The invention provides a human cytochrome P450 (HUCYP) and polynucleotides which identify and encode HUCYP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of HUCYP.

FIELD OF THE INVENTION 
This invention relates to nucleic acid and amino acid sequences of a human 
cytochrome P450 and to the use of these sequences in the diagnosis, 
treatment, and prevention of cell proliferative, developmental, 
autoimmune/inflammatory, and metabolic disorders. 
BACKGROUND OF THE INVENTION 
Members of the cytochrome P450 superfamily of enzymes catalyze the 
oxidative metabolism of a variety of substrates, including natural 
compounds such as steroids, fatty acids, prostaglandins, leukotrienes, and 
vitamins, as well as drugs, carcinogens, mutagens, and xenobiotics. 
Cytochrome P450s, also known as P450 heme-thiolate proteins, usually act 
as terminal oxidases in multi-component electron transfer chains, called 
P450-containing monooxygenase systems. Specific reactions catalyzed 
include hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, 
and O-dealkylations, desulfation, deamination, and reduction of azo, 
nitro, and N-oxide groups. These reactions are involved in steroidogenesis 
of glucocorticoids, cortisols, estrogens, and androgens in animals; 
insecticide resistance in insects; herbicide resistance and flower 
coloring in plants; and environmental bioremediation by microorganisms. 
Cytochrome P450 actions on drugs, carcinogens, mutagens, and xenobiotics 
can result in detoxification or in conversion of the substance to a more 
toxic product. Cytochrome P450s are abundant in the liver, but also occur 
in other tissues. (See ExPASY ENZYME EC 1.14.14.1; Prosite PDOC00081 
Cytochrome P450 cysteine heme-iron ligand signature; PRINTS EP450I E-Class 
P450 Group I signature; and Graham-Lorence, S. and Peterson, J. A. (1996) 
FASEB J. 10:206-214.) 
Four hundred cytochrome P450s have been identified in diverse organisms 
including bacteria, fungi, plants, and animals (Graham-Lorence, supra). 
The B-class is found in prokaryotes and fungi, while the E-class is found 
is found in bacteria, plants, insects, vertebrates, and mammals. Five 
subclasses or groups are found within the larger family of E-class 
cytochrome P450s (PRINTS EP450I E-Class P450 Group I signature). 
All cytochrome P450s use a heme cofactor and share structural attributes. 
Most cytochrome P450s are 400 to 530 amino acids in length. The secondary 
structure of the enzyme is about 70% alpha-helical and about 22% 
beta-sheet. The region around the heme-binding site in the C-terminal part 
of the protein is conserved among cytochrome P450s. A ten amino acid 
signature sequence in this hemeiron ligand region has been identified 
which includes a conserved cysteine involved in binding the heme iron in 
the fifth coordination site. In eukaryotic cytochrome P450s, a 
membrane-spanning region is usually found in the first 15-20 amino acids 
of the protein, generally consisting of approximately 15 hydrophobic 
residues followed by a positively charged residue. (See Prosite PDOC0081, 
supra; Graham-Lorence, supra.) 
Cytochrome P450 enzymes are involved in cell proliferation and development. 
The enzymes have roles in chemical mutagenesis and carcinogenesis by 
metabolizing chemicals to reactive intermediates that form adducts with 
DNA (Nebert, D. W. and Gonzalez, F. J. (1987) Ann. Rev. Biochem. 
56:945-993). These adducts can cause nucleotide changes and DNA 
rearrangements that lead to oncogenesis. Cytochrome P450 expression in 
liver and other tissues is induced by xenobiotics such as polycyclic 
aromatic hydrocarbons, peroxisomal proliferators, phenobarbital, and the 
glucocorticoid dexamethasone (Dogra, S. C. et al. (1998) Clin. Exp. 
Pharmacol. Physiol. 25:1-9). A cytochrome P450 protein may participate in 
eye development as mutations in the P450 gene CYP1B1 cause primary 
congenital glaucoma (Online Mendelian Inheritance in Man (OMIM) *601771 
Cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1; CYP1B1). 
Cytochrome P450s are also involved in drug interactions, because the 
induction of a cytochrome P450 by one drug may affect the metabolism of 
another drug by the enzyme (Katzung, B. G. (1995) Basic and Clinical 
Pharmacoloy, Appleton & Lange, Norwalk Conn., pp. 48-59). 
Cytochrome P450s are associated with inflammation and infection. Hepatic 
cytochrome P450 activities are profoundly affected by various infections 
and inflammatory stimuli, some of which are suppressed and some induced 
(Morgan, E. T. (1997) Drug Metab. Rev. 29:1129-1188). Effects observed in 
vivo can be mimicked by proinflammatory cytokines and interferons. 
Autoantibodies to two cytochrome P450 proteins were found in patients with 
autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), 
a polyglandular autoimmune syndrome (OMIM *240300 Autoimmune 
polyendocrinopathy-candidiasis-ectodermal dystrophy). 
Mutations in cytochrome P450s have been linked to metabolic disorders, 
including congenital adrenal hyperplasia, the most common adrenal disorder 
of infancy and childhood; pseudovitamin D-deficiency rickets; 
cerebrotendinous xanthomatosis, a lipid storage disease characterized by 
progressive neurologic dysfunction, premature atherosclerosis, and 
cataracts; and an inherited resistance to the anticoagulant drugs coumarin 
and warfarin (Isselbacher, K. J. et al. (1994) Harrison's Principles of 
Internal Medicine, McGraw-Hill, Inc. New York, N.Y., pp. 1968-1970; 
Takeyama, K. et al. (1997) Science 277:1827-1830; Kitanaka, S. et al. 
(1998) N. Engl. J. Med. 338:653-661; OMIM *213700 Cerebrotendinous 
xanthomatosis; and OMIM #122700 Coumarin resistance). Extremely high 
levels of expression of the cytochrome P450 protein aromatase were found 
in a fibrolamellar hepatocellular carcinoma from a boy with severe 
gynecomastia (feminization) (Agarwal, V. R. (1998) J. Clin. Endocrinol. 
Metab. 83:1797-1800). 
The discovery of a new human cytochrome P450 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 cell 
proliferative, developmental, autoimmune/inflammatory, and metabolic 
disorders. 
SUMMARY OF THE INVENTION 
The invention is based on the discovery of a new human cytochrome P450 
(HUCYP), the polynucleotides encoding HUCYP, and the use of these 
compositions for the diagnosis, treatment, or prevention of cell 
proliferative, developmental, autoimmune/inflammatory, and metabolic 
disorders. 
The invention features a substantially purified polypeptide comprising the 
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. 
The invention further provides a substantially purified variant having at 
least 90% amino acid sequence identity to the amino acid sequence of SEQ 
ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an 
isolated and purified polynucleotide encoding the polypeptide comprising 
the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The 
invention also includes an isolated and purified polynucleotide variant 
having at least 70% polynucleotide sequence identity to the polynucleotide 
encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 
or a fragment of SEQ ID NO:1. 
The invention further provides an isolated and purified polynucleotide 
which hybridizes under stringent conditions to the polynucleotide encoding 
the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a 
fragment of SEQ ID NO:1, as well as an isolated and purified 
polynucleotide having a sequence which is complementary to the 
polynucleotide encoding the polypeptide comprising the amino acid sequence 
of SEQ ID NO:1 or a fragment of SEQ ID NO:1. 
The invention also provides a method for detecting a polynucleotide in a 
sample containing nucleic acids, the method comprising the steps of (a) 
hybridizing the complement of the polynucleotide sequence to at least one 
of the polynucleotides of the sample, thereby forming a hybridization 
complex; and (b) detecting the hybridization complex, wherein the presence 
of the hybridization complex correlates with the presence of a 
polynucleotide in the sample. In one aspect, the method further comprises 
amplifying the polynucleotide prior to hybridization. 
The invention also provides an isolated and purified polynucleotide 
comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ 
ID NO:2, and an isolated and purified polynucleotide variant having at 
least 70% polynucleotide sequence identity to the polynucleotide 
comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ 
ID NO:2. The invention also provides an isolated and purified 
polynucleotide having a sequence complementary to the polynucleotide 
comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ 
ID NO:2. 
The invention further provides an expression vector containing at least a 
fragment of the polynucleotide encoding the polypeptide comprising the 
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, 
the expression vector is contained within a host cell. 
The invention also provides a method for producing a polypeptide, the 
method comprising the steps of: (a) culturing the host cell containing an 
expression vector containing at least a fragment of a polynucleotide 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 a 
substantially purified polypeptide having the sequence of SEQ ID NO:1 or a 
fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical 
carrier. 
The invention further includes a purified antibody which binds to a 
polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID 
NO:1, as well as a purified agonist and a purified antagonist of the 
polypeptide. 
The invention also provides a method for treating or preventing a disorder 
associated with decreased expression or activity of HUCYP, the method 
comprising administering to a subject in need of such treatment an 
effective amount of a pharmaceutical composition comprising a 
substantially purified polypeptide having the amino acid sequence of SEQ 
ID NO:1 or a fragment of SEQ ID NO:1, in conjunction with a suitable 
pharmaceutical carrier. 
The invention also provides a method for treating or preventing a disorder 
associated with increased expression or activity of HUCYP, the method 
comprising administering to a subject in need of such treatment an 
effective amount of an antagonist of the polypeptide having the amino acid 
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

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 machines, materials and methods 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, a reference to "a 
host cell" includes a plurality of such host cells, and a reference to "an 
antibody" is a reference to one or more antibodies and equivalents thereof 
known to those skilled in the 14 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 machines, materials, 
and methods similar or equivalent to those described herein can be used to 
practice or test the present invention, the preferred machines, materials 
and methods are now described. All publications mentioned herein are cited 
for the purpose of describing and disclosing the cell lines, protocols, 
reagents and vectors which are reported in the publications and 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 
"HUCYP" refers to the amino acid sequences of substantially purified HUCYP 
obtained from any species, particularly a mammalian species, including 
bovine, ovine, porcine, murine, equine, and preferably the human species, 
from any source, whether natural, synthetic, semi-synthetic, or 
recombinant. 
The term "agonist" refers to a molecule which, when bound to HUCYP, 
increases or prolongs the duration of the effect of HUCYP. Agonists may 
include proteins, nucleic acids, carbohydrates, or any other molecules 
which bind to and modulate the effect of HUCYP. 
An "allelic variant" is an alternative form of the gene encoding HUCYP. 
Allelic variants may result from at least one mutation in the nucleic acid 
sequence and may result in altered mRNAs or in 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 allelic variants 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 HUCYP include those sequences 
with deletions, insertions, or substitutions of different nucleotides, 
resulting in a polypeptide the same as HUCYP or a polypeptide with at 
least one functional characteristic of HUCYP. Included within this 
definition are polymorphisms which may or may not be readily detectable 
using a particular oligonucleotide probe of the polynucleotide encoding 
HUCYP, and improper or unexpected hybridization to allelic variants, with 
a locus other than the normal chromosomal locus for the polynucleotide 
sequence encoding HUCYP. The encoded protein may also be "altered," and 
may contain deletions, insertions, or substitutions of amino acid residues 
which produce a silent change and result in a functionally equivalent 
HUCYP. 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 HUCYP 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. 
The terms "amino acid" or "amino acid sequence" refer to an oligopeptide, 
peptide, polypeptide, or protein sequence, or a fragment of any of these, 
and to naturally occurring or synthetic molecules. In this context, 
"fragments," "immunogenic fragments," or "antigenic fragments" refer to 
fragments of HUCYP which are preferably at least 5 to about 15 amino acids 
in length, most preferably at least 14 amino acids, and which retain some 
biological activity or immunological activity of HUCYP. Where "amino acid 
sequence" is recited 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" relates to the production of additional copies of a nucleic 
acid sequence. Amplification is generally carried out using polymerase 
chain reaction (PCR) technologies well known in the art. 
The term "antagonist" refers to a molecule which, when bound to HUCYP, 
decreases the amount or the duration of the effect of the biological or 
immunological activity of HUCYP. Antagonists may include proteins, nucleic 
acids, carbohydrates, antibodies, or any other molecules which decrease 
the effect of HUCYP. 
The term "antibody" refers to intact molecules as well as to fragments 
thereof, such as Fab, F(ab').sub.2, and Fv fragments, which are capable of 
binding the epitopic determinant. Antibodies that bind HUCYP polypeptides 
can be prepared using intact polypeptides or using fragments containing 
small peptides of interest as the immunizing antigen. The polypeptide or 
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a 
rabbit) 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, thyroglobulin, and keyhole limpet hemocyanin (KLH). 
The coupled peptide is then used to immunize the animal. 
The term "antigenic determinant" refers to that fragment of a molecule 
(i.e., an epitope) that makes contact with a particular antibody. When a 
protein or a 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 antigenic determinants (given regions or 
three-dimensional structures on the protein). 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" refers to any composition containing a nucleic acid 
sequence which is complementary to the "sense" strand of a specific 
nucleic acid sequence. Antisense molecules 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 to block either transcription or translation. 
The designation "negative" can refer to the antisense strand, and the 
designation "positive" can refer to the sense strand. 
The term "biologically active," 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 HUCYP, or of 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" refer to the natural binding 
of polynucleotides by base pairing. For example, the sequence "5' A-G-T 
3'" bonds to the complementary sequence "3' T-C-A 5'." Complementarity 
between two single-stranded molecules may be "partial," such that only 
some of the nucleic acids bind, or it may be "complete," such that 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 the hybridization between the 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 peptide nucleic acid (PNA) molecules. 
A "composition comprising a given polynucleotide sequence" or a 
"composition comprising a given amino acid sequence" refer broadly to any 
composition containing the given polynucleotide or amino acid sequence. 
The composition may comprise a dry formulation or an aqueous solution. 
Compositions comprising polynucleotide sequences encoding HUCYP or 
fragments of HUCYP 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., 
sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's 
solution, dry milk, salmon sperm DNA, etc.). "Consensus sequence" refers 
to a nucleic acid sequence which has been resequenced to resolve uncalled 
bases, extended using XL-PCR kit (Perkin-Elmer, Norwalk Conn.) in the 5' 
and/or the 3' direction, and resequenced, or which has been assembled from 
the overlapping sequences of more than one Incyte Clone using a computer 
program for fragment assembly, such as the GELVIEW 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" indicates that 
the detection of the presence of nucleic acids, the same or related to a 
nucleic acid sequence encoding HUCYP, by northern analysis is indicative 
of the presence of nucleic acids encoding HUCYP in a sample, and thereby 
correlates with expression of the transcript from the polynucleotide 
encoding HUCYP. 
A "deletion" refers to a change in the amino acid or nucleotide sequence 
that results in the absence of one or more amino acid residues or 
nucleotides. 
The term "derivative" refers to the chemical modification of a polypeptide 
sequence, or a polynucleotide sequence. Chemical modifications of a 
polynucleotide sequence can include, for example, replacement of hydrogen 
by an alkyl, acyl, or amino group. A derivative polynucleotide encodes a 
polypeptide which retains at least one biological or immunological 
function of the natural molecule. A derivative polypeptide is one modified 
by glycosylation, pegylation, or any similar process that retains at least 
one biological or immunological function of the polypeptide from which it 
was derived. 
The term "similarity" refers to a degree of complementarity. There may be 
partial similarity or complete similarity. The word "identity" may 
substitute for the word "similarity." A partially complementary sequence 
that at least partially inhibits an identical sequence from hybridizing to 
a target nucleic acid is referred to as "substantially similar." 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 reduced stringency. A substantially similar sequence or hybridization 
probe will compete for and inhibit the binding of a completely similar 
(identical) sequence to the target sequence under conditions of reduced 
stringency. This is not to say that conditions of reduced stringency are 
such that non-specific binding is permitted, as reduced stringency 
conditions require that the binding of two sequences to one another be a 
specific (i.e., a 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% 
similarity or identity). In the absence of non-specific binding, the 
substantially similar sequence or probe will not hybridize to the second 
non-complementary target sequence. 
The phrases "percent identity" or "% identity" refer to the percentage of 
sequence similarity found in a comparison of two or more amino acid or 
nucleic acid sequences. Percent identity can be determined electronically, 
e.g., by using the MEGALIGN program (DNASTAR) which creates alignments 
between two or more sequences according to methods selected by the user, 
e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp 
(1988) Gene 73:237-244.) The clustal algorithm groups sequences into 
clusters by examining the distances between all pairs. The clusters are 
aligned pairwise and then in groups. The percentage similarity between two 
amino acid sequences, e.g., sequence A and sequence B, is calculated by 
dividing the length of sequence A, minus the number of gap residues in 
sequence A, minus the number of gap residues in sequence B, into the sum 
of the residue matches between sequence A and sequence B, times one 
hundred. Gaps of low or of no similarity between the two amino acid 
sequences are not included in determining percentage similarity. Percent 
identity between nucleic acid sequences can also be counted or calculated 
by other methods known in the art, e.g., the Jotun Hein method. (See, 
e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between 
sequences can also be determined by other methods known in the art, e.g., 
by varying hybridization conditions. 
"Human artificial chromosomes" (HACs) are linear microchromosomes which may 
contain DNA sequences of about 6 kb to 10 Mb in size, and which contain 
all of the elements required for stable mitotic chromosome segregation and 
maintenance. 
The term "humanized antibody" refers to antibody molecules in which the 
amino acid sequence in the non-antigen binding regions has been altered so 
that the antibody more closely resembles a human antibody, and still 
retains its original binding ability. 
"Hybridization" refers to any process by which a strand of nucleic acid 
binds with a complementary strand through base pairing. 
The term "hybridization complex" refers to a complex formed between two 
nucleic acid sequences by virtue of the formation of hydrogen bonds 
between complementary bases. A hybridization complex may be formed in 
solution (e.g., C.sub.0 t or R.sub.0 t analysis) or formed 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). 
The words "insertion" or "addition" refer to changes in an amino acid or 
nucleotide sequence resulting in the addition of one or more amino acid 
residues or nucleotides, respectively, to the sequence found in the 
naturally occurring molecule. 
"Immune response" can refer to conditions associated with inflammation, 
trauma, immune disorders, or infectious or genetic disease, etc. These 
conditions can be characterized by expression of various factors, e.g., 
cytokines, chemokines, and other signaling molecules, which may affect 
cellular and systemic defense systems. 
The term "microarray" refers to an arrangement of distinct polynucleotides 
on a substrate. 
The terms "element" or "array element" in a microarray context, refer to 
hybridizable polynucleotides arranged on the surface of a substrate. 
The term "modulate" refers to a change in the activity of HUCYP. For 
example, modulation may cause an increase or a decrease in protein 
activity, binding characteristics, or any other biological, functional, or 
immunological properties of HUCYP. 
The phrases "nucleic acid" or "nucleic acid sequence" refer to a 
nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. 
These phrases also refer to DNA or RNA of genomic or synthetic origin 
which may be single-stranded or double-stranded and may represent the 
sense or the antisense strand, to peptide nucleic acid (PNA), or to any 
DNA-like or RNA-like material. In this context, "fragments" refers to 
those nucleic acid sequences which, when translated, would produce 
polypeptides retaining some functional characteristic, e.g., antigenicity, 
or structural domain characteristic, e.g., ATP-binding site, of the 
full-length polypeptide. 
The terms "operably associated" or "operably linked" refer to functionally 
related nucleic acid sequences. A promoter is operably associated or 
operably linked with a coding sequence if the promoter controls the 
translation of the encoded polypeptide. While operably associated or 
operably linked nucleic acid sequences can be contiguous and in the same 
reading frame, certain genetic elements, e.g., repressor genes, are not 
contiguously linked to the sequence encoding the polypeptide but still 
bind to operator sequences that control expression of the polypeptide. 
The term "oligonucleotide" refers to a nucleic acid sequence of at least 
about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 
nucleotides, and most preferably about 20 to 25 nucleotides, which can be 
used in PCR amplification or in a hybridization assay or microarray. 
"Oligonucleotide" is substantially equivalent to the terms "amplimer," 
"primer," "oligomer," and "probe," as these terms are commonly defined in 
the art. 
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene 
agent which comprises an oligonucleotide of at least about 5 nucleotides 
in length linked to a peptide backbone of amino acid residues ending in 
lysine. The terminal lysine confers solubility to the composition. PNAs 
preferentially bind complementary single stranded DNA or RNA and stop 
transcript elongation, and may be pegylated to extend their lifespan in 
the cell. 
The term "sample" is used in its broadest sense. A sample suspected of 
containing nucleic acids encoding HUCYP, or fragments thereof, or HUCYP 
itself, may comprise a bodily fluid; an 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 substrate; a tissue; a tissue print; etc. 
The terms "specific binding" or "specifically binding" refer to that 
interaction between a protein or peptide and an agonist, an antibody, or 
an antagonist. The interaction is dependent upon the presence of a 
particular structure of the protein, e.g., the antigenic determinant or 
epitope, recognized by the binding molecule. For example, if an antibody 
is specific for epitope "A," the presence of a polypeptide containing the 
epitope A, or the presence of free unlabeled A, in a reaction containing 
free labeled A and the antibody will reduce the amount of labeled A that 
binds to the antibody. 
The term "stringent conditions" refers to conditions which permit 
hybridization between polynucleotides and the claimed polynucleotides. 
Stringent conditions can be defined by salt concentration, the 
concentration of organic solvent, e.g., formamide, temperature, and other 
conditions well known in the art. In particular, stringency can be 
increased by reducing the concentration of salt, increasing the 
concentration of formamide, or raising the hybridization temperature. 
The term "substantially purified" refers to nucleic acid or amino acid 
sequences that are removed from their natural environment and are isolated 
or separated, and are at least about 60% free, preferably about 75% free, 
and most preferably about 90% free from other components with which they 
are naturally associated. 
A "substitution" refers to the replacement of one or more amino acids or 
nucleotides by different amino acids or nucleotides, respectively. 
"Substrate" refers to any suitable rigid or semi-rigid support including 
membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic 
beads, gels, tubing, plates, polymers, microparticles and capillaries. The 
substrate can have a variety of surface forms, such as wells, trenches, 
pins, channels and pores, to which polynucleotides or polypeptides are 
bound. 
"Transformation" describes a process by which exogenous DNA enters and 
changes a recipient cell. Transformation may occur under natural or 
artificial conditions according to various methods well known in the art, 
and may rely on any known method for the insertion of foreign nucleic acid 
sequences into a prokaryotic or eukaryotic host cell. The method for 
transformation 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. The 
term "transformed" cells includes 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, as well as 
transiently transformed cells which express the inserted DNA or RNA for 
limited periods of time. 
A "variant" of HUCYP polypeptides refers to an amino acid sequence that is 
altered by one or more amino acid residues. 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 glycine with 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, 
LASERGENE software (DNASTAR). 
The term "variant," when used in the context of a polynucleotide sequence, 
may encompass a polynucleotide sequence related to HUCYP. This definition 
may also include, for example, "allelic" (as defined above), "splice," 
"species," or "polymorphic" variants. A splice variant may have 
significant identity to a reference molecule, but will generally have a 
greater or lesser number of polynucleotides due to alternate splicing of 
exons during mRNA processing. The corresponding polypeptide may possess 
additional functional domains or an absence of domains. Species variants 
are polynucleotide sequences that vary from one species to another. The 
resulting polypeptides generally will have significant amino acid identity 
relative to each other. A polymorphic variant is a variation in the 
polynucleotide sequence of a particular gene between individuals of a 
given species. Polymorphic variants also may encompass "single nucleotide 
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one 
base. The presence of SNPs may be indicative of, for example, a certain 
population, a disease state, or a propensity for a disease state. 
The Invention 
The invention is based on the discovery of a new human cytochrome P450 
(HUCYP), the polynucleotides encoding HUCYP, and the use of these 
compositions for the diagnosis, treatment, or prevention of cell 
proliferative, developmental, autoimmune/inflammatory, and metabolic 
disorders. 
Nucleic acids encoding the HUCYP of the present invention were identified 
in Incyte Clone 991729H1from the colon cDNA library (COLNNOT11) using a 
computer search for nucleotide and/or 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 
2812042H1 (OVARNOT10), 4502047H1 (BRAVTXT02), 2100169H1 (BRAITUT02), 
991729R6 and 991729H1 (COLNNOT11), 038338R6 (HUVENOB01), 1996448H1 
(BRSTTUT03), 687353H1 (UTRSNOT02), 1700412F6 (BLADTUT05), 4538247H1 
(OVARNOT12), and 2381571F6 (ISLTNOT01). 
In one embodiment, the invention encompasses a polypeptide comprising the 
amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C, 1D, and 
1E. HUCYP is 462 amino acids in length and has three potential 
N-glycosylation sites at residues N94, N217, and N246; eleven potential 
casein kinase 2 phosphorylation sites as residues T36, S53, S116, S155, 
S183, T184, T293, T294, T442, S449, and S450; and seven potential protein 
kinase C phosphorylation sites at residues S134, T219, T281, T333, T336, 
S449, and S459. PFAM analysis indicates that HUCYP resembles P450 proteins 
from I32 through R46 1. PRINTS analysis indicates that HUCYP resembles 
E-class P450 Group I proteins from H62 through L81, from C285 through 
G311, from A367 through D391; resembles E-class P450 Group II proteins 
from Q265 through T293, from T294 through G311, and from T377 through 
E392; resembles E-class P450 Group IV proteins from E56 through V79, from 
L372 through D390, and from C409 through V427; resembles P450 superfamily 
proteins from T294 through N307; and resembles B-class P450 proteins from 
Q373 through R388. HUCYP shares conserved residues with cytochrome P450s 
in the potential heme-iron ligand region at F403, S404, G405, C409, and 
P410. C409 is the potential heme-binding residue. Like other eukaryotic 
cytochrome P450s, HUCYP contains a potentially membrane-spanning region in 
its first 15 to 20 residues. SPScan and HMM indicate that HUCYP has a 
potential signal peptide from M1 through about A18 or a potential 
transmembrane sequence from M1 through about Y23. A fragment of SEQ ID 
NO:2 from about nucleotide 109 to about nucleotide 153 is useful in 
hybridization or amplification technologies to identify SEQ ID NO:2 and to 
distinguish between SEQ ID NO:2 and a related sequence. Northern analysis 
shows the expression of this sequence in various libraries, 72% of which 
are associated with cancer and cell proliferation, 28% of which are 
associated with inflammation and immune response, 25% of which are 
reproductive tissues, 19% of which are nervous tissues, 19% of which are 
gastrointestinal tissues, and 16% of which are cardiovascular tissues. Of 
particular note is the expression of HUCYP in cancers of the liver, colon, 
uterus, testes, lung, breast, brain, and bladder, and in leukemia. 
The invention also encompasses HUCYP variants. A preferred HUCYP variant is 
one which has at least about 80%, more preferably at least about 90%, and 
most preferably at least about 95% amino acid sequence identity to the 
HUCYP amino acid sequence, and which contains at least one functional or 
structural characteristic of HUCYP. 
The invention also encompasses polynucleotides which encode HUCYP. In a 
particular embodiment, the invention encompasses a polynucleotide sequence 
comprising the sequence of SEQ ID NO:2, which encodes HUCYP. 
The invention also encompasses a variant of a polynucleotide sequence 
encoding HUCYP. In particular, such a variant polynucleotide sequence will 
have at least about 70%, more preferably at least about 85%, and most 
preferably at least about 95% polynucleotide sequence identity to the 
polynucleotide sequence encoding HUCYP. A particular aspect of the 
invention encompasses a variant of SEQ ID NO:2 which has at least about 
70%, more preferably at least about 85%, and most preferably at least 
about 95% polynucleotide sequence identity to SEQ ID NO:2. Any one of the 
polynucleotide variants described above can encode an amino acid sequence 
which contains at least one functional or structural characteristic of 
HUCYP. 
It will be appreciated by those skilled in the art that as a result of the 
degeneracy of the genetic code, a multitude of polynucleotide sequences 
encoding HUCYP, some bearing minimal similarity to the polynucleotide 
sequences of any known and naturally occurring gene, may be produced. 
Thus, the invention contemplates each and every possible variation of 
polynucleotide 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 polynucleotide 
sequence of naturally occurring HUCYP, and all such variations are to be 
considered as being specifically disclosed. 
Although nucleotide sequences which encode HUCYP and its variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring HUCYP under appropriately selected conditions of 
stringency, it may be advantageous to produce nucleotide sequences 
encoding HUCYP or its derivatives possessing a substantially different 
codon usage, e.g., inclusion of non-naturally occurring codons. 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 HUCYP 
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 which encode 
HUCYP and HUCYP derivatives, or fragments thereof, 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 well known in the art. Moreover, synthetic chemistry may be used 
to introduce mutations into a sequence encoding HUCYP or any fragment 
thereof. 
Also encompassed by the invention are polynucleotide sequences that are 
capable of hybridizing to the claimed polynucleotide sequences, and, in 
particular, to those shown in SEQ ID NO:2, or to a fragment of SEQ ID 
NO:2, under various conditions of stringency. (See, e.g., Wahl, G. M. and 
S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) 
Methods Enzymol. 152:507-511.) For example, stringent salt concentration 
will ordinarily be less than about 750 mM NaCl and 75 mM trisodium 
citrate, preferably less than about 500 mM NaCl and 50 mM trisodium 
citrate, and most preferably less than about 250 mM NaCl and 25 mM 
trisodium citrate. Low stringency hybridization can be obtained in the 
absence of organic solvent, e.g., formamide, while high stringency 
hybridization can be obtained in the presence of at least about 35% 
formamide, and most preferably at least about 50% formamide. Stringent 
temperature conditions will ordinarily include temperatures of at least 
about 30.degree. C., more preferably of at least about 37.degree. C., and 
most preferably of at least about 42.degree. C. Varying additional 
parameters, such as hybridization time, the concentration of detergent, 
e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of 
carrier DNA, are well known to those skilled in the art. Various levels of 
stringency are accomplished by combining these various conditions as 
needed. In a preferred embodiment, hybridization will occur at 30.degree. 
C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more 
preferred embodiment, hybridization will occur at 37.degree. C. in 500 mM 
NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml 
denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, 
hybridization will occur at 42.degree. C. in 250 mM NaCl, 25 mM trisodium 
citrate, 1% SDS, 50% formamide, and 200 .mu.g/ml ssDNA. Useful variations 
on these conditions will be readily apparent to those skilled in the art. 
The washing steps which follow hybridization can also vary in stringency. 
Wash stringency conditions can be defined by salt concentration and by 
temperature. As above, wash stringency can be increased by decreasing salt 
concentration or by increasing temperature. For example, stringent salt 
concentration for the wash steps will preferably be less than about 30 mM 
NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM 
NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for 
the wash steps will ordinarily include temperature of at least about 
25.degree. C., more preferably of at least about 42.degree. C., and most 
preferably of at least about 68.degree. C. In a preferred embodiment, wash 
steps will occur at 25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, 
and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 
42.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a 
most preferred embodiment, wash steps will occur at 68.degree. C. in 15 mM 
NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on 
these conditions will be readily apparent to those skilled in the art. 
Methods for DNA sequencing are well known 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 (US 
Biochemical, Cleveland Ohio), Taq polymerase (Perkin-Elmer), thermostable 
T7 polymerase (Amersham Pharmacia Biotech, Piscataway N.J.), or 
combinations of polymerases and proofreading exonucleases such as those 
found in the ELONGASE amplification system (Life Technologies, 
Gaithersburg Md.). Preferably, sequence preparation is automated with 
machines such as the Robbins Hydra microdispenser (Robbins Scientific, 
Sunnyvale Calif.), Hamilton MICROLAB 2200 (Hamilton, Reno Nev.), Peltier 
Thermal Cycler 200 (PTC200; M J Research, Watertown Mass.) and the ABI 
CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using the ABI 
373 or 377 DNA sequencing systems (Perkin-Elmer), the MEGABACE 1000 DNA 
sequencing system (Molecular Dynamics, Sunnyvale Calif.), or other systems 
known in the art. The resulting sequences are analyzed using a variety of 
algorithms which are well known in the art. (See, e.g., Ausubel, F. M. 
(1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York 
N.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, 
Wiley VCH, New York N.Y., pp. 856-853.) 
The nucleic acid sequences encoding HUCYP may be extended utilizing a 
partial nucleotide sequence and employing various PCR-based 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 and nested primers to amplify unknown sequence from 
genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR 
Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that 
extend in divergent directions to amplify unknown sequence from a 
circularized template. The template is derived from restriction fragments 
comprising a known genomic locus and surrounding sequences. (See, e.g., 
Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, 
capture PCR, involves PCR amplification of DNA fragments adjacent to known 
sequences in human and yeast artificial chromosome DNA. (See, e.g., 
Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this 
method, multiple restriction enzyme digestions and ligations may be used 
to insert an engineered double-stranded sequence into a region of unknown 
sequence before performing PCR. Other methods which may be used to 
retrieve unknown sequences are known in the art. (See, e.g., Parker, J. D. 
et al. (1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use 
PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto 
Calif.) to walk genomic DNA. This procedure avoids the need to screen 
libraries and is useful in finding intron/exon junctions. For all 
PCR-based methods, primers may be designed using commercially available 
software, such as OLIGO 4.06 primer analysis software (National 
Biosciences, Plymouth Minn.) or another appropriate program, to be about 
22 to 30 nucleotides in length, to have a GC content of about 50% or more, 
and to anneal to the template at temperatures of about 68.degree. C. to 
72.degree. C. 
When screening for full-length cDNAs, it is preferable to use libraries 
that have been size-selected to include larger cDNAs. In addition, 
random-primed libraries, which often include sequences containing the 5' 
regions of genes, are 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 
nucleotide-specific, laser-stimulated fluorescent dyes, and a charge 
coupled device camera for detection of the emitted wavelengths. 
Output/light intensity may be converted to electrical signal using 
appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, 
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 sequencing small DNA 
fragments which may be present in limited amounts in a particular sample. 
In another embodiment of the invention, polynucleotide sequences or 
fragments thereof which encode HUCYP may be cloned in recombinant DNA 
molecules that direct expression of HUCYP, or 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 used to express HUCYP. 
The nucleotide sequences of the present invention can be engineered using 
methods generally known in the art in order to alter HUCYP-encoding 
sequences for a variety of purposes including, but not limited to, 
modification of 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, oligonucleotide-mediated site-directed 
mutagenesis may be used to introduce mutations that create new restriction 
sites, alter glycosylation patterns, change codon preference, produce 
splice variants, and so forth. 
In another embodiment, sequences encoding HUCYP may be synthesized, in 
whole or in part, using chemical methods well known in the art. (See, 
e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 
(7):215-223, and Horn, T. et al. (1980) Nucl. Acids Symp. Ser. 
(7):225-232.) Alternatively, HUCYP itself or a fragment thereof may be 
synthesized using chemical methods. For example, peptide synthesis can be 
performed using various solid-phase techniques. (See, e.g., Roberge, J. Y. 
et al. (1995) Science 269:202-204.) Automated synthesis may be achieved 
using the ABI 431A peptide synthesizer (Perkin-Elmer). Additionally, the 
amino acid sequence of HUCYP, or any part thereof, may be altered during 
direct synthesis and/or combined with sequences from other proteins, or 
any part thereof, to produce a variant polypeptide. 
The peptide may be substantially purified by preparative high performance 
liquid chromatography. (See, e.g, Chiez, R. M. and F. Z. Regnier (1990) 
Methods Enzymol. 182:392-421.) The composition of the synthetic peptides 
may be confirmed by amino acid analysis or by sequencing. (See, e.g., 
Creighton, T. (1984) Proteins. Structures and Molecular Properties, W H 
Freeman, New York N.Y.) 
In order to express a biologically active HUCYP, the nucleotide sequences 
encoding HUCYP or derivatives thereof may be inserted into an appropriate 
expression vector, i.e., a vector which contains the necessary elements 
for transcriptional and translational control of the inserted coding 
sequence in a suitable host. These elements include regulatory sequences, 
such as enhancers, constitutive and inducible promoters, and 5' and 3' 
untranslated regions in the vector and in polynucleotide sequences 
encoding HUCYP. Such elements may vary in their strength and specificity. 
Specific initiation signals may also be used to achieve more efficient 
translation of sequences encoding HUCYP. Such signals include the ATG 
initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases 
where sequences encoding HUCYP and its initiation codon and upstream 
regulatory 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 an 
in-frame ATG initiation codon should be provided by the vector. 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 appropriate for the particular host cell 
system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell 
Differ. 20:125-162.) 
Methods which are well known to those skilled in the art may be used to 
construct expression vectors containing sequences encoding HUCYP and 
appropriate transcriptional and translational control elements. These 
methods include in vitro recombinant DNA techniques, synthetic techniques, 
and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) 
Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, 
Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current 
Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., ch. 9, 
13, and 16.) 
A variety of expression vector/host systems may be utilized to contain and 
express sequences encoding HUCYP. 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 viral expression vectors (e.g., baculovirus); plant cell systems 
transformed with viral expression vectors (e.g., cauliflower mosaic virus, 
CaMV, or 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. 
In bacterial systems, a number of cloning and expression vectors may be 
selected depending upon the use intended for polynucleotide sequences 
encoding HUCYP. For example, routine cloning, subcloning, and propagation 
of polynucleotide sequences encoding HUCYP can be achieved using a 
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla 
Calif.) or pSPORT1 plasmid (Life Technologies). Ligation of sequences 
encoding HUCYP into the vector's multiple cloning site disrupts the lacZ 
gene, allowing a colorimetric screening procedure for identification of 
transformed bacteria containing recombinant molecules. In addition, these 
vectors may be useful for in vitro transcription, dideoxy sequencing, 
single strand rescue with helper phage, and creation of nested deletions 
in the cloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster 
(1989) J. Biol. Chem. 264:5503-5509.) When large quantities of HUCYP are 
needed, e.g. for the production of antibodies, vectors which direct high 
level expression of HUCYP may be used. For example, vectors containing the 
strong, inducible T5 or T7 bacteriophage promoter may be used. 
Yeast expression systems may be used for production of HUCYP. A number of 
vectors containing constitutive or inducible promoters, such as alpha 
factor, alcohol oxidase, and PGH promoters, may be used in the yeast 
Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors 
direct either the secretion or intracellular retention of expressed 
proteins and enable integration of foreign sequences into the host genome 
for stable propagation. (See, e.g., Ausubel, 1995, supra; Grant et al. 
(1987) Methods Enzymol. 153:516-54; and Scorer, C. A. et al. (1994) 
Bio/Technology 12:181-184.) 
Plant systems may also be used for expression of HUCYP. Transcription of 
sequences encoding HUCYP may be driven by viral promoters, e.g., the 35S 
and 19S promoters of CaMV 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. (See, e.g., 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. (See, e.g., The McGraw Hill Yearbook of 
Science and Technology (1992) McGraw Hill, New York N.Y., pp. 191-196.) 
In mammalian cells, a number of viral-based expression systems may be 
utilized. In cases where an adenovirus is used as an expression vector, 
sequences encoding HUCYP 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 infective virus which expresses 
HUCYP in host cells. (See, e.g., Logan, J. and T. Shenk (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. SV40 or EBV-based vectors may also be used for 
high-level protein expression. 
Human artificial chromosomes (HACs) may also be employed to deliver larger 
fragments of DNA than can be contained in and expressed from a plasmid. 
HACs of about 6 kb to 10 Mb are constructed and delivered via conventional 
delivery methods (liposomes, polycationic amino polymers, or vesicles) for 
therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997) Nat 
Genet. 15:345-355.) 
For long term production of recombinant proteins in mammalian systems, 
stable expression of HUCYP in cell lines is preferred. For example, 
sequences encoding HUCYP can be transformed into cell lines 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 about 1 to 2 days in enriched media before being 
switched to selective media. The purpose of the selectable marker is to 
confer resistance to a selective agent, and its presence allows growth and 
recovery of cells which successfully express the introduced sequences. 
Resistant clones of stably transformed cells may be propagated 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 and adenine phosphoribosyltransferase genes, for use in 
tk.sup.- or apr.sup.- cells, respectively. (See, e.g., Wigler, M. et al. 
(1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, 
antimetabolite, antibiotic, or herbicide resistance can be used as the 
basis for selection. For example, dhfr confers resistance to methotrexate; 
neo confers resistance to the aminoglycosides, neomycin and G-418; and als 
or pat confer resistance to chlorsulfuron and phosphinotricin 
acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) 
Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. 
Mol. Biol. 150:1-14.) Additional selectable genes have been described, 
e.g., trpB and hisD, which alter cellular requirements for metabolites. 
(See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. 
Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent 
proteins (GFP; Clontech), .beta. glucuronidase and its substrate 
.beta.-glucuronide, or luciferase and its substrate luciferin may be used. 
These markers can be used not only to identify transformants, but also to 
quantify the amount of transient or stable protein expression attributable 
to a specific vector system. (See, e.g., Rhodes, C. A. (1995) Methods Mol. 
Biol. 55:121-131.) 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, the presence and expression of the gene 
may need to be confirmed. For example, if the sequence encoding HUCYP is 
inserted within a marker gene sequence, transformed cells containing 
sequences encoding HUCYP can be identified by the absence of marker gene 
function. Alternatively, a marker gene can be placed in tandem with a 
sequence encoding HUCYP 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. 
In general, host cells that contain the nucleic acid sequence encoding 
HUCYP and that express HUCYP 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, PCR amplification, 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 sequences. 
Immunological methods for detecting and measuring the expression of HUCYP 
using either specific polyclonal or monoclonal antibodies are known in the 
art. Examples of such techniques include enzyme-linked immunosorbent 
assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell 
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing 
monoclonal antibodies reactive to two non-interfering epitopes on HUCYP is 
preferred, but a competitive binding assay may be employed. These and 
other assays are well known in the art. (See, e.g., Hampton, R. et al. 
(1990) Serological Methods, a Laboratory Manual, APS Press, St Paul Minn., 
Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology, 
Greene Pub. Associates and Wiley-Interscience, New York N.Y.; and Pound, 
J. D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.). 
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 HUCYP include 
oligolabeling, nick translation, end-labeling, or PCR amplification using 
a labeled nucleotide. Alternatively, the sequences encoding HUCYP, 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, such as those provided by Amersham Pharmacia Biotech, 
Promega (Madison Wis.), and US Biochemical. 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 HUCYP 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 retained 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 HUCYP may be 
designed to contain signal sequences which direct secretion of HUCYP 
through a prokaryotic or eukaryotic cell membrane. 
In addition, a host cell strain may be chosen for its ability to modulate 
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 specify 
protein targeting, folding, and/or activity. Different host cells which 
have specific cellular machinery and characteristic mechanisms for 
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), 
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. 
In another embodiment of the invention, natural, modified, or recombinant 
nucleic acid sequences encoding HUCYP may be ligated to a heterologous 
sequence resulting in translation of a fusion protein in any of the 
aforementioned host systems. For example, a chimeric HUCYP protein 
containing a heterologous moiety that can be recognized by a commercially 
available antibody may facilitate the screening of peptide libraries for 
inhibitors of HUCYP activity. Heterologous protein and peptide moieties 
may also facilitate purification of fusion proteins using commercially 
available affinity matrices. Such moieties include, but are not limited 
to, glutathione S-transferase (GST), maltose binding protein (MBP), 
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, 
and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification 
of their cognate fusion proteins on immobilized glutathione, maltose, 
phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. 
FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of 
fusion proteins using commercially available monoclonal and polyclonal 
antibodies that specifically recognize these epitope tags. A fusion 
protein may also be engineered to contain a proteolytic cleavage site 
located between the HUCYP encoding sequence and the heterologous protein 
sequence, so that HUCYP may be cleaved away from the heterologous moiety 
following purification. Methods for fusion protein expression and 
purification are discussed in Ausubel (1995, supra, ch 10). A variety of 
commercially available kits may also be used to facilitate expression and 
purification of fusion proteins. 
In a further embodiment of the invention, synthesis of radiolabeled HUCYP 
may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat 
germ extract systems (Promega). These systems couple transcription and 
translation of protein-coding sequences operably associated with the T7, 
T3, or SP6 promoters. Translation takes place in the presence of a 
radiolabeled amino acid precursor, preferably .sup.35 S-methionine. 
Fragments of HUCYP may be produced not only by recombinant production, but 
also by direct peptide synthesis using solid-phase techniques. (See, e.g., 
Creighton, supra pp. 55-60.) Protein synthesis may be performed by manual 
techniques or by automation. Automated synthesis may be achieved, for 
example, using the ABI 431A peptide synthesizer (Perkin-Elmer). Various 
fragments of HUCYP may be synthesized separately and then combined to 
produce the full length molecule. 
Therapeutics 
Chemical and structural similarity, e.g., in the context of sequences and 
motifs, exists between regions of HUCYP and cytochrome P450 proteins. In 
addition, the expression of HUCYP is closely associated with cancer and 
cell proliferation, inflammation and immune response, reproductive 
tissues, nervous tissues, gastrointestinal tissues, and cardiovascular 
tissues. Therefore, HUCYP appears to play a role in cell proliferative, 
developmental, autoimmune/inflammatory, and metabolic disorders. In the 
treatment of disorders associated with increased HUCYP expression or 
activity, it is desirable to decrease the expression or activity of HUCYP. 
In the treatment of disorders associated with decreased HUCYP expression 
or activity, it is desirable to increase the expression or activity of 
HUCYP. 
Therefore, in one embodiment, HUCYP or a fragment or derivative thereof may 
be administered to a subject to treat or prevent a disorder associated 
with decreased expression or activity of HUCYP. Examples of such disorders 
include, but are not limited to, a cell proliferative disorder such as 
actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, 
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, 
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary 
thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, 
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, 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; a developmental 
disorder such as renal tubular acidosis, anemia, Cushing's syndrome, 
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, 
epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, 
genitourinary abnormalities, and mental retardation), Smith-Magenis 
syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, 
hereditary keratodermas, hereditary neuropathies such as 
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, 
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral 
palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, 
cataract, and sensorineural hearing loss; an autoimmune/inflammatory 
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's 
disease, adult respiratory distress syndrome, allergies, ankylosing 
spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune 
hemolytic anemia, autoimmune thyroiditis, autoimmune 
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, 
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, 
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with 
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic 
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' 
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel 
syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial 
inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, 
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's 
syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic 
sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner 
syndrome, complications of cancer, hemodialysis, and extracorporeal 
circulation, viral, bacterial, fungal, parasitic, protozoal, and 
helminthic infections, and trauma; and a metabolic disorder such as 
Addison's disease, cystic fibrosis, diabetes, fatty hepatocirrhosis, 
galactosemia, goiter, hyperadrenalism, hypoadrenalism, 
hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, 
hyperthyroidism, hypothyroidism hyperlipidemia, hyperlipemia, lipid 
myopathies, obesity, lipodystrophies, and phenylketonuria, congenital 
adrenal hyperplasia, pseudovitamin D-deficiency rickets, cerebrotendinous 
xanthomatosis, and coumarin resistance. 
In another embodiment, a vector capable of expressing HUCYP or a fragment 
or derivative thereof may be administered to a subject to treat or prevent 
a disorder associated with decreased expression or activity of HUCYP 
including, but not limited to, those described above. 
In a further embodiment, a pharmaceutical composition comprising a 
substantially purified HUCYP in conjunction with a suitable pharmaceutical 
carrier may be administered to a subject to treat or prevent a disorder 
associated with decreased expression or activity of HUCYP including, but 
not limited to, those provided above. 
In still another embodiment, an agonist which modulates the activity of 
HUCYP may be administered to a subject to treat or prevent a disorder 
associated with decreased expression or activity of HUCYP including, but 
not limited to, those listed above. 
In a further embodiment, an antagonist of HUCYP may be administered to a 
subject to treat or prevent a disorder associated with increased 
expression or activity of HUCYP. Such disorders may include, but are not 
limited to, cell proliferative, developmental, autoimmune/inflammatory, 
and metabolic disorders as listed above. In one aspect, an antibody which 
specifically binds HUCYP 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 HUCYP. 
In an additional embodiment, a vector expressing the complement of the 
polynucleotide encoding HUCYP may be administered to a subject to treat or 
prevent a disorder associated with increased expression or activity of 
HUCYP including, but not limited to, those described 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. 
An antagonist of HUCYP may be produced using methods which are generally 
known in the art. In particular, purified HUCYP may be used to produce 
antibodies or to screen libraries of pharmaceutical agents to identify 
those which specifically bind HUCYP. Antibodies to HUCYP may also be 
generated using methods that are well known in the art. Such antibodies 
may include, but are not limited to, polyclonal, monoclonal, chimeric, and 
single chain antibodies, 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 HUCYP or 
with 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, KLH, 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 HUCYP have an amino acid sequence consisting of at 
least about 5 amino acids, and, more preferably, of at least about 10 
amino acids. It is also preferable that these oligopeptides, peptides, or 
fragments are identical to a portion of the amino acid sequence of the 
natural protein and contain the entire amino acid sequence of a small, 
naturally occurring molecule. Short stretches of HUCYP amino acids may be 
fused with those of another protein, such as KLH, and antibodies to the 
chimeric molecule may be produced. 
Monoclonal antibodies to HUCYP 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. (See, e.g., 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; and Cole, S. P. et al. (1984) 
Mol. Cell Biol. 62:109-120.) 
In addition, techniques developed for the production of "chimeric 
antibodies," such as the splicing of mouse antibody genes to human 
antibody genes to obtain a molecule with appropriate antigen specificity 
and biological activity, can be used. (See, e.g., Morrison, S. L. et al. 
(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) 
Nature 312:604-608; and 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 
HUCYP-specific single chain antibodies. Antibodies with related 
specificity, but of distinct idiotypic composition, may be generated by 
chain shuffling from random combinatorial immunoglobulin libraries. (See, 
e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:10134-10137.) 
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. (See, 
e.g., 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 HUCYP may also 
be generated. For example, such fragments include, but are not limited to, 
F(ab')2 fragments produced by pepsin digestion of the antibody molecule 
and Fab fragments 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. (See, e.g., Huse, W. D. et al. 
(1989) Science 246:1275-1281.) 
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 HUCYP and its specific antibody. A two-site, monoclonal-based 
immunoassay utilizing monoclonal antibodies reactive to two 
non-interfering HUCYP epitopes is preferred, but a competitive binding 
assay may also be employed (Pound, supra). 
Various methods such as Scatchard analysis in conjunction with 
radioimmunoassay techniques may be used to assess the affinity of 
antibodies for HUCYP. Affinity is expressed as an association constant, 
K.sub.a, which is defined as the molar concentration of HUCYP-antibody 
complex divided by the molar concentrations of free antigen and free 
antibody under equilibrium conditions. The K.sub.a determined for a 
preparation of polyclonal antibodies, which are heterogeneous in their 
affinities for multiple HUCYP epitopes, represents the average affinity, 
or avidity, of the antibodies for HUCYP. The K.sub.a determined for a 
preparation of monoclonal antibodies, which are monospecific for a 
particular HUCYP epitope, represents a true measure of affinity. 
High-affinity antibody preparations with K.sub.a ranging from about 
10.sup.9 to 10.sup.12 l/mole are preferred for use in immunoassays in 
which the HUCYP-antibody complex must withstand rigorous manipulations. 
Low-affinity antibody preparations with K.sub.a ranging from about 
10.sup.6 to 10.sup.7 l/mole are preferred for use in immunopurification 
and similar procedures which ultimately require dissociation of HUCYP, 
preferably in active form, from the antibody (Catty, D. (1988) Antibodies, 
Volume I: A Practical Approach, IRL Press, Washington D.C.; Liddell, J. E. 
and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John 
Wiley & Sons, New York N.Y.). 
The titer and avidity of polyclonal antibody preparations may be further 
evaluated to determine the quality and suitability of such preparations 
for certain downstream applications. For example, a polyclonal antibody 
preparation containing at least 1-2 mg specific antibody/ml, preferably 
5-10 mg specific antibody/ml, is preferred for use in procedures requiring 
precipitation of HUCYP-antibody complexes. Procedures for evaluating 
antibody specificity, titer, and avidity, and guidelines for antibody 
quality and usage in various applications, are generally available. (See, 
e.g., Catty, supra, and Coligan et al. supra.) 
In another embodiment of the invention, the polynucleotides encoding HUCYP, 
or any fragment or complement thereof, may be used for therapeutic 
purposes. In one aspect, the complement of the polynucleotide encoding 
HUCYP 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 HUCYP. Thus, 
complementary molecules or fragments may be used to modulate HUCYP 
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 HUCYP. 
Expression vectors derived from retroviruses, adenoviruses, or 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 to express nucleic acid sequences 
complementary to the polynucleotides encoding HUCYP. (See, e.g., Sambrook, 
supra; Ausubel, 1995, supra.) 
Genes encoding HUCYP can be turned off by transforming a cell or tissue 
with expression vectors which express high levels of a polynucleotide, or 
fragment thereof, encoding HUCYP. 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 may last 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 HUCYP. 
Oligonucleotides derived from the transcription initiation site, e.g., 
between about 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. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. 
Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco 
N.Y., pp. 163-177.) A 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. For example, engineered 
hammerhead motif ribozyme molecules may specifically and efficiently 
catalyze endonucleolytic cleavage of sequences encoding HUCYP. 
Specific ribozyme cleavage sites within any potential RNA target are 
initially identified by scanning the target molecule for ribozyme cleavage 
sites, including 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 HUCYP. 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 by 
polycationic amino polymers may be achieved using methods which are well 
known in the art. (See, e.g., Goldman, C. K. et al. (1997) Nature 
Biotechnology 15:462-466.) 
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 or sterile composition, in conjunction with a 
pharmaceutically acceptable carrier, for any of the therapeutic effects 
discussed above. Such pharmaceutical compositions may consist of HUCYP, 
antibodies to HUCYP, and mimetics, agonists, antagonists, or inhibitors of 
HUCYP. The compositions may be administered alone or in combination with 
at least one other agent, such as a 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, 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 combining 
active compounds with solid excipient and processing the resultant mixture 
of granules (optionally, after grinding) to obtain tablets or dragee 
cores. Suitable auxiliaries can be added, if desired. Suitable excipients 
include carbohydrate or protein fillers, such as sugars, including 
lactose, sucrose, mannitol, and 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, and 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 fillers 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' 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, 
triglycerides, or liposomes. Non-lipid polycationic amino polymers may 
also be used for delivery. Optionally, the suspension may also contain 
suitable stabilizers or agents to increase the solubility of the compounds 
and 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, and succinic acids. 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 mM to 
50 mM histidine, 0.1% to 2% sucrose, and 2% to 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 HUCYP, 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 such as mice, rats, rabbits, dogs, or pigs. An 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 HUCYP or fragments thereof, antibodies of HUCYP, 
and agonists, antagonists or inhibitors of HUCYP, which ameliorates the 
symptoms or condition. Therapeutic efficacy and toxicity may be determined 
by standard pharmaceutical procedures in cell cultures or with 
experimental animals, such as by calculating the ED.sub.50 (the dose 
therapeutically effective in 50% of the population) or LD.sub.50 (the dose 
lethal to 50% of the population) statistics. The dose ratio of toxic to 
therapeutic effects is the therapeutic index, and it can be expressed as 
the LD.sub.50 /ED.sub.50 ratio. Pharmaceutical compositions which exhibit 
large therapeutic indices are preferred. The data obtained from cell 
culture assays and animal studies are used to formulate a range of dosage 
for human use. The dosage contained in such compositions is preferably 
within a range of circulating concentrations that includes the ED.sub.50 
with little or no toxicity. The dosage varies within this range depending 
upon the dosage form employed, the 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 requiring 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, the general health of 
the subject, the age, weight, and gender of the subject, time and 
frequency of administration, drug combination(s), reaction sensitivities, 
and response to therapy. Long-acting pharmaceutical compositions may be 
administered every 3 to 4 days, every week, or biweekly depending on the 
half-life and clearance rate of the particular formulation. 
Normal dosage amounts may vary from about 0.1 .mu.g to 100,000 .mu.g, up to 
a total dose of about 1 gram, 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 HUCYP may be used 
for the diagnosis of disorders characterized by expression of HUCYP, or in 
assays to monitor patients being treated with HUCYP or agonists, 
antagonists, or inhibitors of HUCYP. Antibodies useful for diagnostic 
purposes may be prepared in the same manner as described above for 
therapeutics. Diagnostic assays for HUCYP include methods which utilize 
the antibody and a label to detect HUCYP in human body fluids or in 
extracts of cells or tissues. The antibodies may be used with or without 
modification, and may be labeled by covalent or non-covalent attachment of 
a reporter molecule. A wide variety of reporter molecules, several of 
which are described above, are known in the art and may be used. 
A variety of protocols for measuring HUCYP, including ELISAs, RIAs, and 
FACS, are known in the art and provide a basis for diagnosing altered or 
abnormal levels of HUCYP expression. Normal or standard values for HUCYP 
expression are established by combining body fluids or cell extracts taken 
from normal mammalian subjects, preferably human, with antibody to HUCYP 
under conditions suitable for complex formation. The amount of standard 
complex formation may be quantitated by various methods, preferably by 
photometric means. Quantities of HUCYP expressed in subject samples, 
control and disease 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 HUCYP 
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 HUCYP may be 
correlated with disease. The diagnostic assay may be used to determine 
absence, presence, and excess expression of HUCYP, and to monitor 
regulation of HUCYP levels during therapeutic intervention. 
In one aspect, hybridization with PCR probes which are capable of detecting 
polynucleotide sequences, including genomic sequences, encoding HUCYP or 
closely related molecules may be used to identify nucleic acid sequences 
which encode HUCYP. The specificity of the probe, whether it is made from 
a highly specific region, e.g., the 5' regulatory region, or from a less 
specific region, e.g., a conserved motif, and the stringency of the 
hybridization or amplification (maximal, high, intermediate, or low), will 
determine whether the probe identifies only naturally occurring sequences 
encoding HUCYP, allelic variants, or related sequences. 
Probes may also be used for the detection of related sequences, and should 
preferably have at least 50% sequence identity to any of the HUCYP 
encoding sequences. The hybridization probes of the subject invention may 
be DNA or RNA and may be derived from the sequence of SEQ ID NO:2 or from 
genomic sequences including promoters, enhancers, and introns of the HUCYP 
gene. 
Means for producing specific hybridization probes for DNAs encoding HUCYP 
include the cloning of polynucleotide sequences encoding HUCYP or HUCYP 
derivatives into vectors for the production of mRNA probes. Such vectors 
are known in the art, are 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, by 
radionuclides such as .sup.32 P or 35S, or by enzymatic labels, such as 
alkaline phosphatase coupled to the probe via avidin/biotin coupling 
systems, and the like. 
Polynucleotide sequences encoding HUCYP may be used for the diagnosis of 
disorders associated with expression of HUCYP. Examples of such disorders 
include, but are not limited to, a cell proliferative disorder such as 
actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, 
hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, 
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary 
thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, 
melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, 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; a developmental 
disorder such as renal tubular acidosis, anemia, Cushing's syndrome, 
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, 
epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, 
genitourinary abnormalities, and mental retardation), Smith-Magenis 
syndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia, 
hereditary keratodermas, hereditary neuropathies such as 
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism, 
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral 
palsy, spina bifida, anencephaly, craniorachischisis, congenital glaucoma, 
cataract, and sensorineural hearing loss; an autoimmune/inflammatory 
disorder such as acquired immunodeficiency syndrome (AIDS), Addison's 
disease, adult respiratory distress syndrome, allergies, ankylosing 
spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune 
hemolytic anemia, autoimmune thyroiditis, autoimmune 
polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, 
cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, 
dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with 
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic 
gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' 
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel 
syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial 
inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, 
psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's 
syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic 
sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner 
syndrome, complications of cancer, hemodialysis, and extracorporeal 
circulation, viral, bacterial, fungal, parasitic, protozoal, and 
helminthic infections, and trauma; and a metabolic disorder such as 
Addison's disease, cystic fibrosis, diabetes, fatty hepatocirrhosis, 
galactosemia, goiter, hyperadrenalism, hypoadrenalism, 
hyperparathyroidism, hypoparathyroidism, hypercholesterolemia, 
hyperthyroidism, hypothyroidism hyperlipidemia, hyperlipemia, lipid 
myopathies, obesity, lipodystrophies, and phenylketonuria, congenital 
adrenal hyperplasia, pseudovitamin D-deficiency rickets, cerebrotendinous 
xanthomatosis, and coumarin resistance. The polynucleotide sequences 
encoding HUCYP may be used in Southern or northern analysis, dot blot, or 
other membrane-based technologies; in PCR technologies; in dipstick, pin, 
and multiformat ELISA-like assays; and in microarrays utilizing fluids or 
tissues from patients to detect altered HUCYP expression. Such qualitative 
or quantitative methods are well known in the art. 
In a particular aspect, the nucleotide sequences encoding HUCYP may be 
useful in assays that detect the presence of associated disorders, 
particularly those mentioned above. The nucleotide sequences encoding 
HUCYP 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 patient sample is significantly altered in 
comparison to a control sample then the presence of altered levels of 
nucleotide sequences encoding HUCYP in the sample indicates the presence 
of the associated disorder. Such assays may also be used to evaluate the 
efficacy of a particular therapeutic treatment regimen in animal studies, 
in clinical trials, or to monitor the treatment of an individual patient. 
In order to provide a basis for the diagnosis of a disorder associated with 
expression of HUCYP, 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, encoding HUCYP, under conditions suitable 
for hybridization or amplification. Standard hybridization may be 
quantified by comparing the values obtained from normal subjects with 
values from an experiment in which a known amount of a substantially 
purified polynucleotide is used. Standard values obtained in this manner 
may be compared with values obtained from samples from patients who are 
symptomatic for a disorder. Deviation from standard values is used to 
establish the presence of a disorder. 
Once the presence of a disorder is established and a treatment protocol is 
initiated, hybridization assays may be repeated on a regular basis to 
determine if the level of expression in the patient begins to approximate 
that which is observed in the normal subject. 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 an abnormal amount of transcript 
(either under- or over-expressed) 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 HUCYP may involve the use of PCR. These oligomers may be 
chemically synthesized, generated enzymatically, or produced in vitro. 
Oligomers will preferably contain a fragment of a polynucleotide encoding 
HUCYP, or a fragment of a polynucleotide complementary to the 
polynucleotide encoding HUCYP, and will be employed under optimized 
conditions for identification of a specific gene or condition. Oligomers 
may also be employed under less stringent conditions for detection or 
quantitation of closely related DNA or RNA sequences. 
Methods which may also be used to quantitate the expression of HUCYP 
include radiolabeling or biotinylating nucleotides, coamplification of a 
control nucleic acid, and interpolating results from standard curves. 
(See, e.g., 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 colorimetric response gives rapid 
quantitation. 
In further embodiments, oligonucleotides or longer fragments derived from 
any of the polynucleotide sequences described herein may be used as 
targets in a microarray. The microarray can be used to monitor the 
expression level of large numbers of genes simultaneously and to identify 
genetic variants, mutations, and polymorphisms. This information may be 
used to determine gene function, to understand the genetic basis of a 
disorder, to diagnose a disorder, and to develop and monitor the 
activities of therapeutic agents. 
Microarrays may be prepared, used, and analyzed using methods known in the 
art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; 
Schena, M. et al. (1996) Proc. Natl. Acad. Sci. 93:10614-10619; 
Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et 
al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. 
Natl. Acad. Sci. 94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. 
No. 5,605,662.) 
In another embodiment of the invention, nucleic acid sequences encoding 
HUCYP may be used to generate hybridization probes useful in 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, e.g., human artificial chromosomes 
(HACs), yeast artificial chromosomes (YACs), bacterial artificial 
chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA 
libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat Genet. 
15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J. 
(1991) Trends Genet. 7:149-154.) 
Fluorescent in situ hybridization (FISH) may be correlated with other 
physical chromosome mapping techniques and genetic map data. (See, e.g., 
Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of 
genetic map data can be found in various scientific journals or at the 
Online Mendelian Inheritance in Man (OMIM) site. Correlation between the 
location of the gene encoding HUCYP on a physical chromosomal map and a 
specific disorder, or a predisposition to a specific disorder, may help 
define the region of DNA associated with that disorder. The nucleotide 
sequences of the invention may be used to detect differences in gene 
sequences among normal, carrier, and 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 
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, e.g., 
ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may 
represent associated or regulatory genes for further investigation. (See, 
e.g., Gatti, R. A. et al. (1988) Nature 336:577-580.) 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, HUCYP, 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 HUCYP and the agent being tested 
may be measured. 
Another technique for drug screening provides for high throughput screening 
of compounds having suitable binding affinity to the protein of interest. 
(See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this 
method, large numbers of different small test compounds are synthesized on 
a solid substrate. The test compounds are reacted with HUCYP, or fragments 
thereof, and washed. Bound HUCYP is then detected by methods well known in 
the art. Purified HUCYP 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 HUCYP specifically 
compete with a test compound for binding HUCYP. In this manner, antibodies 
can be used to detect the presence of any peptide which shares one or more 
antigenic determinants with HUCYP. 
In additional embodiments, the nucleotide sequences which encode HUCYP 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. cDNA Library Construction 
The COLNNOT11 cDNA library was constructed from microscopically normal 
colon tissue obtained from a 60-year-old Caucasian male who had undergone 
a left hemicolectomy to remove grade 3 (of 4) adenocarcinoma in a 
different part of his bowel. The patient history reported previous 
diagnoses of depressive disorder and thrombophlebitis, accompanied by 
inflammatory polyarthropathies and inflammatory disease of the prostate. 
The frozen tissue was homogenized and lysed using a Polytron PT-3000 
homogenizer (Brinkmann Instruments, Westbury N.J.) in guanidinium 
isothiocyanate solution. The lysate was centrifuged over a 5.7 M CsCl 
cushion using a SW28 rotor in a L8-70M ultracentrifuge (Beckman 
Instruments, Fullerton Calif.) for 18 hours at 25,000 rpm at ambient 
temperature. The RNA was extracted with acid phenol pH 4.0, precipitated 
using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in 
RNAse-free water and treated with DNase at 37.degree. C. The RNA 
extraction and precipitation were repeated as before. The mRNA was 
isolated using an OLIGOTEX kit (QIAGEN, Valencia Calif.) and used to 
construct the cDNA library. 
The mRNA was handled according to the recommended protocols in the 
SUPERSCRIPT plasmid system (Life Technologies). cDNAs were fractionated on 
a SEPHAROSE CL4B column (Amersham Pharmacia Biotech), and those cDNAs 
exceeding 400 bp were ligated into pSPORT1 plasmid (Life Technologies). 
The plasmid pSport I was subsequently transformed into DH5.alpha. 
competent cells (Life Technologies). 
II. Isolation of cDNA Clones 
Plasmid DNA was released from the cells and purified using the R.E.A.L. 
PREP 96 plasmid kit from QIAGEN. This kit enables the simultaneous 
purification of 96 samples in a 96-well block using multi-channel reagent 
dispensers. The recommended protocol was employed except for the following 
changes: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth 
(Life Technologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) 
after inoculation, the cultures were incubated for 19 hours and at the end 
of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 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 96-well block for storage at 
4.degree. C. 
III. Sequencing and Analysis 
The cDNAs were prepared for sequencing using the ABI CATALYST 800 
(Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) or 
MICROLAB 2200 (Hamilton) systems in combination with PTC-200 thermal 
cyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373 or 
377 sequencing systems (Perkin-Elmer) and standard ABI protocols, base 
calling software, and kits. In one alternative, cDNAs were sequenced using 
the MEGABACE 1000 DNA sequencing system (Molecular Dynamics). In another 
alternative, the cDNAs were amplified and sequenced using the ABI PRISM 
BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer). In 
yet another alternative, cDNAs were sequenced using solutions and dyes 
from Amersham Pharmacia Biotech. Reading frames for the ESTs were 
determined using standard methods (reviewed in Ausubel, 1997, supra, unit 
7.7). Some of the cDNA sequences were selected for extension using the 
techniques disclosed in Example V. 
The polynucleotide sequences derived from cDNA, extension, and shotgun 
sequencing were assembled and analyzed using a combination of software 
programs which utilize algorithms well known to those skilled in the art. 
Table 1 summarizes the tools, programs, and algorithms used and provides 
descriptions, references, and threshold parameters when applicable. The 
first column of Table 1 shows the tools, programs, and algorithms used, 
the second column provides brief descriptions, the third column presents 
the appropriate references, all of which are incorporated by reference 
herein in their entirety, and the fourth column presents, where 
applicable, the scores, probability values, and other parameters used to 
evaluate the strength of a match between two sequences (the higher the 
score, the greater the homology between two sequences). Sequences were 
analyzed using MACDNASIS PRO software (Hitachi Software Engineering) and 
LASERGENE software (DNASTAR). 
The polynucleotide sequences were validated by removing vector, linker, and 
polyA sequences and by masking ambiguous bases, using algorithms and 
programs based on BLAST, dynamic programming, and dinucleotide nearest 
neighbor analysis. The sequences were then queried against a selection of 
public databases, such as the GenBank primate, rodent, mammalian, 
vertebrate, and eukaryote databases, and BLOCKS to acquire annotation, 
using programs based on BLAST, FASTA, and BLIMPS. The sequences were 
assembled into full length polynucleotide sequences using programs based 
on Phred, Phrap, and Consed, and were screened for open reading frames 
using programs based on GeneMark, BLAST, and FASTA. The full length 
polynucleotide sequences were translated to derive the corresponding full 
length amino acid sequences, and these full length sequences were 
subsequently analyzed by querying against databases such as the GenBank 
databases, SwissProt, BLOCKS, PRINTS, PFAM, and Prosite. 
The programs described above for the assembly and analysis of full length 
polynucleotide and amino acid sequences were used to identify 
polynucleotide sequence fragments from SEQ ID NO:2. 
Fragments from about 20 to about 4000 nucleotides which are useful in 
hybridization and amplification technologies were described in the 
Invention section above. 
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. (See, e.g., Sambrook, supra, ch. 7; 
Ausubel, 1995, supra, ch. 4 and 16.) 
Analogous computer techniques applying BLAST were used to search for 
identical or related molecules in nucleotide databases such as the GenBank 
or LIFESEQ databases (Incyte Pharmaceuticals, Palo Alto Calif.). 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 similar. 
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% to 2% error, and, 
with a product score of 70, the match will be exact. Similar 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 analyses reported a percentage distribution of 
libraries in which the transcript encoding HUCYP occurred. Analysis 
involved the categorization of cDNA libraries by organ/tissue and disease. 
The organ/tissue categories included cardiovascular, dermatologic, 
developmental, endocrine, gastrointestinal, hematopoietic/immune, 
musculoskeletal, nervous, reproductive, and urologic. The disease 
categories included cancer, inflammation/trauma, fetal, neurological, and 
pooled. For each category, the number of libraries expressing the sequence 
of interest was counted and divided by the total number of libraries 
across all categories. Percentage values of tissue-specific and disease 
expression are reported in the description of the invention. 
V. Extension of HUCYP Encoding Polynucleotides 
The full length nucleic acid sequence of SEQ ID NO:2 was produced by 
extension of an appropriate fragment of the full length molecule using 
oligonucleotide primers designed from this fragment. One primer was 
synthesized to initiate 5' extension of the known fragment, and the other 
primer, to initiate 3' extension of the known fragment. The initial 
primers were designed using OLIGO 4.06 software (National Biosciences), or 
another appropriate program, to be about 22 to 30 nucleotides in length, 
to have a GC content of about 50% or more, and to anneal to the target 
sequence at temperatures of about 68.degree. C. 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 were used to extend the sequence. If more 
than one extension was necessary or desired, additional or nested sets of 
primers were designed. 
High fidelity amplification was obtained by PCR using methods well known in 
the art. PCR was performed in 96-well plates using the PTC-200 thermal 
cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 
nmol of each primer, reaction buffer containing Mg.sup.2+, 
(NH.sub.4).sub.2 SO.sub.4, and .beta.-mercaptoethanol, Taq DNA polymerase 
(Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu 
DNA polymerase (Stratagene), with the following parameters for primer pair 
PCI A and PCI B: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15 
sec; Step 3: 60.degree. C., 1 min; Step 4: 68.degree. C., 2 min; Step 5: 
Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C., 5 min; Step 7: 
storage at 4.degree. C. In the alternative, the parameters for primer pair 
T7 and SK+ were as follows: Step 1: 94.degree. C., 3 min; Step 2: 
94.degree. C., 15 sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. 
C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. 
C., 5 min; Step 7: storage at 4.degree. C. 
The concentration of DNA in each well was determined by dispensing 100 
.mu.l PICO GREEN quantitation reagent (0.25% (v/v) PICO GREEN; Molecular 
Probes, Eugene Org.) dissolved in 1X TE and 0.5 .mu.l of undiluted PCR 
product into each well of an opaque fluorimeter plate (Corning Costar, 
Acton Mass.), allowing the DNA to bind to the reagent. The plate was 
scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure 
the fluorescence of the sample and to quantify the concentration of DNA. A 
5 .mu.l to 10 .mu.l aliquot of the reaction mixture was analyzed by 
electrophoresis on a 1% agarose mini-gel to determine which reactions were 
successful in extending the sequence. 
The extended nucleotides were desalted and concentrated, transferred to 
384-well plates, digested with CviJI cholera virus endonuclease (Molecular 
Biology Research, Madison Wis.), and sonicated or sheared prior to 
religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun 
sequencing, the digested nucleotides were separated on low concentration 
(0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with 
Agar ACE (Promega). Extended clones were religated using T4 ligase (New 
England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham Pharmacia 
Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in 
restriction site overhangs, and transfected into competent E. coli cells. 
Transformed cells were selected on antibiotic-containing media, individual 
colonies were picked and cultured overnight at 37.degree. C. in 384-well 
plates in LB/2x carb liquid media. 
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase 
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the 
following parameters: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 
15 sec; Step 3: 60.degree. C., I min; Step 4: 72.degree. C., 2 min; Step 
5: step 2, 3, and 4 repeated 29 times; Step 6: 72.degree. C., 5 min; Step 
7: storage at 4.degree. C. DNA was quantified by PICOGREEN reagent 
(Molecular Probes) as described above. Samples with low DNA recoveries 
were reamplified using the same conditions as described above. Samples 
were diluted with 20% dimethysulphoxide (1:2, v/v), and sequenced using 
DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit 
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle 
sequencing ready reaction kit (Perkin-Elmer). 
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 such 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 software (National Biosciences) and labeled by combining 50 
pmol of each oligomer, 250 .mu.Ci of [.sup.32 P]-adenosine triphosphate 
(Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, 
Boston Mass.). The labeled oligonucleotides are substantially purified 
using a SEPHADEX G-25 superfine size exclusion dextran bead column 
(Amersham Pharmacia Biotech). An 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, Xbal, or Pvu II 
(DuPont NEN). 
The DNA from each digest is fractionated on a 0.7% 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. Hybridization patterns are 
visualized and compared using autoradiography. 
VII. Microarrays 
A chemical coupling procedure and an ink jet device can be used to 
synthesize array elements on the surface of a substrate. (See, e.g., 
Baldeschweiler, supra.) An array analogous to a dot or slot blot may also 
be used to arrange and link elements to the surface of a substrate using 
thermal, UV, chemical, or mechanical bonding procedures. A typical array 
may be produced by hand or using available methods and machines and 
contain any appropriate number of elements. After hybridization, 
nonhybridized probes are removed and a scanner used to determine the 
levels and patterns of fluorescence. The degree of complementarity and the 
relative abundance of each probe which hybridizes to an element on the 
microarray may be assessed through analysis of the scanned images. 
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may 
comprise the elements of the microarray. Fragments suitable for 
hybridization can be selected using software well known in the art such as 
LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments 
thereof corresponding to one of the nucleotide sequences of the present 
invention, or selected at random from a cDNA library relevant to the 
present invention, are arranged on an appropriate substrate, e.g., a glass 
slide. The cDNA is fixed to the slide using, e.g., UV cross-linking 
followed by thermal and chemical treatments and subsequent drying. (See, 
e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. 
(1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used 
for hybridization to the elements on the substrate. The substrate is 
analyzed by procedures described above. 
VIII. Complementary Polynucleotides 
Sequences complementary to the HUCYP-encoding sequences, or any parts 
thereof, are used to detect, decrease, or inhibit expression of naturally 
occurring HUCYP. Although use of oligonucleotides comprising from about 15 
to 30 base pairs is described, essentially the same procedure is used with 
smaller or with larger sequence fragments. Appropriate oligonucleotides 
are designed using OLIGO 4.06 software (National Biosciences) and the 
coding sequence of HUCYP. 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 HUCYP-encoding transcript. 
IX. Expression of HUCYP 
Expression and purification of HUCYP are achieved using bacterial or 
virus-based expression systems. For expression of HUCYP in bacteria, cDNA 
is subcloned into an appropriate vector containing an antibiotic 
resistance gene and an inducible promoter that directs high levels of cDNA 
transcription. Examples of such promoters include, but are not limited to, 
the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter 
in conjunction with the lac operator regulatory element. Recombinant 
vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). 
Antibiotic resistant bacteria express HUCYP upon induction with isopropyl 
beta-D-thiogalactopyranoside (IPTG). Expression of HUCYP in eukaryotic 
cells is achieved by infecting insect or mammalian cell lines with 
recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), 
commonly known as baculovirus. The nonessential polyhedrin gene of 
baculovirus is replaced with cDNA encoding HUCYP by either homologous 
recombination or bacterial-mediated transposition involving transfer 
plasmid intermediates. Viral infectivity is maintained and the strong 
polyhedrin promoter drives high levels of cDNA transcription. Recombinant 
baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in 
most cases, or human hepatocytes, in some cases. Infection of the latter 
requires additional genetic modifications to baculovirus. (See Engelhard, 
E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-25 3227; Sandig, V. 
et al. (1996) Hum. Gene Ther. 7:1937-1945.) 
In most expression systems, HUCYP is synthesized as a fusion protein with, 
e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as 
FLAG or 6-His, permitting rapid, single-step, affinity-based purification 
of recombinant fusion protein from crude cell lysates. GST, a 
26-kilodalton enzyme from Schistosoma japonicum, enables the purification 
of fusion proteins on immobilized glutathione under conditions that 
maintain protein activity and antigenicity (Amersham Pharmacia Biotech). 
Following purification, the GST moiety can be proteolytically cleaved from 
HUCYP at specifically engineered sites. FLAG, an 8-amino acid peptide, 
enables immunoaffinity purification using commercially available 
monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a 
stretch of six consecutive histidine residues, enables purification on 
metal-chelate resins (QIAGEN). Methods for protein expression and 
purification are discussed in Ausubel (1995, supra, ch 10 and 16). 
Purified HUCYP obtained by these methods can be used directly in the 
following activity assay. 
X. Demonstration of HUCYP Activity 
HUCYP activity is demonstrated as the ability to hydroxylate the steroid 
testosterone (Waxman, D. J. (1991) Methods Enzymol. 206:462-476). 
4-.sup.14 C-testosterone is diluted with unlabeled testosterone dissolved 
in toluene to give a working solution of 5-7 mCi/mmol. Ten nanomoles of 
the working solution of 4-.sup.14 C-testosterone (final assay 
concentration 50 .mu.M) are aliquoted to a 13.times.100 mm assay tube, and 
the solvent is evaporated under N.sub.2. HUCYP, 0.1 M HEPES buffer, pH 
7.4, and 0.1 mM EDTA are added to the tube, on ice, to give a final volume 
of 175 .mu.l. A control tube is prepared that lacks HUCYP. Samples are 
vortexed briefly, and the tubes are transferred to a shaking 37.degree. C. 
water bath. Reactions are initiated 4 minutes later by the addition of 
NADPH dissolved in 25 .mu.l 0.1 M HEPES buffer, pH 7.4 to give a final 
NADPH concentration of 0.3 mM. Reactions are terminated after 10-20 
minutes by the addition of 1 ml ethyl acetate and vortexed for 30 seconds. 
The hydroxylated products are separated from testosterone by thin layer 
chromatography and detected by autoradiography. Product migrations are 
compared with those of testosterone and of hydroxylated steroid standards 
to identify the products. Reaction products are cut out of the thin layer 
chromatography plate and counted in a liquid scintillation counter. The 
amount of hydroxylated steroid products found is proportional to HUCYP 
activity. 
XI. Functional Assays 
HUCYP function is assessed by expressing the sequences encoding HUCYP at 
physiologically elevated levels in mammalian cell culture systems. cDNA is 
subcloned into a mammalian expression vector containing a strong promoter 
that drives high levels of cDNA expression. Vectors of choice include pCMV 
SPORT (Life Technologies) and pCR3.1 (Invitrogen, Carlsbad Calif.), both 
of which contain the cytomegalovirus promoter. 5-10 .mu.g of recombinant 
vector are transiently transfected into a human cell line, preferably of 
endothelial or hematopoietic origin, using either liposome formulations or 
electroporation. 1-2 .mu.g of an additional plasmid containing sequences 
encoding a marker protein are co-transfected. Expression of a marker 
protein provides a means to distinguish transfected cells from 
nontransfected cells and is a reliable predictor of cDNA expression from 
the recombinant vector. Marker proteins of choice include, e.g., Green 
Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. 
Flow cytometry (FCM), an automated, laser optics-based technique, is used 
to identify transfected cells expressing GFP or CD64-GFP, and to evaluate 
cellular properties, for example, their apoptotic state. FCM detects and 
quantifies the uptake of fluorescent molecules that diagnose events 
preceding or coincident with cell death. These events include changes in 
nuclear DNA content as measured by staining of DNA with propidium iodide; 
changes in cell size and granularity as measured by forward light scatter 
and 90 degree side light scatter; down-regulation of DNA synthesis as 
measured by decrease in bromodeoxyuridine uptake; alterations in 
expression of cell surface and intracellular proteins as measured by 
reactivity with specific antibodies; and alterations in plasma membrane 
composition as measured by the binding of fluorescein-conjugated Annexin V 
protein to the cell surface. Methods in flow cytometry are discussed in 
Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York N.Y. 
The influence of HUCYP on gene expression can be assessed using highly 
purified populations of cells transfected with sequences encoding HUCYP 
and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the 
surface of transfected cells and bind to conserved regions of human 
immunoglobulin G (IgG). Transfected cells are efficiently separated from 
nontransfected cells using magnetic beads coated with either human IgG or 
antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified 
from the cells using methods well known by those of skill in the art. 
Expression of mRNA encoding HUCYP and other genes of interest can be 
analyzed by northern analysis or microarray techniques. 
XII. Production of HUCYP Specific Antibodies 
HUCYP substantially purified using polyacrylamide gel electrophoresis 
(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), 
or other purification techniques, is used to immunize rabbits and to 
produce antibodies using standard protocols. 
Alternatively, the HUCYP amino acid sequence is analyzed using LASERGENE 
software (DNASTAR) 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. Methods for selection of 
appropriate epitopes, such as those near the C-terminus or in hydrophilic 
regions are well described in the art. (See, e.g., Ausubel, 1995, supra, 
ch. 11.) 
Typically, oligopeptides 15 residues in length are synthesized using an ABI 
431A Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled 
to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction with 
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase 
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized 
with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting 
antisera are tested for antipeptide activity by, for example, binding the 
peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, 
washing, and reacting with radio-iodinated goat anti-rabbit IgG. 
XIII. Purification of Naturally Occurring HUCYP Using Specific Antibodies 
Naturally occurring or recombinant HUCYP is substantially purified by 
immunoaffinity chromatography using antibodies specific for HUCYP. An 
immunoaffinity column is constructed by covalently coupling anti-HUCYP 
antibody to an activated chromatographic resin, such as CNBr-activated 
SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is 
blocked and washed according to the manufacturer's instructions. 
Media containing HUCYP are passed over the immunoaffinity column, and the 
column is washed under conditions that allow the preferential absorbance 
of HUCYP (e.g., high ionic strength buffers in the presence of detergent). 
The column is eluted under conditions that disrupt antibody/HUCYP binding 
(e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, 
such as urea or thiocyanate ion), and HUCYP is collected. 
XIV. Identification of Molecules Which Interact with HUCYP 
HUCYP, or biologically active fragments thereof, are labeled with .sup.125 
I Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 
133:529-539.) Candidate molecules previously arrayed in the wells of a 
multi-well plate are incubated with the labeled HUCYP, washed, and any 
wells with labeled HUCYP complex are assayed. Data obtained using 
different concentrations of HUCYP are used to calculate values for the 
number, affinity, and association of HUCYP with the candidate molecules. 
Various modifications and variations of the described methods and systems 
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 
- &lt;160&gt; NUMBER OF SEQ ID NOS: 2 
- &lt;210&gt; SEQ ID NO 1 
&lt;211&gt; LENGTH: 462 
&lt;212&gt; TYPE: PRT 
&lt;213&gt; ORGANISM: Homo sapiens 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: 991729 
- &lt;400&gt; SEQUENCE: 1 
- Met Leu Asp Phe Ala Ile Phe Ala Val Thr Ph - #e Leu Leu Ala Leu 
# 15 
- Val Gly Ala Val Leu Tyr Leu Tyr Pro Ala Se - #r Arg Gln Ala Ala 
# 30 
- Gly Ile Pro Gly Ile Thr Pro Thr Glu Glu Ly - #s Asp Gly Asn Leu 
# 45 
- Pro Asp Ile Val Asn Ser Gly Ser Leu His Gl - #u Phe Leu Val Asn 
# 60 
- Leu His Glu Arg Tyr Gly Pro Val Val Ser Ph - #e Trp Phe Gly Arg 
# 75 
- Arg Leu Val Val Ser Leu Gly Thr Val Asp Va - #l Leu Lys Gln His 
# 90 
- Ile Asn Pro Asn Lys Thr Leu Asp Pro Phe Gl - #u Thr Met Leu Lys 
# 105 
- Ser Leu Leu Arg Tyr Gln Ser Gly Gly Gly Se - #r Val Ser Glu Asn 
# 120 
- His Met Arg Lys Lys Leu Tyr Glu Asn Gly Va - #l Thr Asp Ser Leu 
# 135 
- Lys Ser Asn Phe Ala Leu Leu Leu Lys Leu Se - #r Glu Glu Leu Leu 
# 150 
- Asp Lys Trp Leu Ser Tyr Pro Glu Thr Gln Hi - #s Val Pro Leu Ser 
# 165 
- Gln His Met Leu Gly Phe Ala Met Lys Ser Va - #l Thr Gln Met Val 
# 180 
- Met Gly Ser Thr Phe Glu Asp Asp Gln Glu Va - #l Ile Arg Phe Gln 
# 195 
- Lys Asn His Gly Thr Val Trp Ser Glu Ile Gl - #y Lys Gly Phe Leu 
# 210 
- Asp Gly Ser Leu Asp Lys Asn Met Thr Arg Ly - #s Lys Gln Tyr Glu 
# 225 
- Asp Ala Leu Met Gln Leu Glu Ser Val Leu Ar - #g Asn Ile Ile Lys 
# 240 
- Glu Arg Lys Gly Arg Asn Phe Ser Gln His Il - #e Phe Ile Asp Ser 
# 255 
- Leu Val Gln Gly Asn Leu Asn Asp Gln Gln Il - #e Leu Glu Asp Ser 
# 270 
- Met Ile Phe Ser Leu Ala Ser Cys Ile Ile Th - #r Ala Lys Leu Cys 
# 285 
- Thr Trp Ala Ile Cys Phe Leu Thr Thr Ser Gl - #u Glu Val Gln Lys 
# 300 
- Lys Leu Tyr Glu Glu Ile Asn Gln Val Phe Gl - #y Asn Gly Pro Val 
# 315 
- Thr Pro Glu Lys Ile Glu Gln Leu Arg Tyr Cy - #s Gln His Val Leu 
# 330 
- Cys Glu Thr Val Arg Thr Ala Lys Leu Thr Pr - #o Val Ser Ala Gln 
# 345 
- Leu Gln Asp Ile Glu Gly Lys Ile Asp Arg Ph - #e Ile Ile Pro Arg 
# 360 
- Glu Thr Leu Val Leu Tyr Ala Leu Gly Val Va - #l Leu Gln Asp Pro 
# 375 
- Asn Thr Trp Pro Ser Pro His Lys Phe Asp Pr - #o Asp Arg Phe Asp 
# 390 
- Asp Glu Leu Val Met Lys Thr Phe Ser Ser Le - #u Gly Phe Ser Gly 
# 405 
- Thr Gln Glu Cys Pro Glu Leu Arg Phe Ala Ty - #r Met Val Thr Thr 
# 420 
- Val Leu Leu Ser Val Leu Val Lys Arg Leu Hi - #s Leu Leu Ser Val 
# 435 
- Glu Gly Gln Val Ile Glu Thr Lys Tyr Glu Le - #u Val Thr Ser Ser 
# 450 
- Arg Glu Glu Ala Trp Ile Thr Val Ser Lys Ar - #g Tyr 
# 460 
- &lt;210&gt; SEQ ID NO 2 
&lt;211&gt; LENGTH: 1648 
&lt;212&gt; TYPE: DNA 
&lt;213&gt; ORGANISM: Homo sapiens 
&lt;220&gt; FEATURE: 
&lt;223&gt; OTHER INFORMATION: 991729 
- &lt;400&gt; SEQUENCE: 2 
- gccgatccga gacgtggctc cctgggcggc agaaccatgt tggacttcgc ga - #tcttcgcc 
60 
- gttaccttct tgctggcgtt ggtgggagcc gtgctctacc tctatccggc tt - #ccagacaa 
120 
- gctgcaggaa ttccagggat tactccaact gaagaaaaag atggtaatct tc - #cagatatt 
180 
- gtgaatagtg gaagtttgca tgagttcctg gttaatttgc atgagagata tg - #ggcctgtg 
240 
- gtctccttct ggtttggcag gcgcctcgtg gttagtttgg gcactgttga tg - #tactgaag 
300 
- cagcatatca atcccaataa gacattggac ccttttgaaa ccatgctgaa gt - #cattatta 
360 
- aggtatcaat ctggtggtgg cagtgtgagt gaaaaccaca tgaggaaaaa at - #tgtatgaa 
420 
- aatggtgtga ctgattctct gaagagtaac tttgccctcc tcctaaagct tt - #cagaagaa 
480 
- ttattagata aatggctctc ctacccagag acccagcacg tgcccctcag cc - #agcatatg 
540 
- cttggttttg ctatgaagtc tgttacacag atggtaatgg gtagtacatt tg - #aagatgat 
600 
- caggaagtca ttcgcttcca gaagaatcat ggcacagttt ggtctgagat tg - #gaaaaggc 
660 
- tttctagatg ggtcacttga taaaaacatg actcggaaaa aacaatatga ag - #atgccctc 
720 
- atgcaactgg agtctgtttt aaggaacatc ataaaagaac gaaaaggaag ga - #acttcagt 
780 
- caacatattt tcattgactc cttagtacaa gggaacctta atgaccaaca ga - #tcctagaa 
840 
- gacagtatga tattttctct ggccagttgc ataataactg caaaattgtg ta - #cctgggca 
900 
- atctgttttt taaccacctc tgaagaagtt caaaaaaaat tatatgaaga ga - #taaaccaa 
960 
- gtttttggaa atggtcctgt tactccagag aaaattgagc agctcagata tt - #gtcagcat 
1020 
- gtgctttgtg aaactgttcg aactgccaaa ctgactccag tttctgccca gc - #ttcaagat 
1080 
- attgaaggaa aaattgaccg atttattatt cctagagaga ccctcgtcct tt - #atgccctt 
1140 
- ggtgtggtac ttcaggatcc taatacttgg ccatctccac acaagtttga tc - #cagatcgg 
1200 
- tttgatgatg aattagtaat gaaaactttt tcctcacttg gattctcagg ca - #cacaggag 
1260 
- tgtccagagt tgaggtttgc atatatggtg accacagtac ttcttagtgt at - #tggtgaag 
1320 
- agactgcacc tactttctgt ggagggacag gttattgaaa caaagtatga ac - #tggtaaca 
1380 
- tcatcaaggg aagaagcttg gatcactgtc tcaaagagat attaaaattt ta - #tacattta 
1440 
- aaatcattgt taaattgatt gaggaaaaca accatttaaa aaaaatctat gt - #tgaatcct 
1500 
- tttataaacc agtatcactt tgtaatataa acacctattt gtacttaatt tt - #gtaaattt 
1560 
- ggatttttat atatcatatt ttcttaattc attgtacaca tttgacttac tg - #cacagtat 
1620 
# 1648 ggaa aaaaaaaa 
__________________________________________________________________________