The present invention provides a human phospholemman homolog protein (HPLMH) and polynucleotides which identify and encode HPLMH. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding HPLMH. The invention also provides for the use of HPLMH, and antibodies, or agonists or antagonists specifically binding HPLMH, in the prevention and treatment of diseases associated with expression of HPLMH. Additionally the invention provides for the use of antisense molecules to polynucleotides encoding HPLMH for the treatment of diseases associated with the expression of HPLMH. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding HPLMH.

FIELD OF THE INVENTION 
This invention relates to nucleic acid and amino acid sequences of a novel 
human phospholemman homolog protein and to the use of these sequences in 
the diagnosis, prevention, and treatment of diseases of the central 
nervous system, cardiovascular system, prostate, and disorders related to 
smooth muscle function. 
BACKGROUND OF THE INVENTION 
Phospholemman (PLM) is the major plasmalemmal substrate for cAMP-dependent 
protein kinase (cAMPK) and protein kinase C (PKC). Canine and murine PLM 
are expressed at high levels in heart, skeletal muscle, and liver, and at 
low levels in breast, brain, lung, stomach, kidney, and colon (Palmer C. 
et al. (1991) J Biol Chem 266:11126-11130; Moorman J. R. et al. (1992) J 
Biol Chem 267:14551-14554). PLM is a membrane protein which consists of 72 
amino acids and has a calculated molecular weight of 8409. The native 
protein has an apparent molecular weight of 15 kDa as determined following 
polyacrylamide gel electrophoresis. A distinguishing feature of PLM is its 
highly basic nature, with a calculated isoelectric point of 9.7 (Palmer C. 
et al., supra). PLM consists of an acidic extracellular amino-terminal 
domain, a single uncharged transmembrane domain, and an extremely basic 
cytoplasmic carboxy-terminal domain. The cytoplasmic domain contains 
consensus cAMPK and PKC phosphorylation sites. The phosphorylation of PLM 
by PKC and cAMPK is regulated by insulin and adrenaline, respectively 
(Walaas S. et al. (1994) Biochem J 304:635-640). PLM phosphorylation in 
cardiac muscle occurs after activation of either .alpha.- or .beta. 
adrenergic receptors, and correlates with an increase in contractility 
(Lindemann JP (1986) J Biol Chem 261:4860-4867). 
Expression of PLM in Xenopus oocytes injected with PLM mRNA coincides with 
the appearance of voltage-activated chloride currents (Moorman et al., 
supra). Immunoaffinity-purified recombinant PLM added to planar 
phospholipid bilayers produces unitary anion currents (Moorman J. R. et 
al. (1995) Nature 377:737-740). The high selectivity of the PLM channel 
for the sulfonic amino acid taurine suggests that PLM channels link signal 
transduction cascades to cell volume regulation. PLM is the smallest 
membrane protein known to form an ion channel (Moorman et al. (1995), 
supra). 
Mat-8, an 8-kDa transmembrane protein related to PLM, is expressed in 
murine breast tumor lines transformed by Neu or Ras oncoproteins. Morrison 
B. W. et al. ((1994) Oncogene 9:3417-3426) proposed that Mat-8 is a marker 
of the cell type preferentially transformed by neu or v-Ha-ras oncogenes. 
A human Mat-8 homolog is expressed both in primary breast tumors and in 
breast tumor cell lines. Murine Mat-8 is also expressed in uterus, 
stomach, colon, and at low levels in virgin breast, ovary, lung, small 
intestine and thymus. In contrast to PLM, Mat-8 is not expressed in liver, 
heart or skeletal muscle, which suggests distinct cellular functions for 
the two molecules (Morrison B. W. et al. (1995) J Biol Chem 
270:2176-2182). 
The extracellular and transmembrane domains of Mat-8 are homologous to 
those of PLM. However, the cytoplasmic domain of Mat-8 is unrelated to PLM 
and contains no consensus phosphorylation sites for PKC or cAMPK. 
Expression of Mat-8 in Xenopus oocytes induces voltage-activated Cl.sup.- 
currents similar to those induced by expression of PLM (Morrison et al. 
(1995), supra), but direct ion channel formation by Mat-8 has not been 
reported. The ability of Mat-8 protein to induce Cl.sup.- channel 
activity, together with its tissue distribution (see above), suggests that 
this protein may be involved in the regulation of transepithelial 
transport in tissues containing absorptive or secretory epithelia. 
Additional proteins similar in structure to PLM and Mat-8 have been found 
to induce ion channel activity when expressed in Xenopus oocytes. 
Channel-inducing factor (CHIF), found in colon and kidney, consists of a 
single transmembrane domain and exhibits 50% sequence similarity to PLM 
(Attali B. et al. (1995) Proc Natl Acad Sci USA 92:6092-6096). Xenopus 
oocytes injected with CHIF mRNA exhibit K.sup.+ specific channel activity. 
Slow-activating voltage dependent potassium ion channel (IsK; Takumi T. et 
al. (1988) Science 242:1042-1045) is a single transmembrane domain 
glycoprotein present in epithelial cells, heart, uterus and lymphocytes 
(Attali B. et al. (1993) Nature 365:850-852). IsK induces both K.sup.+ and 
Cl.sup.- currents when expressed in Xenopus oocytes and HEK293 cells. The 
accumulated evidence suggests that CHIF and IsK act as regulatory subunits 
of pre-existing channel complexes rather than as channels per se (Attali 
B. et al. (1995), supra; Ben-Efraim I. et al. (1996) J Biol Chem 
271:8768-8771). 
The sodium, potassium ATPase (Na,K-ATPase) .gamma.-subunit, formerly known 
as the Na,K-ATPase proteolipid, is a small membrane protein that 
co-purifies with the .alpha.- and .beta.-subunits of Na,K-ATPase (Mercer 
R. W. et al. (1993) J Cell Biol 121:579-586). The .gamma.-subunit contains 
58 amino acids with a single transmembrane domain. This transmembrane 
domain is structurally related to the transmembrane domains of other 
PLM-like proteins. The .gamma.-subunit may act as a regulator of the 
ATP-dependent ion channel activity of Na,K-ATPase. 
The polynucleotide sequence and polypeptides encoding a human phospholemman 
homolog protein associated with neurotransmitter release, transepithelial 
transport, membrane potential stabilization, signal transduction, and cell 
volume regulation would satisfy a need in the art by providing a new means 
for the diagnosis, prevention, or treatment of diseases of the central 
nervous system such as Alzheimer's disease, Parkinson's disease, 
Huntington's disease, Creutzfeld-Jacob disease, amyotrophic lateral 
sclerosis, and hydrocephalus; diseases of the cardiovascular system such 
as angina, cardiac hypertrophy, congestive heart failure, 
vasoconstriction, and hypertension, prostate hypertrophy, and disorders 
related to smooth muscle function such as bladder and sphincter 
dysfunction, bronchial constriction, and asthma. 
SUMMARY OF THE INVENTION 
The present invention features a novel human phospholemman homolog protein 
hereinafter designated HPLMH and characterized by having homology to 
canine PLM, MAT-8, CHIF, and (Na,K-ATPase) .gamma.-subunit and acting as 
an ion channel with preference for chloride ions and/or taurine. 
Accordingly, the invention features a substantially purified human 
phospholemman homolog protein having ion channel activity, an 
amino-terminal signal sequence, and a single transmembrane domain and 
having the amino acid sequence of SEQ ID NO:1. 
One aspect of the invention features isolated and substantially purified 
polynucleotides that encode HPLMH. In a particular aspect, the 
polynucleotide is the nucleotide sequence of SEQ ID NO:2. 
The invention also relates to a polynucleotide sequence comprising the 
complement of SEQ ID NO:2 or variants thereof. In addition, the invention 
features polynucleotide sequences which hybridize under stringent 
conditions to SEQ ID NO:2. 
The invention additionally features nucleic acid sequences encoding 
polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, 
portions or antisense molecules thereof, and expression vectors and host 
cells comprising polynucleotides that encode HPLMH. The present invention 
also features antibodies which bind specifically to HPLMH and 
pharmaceutical compositions comprising substantially purified HPLMH. The 
invention also features agonists and antagonists of HPLMH and the use 
thereof

DESCRIPTION OF THE INVENTION 
Before the present protein, nucleotide sequence, and methods are described, 
it is understood that this invention is not limited to the particular 
methodology, protocols, cell lines, vectors, and reagents described as 
these may, of course, vary. It is also to be understood that the 
terminology used herein is for the purpose of describing particular 
embodiments only, and is not intended to limit the scope of the present 
invention which will be limited only by the appended claims. 
It must be noted that as used herein and in the appended claims, the 
singular forms "a", "an", and "the" include plural reference unless the 
context clearly dictates otherwise. Thus, for example, reference to "a 
host cell" includes a plurality of such host cells, reference to the 
"antibody" is a reference to one or more antibodies and equivalents 
thereof known to those skilled in the art, and so forth. 
Unless defined otherwise, all technical and scientific terms used herein 
have the same meanings as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Although any methods and 
materials similar or equivalent to those described herein can be used in 
the practice or testing of the present invention, the preferred methods, 
devices, and materials are now described. All publications mentioned 
herein are incorporated herein by reference for the purpose of describing 
and disclosing the cell lines, vectors, and methodologies which are 
reported in the publications which might be used in connection with the 
invention. Nothing herein is to be construed as an admission that the 
invention is not entitled to antedate such disclosure by virtue of prior 
invention. 
DEFINITIONS 
"Nucleic acid sequence" as used herein refers to an oligonucleotide, 
nucleotide, or polynucleotide, and fragments or portions thereof, and to 
DNA or RNA of genomic or synthetic origin which may be single- or 
double-stranded, and represent the sense or antisense strand. Similarly, 
"amino acid sequence" as used herein refers to an oligopeptide, peptide, 
polypeptide, or protein sequence and fragments or portions thereof, of a 
naturally occurring or synthetic molecule. 
Where "amino acid sequence" is recited herein to refer to an amino acid 
sequence of a naturally occurring protein molecule, "amino acid sequence" 
and like terms, such as "polypeptide" or "protein" are not meant to limit 
the amino acid sequence to the complete, native amino acid sequence 
associated with the recited protein molecule. 
"Peptide nucleic acid", as used herein, refers to a molecule which 
comprises an oligomer to which an amino acid residue, such as lysine, and 
an amino group have been added. These small molecules, also designated 
anti-gene agents, stop transcript elongation by binding to their 
complementary strand of nucleic acid (Nielsen et al. (1993) Anticancer 
Drug Des 8:53-63). 
HPLMH, as used herein, refers to the amino acid sequences of substantially 
purified HPLMH obtained from any species, particularly mammalian, 
including bovine, ovine, porcine, murine, equine, and preferably human, 
from any source whether natural, synthetic, semi-synthetic, or 
recombinant. 
"Consensus", as used herein, refers to a nucleic acid sequence which has 
been resequenced to resolve uncalled bases, or which has been extended 
using XL-PCR.TM. (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 the GCG Fragment 
Assembly.TM. system (GCG, Madison Wis.), or which has been both extended 
and assembled. 
A "variant" of HPLMH, as used herein, refers to an amino acid sequence that 
is altered by one or more amino acids. The variant may have "conservative" 
changes, wherein a substituted amino acid has similar structural or 
chemical properties, e.g., replacement of leucine with isoleucine. More 
rarely, a variant may have "nonconservative" changes, e.g., replacement of 
a glycine with a tryptophan. Similar minor variations may also include 
amino acid deletions or insertions, or both. Guidance in determining which 
amino acid residues may be substituted, inserted, or deleted without 
abolishing biological or immunological activity may be found using 
computer programs well known in the art, for example, DNASTAR software. 
A "deletion", as used herein, refers to a change in either amino acid or 
nucleotide sequence in which one or more amino acid or nucleotide 
residues, respectively, are absent. 
An "insertion" or "addition", as used herein, refers to a change in an 
amino acid or nucleotide sequence resulting in the addition of one or more 
amino acid or nucleotide residues, respectively, as compared to the 
naturally occurring molecule. 
A "substitution", as used herein, refers to the replacement of one or more 
amino acids or nucleotides by different amino acids or nucleotides, 
respectively. 
The term "biologically active", as used herein, refers to a protein having 
structural, regulatory, or biochemical functions of a naturally occurring 
molecule. Likewise, "immunologically active" refers to the capability of 
the natural, recombinant, or synthetic HPLMH, or any oligopeptide thereof, 
to induce a specific immune response in appropriate animals or cells and 
to bind with specific antibodies. 
The term "agonist", as used herein, refers to a molecule which, when bound 
to HPLMH, causes a change in HPLMH which modulates the activity of HPLMH. 
Agonists may include proteins, nucleic acids, carbohydrates, or any other 
molecules which bind to HPLMH. 
The terms "antagonist" or "inhibitor", as used herein, refer to a molecule 
which, when bound to HPLMH, blocks the binding of an agonist to HPLMH, 
which prevents the agonist-induced change in the biological activity of 
HPLMH. Antagonists may include proteins, nucleic acids, carbohydrates, or 
any other molecules which bind to HPLMH. 
The term "modulate", as used herein, refers to a change or an alteration in 
the biological activity of HPLMH. Modulation may be an increase or a 
decrease in biological activity, a change in binding characteristics, or 
any other change in the biological properties of HPLMH. 
The term "mimetic", as used herein, refers to a molecule, the structure of 
which is developed from knowledge of the structure of human phospholemman 
homolog protein or portions thereof and, as such, is able to effect some 
or all of the actions of human phospholemman homolog protein. 
The term "derivative", as used herein, refers to the chemical modification 
of a nucleic acid encoding HPLMH or the encoded HPLMH. Illustrative of 
such modifications would be replacement of hydrogen by an alkyl, acyl, or 
amino group. A nucleic acid derivative would encode a polypeptide which 
retains essential biological characteristics of the natural molecule. 
The term "substantially purified", as used herein, refers to nucleic or 
amino acid sequences that are removed from their natural environment, 
isolated or separated, and are at least 60% free, preferably 75% free, and 
most preferably 90% free from other components with which they are 
naturally associated. 
"Amplification" as used herein refers to the production of additional 
copies of a nucleic acid sequence and is generally carried out using 
polymerase chain reaction (PCR) technologies well known in the art 
(Dieffenbach et al. (1995) PCR Primer, a Laboratory Manual, Cold Spring 
Harbor Press, Plainview, N.Y.). 
The term "hybridization", as used herein, refers to any process by which a 
strand of nucleic acid binds with a complementary strand through base 
pairing. 
The term "hybridization complex", as used herein, refers to a complex 
formed between two nucleic acid sequences by virtue of the formation of 
hydrogen bonds between complementary G and C bases and between 
complementary A and T bases; these hydrogen bonds may be further 
stabilized by base stacking interactions. The two complementary nucleic 
acid sequences hydrogen bond in an antiparallel configuration. A 
hybridization complex may be formed in solution (e.g., C.sub.0 t or 
R.sub.0 t analysis) or between one nucleic acid sequence present in 
solution and another nucleic acid sequence immobilized on a solid support 
(e.g., membranes, filters, chips, pins or glass slides to which cells have 
been fixed for in situ hybridization). 
The terms "complementary" or "complementarity", as used herein, refer to 
the natural binding of polynucleotides under permissive salt and 
temperature conditions by base-pairing. For example, for the sequence 
"A-G-T" bonds to the complementary sequence "T-C-A". Complementarity 
between two single-stranded molecules may be "partial", in which only some 
of the nucleic acids bind, or it may be complete when total 
complementarity exists between the single-stranded molecules. The degree 
of complementarity between nucleic acid strands has significant effects on 
the efficiency and strength of hybridization between nucleic acid strands. 
This is of particular importance in amplification reactions, which depend 
upon binding between the nucleic acids strands. 
The term "homology", as used herein, refers to a degree of complementarity. 
There may be partial homology or complete homology (i.e., identity). A 
partially complementary sequence is one that at least partially inhibits 
an identical sequence from hybridizing to a target nucleic acid; it is 
referred to using the functional term "substantially homologous." The 
inhibition of hybridization of the completely complementary sequence to 
the target sequence may be examined using a hybridization assay (Southern 
or northern blot, solution hybridization and the like) under conditions of 
low stringency. A substantially homologous sequence or probe will compete 
for and inhibit the binding (i.e., the hybridization) of a completely 
homologous sequence or probe to the target sequence under conditions of 
low stringency. This is not to say that conditions of low stringency are 
such that non-specific binding is permitted; low stringency conditions 
require that the binding of two sequences to one another be a specific 
(i.e., selective) interaction. The absence of non-specific binding may be 
tested by the use of a second target sequence which lacks even a partial 
degree of complementarity (e.g., less than about 30% identity); in the 
absence of non-specific binding the probe will not hybridize to the second 
non-complementary target sequence. 
As known in the art, numerous equivalent conditions may be employed to 
comprise either low or high stringency conditions. Factors such as the 
length and nature (DNA, RNA, base composition) of the sequence, nature of 
the target (DNA, RNA, base composition, presence in solution or 
immobilization, etc.), and the concentration of the salts and other 
components (e.g., the presence or absence of formamide, dextran sulfate 
and/or polyethylene glycol) are considered and the hybridization solution 
may be varied to generate conditions of either low or high stringency 
different from, but equivalent to, the above listed conditions. 
The term "stringent conditions", as used herein, is the "stringency" which 
occurs within a range from about Tm-5.degree. C. (5.degree. C. below the 
melting temperature (Tm) of the probe) to about 20.degree. C. to 
25.degree. C. below Tm. As will be understood by those of skill in the 
art, the stringency of hybridization may be altered in order to identify 
or detect identical or related polynucleotide sequences. 
The term "antisense", as used herein, refers to RNA sequences which are 
complementary to a specific RNA sequence (e.g., mRNA). Antisense RNA may 
be produced by any method, including synthesis by ligating the gene(s) of 
interest in a reverse orientation to a viral promoter which permits the 
synthesis of a complementary-coding strand. Once introduced into a cell, 
this transcribed strand combines with natural mRNA produced by the cell to 
form duplexes. These duplexes then block the further translation of the 
mRNA. In this manner, mutant phenotypes may be generated. The term 
"antisense strand" is used in reference to a nucleic acid strand that is 
complementary to the "sense" strand. The designation "negative" is 
sometimes used in reference to the antisense strand, and "positive" is 
sometimes used in reference to the sense strand. 
The term "portion", as used herein, with regard to a protein (as in "a 
portion of a given protein") refers to fragments of that protein. The 
fragments may range in size from four amino acid residues to the entire 
amino acid sequence minus one amino acid. Thus, a protein "comprising at 
least a portion of the amino acid sequence of SEQ ID NO:1 encompasses the 
full-length human HPLMH and fragments thereof. 
The term "antigenic determinant", as used herein, refers to that portion of 
a molecule that makes contact with a particular antibody (i.e., an 
epitope). When a protein or fragment of a protein is used to immunize a 
host animal, numerous regions of the protein may induce the production of 
antibodies which bind specifically to a given region or three-dimensional 
structure on the protein; these regions or structures are referred to as 
antigenic determinants. An antigenic determinant may compete with the 
intact antigen (i.e., the immunogen used to elicit the immune response) 
for binding to an antibody. 
The terms "specific binding" or "specifically binding", as used herein, in 
reference to the interaction of an antibody and a protein or peptide, mean 
that the interaction is dependent upon the presence of a particular 
structure (i.e., the antigenic determinant or epitope) on the protein; in 
other words, the antibody is recognizing and binding to a specific protein 
structure rather than to proteins in general. For example, if an antibody 
is specific for epitope "A", the presence of a protein containing epitope 
A (or free, unlabeled A) in a reaction containing labeled "A" and the 
antibody will reduce the amount of labeled A bound to the antibody. 
The term "sample", as used herein, is used in its broadest sense. A 
biological sample suspected of containing nucleic acid encoding HPLMH or 
fragments thereof may comprise a cell, chromosomes isolated from a cell 
(e.g., a spread of metaphase chromosomes), genomic DNA (in solution or 
bound to a solid support such as for Southern blot analysis), RNA (in 
solution or bound to a solution or bound to a solid support such as for 
northern blot analysis), cDNA (in solution or bound to a solid support), 
an extract from cells or a tissue containing one or more proteins, and the 
like. 
The term "correlates with expression of a polynucleotide", as used herein, 
indicates that the detection of the presence of ribonucleic acid that is 
complementary to SEQ ID NO:2 by northern analysis hybridization assays is 
indicative of the presence of mRNA encoding HPLMH in a sample and thereby 
correlates with expression of the transcript from the gene encoding the 
protein. 
"Alterations" in the polynucleotide of SEQ ID NO:2, as used herein, 
comprise any alteration in the sequence of polynucleotides encoding HPLMH 
including deletions, insertions, and point mutations that may be detected 
using hybridization assays. Included within this definition is the 
detection of alterations to the genomic DNA sequence which encodes HPLMH 
(e.g., by alterations in the pattern of restriction enzyme fragments 
capable of hybridizing to SEQ ID NO:2), the inability of a selected 
fragment of SEQ ID NO:2 to hybridize to a sample of genomic DNA (e.g., 
using allele-specific oligonucleotide probes), and improper or unexpected 
hybridization, such as hybridization to a locus other than the normal 
chromosomal locus for the gene encoding HPLMH (e.g., using FISH to 
metaphase chromosomes spreads). 
The terms "transformed" and "transformation", as used herein, refer to any 
known method for the insertion of foreign DNA or RNA sequences into host 
prokaryotic or eukaryotic cells. It may occur under natural or artificial 
conditions using various methods well known in the art. Such transformed 
cells include cells in which the inserted DNA is capable of replication 
either as an autonomously replicating plasmid or as part of the host 
chromosome in the host cell. They also include cells which transiently 
express the inserted DNA or RNA for limited periods of time. The 
transformation method depends on the host cell being transformed. Methods 
of transformation are well known in the art and include, but are not 
limited to, viral infection, electroporation, lipofection, and calcium 
phosphate mediated direct uptake. 
As used herein, the term "antibody" refers to intact molecules as well as 
fragments thereof, such as Fa, F(ab').sub.2, and Fv, which are capable of 
binding the epitopic determinant. Antibodies that bind HPLMH polypeptides 
can be prepared using intact polypeptides or fragments containing small 
peptides of interest as the immunizing antigen. The polypeptide or peptide 
used to immunize an animal can be derived from translated cDNA or 
chemically synthesized, and can be conjugated to a carrier protein, if 
desired. Commonly used carriers that are chemically coupled to peptides 
include bovine serum albumin and thyroglobulin. The coupled peptide is 
then used to immunize the animal (e.g., a mouse, a rat, or a rabbit). 
The term "humanized antibody", as used herein, refers to antibody molecules 
in which amino acids have been replaced in the non-antigen binding regions 
in order to more closely resemble a human antibody, while still retaining 
the original binding ability. 
THE INVENTION 
The invention is based on the discovery of the human phospholemman homolog 
protein, HPLMH, the polynucleotides encoding HPLMH, and the use of these 
compositions for the diagnosis, prevention or treatment of diseases of the 
central nervous system, cardiovascular system, prostate, and disorders 
related to smooth muscle function. 
Nucleic acids encoding the HPLMH of the present invention were first 
identified in cDNA in Incyte Clone 786812 (SEQ ID NO:2) from a normal 
human prostate cDNA library (PROSNOT05) through a computer-generated 
search for amino acid sequence alignments. 
In one embodiment, the invention encompasses human phospholemman homolog, a 
protein comprising the amino acid sequence of SEQ ID NO:1, as shown in 
FIG. 1. HPLMH is 92 amino acids in length. HPLMH has chemical and 
structural homology with PLM (SEQ ID NO:3; GI 108084), MAT-8 (SEQ ID NO:4; 
GI 1085026), CHIF (SEQ ID NO:5; GI 951423), and Na,K-ATPase 
.gamma.-subunit (SEQ ID NO:6; GI 51112). PLM, Mat-8, CHIF, and Na,K-ATPase 
.gamma.-subunit have, respectively, 90%, 33%, 22%, and 25% sequence 
identity to HPLMH. The amino acid identity between HPLMH (SEQ ID NO:1) and 
PLM (SEQ ID NO:3) increases to 94% when the signal peptide sequence is 
excluded from the comparison; the remaining 4 differences between HPLMH 
and PLM are conservative amino acid replacements. 
From the amino acid sequence alignments (FIG. 2) and the hydrophobicity 
plot for HPLMH (FIG. 3), the HPLMH signal peptide is predicted to extend 
from residue 1 to residue 20. A single transmembrane domain is predicted 
to extend from residue 38 to residue 57 of HPLMH, using the numbering of 
SEQ ID NO:1, and terminates in a positively-charged membrane stop transfer 
sequence (RRCRCK) at residue 58 to residue 63. Both HPLMH and PLM are 
highly basic proteins with predicted isoelectric points of 9.45 (FIG. 4) 
and 9.7 (Palmer C. et al., supra), respectively, which suggests that they 
have a similar structure. 
The invention also encompasses HPLMH variants. A preferred HPLMH variant is 
one having at least 80%, and more preferably 90%, amino acid sequence 
similarity to the HPLMH amino acid sequence (SEQ ID NO:1). A most 
preferred HPLMH variant is one having at least 95% amino acid sequence 
similarity to SEQ ID NO:1. 
The invention also encompasses polynucleotides which encode HPLMH. 
Accordingly, any nucleic acid sequence which encodes the amino acid 
sequence of HPLMH can be used to generate recombinant molecules which 
express HPLMH. In a particular embodiment, the invention encompasses the 
polynucleotide comprising the nucleic acid of SEQ ID NO:2 as shown in FIG. 
1. 
It will be appreciated by those skilled in the art that as a result of the 
degeneracy of the genetic code, a multitude of nucleotide sequences 
encoding HPLMH, some bearing minimal homology to the nucleotide sequences 
of any known and naturally occurring gene, may be produced. Thus, the 
invention contemplates each and every possible variation of nucleotide 
sequence that could be made by selecting combinations based on possible 
codon choices. These combinations are made in accordance with the standard 
triplet genetic code as applied to the nucleotide sequence of naturally 
occurring HPLMH, and all such variations are to be considered as being 
specifically disclosed. 
Although nucleotide sequences which encode HPLMH and its variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring HPLMH under appropriately selected conditions of 
stringency, it may be advantageous to produce nucleotide sequences 
encoding HPLMH or its derivatives possessing a substantially different 
codon usage. Codons may be selected to increase the rate at which 
expression of the peptide occurs in a particular prokaryotic or eukaryotic 
expression host in accordance with the frequency with which particular 
codons are utilized by the host. Other reasons for substantially altering 
the nucleotide sequence encoding HPLMH 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 a DNA sequence, or portions 
thereof, which encode HPLMH and its derivatives, entirely by synthetic 
chemistry. After production, the synthetic gene may be inserted into any 
of the many available DNA vectors and cell systems using reagents that are 
well known in the art at the time of the filing of this application. 
Moreover, synthetic chemistry may be used to introduce mutations into a 
sequence encoding HPLMH or any portion thereof. 
Also encompassed by the invention are polynucleotide sequences that are 
capable of hybridizing to the claimed nucleotide sequences, and in 
particular, those shown in SEQ ID NO:2 under various conditions of 
stringency. Hybridization conditions are based on the melting temperature 
(Tm) of the nucleic acid binding complex or probe, as taught in Berger and 
Kimmel 1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, 
Vol 152, Academic Press, San Diego, Calif.), and may be used at a defined 
stringency. 
Altered nucleic acid sequences encoding HPLMH which are encompassed by the 
invention include deletions, insertions, or substitutions of different 
nucleotides resulting in a polynucleotide that encodes the same or a 
functionally equivalent HPLMH. The encoded protein may also contain 
deletions, insertions, or substitutions of amino acid residues which 
produce a silent change and result in a functionally equivalent HPLMH. 
Deliberate amino acid substitutions may be made on the basis of similarity 
in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or 
the amphipathic nature of the residues as long as the biological activity 
of HPLMH 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; phenylalanine and tyrosine. 
Also included within the scope of the present invention are encoded alleles 
of the gene encoding HPLMH. As used herein, an "allele" or "allelic 
sequence" is an alternative form of the gene for HPLMH which may result 
from a mutation in the nucleic acid sequence. Alleles may result in 
altered mRNAs or polypeptides whose structure or function may or may not 
be altered. Any given gene may have none, one, or many allelic forms. 
Common mutational changes which give rise to alleles are generally 
ascribed to natural deletions, additions, or substitutions of nucleotides. 
Each of these types of changes may occur alone, or in combination with the 
others, one or more times in a given sequence. 
Methods for DNA sequencing which are well known and generally available in 
the art may be used to practice any embodiments of the invention. The 
methods may employ such enzymes as the Klenow fragment of DNA polymerase 
I, Sequenase.RTM. (US Biochemical Corp, Cleveland, Ohio), Taq polymerase 
(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), or 
combinations of recombinant polymerases and proofreading exonucleases such 
as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg, 
Md.). Preferably, the process is automated with machines such as the 
Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler 
(PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNA sequencers 
(Perkin Elmer). 
The polynucleotide sequence encoding HPLMH may be extended utilizing a 
partial nucleotide sequence and employing various methods known in the art 
to detect upstream sequences such as promoters and regulatory elements. 
For example, one method which may be employed, "restriction-site" PCR, 
uses universal primers to retrieve unknown sequence adjacent to a known 
locus (Sarkar G. et al. (1993); PCR Methods Applic 2:318-322). In 
particular, genomic DNA is first amplified in the presence of primer to a 
linker sequence and a primer specific to the known region. The amplified 
sequences are then subjected to a second round of PCR with the same linker 
primer and another specific primer internal to the first one. Products of 
each round of PCR are transcribed with an appropriate RNA polymerase and 
sequenced using reverse transcriptase. 
Inverse PCR may also be used to amplify or extend sequences using divergent 
primers based on a known region (Triglia et al. (1988) Nucleic Acids Res 
16:8186). The primers may be designed using OLIGO.RTM. 4.06 Primer 
Analysis Software (1992; National Biosciences Inc., Plymouth, Minn.), or 
another appropriate program, to be 22-30 nucleotides in length, to have a 
GC content of 50% or more, and to anneal to the target sequence at 
temperatures about 68.degree.-72.degree. C. The method uses several 
restriction enzymes to generate a suitable fragment in the known region of 
a gene. The fragment is then circularized by intramolecular ligation and 
used as a PCR template. 
Another method which may be used is capture PCR which involves PCR 
amplification of DNA fragments adjacent to a known sequence in human and 
yeast artificial chromosome DNA (Lagerstrom et al. (1991) PCR Methods 
Applic 1:111-119). In this method, multiple restriction enzyme digestions 
and ligations may also be used to place an engineered double-stranded 
sequence into an unknown portion of the DNA molecule before performing 
PCR. 
Another method which may be used to retrieve unknown sequences is that of 
Parker et al. (1991; Nucleic Acids Res 19:3055-3060). Additionally, one 
may use PCR, nested primers, and PromoterFinder.TM. libraries to walk in 
genomic DNA (Clontech, Palo Alto, Calif.). This process avoids the need to 
screen libraries and may be useful in finding intron/exon junctions. 
When screening for full-length cDNAs, it is preferable to use libraries 
that have been size-selected to include larger cDNAs. Also, random-primed 
libraries are preferable in that they will contain more sequences which 
contain the 5' regions of the mRNA. Use of a randomly primed library may 
be especially preferable for situations in which an oligo d(T) library 
does not yield a full-length cDNA. Genomic libraries may be useful for 
extension of sequence into the 5' and 3' regulatory regions. 
Capillary electrophoresis systems which are commercially available may be 
used to analyze the size or confirm the nucleotide sequence of sequencing 
or PCR products. In particular, capillary sequencing may employ flowable 
polymers for electrophoretic separation, four different fluorescent dyes 
(one for each nucleotide) which are laser activated, and detection of the 
emitted wavelengths by a charge coupled devise camera. Output/light 
intensity may be converted to electrical signal using appropriate software 
(e.g., Genotyper.TM. and Sequence Navigator.TM. from Perkin Elmer) and the 
entire process from loading of samples to computer analysis and electronic 
data display may be computer controlled. Capillary electrophoresis is 
especially preferable for the sequencing of small pieces of DNA which 
might be present in limited amounts in a particular sample. 
In another embodiment of the invention, polynucleotide sequences or 
fragments thereof which encode HPLMH, or fusion proteins or functional 
equivalents thereof, may be used in recombinant DNA molecules to direct 
expression of HPLMH in appropriate host cells. Due to the inherent 
degeneracy of the genetic code, other DNA sequences which encode 
substantially the same or a functionally equivalent amino acid sequence 
may be produced and these sequences may be used to clone and express 
HPLMH. 
As will be understood by those of skill in the art, it may be advantageous 
to produce HPLMH-encoding nucleotide sequences possessing non-naturally 
occurring codons. For example, codons preferred by a particular 
prokaryotic or eukaryotic host can be selected to increase the rate of 
HPLMH expression or to produce a recombinant RNA transcript having 
desirable properties, such as a half-life which is longer than that of a 
transcript generated from the naturally occurring sequence. 
The nucleotide sequences of the present invention can be engineered using 
methods generally known in the art in order to alter the HPLMH coding 
sequence for a variety of reasons, including but not limited to, 
alterations which modify the cloning, processing, and/or expression of the 
gene product. DNA shuffling by random fragmentation and PCR reassembly of 
gene fragments and synthetic oligonucleotides may be used to engineer the 
nucleotide sequence. For example, site-directed mutagenesis may be used to 
insert new restriction sites, alter glycosylation patterns, to change 
codon preference, to produce splice variants, or other mutations, and so 
forth. 
In another embodiment of the invention, a natural, modified, or recombinant 
polynucleotide encoding HPLMH may be ligated to a heterologous sequence to 
encode a fusion protein. For example, to screen peptide libraries for 
inhibitors of HPLMH activity, it may be useful to encode a chimeric HPLMH 
protein that can be recognized by a commercially available antibody. A 
fusion protein may also be engineered to contain a cleavage site located 
between a HPLMH encoding sequence and the heterologous protein sequence, 
so that the HPLMH may be cleaved and purified away from the heterologous 
moiety. 
In another embodiment, the coding sequence of HPLMH may be synthesized, in 
whole or in part, using chemical methods well known in the art (see 
Caruthers et al. (1980) Nucleic Acids Res Symp Ser 215-223, Horn et al. 
(1980) Nucleic Acids Res Symp Ser 225-232, etc.). Alternatively, the 
protein itself may be produced using chemical methods to synthesize the 
HPLMH amino acid sequence, or a portion thereof. For example, peptide 
synthesis can be performed using various solid-phase techniques (Roberge 
et al. (1995) Science 269:202-204) and automated synthesis may be 
achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin 
Elmer). 
The newly synthesized peptide may be substantially purified by preparative 
high performance liquid chromatography (e.g., Creighton (1983) Proteins, 
Structures and Molecular Principles, WH Freeman and Co., New York, N.Y.). 
The composition of the synthetic peptides may be confirmed by amino acid 
analysis or sequencing (e.g., the Edman degradation procedure; Creighton, 
supra). Additionally, the amino acid sequence of HPLMH, or any part 
thereof, may be altered during direct synthesis and/or combined using 
chemical methods with sequences from other proteins, or any part thereof, 
to produce a variant polypeptide. 
In order to express a biologically active HPLMH, the nucleotide sequence 
encoding HPLMH or functional equivalents, may be inserted into an 
appropriate expression vector, i.e., a vector which contains the necessary 
elements for the transcription and translation of the inserted coding 
sequence. 
Methods which are well known to those skilled in the art may be used to 
construct expression vectors containing a HPLMH coding sequence and 
appropriate transcriptional and translational control elements. These 
methods include in vitro recombinant DNA techniques, synthetic techniques, 
and in vivo recombination or genetic recombination. Such techniques are 
described in Sambrook et al. (1989) Molecular Cloning, A Laboratory 
Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel et al. 
(1989) Current Protocols in Molecular Biology, John Wiley & Sons, New 
York, N.Y. 
A variety of expression vector/host systems may be utilized to contain and 
express a HPLMH coding sequence. These include, but are not limited to, 
microorganisms such as bacteria transformed with recombinant 
bacteriophage, plasmid, or cosmid DNA expression vectors; yeast 
transformed with yeast expression vectors; insect cell systems infected 
with virus expression vectors (e.g., baculovirus); plant cell systems 
transformed with virus expression vectors (e.g., cauliflower mosaic virus, 
CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression 
vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. 
The "control elements" or "regulatory sequences" are those non-translated 
regions of the vector, enhancers, promoters, 5' and 3' untranslated 
regions, which interact with host cellular proteins to carry out 
transcription and translation and they may vary in their strength and 
specificity. Depending on the vector system and host utilized, any number 
of suitable transcription and translation elements, including constitutive 
and inducible promoters, may be used. For example, when cloning in 
bacterial systems, inducible promoters such as the hybrid lacZ promoter of 
the Bluescript.RTM. phagemid (Stratagene, La Jolla, Calif.) or pSport1 
(Gibco BRL) and ptrp-lac hybrids, and the like may be used. The 
baculovirus polyhedrin promoter may be used in insect cells. Promoters or 
enhancers derived from the genomes of plant cells (e.g., heat shock, 
RUBISCO; and storage protein genes) or from plant viruses (e.g., viral 
promoters or leader sequences) may be cloned into the vector. In mammalian 
cell systems, promoters from mammalian genes or from mammalian viruses are 
preferable. If it is necessary to generate a cell line that contains 
multiple copies of HPLMH, vectors based on SV40 or EBV may be used with an 
appropriate selectable marker. 
In bacterial systems, a number of expression vectors may be selected 
depending upon the use intended for HPLMH. For example, when large 
quantities of HPLMH are needed for the induction of antibodies, vectors 
which direct high level expression of fusion proteins that are readily 
purified may be used. Such vectors include, but are not limited to, the 
multifunctional E. coli cloning and expression vectors such as 
Bluescript.RTM. (Stratagene), in which the HPLMH coding sequence may be 
ligated into the vector in frame with sequences for the amino-terminal Met 
and the subsequent 7 residues of .beta.-galactosidase so that a hybrid 
protein is produced; pIN vectors (Van Heeke and Schuster (1989) J Biol 
Chem 264:5503-5509); and the like. pGEX vectors (Promega, Madison, Wis.) 
may also be used to express foreign polypeptides as fusion proteins with 
glutathione S-transferase (GST). In general, such fusion proteins are 
soluble and can easily be purified from lysed cells by adsorption to 
glutathione-agarose beads followed by elution in the presence of free 
glutathione. Proteins made in such systems may be designed to include 
heparin, thrombin, or factor XA protease cleavage sites so that the cloned 
polypeptide of interest can be released from the GST moiety at will. 
In the yeast, Saccharomyces cerevisiae, a number of vectors containing 
constitutive or inducible promoters such as alpha factor, alcohol oxidase, 
and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et 
al. (1987) Methods in Enzymol 153:516-544. 
In cases where plant expression vectors are used, the expression of a 
sequence encoding HPLMH may be driven by any of a number of promoters. For 
example, viral promoters such as the 35S and 19S promoters of CaMV may be 
used alone or in combination with the omega leader sequence from TMV 
(Takamatsu et al. (1987) EMBO J 6:307-311; Brisson et al. (1984) Nature 
310:511-514). Alternatively, plant promoters such as the small subunit of 
RUBISCO; or heat shock promoters may be used (Coruzzi et al. (1984) EMBO J 
3:1671-1680; Broglie et al. (1984) Science 224:838-843; Winter et al. 
(1991) Results Probl Cell Differ 17:85-105). These constructs can be 
introduced into plant cells by direct DNA transformation or 
pathogen-mediated transfection. Such techniques are described in a number 
of generally available reviews (see, for example, Hobbs, S or Murry, LE in 
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New 
York, NY; pp. 191-196 or Weissbach and Weissbach (1988) Methods for Plant 
Molecular Biology, Academic Press, New York, N.Y.; pp. 421-463). 
An insect system may also be used to express HPLMH. For example, in one 
such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is 
used as a vector to express foreign genes in Spodoptera frugiperda cells 
or in Trichoplusia larvae. The HPLMH coding sequence may be cloned into a 
non-essential region of the virus, such as the polyhedrin gene, and placed 
under control of the polyhedrin promoter. Successful insertion of HPLMH 
will render the polyhedrin gene inactive and produce recombinant virus 
lacking coat protein. The recombinant viruses may then be used to infect, 
for example, S. frugiperda cells or Trichoplusia larvae in which HPLMH may 
be expressed (Smith et al. (1983) J Virol 46:584; Engelhard et al. (1994) 
Proc Nat Acad Sci 91:3224-3227). 
In mammalian host cells, a number of viral-based expression systems may be 
utilized. In cases where an adenovirus is used as an expression vector, a 
HPLMH coding sequence may be ligated into an adenovirus 
transcription/translation complex consisting of the late promoter and 
tripartite leader sequence. Insertion in a non-essential E1 or E3 region 
of the viral genome may be used to obtain a viable virus which is capable 
of expressing HPLMH in infected host cells (Logan and 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. 
Specific initiation signals may also be used to achieve more efficient 
translation of a HPLMH sequence. Such signals include the ATG initiation 
codon and adjacent sequences. 
In cases where sequences encoding HPLMU, its initiation codon, and upstream 
sequences are inserted into the appropriate expression vector, no 
additional transcriptional or translational control signals may be needed. 
However, in cases where only coding sequence, or a portion thereof, is 
inserted, exogenous translational control signals including the ATG 
initiation codon should be provided. Furthermore, the initiation codon 
should be in the correct reading frame to ensure translation of the entire 
insert. Exogenous translational elements and initiation codons may be of 
various origins, both natural and synthetic. The efficiency of expression 
may be enhanced by the inclusion of enhancers which are appropriate for 
the particular cell system which is used, such as those described in the 
literature (Scharf D. et al. (1994) Results Probl Cell Differ 20:125-162; 
Bittner et al. (1987) Methods in Enzymol 153:516-544). 
In addition, a host cell strain may be chosen for its ability to modulate 
the expression of the inserted sequences or to process the expressed 
protein in the desired fashion. Such modifications of the polypeptide 
include, but are not limited to, acetylation, carboxylation, 
glycosylation, phosphorylation, lipidation, and acylation. 
Post-translational processing which cleaves a "prepro" form of the protein 
may also be used to facilitate correct insertion, folding and/or function. 
Different host cells such as CHO, HeLa, MDCK, HEK293, WI38, which have 
specific cellular machinery and characteristic mechanisms for such 
post-translational activities, may be chosen to ensure the correct 
modification and processing of the introduced foreign protein. 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express 
HPLMH may be transformed using expression vectors which may contain viral 
origins of replication and/or endogenous expression elements and a 
selectable marker gene on the same or separate vector. Following the 
introduction of the vector, cells may be allowed to grow for 1-2 days in 
an enriched media before they are switched to selective media. The purpose 
of the selectable marker is to confer resistance to selection, and its 
presence allows growth and recovery of cells which successfully express 
the introduced sequences. Resistant clones of stably transformed cells may 
be proliferated using tissue culture techniques appropriate to the cell 
type. 
Any number of selection systems may be used to recover transformed cell 
lines. These include, but are not limited to, the herpes simplex virus 
thymidine kinase and adenine phosphoribosyltransferase genes which can be 
employed in tk.sup.- or aprt.sup.- cells, respectively (Wigler M. et al. 
(1977) Cell 11:223-232 and Lowy et al. (1980) Cell 22:817-823). Also, 
antimetabolite, antibiotic, or herbicide resistance may be used as the 
basis for selection. For example, dhfr, which confers resistance to 
methotrexate, npt, which confers resistance to the aminoglycosides 
neomycin and G-418, and als or pat, which confer resistance to 
chlorsulfuron and phosphinotricin acetyltransferase, may be used (Wigler 
M. et al. (1980) Proc Natl Acad Sci 77:3567-3570 and Colbere-Garapin et 
al. (1981) J Mol Biol 150:1-14). Additional selectable genes may be used, 
for example, trpB, which allows cells to utilize indole in place of 
tryptophan, or hisD, which allows cells to utilize histinol in place of 
histidine (Hartman et al. (1988) Proc Natl Acad Sci 85:8047-8051). Also, 
visible markers such as green fluorescent protein, anthocyanins, .beta. 
glucuronidase, and its substrate, GUS, and luciferase and its substrate, 
luciferin, may 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 (Rhodes C. et al. (1995) Methods Mol Biol 
55:121-131). 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, its presence and expression may be 
confirmed. For example, if the sequence encoding HPLMH is inserted within 
a marker gene sequence, recombinant cells containing sequences encoding 
HPLMH can be identified by the absence of marker gene function. 
Alternatively, a marker gene can be placed in tandem with a HPLMH sequence 
under the control of a single promoter. Expression of the marker gene in 
response to induction or selection usually indicates expression of the 
tandem HPLMH as well. Alternatively, host cells which contain the coding 
sequence for HPLMH and express HPLMH 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, fluorescent 
activated cell sorting and protein bioassay or immunoassay techniques 
which include membrane, solution, or chip based technologies for the 
detection and/or quantification of the nucleic acid or protein. 
The presence of the polynucleotide sequence encoding HPLMH can be detected 
by DNA-DNA or DNA-RNA hybridization or amplification using probes or 
portions or fragments of polynucleotides encoding HPLMH. Nucleic acid 
amplification based assays involve the use of oligonucleotides or 
oligomers based on the HPLMH-encoding sequence to detect transformants 
containing DNA or RNA encoding HPLMH. As used herein "oligonucleotides" or 
"oligomers" refer to a nucleic acid sequence of at least about 10 
nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 
nucleotides, and more preferably about 20-25 nucleotides, which can be 
used as a probe or amplimer. 
A variety of protocols for detecting and measuring the expression of HPLMH, 
using either polyclonal or monoclonal antibodies specific for the protein 
are known in the art. Examples include enzyme-linked immunosorbent assay 
(ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting 
(FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal 
antibodies reactive to two non-interfering epitopes on HPLMH is preferred, 
but a competitive binding assay may be employed. These and other assays 
are described, among other places, in Hampton et al. (1990, Serological 
Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox et al. 
(1983) J Exp Med 158:1211). 
A wide variety of labels and conjugation techniques are known by those 
skilled in the art and 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 HPLMH include 
oligolabeling, nick translation, end-labeling or PCR amplification using a 
labeled nucleotide. Alternatively, the sequence encoding HPLMH, or any 
portion of it, may be cloned into a vector for the production of an mRNA 
probe. Such vectors are known in the art, are commercially available, and 
may be used to synthesize RNA probes in vitro by addition of an 
appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. 
These procedures may be conducted using a variety of commercially 
available kits (Pharmacia and Upjohn, Kalamazoo, Mich., Promega, Madison, 
Wis., and U.S. Biochemical Corp., Cleveland, Ohio). Suitable reporter 
molecules or labels, which may be used, include radionuclides, enzymes, 
fluorescent, chemiluminescent, or chromogenic agents as well as 
substrates, cofactors, inhibitors, magnetic particles, and the like. 
Host cells transformed with a nucleotide sequence encoding HPLMH may be 
cultured under conditions suitable for the expression and recovery of the 
encoded protein from cell culture. The protein produced by a recombinant 
cell may be secreted or contained intracellularly depending on the 
sequence and/or the vector used. As will be understood by those of skill 
in the art, expression vectors containing polynucleotides which encode 
HPLMH may be designed to contain signal sequences which direct secretion 
of HPLMH through a prokaryotic or eukaryotic cell membrane. Other 
recombinant constructions may be used to join sequences encoding HPLMH to 
nucleotide sequence encoding a polypeptide domain which will facilitate 
purification of soluble proteins. Such purification facilitating domains 
include, but are not limited to, metal chelating peptides such as 
histidine-tryptophan modules that allow purification on immobilized 
metals, protein A domains that allow purification on immobilized 
immunoglobulin, and the domain utilized in the FLAGS extension/affinity 
purification system (Immunex Corp., Seattle, Wash.). The inclusion of a 
cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, 
San Diego, Calif.) between the purification domain and HPLMH may be used 
to facilitate purification. One such expression vector which may be used 
provides for expression of a fusion protein containing a HPLMH and a 
nucleic acid encoding 6 histidine residues followed by thioredoxin and an 
enterokinase cleavage site. The histidine residues facilitate purification 
on IMIAC (immobilized metal ion affinity chromatography as described in 
Porath et al. (1992) Protein Expression and Purification 3:263-281) while 
the enterokinase cleavage site provides a means for purifying HPLMH from 
the fusion protein. A discussion of vectors which contain fusion proteins 
is provided in Kroll et al. (1993) DNA Cell Biol 12:441-453. 
In addition to recombinant production, fragments of HPLMH may be produced 
by direct peptide synthesis using solid-phase techniques (cf Stewart et 
al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, 
Calif.; Maryfield J. (1963) J Am Chem Soc 85:2149-2154). In vitro protein 
synthesis may be performed using manual techniques or by automation. 
Automated synthesis may be achieved, for example, using Applied Biosystems 
431A Peptide Synthesizer (Perkin Elmer). Various fragments of HPLMH may be 
chemically synthesized separately and combined using chemical methods to 
produce the full length molecule. 
THERAPEUTICS 
Chemical and structural homology exists among HPLMH protein, canine PLM 
(SEQ ID NO:3; GI 108084), human MAT-8 (SEQ ID NO:4; GI 1085026), rat CHIF 
(SEQ ID NO:5; GI 951423), and mouse Na,K-ATPase .gamma.-subunit (SEQ ID 
NO:6; GI 51112) (FIG. 2). In addition, northern analysis demonstrates that 
HPLMH molecules are expressed in prostate, nervous system tissues, muscle, 
uterus, breast, and lung. 
From the homology and expression information provided above, and the known 
associations and functions of PLM-like transmembrane proteins in heart and 
other tissues, it appears that HPLMH plays a role in modifying or 
regulating ion currents, including those associated with Cl.sup.- and 
Ca.sup.2+ ions, and/or taurine influx and efflux. Therefore, in another 
embodiment of the invention, HPLMH or fragments thereof may be used for 
therapeutic purposes. Altered or mutated activity, or altered expression 
of HPLMH may be associated with diseases and conditions relating, but are 
not limited, to defective ion transport, defects in signal transmission, 
membrane potential generation, or fluid volume regulation. Accordingly, 
HPLMH or derivatives thereof, may be used to treat central nervous system 
diseases such as Alzheimer's disease, Parkinson's disease, Huntington's 
disease, Creutzfeld-Jacob disease, amyotrophic lateral sclerosis, and 
hydrocephalus, cardiovascular diseases such as angina, cardiac 
hypertrophy, congestive heart failure, vasoconstriction, and hypertension, 
prostate hypertrophy, and smooth muscle disorders such as bladder and 
sphincter dysfunction, bronchial constriction, and asthma. 
In another embodiment, antagonists which block or modulate the effect of 
HPLMH may be used in those situations where such inhibition is 
therapeutically desirable. Such antagonists or inhibitors may be produced 
using methods which are generally known in the art, and include 
particularly the use of purified HPLMH to produce antibodies or to screen 
libraries of pharmaceutical agents for those which specifically bind 
HPLMH. For example, in one aspect, antibodies which are specific for HPLMH 
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 HPLMH. 
The antibodies may be generated using methods that are well known in the 
art. Such antibodies may include, but are not limited to, polyclonal, 
monoclonal, chimeric, single chain, Fab fragments, and fragments produced 
by a Fab expression library. Neutralizing antibodies, (i.e., those which 
inhibit dimer formation) are especially preferred for therapeutic use. 
For the production of antibodies, various hosts including goats, rabbits, 
rats, mice, humans, and others, may be immunized by injection with HPLMH 
or any fragment or oligopeptide thereof which has immunogenic properties. 
Depending on the host species, various adjuvants may be used to increase 
immunological response. Such adjuvants include, but are not limited to, 
Freund's, mineral gels such as aluminum hydroxide, and surface active 
substances such as lysolecithin, pluronic polyols, polyanions, peptides, 
oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among 
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and 
Corynebacterium parvum are especially preferable. 
It is preferred that the peptides, fragments, or oligopeptides used to 
induce antibodies to HPLMH have an amino acid sequence consisting of at 
least five amino acids, and more preferably at least 10 amino acids. It is 
also preferable that they are identical to a portion of the amino acid 
sequence of the natural protein, and they may contain the entire amino 
acid sequence of a small, naturally occurring molecule. Short stretches of 
HPLMH amino acids may be fused with those of another protein such as 
keyhole limpet hemocyanin and antibody produced against the chimeric 
molecule. 
Monoclonal antibodies to HPLMH 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 (Koehler et al. (1975) Nature 256:495-497; Kosbor et al. (1983) 
Immunol Today 4:72; Cote et al. (1983) Proc Natl Acad Sci 80:2026-2030; 
Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss 
Inc., New York, NY, pp. 77-96). 
In addition, techniques developed for the production of "chimeric 
antibodies", the splicing of mouse antibody genes to human antibody genes 
to obtain a molecule with appropriate antigen specificity and biological 
activity, can be used (Morrison et al. (1984) Proc Natl Acad Sci 
81:6851-6855; Neuberger et al. (1984) Nature 312:604-608; Takeda et al. 
(1985) Nature 314:452-454). Alternatively, techniques described for the 
production of single chain antibodies may be adapted, using methods known 
in the art, to produce HPLMH-specific single chain antibodies. Antibodies 
with related specificity but of distinct idiotypic composition may be 
generated by chain shuffling from random combinatorial immnunoglobin 
libraries (Burton D. R. (1991) Proc Natl Acad Sci 88:11120-3). 
Antibodies may also be produced by inducing in vivo production in the 
lymphocyte population or by screening recombinant immunoglobulin libraries 
or panels of highly specific binding reagents as disclosed in the 
literature (Orlandi et al. (1989) Proc Natl Acad Sci 86:3833-3837; Winter 
et al. (1991), Nature 349:293-299). 
Antibody fragments which contain specific binding sites for HPLMH may also 
be generated. For example, such fragments include, but are not limited to, 
the F(ab')2 fragments which can be produced by pepsin digestion of the 
antibody molecule and the Fab fragments which can be generated by reducing 
the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab 
expression libraries may be constructed to allow rapid and easy 
identification of monoclonal Fab fragments with the desired specificity 
(Huse et al. (1989) Science 256:1275-1281). 
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 HPLMH and its specific antibody. A two-site, monoclonal-based 
immunoassay utilizing monoclonal antibodies reactive to two 
non-interfering epitopes on a specific HPLMH protein is preferred, but a 
competitive binding assay may also be employed (Maddox et al. (1983) J Exp 
Med 158:1211). 
In another embodiment of the invention, the polynucleotides encoding HPLMH, 
or any fragment thereof or antisense sequences, may be used for 
therapeutic purposes. In one aspect, antisense to the polynucleotide 
encoding HPLMH may be used in situations in which it would be desirable to 
block the synthesis of HPLMH. In particular, cells may be transformed with 
antisense sequences to polynucleotides encoding HPLMH. Thus, antisense 
sequences may be used to achieve regulation of gene function. Such 
technology is now well known in the art, and sense or antisense oligomers 
or larger fragments, can be designed from various locations along the 
coding or control regions. 
Expression vectors derived from retroviruses, adenovirus, herpes or 
vaccinia viruses, or from various bacterial plasmids may be used for 
delivery of nucleotide sequences to the targeted organ, tissue or cell 
population. Methods which are well known to those skilled in the art can 
be used to construct recombinant vectors which will express antisense 
polynucleotides of the gene encoding HPLMH. See, for example, the 
techniques described in Sambrook et al. (supra) and Ausubel et al. 
(supra). 
Genes encoding HPLMH can be turned off by transforming a cell or tissue 
with expression vectors which express high levels of a polynucleotide or 
fragment thereof which encodes HPLMH. 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 all copies are disabled by endogenous 
nucleases. Transient expression may last for a month or more with a 
non-replicating vector and even longer if appropriate replication elements 
are part of the vector system. 
As mentioned above, modifications of gene expression can be obtained by 
designing antisense molecules, DNA, RNA, or PNA, to the control regions of 
the gene encoding HPLMH, i.e., the promoters, enhancers, and introns. 
Oligonucleotides derived from the transcription initiation site, e.g., 
between positions -10 and +10 from the 5' end of the transcript, are 
preferred. Similarly, inhibition can be achieved using "triple helix" 
base-pairing methodology. Triple helix pairing is useful because it causes 
inhibition of the ability of the double helix to open sufficiently for the 
binding of polymerases, transcription factors, or regulatory molecules. 
Recent therapeutic advances using triplex DNA have been described in the 
literature (Gee et al., in Huber and Carr (1994) Molecular and Immunologic 
Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Antisense molecules 
may also be designed to block translation of mRNA by preventing the 
transcript from binding to ribosomes. 
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the 
specific cleavage of RNA. The mechanism of ribozyme action involves 
sequence-specific hybridization of the ribozyme molecule to complementary 
target RNA, followed by endonucleolytic cleavage. Examples which may be 
used include engineered hammerhead motif ribozyme molecules that can 
specifically and efficiently catalyze endonucleolytic cleavage of 
sequences encoding HPLMH. 
Specific ribozyme cleavage sites within any potential RNA target are 
initially identified by scanning the target molecule for ribozyme cleavage 
sites which include the following sequences: GUA, GUU, and GUC. Once 
identified, short RNA sequences of between 15 and 20 ribonucleotides 
corresponding to the region of the target gene containing the cleavage 
site may be evaluated for secondary structural features which may render 
the oligonucleotide inoperable. The suitability of candidate targets may 
also be evaluated by testing accessibility to hybridization with 
complementary oligonucleotides using ribonuclease protection assays. 
Antisense molecules and ribozymes of the invention may be prepared by any 
method known in the art for the synthesis of RNA molecules. These include 
techniques for chemically synthesizing oligonucleotides such as solid 
phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may 
be generated by in vitro and in vivo transcription of DNA sequences 
encoding HPLMH. Such DNA sequences may be incorporated into a wide variety 
of vectors with suitable RNA polymerase promoters such as T7 or SP6. 
Alternatively, cDNA constructs that synthesize antisense RNA 
constitutively or inducibly can be introduced into cell lines, cells, or 
tissues. 
RNA molecules may be modified to increase intracellular stability and 
half-life. Possible modifications include, but are not limited to, the 
addition of flanking sequences at the 5' and/or 3' ends of the molecule or 
the use of phosphorothioate or 2'-O-methyl-ribose within the 
phosphodiester backbone of the molecule. This concept is inherent in the 
production of PNAs and can be extended in all of these molecules by the 
inclusion of nontraditional bases such as inosine, queosine, and 
wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified 
forms of adenine, cytidine, guanine, thymine, and uridine which are not as 
easily recognized by endogenous endonucleases. 
Methods for introducing vectors into cells or tissues include those methods 
discussed above. These methods are equally suitable for use in in vivo, in 
vitro and ex vivo therapy. 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 
transformation and by liposome injections may be achieved using methods 
which are well known in the art. 
Any of the therapeutic methods described above may be applied to any 
suitable subject including, for example, mammals such as dogs, cats, cows, 
horses, rabbits, monkeys, and most preferably, humans. 
An additional embodiment of the invention relates to the administration of 
a pharmaceutical composition, in conjunction with a pharmaceutically 
acceptable carrier, for any of the therapeutic effects discussed above. 
Such pharmaceutical compositions may consist of HPLMH, antibodies to 
HPLMH, mimetics, agonists, antagonists, or inhibitors of HPLMH. The 
compositions may be administered alone or in combination with at least one 
other agent, such as stabilizing compound, which may be administered in 
any sterile, biocompatible pharmaceutical carrier, including, but not 
limited to, saline, buffered saline, dextrose, and water. The compositions 
may be administered to a patient alone, or in combination with other 
agents, drugs or hormones. 
The pharmaceutical compositions utilized in this invention may be 
administered by any number of routes including, but not limited to, oral, 
intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, 
intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, 
enteral, topical, sublingual, or rectal means. 
In addition to the active ingredients, these pharmaceutical compositions 
may contain suitable pharmaceutically-acceptable carriers comprising 
excipients and auxiliaries which facilitate processing of the active 
compounds into preparations which can be used pharmaceutically. Further 
details on techniques for formulation and administration may be found in 
the latest edition of Remington's Pharmaceutical Sciences (Maack 
Publishing Co., Easton, Pa.). 
Pharmaceutical compositions for oral administration can be formulated using 
pharmaceutically acceptable carriers well known in the art in dosages 
suitable for oral administration. Such carriers enable the pharmaceutical 
compositions to be formulated as tablets, pills, dragees, capsules, 
liquids, gels, syrups, slurries, suspensions, and the like, for ingestion 
by the patient. 
Pharmaceutical preparations for oral use can be obtained through 
combination of active compounds with solid excipient, optionally grinding 
a resulting mixture, and processing the mixture of granules, after adding 
suitable auxiliaries, if desired, to obtain tablets or dragee cores. 
Suitable excipients are carbohydrate or protein fillers, such as sugars, 
including lactose, sucrose, mannitol, or sorbitol; starch from corn, 
wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, 
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums 
including arabic and tragacanth; and proteins such as gelatin and 
collagen. If desired, disintegrating or solubilizing agents may be added, 
such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a 
salt thereof, such as sodium alginate. 
Dragee cores may be used in conjunction with suitable coatings, such as 
concentrated sugar solutions, which may also contain gum arabic, talc, 
polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium 
dioxide, lacquer solutions, and suitable organic solvents or solvent 
mixtures. Dyestuffs or pigments may be added to the tablets or dragee 
coatings for product identification or to characterize the quantity of 
active compound, i.e., dosage. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a coating, such as glycerol or sorbitol. Push-fit capsules can contain 
active ingredients mixed with a filler or binders, such as lactose or 
starches, lubricants, such as talc or magnesium stearate, and, optionally, 
stabilizers. In soft capsules, the active compounds may be dissolved or 
suspended in suitable liquids, such as fatty oils, liquid paraffin or 
liquid polyethylene glycol with or without stabilizers. 
Pharmaceutical formulations suitable for parenteral administration may be 
formulated in aqueous solutions, preferably in physiologically compatible 
buffers such as Hanks's solution, Ringer's solution, or physiologically 
buffered saline. Aqueous injection suspensions may contain substances 
which increase the viscosity of the suspension, such as sodium 
carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions 
of the active compounds may be prepared as appropriate oily injection 
suspensions. Suitable lipophilic solvents or vehicles include fatty oils 
such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate 
or triglycerides, or liposomes. Optionally, the suspension may also 
contain suitable stabilizers or agents which increase the solubility of 
the compounds to allow for the preparation of highly concentrated 
solutions. 
For topical or nasal administration, penetrants appropriate to the 
particular barrier to be permeated are used in the formulation. Such 
penetrants are generally known in the art. 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that is known in the art, e.g., by means of 
conventional mixing, dissolving, granulating, dragee-making, levigating, 
emulsifying, encapsulating, entrapping, or lyophilizing processes. 
The pharmaceutical composition may be provided as a salt that can be formed 
with either an acid or base, depending on the nature of the therapeutic 
compound. Such acids include, but are not limited to, hydrochloric, 
sulfuric, acetic, lactic, tartaric, malic, and succinic; bases include, 
but are not limited to, sodium hydroxide and potassium hydroxide. Salts 
tend to be more soluble in aqueous or other protonic solvents than are the 
corresponding free acid or base forms. In other cases, the preferred 
preparation may be a lyophilized powder which may contain any or all of 
the following: 1 mM-50 mM histidine, 0.1%-2% sucrose, and 2%-7% mannitol, 
in a pH range of 4.5 to 5.5, that is/are 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 HPLMH, such labeling would include 
amount, frequency, and method of administration. 
Pharmaceutical compositions suitable for use in the invention include 
compositions wherein the active ingredients are contained in an effective 
amount to achieve the intended purpose. The determination of an effective 
dose is well within the capability of those skilled in the art. 
For any compound, the therapeutically effective dose can be estimated 
initially either in cell culture assays, e.g., of neoplastic cells, or in 
animal models, usually mice, rabbits, dogs or pigs. The animal model may 
also be used to determine the appropriate concentration range and route of 
administration. Such information can then be used to determine useful 
doses and routes for administration in humans. 
A therapeutically effective dose refers to that amount of active 
ingredient, for example HPLMH or fragments thereof, antibodies to HPLMH or 
agonists, antagonists or inhibitors of HPLMH, which ameliorates the 
symptoms or condition. Therapeutic efficacy and toxicity may be determined 
by standard pharmaceutical procedures in cell cultures or experimental 
animals, e.g., ED50 (the dose therapeutically effective in 50% of the 
population) and LD50 (the dose lethal to 50% of the population). The dose 
ratio between therapeutic and toxic effects is the therapeutic index, and 
it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions 
which exhibit large therapeutic indices are preferred. The data obtained 
from cell culture assays and animal studies is used in formulating a range 
of dosage for human use. The dosage contained in such compositions is 
preferably within a range of circulating concentrations that include the 
ED50 with little or no toxicity. The dosage varies within this range 
depending upon the dosage form employed, sensitivity of the patient, and 
the route of administration. 
The exact dosage will be determined by the practitioner, in light of 
factors related to the subject which requires treatment. Dosage and 
administration are adjusted to provide sufficient levels of the active 
moiety or to maintain the desired effect. Factors which may be taken into 
account include the severity of the disease state, general health of the 
subject, age, weight, and gender of the subject, diet, time and frequency 
of administration, drug combination(s), reaction sensitivities, and 
tolerance/response to therapy. Long-acting pharmaceutical compositions may 
be administered every 3 to 4 days, every week, or once every two weeks 
depending on half-life and clearance rate of the particular formulation. 
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a 
total dose of about 1 g, depending upon the route of administration. 
Guidance as to particular dosages and methods of delivery is provided in 
the literature and generally available to practitioners in the art. Those 
skilled in the art will employ different formulations for nucleotides than 
for proteins or their inhibitors. Similarly, delivery of polynucleotides 
or polypeptides will be specific to particular cells, conditions, 
locations, etc. 
DIAGNOSTICS 
In another embodiment, antibodies which are specific for HPLMH may be used 
for the diagnosis of conditions or diseases characterized by expression of 
HPLMH, or in assays to monitor patients being treated with HPLMH, 
agonists, antagonists or inhibitors. The antibodies useful for diagnostic 
purposes may be prepared in the same manner as those described above for 
therapeutics. Diagnostic assays for HPLMH include methods which utilize 
the antibody and a label to detect HPLMH in human body fluids or extracts 
of cells or tissues. The antibodies may be used with or without 
modification, and may be labeled by joining them, either covalently or 
non-covalently, with a reporter molecule. A wide variety of reporter 
molecules which are known in the art may be used, several of which are 
described above. 
A variety of protocols for measuring HPLMH, using either polyclonal or 
monoclonal antibodies specific for the respective protein are known in the 
art. Examples include enzyme-linked immunosorbent assay (ELISA), 
radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). A 
two-site, monoclonal-based immunoassay utilizing monoclonal antibodies 
reactive to two non-interfering epitopes on HPLMH is preferred, but a 
competitive binding assay may be employed. 
In order to provide a basis for diagnosing abnormal levels of HPLMH 
expression, normal or standard values for HPLMH expression are 
established. This may be accomplished by combining body fluids or cell 
extracts taken from normal mammalian subjects, preferably human, with 
antibody to HPLMH under conditions suitable for complex formation which 
are well known in the art. The amount of standard complex formation may be 
quantified by comparing various artificial membranes containing known 
quantities of HPLMH with both control and disease samples from biopsied 
tissues. Then, standard values obtained from normal samples may be 
compared with values obtained from samples from subjects that are 
symptomatic for the disease. Deviation between standard and subject values 
establishes the parameters for diagnosing the disease. 
In another embodiment of the invention, the polynucleotides encoding HPLMH 
may be used for diagnostic purposes. The polynucleotides which may be used 
include oligonucleotide sequences, antisense RNA and DNA molecules, and 
PNAs. The polynucleotides may be used to detect and quantitate gene 
expression in biopsied tissues in which expression of HPLMH may be 
implicated. The diagnostic assay may be used to distinguish between 
absence, presence, and excess expression of HPLMH, and to monitor 
regulation of HPLMH levels during therapeutic intervention. 
In one aspect, hybridization or PCR probes which are capable of detecting 
polynucleotide sequences, including genomic sequences, encoding HPLMH or 
closely related molecules, may be used to identify nucleic acid sequences 
which encode HPLMH. The specificity of the probe, whether it is made from 
a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory 
region, or a less specific region, e.g., especially in the 3' region, and 
the stringency of the hybridization or amplification (maximal, high, 
intermediate or low) will determine whether the probe identifies only 
naturally occurring sequences encoding HPLMH, alleles or related 
sequences. 
Probes may also be used for the detection of related sequences, and should 
preferably contain at least 50% of the nucleotides from any of those HPLMH 
encoding sequences. The hybridization probes of the subject invention may 
be derived from the nucleotide sequence of SEQ ID NO:2 or from genomic 
sequence including promoter, enhancer elements, and introns of the 
naturally occurring HPLMH. 
Other means for producing specific hybridization probes for DNAs encoding 
HPLMH include the cloning of nucleic acid sequences encoding HPLMH or 
HPLMH derivatives into vectors for the production of RNA probes. Such 
vectors are known in the art, commercially available, and may be used to 
synthesize RNA probes in vitro by means of the addition of the appropriate 
RNA polymerase, such as T7 or SP6 RNA polymerase, and the appropriate 
radioactively labeled nucleotides. Hybridization probes may be labeled by 
a variety of reporter groups, for example, radionuclides such as .sup.32 P 
or .sup.35 S, or enzymatic labels, such as alkaline phosphatase coupled to 
the probe via avidin/biotin coupling systems, and the like. 
Polynucleotide sequences encoding HPLMH may be used for the diagnosis of 
conditions or diseases which are associated with expression of HPLMH. 
Examples of such conditions or diseases include central nervous system 
diseases such as Alzheimer's disease, Parkinson's disease, Huntington's 
disease, Creutzfeld-Jacob disease, amyotrophic lateral sclerosis, and 
hydrocephalus, cardiovascular diseases such as angina, cardiac 
hypertrophy, congestive heart failure, vasoconstriction, and hypertension, 
prostate hypertrophy, disorders related to smooth muscle function such as 
bladder and sphincter dysfunction, bronchial constriction, and asthma. The 
polynucleotide sequences encoding HPLMH may be used in hybridization or 
PCR assays of fluids or tissues from patient biopsies to detect HPLMH 
expression. The form of such qualitative or quantitative methods may 
include Southern or northern analysis, dot blot, or other membrane-based 
technologies PCR technologies, dip stick, pin, chip, and ELISA, all 
methods which are well known in the art. 
In order to provide a basis for the diagnosis of disease associated with 
expression of HPLMH, 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 
polynucleotides encoding HPLMH, or a fragment thereof, under conditions 
suitable for hybridization or amplification. Standard hybridization may be 
quantified by comparing the values obtained from normal subjects with a 
dilution series of polynucleotides encoding HPLMH measured in the same 
experiment, where a known amount of a substantially purified 
polynucleotides encoding HPLMH is used. Standard values obtained from 
normal samples may be compared with values obtained from samples from 
patients who are symptomatic for disease associated with HPLMH. Deviation 
between standard and subject values is used to establish the presence of 
disease. 
Once disease is established and a treatment protocol is initiated, 
hybridization assays may be repeated on a regular basis to evaluate 
whether the level of expression in the patient begins to approximate that 
which is observed in the normal patient. The results obtained from 
successive assays may be used to show the efficacy of treatment over a 
period ranging from several days to months. 
Additional diagnostic uses for oligonucleotides encoding HPLMH may involve 
the use of PCR. Such oligomers may be chemically synthesized, generated 
enzymatically, or produced from a recombinant source. Oligomers will 
preferably consist of two nucleotide sequences, one with sense orientation 
(5'.fwdarw.3') and another with antisense (3'.rarw.5'), employed under 
optimized conditions for identification of a specific gene or condition. 
The same two oligomers, nested sets of oligomers, or even a degenerate 
pool of oligomers may be employed under less stringent conditions for 
detection and/or quantitation of closely related DNA or RNA sequences. 
Methods which may also be used to quantitate the expression of HPLMH 
include radiolabeling or biotinylating nucleotides, coamplification of a 
control nucleic acid, and standard curves onto which the experimental 
results are interpolated (Melby P. C. et al. (1993) J Immunol Methods, 
159:235-244; Duplaa C. et al. (1993) Anal Biochem 229-236.) The speed of 
quantitation of multiple samples may be accelerated by running the assay 
in an ELISA format where the oligomer of interest is presented in various 
dilutions and a spectrophotometric or colorimetric response gives rapid 
quantitation. 
In other embodiments of the invention, the nucleotide sequences of the 
invention may be used in molecular biology techniques that have not yet 
been developed, provided the new techniques rely on properties of 
nucleotide sequences that are currently known, such as the triplet genetic 
code, specific base pair interactions, and the like. 
In another embodiment of the invention, the nucleic acid sequence which 
encodes HPLMH may also be used to generate hybridization probes which are 
useful for mapping the naturally occurring genomic sequence. The sequence 
may be mapped to a particular chromosome or to a specific region of the 
chromosome using well known techniques. Such techniques include in situ 
hybridization to chromosomal spreads, flow-sorted chromosomal 
preparations, or artificial chromosome constructions, such as yeast 
artificial chromosomes, bacterial artificial chromosomes, bacterial PI 
constructions or single chromosome cDNA libraries as reviewed in Price C. 
M. (1993) Blood Rev 7:127-134, and Trask B. J. (1991) Trends Genet 
7:149-154. 
The technique of fluorescent in situ hybridization of chromosome spreads, 
as described in Vera et al. (1988) Human Chromosomes: A Manual of Basic 
Techniques, Pergamon Press, New York, N.Y., may also be used. Fluorescent 
in situ hybridization of chromosomal preparations and other physical 
chromosome mapping techniques may be correlated with additional genetic 
map data. Examples of genetic map data can be found in the 1994 Genome 
Issue of Science (265:1981f). Correlation between the location of the gene 
encoding HPLMH on a physical chromosomal map and a specific disease (or 
predisposition to a specific disease) may help delimit the region of DNA 
associated with that genetic disease. The nucleotide sequences of the 
subject invention may be used to detect differences in gene sequences 
between normal, carrier or affected individuals. 
In situ hybridization of chromosomal preparations and physical mapping 
techniques such as linkage analysis using established chromosomal markers 
may be used for extending genetic maps. Often the placement of a gene on 
the chromosome of another mammalian species, such as mouse, may reveal 
associated markers even if the number or arm of a particular human 
chromosome is not known. New sequences can be assigned to chromosomal 
arms, or parts thereof, by physical mapping. This provides valuable 
information to investigators searching for disease genes using positional 
cloning or other gene discovery techniques. Once the disease or syndrome 
has been crudely localized by genetic linkage to a particular genomic 
region, for example, AT to 11q22-23 (Gatti et al. (1988) Nature 
336:577-580), any sequences mapping to that area may represent associated 
or regulatory genes for further investigation. The nucleotide sequence of 
the subject invention may also be used to detect differences in the 
chromosomal location due to translocation, inversion, etc. among normal, 
carrier, or affected individuals. 
In another embodiment of the invention, HPLMH, its functional, 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 a test may be free in solution, affixed to a 
solid support, borne on a cell surface, or located intracellularly. The 
formation of binding complexes, between HPLMH and the agent being tested, 
may be measured. 
Another technique for drug screening which may be used provides for high 
throughput screening of compounds having suitable binding affinity to the 
protein of interest as described in published PCT application WO84/03564. 
In this method, as applied to HPLMH, large numbers of different small test 
compounds are synthesized on a solid substrate, such as plastic pins or 
some other surface. The test compounds are reacted with HPLMH, or 
fragments thereof, and washed. Bound HPLMH is then detected by methods 
well known in the art. Purified HPLMH 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 HPLMH specifically 
compete with a test compound for binding to HPLMH. In this manner, the 
antibodies can be used to detect the presence of any peptide which shares 
one or more antigenic determinants with HPLMH. 
In additional embodiments, the nucleotide sequences which encode HPLMH 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 Construction of PROSNOT05 cDNA Library 
The PROSNOT05 cDNA library was constructed from a non-tumorous prostate 
tissue removed from a 67-year-old Caucasian male (specimen #0036B; Mayo 
Clinic, Rochester, Minn.) by radical prostatectomy. The pathology report 
indicated Mayo grade 3 (of 4) adenocarcinoma (Gleason grade 3+3) in the 
periphery of the prostate. Perineural invasion was present as was 
involvement of periprostatic tissue. Non-tumorous portions of the prostate 
exhibited adenofibromatous hyperplasia. The patient had elevated levels of 
prostate specific antigen (PSA). Pelvic lymph nodes were negative for 
tumor. A prior stomach ulcer and atherosclerosis were reported in the 
patient history; however, the patient was not on any medication at the 
time of surgery. 
The frozen tissue was homogenized and lysed using a Brinkmann Homogenizer 
Polytron-PT 3000 (Brinkmann Instruments, Inc., Westbury, N.Y.) in 
guanidinium isothiocyanate solution. The lysate was extracted once with 
acid phenol at pH 4.0 per Stratagene's RNA isolation protocol (Stratagene 
Inc., San Diego, Calif.). The lysate was reextracted once more with phenol 
chloroform at pH 4.0. The RNA was then precipitated using 0.3M sodium 
acetate and 2.5 volumes of ethanol, resuspended in DEPC-treated water and 
DNase treated for 25 min at 37.degree. C. The RNA was again extracted once 
with an equal volume of acid phenol, and reprecipitated using conditions 
described above. The mRNA was isolated using the Qiagen Oligotex kit 
(QIAGEN Inc., Chatsworth, Calif.) and used to construct the cDNA library. 
The RNA was handled according to the recommended protocols in the 
SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (catalog 
#18248-013; Gibco BRL). cDNAs were fractionated on a Sepharose CL4B column 
(catalog #275105, Pharmacia), and those cDNAs exceeding 400 bp were 
ligated into pSport I. The plasmid pSport I was subsequently transformed 
into DH5a.TM. competent cells (Cat. #18258-012, Gibco B.L.). 
II Isolation and Sequencing of cDNA Clones Plasmid DNA was released from 
the cells and purified using the REAL Prep 96 Plasmid Kit for Rapid 
Extraction Alkaline Lysis Plasmid Minipreps (Cat. #26173). 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 (Catalog #22711, LIFE TECHNOLOGIES.TM.) 
with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the cultures were 
incubated for 19 hours after the wells were inoculated and then lysed with 
0.3 ml of lysis buffer; 3) following isopropanol precipitation, the 
plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the 
last step in the protocol, samples were transferred to a Beckman 96-well 
block for storage. 
The cDNAs were sequenced by the method of Sanger F. and Coulson A. R. 
(1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, 
Reno, Nev.) in combination with Peltier Thermal Cyclers (PTC200 from MJ 
Research, Watertown, Ma.) and Applied Biosystems 377 DNA Sequencing 
Systems; and the reading frame was determined. 
III Homology Searching of CDNA Clones and Their Deduced Proteins 
Each cDNA was compared to sequences in GenBank using a search algorithm 
developed by Applied Biosystems and incorporated into the INHERIT.TM.670 
Sequence Analysis System. In this algorithm, Pattern Specification 
Language (TRW Inc., Los Angeles, Calif.) was used to determine regions of 
homology. The three parameters that determine how the sequence comparisons 
run were window size, window offset, and error tolerance. Using a 
combination of these three parameters, the DNA database was searched for 
sequences containing regions of homology to the query sequence, and the 
appropriate sequences were scored with an initial value. Subsequently, 
these homologous regions were examined using dot matrix homology plots to 
distinguish regions of homology from chance matches. Smith-Waterman 
alignments were used to display the results of the homology search. 
Peptide and protein sequence homologies were ascertained using the 
INHERIT-670 Sequence Analysis System using the methods similar to those 
used in DNA sequence homologies. Pattern Specification Language and 
parameter windows were used to search protein databases for sequences 
containing regions of homology which were scored with an initial value. 
Dot-matrix homology plots were examined to distinguish regions of 
significant homology from chance matches. 
BLAST, which stands for Basic Local Alignment Search Tool (Altschul S. F. 
(1993) J Mol Evol 36:290-300; Altschul et al. (1990) J Mol Biol 
215:403-410), was used to search for local sequence alignments. BLAST 
produces alignments of both nucleotide and amino acid sequences to 
determine sequence similarity. Because of the local nature of the 
alignments, BLAST is especially useful in determining exact matches or in 
identifying homologs. BLAST is useful for matches which do not contain 
gaps. The fundamental unit of BLAST algorithm output is the High-scoring 
Segment Pair (HSP). 
An HSP consists of two sequence fragments of arbitrary but equal lengths 
whose alignment is locally maximal and for which the alignment score meets 
or exceeds a threshold or cutoff score set by the user. The BLAST approach 
is to look for homology between a query sequence and a database sequence, 
to evaluate the statistical significance of any matches found, and to 
report only those matches which satisfy the user-selected threshold of 
significance. The parameter E establishes the statistically significant 
threshold for reporting database sequence matches. E is interpreted as the 
upper bound of the expected frequency of chance occurrence of an HSP (or 
set of HSPs) within the context of the entire database search. Any 
database sequence whose match satisfies E is reported in the program 
output. 
IV Northern Analysis 
Northern analysis is a laboratory technique used to detect the presence of 
a transcript of a gene and involves the hybridization of a labeled 
nucleotide sequence to a membrane on which RNAs from a particular cell 
type or tissue have been bound (Sambrook et al., supra). 
Analogous computer techniques using BLAST (Altschul S. F. 1993 and 1990, 
supra) are used to search for identical or related molecules in nucleotide 
databases such as GenBank or the LIFESEQ.TM. database (Incyte 
Pharmaceuticals, Inc., 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 homologous. 
The basis of the search is the product score which is defined as: 
##EQU1## 
and it takes into account both the degree of similarity between two 
sequences and the length of the sequence match. For example, with a 
product score of 40, the match will be exact within a 1-2% error; and at 
70, the match will be exact. Homologous molecules are usually identified 
by selecting those which show product scores between 15 and 40, although 
lower scores may identify related molecules. 
V Extension of HPLMH-Encoding Polynucleotides to Full Length or to Recover 
Regulatory Elements 
Full length HPLMH-encoding nucleic acid sequence (SEQ ID NO:2) is used to 
design oligonucleotide primers for extending a partial nucleotide sequence 
to full length or for obtaining 5' sequences from genomic libraries. One 
primer is synthesized to initiate extension in the antisense direction 
(XLR) and the other is synthesized to extend sequence in the sense 
direction (XLF). Primers are used to facilitate the extension of the known 
HPLMH-encoding sequence "outward" generating amplicons containing new, 
unknown nucleotide sequence for the region of interest. The initial 
primers are designed from the cDNA using OLIGO.RTM. 4.06 Primer Analysis 
Software (National Biosciences), or another appropriate program, to be 
22-30 nucleotides in length, to have a GC content of 50% or more, and to 
anneal to the target sequence at temperatures about 68.degree.-72.degree. 
C. Any stretch of nucleotides which would result in hairpin structures and 
primer-primer dimerizations is avoided. 
The original, selected cDNA libraries, or a human genomic library are used 
to extend the sequence; the latter is most useful to obtain 5' upstream 
regions. If more extension is necessary or desired, additional sets of 
primers are designed to further extend the known region. 
By following the instructions for the XL-PCR kit (Perkin Elmer) and 
thoroughly mixing the enzyme and reaction mix, high fidelity amplification 
is obtained. Beginning with 40 pmol of each primer and the recommended 
concentrations of all other components of the kit, PCR is performed using 
the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Ma.) and the 
following parameters: 
______________________________________ 
Step 1 94.degree. C. for 1 min (initial denaturation) 
Step 2 65.degree. C. for 1 min 
Step 3 68.degree. C. for 6 min 
Step 4 94.degree. C. for 15 sec 
Step 5 65.degree. C. for 1 min 
Step 6 68.degree. C. for 7 min 
Step 7 Repeat step 4-6 for 15 additional cycles 
Step 8 94.degree. C. for 15 sec 
Step 9 65.degree. C. for 1 min 
Step 10 68.degree. C. for 7:15 min 
Step 11 Repeat step 8-10 for 12 cycles 
Step 12 72.degree. C. for 8 min 
Step 13 4.degree. C. (and holding) 
______________________________________ 
A 5-10 .mu.l aliquot of the reaction mixture is analyzed by electrophoresis 
on a low concentration (about 0.6-0.8%) agarose mini-gel to determine 
which reactions were successful in extending the sequence. Bands thought 
to contain the largest products are selected and cut out of the gel. 
Further purification involves using a commercial gel extraction method 
such as QIAQuick.TM. (QIAGEN Inc., Chatsworth, Calif.). After recovery of 
the DNA, Klenow enzyme is used to trim single-stranded, nucleotide 
overhangs creating blunt ends which facilitate religation and cloning. 
After ethanol precipitation, the products are redissolved in 13 .mu.of 
ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1 .mu.l T4 
polynucleotide kinase are added, and the mixture is incubated at room 
temperature for 2-3 hours or overnight at 16.degree. C . Competent E. coli 
cells (in 40 .mu.l of appropriate media) are transformed with 3 .mu.l of 
ligation mixture and cultured in 80 .mu.l of SOC medium (Sambrook et al., 
supra). After incubation for one hour at 37.degree. C., the whole 
transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook et 
al., supra) containing 2.times.Carb. The following day, several colonies 
are randomly picked from each plate and cultured in 150 .mu.l of liquid 
LB/2.times.Carb medium placed in an individual well of an appropriate, 
commercially-available, sterile 96-well microtiter plate. The following 
day, 5 .mu.l of each overnight culture is transferred into a non-sterile 
96-well plate and after dilution 1:10 with water, 5 .mu.l of each sample 
is transferred into a PCR array. 
For PCR amplification, 18 .mu.l of concentrated PCR reaction mix 
(3.3.times.) containing 4 units of rTth DNA polymerase, a vector primer, 
and one or both of the gene specific primers used for the extension 
reaction are added to each well. Amplification is performed using the 
following conditions: 
______________________________________ 
Step 1 94.degree. C. for 60 sec 
Step 2 94.degree. C. for 20 sec 
Step 3 55.degree. C. for 30 sec 
Step 4 72.degree. C. for 90 sec 
Step 5 Repeat steps 2-4 for an additional 29 cycles 
Step 6 72.degree. C. for 180 sec 
Step 7 4.degree. C. (and holding) 
______________________________________ 
Aliquots of the PCR reactions are run on agarose gels together with 
molecular weight markers. The sizes of the PCR products are compared to 
the original partial cDNAs, and appropriate clones are selected, ligated 
into plasmid, and sequenced. 
VI Labeling and Use of 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 cDNA fragments. Oligonucleotides 
are designed using state-of-the-art software such as OLIGO 4.06 (National 
Biosciences), labeled by combining 50 pmol of each oligomer and 250 .mu.Ci 
of .gamma.-.sup.32 P! adenosine triphosphate (Amersham, Chicago, Ill.) 
and T4 polynucleotide kinase (DuPont NEN.RTM., Boston, Ma.). The labeled 
oligonucleotides are substantially purified with Sephadex G-25 superfine 
resin column (Pharmacia). A portion containing 10.sup.7 counts per minute 
of each of the sense and antisense oligonucleotides is used in a typical 
membrane based hybridization analysis of human genomic DNA digested with 
one of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1 or 
Pvu II; DuPont NEN.RTM.). 
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. After XOMAT AR.TM. film 
(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimager 
cassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours, 
hybridization patterns are compared visually. 
VII Antisense Molecules 
The HPLMH-encoding sequence, or any part thereof, is used to inhibit in 
vivo or in vitro expression of naturally occurring HPLMH. Although use of 
antisense oligonucleotides, comprising about 20 base-pairs, is 
specifically described, essentially the same procedure is used with larger 
cDNA fragments. An oligonucleotide based on the coding sequences of HPLMH, 
as shown in FIG. 1, is used to inhibit expression of naturally occurring 
HPLMH. The complementary oligonucleotide is designed from the most unique 
5' sequence as shown in FIG. 1 and used either to inhibit transcription, 
by binding to the DNA near the transcription initiation site, or 
translation of an HPLMH-encoding transcript by preventing the ribosome 
from binding to the 5' untranslated region. Using an appropriate portion 
of the 5' sequence of SEQ ID NO:2, an effective antisense oligonucleotide 
includes any 15-20 nucleotides spanning the region which is translated 
into the amino terminus of the polypeptide as shown in FIGS. 1 and 2. 
VIII Expression of HPLMH 
Expression of the HPLMH is accomplished by subcloning the cDNAs into 
appropriate vectors and transforming the vectors into host cells. In this 
case, the cloning vector, pSport, previously used for the generation of 
the cDNA library is used to express HPLMH in E. coli. Upstream of the 
cloning site, this vector contains a promoter for .beta.-galactosidase, 
followed by sequence containing the amino-terminal Met, and the subsequent 
seven residues of B3-galactosidase. Immediately following these eight 
residues is a bacteriophage promoter useful for transcription and a linker 
containing a number of unique restriction sites. 
Induction of an isolated, transformed bacterial strain with IPTG using 
standard methods produces a fusion protein which consists of the first 
seven residues of .beta.-galactosidase, about 5 to 15 residues of linker, 
and the full length HPLMH-encoding sequence. The signal sequence directs 
the secretion of HPLMH into the bacterial growth media which can be used 
directly in the following assay for activity. 
IX Demonstration of HPLMH Activity 
The channel-forming ability of HPLMH is assayed by monitoring efflux of 
Cl.sup.- ions from vesicles containing HPLMH and subjected to a 
transmembrane ion potential. HPLMH and mitochondrial cytochrome C oxidase, 
a proton pump, are reconstituted into lipid vesicles by sonication. 
.sup.36 Cl.sup.- is then incorporated into the vesicles by passive 
diffusion, incubating the vesicles in a solution containing .sup.36 
Cl!-potassium chloride for several hours. The vesicles are then dispersed 
in an appropriate reaction buffer. Addition of ascorbate and cytochrome C 
initiates proton uptake into the vesicles generating an interior-positive 
membrane potential. The voltage generated across the membrane activates 
gating of the HPLMH ion channel. At predetermined times, aliquots of the 
vesicle-containing solution are removed from the reaction buffer and 
filtered through 0.2 m.mu. membrane filters (Millipore, Marlborough, Ma.). 
The vesicles are retained on the filters. The filters are rinsed and 
dried. Radioactivity on the filters is measured in a scintillation 
counter. The decrease in radioactivity on the filters as a function of 
reaction time gives a measure of the rate of Cl.sup.- efflux through the 
voltage-activated HPLMH ion channel. 
Alternatively, Xenopus oocytes are microinjected with RNA, corresponding to 
the message sequence encoding HPLMH which can be synthesized in vitro by 
techniques well known to those skilled in the art. Oocytes injected with 
HPLMH RNA are compared with mock-injected oocytes for .sup.3 H!taurine 
fluxes as described in detail (Moorman J. R. et al., supra). 
A third method is to measure currents regulated or modulated by HPLMH 
directly by introducing in vitro expressed HPLMH protein into synthetic 
phospholipid bilayers and measuring ion flux using standard 
electrophysiological techniques well known to those versed in the art. 
Alternatively, ion currents can be measured electrophysiologically in 
Xenopus oocytes that are microinjected with in vitro synthesized HPLMH RNA 
using techniques well known in the art. 
X Production of HPLMH Specific Antibodies 
HPLMH that is substantially purified using PAGE electrophoresis (Sambrook, 
supra), or other purification techniques, is used to immunize rabbits and 
to produce antibodies using standard protocols. The amino acid sequence 
translated from HPLMH is analyzed using DNASTAR software (DNASTAR Inc.) to 
determine regions of high immunogenicity and a corresponding 
oligopolypeptide is synthesized and used to raise antibodies by means 
known to those of skill in the art. Analysis, such as that described by 
Ausubel et al. (supra), may be used to select appropriate epitopes, such 
as those near the C-terminus or in hydrophilic regions. 
Typically, the oligopeptides are 15 residues in length, synthesized using 
an Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry, 
and coupled to keyhole limpet hemocyanin (KLH, Sigma, St. Louis, Mo.) by 
reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel 
et al., supra). Rabbits are immunized with the oligopeptide-KLH complex in 
complete Freund's adjuvant. The resulting antisera are tested for 
antipeptide activity, for example, by binding the peptide to plastic, 
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting 
with radioiodinated, goat anti-rabbit IgG. 
XI Purification of Naturally Occurring HPLMH Using Specific Antibodies 
Naturally occurring or recombinant HPLMH is substantially purified by 
immunoaffinity chromatography using antibodies specific for HPLMH. An 
immunoaffinity column is constructed by covalently coupling HPLMH antibody 
to an activated chromatographic resin, such as CnBr-activated Sepharose 
(Pharmacia Biotech). After the coupling, the resin is blocked and washed 
according to the manufacturer's instructions. 
Media containing HPLMH is passed over the immunoaffinity column, and the 
column is washed under conditions that allow the preferential absorbance 
of HPLMH (e.g., high ionic strength buffers in the presence of detergent). 
The column is eluted under conditions that disrupt antibody/HPLMH binding 
(e.g., a buffer of pH 2-3 or a high concentration of a chaotrope, such as 
urea or thiocyanate ion), and HPLMH is collected. 
XII Identification of Molecules Which Interact with HPLMH 
HPLMH, or biologically active fragments thereof, is labeled with .sup.125 I 
Bolton-Hunter reagent (Bolton et al. (1973) Biochem J 133:529). Candidate 
molecules previously arrayed in the wells of a multi-well plate are 
incubated with the labeled HPLMH, washed and any wells with labeled HPLMH 
complex are assayed. Data obtained using different concentrations of HPLMH 
are used to calculate values for the number, affinity, and association of 
HPLMH with the candidate molecules. 
All publications and patents mentioned in the above specification are 
herein incorporated by reference. Various modifications and variations of 
the described method and system of the invention will be apparent to those 
skilled in the art without departing from the scope and spirit of the 
invention. Although the invention has been described in connection with 
specific preferred embodiments, it should be understood that the invention 
as claimed should not be unduly limited to such specific embodiments. 
Indeed, various modifications of the described modes for carrying out the 
invention which are obvious to those skilled in molecular biology or 
related fields are intended to be within the scope of the following 
claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 6 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 92 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PROSNOT05 
(B) CLONE: 786812 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
#Cys Val Gly Leu Leu Thris Ile Leu Val Phe 
# 15 
#Asp Pro Phe Thr Tyr Asper Pro Lys Glu His 
# 30 
#Ile Ala Gly Ile Leu Phele Gly Gly Leu Val 
# 45 
#Arg Cys Arg Cys Lys Phele Val Leu Ser Arg 
# 60 
#Glu Glu Glu Gly Thr Phehr Gly Glu Pro Asp 
# 80 
#Arg Arger Ser Ile Arg Arg Leu Ser Thr Arg 
# 90 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 554 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: PROSNOT05 
(B) CLONE: 786812 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
#AGCAGGACGT 60GGGCCAG GGGGTCCAAA GTGCTCAGCC CCCGGGGCAC 
#TCTTGGCCAC 120TTCAGCA GGGGACAGCC CGATTGGGGA CAATGGCGTC 
#AAAGGAACAC 180GTGTGGG TCTCCTCACC ATGGCCAAGG CAGAAAGTCC 
#CGCCGGGATC 240ACGACTA CCAGTCCCTG CAGATCGGAG GCCTCGTCAT 
#GTTCAACCAG 300GCATCCT CATCGTGCTG AGCAGAAGAT GCCGGTGCAA 
#CATCCGCCGT 360GGGAACC CGATGAAGAG GAGGGAACTT TCCGCAGCTC 
#GACTKCCCTG 420GGCGGTA GAAACACCTG GAGCGATGGA ATCCGGCCAG 
#GCCCTTTCCC 480CCACGGT CCACCTGCGC GKCCACCKWY CCCTTCGNCG 
#TTTCTTTTGA 540CAGATTC CCCCTTGCCG CAAGGGTTTC CATAAAGTGG 
# 554 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 92 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 108084 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
#Cys Val Gly Phe Leu Thris Ile Leu Val Leu 
# 15 
#Asp Pro Phe Thr Tyr Aspla Pro Gln Glu His 
# 30 
#Ile Ala Gly Ile Leu Phele Gly Gly Leu Ile 
# 45 
#Arg Cys Arg Cys Lys Phele Val Leu Ser Arg 
# 60 
#Glu Glu Glu Gly Thr Phehr Gly Glu Pro Asp 
# 80 
#Arg Arger Ser Ile Arg Arg Leu Ser Thr Arg 
# 90 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 87 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 1085026 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
#Phe Leu Ala Gly Phe Proeu Gly Leu Leu Val 
# 15 
#Asn Ser Pro Phe Tyr Tyrsp Leu Glu Asp Lys 
# 30 
#Ile Cys Ala Gly Val Leuln Val Gly Gly Leu 
# 45 
#Ala Lys Cys Lys Cys Lysle Ile Val Met Ser 
# 60 
#Glu Thr Pro Pro Leu Ilely His His Pro Gly 
# 80 
- Thr Pro Gly Ser Ala Gln Ser 
85 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 87 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 951423 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
#Val Leu Ala Gly Leu Proys Ala Phe Leu Leu 
# 15 
#Gly Ser Pro Phe Tyr Tyrly Pro Val Asp Lys 
# 30 
#Ile Phe Gly Gly Leu Leuln Leu Gly Gly Met 
# 45 
#Gly Lys Cys Lys Cys Argla Met Ala Leu Ser 
# 60 
#Lys Val Thr Pro Leu Ileer Ser Leu Pro Glu 
# 80 
- Thr Pro Gly Ser Ala Ser Thr 
85 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 58 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 51112 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
#Phe Glu Tyr Asp Tyr Gluly Thr Glu Asn Pro 
# 15 
#Gly Leu Ala Phe Val Vally Leu Ile Phe Ala 
# 30 
#Arg Cys Gly Gly Gly Lyseu Ser Lys Arg Phe 
# 45 
- Lys His Arg Gln Val Asn Glu Asp Glu Leu 
# 55 
__________________________________________________________________________