Note: Descriptions are shown in the official language in which they were submitted.
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MAMMALIAN LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE
Technical Field of the Invention
This present invention provides cDNA sequences and polypeptides having the
enzyme lysophosphatidic acid acyltransferase (LPAAT) activity. LPAAT is also
known
as 1-acyl sn-glycerol-3-phosphate acyltransferase. The present invention
further provides
for isolation and production of polypeptides involved in phosphatidic acid
metabolism and
signaling in mammalian cells, in particular, the production of purified forms
of LPAAT.
Background of the Invention
Originally regarded as intermediates in lipid biosynthesis {Kent, Anal. Rev.
Biochem. 64:31 S-343, 1995), phosphatidic acid (PA) and one of its precursors,
lysophosphatidic acid (LPA), have also been identified as phospholipid
signaling
molecules that affect a wide range of biological responses (McPhail et al.,
Proc. Natl.
Acad. Sci. USA 92:7931-7935, 1995; Williger et al., J. Biol. Chem. 270:29656-
29659,
1995; Moolenaar, Curr. Opin. Cell Biol. 7:203-210, 1995).
Cellular activation in monocytic and lymphoid cells is associated with rapid
upregulation of synthesis of phospholipids (PL) that includes phosphatidic
acid (PA),
diacylglycerol (DAG) and glycan phosphatidylinositol (PI). Phosphatidic acids
{PA) are a
molecularly diverse group of phospholipid second messengers coupled to
cellular
activation and mitogenesis (Singer et al., Exp. Opin. Invest. Drugs 3:631-643,
1994).
Compounds that would block PA generation and hence diminish the signal
involved in cell
activation may therefore be of therapeutic interest in the area of
inflammation and
oncology. Lysofylline {1-(R)-(5-hydroxyhexyl)-3,7-dimethylxanthine) (Singer et
al., Exp.
Opin. Invest. Drugs 3:631-643, 1994; and Rice et al., Proc. Natl. Acad. Sci.
USA 91:3857-
3861, 1994) has been found to be an effective inhibitor of cellular activation
by blocking
the synthesis of a specific phosphatidic acid (PA) species produced by
lysophosphatidic
acid acyltransferase (LPAAT) in activated monocytic cells (Rice et al., Proc.
Natl. Acad.
Sci. USA 91:3857-3861, 1994). PA can be generated through hydrolysis of
phosphatidycholine (PC) (Exton, Biochim. Biophys. Acta 1212:26-42, 1994) or
glycan PI
(Eardley et al., Science 251:78-81, 1991; Merida et al., DNA Cell Biol. 12:473-
479, 1993),
through phosphorylation of DAG by DAG kinase (Kanoh et al., Trends Biochem.
Sci.
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15:47-50, 1990) or through acylation of LPA at the SN2 position (Bursten et
al., Am. J.
Physiol. 266:CI093-C1 I04, 1994). Compounds that would block PA generation and
hence diminish lipid biosynthesis and the signal involved in cell activation
may therefore
be of therapeutic interest in the area of inflammation and oncology as well as
obesity
treatment.
The genes coding for LPAAT have been isolated in bacteria (Coleman, Mol. Gen.
Genet. 232:295-303, 1992), in yeast (Nagiec et al., J. Biol. Chem. 268:22156-
22163, 1993)
and in plants {Brown et al., Plant Mol. Biol. 26:211-223, 1994; and Hanke et
al., Eur J.
Biochem. 232:806-810, 1995) using genetic complementation techniques. The
cloning of
a mammalian version of LPAAT has not been reported. Homology among the
bacterial,
yeast and plant LPAAT is only found in a very few block of three or at most
four amino
acids scattered throughout the sequences {Brown et al., Plant Mol. Biol.
26:211-223,
1994). Further, there is a need in the art for recombinant LPAAT from a
mammalian
source to enable compound screening for LPAAT inhibitors for the development
of
specific compounds that would inhibit this enzyme.
Summary of the Invention
The present invention provides a cDNA sequence, polypeptide sequence, and
transformed cells for producing isolated recombinant mammalian LPAAT. The
present
invention provides two novel human polypeptides, and fragments thereof, having
LPAAT
activity. The polypeptides discovered herein is novel and will be called
hLPAAT with the
f rst one discovered designated hLPAATa and the second one discovered called
hLPAAT(3. LPAAT catalyzes the acylation of lysophosphatidic acid (LPA) to
phosphatidic acid (PA) by acylating the sn-2 position of LPA with a fatty acid
acyl-chain
moiety.
The present invention further provides nucleic acid sequences coding for
expression of the novel LPAAT polypeptides and active fragments thereof. The
invention
further provides purified LPAATs and antisense oligonucleotides for modulation
of
expression of the genes coding for LPAAT polypeptides. Assays for screening
test
compounds for their ability to inhibit LPAATs are also provided.
Recombinant LPAAT is useful for screening candidate drug compounds that
inhibit LPAAT activity. The present invention provides cDNA sequences encoding
a
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Figure 2 shows amino acid sequence alignment of the human LPAATa coding
sequence, the yeast LPAAT coding sequence, E. coli LPAAT coding sequence, and
the
maize LPAAT coding sequence. This comparison shows that human LPAATa has the -
greatest extended homology with yeast or E. coli LPAAT than with the plant
LPAAT.
Figure 3 shows the DNA sequence of the cDNA insert pSP.LPAT3 encoding
hLPAAT~i. The nucleotide sequence analysis and restriction mapping of the cDNA
clone
revealed a 5' untranslated region of 39 base pairs and an open reading frame
encoding a
278 amino acid polypeptide that spans positions 40-876. It also shows a 3'
untranslated
region of 480 base pairs from pSP.LPAT3. The initiation site for translation
was localized
at nucleotide positions 40-42 and fulfilled the requirement for an adequate
initiation site
(Kozak, Critical Rev. Biochem. Mol. Biol. 27:385-402, 1992).
Figure 4 shows the sequence of the hLPAAT~3 278 amino acid open reading frame.
The amino acid sequence was used as the query sequence to search for
homologous
sequences in protein databases. Search of the database based on Genbank
Release 92 from
the National Center for Biotechnology Information (NCBI) using the blastp
program
showed that this protein was most homologous to the yeast, bacterial and plant
LPAATs.
Figure 5 shows amino acid sequences alignment of this putative human LPAAT(3
coding sequence, human LPAATa coding, the yeast LPAAT coding sequence, the
bacterial (E. coli, H. influenzae, and S. typhimurium) LPAAT coding sequences,
and the
plant (L. douglassi and C. nucifera) LPAAT coding sequences, revealing that
the human
LPAAT coding sequences have a much more extended homology with the yeast or
the
bacterial LPAAT than with the plant LPAAT.
Figure 6 shows a comparison of LPAAT activity in A549 cells transfected with
pCE9.LPAAT 1 DNA, or no DNA using a TLC (thin layer chromatography) assay.
These
data are described in more detail in examples 3 and 4.
Figures 7 and 8 show a comparison of the production of TNF (Figure 7) and IL-6
(Figure 8) between A549 cells transfected with pCE9.LPAATI and control A549
cells
after stimulation with IL-1 (i and marine TNF. These data show A549
overexpressing
LPAAT produces >5 fold more TNF and >10 fold more IL-6 relative to
untransfected
A549 cells, suggesting that overexpression of LPAAT enhances the cytokine
signaling
response in cells.
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poIypeptide having LPAAT activity and comprising the DNA sequence set forth in
SEQ
ID NO. 1 of SEQ ID NO. 7, shortened fragments thereof, or additional cDNA
sequences
which due to the degeneracy of the genetic code encode a polypeptide of SEQ ID
NO. 2 0~-
SEQ. ID NO. 8 or biologically active fragments thereof or a sequence capable
of
hybridizing thereto under high stringency conditions. The present invention
further
provides a polypeptide having LPAAT activity and comprising the amino acid
sequence of
SEQ ID NO. 2 or SEQ ID NO. 8 or biologically active fragments thereof.
Also provided by the present invention are vectors containing a DNA sequence
encoding a mammalian LPAAT enzyme in operative association with an expression
control sequence. Host cells, transformed with such vectors for use in
producing
recombinant LPAAT , are also provided with the present invention. The
inventive vectors
and transformed cells are employed in a process for producing recombinant
mammalian
LPAAT. In this process, a cell line transformed with a DNA sequence encoding
on
expression for a LPAAT enzyme in operative association with an expression
control
sequence, is cultured. The claimed process may employ a number of known cells
as host
cells for expression of the LPAAT polypeptide, including, for example,
mammalian cells,
yeast cells, insect cells and bacterial cells.
Another aspect of this invention provides a method for identifying a
pharmaceutically-active compound by determining if a selected compound is
capable of
inhibiting the activity of LPAAT for acylating LPA to PA. A compound capable
of such
activity is capable of indirectly inhibiting SAPkinase and being a
pharmaceutical
compound useful for augmenting trilineage hematopoiesis after cytoreductive
therapy and
for anti-inflammatory activity in inhibiting the inflammatory cascade
following hypoxia
and reoxygenation injury (e.g., sepsis, trauma, ARDS, etc.).
The present invention further provides a transformed cell that expresses
active
mammalian LPAAT and further comprises a means for determining if a drug
candidate
compound is therapeutically active by inhibiting recombinant LPAAT activity.
Brief Description of the Drawings
Figure 1 shows the DNA sequence of the cDNA insert of pZplat.l 1 encoding
hLPAAToc.
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Detailed Description of the Invention
The present invention provides novel, isolated, biologically active mammalian
LPAAT enzymes. The term "isolated" means any LPAAT polypeptide of the present
invention, or any other gene encoding LPAAT polypeptide, which is essentially
free of
other polypeptides or genes, respectively, or of other contaminants with which
the LPAAT
polypeptide of gene might normally be found in nature.
The invention includes a functional polypeptide, LPAAT, and functional
fragments
thereof. As used herein, the term "functional polypeptide" refers to a
polypeptide which
possesses a biological function or activity which is identified through a
biological assay,
preferably cell-based, and which results in the formation of PA species from
LPA. A
"functional polynucleotide" denotes a polynucleotide which encodes a
functional
polypeptide. Minor modification of the hLPAATa primary amino acid sequence may
result in proteins which have substantially equivalent activity as compared to
the
sequenced hLPAATa polypeptide described herein. Such modifications may be
deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the
polypeptides produced by these modifications are included herein as long as
the
acyltransferase activity of LPAAT is present. This can lead to the development
of a
smaller active molecule which would have broader utility. For example, it is
possible to
remove amino or carboxy terminal amino acids which may not be required for
LPAAT
activity.
The hLPAATa and hLPAAT~3 polypeptide of the present invention also includes
conservative variations of the polypeptide sequence. The term "conservative
variation"
denotes the replacement of an amino acid residue by another, biologically
active similar
residue. Examples of conservative variations include the substitution of one
hydrophobic
residue, such as isoleucine, vaiine, leucine or methionine for another, or the
substitution of
one polar residue for another, such as the substitution of arginine for
lysine, glutamic for
aspartic acids, or giutamine for asparagine, and the like. The term
"conservative variation"
also includes the use of a substituted amino acid in place of an unsubstituted
parent amino
acid provided that antibodies raised to the substituted polypeptide also
immunologically
react with the unsubstituted polypeptide.
Polypeptides of the present invention can be synthesized by such commonly used
methods as t-BOC or FMOC protection of alpha-amino groups. Both methods
involve
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step-wise syntheses whereby a single amino acid is added at each step starting
from the C
terminus of the peptide (Coligan et al., Current Protocols in Immunology,
Wiley
lnterscience, Unit 9, 1991). In addition, polypeptide of the present invention
can also be
synthesized by solid phase synthesis methods (e.g., Mernfield, J. Am. Chem.
Soc. 85:2149,
1962; and Steward and Young, Solid Phase Peptide Synthesis, Freeman, San
Francisco pp.
27-62, 1969) using copolyol (styrene-divinylbenzene} containing 0.1-1.0 mM
amines/g
polymer. On completion of chemical synthesis, the ploypeptides can be
deprotected and
cleaved from the polymer by treatment with liquid HF 10% anisole for about 15-
60 min at
0 °C. After evaporation of the reagents, the peptides are extracted
from the polymer with
1 % acetic acid solution, which is then lyophilized to yield crude material.
This can
normally be purified by such techniques as gel filtration of Sephadex G-15
using 5%
acetic acid as a solvent. Lyophiiization of appropriate fractions of the
column will yield a
homogeneous polypeptide or polypeptide derivatives, which are characterized by
such
standard techniques as amino acid analysis, thin layer chromatography, high
performance
liquid chromatography, ultraviolet absorption spectroscopsy, molar rotation,
solubility and
quantitated by solid phase Edman degradation.
The invention also provides polynucleotides which encode the hLPAAT
polypeptide of the invention. As used herein, "polynucleotide" refers to a
polymer of
deoxyribonucleotides or ribonucleotides in the form of a separate fragment or
as a
component of a larger construct. DNA encoding the polypeptide of the invention
can be
assembled from cDNA fragments or from oligonucleotides which provide a
synthetic gene
which is capable of being expressed in a recombinant transcriptional unit.
Polynucleotide
sequences of the invention include DNA, RNA and cDNA sequences. Preferably,
the
nucleotide sequence encoding hLPAAT is the sequence of SEQ m NO. 1 for hLPAATa
or SEQ ID NO. 7 for LPAAT(3. DNA sequences of the present invention can be
obtained
by several methods. For example, the DNA can be isolated using hybridization
procedures
which are known in the art. Such hybridization procedures include, for
example,
hybridization of probes to genomic of cDNA libraries to detect shared
nucleotide
sequences, antibody screening of expression libraries to detect shared
structural features,
such as a common antigenic epitope, and synthesis by the polymerase chain
reaction
(PCR).
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Hybridization procedures are useful for screening of recombinant clones by
using
labeled mixed synthetic oligonucleotides probes, wherein each probe is
potentially the
complete complement of a specific DNA sequence in a hybridization sample which
incIudes a heterogeneous mixture of denatured double-stranded DNA. For such
screening,
hybridization is preferably performed on either single-stranded DNA or
denatured double-
stranded DNA. Hybridization is particularly useful for detection of cDNA
clones derived
from sources where an extremely low amount of mRNA sequences relating to the
polypeptide of interest are present. Using stringent hybridization conditions
directed to
avoid non-specific binding, it is possible to allow an autoradiographic
visualization of a
specific cDNA clone by the hybridization of the target DNA to that single
probe in the
mixture, which is its complement (Wallace et al. Nucl. Acid Res. 9:879, 1981
). The
development of specific DNA sequences encoding hLPAAT can also be obtained by
isolation of double-stranded DNA sequences from the genomic DNA, chemical
manufacture of a DNA sequence to provide the necessary codons for the
polypeptide of
interest, and in vitro synthesis of a double-stranded DNA sequence by reverse
transcription
of mRNA isolated for a eukaryotic donor cell In the latter case, a double-
stranded DNA
complement of mRNA is eventually formed which is generally referred to as
cDNA. Of
these three methods for developing specific DNA sequences for use in
recombinant
procedures, the isolation of genomic DNA isolates is the least common. This is
especially
true when it is desirable to obtain the microbial expression of mammalian
polypeptides
due to the presence of introns.
The synthesis of DNA sequences is frequently a method that is preferred when
the
entire sequence of amino acids residues of the desired polypeptide product is
known.
When the entire sequence of amino acid residues of the desired polypeptide is
not known,
direct synthesis of DNA sequences is not possible and it is desirable to
synthesize cDNA
sequences. cDNA sequence isolation can be done, for example, by formation of
plasmid-
or phage-carrying cDNA libraries which are derived from reverse transcription
of mRNA.
mRNA is abundant in donor cells that have high levels of genetic expression.
In the event
of lower levels of expression, PCR techniques are preferred. When a
significant portion of
the amino acid sequence is known, production of labeled single or double
stranded DNA
or RNA probe sequences duplicating a sequence putatively present in the target
cDNA
may be employed in DNA/DNA hybridization procedures, carried out on cloned
copies of
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the cDNA (denatured into a single-stranded form) (Jay et al., Nucl. Acid Res.
11:2325,
1983).
A cDNA expression library, such as lambda gtl l, can be screened indirectly
for -
hLPAATa or hLPAAT(3 polypeptide having at least one epitope, using antibodies
specific
for hLPAATa or hLPAAT~i. Such antibodies can be either polycionally or
monoclonally
derived and used to detect expression product indicative of the presence of
hLPAATa or
hLPAAT~i cDNA.
A polynucleotide sequence can be deduced from the genetic code, however the
degeneracy of the code must be taken into account. Polynucleotides of this
invention
i 0 include sequences which are degenerate as a result of the genetic code.
The
polynucleotides of this invention also include sequences that are degenerate
as a result of
the genetic code. There are 20 natural amino acids, most of which are
specified by more
that one codon (a three base sequence). Therefore, as long as the amino acid
sequences of
hLPAATa and hLPAAT(i results in a functional polypeptide (at least, in the
case of the
sense polynucleotide strand), all degenerate nucleotide sequences are included
in the
invention. The polynucleotide sequence for hLPAATa and hLPAAT(3 also includes
sequences complementary to the polynucleotides encoding hLPAATa and hLPAAT~i
(antisense sequences). Antisense nucleic acids are DNA and RNA molecules that
are
complementary to at least a portion of a specific mRNA molecule (Weintraub,
Sci. Amer.
262:40, 1990). The invention embraces all antisense polynucleotides capable of
inhibiting
the production of hLPAATa and hLPAAT/3 polypeptide. In the cell, the antisense
nucleic
acids hybridize to the corresponding mRNA, forming a double-stranded molecule.
The
antisense nucleic acids interfere with the translation of mRNA since the cell
cannot
translate mRNA that is double-stranded. Antisense oligomers of about 15
nucleotides are
preferred, since they are easily synthesized and are less likely to cause
problems than
larger molecules when introduced into the target hLPAATa and hLPAAT~3-
producing
cell. The use of antisense methods to inhibit translation of genes is known
{e.g., Marcus-
Sakura, Anal. Biochem. 172:289, 1988).
In addition, ribozyme nucleotide sequences for hLPAATa and hLPAAT(3 are
included in this invention. Ribozymes are RNA molecules possessing an ability
to
specifically cleave other single-stranded RNA in a manner analogous to DNA
restriction
endonucleases. Through the modification of nucleotide sequences which encode
such
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RNAs, it is possible to engineer molecules that recognize specific nucleotide
sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn. 260:3030, 1988}. An
advantage of this approach is that only mRNAs with particular sequences are
inactivated-
- because they are sequence-specific.
S There are two basic types of ribozymes, tetrahymena-type (Hasselhoff, Nature
334:S8S, 1988) and "hammerhead-type". Tetrahymena-type ribozymes recognize
sequences which are four bases in length, while "hammerhead-type" ribozymes
recognize
base sequences 11-18 bases in length. The longer the recognition sequence, the
greater the
likelihood that the sequence will occur exclusively in the target mRNA
species.
Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes
for inactivating a specific mRNA species.
Polynucleotide sequences encoding the hLPAATa and hLPAATø polypeptides of
the invention can be expressed in either prokaryotes or eukaryotes. Hosts can
include
microbial {bacterial), yeast, insect and mammalian organisms. Methods of
expressing
1 S DNA sequences having eukaryotic or viral sequences in prokaryotes are
known in the art.
Biologically functional viral and plasmid DNA vectors capable of expression
and
replication in a host are known in the art. Such vectors are used to
incorporate DNA
sequences of the invention. DNA sequences encoding the inventive polypeptides
can be
expressed in vitro by DNA transfer into a suitable host using known methods of
transfection.
The hLPAAToc and hLPAAT(3 DNA sequences may be inserted into a recombinant
expression vector. The term "recombinant expression vector" refers to a
plasmid, virus or
other vehicle that has been manipulated by insertion or incorporation of the
genetic
sequences. Such expression vectors contain a promoter sequence which
facilitates
efficient transcription of the inserted genetic sequence of the host. The
expression vector
typically contains an origin of replication, a promoter, as well as specific
genes which
allow phenotypic selection of the transformed cells. Vectors suitable for use
in the present
invention include, for example, with bacterial promoter and ribosome binding
site
expression vector for expression in bacteria (Gold, Meth. Enzymol. 185:11,
1990),
expression vector with animal promoter and enhancer for expression in
mammalian cells
(Kaufrnan, Meth. Enrymol. 185:487, 1990) and baculovirus-derived vectors for
expression
in insect cells {Luckow et al., J. Viro1.67:4566, 1993). The DNA segment can
be present
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in the vector operably linked to regulatory elements, for example, a promoter
(e.g., T7,
metallothionein I, or polyhedren promoters).
The vector may include a phenotypically selectable marker to identify host
cells
which contain the expression vector. Examples of markers typically used in
prokaryotic
expression vectors include antibiotic resistance genes for ampicillin ((3-
lactamases),
tetracycline and chloramphenicol (chloramphenicol acetyltransferase). Examples
of such
markers typically used in mammalian expression vectors include the gene for
adenosine
deaminase (ADA), aminoglycoside phosphotransferase (neo, G418), dihydrofolate
reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase
(TK), and
i 0 xanthine guanine phosphoriboseyltransferase (XGPRT, gpt). .
In another preferred embodiment, the expression system used is one driven by
the
baculovirus polyhedrin promoter. The gene encoding the polypeptide can be
manipulated by
standard techniques in order to facilitate cloning into the baculovirus
vector. See Ausubel et
al., supra. A preferred baculovirus vector is the pBlueBac vector (Invitrogen,
Sorrento, CA).
The vector carrying the gene for the polypeptide is transfected into
Spodoptera frugiperda
(Sf~) cells by standard protocols, and the cells are cultured and processed to
produce the
recombinant polypeptide. See Summers et al., A Manual for Methods of
Baculovirus
Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental
Station.
Once the entire coding sequence of the gene for the polypeptides has been
determined, the gene can be inserted into an appropriate expression system.
The gene can be
expressed in any number of different recombinant DNA expression systems to
generate large
amounts of polypeptide. Included within the present invention are polypeptides
having
native glycosylation sequences, and deglycosylated or unglycosylated
polypeptides prepared
by the methods described below. Examples of expression systems known to the
skilled
practitioner in the art include bacteria such as E. toll, yeast such as Pichia
pastoris,
baculovirus, and mammalian expression systems such as in Cos or CHO cells.
The gene or gene fragment encoding the desired polypeptide can be inserted
into an
expression vector by standard subcloning techniques. In a preferred
embodiment, an E. toll
expression vector is used which produces the recombinant protein as a fusion
protein,
allowing rapid affinity purification of the protein. Examples of such fusion
protein
expression systems are the glutathione S-transferase system (Pharmacia,
Piscataway, N~, the
maltose binding protein system (NEB, Beverley, MA), the thiofusion system
(Invotrogen,
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San Diego, CA), the FLAG system (IBI, New Haven, CT), and the 6xHis system
(Qiagen,
Chatsworth, CA). Some of these systems produce recombinant polypeptides
bearing only a
small number of additional amino acids, which are unlikely to affect the LPAAT
ability of
the recombinant polypeptide. For example, both the FLAG system and the 6xHis
system
S add only short sequences, both of which are known to be poorly antigenic and
which do not
adversely affect folding of the polypeptide to its native conformation. Other
fusion systems
produce proteins where it is desirable to excise the fusion partner from the
desired protein.
In a preferred embodiment, the fusion partner is linked to the recombinant
polypeptide by a
peptide sequence containing a specific recognition sequence for a protease.
Examples of
suitable sequences are those recognized by the Tobacco Etch Virus protease
(Life
Technologies, Gaithersburg, MD) or Factor Xa (New England Biolabs, Beverley,
MA) or
enterokinase (Invotrogen, San Diego, CA).
Production of Polypeptides
In a preferred embodiment, recombinant proteins are expressed in E. coli and
in
i 5 baculovirus expression systems. The complete gene for the polypeptide can
be expressed or,
alternatively, fragments of the gene encoding antigenic determinants can be
produced. In a
first preferred embodiment, the gene sequence encoding the polypeptide is
analyzed to detect
putative transmembrane sequences. Such sequences are typically very
hydrophobic and are
readily detected by the use of standard sequence analysis software, such as
MacDNASIS
(Hitachi, San Bn.mo, CA). The presence of transmembrane sequences is often
deleterious
when a recombinant protein is synthesized in many expression systems,
especially E. coli, as
it leads to the production of insoluble aggregates which are difficult to
renature into the
native conformation of the polypeptide. Deletion of transmembrane sequences
typically
does not significantly alter the conformation of the remaining polypeptide
structure.
Moreover, transmembrane sequences, being by definition embedded within a
membrane, are
inaccessible as antigenic determinants to a host immune system. Antibodies to
these
sequences will not, therefore, provide immunity to the host and, hence, little
is lost in terms
of immunity by omitting such sequences from the recombinant polypeptides of
the
invention. Deletion of transmembrane-encoding sequences from the genes used
for
expression can be achieved by standard techniques. See Ausubel et al., supra,
Chapter 8.
For example, fortuitously-placed restriction enzyme sites can be used to
excise the desired
gene fragment, or the PCR can be used to amplify only the desired part of the
gene.
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Transformation of a host cell with recombinant DNA may be carried out by
conventional techniques. When the host is prokaryotic, such as E. coli,
competent cells
which are capable of DNA uptake can be prepared from cells harvested after
exponential
growth phases and subsequently treated by a CaCI, method using standard
procedures.
Alternatively, MgCI= or RbCI can be used. Transformation can also be performed
after
forming a protoplast of the host cell or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium
phosphate co-precipitates, conventional mechanical procedures, such as
microinjection,
electroporation, insertion of a plasmid encased in a liposome, or virus
vectors may be
used. Eukaryotic cells can also be cotransformed with DNA sequences encoding
the
hLPAATa and hLPAAT(3 poiypeptides of the invention, and a second foreign DNA
molecule encoding a selectable phenotype, such as the herpes simplex thymidine
kinase
gene. Another method uses a eukaryotic viral vector, such as simian virus 40
(SV40) or
bovine papilloma virus to transiently infect or transform eukaryotic cells and
express the
hLPAATa and hLPAAT~i polypeptides.
Expression vectors that are suitable for production of LPAAT polypeptides
typically
contain (1) prokaryotic DNA elements coding for a bacterial replication origin
and an
antibiotic resistance marker to provide for the growth and selection of the
expression vector
in a bacterial host; (2) eukaryotic DNA elements that control initiation of
transcription, such
as a promoter; and (3) DNA elements that control the processing of
transcripts, such as a
transcription termination/polyadenylation sequence. LPAAT polypeptides of the
present
invention preferably is expressed in eukaryotic cells, such as mammalian,
insect and yeast
cells. Mammalian cells are especially preferred eukaryotic hosts because
mammalian cells
provide suitable post-translational modifications such as glycosylation.
Examples of
mammalian host cells include Chinese hamster ovary cells {CHO-Kl; ATCC CCL61),
rat
pituitary cells (GH,; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma
cells (H-
4-II-E; ATCC CRL1548) SV40-transformed monkey kidney cells (COS-1; ATCC CRL
1650) and marine embryonic cells (NIH-3T3; ATCC CRL 1658). For a mammalian
host,
the transcriptional and transiational regulatory signals may be derived from
viral sources,
such as adenovirus, bovine papilloma virus, simian virus, or the like, in
which the regulatory
signals are associated with a particular gene which has a high level of
expression. Suitable
12
CA 02291516 1999-11-26
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transcriptional and translational regulatory sequences also can be obtained
from mammalian
genes, such as actin, collagen, myosin, and metallothionein genes.
Transcriptional regulatory sequences include a promoter region sufficient to
direct
the initiation of RNA synthesis. Suitable eukaryotic promoters include the
promoter of the
mouse metallothionein I gene (Hamer et al., J. Molec. Appl. Genet.
1:273,1982); the TK
promoter of Herpes virus (McKnight, Cell 31: 355, 1982); the SV40 early
promoter (Benoist
et al., Nature 290:304, 1981}; the Rous sarcoma virus promoter (Gonnan et al.,
Proc. Nat'I.
Acid. Sci. USA 79:6777, 1982); and the cytomegalovirus promoter (Foecking et
al., Gene
45:1 Ol, 1980). Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA
I 0 polymerise promoter, can be used to control fusion gene expression if the
prokaryotic
promoter is regulated by a eukaryotic promoter (Zhou et al., Mol. Cell. Biol.
10:4529, 1990;
Kaufinan et aL, Nucl. Acids Res. 19:4485, 1991 ).
An expression vector can be introduced into host cells using a variety of
techniques
including calcium phosphate transfection, liposome-mediated transfection,
electroporation,
15 and the like. Preferably, transfected cells are selected and propagated
wherein the expression
vector is stably integrated in the host cell genome to produce stable
transformants.
Techniques for introducing vectors into eukaryotic cells and techniques for
selecting stable
transformants using a dominant selectable marker are described, for example,
by Ausubel
and by Murray {ed.), Gene Transfer and Expression Protocols (Humana Press 1991
).
20 Examples of mammalian host cells include COS, BHK, 293 and CHO cells.
Purification of Recombinant Polypeptides.
The polypeptide expressed in any of a number of different recombinant DNA
expression systems can be obtained in large amounts and tested for biological
activity. The
recombinant bacterial cells, for example E. toll, are grown in any of a number
of suitable
25 media, for example LB, and the expression of the recombinant polypeptide
induced by
adding IPTG to the media or switching incubation to a higher temperature.
After culturing
the bacteria for a further period of between 2 and 24 hours, the cells are
collected by
centrifugation and washed to remove residual media. The bacterial cells are
then lysed, for
example, by disruption in a cell homogenizer and centrifuged to separate the
dense inclusion
30 bodies and cell membranes from the soluble cell components. This
centrifugation can be
performed under conditions whereby the dense inclusion bodies are selectively
enriched by
incorporation of sugars such as sucrose into the buffer and centrifugation at
a selective speed.
13
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If the recombinant polypeptide is expressed in the inclusion, these can be
washed in any of
several solutions to remove some of the contaminating host proteins, then
solubilized in
solutions containing high concentrations of urea (e.g., 8 M) or chaotropic
agents such as
guanidine hydrochloride in the presence of reducing agents such as 13-
mercaptoethanol or
DTT (dithiothreitol). At this stage it may be advantageous to incubate the
polypeptide for
several hours under conditions suitable for the polypeptide to undergo a
refolding process
into a conformation which more closely resembles that of the native
polypeptide. Such
conditions generally include low polypeptide (concentrations less than 500
mg/ml), low
levels of reducing agent, concentrations of urea less than 2 M and often the
presence of
reagents such as a mixture of reduced and oxidized glutathione which
facilitate the
interchange of disulphide bonds within the protein molecule. The refolding
process can be
monitored, for example, by SDS-PAGE or with antibodies which are specific for
the native
molecule. Following refolding, the polypeptide can then be purified further
and separated
from the refolding mixture by chromatography on any of several supports
including ion
exchange resins, gel permeation resins or on a variety of affinity columns.
Isolation and purif cation of host cell expressed polypeptide, or fragments
thereof
may be carried out by conventional means including, but not limited to,
preparative
chromatography and immunological separations involving monoclonal or
polyclonal
antibodies.
These polypeptides may be produced in a variety of ways, including via
recombinant DNA techniques, to enable large scale production of pure, active
hLPAATa
and hLPAAT(3 useful for screening compounds for trilineage hematopoietic and
anti-
inflammatory therapeutic applications, and developing antibodies for
therapeutic,
diagnostic and research use.
The hLPAATa and hLPAAT(3 polypeptides of the present invention are useful in a
screening methodology for identifying compounds or compositions which affect
cellular
signaling of an inflammatory response. This method comprises incubating the
hLPAATa
and hLPAAT(3 polypeptides or a cell transfected with cDNA encoding hLPAATa and
hLPAAT(3 under conditions sufficient to allow the components to interact, and
then
measuring the effect of the compound or composition on hLPAATa and hLPAAT(3
activity. The observed effect on hLPAATa and hLPAAT[3 may be either inhibitory
or
stimulatory.
14
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WO 98/54303 PCT/US97105360
hLPAATa
Search of the Genbank database of expressed sequence tag (dbest) using either
the
yeast or plant LPAAT protein sequences as probe came up with several short
stretches of
cDNA sequences with homology to the yeast or plant LPAAT protein sequence.
These
cDNA sequences of interest were derived from single-run partial sequencing of
random
- human cDNA clones projects carried out by either the WashU-Merck EST or the
Genexpress-Genethon program. An example of the amino acids sequence homology
between the yeast LPAAT and a human cDNA clone (dbest#102250) is shown below
by
comparing SEQ ID NO. 3 (top amino acid sequence) with SEQ ID NO 4 (bottom
amino
IO acid sequence):
PFKKGAFHLAQQGKIPIVPVVVSNTSTLVSPKYGVFNRGCMIVRILKPIST
E
* ****** * **** * * * * * ** * * **
PSNCGAFHLAVQAQVPIVPIVMSSYQDFYCKKERRFTSGQCQVRVLPPVPT
IS E
The top line refers to the yeast LPAAT sequence from amino acids 169 to 220
and
the bottom line refers to the homologous region from the dbest clone#102250.
Identical
amino acids between these two sequences are shown in block letters with
asterisks in
between
20 Accordingly, a synthetic oligonucleotide (o.BLPAT.2R), S'-
TGCAAGATGGAAGGCGCC-3' (SEQ ID NO. 5), was made based on the complement
sequence of the conserved amino acids region, GAFHLA (SEQ >D NO. 6), of
clone#102250. o.BPLAT.2R was radiolabeled at its 5'-end using Y-32p-ATP and T4
polynucleotide kinase as a probe in screening a ,zap human brain cDNA library
25 (Stratagene).
Screening of the cDNA library was accomplished by filter hybridization using
standard methods (Current Protocols in Molecular Biology, John Wiley & Sons,
Inc.,
1995). Duplicate filters containing DNA derived from ~, phage plagues were
prehybridized at 60 °C for 2 hr in 6X SSC (I X SSC is 0.15 M NaCI,
0.015 M sodium
30 citrate, pH 7.0), SX Denhardt's solution (1X Denhardt's solution is 0.02%
Ficoll, 0.02%
bovine serum albumin, and 0.02% polyvinyl-pyrrolidone), 0.1% sodium dodecyl
sulfate
CA 02291516 1999-11-26
WO 98/54303 PCT/US97/05360
(SDS), 50 mg/ml sonicated and denatured salmon sperm DNA. Hybridization was
carried
out in the same buffer as used for prehybridzation. After hybridization, the
filters were
washed in 6 X SSC at 42 °C, and autoradiographed.
Of the approximately 1 X 106 clones from the human brain cDNA library that
were screened, twelve clones were identified that hybridized with the probe in
duplicate
filters. Eleven out twelve clones were enriched and recovered after a
secondary screen.
Ten enriched phage samples were then converted to plasmid transformed cells by
co-
infecting E. coli XLl-Blue with the helper phage 8408 using Stratagene's
recommended
procedure. Colony filter hybridization was performed and identified those
colonies that
"lit up" with the probe. Seven out of the ten pools of colonies contained
positive clones.
Two out of these seven clones, pZlpat.l0 and pZlpat.l l, contained inserts >2
kb.
Restriction mapping using a combination of Sst I, Pst I and BamHI digests
showed these
two clones contained many common fragments with respect to each other.
Nucleotide sequencing of the cDNA inserts in pZlpat.l0 and pZlpat.l 1 was
i 5 performed. Figure 1 shows the DNA sequence of the cDNA insert of pZplat. l
l . The
nucleotide sequence analysis and restriction mapping of the cDNA clone
revealed a 5'-
untranslated region of >300 bp, an open reading frame capable of encoding a
283 amino
acid polypeptide, and a 3'-untranslated region of >800 bp. The initiation site
for
translation was localized at nucleotide positions 319-321 and fulfilled the
requirement for
an adequate initiation site according to Kozak (Kozak, Critical Rev. Biochem.
Mol. Biol.
27:385-402, 1992). There was another upstream ATG at positions 131-133 with an
in-
phase stop codon at positions 17b-178. Except with a shorter S'-untranslated
region, the
cDNA insert of pZplat.10 has the same DNA sequence as that of pZplat. l l .
The sequence of the 283 amino acid open reading frame in pZplat. l I was used
as
the query sequence to search for homologous sequences in protein databases.
Search of
the database based on Genbank Release 90 from the National Center for
Biotechnology
Information (NCBI) using the blastp program showed that the protein encoded by
pZplat.l 1 was most homologous to the yeast and bacterial LPAATs. Figure 2
shows
amino acid sequences alignment of the putative human LPAATa coding sequence,
the
yeast LPA.AT coding sequence, the E. toll LPAAT coding sequence, and the maize
LPAAT coding sequence, revealing that human LPAATa has a much more extended
homology with the yeast or the E. toll LPAAT than with the plant LPAAT.
16
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WO 98/54303 PCT/US97/05360
hLPAAT
Search of the Genbank database (Boguski, et al., Science 265:1993-1994, 1994)
of
expressed sequence tag (dbEST) using either the yeast or plant LPAAT protein
sequences
as probe came up with several short stretches of cDNA sequences with homology
to the
yeast or plant LPAAT protein sequence. These cDNA sequences of interest were
derived
from single-run partial sequencing of random human cDNA clones projects
carried out
mainly by LM.A.G.E. Consortium [LLNL] cDNA clones program. An example of the
amino acids sequence homology between the yeast LPAAT and a human cDNA clone
(dbEST#363498) is shown below:
180 190 200 210 220 230
QQGKIPIVPVWSNTSTLVSPKYGVFNRGCMIVRILKPISTENLTKDKIGEFAEKVRDQM
VRENVPIVPVVYSSFSSFYNTKKKFFTSGTVTVQVLEAIPTSGLTAADVPALRGTPATGP
70 80 90 100 110
120
The top line refers to the yeast LPAAT sequence from amino acids 171 to 230
(SEQ ID NO. 9) and the bottom line refers to the homologous region from the
dbest
clone#363498 using the +1 reading frame (SEQ ID NO. 10). Identical and
conserved
amino acids between these two sequences are shown with double dots and single
dot,
respectively, in between. In order to find out if such cDNA clones with
limited homology
to yeast LPAAT sequence indeed encode human LPAAT[3 sequence, it was necessary
to
isolate the full-length cDNA clone, insert it into an expression vector, and
to test if cells
transformed or transfected with the cDNA expression vector produced more LPAAT
activity.
Accordingly, two synthetic oligonucleotides, 5'-CCTCAAAGTG
TGGATCTATC-3' (o.LPAT3.F) {SEQ ID NO. 11 ) and S'-GGAAGAGTAC
ACCACGGGGA C-3' (o.LPAT3.R), {SEQ ID NO. 12) were ordered (Life Technologies,
Gaithersburg, MD) based on, respectively, the coding and the complement
sequence of
clone#363498. o.LPAT3.R was used in combination with a forward vector primer
(o.sport.l), 5'- GACTCTAGCC TAGGCTTTTG C-3'(SEQ ID NO. 13) for amplification
of the 5'-region, while o.LPAT3.F was used in combination with a reverse
vector primer
(o.sport.lR), 5'-CTAGCTTATA ATACGACTCA C-3' (SEQ ID NO. 14), for
amplification of the 3'-region of potential LPAAT[3 sequences from a
pCMV.SPORT
human leukocyte cDNA library (Life Technologies, Gaithersburg, MD). A 700 by
PCR
17
CA 02291516 1999-11-26
WO 98/54303 PCTIUS97/05360
fragment derived from o.sport.l and o.LPAT3.R amplification was cut with EcoR
I before
inserting in between the Sma I and EcoR I of pBluescript(II)SK(-) (Stratagene,
LaJolla,
CA) to generate pLPAT3.5'. A 900 by PCR fragment derived from o.sport.lR and
o.LPAT3.F amplification was cut with Xba I before inserting in between the Sma
I and
Xba I of pBluescript(II)SK(-) (Stratagene, LaJolla, CA) to generate pLPAT3.3'.
Nucleotide sequencing analysis of the cDNA inserts from these two plasmids
showed they
contained overlapping sequences with each other, sequences that matched with
the
dbEST#363498 as well as extensive homology with the yeast LPAAT amino acids
sequence (Nagiec et al., .l. Biol. Chem. 268:22156-22163, 1993). To assemble
the two
halves of the cDNA into a full-length clone, the 560 by Nco I - Nar I fragment
from
pLPAT3.5' and the 780 by Nar I - Xba I fragment from pLPAT3.3' were inserted
into the
Nco I l Xba I vector prepared from pSP-luc+ (promega, Madison, WI) via a three-
part
ligation to generate pSP.LPAT3.
Figure 3 shows the DNA sequence ID of the cDNA insert of pSP.LPAT3. The
I S nucleotide sequence analysis and restriction mapping of the cDNA clone
revealed a S'-
untranslated region of 39 bp, an open reading frame capable of encoding a 278
amino
acids polypeptide that spans nucleotide positions 40 to 876 and a 3'-
untranslated region of
480 by (Figure 3). The initiation site for translation was localized at
nucleotide positions
40-42 and fulfilled the requirement for an adequate initiation site according
to Kozak
(Kozak, Critical Rev. Biochem. Mol. Biol. 27:385-402, 1992).
The sequence of the 278 amino acid open reading frame (Figure 4) was used as
the
query sequence to search for homologous sequences in protein databases. Search
of the
database based on Genbank Release 92 from the National Center for
Biotechnology
Information (NCBI) using the blastp program showed that this protein was most
homologous to the yeast, bacterial and plant LPAATs. Figure 5 shows amino acid
sequences alignment of this putative human LPAAT~i coding sequence, human
LPAATa.
coding, the yeast LPAAT coding sequence, the bacterial (E. toll, H. in,
fluenzae, and S.
typhimurium) LPAAT coding sequences, and the plant (L. douglassi and C.
nucifera)
LPAAT coding sequences, revealing that the human LPAAT coding sequences have a
much more extended homology with the yeast or the bacterial LPAAT than with
the plant
LPAAT.
Characterization of the Invention
i8
CA 02291516 1999-11-26
WO 98/54303 PCT/US97/05360
Accordingly, human LPAATa is characterized by the 283 amino acids of SEQ ID
NO. 2. The present invention further includes allelic variations {naturally-
occurnng base
changes in the species population which may or may not result in an amino acid
change)
of the DNA sequences herein encoding active LPAAT polypeptides and active
fragments
thereof. The inventive DNA sequences further comprise those sequences which
hybridize
under stringent conditions (see, for example, Maniatis et al, Molecular
Coining (A
Laboratory Manual), Cold Spring Harbor Laboratory, pages 387-389, 1982) to the
coding
region (e.g., nucleotide #319 to nucleotide #1167). One such stringent
hybridization
condition is, for example, 4 X SSC at 65 °C, followed by washing in 0.1
X SSC at 65 °C
for thirty minutes. Alternatively, another stringent hybridization condition
is in 50%
formamide, 4 X SSC at 42 °C. The present invention further includes DNA
sequences
which code for LPAAT polypeptides having LPAAT activity but differ in codon
sequence
due to degeneracy of the genetic code. Variations in the DNA sequences which
are caused
by point mutations or by induced modifications of the sequence of SEQ ID NO.
l, which
enhance the activity of the encoded polypeptide or production of the encoded
LPAAT
polypeptide are also encompassed by the present invention.
Definitions
In the description that follows, a number of terms are utilized extensively.
Definitions are provided to facilitate understanding of the invention.
The term "isolated" applied throughout the specification to polypeptides
refers to that
level of purity in which the polypeptide is sufficiently free of other
materials endogenous to
the host from which the polypeptide is isolated such that any remaining
materials do not
materially affect the biological properties of the polypeptide.
The term "derived" as used throughout the specification in relation to the
polypeptides of the present invention, encompasses polypeptides obtained by
isolation and
purification from host cells, as well as polypeptides obtained by manipulation
and expression
of nucleotide sequences prepared from host cells. It also encompasses
nucleotide sequences
including genomic DNA, mRNA, cDNA synthesized from mRNA, and synthetic
oligonucleotides having sequences corresponding to the inventive nucleotide
sequences. It
further encompasses synthetic polypeptide antigens prepared on the basis of
the known
amino acid sequences of the proteins of the present invention.
The term "expression product" as used throughout the specification refers to
19
CA 02291516 1999-11-26
WO 98/54343 PCT/US97/05360
materials produced by recombinant DNA techniques.
Peptide seauencing of polypeptides
Purified polypeptides prepared by the methods described above can be sequenced
using methods well known in the art, for example using a gas phase peptide
sequencer
(Applied Biosystems, Foster City, CA). Because the proteins of the present
invention may
be glycosylated, it is preferred that the carbohydrate groups are removed from
the proteins
prior to sequencing. This can be achieved by using glycosidase enzymes.
Preferably,
glycosidase F (Boehringer-Mannheim, Indianapolis, Il~ is used. To determine as
much of
the polypeptide sequence as possible, it is preferred that the polypeptides of
the present
invention be cleaved into smaller fragments more suitable for gas-phase
sequence analysis.
This can be achieved by treatment of the polypeptides with selective
peptidases, and in a
particularly preferred embodiment, with endoproteinase lys-C (Boehringer). The
fragments
so produced can be separated by reversed-phase HPLC chromatography.
Substitutional variants typically contain the exchange of one amino acid for
another
1 S at one or more sites within the protein, and are designed to modulate one
or more properties
of the polypeptides such as stability against proteolytic cleavage.
Substitutions preferably
are conservative, that is, one amino acid is replaced with one of similar
shape and charge.
Conservative substitutions are well known in the art and include, for example,
the changes
of alanine to serine; arginine to lysine; asparigine to glutamine or
histidine; aspartate to
glutamate; cysteine to serine; glutamine to asparigine; glutamate to
aspartate; glycine to
praline; histidine to asparigine or glutamine; isoleucine to leucine or
valine; leucine to valine
or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to
leucine or
isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to
threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and
valine to
isoleucine or leucine. Insertional variants contain fusion proteins such as
those used to allow
rapid purification of the polypeptide and also can include hybrid polypeptides
containing
sequences from other proteins and polypeptides which are homologues of the
inventive
polypeptide. For example, an insertional variant could include portions of the
amino acid
sequence of the polypeptide from one species, together with portions of the
homologous
polypeptide from another species. Other insertional variants can include those
in which
additional amino acids are introduced within the coding sequence of the
polypeptides. These
CA 02291516 1999-11-26
WO 98/54303 PCT/US97I05360
typically are smaller insertions than the fusion proteins described above and
are introduced,
far example, to disrupt a protease cleavage site.
Antibodies to human LPAAT protein can be obtained using the product of an
LPAAT expression vector or synthetic peptides derived from the LPAAT coding
sequence
coupled to a carrier (Pasnett et al., J. Biol. Chem. 263:1728, 1988) as an
antigen. The
preparation of polyclonal antibodies is well-known to those of skill in the
art. See, for
example, Green et al., "Production of Polycional Antisera," in Immunochemical
Protocols
(Manson, ed.), pages 1-5 (Humana Press 1992). Alternatively, an LPAAT antibody
of the
present invention may be derived from a rodent monoclonal antibody (MAb).
Rodent
monoclonal antibodies to specific antigens may be obtained by methods known to
those
skilled in the art. See, for example, Kohler and Milstein, Nature 256:495,
1975, and Coligan
et al. (eds.), Current Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley &
Sons 1991).
Briefly, monoclonal antibodies can be obtained by injecting mice with a
composition
comprising an antigen, verifying the presence of antibody production by
removing a serum
sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes
with
myeloma cells to produce hybridomas, cloning the hybridomas, selecting
positive clones
which produce antibodies to the antigen, culturing the clones that produce
antibodies to the
antigen, and isolating the antibodies from the hybridoma cultures.
MAbs can be isolated and purified from hybridoma cultures by a variety of well-
established techniques. Such isolation techniques include affinity
chromatography with
Protein-A Sepharose, size-exclusion chromatography, and ion-exchange
chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also,
see Baines et
al., "Purification of Immunoglobulin G (IgG)," in Methods in Molecular
Biology, 10:79-104
Humana Press, Inc. 1992. An LPAAT antibody of the present invention may also
be derived
from a subhuman primate antibody. General techniques for raising
therapeutically useful
antibodies in baboons may be found, for example, in Goldenberg et a1,
international patent
publication No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer
46:310, 1990.
Alternatively, a therapeutically useful LPAAT antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies are produced
by
transferring mouse complementary determining regions from heavy and light
variable chains
of the mouse immunoglobulin into a human variable domain, and then,
substituting human
residues in the framework regions of the marine counterparts. The use of
antibody
21
CA 02291516 1999-11-26
WO 98154303 PCT/US97/05360
components derived from humanized monoclonal antibodies obviates potential
problems
associated with the immunogenicity of marine constant regions. General
techniques for
cloning marine immunoglobulin variable domains are described, for example, by
the
publication of Orlandi et al., Proc. Nat'l. Acad. Sci. USA 86:3833, 1989.
Techniques for
producing humanized MAbs are described, for example, by Jones et al., Nature
321:522,
1986, Riechmann et al., Nature 332:323, 1988, Verhoeyen et al., Science
239:1534, 1988,
Carter et al., Proc. ~Vat'I Acad. Sci. USA 89:4285, 1992, Sandhu, Crit. Rev.
Biotech. 12: 437,
1992, and Singer et al., J. Immun. 150:2844, 1993, each of which is hereby
incorporated by
reference.
I O As an alternative, an LPAAT antibody of the present invention may be
derived from
human antibody fragments isolated from a combinatorial immunoglobulin library.
See, for
example, Barbas et al., METHODS. A Companion to Methods in Enzymology 2:119
1991,
and Winter et al., Ann. Rev. Immunol. 12:433, 1994, which are incorporated by
reference.
Cloning and expression vectors that are useful for producing a human
immunoglobulin
phage library can be obtained, for example, from STRATAGENE Cloning Systems
(La
Jolla, CA). In addition, an LPAAT antibody of the present invention may be
derived from a
human monoclonal antibody. Such antibodies are obtained from transgenic mice
that have
been "engineered" to produce specific human antibodies in response to
antigenic challenge.
In this technique, elements of the human heavy and light chain locus are
introduced into
strains of mice derived from embryonic stem cell lines that contain targeted
disruptions of
the endogenous heavy chain and light chain loci. The transgenic mice can
synthesize human
antibodies specific for human antigens, and the mice can be used to produce
human
antibody-secreting hybridomas. Methods for obtaining human antibodies from
transgenic
mice are described by Green et al., Nature Genet. 7:13, 1994; Lonberg et al.,
Nature
368:856, 1994, and Taylor et al., Int. Immun. 6:579, 1994.
Example 1
This example illustrates an experiment to determine if the human LPAATa clone
encodes a protein with LPAAT activity, an E. coli vector expressing the human
LPAATa.
as a fusion protein with (3-galactosidase was transformed into a LPAAT minus
strain ofE.
toll to see if it would complement the defect in E. toll. Specifically, the
840 by Bgl II-
Nco I fragment, which spans the coding region of human LPAATcc from amino acid
68 to
22
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WO 98/54303 PCT/US97/05360
beyond the stop codon, derived from pZplat. l l was inserted into a Bgl II l
Nco I digested
cloning vector pLitmus28 (Evans et al., BioTechniques 19:130-I35, 1995) to
generate the
plasmid p28BgN. This plaslilid is expected to express the human LPAATa as a
fusion
protein containing the first 16 amino acids of ~i-galactosidase and the last
216 residues of
the human LPAATa coding sequence using the lac promoter in pLitmus28. This
plasmid
was transformed into the E. coli strain JC201 (obtained from Dr. Jack Coleman,
Louisiana
State University). JC201 (Coleman, Mol. Gen. Genet. 232:295-303, 1992; Nagiec
et al., J.
Biol. Chem. 268:22156-22163, 1993; and Brown et al., Plant Mol. Biol. 26:211-
223, 1994)
is deficient in LPAAT activity due to mutation in the plsC locus. This
mutation leads to a
temperature-sensitive phenotype that causes JC201 to grow slowly at 37
°C, almost not at
all at 42 °C, and not at all at 44 °C. JC201 transformed with
p28BgN was able to grow
normally at 44 °C when compared to the wild type strain JC200 {pisC+),
while JC201
transformed with pLitmus28 vector was not able to support growth at 44
°C. These data
suggest that the putative human LPAATa cDNA isolated here does possess LPAAT
activity, as the Iast 216 amino acids of this cDNA is sufficient to complement
the defective
LPAAT gene (plsC) in JC201.
Example 2
To see if the putative human LPAAT(3 clone encodes a protein with LPAAT
activity, an E. coli vector expressing this human LPAAT/3 as a direct product
was
transformed into a LPAAT minus strain of E. coli to see if it would complement
the defect
in E. coli. Specifically, the 1350 by Nco I - Xba I fragment from pSP.LPAT3,
which spans
the entire coding region from amino acid 1 to beyond the stop codon, was
inserted into a
Nco I l Xba I digested cloning vector pKK388-1 (Clontech, Palo Alto, CA) to
generate the
plasmid pTrc.LPAT3. This plasmid was transformed into the E. coli strain JC201
(obtained from Dr. Jack Coleman, Louisiana State University). JC201 (Coleman,
Mol.
Gen. Genet. 232:295-303, 1992) is deficient in LPAAT activity due to mutation
in the
plsC locus. This mutation leads to a temperature-sensitive phenotype that
causes JC201 to
grow slowly at 37 °C, almost not at all at 42 °C, and not at all
at 44 °C. JC201
transformed with pTrc.LPAT3 was able to grow normally at 44 °C when
compared to the
wild type strain JC200 (plsC+), while JC201 transformed with pKK388-1 vector
was not
able to support growth at 44 °C. These data suggest that the putative
human LPAAT(3
23
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WO 98/54303 PCTlUS97/05360
cDNA isolated here does possess LPAAT activity, as the putative protein
product of this
cDNA is able to complement the defective LPAAT gene (plsC) in JC201.
Example 3
This example illustrates a group of experiments to see if overexpression of
this
human LPAATa, would have any effect on mammalian cells. The entire cDNA insert
(2,300 bp) from pZplat.l l was cleaved with Asp718 I and Xho I for insertion
into the
mammalian expression vector pCE9 to generate pCE9.LPAAT 1. pCE9 was derived
from
pCE2 with two modifications. The 550 by BstY I fragment within the elongation
factor-
1 a (EF-1 a) intron of pCE2 was deleted. The multiple cloning region of pCE2
between the
Asp718 I and BamH I site was replaced with the multiple cloning region
spanning the
Asp718 I and Bgl II sites from pLitmus28. The plasmid pCE2 was derived from
pREP7b
(Leung, et al., Proc. Natl. Acad. Sci. USA, 92: 4813-4817, 1995) with the RSV
promoter
region replaced by the CMV enhancer and the elongation factor-1 a (EF-1 a)
promoter and
intron. The CMV enhancer came from a 380 by Xba I-Sph I fragment produced by
PCR
from pCEP4 (Invitrogen, San Diego, CA) using the primers 5'-GGCTCTAGAT
ATTAATAGTA ATCAATTAC-3' and 5'-CCTCACGCAT GCACCATGGT AATAGC-
3'. The EF-1 a promoter and intron (LJetsuki, et al., J. Biol. Chem., 264:
5791-5798, 1989)
came from a 1200 by Sph I-Asp718 I fragment produced by PCR from human genomic
DNA using the primers 5'-GGTGCATGCG TGAGGCTCCG GTGC-3' and 5'-
GTAGTTTTCA CGGTACCTGA AATGGAAG-3'. These 2 fragments were ligated into a
Xba I/Asp718 I digested vector derived from pREP7b'to generate pCE2.
pCE9.LPAATI DNA was transfected into several mammalian cell lines, including
A549 cells, ECV304 cells (American Type Culture Collection, Rockville, MD),
two
human cell line that would produce IL-6 and TNF upon stimulation with IL-Ib
and murine
TNF and 293-EBNA cells (Invitrogen, San Diego, CA). pCE9.LPAAT1 was digested
with
BspH I before electroporating into these cell lines with a Cell-PoratorTM
(Life
Technologies, Gaithersburg,1VID) using conditions described previously
(Cachianes, et al.,
Biotechniques 15:255-259, 1993). After adherence of the transfected cells 24
hours later,
the cells were grown in the presence of 200 pg / ml Hygromycin B (Hyg)
(Calbiochem, La
Jolla, CA) to select for cells that had incorporated both plasmids. Hyg-
resistant clones that
24
CA 02291516 1999-11-26
WO 98/54303 PCT/LJS97/05360
expressed LPAAT mRNA at a level more than 20 fold higher relative to
untranfected cells
based on Northern Blot analysis (Kroczek, et al., Anal. Biochem. 184: 90-95,
1990) were
selected for further study.
Figure 6 compares the LPAAT activity in A549 cells and in A549 cells
transfected
with pCE9.LPAAT1 DNA using aTLC assay. This screening assay for LPAAT activity
in
cell extracts was based on a fluorecent assay using fluorecent lipid
substrates (Ella, et al.,
Anal. Biochem. 218: 136-142, 1994). Instead of using the PC-substrate, BPC
(Molecular
Probes, Eugene, OR), a synthetic PC that contains an ether linkage at the SN1
position
with a fluorescent Bodipy moiety incorporated into the end of the alkyl-chain
at the SN1
position, BPC was converted to Bodipy-PA using cabbage phospholipase D (Sigma,
St.
Louis, MO). Bodipy-PA was then converted to Bodipy-LPA using snake venom
phospholipase A2. The Bodipy-LPA obtained was purif ed by preparative TLC for
use in
the LPAAT assay. The assay was carried out in total cell extracts resuspended
in lysis
buffer (Ella, et al., Anal. Biochem. 218: 136-142, 1994) supplemented with 0.5
mM ATP,
0.3 mM MgClz, 100 pM oleoyl-CoA and 10 uM Bodipy LPA. The samples were
incubated for 30 min before loading onto TLC plates.
Lane 1 refers to Bodipy LPA incubated with buffer only without any cell
extract
added. Lane 9 refers to BPC treated with cabbage phospholipase D for
generateing a
Bodipy-PA marker. Lanes 2 and 4 refer to to Bodipy LPA incubated with control
A549
cell extracts with or without lipid A, respectively. Lanes 3 and 5 refer to
Bodipy LPA
incubated with A549 cell extracts transfected with pCE9.LPAAT1 DNA with or
without
lipid A, respectively. Figure 3 shows A549 cells transfected with the LPAAT
cDNA
(lanes 3 and 5) contain much more LPAAT activity than those of control cells
(lanes 2 and
4) as evidenced by the increased conversion of Bodipy-LPA to Bodipy-PA.
Addition of
lipid A to the cell extracts has little effect on LPAAT activity (lanes 2 vs 4
and 3 vs 5).
A549 cell extract also contains a phosphohydrolase activiity that converts
Bodipy-LPA to
Bodipy-monoalkylglycerol (lanes 2 to S). Interestingly, A549 cells
overexpressing
LPAAT (lanes 3 and 5) have less of this activiity compared to control cells
(lanes 2 and 4),
suggesting this phosphohydrolase prefers LPA to PA as substrate. There is also
an
increase of DAG in transfected cells (lanes 3 and 5) compared to control cells
(lanes 2 and
CA 02291516 1999-11-26
WO 98/54303 PCTIUS97/05360
4) possibly due to partial conversion of the PA formed to DAG from this
endogenous
phosphohydrolase.
Example 4
To see if the expressed LPAAT cDNA clone described here would also use other
glycerol-lipids that contain a free-hydroxyl group at the SN2 position, the
cell extracts
were incubated with the substrates NBD-IysoPC (lanes 6 and 7} and NBD-
monoacylglycerol (MAG) (lanes 10 and I I) to see if there is increased
conversion to
lysoPC and DAG, respectively. Lane 8 and 12 refer, respectively, to NBD-lysoPC
and
NBD-MAG incubated with buffer only without any cell extract added. TLC
analysis
shows little difference in the lipid profile between the transfected and
control cells (lanes 7
vs 6, lanes I lvs 10), suggesting the cloned LPAAT enzyme uses LPA as the
preferred
substrate. It is likely that the acyltransferases for lysoPC (Fyrst, et al.,
Biochem. J.
306:793-799, 1995) and for MAG (Bhat, et al., Biochemistry 34: 11237-11244,
1995)
represent different enzymes from the LPAAT described here.
Example 5
pCE9.LPAAT1 DNA was transfected into A549 cells (American Type Culture
Collection, Rockville, MD), a human cell line that would produce IL-6 and TNF
upon
stimulation with IL-1 (3 and marine TNF. pCE9.LPAAT 1 was digested with BspH I
before
electroporating into A549 cells with a Cell-PoratorTM (Life Technologies,
Gaithersburg,
MD) using conditions described previously (Cachianes, et al., Biotechniques
15:255-259,
1993). After adherence of the transfected cells 24 hours later, the cells were
grown in the
presence of 200 ug/ml Hygromycin B (Hyg) (Calbiochem, La Jolla, CA) to select
for cells
that had incorporated both plasmids. A Hyg-resistant clone that expressed
LPAAT mRNA
at a level more than 20 fold higher relative to untranfected A549 cells based
on Northern
Blot analysis (Kroczek et al., Anal. Biochem. 184:90-95, 1990) was selected
for further
study.
A comparison of the production of TNF (Figure 7) and IL-6 (Figure 8) between
A549 cells transfected with pCE9.LPAAT1 and control A549 cells after
stimulation with
IL-1~3 and marine TNF shows A549 overexpressing LPAAT produces >5 fold more
TNF
26
CA 02291516 1999-11-26
WO 98154303 PCT/US97/05360
and > 10 fold more IL-6 relative to untransfected A549 cells, suggesting that
overexpression of LPAAT would enhance the cytokine signaling response in
cells.
Development of compounds that would modulate LPAAT activity should therefore
be of
therapeutic interest in the field of inflammation.
SEQUENCE LISTING
(1) GENERAL
INFORMATION:
(i) APPLICANTS: Leung, David W.
West, James
Tompkins, Christopher
(ii) TITLE OF INVENTION: MAMMALIAN LYSOPHOSPHATIDIC ACID
ACYL TRANSFERASE
(iii ) NUMBER OF SEQUENCES: 18
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Cell Therapeutics, Inc.
{B) STREET: 201 Elliott Avenue West
(C) CITY: Seattle
(D) STATE: Washington
(E) COUNTRY: U.S.A.
(F) ZIP 98119
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5" disk, 1.44Mb, double
side, high density
(B) COMPUTER: PC Clone (486 microprocessor)
(C) OPERATING SYSTEM: MS-DOS Version 6.1,
Windows 3.1
(D) SOFTWARE: WORD 6.0
(vi) CURRENT APPLICATION DATA .
(A) APPLICATION NUMBER:
(B) FILING DATE: 15-Dec-1995
{vii i) ATTORNEY/AGENT INFORMATION:
(A) NAME: Oster, Jeffrey B.
(B) REGISTRATION NUMBER: 32,585
{C) REFERENCE/DOCKET NUMBER:1801
(ix} TELECOMMUNICATION INFORMATION:
(A) TELEPHONE:(206)282-7100
(B) TELEFAX:(206)284-6206
(2) INFORMATION
FOR SEQ
ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:2242
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL:
no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi} ORIGINAL SOURCE:
{A) ORGANISM: homo sapien
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
27
CA 02291516 1999-11-26
WO 98/54303 PCT/US97105360
(D) DEVELOPMENTAL STAGE:
( E ) HAPLOTYPE
(F) TISSUE TYPE: brain
(G) CELL TYPE: -
(H) CELL LINE
(I) ORGANELLE:
( ix) FEATURE
(A) NAME/KEY: hLPAATa
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
1 GGAAGTCAGCAGGCGTTGGGGAGGGGTGGCGGGGGAATAGCGGCGGCAGC
51 AGCCCCAGCCCTCAGAGAGACAGCAGAAAGGGAGGGAGGGAGGGTGCTGG
101 GGGGACAGCCCCCCACCATTCCTACCGCTATGGGCCCAACCTCCCACTCC
151 CACCTCCCCTCCATCGGCCGGGGCTAGGACACCCCCAAATCCCGTCGCCC
201 CCTTGGCACCGACACCCCGACAGAGACAGAGACACAGCCATCCGCCACCA
251 CCGCTGCCGCAGCCTGGCTGGGGAGGGGGCCAGCCCCCCAGGCCCCCTAC
301 CCCTCTGAGGTGGCCAGA ATG GAT TTG TGG CCA GGG GCA TGG
343 ATG CTG CTG CTG CTG CTC TTC CTG CTG CTG CTC TTC C
380 TG CTG CCC ACC CTG TGG TTC TGC AGC CCC AGT GCC AAG
418 TAC TTC TTC AAG ATG GCC TTC TAC AAT GGC TGG ATC C
455 TC TTC CTG GCT GTG CTC GCC ATC CCT GTG TGT GCC GTG
493 CGA GGA CGC AAC GTC GAG AAC ATG AAG ATC TTG CGT C
530 TA ATG CTG CTC CAC ATC AAA TAC CTG TAC GGG ATC CGA
568 GTG GAG GTG CGA GGG GCT CAC CAC TTC CCT CCC TCG C
605 AG CCC TAT GTT GTT GTC TCC AAC CAC CAG AGC TCT CTC
643 GAT CTG CTT GGG ATG ATG GAG GTA CTG CCA GGC CGC T
680 GT GTG CCC ATT GCC AAG CGC GAG CTA CTG TGG GCT GGC
718 TCT GCC GGG CTG GCC TGC TGG CTG GCA GGA GTC ATC T
755 TC ATC GAC CGG AAG CGC ACG GGG GAT GCC ATC AGT GTC
793 ATG TCT GAG GTC GCC CAG ACC CTG CTC ACC CAG GAC G
830 TG AGG GTC TGG GTG TTT CCT GAG GGA ACG AGA AAC CAC
868 AAT GGC TCC ATG CTG CCC TTC AAA CGT GGC GCC TTC C
905 AT CTT GCA GTG CAG GCC CAG GTT CCC ATT GTC CCC ATA
943 GTC ATG TCC TCC TAC CAA GAC TTC TAC TGC AAG AAG G
980 AG CGT CGC TTC ACC TCG GGA CAA TGT CAG GTG CGG GTG
1018 CTG CCC CCA GTG CCC ACG GAA GGG CTG ACA CCA GAT G
1055 AC GTC CCA GCT CTG GCT GAC AGA GTC CGG CAC TCC ATG
1093 CTC ACT GTT TTC CGG GAA ATC TCC ACT GAT GGC CGG G
1130 GT GGT GGT GAC TAT CTG AAG AAG CCT GGG GGC GGT GGG
1168 TGA ACCCTGGCTCTGAGCTCTCCTCCCATCTGTCCCCATCTTCCTCCC
1216 CACACCTACCCACCCAGTGGGCCCTGAAGCAGGGCCAAACCCTCTTCCTT
1266 GTCTCCCCTCTCCCCACTTATTCTCCTCTTTGGAATCTTCA.ACTTCTGAA
28
CA 02291516 1999-11-26
WO 98/54303 PCT/U597105360
1316 GTGAATGTGGATACAGCGCCACTCCTGCCCCCTCTTGGCCCCATCCATGG
1366 ACTCTTGCCTCGGTGCAGTTTCCACTCTTGACCCCCACCTCCTACTGTCT
1416 TGTCTGTGGGACAGTTGCCTCCCCCTCATCTCCAGTGACTCAGCCTACAC
1466 AAGGGAGGGGAACATTCCATCCCCAGTGGAGTCTCTTCCTATGTGGTCTT
1516 CTCTACCCCTCTACCCCCACATTGGCCAGTGGACTCATCCATTCTTTGGA
1566 ACAAATCCCCCCCCACTCCAAAGTCCATGGATTCAATGGACTCATCCATT
1616 TGTGAGGAGGACTTCTCGCCCTCTGGCTGGAAGCTGATACCTGAAGCACT
1666 CCCAGGCTCATCCTGGGAGCTTTCCTCAGCACCTTCACCTTCCCTCCCAG
1716 TGTAGCCTCCTGTCAGTGGGGGCTGGACCCTTCTAATTCAGAGGTCTCAT
1766 GCCTGCCCTTGCCCAGATGCCCAGGGTCGTGCACTCTCTGGGATACCAGT
1816 TCAGTCTCCACATTTCTGGTTTTCTGTCCCCATAGTACAGTTCTTCAGTG
1866 GACATGACCCCACCCAGCCCCCTGCAGCCCTGCTGACCATCTCACCAGAC
1916 ACAAGGGGAAGAAGCAGACATCAGGTGCTGCACTCACTTCTGCCCCCTGG
1966 GGAGTTGGGGAAAGGAACGAACCCTGGCTGGAGGGGATAGGAGGGCTTTT
2016 AATTTATTTCTTTTTCTGTTGAGGCTTCCCCCTCTCTGAGCCAGTTTTCA
2066 TTTCTTCCTGGTGGCATTAGCCACTCCCTGCCTCTCACTCCAGACCTGTT
2116 CCCACAACTGGGGAGGTAGGCTGGGAGCAAAAGGAGAGGGTGGGACCCAG
2166 TTTTGCGTGGTTGGTTTTTATTAATTATCTGGATAACAGCAAAAAAACTG
2216 AAAATAAAGAGAGAGAG
(2) INFORMATION
FOR SEQ
ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:283
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(iii ) HYPOTHETICAL: no
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapien
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE: brain
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A} NAME/KEY: hLPAATa
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
1 Met Asp
Leu Trp
Pro Gly
Ala Trp
Met Leu
Leu Leu
Leu Leu
Phe
29
CA 02291516 1999-11-26
WO 98/54303 PCTIUS97/05360
16 Leu Leu Leu Leu Phe Leu Leu Pro Thr Leu Trp Phe Cys Ser
Pro
31 Ser Ala Lys Tyr Phe Phe Lys Met Ala Phe Tyr Asn Gly Trp
Ile -
46 Leu Phe Leu Ala Val Leu Ala Ile Pro Val Cys Ala Val Arg
Gly
61 Arg Asn Val Glu Asn Met Lys Ile Leu Arg Leu Met Leu Leu
His
76 Ile Lys Tyr Leu Tyr Gly Ile Arg Val Glu Val Arg Gly Ala
His
91 His Phe Pro Pro Ser Gln Pro Tyr Val Val Val Ser Asn His
Gln
106 Ser Ser Leu Asp Leu Leu Gly Met Met Glu Val Leu Pro Gly
Arg
121 Cys Val Pro Ile Ala Lys Arg Glu Leu Leu Trp Ala Gly Ser
Ala
136 Gly Leu Ala Cys Trp Leu Ala Gly Val Ile Phe Ile Asp Arg
Lys
151 Arg Thr Gly Asp Ala Ile Ser Val Met Ser Glu Val Ala Gln
Thr
166 Leu Leu Thr Gln Asp Val Arg Val Trp Val Phe Pro Glu Gly
Thr
181 Arg Asn His Asn Gly Ser Met Leu Pro Phe Lys Arg Gly Ala
Phe
196 His Leu Ala Val Gln Ala Gln Val Pro Ile Val Pro Ile Val
Met
211 Ser Ser Tyr Gln Asp Phe Tyr Cys Lys Lys Glu Arg Arg Phe
Thr
226 Ser Gly Gln Cys Gln Val.Arg Val Leu Pro Pro Val Pro Thr
Glu
241 Gly Leu Thr Pro Asp Asp Val Pro Ala Leu Ala Asp Arg Val
Arg
256 His Ser Met Leu Thr Val Phe Arg Glu Ile Ser Thr Asp Gly
Arg
271 Gly Gly Gly Asp Tyr Leu Lys Lys Pro Gly Gly Gly Gly ***
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:52
(B) TYPE: AMINO acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: yeast
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
{D) DEVELOPMENTAL STAGE:
(E} HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
{H) CELL LINE
(I) ORGANELLE:
( ix) FEATURE
(A} NAME/KEY: LPAAT fragment
CA 02291516 1999-11-26
WO 98/54303 PCT/US97105360
(B) LOCATION:169-220
(C) IDENTIFICATION METHOD:
(J) PUBLICATION DATE:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: _
1 PFKKGAFHLAQQGKIPIVPVWSNTSTLVSPKYGVFNRGCMIVRILKPISTE 52
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:52
(B) TYPE : amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(iii) HYPOTHETICAL: no
1$ (iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapien
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
{E} HAPLOTYPE:
{F) TISSUE TYPE: placenta
{G) CELL TYPE:
{H) CELL LINE:
(I) ORGANELLE:
( ix ) FEATURE
(A) NAME/KEY: dbest clone #102250
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
1 PSNCGAFHLAVQAQVPIVPIVMSSYQDFYCKKERRFTSGQCQVRVLPPVPTE 52
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:18
(B) TYPE: nucleotide
(C) STRANDEDNESS: single
{D} TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide fragment
(iii)
HYPOTHETICAL:
no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE
( I ) ORGANELLE
( ix) FEATURE
(A) NAME/KEY: o.BLPAT.2R
(B) LOCATION:
(C) IDENTIFICATION METHOD:
31
CA 02291516 1999-11-26
WO 98/54303 PCT/US97105360
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
1 TGCAAGATGGAAGGCGCC 18
{2) INFORMATION
FOR SEQ
ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:6
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide fragment
(iii ) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(v} FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY: o.BLPAT.2R
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
1 GAFHLA
6
(2) INFORMATION
FOR SEQ
ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:1373
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA to mRNA
(iii ) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
{v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapien
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY: hLPAAT~i
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
SS 1 GGAGCGAGCT
GGCGGCGCCG
TCGGGCGCCG
GGCCGGGCCA
TGGAGCTGTG
51 GCCGCGGCGC TGCTGTTGCT GCTGCTGCTG GTGCAGCTGA GCCGCGCGGC
32
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WO 98/54303 PCTIUS97I05360
101 CGAGTTCTAC GCCAAGGTCG CCCTGTACTG CGCGCTGTGC TTCACGGTGT
151 CCGCCGTGGC CTCGCTCGTC TGCCTGCTGT GCCACGGCGG CCGGACGGTG
201 GAGAACATGA GCATCATCGG CTGGTTCGTG CGAAGCTTCA AGTACTTTTA
251 CGGGCTCCGC TTCGAGGTGC GGGACCCGCG CAGGCTGCAG GAGGCCCGTC
301 CCTGTGTCAT CGTCTCCAAC CACCAGAGCA TCCTGGACAT GATGGGCCTC
351 ATGGAGGTCC TTCCGGAGCG CTGCGTGCAG ATCGCCAAGC GGGAGCTGCT
401 CTTCCTGGGG CCCGTGGGCC TCATCATGTA CCTCGGGGGC GTCTTCTTCA
451 TCAACCGGCA GCGCTCTAGC ACTGCCATGA CAGTGATGGC CGACCTGGGC
501 GAGCGCATGG TCAGGGAGAA CCTCAAAGTG TGGATCTATC CCGAGGGTAC
551 TCGCAACGAC AATGGGGACC TGCTGCCTTT TAAGAAGGGC GCCTTCTACC
601 TGGCAGTCCA GGCACAGGTG CCCATCGTCC CCGTGGTGTA CTCTTCCTTC
651 TCCTCCTTCT ACAACACCAA GAAGAAGTTC TTCACTTCAG GAACAGTCAC
701 AGTGCAGGTG CTGGAAGCCA TCCCCACCAG CGGCCTCACT GCGGCGGACG
751 TCCCTGCGCT CGTGGACACC TGCCACCGGG CCATGAGGAC CACCTTCCTC
801 CACATCTCCA AGACCCCCCA GGAGAACGGG GCCACTGCGG GGTCTGGCGT
851 GCAGCCGGCC CAGTAGCCCA GACCACGGCA GGGCATGACC TGGGGAGGGC
901 AGGTGGAAGC CGATGGCTGG AGGATGGGCA GAGGGGACTC CTCCCGGCTT
951 CCAAATACCA CTCTGTCCGG CTCCCCCAGC TCTCACTCAG CCCGGGAAGC
1001 AGGAAGCCCC TTCTGTCACT GGTCTCAGAC ACAGGCCCCT GGTGTCCCCT
1051 GCAGGGGGCT CAGCTGGACC CTCCCCGGGC TCGAGGGCAG GGACTCGCGC
1101 CCACGGCACC TCTGGGNGCT GGGNTGATAA AGATGAGGCT TGCGGCTGTG
1151 GCCCGCTGGT GGGCTGAGCC ACAAGGCCCC CGATGGCCCA GGAGCAGATG
1201 GGAGGACCCC GAGGCCAGGA GTCCCAGACT CACGCACCCT GGGCCACAGG
1251 GAGCCGGGAA TCGGGGCCTG CTGCTCCTGC TGGCCTGAAG AATCTGTGGG
1301 GTCAGCACTG TACTCCGTTG CTGTTTTTTT ATAAACACAC TCTTGGAAAA
1351 AAA..1373
{2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:274
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE:
{vi) ORIGINAL SOURCE:
{A) ORGANISM: homo sapien
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
{F) TISSUE TYPE:
(G) CELL TYPE
(H) CELL LINE:
(I) ORGANELLE:
( ix) FEATURE
(A) NAME/KEY: hLPAAT~i
{B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
1 Met Glu Leu Trp Cys Leu Ala Ala Ala Leu Leu Leu Leu Leu
Leu
16 Leu Val Gln Ser Arg Ala Ala Glu Phe Tyr AIa Lys Val Ala
Leu
33
CA 02291516 1999-11-26
WO 98!54303 PCT/US97/05360
31 Tyr Cys Leu Cys Phe ThrVal Ser Ala Val Ala Ser Leu Val
Cys
46 Leu Cys His Gly Gly ArgThr Val Glu Asn Met Ser Ile Ile
Gly -
61 Trp Phe Val Arg Ser PheLys Tyr Phe Tyr Gly Leu Arg Phe
Glu
76 Val Arg Asp Pro Arg ArgLeu Gln Glu Ala Arg Pro Cys Val
Ile
91 Val Ser Asn His Gln SerIle Leu Asp Met Met Gly Leu Met
10Glu
106 Val Leu Pro Glu Arg CysVal Gln Ile Ala Lys Arg Glu Leu
Leu
121 Phe Leu GIy Pro Val GlyLeu Ile Met Tyr Leu Gly Gly Val
Phe
15136 Phe Ile Asn Arg Gln ArgSer Ser Thr Ala Met Thr Val Met
Ala
151 Asp Leu Gly Glu Arg MetVal Arg Glu Asn Leu Lys Val Trp
Ile
166 Tyr Pro Glu Gly Thr ArgAsn Asp Asn Gly Asp Leu Leu Pro
20Phe
181 Lys Lys Gly Ala Phe TyrLeu Ala Val Gln Ala Gln Val Pro
Ile
196 Val Pro Val Val Tyr SerSer Phe Ser Ser Phe Tyr Asn Thr
Lys
25211 Lys Lys Phe Phe Thr SerGly Thr Val Thr Val Gln Val Leu
Glu
226 Ala Ile Pro Thr Ser GlyLeu Thr Ala Ala Asp Val Pro Ala
Leu
241 Val Asp Thr Cys His ArgAla Met Arg Thr Thr Phe Leu His
30Ile
256 Ser Lys Thr Pro Gln GluAsn Gly Ala Thr Ala Gly Ser Gly
Val
271 Gln Pro Ala Gln *** 274
35 (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:60
(B) TYPE: amino acid
(C) STRANDEDNESS: single
40 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: NO
(v) FRAGMENT
TYPE:
45 (vi ) ORI GINAL SOURCE
(A) ORGANISM: yeast
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
50 (E) HAPLOTYPE:
(F) TISSUE TYPE:
{G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
55 ( ix) FEATURE
(A) NAME/KEY: LPAAT fragment
(B) LOCATION:171-230
(C) IDENTIFICATION METHOD:
34
CA 02291516 1999-11-26
WO 98!54303 PCT/US97/05360
(J) PUBLICATION DATE:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
I
QQGKIPIVPVWSNTSTLVSPKYGVFNRGCMIVRILKPISTENLTKDKIGEFAEKVRDQM~
(2) INFORMATION
FOR SEQ
ID NO:IO:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:60
10 (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: polypeptide
(iii} HYPOTHETICAL:
no
15 (iv} ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: homo sapien
(B) STRAIN:
20 (C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
25 (H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY: dbest clone #363498
(B) LOCATION:
30 (C} IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
1
VRENVPIVPVVYSSFSSFYNTKKKFFTSGTVTVQVLEAIPTSGLTAADVPALRGTPATGP
35 60
(2) INFORMATION
FOR SEQ
ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:20
40 (B) TYPE: nucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide fragment
(iii ) HYPOTHETICAL: no
45 (iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
50 (C} INDIVIDUAL ISOLATE:
(D} DEVELOPMENTAL STAGE:
(E} HAPLOTYPE:
(F) TISSUE TYPE:
(G} CELL TYPE:
55 (H} CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY: o. LPAT.3F
CA 02291516 1999-11-26
WO 98/54303 PCT/US97105360
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: -
1 CCTCAAAGTGTGGATCTATC
20
(2) INFORMATION
FOR SEQ
ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:21
(B) TYPE: nucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
{iii ) HYPOTHETICAL: no
{iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
( E ) HAPLOTYPE
(F) TISSUE TYPE:
{G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY: o.LPAT3.R
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
1 GGAAGAGTACACCACGGGGAC
21
(2) INFORMATION
FOR SEQ
ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH:21
(B) TYPE: nucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
( i i i
) HYPOTHET
I CAL
: no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE
(vi ) ORIGINAL SOURCE
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY: o.sport.l
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
36
CA 02291516 1999-11-26
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
1 GACTCTAGCCTAGGCTTTTGC 21
(2) INFORMATION
FOR SEQ
ID N0:14:
S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:21
(B) TYPE: nucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL:
no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
{vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
{C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
{F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY: o.sport.lR
{B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
1 GCTAGCTTATAATACGACTCAC
21
(2) INFORMATION
FOR SEQ
ID NO:15:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:29
{B) TYPE: nucleotide
(C) STRANDEDNESS: single
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii ) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORTGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
37
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
1 GGCTCTAGAT ATTAATAGTA ATCAATTAC 29
(2) INFORMATION
FOR SEQ
ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:26
(B) TYPE: nucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii ) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
( I ) ORGANELLE
( i x ) FEATURE
{A} NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
1 CCTCACGCAT
GCACCATGGT
AATAGC
26
(2) INFORMATION
FOR SEQ
ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:24
(B) TYPE: nucleotide
{C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL:
no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
{A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E} HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE
{I) ORGANELLE:
( ix) FEATURE
(A) NAME/KEY:
{B) LOCATION:
SS (C) IDENTIFICATION METHOD:
{D) OTHER INFORMATION:
38
CA 02291516 1999-11-26
WO 98/54303 PCT/US97/05360
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
1 GGTGCATGCG TGAGGCTCCG GTGC 24
(2) INFORMATION
FOR SEQ
ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:28
(B) TYPE: nucleotide
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
i0 (ii) MOLECULE TYPE: oligonucleotide
(iii) HYPOTHETICAL:
no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
( E ) HAPLOTYPE
(F) TISSUE TYPE:
(G) CELL TYPE
(H) CELL LINE
(I) ORGANELLE:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
1 GTAGTTTTCA
CGGTACCTGA
AATGGAAG
28
39
