DNA encoding a novel human proteolipid

The present invention provides polynucleotides which identify and encode a novel human proteolipid (PLHu). The invention provides for genetically engineered expression vectors and host cells comprising the nucleic acid sequence encoding PLHu. The invention also provides for the use of substantially purified PLHu and its agonists in the commercial production of recombinant proteins for the treatment of diseases associated with the expression of PLHu. Additionally, the invention provides for the use of antisense molecules to PLHu in the treatment of diseases associated with the expression of PLHu. The invention also describes diagnostic assays which utilize diagnostic compositions comprising the polynucleotides which hybridize with naturally occurring sequences encoding PLHu and antibodies which specifically bind to the protein.

FIELD OF THE INVENTION 
The present invention relates to nucleic acid and amino acid sequences of a 
novel human proteolipid and to the use of these sequences in the 
diagnosis, study, prevention and treatment of disease. 
BACKGROUND OF THE INVENTION 
Proteolipids are a class of hydrophobic membrane proteins characterized in 
part by their capacity to assume conformations compatible with solubility 
in organic solvents and in water (Sapirstein V. S. et al (1983) 
Biochemistry 22:3330-3335). This amphipathic character of proteolipids 
explains their participation in transmembrane ion movement. Proteolipids 
are components of ion channel and transport systems, such as H.sup.+ 
channels (Arai H. et al (1987) J Biol Chem 262:11006-11011), Ca.sup.2+ 
channels (Eytan G. D. et al (1977) J Biol Chem 252: 3208-3213) and the C 
(membrane channel) subunit of the vacuolar H.sup.+ -ATPase (Nelson H. et 
al (1990) J Biol Chem 265: 20390-20393). 
The latter proteolipid, also known as ductin, is also associated with gap 
junctions. Gap junctions are the relatively large pores which allow free 
diffusion of ions across biological membranes (Finbow M. E. et al (1995) 
Bioessays 17:247-255). Altered gap-junction intercellular communication 
(GJIC) may play an essential role in cancer development. A lack of GJIC 
has been observed between transformed and neighboring normal cells (Trosko 
et al (1990) Radiation Res 123:241-251). A decrease in GJIC has also been 
observed within tumor cells (Krutovskikh et al (1991) Carcinogenesis 
12:1701-1706). 
Proteolipids are also involved in membrane vesicular trafficking. Due to 
their lipid-like properties, proteolipids destabilize lipid bilayers and 
promote membrane vesicle fusion. Such proteolipid-assisted events may 
include the fusions and fissions of the nuclear membrane, endoplasmic 
reticulum, Golgi apparatus, and various inclusion bodies (peroxisomes, 
lysosomes, etc). 
Human T-lymphocyte maturation-associated protein (MAL), a 153 amino acid 
proteolipid, has been localized to the endoplasmic reticulum (ER) of 
T-lymphocytes, where it mediates the fusion of ER-derived vesicles and 
Golgi cisterna (Rancano C. et al (1994) J Biol Chem 269:8159-8164). A 
canine MAL homolog, VIP17, is involved in the sorting and targeting of 
proteins between the Golgi complex and the apical plasma membrane 
(Zacchetti D. et al (1995) FEBS Lett 377:465-469). A rat MAL homolog, 
rMAL, is expressed in the myelinating cells of the nervous system 
including oligodendrocytes and Schwann cells. The rMAL protein serves as a 
gap junction component and plays a role in myelin compaction 
(Schaeren-Wiemers N. et al (1995) J. Neurosci 5753-5764). 
Plasmolipin from rat is a proteolipid localized to plasma membranes in 
kidney and brain. It has 157 amino acids and, based on hydropathy plots 
and secondary structure predictions, consists of four alpha-helical 
transmembrane domains (I through IV) of 20-22 amino acids in length. 
Transmembrane domains III and IV contain hydroxyl groups which may 
contribute to an aqueous channel. Domains I through III are connected by 
short hydrophilic segments of 9-11 amino acids in length, and domains III 
and IV are connected by a longer hydrophilic segment of 20 amino acids. 
The small size and high hydrophobicity of plasmolipin constrains the 
distribution of its transmembrane regions such that the four transmembrane 
alpha-helices form an antiparallel bundle, and both the amino- and 
carboxy-termini face the cytoplasm. This structural model defines the 
growing class of small hydrophobic transport-related proteolipids 
containing four-helix transmembrane segments, such as the MAL homologs 
(Rancano et al, supra), and the vacuolar H.sup.+ -ATPase C subunit (Nelson 
et al, supra). 
In rat brain, plasmolipin is localized to myelinated nerve tracts, and its 
expression increases markedly with the onset of myelination (Fischer I. et 
al (1991) Neurochem Res 28:81-89). The distribution of plasmolipin within 
myelin appears to include regions active in membrane recycling. 
Endocytotic coated vesicles isolated from myelinated tracts are enriched 
with plasmolipin (Sapirstein V. S. (1994) J Neurosci Res 37:348-358). 
Incorporation of the purified rat plasmolipin protein into lipid bilayers 
induces voltage-dependent K.sup.+ channel formation, suggesting it may 
function in vivo as a pore or channel (Tosteson M. T. et al (1981) J Membr 
Biol 63:77-84). Channel formation involved the trimerization of the 
plasmolipin molecule. The oligomerization model of the plasmolipin 
molecule portrays transmembrane domains III and IV as walls of the 
channel, consistent with the presence of hydroxyl groups in these domains 
(Sapirstein et al (1983) supra). The putative role of rat plasmolipin in 
transport suggests its function may be in the fluid volume regulation of 
the myelin complex (Fischer et al (1994), supra). 
Proteolipids are involved in membrane trafficking, gap junction formation, 
ion transport and cellular fluid volume regulation. The selective 
modulation of their expression may provide a means for the regulation of 
vesicle trafficking or the formation of channels or gap junctions in 
normal as well as acute and chronic disease situations. 
SUMMARY OF THE INVENTION 
The present invention discloses a novel human proteolipid, hereinafter 
referred to as PLHu, having homology to plasmolipin from rat (Rattus 
norvegicus). Accordingly, the invention features a substantially purified 
proteolipid, encoded by amino acid sequence of SEQ ID NO:1, having 
structural characteristics of the class of proteolipids including 
plasmolipin. 
One aspect of the invention features isolated and substantially purified 
polynucleotides which encode PLHu. In a particular aspect, the 
polynucleotide is the nucleotide sequence of SEQ ID NO:2. In addition, the 
invention features nucleotide sequences which hybridize under stringent 
conditions to SEQ ID NO:2. 
The invention further relates to nucleic acid sequence encoding PLHu, 
oligonucleotides, peptide nucleic acids (PNA), fragments, portions or 
antisense molecules thereof. The present invention also relates to an 
expression vector which includes polynucleotide encoding PLHu and its use 
to transform host cells or organisms. The invention also relates to 
antibodies which bind specifically to the proteolipid of SEQ ID NO:1 and 
to a pharmaceutical composition comprising a substantially purified 
proteolipid of SEQ ID NO:1.

DETAILED DESCRIPTION OF THE 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 peptide or protein sequence. 
"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 
(template) strand of nucleic acid (Nielsen P. E. et al (1993) Anticancer 
Drug Des 8:53-63). 
A "variant" of PLHu is defined as an amino acid sequence that is different 
by one or more amino acid "substitutions". The variant may have 
"conservative" changes, wherein a substituted amino acid has similar 
structural or chemical properties, eg, replacement of leucine with 
isoleucine. More rarely, a variant may have "nonconservative" changes, eg, 
replacement of a glycine with a tryptophan. Similar minor variations may 
also include amino acid deletions or insertions, or both. Guidance in 
determining which and how many amino acid residues may be substituted, 
inserted or deleted without abolishing biological or immunological 
activity may be found using computer programs well known in the art, for 
example, DNAStar software. 
The term "biologically active" refers to a PLHu having structural, 
regulatory or biochemical functions of the naturally occurring PLHu. 
Likewise, "immunologically active" defines the capability of the natural, 
recombinant or synthetic PLHu, or any oligopeptide thereof, to induce a 
specific immune response in appropriate animals or cells and to bind with 
specific antibodies. 
The term "derivative" as used herein refers to the chemical modification of 
a nucleic acid encoding PLHu or the encoded PLHu. 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 natural PLHu. 
As used herein, the term "substantially purified" refers to molecules, 
either 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. 
"Stringency" typically occurs in a range from about Tm-5.degree. C. 
(5.degree. C. below the 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, a stringency hybridization can be used to identify or detect 
identical polynucleotide sequences or to identify or detect similar or 
related polynucleotide sequences. 
The term "hybridization" as used herein shall include "any process by which 
a strand of nucleic acid joins with a complementary strand through base 
pairing" (Coombs J. (1994) Dictionary of Biotechnology, Stockton Press, 
New York N.Y.). Amplification as carried out in the polymerase chain 
reaction technologies is described in Dieffenbach C. W. and G. S. Dveksler 
(1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, 
Plainview N.Y.). 
A "deletion" is defined as a change in either nucleotide or amino acid 
sequence in which one or more nucleotides or amino acid residues, 
respectively, are absent. 
An "insertion" or "addition" is that change in a nucleotide or amino acid 
sequence which has resulted in the addition of one or more nucleotides or 
amino acid residues, respectively, as compared to the naturally occurring 
PLHu. 
A "substitution" results from the replacement of one or more nucleotides or 
amino acids by different nucleotides or amino acids, respectively. 
Description 
The present invention relates to a novel human proteolipid, PLHu, initially 
identified among the partial cDNAs from a human breast library (BRSTNOT03) 
and to the use of the nucleic acid and amino acid sequences disclosed 
herein in the study, diagnosis, prevention and treatment of disease. 
Northern analysis using the LIFESEQ.TM. database (Incyte Pharmaceuticals, 
Palo Alto Calif.) indicates that PLHu-encoding nucleotide sequences are 
also transcribed in heart atrial tissue and in lymphocytes. 
The present invention also encompasses PLHu variants. A preferred PLHu 
variant is one having at least 80% amino acid sequence similarity to the 
PLHu amino acid sequence (SEQ ID NO:1), a more preferred PLHu variant is 
one having at least 90% amino acid sequence similarity to SEQ ID NO:1and a 
most preferred PLHu variant is one having at least 95% amino acid sequence 
similarity to SEQ ID NO:1. 
The nucleic acid sequence encoding a portion of PLHu was first identified 
in the cDNA, Incyte Clone 640699, through a computer-generated search for 
amino acid sequence alignments. The nucleic acid sequence, SEQ ID NO:2, 
disclosed herein (FIGS. 1A and 1B) encodes the amino acid sequence, SEQ ID 
NO:1, designated PLHu. The present invention is based in part on the 
structural homology shown in FIG. 2, among PLHu and other small 
proteolipids including rat plasmolipin (GI 1346732; Fischer et al (1994), 
supra) and human MAL (GI 126719; Rancano et al, supra). 
PLHu consists of 153 amino acids and, based on the hydropathy plot (FIG. 3) 
and secondary structure predictions, is a member of the class of small 
hydrophobic transport-related proteolipids containing four alpha-helical 
transmembrane domains. From its homology to rat plasmolipin (FIG. 2), the 
transmembrane domains I to IV of PLHu are predicted to span residues 
20-40, 50-72, 84-108, and 127-147, respectively. Transmembrane domains III 
and IV of PLHu contain a total of six ser/thr hydroxyl groups, which may 
form an aqueous channel. The N-terminal hydrophilic segment of PLHu is 20 
amino acids, longer than the ten amino acid N-terminal segment of rat 
plasmolipin, and the C-terminal hydrophilic segment of PLHu is 6 amino 
acids, shorter than the 17 amino acid C-terminal segment of rat 
plasmolipin. In accordance with the structural model proposed for other 
small proteolipids, both the amino- and carboxy-termini of PLHu are 
predicted to face the cytoplasm. The short hydrophilic segments connecting 
domains I to II and domains II to III are 9 and 11 amino acids in length, 
respectively. The hydrophilic segment connecting domains III to IV is 18 
amino acids in length. The human plasmolipin PLHu has 43% sequence 
identity with rat plasmolipin and 28% sequence identity with human MAL 
(FIG. 2). 
The PLHu Coding Sequences 
The nucleic acid and amino acid sequences of PLHu are shown in FIGS. 1A and 
1B. In accordance with the invention, any nucleic acid sequence which 
encodes the amino acid sequence of PLHu can be used to generate 
recombinant molecules which express PLHu. In a specific embodiment 
described herein, a partial sequence of PLHu was first isolated as Incyte 
Clone 640699 from a human breast tissue cDNA library (BRSTNOT03). 
It will be appreciated by those skilled in the art that as a result of the 
degeneracy of the genetic code, a multitude of PLHu-encoding nucleotide 
sequences, some bearing minimal homology to the nucleotide sequences of 
any known and naturally occurring gene may be produced. 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 
PLHu, and all such variations are to be considered as being specifically 
disclosed. 
Although nucleotide sequences which encode PLHu and its variants are 
preferably capable of hybridizing to the nucleotide sequence of the 
naturally occurring PLHu under appropriately selected conditions of 
stringency, it may be advantageous to produce nucleotide sequences 
encoding PLHu 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 PLHu 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. 
It is now possible to produce a DNA sequence, or portions thereof, encoding 
a PLHu and its derivatives entirely by synthetic chemistry, after which 
the synthetic gene may be inserted into any of the many available DNA 
vectors and cell systems using reagents that are well known in the art at 
the time of the filing of this application. Moreover, synthetic chemistry 
may be used to introduce mutations into a gene encoding PLHu. 
Also included within the scope of the present invention are polynucleotide 
sequences that are capable of hybridizing to the nucleotide sequence of 
FIGS. 1A and 1B under various conditions of stringency. Hybridization 
conditions are based on the melting temperature (Tm) of the nucleic acid 
binding complex or probe, as taught in Berger and Kimmel (1987, Guide to 
Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic 
Press, San Diego Calif.) incorporated herein by reference, and confer may 
be used at a defined stringency. 
Altered nucleic acid sequences encoding PLHu which may be used in 
accordance with the invention include deletions, insertions or 
substitutions of different nucleotides resulting in a polynucleotide that 
encodes the same or a functionally equivalent PLHu. The protein may also 
show deletions, insertions or substitutions of amino acid residues which 
produce a silent change and result in a functionally equivalent PLHu. 
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 PLHu is retained. For example, negatively charged amino acids include 
aspartic acid and glutamic acid; positively charged amino acids include 
lysine and arginine; and amino acids with uncharged polar head groups 
having similar hydrophilicity values include leucine, isoleucine, valine; 
glycine, alanine; asparagine, glutamine; serine, threonine phenylalanine, 
and tyrosine. 
Included within the scope of the present invention are alleles of PLHu. As 
used herein, an "allele" or "allelic sequence" is an alternative form of 
PLHu. Alleles result from a mutation, ie, a change in the nucleic acid 
sequence, and generally produce altered mRNAs or polypeptides whose 
structure or function may or may not be altered. Any given gene may have 
none, one or many allelic forms. Common mutational changes which give rise 
to alleles are generally ascribed to natural deletions, additions or 
substitutions of amino acids. Each of these types of changes may occur 
alone, or in combination with the others, one or more times in a given 
sequence. 
Methods for DNA sequencing are well known in the art and employ such 
enzymes as the Klenow fragment of DNA polymerase I, Sequenase.RTM. (U.S. 
Biochemical Corp, Cleveland Ohio)), Taq polymerase (Perkin Elmer, Norwalk 
Conn.), 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). 
Extending the Polynucleotide Sequence 
The polynucleotide sequence encoding PLHu may be extended utilizing partial 
nucleotide sequence and various methods known in the art to detect 
upstream sequences such as promoters and regulatory elements. Gobinda et 
al (1993; PCR Methods Applic 2:318-22) disclose "restriction-site" 
polymerase chain reaction (PCR) as a direct method which uses universal 
primers to retrieve unknown sequence adjacent to a known locus. First, 
genomic DNA is amplified in the presence of primer to a linker sequence 
and a primer specific to the known region. The amplified sequences are 
subjected to a second round of PCR with the same linker primer and another 
specific primer internal to the first one. Products of each round of PCR 
are transcribed with an appropriate RNA polymerase and sequenced using 
reverse transcriptase. 
Inverse PCR can be used to amplify or extend sequences using divergent 
primers based on a known region (Triglia T. et al (1988) Nucleic Acids Res 
16:8186). The primers may be designed using OLIGO.RTM. 4.06 Primer 
Analysis Software (1992; National Biosciences Inc, Plymouth Minn.), or 
another appropriate program, to be 22-30 nucleotides in length, to have a 
GC content of 50% or more, and to anneal to the target sequence at 
temperatures about 68.degree.-72.degree. C. The method uses several 
restriction enzymes to generate a suitable fragment in the known region of 
a gene. The fragment is then circularized by intramolecular ligation and 
used as a PCR template. 
Capture PCR (Lagerstrom M. et al (1991) PCR Methods Applic 1:111-19) is a 
method for PCR amplification of DNA fragments adjacent to a known sequence 
in human and yeast artificial chromosome DNA. Capture PCR also requires 
multiple restriction enzyme digestions and ligations to place an 
engineered double-stranded sequence into an unknown portion of the DNA 
molecule before PCR. 
Another method which may be used to retrieve unknown sequences is that of 
Parker J. D. et al (1991; Nucleic Acids Res 19:3055-60). Additionally, one 
can use PCR, nested primers and PromoterFinder libraries to walk in 
genomic DNA (PromoterFinder.TM. Clontech (Palo Alto Calif.). This process 
avoids the need to screen libraries and is useful in finding intron/exon 
junctions. 
Preferred libraries for screening for full length cDNAs are ones that have 
been size-selected to include larger cDNAs. Also, random primed libraries 
are preferred in that they will contain more sequences which contain the 
5' and upstream regions of genes. A randomly primed library may be 
particularly useful if an oligo d(T) library does not yield a full-length 
cDNA. Genomic libraries are useful for extension into the 5' nontranslated 
regulatory region. 
Capillary electrophoresis may be used to analyze the size or confirm the 
nucleotide sequence of sequencing or PCR products. Systems for rapid 
sequencing are available from Perkin Elmer, Beckman Instruments (Fullerton 
Calif.), and other companies. Capillary sequencing may employ flowable 
polymers for electrophoretic separation, four different fluorescent dyes 
(one for each nucleotide) activated, and deactivated, and detection of the 
emitted wavelengths by a charge coupled devise camera. Output/light 
intensity is converted to electrical signal using appropriate software 
(eg. Genotyper.TM. and Sequence Navigator.TM. from Perkin Elmer) and the 
entire process from loading of samples to computer analysis and electronic 
data display is computer controlled. Capillary electrophoresis is 
particularly suited to the sequencing of small pieces of DNA which might 
be present in limited amounts in a particular sample. The reproducible 
sequencing of up to 350 bp of M13 phage DNA in 30 min has been reported 
(Ruiz-Martinez M. C. et al (1993) Anal Chem 65:2851-8). 
Expression of the Nucleotide Sequence 
In accordance with the present invention, polynucleotide sequences which 
encode PLHu, fragments of the polypeptide, fusion proteins or functional 
equivalents thereof may be used in recombinant DNA molecules that direct 
the expression of PLHu in appropriate host cells. Due to the inherent 
degeneracy of the genetic code, other DNA sequences which encode 
substantially the same or a functionally equivalent amino acid sequence, 
may be used to clone and express PLHu. As will be understood by those of 
skill in the art, it may be advantageous to produce PLHu-encoding 
nucleotide sequences possessing non-naturally occurring codons. Codons 
preferred by a particular prokaryotic or eukaryotic host (Murray E. et al 
(1989) Nuc Acids Res 17:477-508) can be selected, for example, to increase 
the rate of PLHu expression or to produce recombinant RNA transcripts 
having desirable properties, such as a longer half-life, than transcripts 
produced from naturally occurring sequence. 
The nucleotide sequences of the present invention can be engineered in 
order to alter a coding sequence of PLHu for a variety of reasons, 
including but not limited to, alterations which modify the cloning, 
processing and/or expression of the gene product. For example, mutations 
may be introduced using techniques which are well known in the art, eg, 
site-directed mutagenesis to insert new restriction sites, to alter 
glycosylation patterns, to change codon preference, to produce splice 
variants, etc. 
In another embodiment of the invention, a natural, modified or recombinant 
nucleotide sequence encoding PLHu may be ligated to a heterologous 
sequence to encode a fusion protein. For example, for screening of peptide 
libraries for inhibitors of PLHu activity, it may be useful to encode a 
chimeric PLHu protein that is recognized by a commercially available 
antibody. A fusion protein may also be engineered to contain a cleavage 
site located between a PLHu sequence and the heterologous protein 
sequence, so that the PLHu may be cleaved and substantially purified away 
from the heterologous moiety. 
In an alternate embodiment of the invention, the coding sequence for PLHu 
may be synthesized, whole or in part, using chemical methods well known in 
the art (see Caruthers M. H. et al (1980) Nuc Acids Res Symp Ser 215-23, 
Horn T. et al (1980) Nuc Acids Res Symp Ser 225-32, etc). Alternatively, 
the protein itself could be produced using chemical methods to synthesize 
a PLHu amino acid sequence, whole or in part. For example, peptide 
synthesis can be performed using various solid-phase techniques (Roberge 
J. Y. et al (1995) Science 269:202-204) and automated synthesis may be 
achieved, for example, using the ABI 431A Peptide Synthesizer (Perkin 
Elmer) in accordance with the instructions provided by the manufacturer. 
The newly synthesized peptide can be substantially purified by preparative 
high performance liquid chromatography (eg, Creighton (1983) Proteins, 
Structures and Molecular Principles, W. H. Freeman and Co, New York N.Y.). 
The composition of the synthetic peptides may be confirmed by amino acid 
analysis or sequencing (eg, the Edman degradation procedure; Creighton, 
supra). Additionally the amino acid sequence of PLHu, or any part thereof, 
may be altered during direct synthesis and/or combined using chemical 
methods with sequences from other proteins, or any part thereof, to 
produce a variant polypeptide. 
Expression Systems 
In order to express a biologically active PLHu, the nucleotide sequence 
encoding PLHu or its functional equivalent, is inserted into an 
appropriate expression vector, ie, a vector which contains the necessary 
elements for the transcription and translation of the inserted coding 
sequence. 
Methods which are well known to those skilled in the art can be used to 
construct expression vectors containing a PLHu coding sequence and 
appropriate transcriptional or translational controls. These methods 
include in vitro recombinant DNA techniques, synthetic techniques and in 
vivo recombination or genetic recombination. Such techniques are described 
in Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold 
Spring Harbor Press, Plainview N.Y. and Ausubel F. M. et al (1989) Current 
Protocols in Molecular Biology, John Wiley & Sons, New York N.Y. 
A variety of expression vector/host systems may be utilized to contain and 
express a PLHu coding sequence. These include but are not limited to 
microorganisms such as bacteria transformed with recombinant 
bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed 
with yeast expression vectors; insect cell systems infected with virus 
expression vectors (eg, baculovirus); plant cell systems transfected with 
virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco 
mosaic virus, TMV) or transformed with bacterial expression vectors (eg, 
Ti or pBR322 plasmid); or animal cell systems. 
The "control elements" or "regulatory sequences" of these systems vary in 
their strength and specificities and are those nontranslated regions of 
the vector, enhancers, promoters, and 3' untranslated regions, which 
interact with host cellular proteins to carry out transcription and 
translation. Depending on the vector system and host utilized, any number 
of suitable transcription and translation elements, including constitutive 
and inducible promoters, may be used. For example, when cloning in 
bacterial systems, inducible promoters such as the hybrid lacZ promoter of 
the Bluescript.RTM. phagemid (Stratagene, LaJolla Calif.) or pSport1 
(Gibco BRL) and ptrp-lac hybrids and the like may be used. The baculovirus 
polyhedrin promoter may be used in insect cells. Promoters or enhancers 
derived from the genomes of plant cells (eg, heat shock, RUBISCO; and 
storage protein genes) or from plant viruses (eg, viral promoters or 
leader sequences) may be cloned into the vector. In mammalian cell 
systems, promoters from the mammalian genes or from mammalian viruses are 
most appropriate. If it is necessary to generate a cell line that contains 
multiple copies of PLHu, 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 PLHu. For example, when large 
quantities of PLHu are needed for the induction of antibodies, vectors 
which direct high level expression of fusion proteins that are readily 
purified may be desirable. Such vectors include, but are not limited to, 
the multifunctional E. coli cloning and expression vectors such as 
Bluescript.RTM. (Stratagene), in which the PLHu coding sequence may be 
ligated into the vector in frame with sequences for the amino-terminal Met 
and the subsequent 7 residues of .beta.-galactosidase so that a hybrid 
protein is produced; pIN vectors (Van Heeke & Schuster (1989) J Biol Chem 
264:5503-5509); and the like. pGEX vectors (Promega, Madison Wis.) may 
also be used to express foreign polypeptides as fusion proteins with 
glutathione S-transferase (GST). In general, such fusion proteins are 
soluble and can easily be purified from lysed cells by adsorption to 
glutathione-agarose beads followed by elution in the presence of free 
glutathione. Proteins made in such systems are designed to include 
heparin, thrombin or factor XA protease cleavage sites so that the cloned 
polypeptide of interest can be released from the GST moiety at will. 
In the yeast, Saccharomyces cerevisiae, a number of vectors containing 
constitutive or inducible promoters such as alpha factor, alcohol oxidase 
and PGH may be used. For reviews, see Ausubel et al (supra) and Grant et 
al (1987) Methods in Enzymology 153:516-544. 
In cases where plant expression vectors are used, the expression of a 
sequence encoding PLHu may be driven by any of a number of promoters. For 
example, viral promoters such as the 35S and 19S promoters of CaMV 
(Brisson et al (1984) Nature 310:511-514) may be used alone or in 
combination with the omega leader sequence from TMV (Takamatsu et al 
(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the 
small subunit of RUBISCO (Coruzzi et al (1984) EMBO J. 3:1671-1680; 
Broglie et al (1984) Science 224:838-843); or heat shock promoters (Winter 
J. and Sinibaldi R. M. (1991) Results Probl Cell Differ 17:85-105) may be 
used. These constructs can be introduced into plant cells by direct DNA 
transformation or pathogen-mediated transfection. For reviews of such 
techniques, see Hobbs S. or Murry L. E. in McGraw Hill Yearbook of Science 
and Technology (1992) McGraw Hill New York N.Y., pp 191-196 or Weissbach 
and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, 
New York N.Y., pp 421-463. 
An alternative expression system which could be used to express PLHu is an 
insect system. In one such system, Autographa californica nuclear 
polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in 
Spodoptera frugiperda cells or in Trichoplusia larvae. The PLHu coding 
sequence may be cloned into a nonessential region of the virus, such as 
the polyhedrin gene, and placed under control of the polyhedrin promoter. 
Successful insertion of the PLHu coding sequence will render the 
polyhedrin gene inactive and produce recombinant virus lacking coat 
protein coat. The recombinant viruses are then used to infect S. 
frugiperda cells or Trichoplusia larvae in which PLHu is expressed (Smith 
et al (1983) J Virol 46:584; Engelhard E. K. et al (1994) Proc Nat Acad 
Sci 91:3224-7). 
In mammalian host cells, a number of viral-based expression systems may be 
utilized. In cases where an adenovirus is used as an expression vector, a 
coding sequence for PLHu may be ligated into an adenovirus 
transcription/translation complex consisting of the late promoter and 
tripartite leader sequence. Insertion in a nonessential E1 or E3 region of 
the viral genome will result in a viable virus capable of expressing PLHu 
in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 
81:3655-59). In addition, transcription enhancers, such as the rous 
sarcoma virus (RSV) enhancer, may be used to increase expression in 
mammalian host cells. 
Specific initiation signals may also be required for efficient translation 
of a PLHu sequence. These signals include the ATG initiation codon and 
adjacent sequences. In cases where nucleic acid encoding PLHu, its 
initiation codon and upstream sequences are inserted into the appropriate 
expression vector, no additional translational control signals may be 
needed. However, in cases where only coding sequence, or a portion 
thereof, is inserted, exogenous transcriptional control signals including 
the ATG initiation codon must be provided. Furthermore, the initiation 
codon must be in the correct reading frame to ensure transcription of the 
entire insert. Exogenous transcriptional elements and initiation codons 
can be of various origins, both natural and synthetic. The efficiency of 
expression may be enhanced by the inclusion of enhancers appropriate to 
the cell system in use (Scharf D. et al (1994) Results Probl Cell Differ 
20:125-62; Bittner et al (1987) Methods in Enzymol 153:516-544). 
In addition, a host cell strain may be chosen for its ability to modulate 
the expression of the inserted sequences or to process the expressed 
protein in the desired fashion. Such modifications of the polypeptide 
include, but are not limited to, acetylation, carboxylation, 
glycosylation, phosphorylation, lipidation and acylation. 
Post-translational processing which cleaves a "prepro" form of the protein 
may also be important for correct insertion, folding and/or function. 
Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc have specific 
cellular machinery and characteristic mechanisms for such 
post-translational activities and may be chosen to ensure the correct 
modification and processing of the introduced, foreign protein. 
For long-term, high-yield production of recombinant proteins, stable 
expression is preferred. For example, cell lines which stably express PLHu 
may be transformed using expression vectors which contain viral origins of 
replication or endogenous expression elements and a selectable marker 
gene. Following the introduction of the vector, cells may be allowed to 
grow for 1-2 days in an enriched media before they are switched to 
selective media. The purpose of the selectable marker is to confer 
resistance to selection, and its presence allows growth and recovery of 
cells which successfully express the introduced sequences. Resistant 
clumps of stably transformed cells can be proliferated using tissue 
culture techniques appropriate to the cell type. 
Any number of selection systems may be used to recover transformed cell 
lines. These include, but are not limited to, the herpes simplex virus 
thymidine kinase (Wigler M. et al (1977) Cell 11:223-32) and adenine 
phosphoribosyltransferase (Lowy I. et al (1980) Cell 22:817-23) genes 
which can be employed in tk- or aprt- cells, respectively. Also, 
antimetabolite, antibiotic or herbicide resistance can be used as the 
basis for selection; for example, dhfr which confers resistance to 
methotrexate (Wigler M. et al (1980) Proc Natl Acad Sci 77:3567-70); npt, 
which confers resistance to the aminoglycosides neomycin and G-418 
(Colbere-Garapin F. et al (1981) J Mol Biol 150:1-14) and als or pat, 
which confer resistance to chlorsulfuron and phosphinotricin 
acetyltransferase, respectively (Murry, supra). Additional selectable 
genes have been described, for example, trpB, which allows cells to 
utilize indole in place of tryptophan, or hisD, which allows cells to 
utilize histinol in place of histidine (Hartman S. C. and R. C. Mulligan 
(1988) Proc Natl Acad Sci 85:8047-51). Recently, the use of visible 
markers has gained popularity with such markers as anthocyanins, .beta. 
glucuronidase and its substrate, GUS, and luciferase and its substrate, 
luciferin, being widely used not only to identify transformants, but also 
to quantify the amount of transient or stable protein expression 
attributable to a specific vector system (Rhodes C. A. et al (1995) 
Methods Mol Biol 55:121-131). 
Identification of Transformants Containing the Polynucleotide Sequence 
Although the presence/absence of marker gene expression suggests that the 
gene of interest is also present, its presence and expression should be 
confirmed. For example, if the PLHu polynucleotide sequence is inserted 
within a marker gene sequence, recombinant cells containing PLHu can be 
identified by the absence of marker gene function. Alternatively, a marker 
gene can be placed in tandem with a PLHu 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 PLHu as well. 
Alternatively, host cells which contain the coding sequence for PLHu and 
express PLHu may be identified by a variety of procedures known to those 
of skill in the art. These procedures include, but are not limited to, 
DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay 
techniques which include membrane, solution, or chip based technologies 
for the detection and/or quantification of the nucleic acid or protein. 
The presence of the polynucleotide sequence encoding PLHu can be detected 
by DNA-DNA or DNA-RNA hybridization or amplification using probes, 
portions or fragments of PLHu-encoding nucleotides. Nucleic acid 
amplification based assays involve the use of oligonucleotides or 
oligomers based on the PLHu sequence to detect transformants containing 
PLHu DNA or RNA. As used herein "oligonucleotides" or "oligomers" refer to 
a nucleic acid sequence of at least about 10 nucleotides and as many as 
about 60 nucleotides, preferably about 15 to 30 nucleotides, and more 
preferably about 20-25 nucleotides which can be used as a probe or 
amplimer. 
A variety of protocols for detecting and measuring the expression of PLHu, 
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 PLHu is preferred, 
but a competitive binding assay may be employed. These and other assays 
are described, among other places, in Hampton R. et al (1990, Serological 
Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox D. E. 
et al (1983, J Exp Med 158:1211). 
A wide variety of labels and conjugation techniques are known by those 
skilled in the art and can be used in various nucleic acid and amino acid 
assays. Means for producing labeled hybridization or PCR probes for 
detecting sequences related to PLHu include oligolabeling, nick 
translation, end-labeling or PCR amplification using a labeled nucleotide. 
Alternatively, the PLHu sequence, or any portion of it, may be cloned into 
a vector for the production of an mRNA probe. Such vectors are known in 
the art, are commercially available, and may be used to synthesize RNA 
probes in vitro by addition of an appropriate RNA polymerase such as T7, 
T3 or SP6 and labeled nucleotides. 
A number of companies such as Pharmacia Biotech (Piscataway N.J.), Promega 
(Madison Wis.), and U.S. Biochemical Corp (Cleveland Ohio) supply 
commercial kits and protocols for these procedures. Suitable reporter 
molecules or labels include those radionuclides, enzymes, fluorescent, 
chemiluminescent, or chromogenic agents as well as substrates, cofactors, 
inhibitors, magnetic particles and the like. Patents teaching the use of 
such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 
3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant 
immunoglobulins may be produced as shown in U.S. Pat. No. 4,816,567 
incorporated herein by reference. 
Purification of PLHu 
Host cells transformed with a PLHu-encoding nucleotide sequence may be 
cultured under conditions suitable for the expression and recovery of the 
encoded protein from cell culture. The protein produced by a recombinant 
cell may be contained intracellularly or secreted depending on the 
sequence and/or the vector used. As will be understood by those of skill 
in the art, expression vectors containing PLHu can be designed for 
efficient production and proper transmembrane insertion of PLHu into a 
prokaryotic or eukaryotic cell membrane. Other recombinant constructions 
may join PLHu to nucleotide sequence encoding a polypeptide domain which 
will facilitate purification of soluble proteins (Kroll D. J. et al (1993) 
DNA Cell Biol 12:441-53; cf discussion of vectors infra containing fusion 
proteins). 
PLHu may also be expressed as a recombinant protein with one or more 
additional polypeptide domains added to facilitate protein purification. 
Such purification facilitating domains include, but are not limited to, 
metal chelating peptides such as histidine-tryptophan modules that allow 
purification on immobilized metals, protein A domains that allow 
purification on immobilized immunoglobulin, and the domain utilized in the 
FLAGS extension/affinity purification system (Immunex Corp, Seattle 
Wash.). The inclusion of a cleavable linker sequences such as Factor XA or 
enterokinase (Invitrogen, San Diego Calif.) between the purification 
domain and PLHu is useful to facilitate purification. One such expression 
vector provides for expression of a fusion protein compromising an PLHu 
and contains 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 the proteolipid from the fusion protein. 
In addition to recombinant production, fragments of PLHu may be produced by 
direct peptide synthesis using solid-phase techniques (cf Stewart et al 
(1969) Solid-Phase Peptide Synthesis, W. H. Freeman Co, San Francisco; 
Merrifield J. (1963) J Am Chem Soc 85:2149-2154). In vitro protein 
synthesis may be performed using manual techniques or by automation. 
Automated synthesis may be achieved, for example, using Applied Biosystems 
431A Peptide Synthesizer (Perkin Elmer, Foster City Calif.) in accordance 
with the instructions provided by the manufacturer. Various fragments of 
PLHu may be chemically synthesized separately and combined using chemical 
methods to produce the full length molecule. 
Uses of PLHu 
The rationale for the use of polynucleotide and polypeptide sequences 
disclosed herein is based in part on the structural homology among the 
novel PLHu and small proteolipids including rat plasmolipin and human MAL. 
Exocytosis facilitated by PLHu may influence membrane trafficking within 
the cell and affect the release of chemokines involved in cell migration, 
proteases which are active in inflammation or other similar activities 
involving endothelial cells, fibroblasts, lymphocytes, etc. Therefore, a 
diagnostic test for altered expression of PLHu can accelerate diagnosis 
and proper treatment of conditions caused by viral or other infections, 
traumatic tissue damage, hereditary diseases such as arthritis or asthma, 
invasive leukemias and lymphomas; or other physiologic/pathologic problems 
associated with abnormal membrane trafficking. 
Gap junctions are important in regulating metabolic communication and 
cooperation among cells. Altered gap-junction intercellular communication 
(GJIC) may play an essential role in cancer development. A lack of GJIC 
has been observed between transformed and neighboring normal cells. In 
addition, a decrease in GJIC has also been observed within tumor cells. A 
diagnostic test for decreased PLHu expression may therefore correlate with 
tumorigenesis. 
In cardiac muscle fibers, gap junctions permit the unimpeded diffusion of 
ions from one cell to another. Thus cardiac cells are so interconnected 
that, for example, when one atrial cell becomes excited, the action 
potential quickly spreads to all of the cells in the atrial syncytium. A 
diagnostic test for abnormal PLHu expression in heart may be correlated 
with abnormal propagation of action potentials in aging cardiac muscle 
fibers, for example in cardiac arrhythmia. In such instances it may be 
advantageous to suppress PLHu expression. PLHu expression could be 
suppressed by administration of PLHu antisense oligonucleotides. 
Alternatively, specific antibodies against PLHu, or inhibitors of PLHu 
such as channel blockers, may be introduced to treat diseases or 
conditions associated with abnormal PLHu expression. 
Due to their lipid-like properties, proteolipids destabilize lipid bilayers 
and promote membrane vesicle fusion. PLHu may therefore be incorporated 
into liposomes or artificial vesicles to promote vesicle fusion for drug 
delivery. 
PLHu Antibodies 
PLHu-specific antibodies are useful for the diagnosis of conditions and 
diseases associated with expression of PLHu. 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, ie, those which inhibit dimer formation, are 
especially preferred for diagnostics and therapeutics. 
PLHu for antibody induction does not require biological activity; however, 
the protein fragment, or oligopeptide must be antigenic. Peptides used to 
induce specific antibodies may have an amino acid sequence consisting of 
at least five amino acids, preferably at least 10 amino acids. Preferably, 
they should mimic a portion of the amino acid sequence of the natural 
protein and may contain the entire amino acid sequence of a small, 
naturally occurring molecule. Short stretches of PLHu amino acids may be 
fused with those of another protein such as keyhole limpet hemocyanin and 
antibody produced against the chimeric molecule. Procedures well known in 
the art can be used for the production of antibodies to PLHu. 
For the production of antibodies, various hosts including goats, rabbits, 
rats, mice, etc may be immunized by injection with PLHu or any portion, 
fragment or oligopeptide which retains immunogenic properties. Depending 
on the host species, various adjuvants may be used to increase 
immunological response. Such adjuvants include but are not limited to, 
Freund's, mineral gels such as aluminum hydroxide, and surface active 
substances such as lysolecithin, pluronic polyols, polyanions, peptides, 
oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (bacilli 
Calmette-Guerin) and Corynebacterium parvum are potentially useful human 
adjuvants. 
Monoclonal antibodies to PLHu may be prepared using any technique which 
provides for the production of antibody molecules by continuous cell lines 
in culture. These include but are not limited to the hybridoma technique 
originally described by Koehler and Milstein (1975 Nature 256:495-497), 
the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 
4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the 
EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and 
Cancer Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96). 
In addition, techniques developed for the production of "chimeric 
antibodies", the splicing of mouse antibody genes to human antibody genes 
to obtain a molecule with appropriate antigen specificity and biological 
activity can be used (Morrison et al (1984) Proc Natl Acad Sci 
81:6851-6855; Neuberger et al (1984) Nature 312:604-608; Takeda et al 
(1985) Nature 314:452-454). Alternatively, techniques described for the 
production of single chain antibodies (U.S. Pat. No. 4,946,778) can be 
adapted to produce PLHu-specific single chain antibodies. 
Antibodies may also be produced by inducing in vivo production in the 
lymphocyte population or by screening recombinant immunoglobulin libraries 
or panels of highly specific binding reagents as disclosed in Orlandi et 
al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G. and Milstein C. 
(1991; Nature 349:293-299). 
Antibody fragments which contain specific binding sites for PLHu may also 
be generated. For example, such fragments include, but are not limited to, 
the F(ab')2 fragments which can be produced by pepsin digestion of the 
antibody molecule and the Fab fragments which can be generated by reducing 
the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab 
expression libraries may be constructed to allow rapid and easy 
identification of monoclonal Fab fragments with the desired specificity 
(Huse W. D. et al (1989) Science 256:1275-1281). 
A variety of protocols for competitive binding or immunoradiometric assays 
using either polyclonal or monoclonal antibodies with established 
specificities are well known in the art. Such immunoassays typically 
involve the formation of complexes between PLHu and its specific antibody 
and the measurement of complex formation. A two-site, monoclonal-based 
immunoassay utilizing monoclonal antibodies reactive to two noninterfering 
epitopes on a specific PLHu protein is preferred, but a competitive 
binding assay may also be employed. These assays are described in Maddox 
D. E. et al (1983, J Exp Med 158:1211). 
Diagnostic Assays Using PLHu Specific Antibodies 
Particular PLHu antibodies are useful for the diagnosis of conditions or 
diseases characterized by expression of PLHu or in assays to monitor 
patients being treated with PLHu, agonists or inhibitors. Diagnostic 
assays for PLHu include methods utilizing the antibody and a label to 
detect PLHu in human body fluids or extracts of cells or tissues. The 
polypeptides and antibodies of the present invention may be used with or 
without modification. Frequently, the polypeptides and antibodies will be 
labeled by joining them, either covalently or noncovalently, with a 
reporter molecule. A wide variety of reporter molecules are known, several 
of which were described above. 
A variety of protocols for measuring PLHu, 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 PLHu is preferred, but a 
competitive binding assay may be employed. These assays are described, 
among other places, in Maddox, D. E. et al (1983, J Exp Med 158:1211). 
In order to provide a basis for diagnosis, normal or standard values for 
PLHu expression must be established. This is accomplished by combining 
body fluids or cell extracts taken from normal subjects, either animal or 
human, with antibody to PLHu 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 PLHu with both control and disease samples 
from biopsied tissues. Then, standard values obtained from normal samples 
may be compared with values obtained from samples from subjects 
potentially affected by disease. Deviation between standard and subject 
values establishes the presence of disease state. 
Drug Screening 
PLHu, its catalytic or immunogenic fragments or oligopeptides thereof, can 
be used for screening therapeutic compounds in any of a variety of drug 
screening techniques. The fragment employed in such a test may be free in 
solution, affixed to a solid support, borne on a cell surface, or located 
intracellularly. The formation of binding complexes, between PLHu and the 
agent being tested, may be measured. 
Another technique for drug screening which may be used for high throughput 
screening of compounds having suitable binding affinity to the PLHu is 
described in detail in "Determination of Amino Acid Sequence Antigenicity" 
by Geysen H. N., WO Application 84/03564, published on Sept. 13, 1984, and 
incorporated herein by reference. In summary, large numbers of different 
small peptide test compounds are synthesized on a solid substrate, such as 
plastic pins or some other surface. The peptide test compounds are reacted 
with fragments of PLHu and washed. Bound PLHu is then detected by methods 
well known in the art. Substantially purified PLHu can also be coated 
directly onto plates for use in the aforementioned drug screening 
techniques. Alternatively, non-neutralizing antibodies can be used to 
capture the peptide and immobilize it on a solid support. 
This invention also contemplates the use of competitive drug screening 
assays in which neutralizing antibodies capable of binding PLHu 
specifically compete with a test compound for binding PLHu. In this 
manner, the antibodies can be used to detect the presence of any peptide 
which shares one or more antigenic determinants with PLHu. 
Uses of the Polynucleotide Encoding PLHu 
A polynucleotide encoding PLHu, or any part thereof, may be used for 
diagnostic and/or therapeutic purposes. For diagnostic purposes, the PLHu 
of this invention may be used to detect and quantitate gene expression in 
biopsied tissues in which expression of PLHu may be implicated. The 
diagnostic assay is useful to distinguish between absence, presence, and 
excess expression of PLHu and to monitor regulation of PLHu levels during 
therapeutic intervention. Included in the scope of the invention are 
oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs. 
Another aspect of the subject invention is to provide for hybridization or 
PCR probes which are capable of detecting polynucleotide sequences, 
including genomic sequences, encoding PLHu or closely related molecules. 
The specificity of the probe, whether it is made from a highly specific 
region, eg, 10 unique nucleotides in the 5' regulatory region, or a less 
specific region, eg, especially in the 3' region, and the stringency of 
the hybridization or amplification (maximal, high, intermediate or low) 
will determine whether the probe identifies only naturally occurring PLHu, 
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 these PLHu 
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 PLHu. Hybridization probes may be labeled by a variety 
of reporter groups, including radionuclides such as .sup.32 P or .sup.35 
S, or enzymatic labels such as alkaline phosphatase coupled to the probe 
via avidin/biotin coupling systems, and the like. 
Other means for producing specific hybridization probes for PLHu DNAs 
include the cloning of nucleic acid sequences encoding PLHu or PLHu 
derivatives into vectors for the production of mRNA probes. Such vectors 
are known in the art and are commercially available and may be used to 
synthesize RNA probes in vitro by means of the addition of the appropriate 
RNA polymerase as T7 or SP6 RNA polymerase and the appropriate 
radioactively labeled nucleotides. 
Diagnostics 
Polynucleotide sequences encoding PLHu may be used for the diagnosis of 
conditions or diseases with which the expression of PLHu is associated. 
For example, polynucleotide sequences encoding PLHu may be used in 
hybridization or PCR assays of fluids or tissues from biopsies to detect 
PLHu expression. The form of such qualitative or quantitative methods may 
include Southern or northern analysis, dot blot or other membrane-based 
technologies; PCR technologies; dip stick, pin, chip and ELISA 
technologies. All of these techniques are well known in the art and are 
the basis of many commercially available diagnostic kits. 
The PLHu nucleotide sequence disclosed herein provide the basis for assays 
that detect activation or induction associated with inflammation or 
disease. The PLHu nucleotide sequence may be labeled by methods known in 
the art and added to a fluid or tissue sample from a patient under 
conditions suitable for the formation of hybridization complexes. After an 
incubation period, the sample is washed with a compatible fluid which 
optionally contains a dye (or other label requiring a developer) if the 
nucleotide has been labeled with an enzyme. After the compatible fluid is 
rinsed off, the dye is quantitated and compared with a standard. If the 
amount of dye in the biopsied or extracted sample is significantly 
elevated over that of a comparable control sample, the nucleotide sequence 
has hybridized with nucleotide sequences in the sample, and the presence 
of elevated levels of PLHu nucleotide sequences in the sample indicates 
the presence of the associated inflammation and/or disease. 
Such assays may also be used to evaluate the efficacy of a particular 
therapeutic treatment regime in animal studies, in clinical trials, or in 
monitoring the treatment of an individual patient. In order to provide a 
basis for the diagnosis of disease, a normal or standard profile for PLHu 
expression must be established. This is accomplished by combining body 
fluids or cell extracts taken from normal subjects, either animal or 
human, with PLHu, or a portion thereof, under conditions suitable for 
hybridization or amplification. Standard hybridization may be quantified 
by comparing the values obtained for normal subjects with a dilution 
series of PLHu run in the same experiment where a known amount of 
substantially purified PLHu is used. Standard values obtained from normal 
samples may be compared with values obtained from samples from patients 
afflicted with PLHu-associated diseases. Deviation between standard and 
subject values is used to establish the presence of disease. 
Once disease is established, a therapeutic agent is administered and a 
treatment profile is generated. Such assays may be repeated on a regular 
basis to evaluate whether the values in the profile progress toward or 
return to the normal or standard pattern. Successive treatment profiles 
may be used to show the efficacy of treatment over a period of several 
days or several months. 
Polymerase Chain Reaction (PCR) as described in U.S. Pat. Nos. 4,683,195 
and 4,965,188 provides additional uses for oligonucleotides based upon the 
PLHu sequence. Such oligomers are generally chemically synthesized, but 
they may be generated enzymatically or produced from a recombinant source. 
Oligomers generally comprise two nucleotide sequences, one with sense 
orientation (5'.fwdarw.3') and one 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. 
Additionally, methods which may be used to quantitate the expression of a 
particular molecule include radiolabeling (Melby P. C. et al 1993 J 
Immunol Methods 159:235-44) or biotinylating (Duplaa C. et al 1993 Anal 
Biochem 229-36) nucleotides, coamplification of a control nucleic acid, 
and standard curves onto which the experimental results are interpolated. 
Quantitation of multiple samples may be speeded up by running the assay in 
an ELISA format where the oligomer of interest is presented in various 
dilutions and a spectrophotometric or colorimetric response gives rapid 
quantitation. A definitive diagnosis of this type may allow health 
professionals to begin aggressive treatment and prevent further worsening 
of the condition. Similarly, further assays can be used to monitor the 
progress of a patient during treatment. Furthermore, the nucleotide 
sequences disclosed herein may be used in molecular biology techniques 
that have not yet been developed, provided the new techniques rely on 
properties of nucleotide sequences that are currently known such as the 
triplet genetic code, specific base pair interactions, and the like. 
Therapeutic Use 
Based upon its homology to the gene encoding rat plasmolipin and its 
expression profile, the PLHu polynucleotide disclosed herein may provide 
the basis for the design of molecules for the treatment of diseases such 
as cardiac arrhythmia. 
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 
PLHu. See, for example, the techniques described in Sambrook et al (supra) 
and Ausubel et al (supra). 
The polynucleotides comprising full length cDNA sequence and/or its 
regulatory elements enable researchers to use PLHu as an investigative 
tool in sense (Youssoufian H. and H. F. Lodish 1993 Mol Cell Biol 
13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60:631-652) 
regulation of gene function. Such technology is now well known in the art, 
and sense or antisense oligomers, or larger fragments, can be designed 
from various locations along the coding or control regions. 
Genes encoding PLHu can be turned off by transfecting a cell or tissue with 
expression vectors which express high levels of a desired PLHu fragment. 
Such constructs can flood cells with untranslatable sense or antisense 
sequences. Even in the absence of integration into the DNA, such vectors 
may continue to transcribe RNA molecules until all copies are disabled by 
endogenous nucleases. Transient expression may last for a month or more 
with a non-replicating vector (Mettler I., personal communication) and 
even longer if appropriate replication elements are part of the vector 
system. 
As mentioned above, modifications of gene expression can be obtained by 
designing antisense molecules, DNA, RNA or PNA, to the control regions of 
PLHu, ie, the promoters, enhancers, and introns. Oligonucleotides derived 
from the transcription initiation site, eg, between -10 and +10 regions of 
the leader sequence, are preferred. The antisense molecules may also be 
designed to block translation of mRNA by preventing the transcript from 
binding to ribosomes. Similarly, inhibition can be achieved using "triple 
helix" base-pairing methodology. Triple helix pairing compromises the 
ability of the double helix to open sufficiently for the binding of 
polymerases, transcription factors, or regulatory molecules. Recent 
therapeutic advances using triplex DNA were reviewed by Gee J. E. et al 
(In: Huber B. E. and B. I. Carr (1994) Molecular and Immunologic 
Approaches, Futura Publishing Co, Mt Kisco N.Y.). 
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific 
cleavage of RNA. The mechanism of ribozyme action involves 
sequence-specific hybridization of the ribozyme molecule to complementary 
target RNA, followed by endonucleolytic cleavage. Within the scope of the 
invention are engineered hammerhead motif ribozyme molecules that can 
specifically and efficiently catalyze endonucleolytic cleavage of RNA 
encoding PLHu. 
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 PLHu. Such DNA sequences may be incorporated into a wide variety 
of vectors with suitable RNA polymerase promoters such as T7 or SP6. 
Alternatively, antisense cDNA constructs that synthesize antisense RNA 
constitutively or inducibly can be introduced into cell lines, cells or 
tissues. 
RNA molecules may be modified to increase intracellular stability and 
half-life. Possible modifications include, but are not limited to, the 
addition of flanking sequences at the 5' and/or 3' ends of the molecule or 
the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase 
linkages within the backbone of the molecule. This concept is inherent in 
the production of PNAs and can be extended in all of these molecules by 
the inclusion of nontraditional bases such as inosine, queosine and 
wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms 
of adenine, cytidine, guanine, thymine, and uridine which are not as 
easily recognized by endogenous endonucleases. 
Methods for introducing vectors into cells or tissues include those methods 
discussed infra and which are equally suitable for in vivo, in vitro and 
ex vivo therapy. For ex vivo therapy, vectors are introduced into stem 
cells taken from the patient and clonally propagated for autologous 
transplant back into that same patient is presented in U.S. Pat. Nos. 
5,399,493 and 5,437,994, disclosed herein by reference. Delivery by 
transfection and by liposome are quite well known in the art. 
Furthermore, the nucleotide sequences for PLHu disclosed herein may be used 
in molecular biology techniques that have not yet been developed, provided 
the new techniques rely on properties of nucleotide sequences that are 
currently known, including but not limited to such properties as the 
triplet genetic code and specific base pair interactions. 
Detection and Mapping of Related Polynucleotide Sequences 
The nucleic acid sequence for PLHu can also be used to generate 
hybridization probes for mapping the naturally occurring genomic sequence. 
The sequence may be mapped to a particular chromosome or to a specific 
region of the chromosome using well known techniques. These include in 
situ hybridization to chromosomal spreads, flow-sorted chromosomal 
preparations, or artificial chromosome constructions such as yeast 
artificial chromosomes, bacterial artificial chromosomes, bacterial P1 
constructions or single chromosome cDNA libraries as reviewed in Price C. 
M. (1993; Blood Rev 7:127-34) and Trask B. J. (1991; Trends Genet 
7:149-54). 
The technique of fluorescent in situ hybridization of chromosome spreads 
has been described, among other places, in Verma et al (1988) Human 
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y. 
Fluorescent in situ hybridization of chromosomal preparations and other 
physical chromosome mapping techniques may be correlated with additional 
genetic map data. Examples of genetic map data can be found in the 1994 
Genome Issue of Science (265:1981f). Correlation between the location of a 
PLHu 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. For example, an STS based map of 
the human genome was recently published by the Whitehead-MIT Center for 
Genomic Research (Hudson T. J. et al (1995) Science 270:1945-1954). Often 
the placement of a gene on the chromosome of another mammalian species 
such as mouse (Whitehead Institute/MIT Center for Genome Research, Genetic 
Map of the Mouse, Database Release 10, Apr. 28, 1995) may reveal 
associated markers even if the number or arm of a particular human 
chromosome is not known. New sequences can be assigned to chromosomal 
arms, or parts thereof, by physical mapping. This provides valuable 
information to investigators searching for disease genes using positional 
cloning or other gene discovery techniques. Once a disease or syndrome, 
such as ataxia telangiectasia (AT), has been crudely localized by genetic 
linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti 
et al (1988) Nature 336:577-580), any sequences mapping to that area may 
represent associated or regulatory genes for further investigation. The 
nucleotide sequence of the subject invention may also be used to detect 
differences in the chromosomal location due to translocation, inversion, 
etc. among normal, carrier or affected individuals. 
Pharmaceutical Compositions 
The present invention relates to pharmaceutical compositions which may 
comprise nucleotides, proteins, antibodies, agonists, antagonists, or 
inhibitors, alone or in combination with at least one other agent, such as 
stabilizing compound, which may be administered in any sterile, 
biocompatible pharmaceutical carrier, including, but not limited to, 
saline, buffered saline, dextrose, and water. Any of these molecules can 
be administered to a patient alone, or in combination with other agents, 
drugs or hormones, in pharmaceutical compositions where it is mixed with 
excipient(s) or pharmaceutically acceptable carriers. In one embodiment of 
the present invention, the pharmaceutically acceptable carrier is 
pharmaceutically inert. 
Administration of Pharmaceutical Compositions 
Administration of pharmaceutical compositions is accomplished orally or 
parenterally. Methods of parenteral delivery include topical, 
intra-arterial (directly to the tumor), intramuscular, subcutaneous, 
intramedullary, intrathecal, intraventricular, intravenous, 
intraperitoneal, or intranasal administration. In addition to the active 
ingredients, these pharmaceutical compositions may contain suitable 
pharmaceutically acceptable carriers comprising excipients and auxiliaries 
which facilitate processing of the active compounds into preparations 
which can be used pharmaceutically. Further details on techniques for 
formulation and administration may be found in the latest edition of 
"Remington's Pharmaceutical Sciences" (Maack Publishing Co, Easton 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; and gums 
including arabic and tragacanth; and proteins such as gelatin and 
collagen. If desired, disintegrating or solubilizing agents may be added, 
such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a 
salt thereof, such as sodium alginate. 
Dragee cores are provided with suitable coatings such as concentrated sugar 
solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, 
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer 
solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or 
pigments may be added to the tablets or dragee coatings for product 
identification or to characterize the quantity of active compound, ie, 
dosage. 
Pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a coating such as glycerol or sorbitol. Push-fit capsules can contain 
active ingredients mixed with a filler or binders such as lactose or 
starches, lubricants such as talc or magnesium stearate, and, optionally, 
stabilizers. In soft capsules, the active compounds may be dissolved or 
suspended in suitable liquids, such as fatty oils, liquid paraffin, or 
liquid polyethylene glycol with or without stabilizers. 
Pharmaceutical formulations for parenteral administration include aqueous 
solutions of active compounds. For injection, the pharmaceutical 
compositions of the invention may be formulated in aqueous solutions, 
preferably in physiologically compatible buffers such as Hanks's solution, 
Ringer's solution, or physiologically buffered saline. Aqueous injection 
suspensions may contain substances which increase the viscosity of the 
suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. 
Additionally, suspensions of the active compounds may be prepared as 
appropriate oily injection suspensions. Suitable lipophilic solvents or 
vehicles include fatty oils such as sesame oil, or synthetic fatty acid 
esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, 
the suspension may also contain suitable stabilizers or agents which 
increase the solubility of the compounds to allow for the preparation of 
highly concentrated solutions. 
For topical or nasal administration, penetrants appropriate to the 
particular barrier to be permeated are used in the formulation. Such 
penetrants are generally known in the art. 
Manufacture and Storage 
The pharmaceutical compositions of the present invention may be 
manufactured in a manner that known in the art, eg, by means of 
conventional mixing, dissolving, granulating, dragee-making, levigating, 
emulsifying, encapsulating, entrapping or lyophilizing processes. 
The pharmaceutical composition may be provided as a salt and can be formed 
with many acids, including but not limited to hydrochloric, sulfuric, 
acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more 
soluble in aqueous or other protonic solvents that are the corresponding 
free base forms. In other cases, the preferred preparation may be a 
lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% 
mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to 
use. 
After pharmaceutical compositions comprising a compound of the invention 
formulated in a acceptable carrier have been prepared, they can be placed 
in an appropriate container and labeled for treatment of an indicated 
condition. For administration of PLHu, such labeling would include amount, 
frequency and method of administration. 
Therapeutically Effective Dose 
Pharmaceutical compositions suitable for use in the present invention 
include compositions wherein the active ingredients are contained in an 
effective amount to achieve the intended purpose. The determination of an 
effective dose is well within the capability of those skilled in the art. 
For any compound, the therapeutically effective dose can be estimated 
initially either in cell culture assays, eg, of neoplastic cells, or in 
animal models, usually mice, rabbits, dogs, or pigs. The animal model is 
also used to achieve a desirable concentration range and route of 
administration. Such information can then be used to determine useful 
doses and routes for administration in humans. 
A therapeutically effective dose refers to that amount of protein or its 
antibodies, antagonists, or inhibitors which ameliorate the symptoms or 
condition. Therapeutic efficacy and toxicity of such compounds can be 
determined by standard pharmaceutical procedures in cell cultures or 
experimental animals, eg, ED50 (the dose therapeutically effective in 50% 
of the population) and LD50 (the dose lethal to 50% of the population). 
The dose ratio between therapeutic and toxic effects is the therapeutic 
index, and it can be expressed as the ratio, 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 of such compounds 
lies preferably within a range of circulating concentrations that include 
the ED50 with little or no toxicity. The dosage varies within this range 
depending upon the dosage form employed, sensitivity of the patient, and 
the route of administration. 
The exact dosage is chosen by the individual physician in view of the 
patient to be treated. Dosage and administration are adjusted to provide 
sufficient levels of the active moiety or to maintain the desired effect. 
Additional factors which may be taken into account include the severity of 
the disease state, eg, tumor size and location; age, weight and gender of 
the patient; diet, time and frequency of administration, drug 
combination(s), reaction sensitivities, and tolerance/response to therapy. 
Long acting pharmaceutical compositions might be administered every 3 to 4 
days, every week, or once every two weeks depending on half-life and 
clearance rate of the particular formulation. 
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a 
total dose of about 1 g, depending upon the route of administration. 
Guidance as to particular dosages and methods of delivery is provided in 
the literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. 
Those skilled in the art will employ different formulations for 
nucleotides than for proteins or their inhibitors. Similarly, delivery of 
polynucleotides or polypeptides will be specific to particular cells, 
conditions, locations, etc. 
The examples below are provided to illustrate the subject invention and are 
not included for the purpose of limiting the invention. 
EXAMPLES 
I cDNA Library Construction 
The BRSTNOT03 cDNA library was constructed from nontumorous breast tissue 
removed from a 54-year-old Caucasian female (specimen #0025B; Mayo Clinic, 
Rochester, Minn.) who had undergone bilateral radical mastectomy following 
diagnosis of residual invasive grade 3 of 4 mammary ductal adenocarcinoma. 
The pathology report indicated that the biopsied fibroadipose tissue from 
the right breast was negative for tumor. Tumor cells forming a nodule 
1.times.0.7.times.0.7 cm were identified in the right breast. The 
remaining breast parenchyma exhibited proliferative fibrocystic changes 
without atypia. The skin, nipple, and fascia were uninvolved. One of 10 
axillary lymph nodes was involved with metastatic tumor, as a microscopic, 
intranodal focus. Prior to surgery, the patient was prescribed estrogen as 
part of postmenopausal hormone replacement therapy. 
The frozen tissue was homogenized and lysed using a Brinkmann Homogenizer 
Polytron PT-3000 (Brinkmann Instruments, Westbury, N.J.) in guanidinium 
isothiocyanate solution. The lysate was centrifuged over a 5.7M CsCl 
cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge 
(Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature. 
The RNA was extracted with acid phenol pH 4.0, precipitated using 0.3M 
sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free 
water, and DNase treated at 37.degree. C. The RNA extraction was repeated 
with acid phenol pH 4.0 and precipitated with sodium acetate and ethanol 
as before. The mRNA was then isolated using the Qiagen Oligotex kit 
(QIAGEN, Inc.; Chatsworth, Calif.) and used to construct the cDNA library. 
The mRNA was handled according to the recommended protocols in the 
SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat. 
#18248-013; Gibco/BRL). cDNAs were fractionated on a Sepharose CL4B column 
(Cat. #275105-01; 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/BRL). 
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 (Catalog 
#26173; QIAGEN, Inc.). This kit enabled 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) after inoculation, the cultures were incubated for 19 hours and at the 
end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 
3) following isopropanol precipitation, the plasmid DNA pellet was 
resuspended in 0.1 ml of distilled water. After the last step in the 
protocol, samples were transferred to a 96-well block for storage at 
40.degree. C. 
The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol Biol 
94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.) in 
combination with Peltier Thermal Cyclers (PTC200 from MJ Research, 
Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems; and 
the reading frame was determined. 
III Homology Searching of cDNA Clones and Their Deduced Proteins 
Each cDNA was compared to sequences in GenBank using a search algorithm 
developed by Applied Biosystems and incorporated into the INHERIT- 670 
Sequence Analysis System. In this algorithm, Pattern Specification 
Language (TRW Inc, Los Angeles Calif.) was used to determine regions of 
homology. The three parameters that determine how the sequence comparisons 
run were window size, window offset, and error tolerance. Using a 
combination of these three parameters, the DNA database was searched for 
sequences containing regions of homology to the query sequence, and the 
appropriate sequences were scored with an initial value. Subsequently, 
these homologous regions were examined using dot matrix homology plots to 
distinguish regions of homology from chance matches. Smith-Waterman 
alignments were used to display the results of the homology search. 
Peptide and protein sequence homologies were ascertained using the 
INHERIT.TM. 670 Sequence Analysis System in a way similar to that used in 
DNA sequence homologies. Pattern Specification Language and parameter 
windows were used to search protein databases for sequences containing 
regions of homology which were scored with an initial value. Dot-matrix 
homology plots were examined to distinguish regions of significant 
homology from chance matches. 
BLAST, which stands for Basic Local Alignment Search Tool (Altschul S. F. 
(1993) J Mol Evol 36:290-300; Altschul, S. F. et al (1990) J Mol Biol 
215:403-10), was used to search for local sequence alignments. BLAST 
produces alignments of both nucleotide and amino acid sequences to 
determine sequence similarity. Because of the local nature of the 
alignments, BLAST is especially useful in determining exact matches or in 
identifying homologs. BLAST is useful for matches which do not contain 
gaps. The fundamental unit of BLAST algorithm output is the High-scoring 
Segment Pair (HSP). 
An HSP consists of two sequence fragments of arbitrary but equal lengths 
whose alignment is locally maximal and for which the alignment score meets 
or exceeds a threshold or cutoff score set by the user. The BLAST approach 
is to look for HSPs between a query sequence and a database sequence, to 
evaluate the statistical significance of any matches found, and to report 
only those matches which satisfy the user-selected threshold of 
significance. The parameter E establishes the statistically significant 
threshold for reporting database sequence matches. E is interpreted as the 
upper bound of the expected frequency of chance occurrence of an HSP (or 
set of HSPs) within the context of the entire database search. Any 
database sequence whose match satisfies E is reported in the program 
output. 
IV 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 labelled 
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 use BLAST (Altschul S. F. 1993 and 1990, 
supra) to search for identical or related molecules in nucleotide 
databases such as GenBank or the LIFESEQ.TM. database (Incyte, 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 PLHu to Full Length or to Recover Regulatory Elements 
The nucleic acid sequence encoding full length PLHu (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 allow the extension of the known 
PLHu nucleotide sequence "outward" generating amplicons containing new, 
unknown nucleotide sequence for the region of interest (U.S. patent 
application Ser. No. 08/487,112, filed Jun. 7, 1995, specifically 
incorporated by reference). 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 Mass.) and the 
following parameters: 
______________________________________ 
Step 1 94.degree. C. for 1 min (initial denaturation) 
Step 2 65.degree. C. for 1 min 
Step 3 68.degree. C. for 6 min 
Step 4 94.degree. C. for 15 sec 
Step 5 65.degree. C. for 1 min 
Step 6 68.degree. C. for 7 min 
Step 7 Repeat step 4-6 for 15 additional cycles 
Step 8 94.degree. C. for 15 sec 
Step 9 65.degree. C. for 1 min 
Step 10 68.degree. C. for 7:15 min 
Step 11 Repeat step 8-10 for 12 cycles 
Step 12 72.degree. C. for 8 min 
Step 13 4.degree. C. (and holding) 
______________________________________ 
A 5-10 .mu.l aliquot of the reaction mixture is analyzed by electrophoresis 
on a low concentration (about 0.6-0.8%) agarose mini-gel to determine 
which reactions were successful in extending the sequence. Bands thought 
to contain the largest products were selected and cut out of the gel. 
Further purification involves using a commercial gel extraction method 
such as QIAQuick.TM. (QIAGEN Inc). After recovery of the DNA, Klenow 
enzyme was used to trim single-stranded, nucleotide overhangs creating 
blunt ends which facilitate religation and cloning. 
After ethanol precipitation, the products are redissolved in 13 .mu.l of 
ligation buffer, 1 .mu.l T4-DNA ligase (15 units) and 1 .mu.l T4 
polynucleotide kinase are added, and the mixture is incubated at room 
temperature for 2-3 hours or overnight at 16.degree. C. Competent E. coli 
cells (in 40 .mu.l of appropriate media) are transformed with 3 .mu.l of 
ligation mixture and cultured in 80 .mu.l of SOC medium (Sambrook J. et 
al, supra). After incubation for one hour at 37.degree. C., the whole 
transformation mixture is plated on Luria Bertani (LB)-agar (Sambrook J. 
et al, supra) containing 2.times.Carb. The following day, several colonies 
are randomly picked from each plate and cultured in 150 .mu.l of liquid 
LB/2.times.Carb medium placed in an individual well of an appropriate, 
commercially-available, sterile 96-well microtiter plate. The following 
day, 5 .mu.l of each overnight culture is transferred into a non-sterile 
96-well plate and after dilution 1:10 with water, 5 .mu.l of each sample 
is transferred into a PCR array. 
For PCR amplification, 18 .mu.l of concentrated PCR reaction mix 
(3.3.times.) containing 4 units of rTth DNA polymerase, a vector primer 
and one or both of the gene specific primers used for the extension 
reaction are added to each well. Amplification is performed using the 
following conditions: 
______________________________________ 
Step 1 94.degree. C. for 60 sec 
Step 2 94.degree. C. for 20 sec 
Step 3 55.degree. C. for 30 sec 
Step 4 72.degree. C. for 90 sec 
Step 5 Repeat steps 2-4 for an additional 29 cycles 
Step 6 72.degree. C. for 180 sec 
Step 7 4.degree. C. (and holding) 
______________________________________ 
Aliquots of the PCR reactions are run on agarose gels together with 
molecular weight markers. The sizes of the PCR products are compared to 
the original partial cDNAs, and appropriate clones are selected, ligated 
into plasmid and sequenced. 
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 mCi of 
.gamma.-.sup.32 P! adenosine triphosphate (Amersham, Chicago Ill.) and T4 
polynucleotide kinase (DuPont NEN.RTM., Boston Mass.). The labeled 
oligonucleotides are substantially purified with Sephadex G-25 super fine 
resin column (Pharmacia). A portion containing 10.sup.7 counts per minute 
of each of the sense and antisense oligonucleotides is used in a typical 
membrane based hybridization analysis of human genomic DNA digested with 
one of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, 
or Pvu II; DuPont NEN.RTM.). 
The DNA from each digest is fractionated on a 0.7 percent agarose gel and 
transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham 
N.H.). Hybridization is carried out for 16 hours at 40.degree. C. To 
remove nonspecific signals, blots are sequentially washed at room 
temperature under increasingly stringent conditions up to 0.1.times. 
saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR.TM. 
film (Kodak, Rochester N.Y.) is exposed to the blots in a Phosphoimager 
cassette (Molecular Dynamics, Sunnyvale Calif.) for several hours, 
hybridization patterns are compared visually. 
VII Antisense Molecules 
The nucleotide sequence encoding PLHu, or any part thereof, is used to 
inhibit in vivo or in vitro expression of naturally occurring PLHu. 
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 
sequence of PLHu as shown in FIGS. 1A and 1B is used to inhibit expression 
of naturally occurring PLHu. The complementary oligonucleotide is designed 
from the most unique 5' sequence as shown in FIGS. 1A and 1B and used 
either to inhibit transcription by preventing promoter binding to the 
upstream nontranslated sequence or translation of an PLHu transcript by 
preventing the ribosome from binding. Using an appropriate portion of the 
leader and 5' sequence of SEQ ID NO:2, an effective antisense 
oligonucleotide includes any 15-20 nucleotides spanning the region which 
translates into the signal or early coding sequence of the polypeptide as 
shown in FIGS. 1A and 1B. 
VIII Expression of PLHu 
Expression of PLHu is accomplished by subcloning the cDNAs into appropriate 
vectors and transfecting the vectors into host cells. In this case, the 
cloning vector, pSport, previously used for the generation of the cDNA 
library is used to express PLHu 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 7 residues 
of .beta.-galactosidase. Immediately following these eight residues is a 
bacteriophage promoter useful for transcription and a linker containing a 
number of unique restriction sites. 
Induction of an isolated, transfected bacterial strain with IPTG using 
standard methods produces a fusion protein which consists of the first 
seven residues of .beta.-galactosidase, about 5 to 15 residues of linker, 
and the full length PLHu. The signal sequence directs the secretion of 
PLHu into the bacterial growth media which can be used directly in the 
following assay for activity. 
IX PLHu Activity 
The pore-forming ability of PLHu is assayed by monitoring its effect on 
transmembrane pH gradients in liposomes. Mitochondrial cytochrome C 
oxidase, a proton pump, is reconstituted into liposomes by sonication. The 
pH-sensitive fluorescent dye pyranine (Eastman Kodak) is then incorporated 
into the proteoliposomes by rapid freeze-thawing and sonication. Excess 
dye is removed by centrifugation and resuspension of the liposomes into an 
appropriate buffer. Addition of ascorbate and cytochrome C initiates 
proton uptake into the liposomes. PLHu is added and proton efflux is 
monitored by the fluorescence changes arising from changes in internal pH 
of the liposomes at excitation and emission wavelengths of 460 nm and 508 
nm, respectively. 
Lipid bilayer destabilization promoted by PLHu, incorporated into membranes 
by expression or by reconstitution, is assayed by measurement of the 
fluorescence polarization of the lipophilic dye 
1,6-diphenyl-1,3,5-hexatriene (Eastman Kodak) inserted into the membranes. 
X Production of PLHu Specific Antibodies 
PLHu substantially purified using PAGE electrophoresis (Sambrook, supra) is 
used to immunize rabbits and to produce antibodies using standard 
protocols. The amino acid sequence translated from PLHu is analyzed using 
DNAStar software (DNAStar Inc) to determine regions of high immunogenicity 
and a corresponding oligopolypeptide is synthesized and used to raise 
antibodies by means known to those of skill in the art. Analysis to select 
appropriate epitopes, such as those near the C-terminus or in hydrophilic 
regions (shown in FIG. 3) is described by Ausubel F. M. et al (supra). 
Typically, the oligopeptides are 15 residues in length, synthesized using 
an Applied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistry, 
and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with 
M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel F. M. et al, 
supra). Rabbits are immunized with the oligopeptide-KLH complex in 
complete Freund's adjuvant. The resulting antisera are tested for 
antipeptide activity, for example, by binding the peptide to plastic, 
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting 
with radioiodinated, goat anti-rabbit IgG. 
XI Purification of Naturally Occurring PLHu Using Specific Antibodies 
Naturally occurring or recombinant PLHu is substantially purified by 
immunoaffinity chromatography using antibodies specific for PLHu. An 
immunoaffinity column is constructed by covalently coupling PLHu 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. 
Cellular fractions from cells containing PLHu are prepared by 
solubilization of the whole cell and isolation of subcellular fractions by 
differential centrifugation, by the addition of detergent, or by other 
methods well known in the art. Alternatively, soluble PLHu containing a 
signal sequence may be secreted in useful quantity into the medium in 
which the cells are grown. 
A fractionated PLHu-containing preparation is passed over the 
immunoaffinity column, and the column is washed under conditions that 
allow the preferential absorbance of PLHu (eg, high ionic strength buffers 
in the presence of detergent). The column is eluted under conditions that 
disrupt antibody/PLHu binding (eg, a buffer of pH 2-3 or a high 
concentration of a chaotrope such as urea or thiocyanate ion), and PLHu is 
collected. 
All publications and patents mentioned in the above specification are 
herein incorporated by reference. Various modifications and variations of 
the described method and system of the invention will be apparent to those 
skilled in the art without departing from the scope and spirit of the 
invention. Although the invention has been described in connection with 
specific preferred embodiments, it should be understood that the invention 
as claimed should not be unduly limited to such specific embodiments. 
Indeed, various modifications of the described modes for carrying out the 
invention which are obvious to those skilled in molecular biology or 
related fields are intended to be within the scope of the following 
claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 153 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: BRSTNOT03 
(B) CLONE: 640699 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetAsnPheSerThrSerSerSerSerPheAlaTyrAspArgGluPhe 
151015 
LeuArgThrLeuProGlyPheLeuIleValAlaGluIleValLeuGly 
202530 
LeuLeuValTrpThrLeuIleAlaGlyThrGluTyrPheArgValPro 
354045 
AlaPheGlyTrpValMetPheValAlaValPheTyrTrpValLeuThr 
505560 
ValPhePheLeuIleIleTyrIleThrMetThrTyrThrArgIlePro 
65707580 
GlnValProTrpThrThrValGlyLeuCysPheAsnGlySerAlaPhe 
859095 
ValLeuTyrLeuSerAlaAlaValValAspAlaSerSerValSerPro 
100105110 
GluArgAspSerHisAsnPheAsnSerTrpAlaAlaSerSerPhePhe 
115120125 
AlaPheLeuValAsnIleCysTyrAlaGlyAsnThrTyrPheSerPhe 
130135140 
IleAlaTrpArgSerArgThrIleGln 
145150 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 853 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: BRSTNOT03 
(B) CLONE: 640699 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CCGCCAGCTCCTTCGGCAATGAACTTCTCCACCAGCAGCAGCAGCTTCGCCTACGACCGG60 
GAGTTCCTCCGCACCCTGCCCGGCTTCCTCATCGTGGCCGAGATCGTTCTGGGGCTGCTG120 
GTATGGACGCTTATTGCTGGAACTGAGTACTTCCGGGTCCCCGCATTTGGCTGGGTCATG180 
TTTGTAGCTGTATTTTACTGGGTCCTCACCGTCTTCTTCCTCATTATCTACATAACAATG240 
ACCTACACCAGGATTCCCCAGGTGCCCTGGACAACAGTGGGCCTGTGCTTTAACGGCAGT300 
GCCTTCGTCTTGTACCTCTCTGCCGCTGTTGTAGATGCATCTTCCGTCTCCCCTGAGAGG360 
GACAGTCACAACTTCAACAGCTGGGCGGCCTCATCGTTCTTTGCCTTCCTGGTCAACATC420 
TGCTACGCTGGAAATACATATTTCAGTTTTATAGCATGGAGATCCAGGACCATACAGTGA480 
TTTACCATTTTGATAATTAAAAGGAAAAAAAAAGGAAGACTCTCACTGTAAAAACAGCTG540 
TAGGTATAATGTATATTCCCAGAGAATTGTATTTAACTAATTAATGTTTTTTATATTCTT600 
AAATTTGCTCACAAATTGTGGTTTGTTACAATTAAACTGGATACTTATTTGCAAAGTGTT660 
GTAGCTTATAATGAACTCTTAAGTATCTTATTAATGTATTAATGTCTTCATAGATCATAT720 
TTTCTTAGACAATGTTTAAATAGATAAATTGCTAATATTGAGAATGTGTCAAGTTTGTAA780 
ACCTAACTTTTAAGATGCCAGATTCTTTTTTGATTAAATGTTGCAAAATCCCAAAAAAAA840 
AAAAAAAAAAAAA853 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 157 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 1346732 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetArgProAspLeuGlyPheValArgSerAlaLeuGlyValLeuAla 
151015 
LeuLeuGlnLeuValLeuGlyLeuLeuValTrpAlaLeuIleAlaAsp 
202530 
ThrProTyrHisLeuTyrProAlaTyrGlyTrpValMetPheValAla 
354045 
ValPheLeuTrpLeuValThrIleValPhePheIleIleTyrLeuPhe 
505560 
GlnLeuHisMetLysLeuTyrMetValProTrpProLeuValLeuLeu 
65707580 
ValPhePheValAlaAlaThrValLeuTyrIleThrAlaPheValAla 
859095 
CysAlaAlaAlaValAspLeuThrSerLeuArgGlySerArgProTyr 
100105110 
AsnGlnArgSerAlaAlaSerPhePheAlaCysLeuValMetIleAla 
115120125 
TyrGlyLeuSerAlaPhePheSerPheGlnAlaTrpArgGlyValGly 
130135140 
SerAsnAlaAlaThrSerGlnMetAlaGlyGlyTyrSer 
145150155 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 153 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(A) LIBRARY: GenBank 
(B) CLONE: 126719 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
MetAlaProAlaAlaAlaThrGlyGlySerThrLeuProSerGlyPhe 
151015 
SerValPheThrThrLeuProAspLeuLeuPheIlePheGluPheIle 
202530 
PheGlyGlyLeuValTrpIleLeuValAlaSerSerLeuValProTrp 
354045 
ProLeuValGlnGlyTrpValMetPheValSerValPheCysPheVal 
505560 
AlaThrThrThrLeuIleIleLeuTyrIleIleGlyAlaHisGlyGly 
65707580 
GluThrSerTrpValThrLeuAspAlaAlaTyrHisCysThrAlaAla 
859095 
LeuPheTyrLeuSerAlaSerValLeuGluAlaLeuAlaThrIleThr 
100105110 
MetGlnAspGlyPheThrTyrArgHisTyrHisGluAsnIleAlaAla 
115120125 
ValValPheSerTyrIleAlaThrLeuLeuTyrValValHisAlaVal 
130135140 
PheSerLeuIleArgTrpLysSerSer 
145150 
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