Note: Descriptions are shown in the official language in which they were submitted.
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COMPOUND HAVING SEQUENCE HOMOLOGY WITH L~ N ASSOClAlED ~ rtl0-
LIPASE A2 (1~PLA2yP~F ~CEl~L HYDROL~SE
The present invention relates to the use of inhibi~ors of a polypeptide in the
therapy. The present invention also relates to the polypeptide, to polynucleotides
encoding the polypeptide, to recombinant host cells transformed with DNA encoding
the polypeptide and to the use of the polypeptide in identifying compounds which are
potentially useful in therapy.
Lipoprotein Associated Phospholipase A2 (Lp-PLA2) is also known in the
art as Platelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase). During
the conversion of LDL to its oxidised form, Lp-PLA2 is responsible for hydrolysing
the sn-2 ester of oxidatively modified phosphatidylcholine to give lyso-
phosphatidylcholine and an oxidatively modified fatty acid. Both of these products of
Lp-PLA2 action are potent chemoattractants for circulating monocytes. As such, this
enzyme is thought to be responsible for the accumulation of cells loaded with
cholesterol ester in the arteries, causing the characteristic 'fatty streak' associated with
the early stages of atherosclerosis. Inhibition of the Lp-PLA2 enzyme would
therefore be expected to stop the build up of this fatty streak (by inhibition of the
formation of lysophosphatidylcholine), and so be useful in the treatment of
atherosclerosis. Lp-PLA2 inhibitors may also have a general application in any
disorder that includes lipid peroxidation in conjunction with the enzyme activity, for
example in addition to conditions such as atherosclerosis and diabetes other
conditions such as rheumatoid arthritis, stroke, myocardial infarction, reperfusion
injury, acute and chronic infl~mm~tion and various neulupsychiatric illnçcces (e.g.,
schizophrenia, ref. Psychopharmacology Bulletin, 31: 159-165, 1995).
The amino acid and DNA sequence of the enzyme lipoprotein associated Lp-
PLA2 is disclosed in W095/00649.
This invention relates to newly identified polynucleotides, polypeptides
encoded by such polynucleotides, the use of such polynucleotides and polypeptides,
as well as the production of such polynucleotides and polypeptides. More
particularly, the polypeptide of the present invention is a novel lipase. The invention
also relates to inhibiting the action of such polypeptides.
In accordance with one aspect of the present invention, there is provided a
novel polypeptide which is a lipase having the amino acid sequence given in SEQ ID
NO 1, or a fragments, analogs or derivative thereof. The polypeptide of the present
invention is of human origin.
Hereinafter the term polypeptide(s) will be used to refer to the lipase and its
fr~gment.c, analogs and derivatives
In accordance with another aspect of the present invention, there are
provided polynucleotides (DNA or RNA) which encode such polypeptides.
- 1 -
CA 02233296 l998-03-27
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In accordance with a preferred aspect of the present invention, there is
provided a polynucleotide which encodes for the polypeptide having the amino acid
sequence of SEQ ID NO 1.
In particular, the invention provides a polynucleotide having the DNA
sequence given in SEQ ID NO 2.
cDNA molecules showing extentlPd identity sections with the cDNA of SEQ
ID NO 2 have been identified in cDNA libraries from the following tissues: foetal
heart, pineal gland, activated T cells, microvascular endothelial cells and secondary
breast tumour The polynucleotide of SEQ ID NO 2 was discovered in a cDNA
lo library derived from prostate (benign possible hyperplasia). It is structurally related
to the lipase family. It contains an open reading frame encoding a protein of about
393 amino acid residues. The protein exhibits the highest degree of homology to Lp-
PLA2 (WO95/00649, WO 95/09912~ with 40% identity and 60% ~imil~rity over a
390 amino acid stretch. Although the overall identity is only 40% the residues
i~lentifit-.d for the catalytic triad in Lp-PLA2 (WO 95/09912) are conserved between
the two polypeptides implying that they are likely to have a similar biochemicalfunction. The positions of the Ser, Asp,and His, are underlined below. SEQ ID NO1, is the lower seqence and Lp-PLA2 the upper sequence. Vertical lines in~ at~P
i~lenti~ residues.
238 DIDHGKPVKNAIRLKFDMEQLKDSIDREKIAVIGH~FGGATVIQTLSEDQ 287
::. 1..1 1 :. :1: 11:.11..::11:11111111.1 .1..:
202 EVTAGQTVFNIFPGGLDLMTLKGNIDMSRVAVMGHSFGGATAILALAKET 251
288 RFRCGIAL~AWMFPLGDEVYSRIPQPLFFINSEYFQYPANIIKMKKCYSP 337
.111::111111111: :.1.: ..1:1111.1 11 ..: 111.:..
252 QFRCAVALDAWMFPLERDFYPKARGPVFFINTEKFQTMESVNLMKKICAQ 301
338 DKERKMITIRGSV~QNFADFTFATGKIIGHML.. KLKGDIDSNAAIDLSN 385
..:.::11: 1111.. .11.1.I1.:11.:: . :1.:1. .: ::
302 HEQSRIITVLGSV~RSQTDFAFVTGNLIGKFFSTETRGSLDPYEGQEVMV 351
Tissue sources of this enzyme suggest a role in the biology of the vasculature
as well as certain c~n~prc As such, an inhibitor of this polypeptide could find utility in
disease states such as atherosclerosis, hypertension, endothelial dysfunction,
myocardial infarction, reperfusion injury, and certain cancers. In addition. thehomology to Lp-PLA2 suggests that this novel enzyme could play similar roles and as
such inhibitors may find utility in atherosclerosis, myocardial infarction, reperfusion
40 injury, acute and chronic in~l~mm~tion, rheumatoid arthritis, stroke, diabetes and
neulop~ychiatric illne.cses
The polynucleotide of the present invention may be in the form of RNA or in
the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
The DNA may be double-stranded or single-stranded, and if single stranded may be
-- 2 -
-
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W O 97/12984
the coding strand or non-coding (anti-sense) strand. The coding sequence which
encodes the polypeptide may be identical to the coding sequence shown in SEQ ID
NO 1 or may be a different coding sequence which, as a result of the rednn-l~n< y or
degeneracy of the genetic code, encodes the same polypeptide as the DNA of SEQ ID
s NO 1.
The present invention includes variants of the hereinabove described
polynucleotides which encode fragmentc, analogs and derivatives of the polypeptide
having the amino acid sequence of SEQ ID NO 1. The variant of the polynucleotidemay be a naturally occurring allelic variant of the polynucleotide or a non-naturally
occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same
polypeptide as shown in SEQ ID NO 1 as well as variants of such polynucleotides
which variants encode for a fragment, derivative or analog of the polypeptide. Such
nucleotide variants include deletion variants, substitution variants and addition or
insertion variants.
The polynucleotide may have a coding sequence which is a naturally
occurring allelic variant of the coding sequence of SEQ ID NO 2. As known in theart, an allelic variant is an alternate form of a polynucleotide sequence which may
have a substitution, deletion or addition of one or more nucleotides, which does not
subst~n~i~lly alter the function of the encoded polypeptide.
The polynucleotide which encodes for the polypeptide of SEQ ID NO 1 may
include: only the coding sequence for the polypeptide; the coding sequence for the
polypeptide and additional coding sequence such as a leader or secretory sequence or
a proprotein sequence; the coding sequence for the polypeptide (and optionally
2s additional coding sequence) and non-coding sequence, such as introns or non-coding
sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encomp~cses a
polynucleotide which includes only coding sequence for the polypeptide as well as a
polynucleotide which jnchl(les additional coding and/or non-coding sequence.
The present invention therefore includes polynucleotides, wherein the coding
sequence for the polypeptide may be fused in the same reading frame to a
polynucleotide sequence which aids in expression and secretion of a polypeptide from
a host cell, for example, a leader sequence which functions as a secretory sequence
for controlling transport of a polypeptide from the cell. The polypeptide having a
3s leader sequence is a preprotein and may have the leader sequence cleaved by the host
cell to form the mature form of the polypeptide. The polynucleotides may also
~ encode for a proprotein which is the mature protein plus additional ~' amino acid
residues. A mature protein having a prosequence is a proprotein and is an inactive
CA 02233296 1998-03-27
WO 97~1~984 PCT/GB95/02320
form of the protein. Once the prosequence is cleaved an active mature protein
remains.
Tnus, for example, the polynucleotide of the present invention may encode
for a mature protein, or for a protein having a prosequence or for a protein having
s both a prosequence and a presequence (leader sequen-~e).
The polynucleotides of the present invention may also have the coding
sequence fused in frame to a marker sequence which allows for pllrifi~ti~n of the
polypeptide of the present invention. The marker sequence may be a hexa-hi~ti~1ine
tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide
10 fused to the marker in the case of a bacterial host, or, for example, the marker
sequence may be a hemagglutinin (HA) tag when a m~mm~ n host, e.g. COS-7
cells, is used. The HA tag corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to
5 the hereinabove-described sequences if there is at least 50% and preferably 70%
identity between the sequences. The present invention particularly relates to
polynucleotides which hybridize under stringent conditions to the hereinabove-
described polynucleotides . As herein used, the term "stringent conditions" means
hybridization will occur only if there is at least 95% and preferably at least 97%
20 identity between the sequences. The polynucleotides which hybridize to the
hereinabove described polynucleotides in a preferred embodiment encode
polypeptides which retain substantially the same biological function or activity as the
polypeptide of SEQ ID NO 1.
The terrns "fragment," "derivative" and "analog" when referring to the
2s polypeptide of SEQ ID NO l, means a polypeptide which retains ecse~ti~lly the same
biological function or activity as such polypeptide. Thus, an analog includes a
proprotein which can be activated by cleavage of the proprotein portion to produce an
active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide,
30 a natural polypeptide or a synthetic polypeptide, preferably a recombinant
polypeptide.
The fragment, derivative or analog of the polypeptide of SEQ ID NO 1 may
be ~i) one in which one or more of the amino acid residues are substituted with a
conserved or non-conserved amino acid residue (preferably a conserved amino acid35 residue) and such substituted amino acid residue may or may not be one encoded by
the genetic code, or (ii) one in which one or more of the amino acid residues includes
a substituent group, or ~iii) one in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the polypeptide (for
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W O 97/12984 PCT/GB95/02320
example, polyethylene glycol), or (iv) one in which the additional amino acids are
fused to the mature polypeptide, such as a leader or secretory sequence or a sequence
which is employed for purification of the mature polypeptide or a proprotein
sequence. Such fragments, derivatives and analogs are ~eemed to be within the scope
s of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably
provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its ori~:in~l
environment (e.g., the natural environment if it is naturally occurring). For eY~mp~
o a naturally-occurring polynucleotide or polypeptide present in a living animal is not
tPd, but the same polynucleotide or polypeptide, separated from some or all of
the coexisting materials in the natural system, is isolated. Such polynucleotides could
be part of a vector and/or such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or composition is not part of its
natural environment. The polypeptide is preferably in purified form. By purifiedform is meant at least 80%, more preferably 90%, still more preferably 95% and most
preferably 99% pure with respect to other protein cont~min~ntc
The DNA of the present invention also makes possible the development by
homologous recombination or "knockout" stategies (Kapecchi, Science, 244,:1288-
1292 (1989) of ~nim~ll.c that fail to express, or express a variant form of this enzyme
The present invention also relates to vectors which include polynucleotides
of the present invention, host cells which are genetically engineered with vectors of
the invention and the production of polypeptides of the invention by recombinanttechniques.
2s In accordance with yet a further aspect of the present invention, there is
therefore provided a process for producing the polypeptide of the invention by
recombinant techniques by expresssing a polynucleotide encoding said polypeptide in
a host and recovering the expressed product. Alternatively, the polypeptides of the
invention can be synthetically produced by conventional peptide synthPsi7Prs.
Host cells are genetically engineered (tr~n~duced or transformed or
transfected) with the vectors of this invention which may be, for example, a cloning
vector or an expression vector. The vector may be, for example, in the form of aplasmid, a cosmid, a phage, etc. The engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating promoters,
selecting transformants or amplifying the genes. The culture conditions, such astemperature, pH and the like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled artisan.
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Suitable expression vectors include chromosomal, nonchromosomal and
synthedc DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of plasmids and
phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and
s pseudorabies. However, any other vector may be used as long as it is replicable and
viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety
of procedures. In general, the DNA sequence is inserted into an applo~ te
restrictiQn endonuclease site(s) by procedures known in the art. Such procedures and
10 others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an
applopriate expression control sequence(s) (promoter) to direct mRNA synthesis. As
representative examples of such promoters, there may be mentioned: LTR or SV40
promoter, the E. coli. Iac or trp, the phage lambda PL promoter and other promoters
15 known to control expression of genes in prokaryotic or eukaryotic cells or their
viruses. The expression vector also contains a ribosome binding site for tr~ncl~tion
initiation and a transcription terminator. The vector may also include applo~liate
sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more selectable
20 marker genes to provide a phenotypic trait for selection of transformed host cells such
as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such
as tetracycline or ampicillin resistance in E. coli.
The gene can be placed under the control of a promoter, ribosome binding
site (for bacterial expression) and, optionally, an operator (collectively referred to
25 herein as "control" elements), so that the DNA sequence encoding the desired protein
is tr~nccrihed into RNA in the host cell transformed by a vector cor t~ining this
e,~lession construction. The coding sequence may or may not contain a signal
peptide or leader sequence. The protein sequences of the present invention can be
expressed using, for example, the E. col~ tac promoter or the protein A gene (spa)
30 promoter and signal sequence. Leader sequences can be removed by the bacterial
host in post-tr~nclslti~nal processing. Promoter regions can be selected from any
desired gene using CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular
named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.
35 Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, earlyand late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the
approp~iate vector and promoter is well within the level of ordinary skill in the art.
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In addition to control sequences, it may be desirable to add regulatory
sequences which allow for regulation of the expression of the prooein sequences
relative to the growth of the host cell. Regulatory sequences are known to those of
skill in the art, and examples include those which cause the expression of a gene to be
5 turned on or off in response to a chemical or physical stimulus, including the presence
of a regulatory compound. Other types of regulatory elements may also be present in
the vector, for example, enhancer sequences.
An expression vector is constructed so that the particular coding sequence is
located in the vector with the appropriate regulatory sequences, the positioning and
10 orientation of the coding sequence with respect to the control sequences being such
that the coding sequence is transcribed under the "control" of the control sequences
(i.e., RNA polymerase which binds to the DNA molecule at the control sequences
t~n~crihes the coding sequence). Mo-iifiç~tion of the coding sequences may be
desirable to achieve this end. For example, in some cases it may be necessslry to
lS modify the sequence so that it may be attached to the control sequences with the
appropriate orientation; i.e., to maint~in the reading frame. The control sequences
and other regulatory sequences may be ligated to the coding sequence prior to
insertion into a vector, such as the cloning vectors described above. ~ltPrnatively, the
coding sequence can be cloned directly into an expression vector which already
20 contains the control sequences and an appropriate restriction site. Morlific~tion of the
coding sequences may also be performed to alter codon usage to suit the chosen host
cell, for enh~nce-l expression.
Generally, recombinant expression vectors will include origins of replication
and selectable markers permitting transformation of the host cell, e.g., the ampicillin
2s resictanre gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a
highly-expressed gene to direct transcription of a downstream structural sequence.
The heterologous structural sequence is assembled in appropriate phase with
translation initiation and termination sequences, and preferably, a leader sequence
capable of directing secretion of tr~n~l~tPd protein into the periplasmic space or
30 extracellular medium. Optionally, the heterologous sequence can encode a fusion
protein including an N-terminal identificS~tion peptide imparting desired chs~racter-
istics, e.g., stabilization or simplified purification of expressed recombinant product.
The vector containing the appropriate DNA sequence as hereinabove
described, as well as an appropriate promoter or control sequence, may be employed
3s to transform an appropriate host to permit the host to express the protein.
Examples of recombinant DNA vectors for cloning and host cells which they
can transform include the bacteriophage ~ (E. coli), pBR322 (E. coli), pACYC177
(E. coli), pKT230 (gram-negative bacteria), pGVl 106 (gram-negative bacteria),
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W O 97/1298~ PCT/GB95/02320
pLAFRl (gram-negative bacteria), pME290 (non-E. coli grarn-negative bacteria),
pHV14 (E. coli and Bacillus subtilis), pBD9 (Rnci~ ), pIJ61 (Streptomyces), pUC6(Streptom~ces), YIp5 (Saccharomyces), a baculovirus insect cell system,, YCpl9
(Saccharomyces). See, generally, "DNA Cloning": Vols. I & II, Glover et al. ed.
s IRL Press Oxford (1985) (1987) and; T. Maniatis et al. ("Molecular Cloning" Cold
Spring Harbor Laboratory (1982).
In some cases, it may be desirable to add sequences which cause the
secretion of the polypeptide from the host org~ni~m, with subsequent cleavage of the
secretory signal.
Yeast e,~ ession vectors are also known in the art. See, e.g., U.S. Patent
Nos. 4,446,235; 4,443,539; 4,430,428; see also European Patent Applir~tion.~
103,409; 100,561; 96,491. pSV2neo (as described in J. Mol. Appl. Genet. 1:327-341)
which uses the SV40 late promoter to drive ~x},l~:ssion in m~mm~ n cells or
pCDNAlneo, a vector derived from pCDNAl(Mol. Cell Biol. 7:4125-29) which uses
1S the CMV promoter to drive expression. Both these latter two vectors can be
employed for transient or stable(using G418 resistance) expression in m~mm~ n
cells. Insect cell ~pl~;ssion systems, e.g., Drosophila, are also useful, see for
example, PCT applications WO 90/06358 and WO 92/06212 as well as EP 290,261-
Bl.
Polypeptides can be expressed in host cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to produce such
proteins using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic
hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2s Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby
incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention
by higher eukaryotes is increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act
on a promoter to increase its transcription. Examples including the SV40 çlnhs~nrer on
the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication origin, and
adenovirls enh~ncer~
In a further aspect, the present invention relates to host cells con~ining the
above-described vectors. The host cell can be a higher eukaryotic cell, such as a
m~mm~ n cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be
a prokaryotic cell, such as a bacterial cell. As representative examples of appropriate
hosts, there may be mentioned: prokaryotes for example bacterial cells, such as E.
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WO 97/12984 PCT/GB95/02320
coli, Streptomyces, Salmonella typhimllrium and eukaryotes for example fungal cells,
such as yeast, insect cells such as Drosophila and Spodoptera frugiperda, m~mm~ n
cells such as CHO, COS or Bowes melanoma, plant cells, etc. The selection of an
appropriate host is deemed to be within the scope of those skilled in the art from the
tPachingc herein.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran m~ rPcl transfection, or electroporation.
(Davis, L., Dibner, M., Battey, I., Basic ~ethods in Molecular Biology, (1986)).Following transformation of a suitable host strain and growth of the host
lo strain to an appropriate cell density, the selected promoter is induced by appropriate
means (e.g., temperature shift or chPmic~l induction) and cells are cultured for an
additional period.
Cells are typically harvested by centrifugation, disrupted by physical or
chemical means, and the resulting crude extract retained for funher purification.
Microbial cells employed in expression of proteins can be disrupted by any
convenient method, including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents, such methods are well know to those skilled in the an.
Various m~mm~ n cell culture systems can also be employed to express
recombinant protein. Examples of m~mm;~ n expression systems include the COS-7
lines of monkey kidney fibroblasts, described by Glll7m~n, Cell, 23:175 (1981), and
other cell lines capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. M~mm~ n expression vectors will comprise
an origin of replication, a suitable promoter and enhancer, and also any n~.ce
ribosome binding sites, polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking nontranscribed sequences.DNA sequences derived from the SV40 splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
Depending on the expression system and host selected, the polypeptide of the
present invention may be produced by growing host cells transformed by an
expression vector described above under conditions whereby the polypeptide of
interest is expressed. The polypeptide is then isolated from the host cells and
purified. If the expression system secretes the polypeptide into growth media, the
polypeptide can be purified directly from the media. If the polypeptide is not
secreted, it is isolated from cell lysates or recovered from the cell membrane fraction.
Where the polypeptide is localized to the cell surface, whole cells or isolated
membranes can be used as an assayable source of the desired gene product.
Polypeptide expressed in bacterial hosts such as E. coli may require isolation from
g
~ - ~
CA 02233296 1998-03-27
W O 97/12984 PCT/GB95/02320
inclusion bodies and refolding. The selection of the appropriate growth conditions
and recovery methods are within the skill of the art.
The polypeptide can be recovered and purified from recombin~nt cell
cultures by methods including ammonium sulfate or ethanol precipitation, acid
s extraction, anion or cation exchange chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity chromatography
hydroxylapatite chromatography and lectin chromatography. Protein refolding steps
can be used, as necess~ry~ in completing configuration of the mature protein. Finally,
high performance liquid chromatography (HPLC) can be employed for final
pllri~ tion steps.
Depending upon the host employed in a recombinant production procedure,
the polypeptides of the present invention may be glycosylated or may be non-
glycosylated. Polypeptides of the invention may also include an initial methionine
amino acid residue.
The polypeptide of the present invention is also useful for identifying other
molecules which have similar biological activity. An example of a screen for this is
isolating the coding region of the lipase gene by using the known DNA sequence to
synthPci7f~ an oligonucleotide probe or as a probe itself. Labeled oligonucleotides
having a sequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determine
which members of the library the probe hybridizes to.
The polypeptides may also be employed in accordance with the present
invention by expression of such polypeptides in vivo, which is often referred to as
"gene therapy."
Thus, for example, cells from a patient may be çngin~ered with a
polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered
cells then being provided to a patient to be treated with the polypeptide. Such
methods are well-known in the art. For ex~mplç, cells may be engineered by
procedures known in the art by use of a retroviral particle cont~ining RNA encoding a
polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in
vivo by, for example, procedures known in the art. As known in the art, a producer
cell for producing a retroviral particle cont~ining RNA encoding the polypeptide of
the present invention may be ~dministered to a patient for engineering cells in vivo
and expression of the polypeptide i~ vivo. These and other methods for ,~riminict~ring
a polypeptide of the present invention by such method should be apparent to those
skilled in the art from the teachings of the present invention. For example, thee~plession vehicle for engineering cells may be other than a retrovirus, for exarnple,
CA 02233296 l99X-03-27
W O 97/12984 PCT/GB95/02320
an adenovirus which may be used to engineer cells in vivo after combination with a
suitable delivery vehicle.
"Recombinant" polypeptides refer to polypeptides produced by recombinant
DNA techniques; i.e., produced from cells transformed by an exogenous DNA
5 construct encoding the desired polypeptide. "Synthetic" polypeptides are those prepared by chemi~l synthesis.
A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that
fun-~tion.c as an autonomous unit of DNA replication in vivo; i.e., capable of
replication under its own control.
A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which
another DNA segment may be attached so as to bring about the replication of the
~,tt~h.~d segment.
A "double-stranded DNA molecule" refers to the polymeric form of
deoxyribonucleotides (bases ~deninç. guanine, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled. This term refers only to the primary and
secondary structure of the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter alia, in linearDNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In
(liscucsing the structure of particular double-stranded DNA molecules, sequences may
be described herein according to the norrnal convention of giving only the sequence
in the 5' to 3' direction along the sense strand of DNA.
A DNA "coding sequence of" or a "nucleotide sequence encoding" a
particular protein, is a DNA sequence which is transcribed and tr~lnsl~tf d into a
polypeptide when placed under the control of appropriate regulatory sequences.
A "promoter sequence" is a DNA regulatory region capable of binding RNA
polymerase in a cell and initi~ting transcription of a downstream (3' direction) coding
sequence. Within the promoter sequence will be found a transcription initiation site
(conveniently defined by mapping with nuclease S1), as well as protein binding
dom~inc (consensus sequences) responsible for the binding of RNA polymerase.
Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT"
boxes.
DNA "control sequences" refers collectively to promoter sequences,
ribosome binding sites, polyadenylation signals, transcription termination sequences,
upstream regulatory dom~ins, enhancers, and the like, which collectively provide for
the expression (i.e., the transcription and translation) of a coding sequence in a host
cell.
A control sequence "directs the expression" of a coding sequence in a cell
when RNA polymerase will bind the promoter sequence and transcribe the coding
CA 02233296 1998-03-27
WO 97/12984 PCT/GB9JJ~2320
sequence into mRNA, which is then translated into the polypeptide encoded by thecoding sequence.
A "host cell" is a cell which has been transformed or ~n.~fectetl, or is
capable of transformation or transfection by an exogenous DNA sequence.
A cell has been "transformed" by exogenous DNA when such exogenous
DNA has been introduced inside the cell membrane. Exogenous DNA may or may
not be integrated (covalently linked) into chromosomal DNA making up the genome
of the cell. In prokaryotes and yeasts, for example, the exogenous DNA may be
m~int~inPd on an episomal element, such as a pl~cmi~l With respect to eukaryoticlo cells, a stably transformed or transfected cell is one in which the exogenous DNA has
become integrated into the chromosome so that it is inherit~od by ~ hter cells
through chromosome replication. This stability is demon~tr~qtf~d by the ability of the
eukaryodc cell to establish cell lines or clones comprised of a population of d~ughter
cell co~t~ining the exogenous DNA.
A "clone" is a population of cells derived from a single cell or common
ancestor by mitocic. A "cell line" is a clone of a primary cell that is capable of stable
growth in vitro for many generations.
Two DNA or polypeptide sequences are "subst~nti~lly homologous" or
"substantially the same" when at least about 85% (preferably at least about 90%, and
most preferably at least about 95%) of the nucleotides or amino acids match over a
deflned length of the molecule and includes allelic variations. As used herein,
substantially homologous also refers to sequences showing identity to the specified
DNA or polypeptide sequence. DNA sequences that are subst~nti~lly homologous
can be i-lentified in a Southern hybri~ tion e~pe~ ent under, for example, stringent
2s conditions, as defined for that particular system. Defining appropriate hybridization
conditions is within the skill of the art. See, e.g., "Current Protocols in Mol. Bio~."
Vol. 1 & II, Wiley In~erscience. Ausbel et al. (ed.) (19g2). Protein sequences that are
subst~nti~lly the same can be i(lentified by proteolytic digestion, gel electrophoresis
and microsequencing.
The term "functionally equivalent" intends that the amino acid sequence of
the subject protein is one that will exhibit enzymadc actdvity of the same kind as that
of the lipase.
A "heterologous" region of a DNA construct is an identifi~ble segment of
DNA within or attached to another DNA molecule that is not found in association -
with the other molecule in nature.
The polypeptides and polynucleotides of the present invendon may be
employed in combination with a suitable pharrn~l~eutic~l carrier. Such composidons
comprise a therapeutically effective amount of the actdve agent, and a
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ph~rm~ceutically acceptable carrier or excipient. Such a carrier includes but is not
limited to saline. buffered saline, dextrose, water, glycerol, ethanol. and combinations
thereof. The formulation should suit the mode of ~-iminictration.
The invention also provides a pharmaceutical pack or kit comprising one or
s more cont~in-ors filled with one or more of the ingredients of the ph~rm~eutical
compositions of the invention. Associated with such container(s) can be a notice in
the form prescribed by a governmental agency regulating the m~nllf~chlre~ use or sale
of ph~rm~ceuticals or biological products, which notice reflects approval by theagency of m~nuf~eture, use or sale for human ?l-lminictration. In addition, the
polypeptides of the present invention may be employed in conjunction with other
therapeutic compounds.
The pharmaceutical compositions may be ~dminictered in a convenient
manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular,
subcutaneous, intranasal or intradermal routes. The polypeptides or polynucleotides
of the present invention is ~minictoted in an amount which is effective for treatment
and/or prophylaxis of the specific indication. The amounts and dosage regimens of
active agent ~rlminictered to a subject will depend on a number of factors such as the
mode of ~rlmini~ctration~ the nature of the condition being treated and the judgment of
the prescribing physician.
The sequences of the present invention are also valuable for chromosome
idPntifir~tion The sequence is specifically targeted to and can hybridi~ with a
particular location on an individual human chromosome. Moreover, there is a current
need for identifying particular sites on the chromosome. Chromosome marking
reagents based on actual sequence data (repeat polymorphisms) are presently
available for m~rking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important first step in
correlating those sequences with genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR
primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is
used to rapidly select primers that do not span more than one exon in the genomic
DNA, thus complicating the amplification process. These primers are then used for
PCR screening of somatic cell hybrids containing individual human chromosomes.
Only those hybrids containing the human gene corresponding to the primer will yield
an amplified &agment.
3s PCR mapping of somatic cell hybrids is a rapid procedure for :~c~igning a
particular DNA to a particular chromosome. Using the present invention with the
same oligonucleotide primers, sublocalization can be achieved with panels of
fragments from specific chromosomes or pools of large genomic clones in an
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analogous manner. Other mapping strategies that can similarly be used to map to its
chromosome include in situ hybridization, prescreening with labeled flow-sorted
chromosomes and preselection by hybridization to construct chromosome specific-
cDNA libraries.
s Fluorescence in situ hybrifli~tion (FISH) of a cDNA clones to a metaphase
chromosomal spread can be used to provide a precise chromosomal location in one
step. This technique can be used with cDNA as short as 500 or 600 bases; however,
clones larger than 2,000 bp have a higher likelihood of binding to a unique
chromosomal location with sufficient signal intensity for simple detection. F~SHrequires use of the clones from which the EST was derived, and the longer the better.
For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not
nece.cs~ry to get good results a reasonable percentage of the time. For a review of this
technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques,
Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with genetic
map data. Such data are found, for example, in V. McKusick, Mendelian Tnh~rit~nce
in Man (available on line through Johns Hopkins University Welch Medical Library).
The relationship between genes and ~iice~çs that have been mapped to the same
chromosomal region are then identified through linkage analysis (coinheritance of
physically adjacent genes).
Next, it is necess~ry to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is observed in
some or all of the affected individuals but not in any normal individuals, then the
2s mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping
techniques, a cDNA precisely localized to a chromosomal region associated with the
disease could be one of between 50 and 500 potential causative genes. (This assumes
1 megabase mapping resolution and one gene per 20 kb).
Comparison of affected and unaffected individuals generally involves first
looking for structural alterations in the chromosomes, such as deletions or
translocations that are visible from chromosome spreads or detectable using PCR
based on that cDNA sequence. Ultimately, complete sequencing of genes from
several individuals is required to confrm the presence of a mutation and to
distinguish mutations from polymorphisms.
The polypeptides of the invention or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be, for example,
polyclonal or monoclonal antibodies. The present invention also includes chimeric,
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single chain, and hum~ni7Pd antibodies, as well as Fab fr~gment.c, or the product of
an Fab expression library. Various procedures known in the art may be used for the
production of such antibodies and fr~g,m.ont.c.
Antibodies generated against the polypeptides of the present invention can be
s obtained by direct injection of the polypeptides into an animal or by ~rlminictering the
polypeptides to an animal, preferably a nnnhllm~n The antibody so obtained will
then bind the polypeptides itself. In this manner, even a sequence encoding only a
fragment of the polypeptides can be used to generate antibodies binding the whole
native polypeptides. Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be used. Examples include
the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the
trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983,
Immunology Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S.
Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic
polypeptide products of this invention.
This invention further provides a method of screening compounds to identify
those compounds which inhibit the polypeptide comprising contacting isolated
polypeptide with a test compound and measuring the rate of turnover of an enzymesubstrate as compared with the rate of turnover in the absence of test compound. The
2s invention also relates to compounds identified thereby.
This invention also provides transgenic non-human ~nim~lc comprising a
polynucleotide encoding a polypeptide of the invention. Also provided are methods
for use of said transgenic ~nim~lc as models for mutation and SAR (structure/activity
relationship) evaluation as well as in drug screens.
The present invention is also directed to inhibitor molecules of the
polypeptides of the present invention, and their use in reducing or elimin~ting the
function of the polypeptide.
An example of an inhibitor is an antibody or in some cases, an
oligonucleotide which binds to the polypeptide.
3s An example of an inhibitor is an antisense construct prepared using ~nticence
technology. Antisense technology can be used to control gene expression through
triple-helix formation or ~ntisence DNA or RNA, both of which methods are based on
binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of
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the polynucleotide sequence, which encodes for the polypeptides of the present
invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40
base pairs in length. A DNA oligonucleotide is designed to be complPm~nt~ry to aregion of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids
s Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al.,
Science, 251: 1360 (1991)), thereby preventing transcription and the production of
polypeptide. The ~nticçnce RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the polypeptide (Okano, J.
Neurochem., 56:~60 (1991); Oligodeoxynucleotides as .AnticenSe Inhibitors of Gene
Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described
above can also be delivered to cells such that the ~ntic.once RNA or DNA may be
expressed in vivo to inhibit production of polypeptide.
Another example of an inhibitor is a small molecule which binds to and
occupies the catalytic site of the polypeptide thereby making the catalytic sitein:~c-ocsjhle to substrate such that normal biological activity is prevented. Examples
of small molecules include but are not limited to small peptides or peptide-likemolecules.
When used in therapy, the inhibitors of the invention are formulated in
accordance with standard pharmaceutical practice.
The inhibitors which are active when given orally can be formulated as
liquids, for example syrups, suspçncions or emulsions, tablets, capsules and, lozenges.
A liquid formulation will generally consist of a suspension or solution of the
compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) forexample, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol,
2s oils, or water with a suspending agent, preservative, flavouring or colouring agent.
A composition in the form of a tablet can be prepared using any suitable
pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples
of such carriers include m~"nesillm stearate, starch, lactose, sucrose and cellulose.
A composition in the form of a capsule can be prepared using routine
çnc~ps~ tion procedures. For example, pellets containing the active ingredient can
be prepared using standard carriers and then filled into a hard gelatin capsule;alternatively, a dispersion or suspension can be prepared using any suitable
pharrnaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and
the dispersion or suspension then filled into a soft gelatin capsule.
3s Typical parenteral compositions consist of a solution or suspension of the
compound or pharmaceutically acceptable salt in a sterile aqueous carrier or
parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone,
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leeithin, arachis oil or sesame oil. Alternatively, the solution ean be lyophilised and
then reeonstituted with a suitable solvent just prior to ~lminictration.
A typieal suppository formulation comprises a compound of formula (I) or a
ph~rm~eutically acceptable salt thereof whieh is aetive when ~tlminictered in this
s way, with a binding and/or lubric~ting agent sueh as polymeric glyeols, gelatins or
eoeoa butter or other low melting vegetable or synthetic waxes or fats.
Preferably the composition is in unit dose form such as a tablet or capsule.
Each dosage unit for oral ~dminictration contains preferably from 1 to 250
mg (and for parenteral ~-lminictration contains preferably from 0.1 to 25 mg) of an
inhibitor of the invention.
The daily dosage regimen for an adult patient may be, for example, an oral
dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an
intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg,
preferably between 0.1 mg and 25 mg, of the eompound of the formula (I) or a
pharrn~eutically acceptable salt thereof calculated as the free base, the compound
being ~riminictPred 1 to 4 times per day. Suitably the eompounds will be
~dminict~red for a period of continuous therapy.
The present invention will be further described with reference to the
following examples; however, it is to be understood that the present invention is not
limited to such examples. All parts or amounts, unless otherwise specified, are by
weight.
In order to facilitate understanding of the following examples certain
frequently occurring methods and/or terms will be described.
"Plasmids" are design~ttod by a lower case p preceded and/or followed by
2s capital letters and/or numbers. The starting plasmids herein are either commercially
available, publicly available on an unrestricted basis, or can be constructed from
available plasmids in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be apparent to the ordinarily
skilled artisan.
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or
two complementary polydeoxynucleotide strands which may be chemieally
synthf~ci7Prl Sueh synthetic oligonucleotides have no 5' phosphate and thus will not
ligate to another oligonueleotide without adding a phosphate with an ATP in the
presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has
3s not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between
two double stranded nucleic acid fragments (M~ni~tic, T., et al., Id., p. 146). Unless
otherwise provided, ligation may be accomplished using known buffers and
,
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PCT/GB95/02320
W O 97/12984
conditions with l0 units to T4 DNA ligase ("ligase") per 0.5 ,ug of approximately
equimolar amounts of the DNA fra~ments to be ligated.
E~AMPLE
GENE C~ONING AND EXPRESSION
cDNA Library construction
Poly A+ (mRNA) was isolated from human prostate (benign possible hyperplasia)
using standard methods (ref Maniatis et al). First strand cDNA was primed using an
oligo dT primer. The cDNA library was constructed with the Stratagene ZAP-cDNA
synthesis kit, packaged with Gigpack 11 gold p~ck~ging extract and amplified in XLl-
blue MRF bacterial cells. The cDNA inserts were cloned unidirectionally into thevector.
DNA Sequencing
The phage clone containing the EST was excised from the ~ Unizap XR
bacteriophage vector into the Bluescript phagemid ~according to the Stratagene
manual) for characterisation. The insert of 1823bp was m~n~l~lly sequenced on both
strands (using the ~m~or~h~m -USB Sequenase 2.0 DNA sequencing kit) by primer
walking (SEQ ID 2). The cDNA has an open reading frame with the potential to
code for a polypeptide of 393 amino acids (SEQ ID 1). The predicted MW for the
full reading frame is 44143Da.
Sequence Data:
SEQIDNO 1
MGVNQSVGFPPVTGPHLVGCGDVMEGQNLQGSFFRLFYPCQKAEETMEQPLWIPRYEYCTGLAEYLQFN
2s KRCAGACCSTWRWDLVACLLAGMAPFKTKDSGYPLIIFSHGLGAFRTLYSAFCMELASRGFVVAVPEPQ
DRSAATTYFCKQAPEENQPTNESLQEEWIPFRR~ K~ HVRNPQVHQRVSECLRVLKILQEVTAGQ
TVFNIFPGGLDLMTLKGNIDMSRVAVMGHSFGGATAILALAKETQFRCAVALDAWMFPLERDFYPKARG
PVFFINTEKFQTMESVNLMKKICAQHEQSRIITVLGSVHRSQTDFAFVTGNLIGKFFSTETRGSLDPYE
GQEVMVRAMLAFLQKHLDLKEDYNQWNNLIEGIGPSLTPGAPHHLSSL
SEQ ID NO 2
GGCACGAGCT TCTGAGGAAT CAGCTTGACT GGCCAGCAAG TTCAGCTCCG
GCAAGTCATT TGATTCACCC GGTGATGAAA TGGGGGTCAA CCAGTCTGTG
GGCTTTCCAC CTGTCACAGG ACCCCACCTC GTAGGCTGTG GGGATGATGA
TGGAGGGGTC AGAATCTCCA GGGGAGCTTC TTTCGACTCT TCTACCCCTG
CCAAAAGGCA GAGGAGACCA TGGAGCAGCC CCTGTGGATT CCCCGCTATG
AGTACTGCAC TGGCCTGGCC GAGTACCTGC AGTTTAATAA GACGACTGCG
GGGGCTTGCT GTTCAACCTG GCGGTGGGAT CTTGTCGCCT GCCTGTTAGC
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WO 97/12984 PCT/GB95/02320
TGGAATGGCC CCCTTTAAGC ACMAAAGGAC TcTGGATAAc CCCATTGATC
AGTCTTCTCC CATGGCCTAG GAGCCTTCAG GACTTTGTAT TCAGCCTTCT
GCATGGAGCT GGCCTACACG TGGCTTTGTG ~ll~G~ GC CAGAGCCACA
GGACCGGTCA GCGGCAACCA CCTATTTCTG CAAGCAGGCC CCAGAAGAGA
ACCAGCCCAC CAATGAATCG CTGCAGGAGG AATGGATCCC TTTCCGTCGA
GTTGAGGAAG GGGAGAAGGA ATTTCATGTT CGGAATCCCC AGGTGCATCA
GCCGGGTAAG CGAGTGTTTA CGGGTGTTGA AGATCCTGCA AGAGGTCACT
GCTGGGCAGA CTGTCTTCAA CATCTTTCCT GGTGGCTTGG ATCTGATGAC
TTTGAAGGGC AACATTGACA TGAGCCGTGT GGCTGTGATG GGACATTCAT
0 TTGGAGGGGC CACAGCTATT CTGGCTTTGG GCCAAGGAAG ACCCAATTTC
TCGTGTGCGG TGGCTCTGGA TGCTTGGATG TTTCCTCTGG AACGTGACTT
TTACCCCAAG GCCCGAGGAC ~'1~'1'~'1"1'~'1"1' TATCAATACT GAGAAATTCC
AGACAATGGA GAGTGTCAAT TTGATGAAGA AGATATGTGC CCAGCATGAA
CAGTCTAGGA TCATAACCGT TCTTGGTTCT GTTCATCGGA GTCAAACTGA
CTTTGCTTTT GTGACTGGCA ACTTGATTGG TAAATTCTTC TCCACTGAAA
CCCGTGGGAG CCTGGACCCC TATGAAGGGC AGGAGGTTAT GGTACGGGCC
ATGTTGGCCT TCCTGCAGAA GCACCTCGAC CTGAAAGAAG ACTATAATCA
ATGGAACAAC CTTATTGAAG GCATTGGACC GTCGCTCACC CCAGGGGCCC
CCCACCCATC TGTCCAGCCT GTAGGCGACA ACTGGCTCAT TTGTAAAGTC
ACTTCAGCCA AGCTTTTCAT TTGGGAGCTA CCCAAGGGCA CCCATGAGCT
CCTATCAAGA AGTGATCAAC GTGACCCCTT TTCACAGATT GAAAGGTGTA
ATCACACTGC TGCTTGGATA ACTGGGTACT TTGATCTTAG ATTTGATCTT
AAAATCACTT TGGGACTGGG ATCCCTTGCT GATTGACAAA CAGACTTTCT
GGGACCTTGA TGGAGTGGGG AACAAGCAGT AGAGTGGGAC TGGGGGAGAC
CCAGGCCCCG GGCTGAGCAC TGTGAGGCCT GGATGTGAAG ACTCAMCCCA
CGAACGCTCA TTCCCTTACC CCCGGCCAGT GCTGCTGCTT CAGTGGAAGA
GATGAAGCCA AAGGTAACAG AATGAAAAAT CCCTACCTTC AGAGACTCTA
GCCCAGCCCA ACACCATCTC TTCCTACCTC TCAGCCTTCT CCCTCCCCAG
GGCCACTTGT TGAGAAGTCT GAGCACTTTA TGTAAATTTC TAGGTGTGAG
CCGTGAAAAA AAAAAAAAAA AAAA
M is defined as either A or C, where the actual base is unclear from either DNA
strand.
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