be used to detect the presence of any peptide which shares 
one or more antigenic determinants with PPT-B. 
In additional embodiments, the nucleotide sequences which encode PPT-B may 
be used in any molecular biology techniques that have yet to be 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. 
The examples below are provided to illustrate the subject invention and are 
not included for the purpose of limiting the invention. 
EXAMPLES 
I BRAITUT03 cDNA Library Construction 
The BRAITUT03 cDNA library was constructed from astrocytoma tissue (left 
frontal lobe) which was obtained from a 17-year-old Caucasian female 
(specimen #0230; Mayo Clinic, Rochester, Minn.) by excision of cerebral 
meningeal lesion. The pathology report indicated a grade IV fibrillary 
giant and small cell astrocytoma. The patient had a history of benign 
hypertension. Dexarnethasone (Merck & Co., West Point, Pa.) was given to 
reduce inflammation of brain tissue. 
The frozen tissue was homogenized and lysed using a Brinkmann Homogenizer 
Polytron-PT 3000 (Brinkmann Instruments, Inc. Westbury, N.Y.) in 
guanidinium isothiocyanate solution. The lysates were extracted once with 
acid phenol at pH 4.0 per Stratagene's RNA isolation protocol (Stratagene 
Inc, San Diego, Calif.). The RNA was extracted twice with an equal volume 
of acid phenol, reprecipitated using 0.3M sodium acetate and 2.5 volumes 
of ethanol, resuspended in DEPC-treated water and DNase treated for 25 min 
at 37.degree. C. mRNAs were isolated using the Qiagen Oligotex kit (QIAGEN 
Inc, Chatsworth, Calif.) and used to construct the cDNA library. 
The RNA was handled according to the recommended protocols in the 
SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat. 
#18248-013; Gibco/BRL, Gaithersburg, Md.). cDNAs were fractionated on a 
Sepharose CL4B column (Cat. #275105, Pharmacia, Alameda, Calif.), and 
those cDNAs exceeding 400 bp were ligated into pSport I. The plasmid 
pSport I was subsequently transformed into DH5.alpha..TM. competent cells 
(Cat. #18258-012, Gibco/BRL, Gaithersburg, Md.). 
II Isolation and Sequencing of cDNA Clones 
Plasmid DNA was released from the cells and purified using the REAL Prep 96 
Plasmid Kit (Catalog #26173; QIAGEN, Inc). The recommended protocol was 
employed except for the following changes: 1) the bacteria were cultured 
in 1 ml of sterile Terrific Broth (Catalog #22711, Gibco/BRL) with 
carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the cultures were 
incubated for 19 hours after the wells were inoculated and then lysed with 
0.3 ml of lysis buffer; 3) following isopropanol precipitation, the 
plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the 
last step in the protocol, samples were transferred to a Beckman 96-well 
block for storage at 4.degree. C. 
The cDNAs were sequenced by the method of Sanger F. and A. R. Coulson 
(1975; J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, 
Reno, Nev.) in combination with Peltier Thermal Cyclers (PTC200 from MJ 
Research, Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing 
Systems; and the reading frame was determined. 
III Homology Searching of cDNA Clones and Their Deduced Proteins 
The nucleotide sequences of the Sequence Listing or amino acid sequences 
deduced from them were used as query sequences against databases such as 
GenBank, SwissProt, BLOCKS, and Pima II. These databases which contain 
previously identified and annotated sequences were searched for regions of 
homology (similarity) using BLAST, which stands for Basic Local Alignment 
Search Tool (Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et 
al. (1990) J. Mol. Biol. 215:403-410). 
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 which may be of prokaryotic (bacterial) or eukaryotic 
(animal, fungal or plant) origin. Other algorithms such as the one 
described in Smith R. F. and T. F. Smith (1992; Protein Engineering 
5:35-51), incorporated herein by reference, can be used when dealing with 
primary sequence patterns and secondary structure gap penalties. As 
disclosed in this application, the sequences have lengths of at least 49 
nucleotides, and no more than 12% uncalled bases (where N is recorded 
rather than A, C, G, or T). 
The BLAST approach, as detailed in Karlin, S. and S. F. Atschul (1993; 
Proc. Nat. Acad. Sci. 90:5873-7) and incorporated herein by reference, 
searches for matches 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. In this application, threshold was set at 10.sup.-25 for 
nucleotides and 10.sup.-14 for peptides. 
Incyte nucleotide sequences were searched against the GenBank databases for 
primate (pri), rodent (rod), and mammalian sequences (mam), and deduced 
amino acid sequences from the same clones are searched against GenBank 
functional protein databases, mammalian (mamp), vertebrate (vrtp) and 
eukaryote (eukp), for homology. The relevant database for a particular 
match were reported as a GIxxx.+-.p (where xxx is pri, rod, etc and if 
present, p=peptide). 
IV Northern Analysis 
Northern analysis is a laboratory technique used to detect the presence of 
a transcript of a gene and involves the hybridization of a labeled 
nucleotide sequence to a membrane on which RNAs from a particular cell 
type or tissue have been bound (Sambrook et al., supra). 
Analogous computer techniques using BLAST (Altschul, S. F. 1993 and 1990, 
supra) are used to search for identical or related molecules in nucleotide 
databases such as GenBank or the LIFESEQ.TM. database (Incyte 
Pharmaceuticals). This analysis is much faster than multiple, 
membrane-based hybridizations. In addition, the sensitivity of the 
computer search can be modified to determine whether any particular match 
is categorized as exact or homologous. 
The basis of the search is the product score which is defined as: 
##EQU1## 
The product score takes into account both the degree of similarity between 
two sequences and the length of the sequence match. For example, with a 
product score of 40, the match will be exact within a 1-2% error; and at 
70, the match will be exact. Homologous molecules are usually identified 
by selecting those which show product scores between 15 and 40, although 
lower scores may identify related molecules. 
The results of northern analysis are reported as a list of libraries in 
which the transcript encoding PPT-B occurs. Abundance and percent 
abundance are also reported. Abundance directly reflects the number of 
times a particular transcript is represented in a cDNA library, and 
percent abundance is abundance divided by the total number of sequences 
examined in the cDNA library. 
V Extension of PPT-B Encoding Polynucleotides 
The nucleic acid sequence of the Incyte Clone 2109906 was used to design 
oligonucleotide primers for extending a partial nucleotide sequence to 
full length. One primer was synthesized to initiate extension in the 
antisense direction, and the other was synthesized to extend sequence in 
the sense direction. Primers were used to facilitate the extension of the 
known sequence "outward" generating amplicons containing new, unknown 
nucleotide sequence for the region of interest. The initial primers were 
designed from the cDNA using OLIGO 4.06 (National Biosciences), or another 
appropriate program, to be about 22 to about 30 nucleotides in length, to 
have a GC content of 50% or more, and to anneal to the target sequence at 
temperatures of about 68.degree. to about 72.degree. C. Any stretch of 
nucleotides which would result in hairpin structures and primer-primer 
dimerizations was avoided. 
Selected human cDNA libraries (Gibco/BRL) were used to extend the sequence 
If more than one extension is necessary or desired, additional sets of 
primers are designed to further extend the known region. 
High fidelity amplification was obtained by following the instructions for 
the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme and 
reaction mix. Beginning with 40 pmol of each primer and the recommended 
concentrations of all other components of the kit, PCR was performed using 
the Peltier Thermal Cycler (PTC200; M. J. 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 inin 
Step 13 4.degree. C. (and holding) 
______________________________________ 
A 5-10 .mu.l aliquot of the reaction mixture was 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. 
Bands thought to contain the largest products were excised from the gel, 
purified using QIAQuick.TM. (QIAGEN Inc., Chatsworth, Calif.), and trimmed 
of overhangs using Klenow enzyme to facilitate religation and cloning. 
After ethanol precipitation, the products were redissolved in 13 .mu.l of 
ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1 .mu.l T4 
polynucleotide kinase were added, and the mixture was 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) were transformed with 3 .mu.l of 
ligation mixture and cultured in 80 .mu.l of SOC medium (Sambrook et al., 
supra). After incubation for one hour at 37.degree. C., the E. coli 
mixture was plated on Luria Bertani (LB)-agar (Sambrook et al., supra) 
containing 2.times.Carb. The following day, several colonies were 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 was transferred into a non-sterile 
96-well plate and after dilution 1:10 with water, 5 .mu.l of each sample 
was 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 were added to each well. Amplification was 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 were run on agarose gels together with 
molecular weight markers. The sizes of the PCR products were compared to 
the original partial cDNAs, and appropriate clones were selected, ligated 
into plasmid, and sequenced. 
In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain 5' 
regulatory sequences using the procedure above, oligonucleotides designed 
for 5' extension, and an appropriate genomic library. 
VI Labeling and Use of Individual Hybridization Probes 
Hybridization probes derived from SEQ ID NO:2 are 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 is used with larger nucleotide 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 .mu.Ci of [.gamma.-.sup.32 P] adenosine triphosphate 
(Amersham) and T4 polynucleotide kinase (DuPont NEN.RTM., Boston, Mass.). 
The labeled oligonucleotides are substantially purified with Sephadex G-25 
superfine resin column (Pharmacia & Upjohn). A aliquot containing 10.sup.7 
counts per minute of the labeled probe 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 RI, 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. 
VII Microarrays 
To produce oligonucleotides for a microarray, the nucleotide sequence 
described herein is examined using a computer algorithm which starts at 
the 3' end of the nucleotide sequence. The algorithm identifies oligomers 
of defined length that are unique to the gene, have a GC content within a 
range suitable for hybridization, and lack predicted secondary structure 
that would interfere with hybridization. The algorithm identifies 20 
sequence-specific oligonucleotides of 20 nucleotides in length (20-mers). 
A matched set of oligonucleotides is created in which one nucleotide in 
the center of each sequence is altered. This process is repeated for each 
gene in the microarray, and double sets of twenty 20 mers are synthesized 
and arranged on the surface of the silicon chip using a light-directed 
chemical process (Chee, M. et al., PCT/WO95/11995, incorporated herein by 
reference). 
In the alternative, a chemical coupling procedure and an ink jet device are 
used to synthesize oligomers on the surface of a substrate 
(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein by 
reference). In another alternative, a "gridded" array analogous to a dot 
(or slot) blot is used to arrange and link cDNA fragments or 
oligonucleotides to the surface of a substrate using a vacuum system, 
thermal, UV, mechanical or chemical bonding procedures. An array may be 
produced by hand or using available materials and machines and contain 
grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots. After 
hybridization, the microarray is washed to remove nonhybridized probes, 
and a scanner is used to determine the levels and patterns of 
fluorescence. The scanned images are examined to determine degree of 
complementarity and the relative abundance of each oligonucleotide 
sequence on the micro-array. 
VIII Complementary Polynucleotides 
Sequence complementary to the PPT-B-encoding sequence, or any part thereof, 
is used to decrease or inhibit expression of naturally occurring PPT-B. 
Although use of oligonucleotides comprising from about 15 to about 30 
base-pairs is described, essentially the same procedure is used with 
smaller or larger sequence fragments. Appropriate oligonucleotides are 
designed using Oligo 4.06 software and the coding sequence of PPT-B, SEQ 
ID NO:1. To inhibit transcription, a complementary oligonucleotide is 
designed from the most unique 5' sequence and used to prevent promoter 
binding to the coding sequence. To inhibit translation, a complementary 
oligonucleotide is designed to prevent ribosomal binding to the 
PPT-B-encoding transcript. 
IX Expression of PPT-B 
Expression of PPT-B is accomplished by subcloning the cDNAs into 
appropriate vectors and transforming the vectors into host cells. In this 
case, the cloning vector is also used to express PPT-B in E. coli. 
Upstream of the cloning site, this vector contains a promoter for 
.beta.-galactosidase, followed by sequence containing the amino-terminal 
Met, and the subsequent seven 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, transformed bacterial strain with IPTG using 
standard methods produces a fusion protein which consists of the first 
eight residues of .beta.-galactosidase, about 5 to 15 residues of linker, 
and the full length protein. The signal residues direct the secretion of 
PPT-B into the bacterial growth media which can be used directly in the 
following assay for activity. 
X Demonstration of PPT-B Activity 
The assay is based on the increase in firefly luciferase activity in the 
reporter cell line A20/NK3-17/3, that has been stably transfected with the 
cDNA for the human neurokinin receptor NK-3, following treatment with the 
proteolytic product of PPT-B, NKB (Stratowa, C. et al. (1995) J. Recept. 
Signal Transduct. Res. 15:617-630). Luciferase activity is induced by 
hydrolysis of endogenous phosphatidylinositol by the phospholipase C- NK-3 
receptor complex. About 10,000 cells/well are seeded into 
light-impermeable 96-well microtiter plates (Microlite.TM., Dynatech 
Laboratories, Burlington, Mass.) and incubated for 18 hours at 37.degree. 
C. Cells are induced with PPT-B (1 .mu.M) for 7 hours to detect activity. 
After incubation the cells are washed twice in phosphate-buffered saline, 
lysed in assay buffer (270 .mu.M coenzyme A, 530 .mu.M ATP in 20 mM 
tris-HCl, pH 7.8, 1.07 mM (MgCO.sub.3).sub.4 Mg(OH).sub.2 5H.sub.2 O, 2.67 
mM MgSO.sub.4, 0.1 mM EDTA disodiurn dihydrate, and 33 mM dithiothreitol) 
containing 5 .mu.M luciferin. Luciferase activity is measured in a 
luminometer (ML-1000, Dynatech Laboratories) and is linear between 10 nM 
and 0.1 mM NKB (Stratowa, C. et al. (1995) supra). 
XI Production of PPT-B Specific Antibodies 
PPT-B that is substantially purified using PAGE electrophoresis (Sambrook, 
supra), or other purification techniques, is used to immunize rabbits and 
to produce antibodies using standard protocols. The amino acid sequence 
deduced from SEQ ID NO:2 is analyzed using DNASTAR software (DNASTAR Inc) 
to determine regions of high immunogenicity and a corresponding 
oligopeptide is synthesized and used to raise antibodies by means known to 
those of skill in the art. Selection of appropriate epitopes, such as 
those near the C-terminus or in hydrophilic regions, is described by 
Ausubel et al. (supra), and others. 
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, St. Louis, Mo.) by 
reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel 
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 radio iodinated, goat anti-rabbit IgG. 
XII Purification of Naturally Occurring PPT-B Using Specific Antibodies 
Naturally occurring or recombinant PPT-B is substantially purified by 
immunoaffinity chromatography using antibodies specific for PPT-B. An 
immunoaffinity column is constructed by covalently coupling PPT-B antibody 
to an activated chromatographic resin, such as CNBr-activated Sepharose 
(Pharmacia & Upjohn). After the coupling, the resin is blocked and washed 
according to the manufacturer's instructions. 
Media containing PPT-B is passed over the immunoaffinity column, and the 
column is washed under conditions that allow the preferential absorbance 
of PPT-B (e.g., high ionic strength buffers in the presence of detergent). 
The column is eluted under conditions that disrupt antibody/PPT-B binding 
(eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as 
urea or thiocyanate ion), and PPT-B is collected. 
XIII Identification of Molecules Which Interact with PPT-B 
PPT-B or biologically active fragments thereof are labeled with .sup.125 I 
Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133:529). 
Candidate molecules previously arrayed in the wells of a multi-well plate 
are incubated with the labeled PPT-B, washed and any wells with labeled 
PPT-B complex are assayed. Data obtained using different concentrations of 
PPT-B are used to calculate values for the number, affinity, and 
association of PPT-B 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: 4 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 122 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: BRAITUT03 
(B) CLONE: 2109906 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - Met Arg Ile Met Leu Leu Phe Thr Ala Ile - #Leu Ala Phe Ser Leu 
Ala 
1 5 - # 10 - # 15 
- - Gln Ser Phe Gly Ala Val Cys Lys Glu Pro - #Gln Glu Glu Val Val Pro 
20 - # 25 - # 30 
- - Gly Gly Gly Arg Ser Lys Arg Asp Pro Asp - #Leu Tyr Gln Leu Leu Gln 
35 - # 40 - # 45 
- - Arg Leu Phe Lys Ser His Ser Ser Leu Glu - #Gly Leu Leu Lys Ala Leu 
50 - # 55 - # 60 
- - Ser Gln Ala Ser Thr Asp Pro Lys Glu Ser - #Thr Ser Pro Glu Lys Arg 
65 - # 70 - # 75 - # 80 
- - Asp Met His Asp Phe Phe Val Gly Leu Met - #Gly Lys Arg Ser Val Gln 
85 - # 90 - # 95 
- - Pro Asp Ser Pro Thr Glu Met Xaa Asn Gln - #Glu Asn Val Pro Ser Phe 
100 - # 105 - # 110 
- - Gly Ile Leu Lys Tyr Pro Pro Arg Ala Glu 
115 - # 120 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 754 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: BRAITUT03 
(B) CLONE: 2109906 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - AGAGCCTTTA TCAGGGAGCT GGGACTGAGT GACTGCAGCC TTCCTAGATC - #CCCTCCACT 
C 60 
- - GGTTTCTCTC TTTGCAGGAG CACCGGCAGC ACCAGTGTGT GAGGGGAGCA - #GGCAGCGGT 
C 120 
- - CTAGCCAGTT CCTTGATCCT GCCAGACCAC CCAGCCCCCG GCACAGAGCT - #GCTCCACAG 
G 180 
- - CACCATGAGG ATCATGCTGC TATTCACAGC CATCCTGGCC TTCAGCCTAG - #CTCAGAGCT 
T 240 
- - TGGGGCTGTC TGTAAGGAGC CACAGGAGGA GGTGGTTCCT GGCGGGGGCC - #GCAGCAAGA 
G 300 
- - GGATCCAGAT CTCTACCAGC TGCTCCAGAG ACTCTTCAAA AGCCACTCAT - #CTCTGGAGG 
G 360 
- - ATTGCTCAAA GCCCTGAGCC AGGCTAGCAC AGATCCTAAG GAATCAACAT - #CTCCCGAGA 
A 420 
- - ACGTGACATG CATGACTTCT TTGTGGGACT TATGGGCAAG AGGAGCGTCC - #AGCCAGACT 
C 480 
- - TCCTACTGAG ATGTNGAATC AAGAGAACGT CCCCAGCTTT GGCATCCTCA - #AGTATCCCC 
C 540 
- - GAGAGCAGAA TAGGTACTCC ACTTCCGGAC TCCTGGACTG CATTAGGAAG - #ACCTCNTTC 
C 600 
- - CTGTCCCAAT CCCCAGGTGC GCACGCTCCT GTTACCCTTT CTCTTCCCTG - #TTCTTTGTA 
A 660 
- - CATTCTTGTG CTTTGACTCC TTCTCCATCT TTTNCTACCT NGACCCTGGG - #TGTGGAAAC 
T 720 
- - TGCATAGTTG AATATNCCCA ACCCCAATGG GCAT - # - 
# 754 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 126 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 163590 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - Met Arg Ser Thr Leu Leu Phe Ala Val Ile - #Leu Ala Leu Ser Ser 
Ala 
1 5 - # 10 - # 15 
- - Arg Ser Leu Gly Ala Val Cys Glu Glu Ser - #Gln Glu Gln Val Val Pro 
20 - # 25 - # 30 
- - Gly Gly Gly His Ser Lys Lys Asp Ser Asn - #Leu Tyr Gln Leu Pro Pro 
35 - # 40 - # 45 
- - Ser Leu Leu Arg Arg Leu Tyr Asp Ser Arg - #Val Val Ser Leu Asp Gly 
50 - # 55 - # 60 
- - Leu Leu Lys Met Leu Ser Lys Ala Ser Val - #Gly Pro Lys Glu Ser Pro 
65 - # 70 - # 75 - # 80 
- - Leu Pro Gln Lys Arg Asp Met His Asp Phe - #Phe Val Gly Leu Met Gly 
85 - # 90 - # 95 
- - Lys Arg Asn Leu Gln Pro Asp Thr Pro Val - #Asp Ile Asn Gln Glu Asn 
100 - # 105 - # 110 
- - Ile Pro Ser Phe Gly Thr Phe Lys Tyr Pro - #Pro Ser Val Glu 
115 - # 120 - # 125 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 116 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 205725 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Met Arg Ser Ala Met Leu Phe Ala Ala Val - #Leu Ala Leu Ser Leu Ala 
1 5 - # 10 - # 15 
- - Trp Thr Phe Gly Ala Ala Cys Glu Glu Pro - #Gln Glu Gln Gly Gly Arg 
20 - # 25 - # 30 
- - Leu Ser Lys Asp Ser Asp Leu Ser Leu Leu - #Pro Pro Pro Leu Leu Arg 
35 - # 40 - # 45 
- - Arg Leu Tyr Asp Ser Arg Ser Ile Ser Leu - #Glu Gly Leu Leu Lys Val 
50 - # 55 - # 60 
- - Leu Ser Lys Ala Ser Val Gly Pro Lys Glu - #Thr Ser Leu Pro Gln Lys 
65 - # 70 - # 75 - # 80 
- - Arg Asp Met His Asp Phe Phe Val Gly Leu - #Met Gly Lys Arg Asn Ser 
85 - # 90 - # 95 
- - Gln Pro Asp Thr Pro Ala Asp Val Val Glu - #Glu Asn Thr Pro Ser Phe 
100 - # 105 - # 110 
- - Gly Val Leu Lys 
115 
__________________________________________________________________________