U.S. patent application number 11/342851 was filed with the patent office on 2006-07-06 for compositions and methods for treating female fertility.
This patent application is currently assigned to SIGMA-TAU INDUSTRIE FARMACEUTICHE RIUNITE S.p.A.. Invention is credited to Alberto Mantovani.
Application Number | 20060148001 11/342851 |
Document ID | / |
Family ID | 33100833 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060148001 |
Kind Code |
A1 |
Mantovani; Alberto |
July 6, 2006 |
Compositions and methods for treating female fertility
Abstract
The PTX3 gene or equivalent PTX3 activity is required for female
fertility. Manipulation of PTX3 activity will regulate female
fertility. The effects of female sterility may be ameliorated,
reproductive ability may be increased or decreased as desired,
female fertility may be enhanced, or combinations thereof. The need
for therapies that affect female fertility is thereby
addressed.
Inventors: |
Mantovani; Alberto; (Milan,
IT) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
SIGMA-TAU INDUSTRIE FARMACEUTICHE
RIUNITE S.p.A.,
Rome
IT
|
Family ID: |
33100833 |
Appl. No.: |
11/342851 |
Filed: |
January 31, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10785427 |
Feb 25, 2004 |
7041648 |
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11342851 |
Jan 31, 2006 |
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10485640 |
Feb 3, 2004 |
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PCT/IT02/00473 |
Jul 18, 2002 |
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10785427 |
Feb 25, 2004 |
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60309472 |
Aug 3, 2001 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 33/689 20130101;
G01N 2333/4756 20130101; A01K 2227/105 20130101; G01N 33/74
20130101; A01K 2267/03 20130101; C12N 2750/14111 20130101; A61K
49/0004 20130101; G01N 2800/367 20130101; C12N 2840/203 20130101;
A01K 67/0276 20130101; A61K 48/00 20130101; A01K 2217/075 20130101;
A61K 38/1709 20130101; C12N 15/8509 20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A method of screening a pharmaceutical compound to asses the
capability of said compound to affect the reproductive ability in a
female subject comprising measuring or detecting the ability or
effectiveness of said compound to increase or decrease the presence
or activity of PTX3 in the extracellular matrix of cumulous
oophorus, or measuring or detecting the ability or effectiveness of
said compound to increase or decrease the presence or activity of
PTX3 expressed from granulosa cells, or measuring or detecting the
ability or effectiveness of said compound to bind or inhibit the
binding of PTX3 to a target receptor or to function.
Description
[0001] The present application is a divisional of application Ser.
No. 10/785,427, filed 25 Feb. 2004 (allowed), which is a
continuation-in-part of application Ser. No. 10/485,640, filed 3
Feb. 2004 (abandoned), which is a 371 U.S. national phase of
International Application PCT/ITO2/00473, filed 18 Jul. 2002, which
designated the U.S., and claims benefit of U.S. Provisional
Application No. 60/309,472, filed 3 Aug. 2001, the entire contents
of each of which is hereby incorporated by reference.
[0002] This invention relates to the requirement of PTX3 activity
for female fertility. The present application demonstrates that a
genetic mutation which reduces PTX3 activity results in female
sterility.
[0003] Pentraxins are a superfamily of proteins, which is
characterized by a cyclic multimeric structure [1]. The classical
short pentraxins C-reactive protein (CRP) and serum amyloid P
component (SAP) are acute phase proteins in man and mouse,
respectively, produced in the liver in response to inflammatory
mediators; in particular, they are directly induced by
interleukin-6 [2-3].
[0004] Long pentraxins share similarities with the classical short
pentraxins, but differ by the presence of an unrelated long
N-terminal domain coupled to the C-terminal pentraxin domain, as
well by genomic organization, chromosomal localization, cellular
source, inducing stimuli, and ligands recognized. Long pentraxin 3
(PTX3) is the first long pentraxin identified as an interleukin-1
(IL-1) inducible gene in endothelial cells [4] and as a tumor
necrosis factor-.alpha. (TNF.alpha.)) inducible gene in fibroblasts
[5]. PTX3 is also produced by macrophages and other cell types and
tissues upon stimulation with primary inflammatory mediators (LPS,
IL-1, TNF.alpha.) [6-8]. PTX3 consists of a C-terminal 203-amino
acid pentraxin-like domain and an N-terminal 178-amino acid
unrelated domain. When secreted, glycosylated PTX3 protomers (45
kDa) assemble to form 10-20 multimers [9]. PTX3 does not bind to
classical pentraxin ligands such as phosphoethanolamine,
phosphocholine, high pyruvate agarose, collagen IV, fibronectin, or
gelatin. In contrast, PTX3 specifically binds with high affinity to
C1q by the pentraxin domain [9]. PTX3 plasma levels are very low in
normal conditions (.ltoreq.2 ng/ml) but increase in several
pathological conditions (10-100 ng/ml) including infections
[10].
[0005] Other long pentraxins cloned after PTX3 include guinea pig
apexin [11, 12] which is expressed in the sperm acrosome, XL-PXN1
from Xenopus laevis [13], rat neuronal pentraxin 1 (NP1) [14],
human NP1 and NP2 [15, 16], mouse NP1 and NP2 [15], Narp [17], and
neuronal pentraxin receptor (NRP), a putative integral membrane
pentraxin [18-9]. The in vivo function of long pentraxins has not
been unequivocally defined.
[0006] PTX3 consists of two structural domains: a N-terminal domain
unrelated to any known molecule and a C-terminal domain similar to
the short pentraxins such as C-reactive protein (Breviario et al.,
J. Biol. Chem., 267:22190-22197, 1992). Substantial similarity has
been found between human PTX3 (hPTX3) and mouse PTX3 (mPTX3). The
degree of identity between human and murine PTX3 genes is 82%, and
reaches 90% if conservative substitutions are considered (Introna
et al., Blood, 87:1862-1872, 1996). The genes are located in
syntenic chromosome locations. The high degree of similarity
between hPTX3 and mPTX3 sequences is a sign of the high degree of
conservation of pentraxins during evolution (Pepys & Baltz,
Adv. Immunol., 34:141-212, 1983). Pentraxins are reviewed by Gewurz
et al. (Curr. Opin. Immunol., 7:54-64, 1995).
[0007] WO 99/32516 describes the use of PTX3 for the therapeutic
treatment of cancer, inflammation, and infectious diseases.
[0008] U.S. Pat. No. 5,767,252 describes a growth factor for
neuronal cells belonging to the pentraxin family.
[0009] WO 02/36151 describes the use of PTX3 for the preparation of
medicament for the prevention and treatment of autoimmune
pathologies.
[0010] In contrast to the foregoing, the study of mice genetically
modified at their PTX3 genetic locus, which were produced by
homologous recombination in embryonic stem cells, and the effects
thereof has revealed the involvement of PTX3 activity in female
fertility.
[0011] It is an objective of the invention to manipulate PTX3
activity and thereby regulate female fertility. The effects of
female sterility may be ameliorated, reproductive ability may be
increased or decreased as desired, female fertility may be
enhanced, or combinations thereof. Other treatments such as in
vitro fertilization require invasive procedures and complicated
technology. The need for therapies that affect female fertility is
thereby addressed. Other advantages and improvements are discussed
below, or would be apparent from the disclosure herein.
[0012] Pharmaceutical compositions, methods for using and making
them, and further objectives are described below.
[0013] An object of the invention is to provide a pharmaceutical
composition which is comprised of an agent which changes PTX3
activity in an amount sufficient to affect female reproductive
ability. The discovery that PTX3 activity is required for
successful oocyte fertilization may be used according to the
present invention as the basis for a conraceptive method to reduce
fertility of a femal patient or animal, and/or as the a basis for a
therapy of a female patient or animal with a defect in
reproduction, and/or for diagnosis of the ability of a female
patient or animal to reproduce, and/or as the basis for a method to
develop drugs or medicines or therapies which reduce or treat
infertility or enhance or treat infertility. One of ordinary skill
will apprecaite from the present disclosure that reduction of the
activity or amount of the function of PTX3 will reduce fertility or
increase infertility of a female patient or animal and enhancement
or increase in the activity or amount of the function of PTX3 will
increase fertility or reduce infertility relating to PTX3
deficiency of a female patient or animal. Such changes or
alterations in the PTX3 levels or activity or affinities are, in
one embodiment of the invention, preferably produced locally, near
the cite of action of oocyte fertilization or potential oocyte
fertilization.
[0014] Examples of such agents include polynucleotides
corresponding to PTX3 genes or encoding PTX3, polypeptides
corresponding to PTX3 proteins encoded thereby, and others that
increase or decrease PTX3 gene expression. This includes the
nucleotide and amino acid sequences listed herein, analogs thereof,
those containing muta-tions or polymorphisms, and other variants
thereof (e.g., partial-length oligo-nucleotides and oligopeptides).
Antibodies and fragments thereof which may antagonize the function
of PTX3 are an example of such agents.
[0015] Hybrids between at least one PTX3 portion and a heterologous
portion (polynucleotide or polypeptide) are considered chimeric
gene or fusion protein variants, respectively. Genetic vectors may
be used to shuttle at least one PTX3 portion into a host or to
express at least one PTX3 portion by transcription and/or
translation in a host or using at least partially purified
components. Activators (e.g., interleukin-6, NF-.kappa.B, receptor
agonists) or inhibitors (e.g., antibody, I.kappa.B, receptor
antagonists) may also be used as agents to modulate PTX3 activity.
The agent may be derived from humans or nonhuman animals (e.g.,
mammals).
[0016] The subject may be a female patient or animal. The
composition may be suitable for systemic administration or adapted
for local administration (i.e., within or around a female
reproductive organ). The composition may be used to treat sterility
or as a contraceptive.
[0017] Another object of the invention is to provide methods of
administering the pharmaceutical composition to a subject in need
of treatment for female sterility or female contraception in an
amount sufficient to increase or decrease, respectively, the
subject's reproductive ability.
[0018] Detecting PTX3 in a female subject and correlating this
amount with her reproductive ability is a further objective of the
invention. Mutations in the human PTX3 genetic locus would map to
chromosome 3q24-q28; mutations in interacting genes would map
outside the PTX3 genetic locus. The function of a PTX3 variant may
be determined by comparison to known PTX3 sequences or other
pentraxin sequences; folding, glycosylation, secretion, or
formation of multimers; receptor binding or signal transduction;
effect on reproductive ability, fertility, or sterility; or
combinations thereof.
[0019] An additional objective of the invention is to screen for at
least one agent which changes PTX3 activity, and thereby affects
female reproductive ability, as well as to obtain an agent by such
processes. Several examples of such agents are disclosed.
[0020] Yet another objective of the invention is to provide
mammalian cells and nonhuman mammals which are genetically mutated
to decrease PTX3 activity. They provide in vitro and in vivo models
for defects in reproductive ability (e.g., sterility). They can be
used for screening or for trials of potential therapeutics.
[0021] Further aspects of the invention will be apparent to a
person skilled in the art from the following description and
claims, and generalizations thereto.
BRIEF DESCRIPTIONS OF THE DRAWINGS AND SEQUENCE LISTING
[0022] FIGS. 1A-1F illustrate the abnormal morphology of cumuli
oophori from PTX3 -/- mice. Cumuli oophori were recovered 14-16 hr
after hCG treatment. They are shown after collection (A and B) or 4
hr later (C and D). In PTX3 +/+mice (A and C), granulosa cells form
a compact and stable cumulus around the oocyte (arrow da mettere).
In PTX3 -/- mice (B and D), they are loosely associated to the
oocyte and the cumulus has completely disappeared in 4 hr.
Histological examination of the ovaries of PTX3 +/+(E) and PTX3 -/-
(F) mice shows normal antral follicles.
[0023] FIGS. 2A-2D show PTX3 mRNA and protein expression in ovarian
tissue. (A) Kinetics of PTX3 expression in ovary after
hormonally-induced superovulation (PMS treatment followed 48 hr
later by hCG treatment) were shown at the mRNA level. Ovaries were
collected at 0, 6, 16, 24 or 48 hr after PMS treatment and then 2,
6, 16, 24 or 48 hr after hCG treatment. Ten .mu.g of total RNA was
loaded in each lane. Ethidium bromide staining of the gel is shown
in the lower panel. (B) In situ hybridization of the ovary:
granulosa cell express PTX3 mRNA only in mature follicles. (C) PTX3
expression by cumuli oophori (C.O.), cumulus oophorus cells (C.O.
cells), and oocytes was detected by Western blotting. Cumuli
oophori were recovered from four PTX3 +/+ and PTX3 -/-
superovulated females; cumulus oophorus cells and oocytes were
obtained from seven and 14 PTX3 +/+super-ovulated females,
respectively. (D) Phase contrast (right panels) and
immunofluorescence analysis (left panels) of cumuli oophori from
PTX3 -/- (lower panels) and PTX3 +/+(upper panels) mice are
illustrated.
[0024] Sequences of a human cDNA and its translated open reading
frame (SEQ ID NOS:1-2, respectively), a mouse cDNA and its
translated open reading frame (SEQ ID NOS:3-4, respectively), human
and mouse upstream regulatory regions (SEQ ID NOS:5-6,
respectively), and PCR primers (SEQ ID NOS:7-10) are shown in the
Sequence Listing. Alignment of human and mouse amino acid sequences
shows 312 of 381 residues are identical (82%) and 351 residues are
at least similar (92%). Both genes contain three exons: the first
encodes for 43 amino acid residues, the second encodes for 135
amino acid residues with no high similarity to known sequence
motifs, and the third encodes 203 amino acid residues with
similarity to pentraxins. A pentraxin-like domain includes two Cys
residues at positions 162 and 254 and a consensus "pentraxin-like"
sequence His-Xaa-Cys-Xaa-Ser/Thr-Trp-Xaa-Ser (SEQ ID NO:11).
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0025] Polynucleotides corresponding to all or part of a PTX3
nucleic acid (e.g., transcripts or genes), which include mutants
and other variants thereof, may be used to increase PTX3 activity
(e.g., in vivo or in vitro expression of PTX3 polypeptide), to
supplement or correct a genetic defect (e.g., transfection,
infection), to decrease PTX3 activity (e.g., antisense, ribozyme,
siRNA), or to detect complementary polynucleotides. Similarly,
polypeptides corresponding to a PTX3 protein, which include mutants
and other variants thereof, may be used directly to provide PTX3
activity if functional; to produce inhibitory anti-bodies,
agonists, and antagonists; and to identify, isolate, or to detect
interacting proteins (e.g., antibodies, receptor agonists or
antagonists) by binding assays.
[0026] Native PTX3 is glycosylated (potential N-linked
glycosylation site at position 203). A multimeric PTX3 complex
eluted in gel filtration with a relative molecular weight of about
900 kDa. It migrated in gel electrophoresis under nondenaturing and
nonreducing conditions as a predominant band of about 440 kDa
(e.g., 9- or 10-mer of about 45 kDa protomers) with two minor bands
in the 540-600 kDa range. Circular dichroism analysis indicated
that PTX3 contained mostly .beta.-sheet structure with some
.alpha.-helical structure. PTX3 polypeptide or a complex thereof
may be identified, isolated, or detected indirectly though a
binding molecule (e.g., antibody, natural or nonnatural peptide
mimetic) for the PTX3 gene product.
[0027] Candidate compounds useful for affecting reproductive
ability may interact with a representative PTX3 polynucleotide or
polypeptide, and be screened for their ability to provide a method
of diagnosis or treatment. These products may be used in assays
(e.g., diagnosis) or for treatment; conveniently, they are packaged
as assay kits or in pharmaceutical form. Binding to C1q was
specific and saturable (one PTX3 protomer bound to one C1q
receptor) with a K.sub.d of 7.4.times.10.sup.-8 M. Kinetic analysis
lead to a calculation of K.sub.on of 2.6.times.10.sup.5 M.sup.-1
s.sup.-1 and K.sub.off of 4.times.10.sup.-4 s.sup.-1. The ligand
for C1q binding is the pentraxin-like domain of PTX3 with
multimerization being required for binding (possibly through an
intramolecular cysteine linkage). Other receptors for PTX3 may be
characterized.
[0028] Another aspect of the invention is a hybrid PTX3
polynucleotide or polypeptide: e.g., a transcriptional chimera or a
translational fusion. In transcriptional chimeras, at least a
transcriptional regulatory region of a heterologous gene is ligated
to a PTX3 polynucleotide or, alternatively, a transcriptional
regulatory region of a PTX3 gene is ligated to at least a
heterologous polynucleotide. The reading frames of a PTX3
polypeptide and at least a heterologous amino acid domain are
joined in register for a translational fusion. If a reporter or
selectable marker is used as the heterologous region or domain,
then the effect of mutating PTX3 nucleotide or amino acid sequences
on PTX3 function may be readily assayed. In particular, a
transcriptional chimera may be used to localize a regulated
promoter of a PTX3 gene and a translational fusion may be used to
localize PTX3 protein in the cell. For example, transcriptional
regulatory regions, ligand-binding domains, or multimerization
domains from PTX3 may be involved in a hybrid molecule.
[0029] "PTX3" refers to human and mouse genes and proteins, mutants
and polymorphisms found in nature, and variant forms thereof (e.g.,
mutants and analogs not found in nature) as well as analogs
thereof. The chemical structure of PTX3 may be a polymer of natural
or nonnatural nucleotides connected by natural or nonnatural
covalent linkages (i.e., polynucleotide) or a polymer of natural or
non-natural amino acids connected by natural or nonnatural covalent
linkages (i.e., polypeptide). See Tables 1-4 of WIPO Standard ST.25
(1998) for a nonlimiting list of natural and nonnatural nucleotides
and amino acids.
[0030] "Mutants" are PTX3 polynucleotides and polypeptides having
at least one function that is more active or less active, an
existing function that is changed or absent, a novel function that
is not naturally present, or combinations thereof. "Polymorphisms"
are PTX3 polynucleotides and polypeptides that are genetically
changed, but the changes do not necessarily have functional
consequences. "Analogs" are PTX3 polynucleotides and polypeptides
with different chemical structures, but substantially equivalent
function as compared to the native gene or protein. PTX3 functions
are described in detail herein. Mutants, polymorphisms, and analogs
can be made by genetic engineering or chemical synthesis, but the
latter is preferred for nonnatural nucleotides, amino acids, or
linkages.
[0031] "Oligonucleotides" and "oligopeptides" are short versions of
polynucleotides and polypeptides (e.g., less than 30, 60, 90 or 180
nucleotides or amino acids). They may be a fragment of a PTX3
nucleotide or amino acid sequence described herein. Generally, they
can be made by chemical synthesis, but cleavage of longer
polynucleotides or polypeptides can also be used. Electrophoresis
and/or reverse phase high-performance liquid chromatography (HPLC)
are suitable biochemical techniques to purify short products.
[0032] A PTX3 gene can be identified using stringent hybridization:
e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or
70.degree. C. for an oligonucleotide; 500 mM NaHPO.sub.4 pH 7.2, 7%
sodium dodecyl sulfate (SDS), 1% bovine serum albumin (BSA), 1 mM
EDTA, 45.degree. C. or 65.degree. C. for a polynucleotide of 50
bases or longer. A PTX3 protein can be identified using an antibody
or other binding protein as a probe using stringent binding: e.g.,
50 mM Tris-HCl pH 7.4, 500 mM NaCl, 0.05% TWEEN 20 surfactant, 1%
BSA, room temperature. Washing conditions may be varied by
adjusting the salt concentration and temperature such that the
signal-to-noise ratio is sufficient for specific hybridization or
binding. Such isolation methods may be used to identify an unknown
PTX3-related nucleic acid or protein using a probe which detects a
known PTX3 nucleic acid or protein, respectively. For example, a
mixture of nucleic acids or proteins may be separated by one or
more physical, chemical, and/or biological properties, and then the
presence or absence of PTX3 nucleic acid or protein may be detected
by specific binding of the probe. The probe may also be used to
detect the presence or absence of a known PTX3 gene or protein, or
to identify a previously unknown PTX3 gene or protein. Blocking and
washing conditions can be varied to obtain a nucleic acid
hybridization or protein binding signal that is target specific
and/or reduces the background.
[0033] An "isolated" product is at least partially purified from
its cell of origin (e.g., human, other mammal, bacterium, yeast) or
manufacturing source. For example, as compared to a lysate of the
cell of origin, the isolated product is at least 50%, 75%, 90%, 95%
or 98% purified from other chemically-similar solutes (e.g., total
nucleic acids for polynucleotides or total proteins for
polypeptides). For a chemically-synthesized polymer of nucleotides
or amino acids, purity is determined by comparison to prematurely
terminated or blocked products and may, as a practical matter, be
considered isolated without purification. Purification may be
achieved by biochemical techniques such as, for example, cell
fractionation, centrifugation, chromatography, electrophoresis,
precipitation, specific binding, or combinations thereof.
Generally, solvent (e.g., water) and functionally inert chemicals
(e.g., salts and buffers) are disregarded when determining purity.
Cloning is often used to isolate the desired product. Therefore, a
pharmaceutical composition may include agents which are responsible
for most if not all of the PTX3 activity.
[0034] The meaning of "heterologous" depends on context. For
example, ligation of heterologous nucleotide regions to form a
chimera means that the regions are not found colinear in nature
(e.g., human-derived PTX3 polynucleotide ligated to a human
non-PTX3 transcriptional regulatory region). Another example is
fusion of amino acid domains which are not found colinear in human
(e.g., human-derived PTX3 polypeptide joined to a human non-PTX3
multimerization domain). Ligation of nucleotide regions or joining
of amino acid domains, one derived from a human and another derived
from an animal, are heterologous because they are derived from
different species. In a further example, transfection of a vector
or expression construct into a heterologous host cell or
transgenesis of a heterologous non-human organism means that the
vector or expression construct is not found in the cell's or
organism's genome in nature. A "recombinant" product is the result
of ligating heterologous regions for a recombinant polynucleotide
or fusing heterologous domains for a recombinant polynucleotide.
Recombination may be genetically engineered in vitro with purified
enzymes or in vivo in a cultured cell.
[0035] According to one aspect of invention, polynucleotides (e.g.,
DNA or RNA, single- or double-stranded) that specifically hybridize
to PTX3 genes and transcripts thereof can be used as probes or
primers. Such polynucleotides could be full length covering the
entire gene or transcribed message (e.g., a recombinant clone in a
phagemid, plasmid, bacteriophage, cosmid, yeast artificial
chromosome or YAC, bacterial artificial chromosome or BAC, or other
vector), an N-terminal "PTX3-unique" or C-terminal "pentraxin-like"
domain, an exon or particular coding region, or a shorter length
sequence which is unique to PTX3 genes or transcripts thereof but
contains only a portion of same. A probe would stably bind its
target to produce a hybridization signal specific for a PTX3
polynucleotide or polypeptide, while a primer may bind its target
less stably because repetitive cycles of polymerization or ligation
will also produce a specific amplification signal. The
polynucleotide may be at least 15, 30, 45, 60, 90, 120, 240, 360,
480, 600, 720, 1200, 2400, 5000, 10K, 20K, 40K, 100K, 250K, or 500K
nucleotides long (including intermediate ranges thereof).
[0036] Typically, a nucleotide sequence may show as little as 85%
sequence identity, and more preferably at least 90% sequence
identity compared to the coding region of SEQ ID NO: 1 or 3,
excluding any deletions or insertions which may be present, and
still be considered related. Amino acid sequences are considered to
be related with as little as 90% sequence identity compared to SEQ
ID NO:2 or 4. But 95% or greater sequence identity is preferred and
98% or greater sequence identity is more preferred.
[0037] Use of complex mathematical algorithms is not required if
sequences can be aligned without introducing many gaps. But such
algorithms are known in the art, and implemented using default
parameters in commercial software package. See Doolittle, Of URFS
and ORFS, University Science Books, 1986; Gribskov and Devereux,
Sequence Analysis Primer, Stockton Press, 1991; and references
cited therein. Percentage identity between a pair of sequences may
be calculated by the algorithm implemented in the BESTFIT computer
program (Smith and Waterman, J. Mol. Biol., 147:195-197, 1981;
Pearson, Genomics, 11:635-650, 1991). Another algorithm that
calculates sequence divergence has been adapted for rapid database
searching and implemented in the BLAST computer program (Altschul
et al., Nucl. Acids Res., 25:3389-3402, 1997).
[0038] Conservative amino acid substitutions (e.g., pair Glu/Asp,
Val/Ile, Ser/Thr, Arg/Lys or Gln/Asn) may also be considered when
making comparisons because the chemical similarity of these pairs
of amino acid residues would be expected to result in functional
equivalency in many cases. Amino acid substitutions that are
expected to conserve the biological function of the polypeptide
would conserve chemical attributes of the substituted amino acid
residues such as hydrophobicity, hydrophilicity, side-chain charge,
or size. Functional equivalency or conservation of biological
function may be evaluated by methods for structural determination
and bioassay as described herein. Thus, amino acid sequences are
considered to be related with as little as 90% sequence similarity
between the two polypeptides; however, 95% or greater sequence
similarity is preferred and 98% or greater sequence similarity is
most preferred.
[0039] The codons used in the native nucleotide sequences may be
adapted for translation in a heterologous host by adopting the
codon preferences of the host. This would accommodate the
translational machinery of the heterologous host without a
substantial change in the chemical structure of the
polypeptide.
[0040] PTX3 polypeptide and its variants (i.e., deletion, domain
shuffling or duplication, insertion, substitution, or combinations
thereof) are also useful for determining structure-function
relationships (e.g., alanine scanning, conservative or
nonconservative amino acid substitution). For example, folding and
processing of PTX3 protein, secretion of PTX3 protomer and
formation of multimers, ligand binding to receptor, signal
transduction, or combinations thereof. See Wells (Bio/Technology,
13:647-651, 1995) and U.S. Pat. No. 5,534,617. Directed evolution
by random mutagenesis or gene shuffling using PTX3 may be used to
acquire new and improved functions in accordance with selection
criteria. Mutant, polymorphic, and analog PTX3 polypeptides are
encoded by suitable mutant, polymorphic, and analog PTX3
polynucleotides. Structure-activity relationships of PTX3 may be
studied (i.e., SAR studies) using variant polypeptides produced by
an expression construct transfected in a host cell with or without
endogenous PTX3. Thus, mutations in discrete domains of the PTX3
polypeptide may be associated with decreasing or even increasing
activity in the protein's function.
[0041] A PTX3 nucleotide sequence can be used to produce a fusion
polypeptide with at least one heterologous peptide domain (e.g., an
affinity or epitope tag). Oligopeptide is useful for producing
specific antibody and epitope mapping of PTX3-specific antibody. A
polypeptide may be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 150, or more amino acids long (including
intermediate ranges thereof). Oligopeptide may be conjugated to one
affinity tag of a specific binding pair (e.g.,
antibody-digoxygenin/hapten/peptide, biotin-avidin/streptavidin,
glutathione S transferase-glutathione, maltose binding
protein-maltose, protein A or G/immunoglobulin,
polyhistidine-nickel). Either a full-length PTX3 polypeptide (e.g.,
SEQ ID NO:2 or 4) or a shorter fragment (e.g., N-terminal or
C-terminal domain) can be produced; optionally including a
heterologous peptide domain. PTX3 polypeptide may be synthesized by
chemical means, purified from natural sources, synthesized in
transfected host cells, or combinations thereof.
[0042] The PTX3 nucleotide sequence or a portion thereof can be
used to monitor PTX3 expression, to determine PTX3 sequence, and/or
to detect PTX3 variants. The invention also provides hybridization
probes and amplification primers (e.g., polymerase chain reaction,
ligation chain reaction, other isothermal amplification reactions).
A pair of such primers may be used for RT-PCR assays to quantitate
PTX3 transcript abundance within cells. Amplification primers may
be between 15 and 30 nucleotides long (preferably about 25
nucleotides), anneal to either sense or antisense strand
(preferably the pair will be complementary to each strand), and
terminate at the 3' end anywhere within SEQ ID NOS:1, 3 and 5-6 or
their complements. Therefore, this invention will be useful for
development and utilization of PTX3 primers and other
oligonucleotides to quantitate cognate RNA and DNA within
cells.
[0043] Binding of polynucleotides or polypeptides may take place in
solution or on a substrate. The assay format may or may not require
separation of bound from not bound. Detectable signals may be
direct or indirect, attached to any part of a bound complex,
measured competitively, amplified, or combinations thereof. A
blocking or washing step may be interposed to improve sensitivity
and/or specificity. Attachment of a polynucleotide or polypeptide,
interacting protein, or binding molecule to a substrate before,
after, or during binding results in capture of an unattached
species. Such immobilization will be stably attached to the
substrate under washing conditions. See U.S. Pat. Nos. 5,143,854
and 5,412,087.
[0044] Changes in gene expression may be manifested in the cell by
affecting transcriptional initiation, transcript stability,
translation of transcript into protein product, protein stability,
glycoprotein processing, rate of folding or secretion, or
combinations thereof. The gene, transcript, or polypeptide can also
be assayed by techniques such as in vitro transcription, in vitro
translation, Northern hybridization, nucleic acid hybridization,
reverse transcription-polymerase chain reaction (RT-PCR), run-on
transcription, Southern hybridization, metabolic protein labeling,
antibody binding, immunoprecipitation (IP), enzyme linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent
labeling or histochemical staining, microscopy and digital image
analysis, and fluorescence activated cell analysis or sorting.
[0045] A reporter or selectable marker gene whose product is easily
assayed may be used for convenient detection. Reporter genes
include, for example, alkaline phosphatase, .beta.-galactosidase
(LacZ), chloramphenicol acetyltransferase (CAT),
.beta.-glucoronidase (GUS), luciferases (LUC), green and red
fluorescent proteins (GFP and RFP, respectively), horseradish
peroxidase (HRP), .beta.-lactamase, and derivatives thereof (e.g.,
blue EBFP, cyan ECFP, yellow-green EYFP, destabilized GFP variants,
stabilized GFP variants, or fusion variants sold as LIVING COLORS
fluorescent proteins by Clontech). Reporter genes would use cognate
substrates that are preferably assayed by a chromogen, fluorescent,
or luminescent signal. Alternatively, assay product may be tagged
with a heterologous epitope (e.g., FLAG, MYC, SV40 T antigen,
glutathione transferase, polyhistidine, maltose binding protein)
for which cognate antibodies or affinity resins are available.
Examples of drugs for which selectable marker genes, which confer
resistance, exist are ampicillin, geneticin/kanamycin/neomycin,
hygromycin, puromycin, and tetracycline. A metabolic enzyme (e.g.,
dihydrofolate reductase, HSV-1 thymidine kinase) may be used as a
selectable marker in sensitive host cells or auxotrophs. For
example, methotrexate can increase the copy number of a
polynucleotide linked to a DHFR selectable marker or gancyclovir
can negatively select for a viral thymidine kinase selectable
marker.
[0046] A polynucleotide may be ligated to a linker oligonucleotide
or conjugated to one member of a specific binding pair (e.g.,
antibody-digoxygenin/hapten/peptide epitope,
biotin-avidin/streptavidin, glutathione S transferase or
GST-glutathione, lectin-sugar, maltose binding protein-maltose,
polyhistidine-nickel, protein A/G-immunoglobulin). The
polynucleotide may be conjugated by ligation of a nucleotide
sequence encoding the binding member. A polypeptide may be joined
to one member of the specific binding pair by producing the fusion
encoded by such a ligated or conjugated polynucleotide or,
alternatively, by direct chemical linkage to a reactive moiety on
the binding member by chemical cross-linking. Such polynucleotides
and polypeptides may be used as an affinity reagent to identify, to
isolate, and to detect interactions that involve specific binding
of a transcript or protein product of the expression vector. Before
or after affinity binding of the transcript or protein product, the
member attached to the polynucleotide or polypeptide may be bound
to its cognate binding member. This can produce a complex in
solution or immobilized to a support. A protease recognition site
(e.g., for enterokinase, Factor Xa, ICE, secretases, thrombin) may
be included between adjoining domains to permit site specific
proteolysis that separates those domains and/or inactivates protein
activity.
[0047] Probes and primers may be used to identify a PTX3 gene or
variant thereof. For example, a probe or primer specific for a
human PTX3 gene identified herein may be used to detect the
presence or absence of the gene, and thereby infer that the source
of the gene is present or absent, respectively. Genetic
polymorphisms and mutations in the PTX3 gene may be specifically
detected by positioning a potentially mismatched base(s) in the
middle portion of a probe or the 3'-end of a primer to stabilize or
to destabilize binding of the probe or primer to its target
depending on whether the target's sequence at that position is
complementary to the base or not, respectively.
[0048] Genetic polymorphisms and mutations may also be detected by
a change in the length of a restriction fragment (RFLP),
nuclease-protected fragment (e.g., S1 nuclease, deoxyribonuclease
I, ribonuclease A, H or T1), or amplified product. For complicated
genetic fingerprints, identification of each component may not be
needed because a side-by-side visual comparison might easily detect
differences (e.g., RAPD). Differences may also be detected by
changes in the molecular weight (MW) or isoelectric point (pI) of
the PTX3 protein by gel electrophoresis or isoelectric focusing,
respectively.
[0049] Presence of PTX3 protein may be used as an indication of
PTX3 activity in human or animal fluids or tissues. The fluid may
be blood, blood product (e.g., plasma, serum), lavage, sputum, or
the like. Exemplary tissues are those of the epithelium (e.g.,
lung) or mucosa (e.g., mouth, vagina), although infection may be
systemic and involve other tissue types as well. Signal may be
detected in situ for solid tissue, on dispersed or homogenized
tissue, in solution (e.g., diluted or undiluted body fluid, wash),
or on a cell smear or touch prep. Oocyes which may be fertilized
can be selected by PTX3 expression.
[0050] Construction of Shuttle or Expression Vectors
[0051] A shuttle or expression vector is a recombinant
polynucleotide that is in chemical form either deoxyribonucleic
acid (DNA) and/or ribonucleic acid (RNA). The physical form of the
vector may be single-stranded or double-stranded; its topology may
be linear or circular. The vector is preferably a double-stranded
deoxyribonucleic acid (dsDNA) or is converted into a dsDNA after
introduction into a cell (e.g., insertion of a retrovirus into a
host genome as a provirus). The vector may include one or more
regions from a mammalian, insect, plant or fungal gene or a virus
(e.g., adenovirus, adeno-associated virus, cytomegalovirus, fowlpox
virus, herpes simplex virus, lentivirus, Moloney leukemia virus,
mouse mammary tumor virus, Rous sarcoma virus, SV40 virus, vaccinia
virus), as well as regions suitable for genetic manipulation (e.g.,
selectable marker, linker with multiple recognition sites for
restriction endonucleases, promoter for in vitro transcription,
primer annealing sites for in vitro replication). The vector may be
associated with proteins and other nucleic acids in a carrier
(e.g., packaged in a viral particle) or condensed with a chemical
(e.g., cationic polymer) to target entry into a cell or tissue,
such as granulosa cells of mature ovarian follicles. Choice of
vector polynucleotides and methods for introducing them into the
female reproductive system (e.g., endometrium, ovary) is within the
skill in the art.
[0052] An expression vector may be further comprised of a
regulatory region for gene expression (e.g., promoter, enhancer,
silencer, splice donor or acceptor site, polyadenylation signal,
cellular localization sequence). Different levels of transcription
can be achieved using an agent with a regulatory system which
responds to the agent (e.g., tetracycline/tetR or FK506/FKBP). The
vector may be further comprised of one or more splice donor and
acceptor sites within an expressed region; Kozak consensus sequence
upstream of an expressed region for initiation of translation; and
downstream of an expressed region, multiple stop codons in the
three forward reading frames to ensure termination of translation,
one or more mRNA degradation signals, a termination of
transcription signal, a polyadenylation signal, and a 3' cleavage
signal. For expressed regions that do not contain an intron (e.g.,
a coding region from a cDNA), a pair of splice donor and acceptor
sites may or may not be preferred. It would be useful, however, to
include mRNA degradation signal(s) if it is desired to express one
or more of the downstream regions only under the inducing
condition.
[0053] A shuttle vector may be further comprised of an origin of
replication (ARS) which allows replication of the vector integrated
in the host genome or as an autonomously replicating episome.
Centromere and telomere sequences can also be included for the
purposes of chromosomal segregation and protecting chromosome ends,
respectively. Random or targeted integration into the host genome
is more likely to ensure maintenance of the vector but episomes can
be maintained by selective pressure or, alternatively, may be
preferred for those applications in which the vector is present
only transiently.
[0054] A vector may be both a shuttle vector and an expression
vector.
[0055] An expressed region may be derived from any gene of
interest, and provided in either orientation with respect to the
promoter. The expressed region in the antisense orientation will be
useful for making antisense polynucleotide or siRNA. The gene may
be derived from the host cell or organism, from the same species
thereof, or designed de novo. Fusions with a domain(s) of genes
that may share a function with PTX3 can be assayed to define the
domain(s) that confers the function or to provide a multifunctional
fusion protein. A fusion may also be made with an epitope tag
(e.g., GFP, GST, HA, MYC). Some genes produce alternative
transcripts, encode subunits that are assembled as homomultimers or
heteromultimers, or produce propeptides that are activated by
protease cleavage. The expressed region may encode a translational
fusion; open reading frames of the regions encoding a polypeptide
and at least one heterologous domain may be ligated in register. If
a reporter or selectable marker is used as the heterologous domain,
then expression of the fusion protein may be readily assayed or
localized. The heterologous domain may be an affinity or epitope
tag.
[0056] Screening of Candidate Compounds
[0057] Other aspects of the invention are chemical or genetic
compounds, derivatives thereof, and compositions including same
that are effective in treatment of sterility or contraception. The
amount that is administered to a subject in need of treatment, its
formulation, and the timing and route of delivery is effective to
reduce fertility, to increase or decrease reproductive ability, or
to enhance fertility. Determination of such amounts, formulations,
and timing and route of drug delivery is within the skill in the
art.
[0058] A screening method may comprise administering a candidate
compound to an organism or incubating a candidate compound with a
cell, and then determining whether or not gene expression is
modulated. Such modulation may be an increase or decrease in
activity that partially or fully compensates for a change that is
associated with or may cause fertility or sterility. Gene
expression may be increased or decreased at the level of rate of
transcriptional initiation or elongation; stability of transcript;
rate of translational initiation or elongation, stability of
protein; rate of protein processing, folding, or secretion;
proportion of protein in active conformation; formation of
multimers; binding to receptor; or combinations thereof. See, for
example, U.S. Pat. Nos. 5,071,773 and 5,262,300. High-throughput
screening assays are possible (e.g., by using parallel processing
and/or robotics).
[0059] The screening method may comprise incubating a candidate
compound with a cell containing a reporter construct, the reporter
construct comprising a transcriptional regulatory region of PTX3
covalently linked in a cis configuration to a downstream gene
encoding an assayable product; and measuring production of the
assayable product. Either a chimera with an upstream region of the
PTX3 gene or a translational fusion in frame with the initiating
ATG codon may be used to provide the transcriptional regulatory
region. For example, any portion of SEQ ID NO:5 or 6 may be used. A
candidate compound which increases production of the assayable
product would be identified as an agent that activates gene
expression while a candidate compound which decreases production of
the assayable product would be identified as an agent that inhibits
gene expression. See, for example, U.S. Pat. Nos. 5,849,493 and
5,863,733.
[0060] Regulation of PTX3 transcription (e.g., transcriptional
regulatory region and cognate transcription factor) has been
characterized for mouse and human genes (Altmeyer et al., J. Biol.
Chem., 270:25584-25590, 1995; Basile et al., J. Biol. Chem.,
272:8172-8178, 1997). PTX3 transcription is specific for certain
cell types. Responsiveness of PTX3 transcription to cytokine
stimulation appears to be mediated through interaction with
NF.kappa.B and I.kappa.B transcription factors, as well as
cell-specific factors.
[0061] The screening method may comprise measuring in vitro
transcription from a reporter construct in the presence or absence
of a candidate compound (the reporter construct comprising a
transcription regulatory region) and then determining whether
transcription is altered by the presence of the candidate compound.
In vitro transcription may be assayed using a cell-free extract,
partially purified fractions of the cell, purified transcription
factors or RNA polymerase, or combinations thereof. See, for
example, U.S. Pat. Nos. 5,453,362; 5,534,410; 5,563,036; 5,637,686;
5,708,158; and 5,710,025.
[0062] Techniques for measuring transcriptional or translational
activity in vivo are known in the art. For example, a nuclear
run-on assay may be employed to measure transcription of a reporter
gene. Translation of the reporter gene may be measured by
determining the activity of the translation product. The activity
of a reporter gene can be measured by determining one or more of
transcription of polynucleotide product (e.g., RT-PCR or
transcript), translation of polypeptide product (e.g., immunoassay
of protein), and biological activity of the reporter protein per
se.
[0063] A compound that increases or decreases PTX3 gene expression
or protein activity could then be assayed for its effect on
reproductive ability, reducing fertility, or enhancing
fertility.
[0064] An epitope-tagged PTX3 protein or antibody specific for PTX3
protein may be used to affinity purify a multimer or other
PTX3-containing complex. Candidate compounds may be screened for
their ability to decrease the abundance (i.e., steady-state level
of complex), rate of assembly, secretion, or biological activity of
the complex. For example, a compound that enhances or inhibits
binding between PTX3 protein and its receptor may be identified.
PTX3 protein can be attached to a substrate as described above. A
candidate compound is incubated with the immobilized PTX3 protein
in the presence of at least one other component of the complex in
at least partially purified form or as a crude mixture. Moreover,
one or more components of the complex can be attached to a
substrate and a candidate compound can be incubated with the
immobilized component in the presence of PTX3 protein with or
without additional components of the complex in at least partially
purified form or as a crude mixture. Examples of conditions for
binding are shown below. After incubation, all non-binding
components can be washed away, leaving one or more components of
the complex bound to the substrate. Complex formation including
PTX3 protein may also take place in solution and then the
PTX3-containing complex may be immobilized or not. Reduction is a
reversible reaction which disassembles PTX3 multimers. The amount
of each component of the complex can then be quantified after
washing and separation of the complex from other proteins (e.g.,
heterogeneous assay) or without separation (e.g., homogeneous
assay). For example, it can be determined using an immunological
assay, such as ELISA, RIA, or Western blotting. Complex formation
may be determined by binding of an antibody to an epitope which is
dependent on formation or an epitope which is masked after
formation. Complex may be immobilized before or after formation by
binding at least one component of the complex to a substrate.
Binding of complex to a substrate may be determined without
separation by proximity detection, such as SPA or BiaCore. The
amount of the one or more bound components of the complex is
determined with and without the candidate compound. A desirable
compound is one which increases or decreases PTX3 abundance,
assembly, secretion, multimer formation, biological activity, or
combinations thereof.
[0065] Genetic Compounds for Treatment
[0066] Activation may be achieved by inducing an expression vector
containing an expressed region which encodes a protein with PTX3
activity or upregulates PTX3 activity (e.g., the full-length coding
region or functional portions of the PTX3 gene; hypermorphic
mutants, homologs, orthologs, or paralogs thereof; acute phase
inducers; positive transcription factors acting on the PTX3 gene)
or which encodes a protein relieving suppression of PTX3 activity
(e.g., at least partially inhibiting expression of a negative
regulator of the PTX3 gene). Overexpression of transcription or
translation, as well as overexpressing protein function, is a more
direct approach to gene activation. Alternatively, the downstream
expressed region may direct homologous recombination into a locus
in the genome and thereby replace an endogenous transcriptional
regulatory region of the gene with an expression cassette or a
particular genetic mutation.
[0067] An expression vector may be introduced into a host cell or
nonhuman animal by a transfection or transgenesis technique using,
for example, one or more chemicals (e.g., calcium phosphate,
DEAE-dextran, lipids, polymers), biolistics, electroporation, naked
DNA technology, microinjection, or viral infection. The introduced
expression vector may integrate into the host genome of the cell or
animal, or be maintained as an episome. Many neutral and charged
lipids, sterols, and other phospholipids to make lipid carriers are
known. For example, neutral lipids are dioleoyl phosphatidylcholine
(DOPC) and dioleoyl phosphatidyl ethanolamine (DOPE); an anionic
lipid is dioleoyl phosphatidyl serine (DOPS); cationic lipids are
dioleoyl trimethyl ammonium propane (DOTAP),
dioctadecyldiamidoglycyl spermine (DOGS), dioleoyl trimethyl
ammonium (DOTMA), and
1,3-dioleoyloxy-2-(6-carboxyspermyl)-propylamide tetraacetate
(DOSPER). Dipalmitoyl phosphatidylcholine (DPPC) can be
incorporated to improve the efficacy and/or stability of delivery.
FUGENE 6, LIPOFECTAMINE, LIPOFECTIN, DMRIE-C, TRANSFECTAM,
CELLFECTIN, PFX-1, PFX-2, PFX-3, PFX-4, PFX-5, PFX-6, PFX-7, PFX-8,
TRANSFAST, TFX-10, TFX-20, TFX-50, and LIPOTAXI lipids are
proprietary formulations. The polymer may be cationic dendrimer,
polyamide, polyamidoamine, polyethylene or polypropylene glycol
(PEG), polyethylenimine (PEI), polylysine, or combinations thereof;
alternatively, polymeric material can be formed into nanoparticle
or microparticle. In naked DNA technology, the vector (usually as a
plasmid) is delivered to a cell or tissue, where it may or may not
become integrated into the host genome, without using chemical
transfecting agents (e.g., lipids, polymers) to condense the vector
prior to its introduction into the cell or tissue.
[0068] An animal, insect, fungal, or bacterial cell may be
transfected; transgenesis may be used with a nonhuman animal. A
homologous region from a gene can be used to direct integration to
a particular genetic locus in the host genome and thereby regulate
expression of the gene at that locus (e.g., homologous
recombination of a promoterless reporter or selectable marker at
the PTX3 genetic locus) or ectopic copies of the PTX3 gene may be
inserted. Polypeptide may also be produced in vitro with a cell
extract or in vivo with a genetically manipulated cell,
[0069] The expression vector may be used to replace function of a
gene that is down regulated or totally defective, supplement
function of a partially defective gene, or compete with activity of
the gene. Thus, the cognate gene activity of the host may be
neomorphic, hypomorphic, hypermorphic, or normal. Replacement or
supplementation of function can be accomplished by the methods
discussed above, and the genetically manipulated cell or organism
may be selected for high or low expression (e.g., assessing the
amount of transcribed or translated product, or the biological
function of either product) of the downstream region. Competition
between the expressed downstream region and a neomorphic,
hypermorphic, or normal gene may be achieved because of the
synthetic interactions present in a multimeric protein complex.
Alternatively, a negative regulator or a single-chain antibody that
inhibits function intracellularly may be encoded by the downstream
region of the expression vector. Therefore, at least partial
inhibition of PTX3 activity may be achieved by antisense, ribozyme,
or RNA interference technology in which the expression vector
contains a downstream region corresponding to the unmodified
antisense molecule, ribozyme, or siRNA molecule corresponding to a
portion of the PTX3 nucleotide sequence.
[0070] A compound that increases or decreases PTX3 gene expression
or protein activity could then be assayed for its effect on
reproductive ability, reducing fertility, or enhancing
fertility.
[0071] Antisense polynucleotides may act by directly blocking
translation by hybridizing to mRNA transcripts or degrading such
transcripts of a gene. The antisense molecule may be recombinantly
made using at least one functional portion of a gene in the
antisense orientation as a region downstream of a promoter in an
expression vector. Chemically modified bases or linkages may be
used to stabilize the antisense polynucleotide by reducing
degradation or increasing half-life in the body (e.g., methyl
phosphonates, phosphorothioate, peptide nucleic acids). The
sequence of the antisense molecule may be complementary to the
translation initiation site (e.g., between -10 and +10 of the
target's nucleotide sequence).
[0072] Ribozymes catalyze specific cleavage of an RNA transcript or
genome. The mechanism of action involves sequence-specific
hybridization to complementary cellular or viral RNA, followed by
endonucleolytic cleavage. Inhibition may or may not be dependent on
ribonuclease H activity. The ribozyme includes one or more
sequences complementary to the target RNA as well as catalytic
sequences responsible for RNA cleavage (e.g., hammerhead, hairpin,
axehead motifs). For example, potential ribozyme cleavage sites
within a subject RNA are initially identified by scanning the
subject RNA for ribozyme cleavage sites which include the following
trinucleotide sequences: GUA, GUU and GUC. Once identified, an
oligonucleotide of between about 15 and about 20 ribonucleotides
corresponding to the region of the subject RNA containing the
cleavage site can be evaluated for predicted structural features,
such as secondary structure, that can render candidate
oligonucleotide sequences unsuitable. The suitability of candidate
sequences can then be evaluated by their ability to hybridize and
cleave target RNA. The ribozyme may be recombinantly produced or
chemically synthesized.
[0073] siRNA refers to double-stranded RNA of at least 20-25
basepairs which mediates RNA interference (RNAi). Duplex siRNA
corresponding to a target RNA may be formed by separate
transcription of the strands, coupled transcription from a pair of
promoters with opposing polarities, or annealing of a single RNA
strand having an at least partially self-complementary sequence.
Alternatively, duplexed oligoribonucleotides of at least about 21
to about 23 basepairs may be chemically synthesized (e.g., a duplex
of 21 ribonucleotides with 3' overhangs of two ribonucleotides)
with some substitutions by modified bases being tolerated.
Mismatches in the center of the siRNA sequence, however, abolishes
interference. The region targeted by RNA interference should be
transcribed, preferably as a coding region of the gene.
Interference appears to be dependent on cellular factors (e.g.,
ribonuclease III) that cleave target RNA at sites 21 to 23 bases
apart; the position of the cleavage site appears to be defined by
the 5' end of the guide siRNA rather than its 3' end. Priming by a
small amount of siRNA may trigger interference after amplification
by an RNA-dependent RNA polymerase.
[0074] Antibody specific for PTX3 can be used for inhibition or
detection. Polyclonal or monoclonal antibodies may be prepared by
immunizing animals (e.g., chicken, hamster, mouse, rat, rabbit,
goat, horse) with antigen, and optionally affinity purified against
the same or a related antigen. Antigen may be native protein,
fragment made by proteolysis or genetic engineering, fusion
protein, or in vitro translated or synthesized protein which
includes at least one or more epitopes bound by the antibody.
Antibody fragments may be prepared by proteolytic cleavage or
genetic engineering; humanized antibody and single-chain antibody
may be prepared by transplanting sequences from antigen binding
domains of an antibody to framework molecules. Other binding
molecules (e.g., agonists or antagonists of ligand-receptor
binding) may be prepared by screening a combinatorial library for a
member which specifically binds antigen (e.g., phage display
library). Antigen may be a full-length protein encoded by the gene
or fragment(s) thereof. The antibody may be specific for PTX3 or it
may cross react with other pentraxins depending on how well the
epitope recognized by the antibody is conserved among different
species. See, for example, U.S. Pat. Nos. 5,403,484; 5,723,286;
5,733,743; 5,747,334; and 5,871,974.
[0075] PTX3-specific binding agents (e.g., polynucleotides,
polypeptides) may be used diagnostically to detect PTX3 nucleic
acid or protein, or for treatment to inhibit PTX3 activity (e.g.,
transcription, translation, processing, secretion, receptor
binding). In particular, agents that affect PTX3 transcription and
PTX3 binding to a receptor are desirable.
[0076] Antibodies or other such PTX3-specific binding agents may be
used, for example, in combination with targeting moieties, such as
liposomes or other carriers bearing further target specific
moieties, such as granulosa cell targeting moieties and/or moeities
which target the PTX3-specific binding agents to the extracellular
matrix of the cumulous oophorus. Preferably, in this embodiment,
the PTX3-binding agent or antagonist is released or otherwise
activiated or made to become active after reaching the target, to
increase to effectiveness and/or localized action of the binding
agent and/or antagonist.
[0077] Formulation of Compositions
[0078] Compounds of the invention or derivatives thereof may be
used as a medicament or used to formulate a pharmaceutical
composition with one or more of the utilities disclosed herein.
[0079] It is therefore an object of the present invention the use
of the recombinant human PTX3 for preparing a medicament for
increasing the reproductive ability in a female subject.
[0080] A further object of the present invention is the use of
virals or plasmids vectors containing the human PTX3 cDNA for the
treatment of female subjects in need of increasing reproductive
ability.
[0081] A further object of the present invention is to provide
compounds, compositons and methods to decrease female fertility,
such as to provide a contraceptive and contraception method.
[0082] Compounds and compositions of the present invention may be
used, for example, as antgonists of PTX3 to redeuce and/or
eliminate PTX3 actions in, preferably, the extracellular matrix of
the cumulous oophorus, and/or to reduce or eliminate the production
of PTX3, preferably, the extracellular matrix of the cumulous
oophorus, alone or in combination with other contraceptive
compounds and/or devices, to reduce female fertility.
[0083] A further object of the present invention is the use of PTX3
protein as diagnostic marker of the reproductive ability in human
female and/or to confirm the infertility of the human or other
mammalian female in which PTX3 expression and action is critical to
oocyte fertiliztion.
[0084] A further object of the present invention is the use of PTX3
as a target protein for the screening of pharmaceutical compounds
to asses their capability to affect the reproductive ability and/or
inability in a female subject.
[0085] The present invention provides an isolated and/or purified
polynucleotide sequence encoding a polypeptide containing an amino
acid sequence of SEQ. ID. NOs: 2 or 4, and complements of the
polynucleotides, functional fragments of the polynucleotides and
functional fragments of the complements of the polynucleotides.
[0086] The present invention further provides a polynucleotide
containing a nucleotide sequence of SEQ ID NOs: 1, 3, 5 or 6, and
complements thereof, as well as RNA equivalents of the noted SEQ ID
NOs: and complements thereof wherein T is U.
[0087] The present invention provides a monoclonal antibody that
binds immunologically to a polypeptide of the invention, or an
antigenic fragment thereof.
[0088] The present invention provides a polyclonal antisera,
antibodies of which bind immunologically to a polypeptide of the
present invention, or an antigenic fragment thereof.
[0089] The present invention provides an expression vector
containing a polynucleotide sequence encoding a polypeptide of the
present invention, wherein the polynucleotide is under control of a
promoter operable in cells. In one embodiment, the promoter is
operable in granulosa cells of mature follicles of a female
subject. Such promoters may be alternatively inducible by an
externally applied compound.
[0090] The present invention provides a host cell, such as a
non-human host cell, transformed with an expression vector of the
present invention.
[0091] The present invention provides a method for producing a
polypeptide of the invention containing the steps of: culturing a
host cell of the invention under conditions suitable for the
expression of the polypeptide; and recovering the polypeptide from
the host cell culture.
[0092] The present invention provides a composition, such as a
pharmaceutical composition, containing a modulator of PTX3
expression dispersed in a pharmaceutically acceptable carrier,
diluent or excipent. In one embodiment, the modulator suppresses
transcription of a PTX3 gene. In a further embodiment, the
modulator suppresses transcription locally in and/or around an area
of granulosa cells of mature follicles and/or the extracellular
matrix of the cumulous oophorus. In a further embodiment, the
modulator of the invention suppresses translation of a PTX3
transcript. In a further embodiment, the modulator suppresses
translation of a PTX3 transcript locally in and/or around an area
of granulosa cells of mature follicles and/or the extracellular
matrix of the cumulous oophorus. In yet a further embodiment, the
modulator alters PTX3 RNA stability by increasing PTX3 RNA
degradation.
[0093] In one embodiment, the modulator enhances transcription of a
PTX3 gene. In a further embodiment, the modulator enhances
transcription locally in and/or around an area of granulosa cells
of mature follicles and/or the extracellular matrix of the cumulous
oophorus. In a further embodiment, the modulator of the invention
enhances translation of a PTX3 transcript. In a further embodiment,
the modulator enhances translation of a PTX3 transcript locally in
and/or around an area of granulosa cells of mature follicles and/or
the extracellular matrix of the cumulous oophorus. In yet a further
embodiment, the modulator alters PTX3 RNA stability by decreasing
PTX3 RNA degradation.
[0094] The modulator of the present invention is or may contain, in
one embodiment, a polypeptide and/or polynucleotide sequence. The
polynucleotide sequence of the modulator may be, when present, DNA
and/or RNA, and further optionally contain an expression vector,
wherein the expression vector contains a promoter and the
polynucleotide sequence, operatively linked. The modulator may
further be an antibody or active fragment thereof.
[0095] The present invention provides a method of identifying
compounds that modulate the activity of PTX3 containing the steps
of: obtaining an isolated PTX3 polypeptide or functional equivalent
thereof; admixing the PTX3 polypeptide or functional equivalent
thereof with a candidate compound; and measuring an effect of the
candidate compound on the activity of PTX3, such as an activity
related to oocyte fertilization.
[0096] A method of producing a modulator of PTX3 activity
containing the steps of: providing a cell expressing an PTX3
polypeptide contacting the cell with a candidate compound;
measuring PTX3 expression; comparing the PTX3 expression in the
presence of the candidate compound with the expression of PTX3
expression in the absence of the candidate compound; wherein a
difference in the expression of PTX3 in the presence of the
candidate compound, as compared with the expression of PTX3 in the
absence of the candidate compound, identifies the candidate
compound as a modulator of PTX3 expression; and producing the
modulator.
[0097] The invention provides a contraceptive method containing the
step of administering to a fertile female animal an inhibitor of
PTX3 activity, in an amount and for a duration which decreases
fertility. The inhibitor may be administered in the method of the
invention in conjunction with other contraceptive compounds,
compositions and/or devices. The inhibitor of the method may be a
modulator of the present invention.
[0098] A vector of the invention may be a bacterial, viral or
mammalian vector. A modulator of the present invention may be
antisense PTX3 RNA, which may optionally be an RNA interference of
PTX3 RNA.
[0099] The present invention further provides a method of enhancing
fertility of a female containing administering to the female an
effective amount of PTX3 and/or a PTX3 enhancer dispersed in a
pharmacologically acceptable carrier, diluent and/or excipient,
wherein the amount is capable of enhancing fertility. A measure of
enhanced fertility according to the present invention is
specifically enhancing the probability of oocyte fertilization in
vivo. A PTX3 enhancer according to the present invention may
include a modulator as described herein.
[0100] The present invention provides a method of diagnosing
infertility containing identifying a mutation in a PTX3 polypeptide
and/or polynucleotide, which preferably effects the constituent
composition of the extracellular matrix of the cumulous oophorus.
In one embodiment, the method contains, optionally, amplification
of polynucleotides, and hybridization of the polynucleotides to a
labeled polynucleotide or other detectable moiety, optionally also
including sequencing of a PTX3 polynucleotide.
[0101] The compounds of the present invention may be administered
in vitro to cells in culture, in vivo to cells in the body, or ex
vivo to cells outside of a subject which may then be returned to
the body of the same subject or another. The subject is a female of
reproductive age; she wants to become pregnant or is at risk for a
pregnancy.
[0102] In a preferred embodiment, compounds and compositions of the
present invention for decreasing female fertility are administered
locally to and/or near a reproductive organ, such as through or
with the aide of a vaginal suppository, condom, cream, gel or
lotion.
[0103] Compounds or derivatives thereof may be used to produce a
medicament or other pharmaceutical compositions. Use of
compositions which further comprise a pharmaceutically acceptable
carrier and compositions which further comprise components useful
for delivering the composition to a subject are known in the art.
Addition of such carriers and other components to the composition
of the invention is well within the level of skill in this art.
[0104] A pharmaceutical composition may be administered as a
formulation which is adapted for direct application to the female
reproductive system (e.g., endometrium, ovary) or suitable for
passage through the gut or blood circulation. Alternatively,
pharmaceutical compositions may be added to the culture medium. In
addition to active compound, such compositions may contain
pharmaceutically-acceptable carriers and other ingredients known to
facilitate administration and/or enhance uptake. The composition
may be administered in a single dose or in multiple doses which are
administered at different times.
[0105] Pharmaceutical compositions may be administered by any known
route. By way of example, the composition may be administered by a
mucosal, pulmonary, topical, or other localized or systemic route
(e.g., enteral and parenteral). In particular, achieving an
effective amount of PTX3 activity in or around the reproductive
system may be desired. This may involve use of local application,
implantation near a reproductive organ, or vaginal suppository. The
term "parenteral" includes subcutaneous, intradermal,
intramuscular, intravenous, intraarterial, intrathecal, and other
injection or infusion techniques, without limitation.
[0106] Suitable choices in amounts and timing of doses,
formulation, and routes of administration can be made with the
goals of achieving a favorable response in the subject (i.e.,
efficacy), and avoiding undue toxicity or other harm thereto (i.e.,
safety). Therefore, "effective" refers to such choices that involve
routine manipulation of conditions to achieve a desired effect:
e.g., affecting reproductive ability, enhancing fertility, or
reducing fertility.
[0107] A bolus of the formulation administered to a female subject
once a day is a convenient dosing schedule. Alternatively, an
effective dose may be administered every other day, once a week, or
once a month. Dosage levels of active ingredients in a
pharmaceutical composition can also be varied so as to achieve a
transient or sustained concentration of the compound or derivative
thereof in a subject and to result in the desired therapeutic
response. But it is also within the skill of the art to start doses
at levels lower than required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved.
[0108] Dosing may be timed relative to the female subject's
reproductive cycle (e.g., menses). As a practical matter, body
temperature or hormone levels may be used as surrogates for events
like ovulation and menstruation in reproduction.
[0109] The amount of compound administered is dependent upon
factors such as, for example, bioactivity and bioavailability of
the compound (e.g., half-life in the body, stability, and
metabolism); chemical properties of the compound (e.g., molecular
weight, hydrophobicity, and solubility); route and scheduling of
administration; and the like. It will also be understood that the
specific dose level to be achieved for any particular subject may
depend on a variety of factors, including age, health, medical
history, weight, combination with one or more other drugs, and
severity of disease.
[0110] The term "treatment" refers to, inter alia, reducing or
alleviating one or more symptoms of sterility in an affected
subject. For a given subject, improvement in a symptom, its
worsening, regression, or progression may be determined by an
objective or subjective measure. Treatment may also involve
combination with other existing modes of treatment and agents
(e.g., superovulation). Thus, combination treatment may be
practiced.
EXAMPLES
[0111] Heterozygous females and males mice genetically modified for
the PTX3 gene are normal and fertile. Breeding inter se yielded the
predicted number of homozygous null mice at a Mendelian frequency.
However, the breeding between homozygous females and males (PTX3
-/-) is completely infertile. Breeding results indicated that
homozygous males are normally fertile when mated with wild type
(PTX3 +/+) or heterozygous (PTX3 +/-) females, while PTX3 -/-
females are always infertile, independently from the male genotype.
Mating experiments indicated that there were no differences between
PTX3 -/- and PTX3 +/+females in the frequency of copulation plugs
after spontaneous mating during a four days period or after
superovulation (Table 1). The number of spontaneously ovulated eggs
(Table 1) (average 7 per mouse, n=4, in PTX3 +/- and 7.8 per mouse,
n=8, in PTX 3 -/- mice) or hormonally induced ovulated eggs
(average 35 per mouse, n=9, in PTX3 +/- and 27 per mouse, n=18, in
PTX3 -/- mice) was comparable in +/+ and -/- mice. Data are from
one representative experiment of four performed. Oocyte and zona
pellucida morphology were normal, and the first polar bodies were
observed in about 50% of oocytes obtained 16 hr after human
chorionic gonadotropin (hCG) treatment from both PTX3 +/+ and PTX3
-/- mice (Table 1). These data indicate that ovulation and oocyte
maturation are normal and are not the cause of infertility. In
contrast, morphological abnormalities of the cumuli oophori
collected from the oviduct of PTX3 -/- mice (FIGS. 1B and 1D) were
consistently observed, since the granulosa cells were loosely
associated to the oocytes and did not form the corona radiata. PTX3
-/- derived cumuli were unstable in vitro and granulosa cells
spontaneously detached from the oocytes in a short time (15-60 min
in PTX3 -/- versus several hours in PTX3 +/+cumuli) after
collection (14-16 hr post hCG, or at day 0.5 after natural mating),
quickly leading to oocyte denudation. TABLE-US-00001 TABLE 1 Normal
mating frequency and ovulation in PTX3 -/- mice PTX3 +/+ PTX3 -/- P
value Mating frequency Spontaneus (a) 1st day 4/9 2/10 NS 2nd day
2/5 2/8 NS 3th day 2/3 2/5 NS After superovulation 4/4 8/8 NS
Ovulation Spontaneus (b): mice ovulating 4/4 5/5 NS eggs per mouse
7 7.8 --# After superovulation: mice ovulating 5/5 6/6 NS eggs per
mouse 37.8 33.3 -- Presence of polar body in 53/98 54/109 NS in
ovulated eggs (c) (54%) (49%) -- (a) Females were housed with males
for a four days period and checked daily for the presence of plugs.
(b) Ovulation was analyzed in females with plugs. (c) The presence
of the first polar body was assessed in oocytes recovered 15 hr
after HCG treatment. NS, not significantly different (p < 0.05)
from control PTX3 +/+ mice by Fischer's exact test. #Numbers refer
to pooled samples from PTX3 +/+ or PTX3 -/- mice. A similar lack of
difference was observed in four experiments with 5-7 mice.
[0112] To understand whether and when pregnancy was interrupted,
zygotes and embryos were collected at different time points after
mating after spontaneous or hormonally-induced ovulation. No
oocytes developing to the two-cell stage in vivo (day 1.5) (Table
2) nor oocytes with two pronuclei (day 0.5) were ever observed,
even if viable sperm were found in the oviduct of deficient mice.
To further identify the cause(s) of infertility, PTX3
+/+blastocysts were transferred to PTX3 -/- pseudopregnant females,
but normal pregnancy and delivery were observed. This excludes
defects in implantation and subsequent processes. TABLE-US-00002
TABLE 2 Fertilization in PTX3 -/- mice Fertilization PTX3 +/+ PTX3
-/- P value In Vivo Eggs fertilized over total (a) Spontaneus
ovulation: 17/28 (60%) 0/39 (0%) <0.0001# After superovulation:
81/162 (50%) 0/192 (0%) <0.0001 In Vitro After zona pellucida
21/27 (77%) 21/31 (68%) NS.sup.+ removal (b) Using intact cumuli
79/189 (41.8%) 68/169 (40%) NS oophori (c) (a) Embryos were
collected at 1.5 days postcoitum, at the two-cell stage. (b) Fusion
was assessed by the dye transfer technique 4 hr after insemination.
(c) Two cells embryos were counted the day after insemination.
#Fischer's exact test. NS, not significantly different (p <
0.05) from control PTX3 +/+ mice.
[0113] To evaluate whether PTX3 -/- oocytes could be fertilized, in
vitro fertilization (IVF) was performed using wild-type sperm from
adult males to inseminate PTX3+/+ or PTX3 -/- oocytes (Table 2).
IVF was first conducted with oocytes freed from the zona pellucida
and stained with the DNA-specific fluorochrome Hoechst 33258 to
observe the fusion. Under these conditions, normal sperm binding to
PTX3 -/- oocyte plasma membrane and comparable fusing ability of
PTX3 +/+(77%) and PTX3 -/- (68%) oocytes with sperm (Table 2) were
observed. These results suggested that sperm-egg binding and fusion
can occur in the absence of PTX3. Intact cumuli collected 13-15 hr
after hCG treatment were inseminated and fertilization of PTX3 -/-
oocytes and progression to the two-cell stage were observed with a
frequency comparable with PTX3 +/+oocytes (Table 2). These data
confirm that oocyte quality is normal in PTX3 deficient mice. Since
the cumulus oophorus plays a critical role for in vivo, but not for
in vitro fertilization, these results suggest that abnormalities in
the cumulus underlie the infertility of PTX3 -/- females.
[0114] The expression of PTX3 mRNA in ovarian tissues has been
investigated by Northern blotting and in situ hybridization. After
hormonally-induced superovulation, PTX3 mRNA expression (assessed
by Northern blotting in whole tissue) starts 2 hr after hCG
treatment and lasts until 12-14 hr (see FIG. 2A), corresponding to
preovulatory expansion until a few hours after ovulation [20].
Granulosa cells obtained by hyaluronidase treatment of cumuli
oophori and separation from oocytes expressed PTX3 transcripts.
[0115] Expression under normal condition in the absence of
superovulation was investigated by in situ hybridization. In situ
hybridization of organs from untreated females (FIG. 2B) confirmed
the expression of PTX3 mRNA in the ovary, confined to granulosa
cells of mature follicles, with no evidence of transcription in
oocytes.
[0116] PTX3 protein expression in ovarian tissues was then
analyzed. Western blotting indicated that PTX3 was associated with
PTX3 +/+cumuli (in particular with extracellular matrix) because
hyaluronidase treatment, which separates cumulus cells from
oocytes, abolished immune reactivity (FIG. 2C). Immunofluorescence
analysis of PTX3 +/+ and -/- cumuli oophori collected after
hormonally-induced superovulation (13-15 hr after hCG) confirmed
the association of PTX3 with cumulus intercellular matrix (FIG.
2D).
[0117] These data suggest that sterility caused by PTX3 deficiency
is due to a lack of oocyte fertilization, as PTX3 deficiency does
not affect other steps of reproduction, from mating to ovulation,
implantation, and pregnancy. PTX3 transcripts are expressed in the
normal ovary exclusively by the granulosa cells of mature
follicles, as well as by separated granulosa cells, but not by
oocytes. PTX3 mRNA expression is induced in total ovarian tissues
following hormonally-induced superovulation. Finally, PTX3 protein
has been identified in the extracellular matrix of isolated cumuli,
presumably produced by granulosa cells. Analysis of PTX3 -/- mice
has identified an abnormal cumulus oophorus as a determinant of
infertility. Cumuli oophori from PTX3 -/- females showed
morphological abnormalities. They lacked a well-defined corona
radiata and, upon in vitro culture, rapidly detached from oocytes.
The "fragility" of PTX3 deficient cumuli may reflect a structural
role of PTX3 in this peculiar matrix or an alteration in regulatory
mechanisms of matrix dissolution. These results identify PTX3 as a
novel constituent of the extracellular matrix of the cumulous
oophorus, playing a key role in fertility. The cumulus oophorus,
though not essential in vitro, plays a key role for in vivo
fertilization. Therefore, the abnormalities of the cumulus oophorus
are likely to be involved in the infertility of PTX3 -/- female
mice.
[0118] Varani et al "Knockout of Pentraxin 3, a Downstream Target
of Growth Differentiation Factor-9, Causes Female Subfertility",
Molecular Endocrinology 16 (6): 1154-1167 (2002), has confirmed
many of the results presented herein. Specifically, the authors
have reported the induction of PTX3 by growth differentiation
factor-9 (GDF-9) in granulosa cells of preovulatory follicles. PTX3
expression in the ovary was observed after the LH surge in the
cumulus granulosa cells adjacent to the oocyte and the authors also
generated knockout mice lacking the PTX3 gene. The authors have
confirmed the present applicants findings that homozygous null
(PTX3-/-) mice develop normally and do not show any gross
abnormalities. The authors further confirmed that whereas PTX3-/-
males are fertile, PTX3-/- females are subfertile due to defects in
the integrity of the cumulus cell-oocyte complex.
[0119] Varani et al concluded that ovaries and cumulus cell-oocyte
complexes within the PTX3 knockout ovaries appear relatively intact
before the breakdown of the follicle wall and suggested that PTX3
becomes a part of the mucoelastic extracellular matrix which
includes the cumulus cells and oocyte. Varani et al hypothesizes
that PTX3 functions to bind to the cumulus cell-oocyte
extracellular matrix to protect the oocyte and extracellular matrix
from proteolytic enzymes present at the apex of the follicle during
the extrusion from the ovary and in the oviductal environment.
Varani et al explain that proteolytic degradation of the
extracellular matrix is a physiological process that starts after
ovulation, leading to progressive oocyte denudation that correlates
with a decline in the ability of the oocyte to be fertilized.
Proteases produced by the preovulatory follicle and present in the
oviductal environment are noted by Varani et al to have been
reported to destabilize the cumulus matrix by degrading proteins
required for hyaluronan stabilization. Varani et al believe that an
untimely release of proteases, as well as a lack of antiprotease
activity in the extracellular matrix (e.g. absence of PTX3), might
account for early oocyte denudation. Thus, Varani et al confirms
that PTX3 appears to protect the cumulus mass that is vital for the
capture of the oocyte by the oviductal fimbria and its efficient
entry into the oviduct. Varani et al offer an alternative
explanation for the decrease in ovulation in the PTX3-/- knockout
mice is that PTX3 on the surface of the cumulus cell-oocyte complex
acts to directly bind the complex to the fimbria of the oviduct to
shuttle the complex into the oviduct.
[0120] Varani et al conclude that the oocytes in PTX3-/- mice
appear to lose their optimal extracellular environment and show a
lower efficiency of ovulation and fertilization.
Materials and Methods
[0121] Generation of PTX3 -/- Mice
[0122] A genomic DNA fragment of 8.5 kb encompassing exons 1
through 2 of the mouse PTX3 gene was used to integrate the
IRES-LacZ cassette followed by the PGK-neomycin resistance gene
from the pWH9 plasmid in exon 1 at a location 71 bp downstream of
the first coding ATG. Methods for the culture, selection, and
identification of ES cells were performed as described [20]. Five
independently targeted R1 ES cell clones were identified by
Southern blot hybridization, using probe A (EcoRI/EcoRV 750 bp
fragment in the second intron). No evidence for random integration
was detected with the probe B (from the neomycin resistance gene).
Two ES cell clones were injected into C57BI/6 blastocysts. For
genotyping of mice, DNA derived from tail biopsies was amplified by
polymerase chain reaction with two primers sets (Primer Set 1:
5'-AGCAATGCACCTCCCTGCGAT-3', SEQ ID NO:7;
5'-TCCTCGGTGGGATGAAGTCCA-3' SEQ ID NO:8; Primer Set 2:
5'-CTGCTCTTTACTGAAGGCTC-3', SEQ ID NO:9; 5'-TCCTCGGTGGGATGAAGT
CCA-3, SEQ ID NO:10) that detected the wild type or targeted
allele, respectively. Phenotypic analysis was performed on the two
lines derived from independent clones, and results were confirmed
in a 129Sv-C57BI/6 mixed and 129Sv inbred genetic background. PTX3
+/+mice were 129Sv-C57BI/6 PTX3 -/- littermates, or 129Sv or
C57BI/6 mice obtained from Charles River, Calco, Italy.
[0123] Procedures involving animals and their care in conformed
with institutional guidelines in compliance with national (4D.L.
N.116, G.U., suppl. 40, 18-2-1992) and international law and
policies (EEC Council Directive 86/609, OJ L 358,1,12-12-1987; NIH
Guide for the Care and Use of Laboratory Animals, U.S. National
Research Council, 1996). All efforts were made to minimize the
number of animals used and their suffering.
[0124] PTX3 mRNA and Protein
[0125] RNA was extracted from cells and purified using TRIZOL
reagent (GIBCO BRL). Northern blotting, probe labeling, and
hybridization (binding and washing) conditions were performed as
described [21].
[0126] In situ hybridation: Cryostat sections (13 .mu.m) recovered
from wildtype and PTX3 -/- ovaries fixed with paraformaldehyde 4%
and frozen in liquid nitrogen were used to perform the in situ
hybridization as described [22]. Briefly, slides pemeabilized with
proteinase K and 0.2N HCl, were incubated at 65.degree. C.
overnight with a radioactively-labelled riboprobe made from PTX3
cDNA containing vector (pBluescript) using a Stratagene RNA
transcription kit. Subsequently, specimens were washed with
formamide-containing buffer, air dried, dipped in photographic
emulsion and incubated at 4.degree. C. in a dark box for at least
10 days. After developing, the slides were counterstained with a
solution of 2 .mu.g/ml Hoechst 33258 dye. For Western blot
analysis, total cell extracts obtained from intact cumuli oophori,
cumulus cells, or oocytes collected from superovulated females were
separated by SDS-polyacrylamide gel electrophoresis (Page),
electroblotted onto nitrocellulose filters (Hybond ECL, Amersham),
and labeled with a purified biotinylated anti-murine PTX3
polyclonal hamster serum (1 .mu.g/ml) followed by streptavidin-HRP
(BIOSPA, Italy). Labeled proteins were detected by enhanced
chemiluminescence (ECL, Amersham).
[0127] Oocyte and Embryo Collection, In Vitro Fertilization, and
Embryo Transfer
[0128] Cumuli oophori, zygotes, and embryos were recovered from the
oviduct or uterus of untreated females after natural mating [20].
Superovulation was induced by treatment with 5 units of pregnant
mare serum (PMS, Folligon, Intervet) and with 5 units of human
chorionic gonadotropin (hCG, Corulon, Intervet) 48 hr later. Cumuli
oophori were collected at different time after mating or 13-15 hr
after hCG treatment. Cumulus cells and oocytes were separated by
hyaluronidase treatment [20].
[0129] In vitro fertilization (IVF) of eggs obtained from
superovulated females was performed with intact cumuli oophori as
described [20] or with zona pellucida free eggs [20] stained with 1
.mu.g/ml Hoechst dye in M16 medium (Sigma) [23] and sperm from BDF
males. Fertilization and sperm-egg fusion were assessed by counting
two-cell stage embryos the day after insemination of intact cumuli
oophori and by counting eggs with fluorescent fertilizing sperm 4
hr after insemination of zona pellucida-free eggs.
[0130] Embryo transfer was performed as described [20], using 3.5
day PTX3 +/+blastocysts implanted in the uterus of 2.5 days
pseudopregnant PTX3 -/- females.
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[0155] Patents, patent applications, and other publications cited
herein are incorporated by reference in their entirety.
[0156] All modifications and substitutions that come within the
meaning of the claims and the range of their legal equivalents are
to be embraced within their scope. A claim using the transition
"comprising" allows the inclusion of other elements to be within
the scope of the claim; the invention is also described by such
claims using the transitional phrase "consisting essentially of"
(i.e., allowing the inclusion of other elements to be within the
scope of the claim if they do not materially affect operation of
the invention) and the transition "consisting" (i.e., allowing only
the elements listed in the claim other than impurities or
inconsequential activities which are ordinarily associated with the
invention) instead of the "comprising" term. Any of the three
transitions can be used to claim the invention.
[0157] It should be understood that an element described in this
specification should not be construed as a limitation of the
claimed invention unless it is explicitly recited in the claims.
Thus, the claims are the basis for determining the scope of legal
protection granted instead of a limitation from the specification
which is read into the claims.
[0158] In contradistinction, the prior art is explicitly excluded
from the invention to the extent of specific embodiments that would
anticipate the claimed invention or destroy novelty. In certain
embodiments, the genus of polynucleotides or polypeptides may be
recited in the claims with the proviso that native nucleic acids or
proteins are excluded (e.g., having a nucleotide or amino acid
sequence which is not given in the sequence listing). For example,
the degeneracy of the genetic code may be used to provide a
polynucleotide having a nucleotide sequence encoding SEQ ID NO:2,
but which is not SEQ ID NO:1. Similarly, a PTX3 polypeptide may be
provided that is functionally equivalent but not identical to the
mouse and/or human protein (e.g., at least 90% identical) by
changing one or more of the amino acid residues of SEQ ID NO:2.
[0159] Moreover, no particular relationship between or among
limitations of a claim is intended unless such relationship is
explicitly recited in the claim (e.g., the arrangement of
components in a product claim or order of steps in a method claim
is not a limitation of the claim unless explicitly stated to be
so). All possible combinations and permutations of the individual
elements disclosed herein are considered to be aspects of the
invention; similarly, generalizations of the invention's
description are considered to be part of the invention.
[0160] From the foregoing, it would be apparent to a person of
skill in this art that the invention can be embodied in other
specific forms without departing from its spirit or essential
characteristics. The described embodiments should be considered
only as illustrative, not restrictive, because the scope of the
legal protection provided for the invention will be indicated by
the appended claims rather than by this specification.
Sequence CWU 0
0
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 11 <210>
SEQ ID NO 1 <211> LENGTH: 1837 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <300> PUBLICATION
INFORMATION: <301> AUTHORS: Breviario et al. <302>
TITLE: Interleukin-1 Inducible Genes in Endothelial Cells
<303> JOURNAL: Journal of Biological Chemistry <304>
VOLUME: 267 <305> ISSUE: 31 <306> PAGES: 22190-22197
<307> DATE: 1992-11-05 <308> DATABASE ACCESSION NUMBER:
X636613 <309> DATABASE ENTRY DATE: 1993-07-29 <400>
SEQUENCE: 1 ctcaaactca gctcacttga gagtctcctc ccgccagctg tggaaagaac
tttgcgtctc 60 tccagcaatg catctccttg cgattctgtt ttgtgctctc
tggtctgcag tgttggccga 120 gaactcggat gattatgatc tcatgtatgt
gaatttggac aacgaaatag acaatggact 180 ccatcccact gaggacccca
cgccgtgcga ctgcggtcag gagcactcgg aatgggacaa 240 gctcttcatc
atgctggaga actcgcagat gagagagcgc atgctgctgc aagccacgga 300
cgacgtcctg cggggcgagc tgcagaggct gcgggaggag ctgggccggc tcgcggaaag
360 cctggcgagg ccgtgcgcgc cgggggctcc cgcagaggcc aggctgacca
gtgctctgga 420 cgagctgctg caggcgaccc gcgacgcggg ccgcaggctg
gcgcgtatgg agggcgcgga 480 ggcgcagcgc ccagaggagg cggggcgcgc
cctggccgcg gtgctagagg agctgcggca 540 gacgcgagcc gacctgcacg
cggtgcaggg ctgggctgcc cggagctggc tgccggcagg 600 ttgtgaaaca
gctattttat tcccaatgcg ttccaagaag atttttggaa gcgtgcatcc 660
agtgagacca atgaggcttg agtcttttag tgcctgcatt tgggtcaaag ccacagatgt
720 attaaacaaa accatcctgt tttcctatgg cacaaagagg aatccatatg
aaatccagct 780 gtatctcagc taccaatcca tagtgtttgt ggtgggtgga
gaggagaaca aactggttgc 840 tgaagccatg gtttccctgg gaaggtggac
ccacctgtgc ggcacctgga attcagagga 900 agggctcaca tccttgtggg
taaatggtga actggcggct accactgttg agatggccac 960 aggtcacatt
gttcctgagg gaggaatcct gcagattggc caagaaaaga atggctgctg 1020
tgtgggtggt ggctttgatg aaacattagc cttctctggg agactcacag gcttcaatat
1080 ctgggatagt gttcttagca atgaagagat aagagagacc ggaggagcag
agtcttgtca 1140 catccggggg aatattgttg ggtggggagt cacagagatc
cagccacatg gaggagctca 1200 gtatgtttca taaatgttgt gaaactccac
ttgaagccaa agaaagaaac tcacacttaa 1260 aacacatgcc agttgggaag
gtctgaaaac tcagtgcata ataggaacac ttgagactaa 1320 tgaaagagag
agttgagacc aatctttatt tgtactggcc aaatactgaa taaacagttg 1380
aaggaaagac attggaaaaa gcttttgagg ataatgttac tagactttat gccatggtgc
1440 tttcagttta atgctgtgtc tctgtcagat aaactctcaa ataattaaaa
aggactgtat 1500 tgttgaacag agggacaatt gttttacttt tctttggtta
attttgtttt ggccagagat 1560 gaattttaca ttggaagaat aacaaaataa
gatttgttgt ccattgttca ttgttattgg 1620 tatgtacctt attacaaaaa
aaatgatgaa aacatattta tactacaagg tgacttaaca 1680 actataaatg
tagtttatgt gttataatcg aatgtcacgt ttttgagaag atagtcatat 1740
aagttatatt gcaaaaggga tttgtattaa tttaagacta tttttgtaaa gctctactgt
1800 aaataaaata ttttataaaa ctaaaaaaaa aaaaaaa 1837 <210> SEQ
ID NO 2 <211> LENGTH: 381 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
SIGNAL PEPTIDE <222> LOCATION: (1)..(17) <223> OTHER
INFORMATION: <220> FEATURE: <221> NAME/KEY: MAT_PEPTIDE
<222> LOCATION: (18)..(381) <300> PUBLICATION
INFORMATION: <301> AUTHORS: Breviario et al. <302>
TITLE: Interleukin-1 Inducible Genes in Endothelial Cells
<303> JOURNAL: Journal of Biological Chemistry <304>
VOLUME: 267 <305> ISSUE: 31 <306> PAGES: 22190-22197
<307> DATE: 1992-11-05 <308> DATABASE ACCESSION NUMBER:
CAA45158 <309> DATABASE ENTRY DATE: 1993-07-29 <400>
SEQUENCE: 2 Met His Leu Leu Ala Ile Leu Phe Cys Ala Leu Trp Ser Ala
Val Leu -15 -10 -5 Ala Glu Asn Ser Asp Asp Tyr Asp Leu Met Tyr Val
Asn Leu Asp Asn -1 1 5 10 15 Glu Ile Asp Asn Gly Leu His Pro Thr
Glu Asp Pro Thr Pro Cys Asp 20 25 30 Cys Gly Gln Glu His Ser Glu
Trp Asp Lys Leu Phe Ile Met Leu Glu 35 40 45 Asn Ser Gln Met Arg
Glu Arg Met Leu Leu Gln Ala Thr Asp Asp Val 50 55 60 Leu Arg Gly
Glu Leu Gln Arg Leu Arg Glu Glu Leu Gly Arg Leu Ala 65 70 75 Glu
Ser Leu Ala Arg Pro Cys Ala Pro Gly Ala Pro Ala Glu Ala Arg 80 85
90 95 Leu Thr Ser Ala Leu Asp Glu Leu Leu Gln Ala Thr Arg Asp Ala
Gly 100 105 110 Arg Arg Leu Ala Arg Met Glu Gly Ala Glu Ala Gln Arg
Pro Glu Glu 115 120 125 Ala Gly Arg Ala Leu Ala Ala Val Leu Glu Glu
Leu Arg Gln Thr Arg 130 135 140 Ala Asp Leu His Ala Val Gln Gly Trp
Ala Ala Arg Ser Trp Leu Pro 145 150 155 Ala Gly Cys Glu Thr Ala Ile
Leu Phe Pro Met Arg Ser Lys Lys Ile 160 165 170 175 Phe Gly Ser Val
His Pro Val Arg Pro Met Arg Leu Glu Ser Phe Ser 180 185 190 Ala Cys
Ile Trp Val Lys Ala Thr Asp Val Leu Asn Lys Thr Ile Leu 195 200 205
Phe Ser Tyr Gly Thr Lys Arg Asn Pro Tyr Glu Ile Gln Leu Tyr Leu 210
215 220 Ser Tyr Gln Ser Ile Val Phe Val Val Gly Gly Glu Glu Asn Lys
Leu 225 230 235 Val Ala Glu Ala Met Val Ser Leu Gly Arg Trp Thr His
Leu Cys Gly 240 245 250 255 Thr Trp Asn Ser Glu Glu Gly Leu Thr Ser
Leu Trp Val Asn Gly Glu 260 265 270 Leu Ala Ala Thr Thr Val Glu Met
Ala Thr Gly His Ile Val Pro Glu 275 280 285 Gly Gly Ile Leu Gln Ile
Gly Gln Glu Lys Asn Gly Cys Cys Val Gly 290 295 300 Gly Gly Phe Asp
Glu Thr Leu Ala Phe Ser Gly Arg Leu Thr Gly Phe 305 310 315 Asn Ile
Trp Asp Ser Val Leu Ser Asn Glu Glu Ile Arg Glu Thr Gly 320 325 330
335 Gly Ala Glu Ser Cys His Ile Arg Gly Asn Ile Val Gly Trp Gly Val
340 345 350 Thr Glu Ile Gln Pro His Gly Gly Ala Gln Tyr Val Ser 355
360 <210> SEQ ID NO 3 <211> LENGTH: 1841 <212>
TYPE: DNA <213> ORGANISM: Mus musculus <300>
PUBLICATION INFORMATION: <301> AUTHORS: Introna et al.
<302> TITLE: Cloning of Mouse PTX3 <303> JOURNAL: Blood
<304> VOLUME: 87 <305> ISSUE: 5 <306> PAGES:
1862-1872 <307> DATE: 1996-03-01 <308> DATABASE
ACCESSION NUMBER: X83601 <309> DATABASE ENTRY DATE:
1996-01-10 <400> SEQUENCE: 3 actcctgcct cacactatct ctcccgggct
caaactcgga tcactgtaga gtctcgcttc 60 ttcccctgcg gctgcgaacg
aaatttcgcc tctccagcaa tgcacctccc tgcgatcctg 120 ctttgtgctc
tctggtctgc agtagtggct gagacctcgg atgactacga gctcatgtat 180
gtgaatttgg acaacgaaat agacaatgga cttcatccca ccgaggaccc cacgccatgc
240 gactgccgcc aggagcactc ggagtgggac aagctgttca tcatgctgga
gaactcgcag 300 atgcgggagg gcatgctgtt gcaggccacc gacgacgtcc
tccgtggaga gctgcagcgg 360 ctgcgggcag agctggggcg gctggcgggc
ggcatggcga ggccgtgcgc agccggtggc 420 cccgcagacg ccaggctggt
gcgggcgctg gagccgctgc tgcaggagag ccgtgacgcg 480 agcctcaggc
tggcgcgcct ggaggacgcg gaggcgcggc gacccgaggc gacagtgcct 540
ggcctaggcg ctgtgctgga ggaactgcgg cggacgcgcg ccgacctgag cgccgtgcag
600 agctgggtcg cccgccactg gctgcccgca ggttgtgaaa cagcaatttt
cttcccaatg 660 cgttcgaaga agatttttgg aagcgtgcat cctgtgagac
caatgaagct tgaatctttt 720 agtacttgca tttgggtcaa agccacagat
gtattaaaca aaaccatcct gttttcttat 780 ggcacaaagt ggaaccccta
tgagattcag ctgtacctca gttcccagtc cctagtgttg 840 gtggtgggtg
gaaaggagaa caagctggct gcagacactg tggtgtccct ggggaggtgg 900
tcccacctgt gtggcacctg gagttcagag caggggagca tgtccctgtg ggcaaacggg
960 gagctggtgg ctaccactgt agagatggcc aaaagtcact ctgttcctga
gggtggactc 1020 ctacagattg gccaagaaaa gaatggttgc tgtgtaggtg
ggggctttga cgaatcatta 1080 gcattttctg gaagaatcac aggcttcaat
atctgggatc gggttctcag cgaggaggag 1140 atacgggcca gtggaggagt
cgaatcctgt cacatccggg gaaatgtcgt cgggtgggga 1200 gtcacagaga
ttcaggcgca cggaggagcc cagtatgttt cttaagtgtt gtgaaaatct 1260
acttgaagcc aaaggagact cacattttaa atatgccagt tggaaaagtc tgaaaacttc
1320 ggtgcgtaat agacgaatga aggagagact tgagattgtc tttgtttatc
ttggcaaaat 1380 actgaataca cagttgaagg gaaggcttga gagagggctc
cgggatgttg ttactaagcc 1440
ttatactgtg gtgctttcag attaatgtct gcctctgtca gataaaccct cagataacta
1500 aacatgactg gactctgaac agagggacga ttgtgtgact tttttttttt
tttattttgg 1560 ttaattttat tttggccaga gacattttta tattggaaga
ataacaaaac aagctctgtt 1620 gcccattgtt cattctttct ggtgtgtatt
ttgtgacaaa agagatgatg agaaaaccat 1680 aattatacca caaagtgact
tattaacgaa cataaatgta gcttacgtgt tataatccaa 1740 tccatttggg
agaaggtagt tgtgtaattt atattgtgaa atgtaattgt attaatttta 1800
tttttgtaaa agtctactgt aaataaattg ttttataaag c 1841 <210> SEQ
ID NO 4 <211> LENGTH: 381 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
SIGNAL PEPTIDE <222> LOCATION: (1)..(17) <223> OTHER
INFORMATION: <220> FEATURE: <221> NAME/KEY: MAT_PEPTIDE
<222> LOCATION: (18)..(381) <223> OTHER INFORMATION:
<300> PUBLICATION INFORMATION: <301> AUTHORS: Introna
et al. <302> TITLE: Cloning of Mouse PTX3 <303>
JOURNAL: Blood <304> VOLUME: 87 <305> ISSUE: 5
<306> PAGES: 1862-1872 <307> DATE: 1996-03-01
<308> DATABASE ACCESSION NUMBER: CAA58580 <309>
DATABASE ENTRY DATE: 1996-01-10 <400> SEQUENCE: 4 Met His Leu
Pro Ala Ile Leu Leu Cys Ala Leu Trp Ser Ala Val Val -15 -10 -5 Ala
Glu Thr Ser Asp Asp Tyr Glu Leu Met Tyr Val Asn Leu Asp Asn -1 1 5
10 15 Glu Ile Asp Asn Gly Leu His Pro Thr Glu Asp Pro Thr Pro Cys
Asp 20 25 30 Cys Arg Gln Glu His Ser Glu Trp Asp Lys Leu Phe Ile
Met Leu Glu 35 40 45 Asn Ser Gln Met Arg Glu Gly Met Leu Leu Gln
Ala Thr Asp Asp Val 50 55 60 Leu Arg Gly Glu Leu Gln Arg Leu Arg
Ala Glu Leu Gly Arg Leu Ala 65 70 75 Gly Gly Met Ala Arg Pro Cys
Ala Ala Gly Gly Pro Ala Asp Ala Arg 80 85 90 95 Leu Val Arg Ala Leu
Glu Pro Leu Leu Gln Glu Ser Arg Asp Ala Ser 100 105 110 Leu Arg Leu
Ala Arg Leu Glu Asp Ala Glu Ala Arg Arg Pro Glu Ala 115 120 125 Thr
Val Pro Gly Leu Gly Ala Val Leu Glu Glu Leu Arg Arg Thr Arg 130 135
140 Ala Asp Leu Ser Ala Val Gln Ser Trp Val Ala Arg His Trp Leu Pro
145 150 155 Ala Gly Cys Glu Thr Ala Ile Phe Phe Pro Met Arg Ser Lys
Lys Ile 160 165 170 175 Phe Gly Ser Val His Pro Val Arg Pro Met Lys
Leu Glu Ser Phe Ser 180 185 190 Thr Cys Ile Trp Val Lys Ala Thr Asp
Val Leu Asn Lys Thr Ile Leu 195 200 205 Phe Ser Tyr Gly Thr Lys Trp
Asn Pro Tyr Glu Ile Gln Leu Tyr Leu 210 215 220 Ser Ser Gln Ser Leu
Val Leu Val Val Gly Gly Lys Glu Asn Lys Leu 225 230 235 Ala Ala Asp
Thr Val Val Ser Leu Gly Arg Trp Ser His Leu Cys Gly 240 245 250 255
Thr Trp Ser Ser Glu Gln Gly Ser Met Ser Leu Trp Ala Asn Gly Glu 260
265 270 Leu Val Ala Thr Thr Val Glu Met Ala Lys Ser His Ser Val Pro
Glu 275 280 285 Gly Gly Leu Leu Gln Ile Gly Gln Glu Lys Asn Gly Cys
Cys Val Gly 290 295 300 Gly Gly Phe Asp Glu Ser Leu Ala Phe Ser Gly
Arg Ile Thr Gly Phe 305 310 315 Asn Ile Trp Asp Arg Val Leu Ser Glu
Glu Glu Ile Arg Ala Ser Gly 320 325 330 335 Gly Val Glu Ser Cys His
Ile Arg Gly Asn Val Val Gly Trp Gly Val 340 345 350 Thr Glu Ile Gln
Ala His Gly Gly Ala Gln Tyr Val Ser 355 360 <210> SEQ ID NO 5
<211> LENGTH: 1531 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
PROMOTER <222> LOCATION: (1)..(1317) <223> OTHER
INFORMATION: <220> FEATURE: <221> NAME/KEY:
PROTEIN_BIND <222> LOCATION: (1222)..(1231) <223> OTHER
INFORMATION: NF-kB <300> PUBLICATION INFORMATION: <301>
AUTHORS: Basile et al. <302> TITLE: Characterization of the
Promoter for the Human Long Pentaxin PTX3 <303> JOURNAL:
Journal of Biological Chemistry <304> VOLUME: 272 <305>
ISSUE: 13 <306> PAGES: 8172-8178 <307> DATE: 1997-03-28
<308> DATABASE ACCESSION NUMBER: X97748 <309> DATABASE
ENTRY DATE: 1997-11-15 <400> SEQUENCE: 5 gaattccccg
gatctccctt ctaactctcc acctttggcc taagctttgc ttccacatgg 60
tcatcaacat ttggtggtta tagaactaat aacccctatc tcacttcact cctatgccag
120 aggggcccta gcatcagctc atgggattgt tgtttttgct ttcctctcta
tctttggctc 180 cgggattttc cccttacttt aatgggagct catctgtacc
ttttaagttt ttattaatat 240 catgtgaaca cagacctgta tatattgtta
gaagcagaaa tctctaagtt tacttttaaa 300 acatgatcct tgcctcgaaa
ccttgtagaa taatataatg tccacataat accaagttat 360 gaaaagaaac
atacctaaat aactaaataa gtatattcct tttttccccc agcttttttt 420
ccccattcta ggttacccag ttgtactgtg ttgtttgtca taggccgggt gaggtggctc
480 acgtctgtaa tcctagcaat ttgggaggcg aaggcgggtg gatcgcctga
ggtcaggagt 540 tcgagaccag cctggctaac atggtgaaac cctgtctcta
ctaaaaatac aaaaattaac 600 tgggtgtggt ggcgggtgcc tgtaattcca
gctacttggg aagctgaggt aggagaatcg 660 cttgaaccca ggatgcggag
gttgcagtga gccgagatca caccattgca ctccagcctg 720 ggcaacaaga
gcgaaattca gtctcaaaaa aaaaaattat ctataaaagt ataggtgcaa 780
ctcctcaagt attaaagaca agatagctcg gattggactt gactttcaga gccataacta
840 ttcttaatat gttggtttat cttggaatca gaccattttc agtttcaacc
tgtaaaacag 900 tgtacaaagg aaacatggaa agttttctat atataaaggg
ttgtgaaata ataacagctc 960 acagaaaatg ctgaaatgat gatttgcttc
agtaccctct gaaatttctc ccctaccacc 1020 cctccttcat ccccattgct
atcaattcaa attacaacag ctaattctca ggagaacagt 1080 agaagcccag
tttctctcct ctttcccctc tgaccctcct ccaattaatc tgactgcagc 1140
gtaaaccttt gcggtttaat attgtgcaac ttccacattt ccctcgctct cccacccagc
1200 cccctccccc accaaattca ggggaactcc cgttaccgca gtgccaccag
cattactcat 1260 tcatccccat tcaggctttc ctcagcattt attaaggact
ctctgctcca gcctctcact 1320 ctcactctcc tccgctcaaa ctcagctcac
ttgagagtct cctcccgcca gctgtggaaa 1380 gaactttgcg tctctccagc
aatgcatctc cttgcgattc tgttttgtgc tctctggtct 1440 gcagtgttgg
ccgagaactc ggatcattat catctcatgt atgtgaattt ggacaacgaa 1500
atagacaatg gactccatcc cactgaggac c 1531 <210> SEQ ID NO 6
<211> LENGTH: 2708 <212> TYPE: DNA <213>
ORGANISM: Mus musculus <220> FEATURE: <221> NAME/KEY:
PROMOTER <222> LOCATION: (1)..(1373) <223> OTHER
INFORMATION: <300> PUBLICATION INFORMATION: <301>
AUTHORS: Altmeyer et al. <302> TITLE: Promoter Structure and
Transcriptional Activation . . . <303> JOURNAL: Journal of
Biological Chemistry <304> VOLUME: 270 <305> ISSUE: 43
<306> PAGES: 25584-15590 <307> DATE: 1995-10-27
<308> DATABASE ACCESSION NUMBER: U33842 <309> DATABASE
ENTRY DATE: 1995-10-27 <400> SEQUENCE: 6 atcccagagg
ctctctgtac tggcattagg acctcacagc accacatcag gtttcttaat 60
gtggactcta gaaactgaac tcgagcccac agccttagga gaaaagcacc ttacaaagct
120 gtggctccac actgcccttt aaacaatatc gtattgtctc atattgccat
cgctttctga 180 tggctttaac ggtttcaaac ataccctgtc tttagccgtg
atctcaaata agtgaagctc 240 ttgagcaggg gcctgatgcc ttttgacttt
gtgttgattc atgcttatga tgccctgttc 300 cctccgtgtc tagctatgtt
taactgtgga ttcaattttt attggtgggt ggattggtac 360 atgcatgtgc
attccagatg cgtgagggca ctcaggccag gaaagccact catgagtctc 420
tgtcaggagc agaggaattt acctatggaa atccaagagc agccttctga gaggcctggc
480 ctgagggtag tacccctccc atcatgatca ggatgtgact ggtaaccctc
cccctccatc 540 tcctttgtat attggagact tgtatcagct caggggtatc
ctctgggagt ggttccctct 600 agatctgtgt agttttttag atcttgcttt
atttggagtt tattctcatg ttttaatttt 660 ttatcactat tattatgact
tatcaacacc tatctaggta cttttcactg ggggaggggg 720 caggttttac
acacacacac acacacacac acacacacac acacacacac acagtcacta 780
atgtaaaatt taaaacaggg accttgatag gatatgtcca agaataccca agcaccctaa
840 agccactata ttcccgccct cactttcctg ttttactggg ttttgaccca
gccatactgt 900 gttttttagt tgctccacca gaggagtcaa gactagttag
tcaagattga cttctagagt 960 cataaaaatt cttaatgggt tattttggag
tcacggaatc attttctata gcttggtctt 1020 gagaaagtat ccaaaggaaa
agtgaaaaaa aaaagttttc cataacttca ggggttgtgg 1080 agtaatgaaa
gctcacacca aatgccaaaa tgataattcg ccctgtacct ctgtgctcct 1140
caccccccaa agcgctagca cttcaggtta cagcaactaa tcctcagggg caccagaaaa
1200 gtccagcttc cctccccttc tccccctgac tcgcctctaa ttaatctgcc
tgcagtgtgg 1260 acctcggtgg tttaacattg tgcaacctct tcagctccct
tgccctccca cccaaccccc 1320 tcccccaaat ccaggggaac tccctcgcgc
tgtgccaccg acattagtca ttcatccgct 1380 catgctttgg agcgtttatt
aagggcttca ctcctgcctc acactatctc tcccgggctc 1440 aaactcggat
cactgtagag tctcgcttct tcccctgcgg gtgcgaagca aatttcggct 1500
ctccagcaat gcacctccct gcgatcctgc tttgtgctct ctggtctgca gtagtggctg
1560 agacctcgga tgactacgag ctcatgtatg tgaatttgga caacgaaata
gacaatggac 1620 ttcatcccac cgaggaccgt aagttcattt ttaactctct
cagcgtatca aaactacata 1680 actcacttct gggggggcgc gattaacata
attaacatag atagccaatg aagcaagcta 1740 aaattatact ttatttgtga
aagcaaggac tgggggaaaa aaggaaagca aggaaatatc 1800 tgagaaaagc
cagaggtttt aaattatttt tgtaacattt atgatgagtt aagttatacg 1860
aaatctttaa ctgtttttag ctatattaat ggcattttct cagttagttt aacatgtcta
1920 taaagaatag tctgtgtcat ctttgagttt acacgcacgc tgttttcaga
gctatcctta 1980 gaaggagagc gttgctgggg acaggctgaa acttggagtc
accaagagtg caacccatgg 2040 ccacccagga caagctgata acacttgtgt
gtgtcctgcg ttctagccac gccatgcgac 2100 tgcgcccagg agcactcgga
gtgggacaag ctgttcatca tgctggagaa ctcgcagatg 2160 cgggagggca
tgctgttgca ggccaccgac gacgtcctcc gtggagagct gcagcggctg 2220
cggtcagagc tgggccggct ggcgggcggc atggcgaggc cgtgcgcagc cggtggcccc
2280 gcagacgcca ggctggtgcg ggcgctggag ccgctgctgc aggagagccg
tgacgcgagc 2340 ctcaggctgg cgcgcctgga ggacgcggag gcgcggcgac
ccgaggcgac agtgcctggc 2400 ctaggcgctg tgctggagga actgcggcgg
acgcgctccg acctgagcgc cgtgcagagc 2460 tgggtcgccc accactggct
gcccgcaggt aagcccacgg tcggctctgt ccctagaggc 2520 aagcttttgt
gggaccctca cactcagagc cccagtactt ttcataggca cactcacaga 2580
gctcacacca cgccaggcag ctcattgcct tttaaaagta tttccaagcc cgaggaaccc
2640 aaaagaaaaa aacgaggatt taaaccatca gtctggaagt tgacgtcaga
ggttcctgat 2700 accggatc 2708 <210> SEQ ID NO 7 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Oligonucleotide Primer <400> SEQUENCE: 7 agcaatgcac
ctccctgcga t 21 <210> SEQ ID NO 8 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Oligonucleotide
Primer <400> SEQUENCE: 8 tcctcggtgg gatgaagtcc a 21
<210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Oligonucleotide Primer <400>
SEQUENCE: 9 ctgctcttta ctgaaggctc 20 <210> SEQ ID NO 10
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Oligonucleotide Primer <400> SEQUENCE: 10
tcctcggtgg gatgaagtcc a 21 <210> SEQ ID NO 11 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Consensus "pentraxin-like" sequence <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (2)..(2)
<223> OTHER INFORMATION: any amino acid <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (4)..(4)
<223> OTHER INFORMATION: any amino acid <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(5)
<223> OTHER INFORMATION: Ser or Thr <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: any amino acid <400> SEQUENCE:
11 His Xaa Cys Xaa Xaa Trp Xaa Ser 1 5
* * * * *