Note: Descriptions are shown in the official language in which they were submitted.
CA 02212991 1997-10-10
INVERTEBRATE MESODERM INDUCTION EARLY RESPONSE (MIER)
GENE FAMILY
FIELD OF THE INVENTION
The present invention relates to a novel family of invertebrate immediate early response genes,
5 the use of members of the family in diagnostic and therapeutic applications, in addition to drug
design and vaccination protocols.
BACKGROUND OF THE INVENTION
Normal growth and differentiation of all org~ni~m~ is dependent on cells responding correctly to
a variety of internal and external signals. Many of these signals produce their effects by
10 ll Itim~ely ch~nging the transcription of specific genes. One of the major goals of developmental
biologists is to define the interactions of gene products and the role they play in regulating
cellular differentiation in time and space. Moreover, it is clear that in~l~ropliate expression of
many genes that control differentiation during embryonic development can lead to oncogenic
transformation. Such genes include members of the growth factor families and components of
15 their signal transduction pathways.
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Polypeptide growth factors are members of a growing family of regulatory molecules that have
been conserved throughout evolution and are known to have pleiotrophic effects which range
from stim~ tion of cell proliferation to control of cell differentiation. Growth factors have been
linked to oncogenesis as many of the known oncogenes have been identified as ov~ essed
5 and/or mutated forms of growth factors, growth factor receptors or components of their
intracellular signal transduction pathways. Oncogenes are thought to be altered such that their
product escapes the normal control mech~ni~m(s), resulting in the signaling pathways being
permanently switched on. The overall result is uncontrolled cell growth.
The family of fibroblast growth factors (FGFs~) consists of nine members related by sequence
10 and their ability to bind heparin (1). FGFs are involved in a number of cellular activities,
including mitogenesis, cell differentiation and angiogenesis (reviewed in 2). In addition,
overexpression of FGF in various cell lines leads to phenotypic transformation (3-5). For
example,f~f-3 was identified by its plo~ y to a pref~ d integration site of the proviral DNA
of the murine m~mm~ry tumour virus (MMTV) in MMTV induced m~mm~ry carcinomas
(Moore, et al., 1986), whilef~-4 was isolated from Kaposi's sarcoma by its ability to transform
NIH 3T3 cells (Delli-Bovi and Basilico, 1987). Some members of the family were identified by
their mitogenic activity such asf,~f-2, which can cause phenotypic transformation when
overexpressed in cultured cells (Sasada, et al., 1988; Neufeld, et al., 1988), thus classifying them
as potential oncogenes.
20 Most ofthe studies to date have focused on FGF's mitogenic and transforming activities,
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however, FGF has also been shown to act as a differentiation factor for embryonic cells (Slack et
al., 1987). For exarnple, FGFs have been shown to induce mesoderm differentiation in Xenopus
embryonic tissue (6) and many of the initial events in the cellular response during induction are
similar to those previously characterized for the FGF-mediated mitogenic response. During
5 mesoderm induction, FGF binds to high affinity cell surface receptors (7) which in turn become
phosphorylated on tyrosine (8). The phosphorylated FGF receptor (FGFR) forms a signaling
complex by binding a number of intracellular substrates (9) which results in activation of several
well-characterized signaling pathways. For instance, protein kinase C becomes activated during
FGF-induced mesoderm differentiation (8) as does MAPK (10).
Previously, growth and differentiation had been thought to be mutually exclusive, i.e. when a cell
begins to differentiate, it stops dividing. Thus, the elucidation of the mech~3ni~m~ that regulate
the differentiation process may provide may provide valuable information about the molecular
signals that are important for arresting cell growth. Further research in this field will contribute
15 to an underst~n(ling of how growth factors, such as FGF function during early embryonic
development to regulate patterning of mesodermal tissues and highlight differences in the
cellular response during growth, differentiation and oncogenesis. It is therefore hoped that by
elucidating the molecular mech~ni~m~ by which genes regulate developmental processes during
embryogenesis, it may be possible to define how misregulation of these genes can lead to cancer.
20 Recent research has focused on finding means for triggering the immune system to attack
cancerous cells, a tactic termed immunotherapy or vaccine therapy. Because h~ lily is a
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systemic reaction, it holds the potential to elimin~te all cancer cells in a patient's body, even
when they migrate away from the original tumor site or reappear after years of clinical remission.
One challenge is that the immune system does not always recognize cancer cells and single them
out for attack. A possible solution is to tag cancer cells with certain genes rendering them more
5 visible to the immune system, which can then destroy them.
The immune response involves many different cells and chemicals that work together to destroy
in several ways invading microbes or damaged cells. In general, abnormal cells sport surface
proteins, called antigens, that differ from those found on healthy cells. When the immune system
is activated, B lymphocytes produce antibodies which circulate through the body and bind to
10 foreign antigens, thereby m~rkin~ the antigen bearers for destruction by other components of the
immune system. Other cells, T lymphocytes, recognize foreign antigens as well; they destroy
cells displaying specific antigens of stimulate other killer T cells to do so. B and T cells
co~ nullicate with one another by way of secreted proteins, cytokines. Other accessory cells,
antigen-presenting cells and dendritic cells, further help T and B lymphocytes detect and respond
15 to antigens on cancerous or infected cells.
One theory of a means of identifying cancer cells entails the abnormal expression of genes that
are normally expressed only very early in development, such as during embryogenesis. If these
types of genes are not expressed in normal, healthy adult cells, but are during cancerous growth,
then proteins could be expressed that could function as an antigenic marker for immlme attack.
20 Immunizing an organism with DNA coding for this antigen, could train or sensitize the immune
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system to attack cells expressing these antigens that are only expressed in during cancerous
growth. Moreover, sensitive diagnostic means using either labelled polynucleotide probes or
antibodies could be developed to detect the polynucleic acid messengers, such as mRNA,
indicating the expression of these genes, hence the transformation into cancerous growth.
5 SUMMARY OF THE INVENTION
The subject invention concerns the MIER invertebrate gene family and its polynucleotide
sequences which encode proteins; members of this gene family are activated in response to
fibroblast growth factor (FGF) in an immediate early sequence. As an exemplary member of the
MIER gene family, erl is an early response gene that encodes a transcription factor found in the
10 cell nucleus and is activated in response to FGF.
Embodiments of this invention pertaining to the MIER gene family comprise:
1) genomic sequences, gene sequences and partial sequences of the members of the
invertebrate MIER gene family;
2) isolated, synthetic MIER gene sequences;
15 3) polynucleotide sequence probes for diagnostic use;
4) polynucleotide sequences for antisense gene therapy;
5) polynucleotide sequences for DNA vaccines;
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6) polynucleotide sequences for gene replacement therapy;
7) cloning vectors comprising invertebrae MIER gene sequences;
8) antibodies to partial invertebrae MER gene sequences;
9) antibodies to peptides encoded by MIER gene sequences;
5 10) diagnostic kits comprising nucleic acid probes; and
11) diagnostic kits comprising antibodies to MIER proteins.
An object of the present invention is to provide a family of invertebrate genes that are transcribed
in the immediate early phase of mesoderm induction following exposure to FGF. In accordance
with an aspect of the present invention there are provided cDNAs encoding members of this
10 MIER gene family.
In accordance with another aspect of the invention there is provided a probe to identify and
isolate similar gene sequences.
In accordance with yet a further aspect of the invention there is provided antisense nucleotides to
block expression of gene products.
15 In one embodiment ofthe subject invention, the proteins encoded by the genes described herein
can be used to raise antibodies which in turn can be used in diagnostic or therapeutic
applications.
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In one aspect, the present invention provides a member of the invertebrate MIER gene family: an
isolated and purified er-l polypeptide. Preferably, the polypeptide is a recombinant polypeptide,
and more preferably comprises the amino acid sequence of FIG. 1.
In another aspect, the present invention provides an isolated and purified polynucleotide that
5 encodes a MIER polypeptide. Preferably, the polynucleotide is a DNA molecule, such as an
isolated and purified polynucleotide comprising the nucleotide base sequence for one member of
the MIER family, erl, shown in FIG. 1.
The present invention also contemplates an expression vector comprising a polynucleotide that
encodes a MIER polypeptide. In a preferred embodiment, the polynucleotide is operatively
10 linked to an enhancer-promoter.
Also contemplated is a recombinant cell transfected with a polynucleotide that encodes a MIER
polypeptide. Preferably, the polynucleotide is under the transcriptional control of regulatory
signals functional in the recombinant cell, and the regulatory signals al)pl~pliately control
expression of the receptor polypeptide in a manner to enable all necessary transcriptional and
15 post-transcriptional modification.
In yet another aspect, the present invention contemplates a process of preparing a MIER
polypeptide, by producing a transformed recombinant cell, and m~ t;.i~ g the transformed
recombinant cell under biological conditions suitable for the expression of the polypeptide.
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The present invention also contemplates an antibody immunoreactive with a MIER
polynucleotide and/or polypeptide. The antibody may be either monoclonal or polyclonal.
Preferably, the antibody is a monoclonal antibody produced by recovering the polynucleotide
and/or polypeptide from a cell host, expressing the polypeptides and then preparing antibody to
5 the polypeptide in a suitable animal host.
In still another aspect, the present invention provides a process of detecting a MIER
polynucleotide and/or polypeptide, which process comprises immunoreacting the polynucleotide
and/or polypeptide with an antibody of the present invention and a diagnostic assay kit for
detecting the presence of a MER polynucleotide and/or polypeptide in a biological sample, the
10 kit comprising a first container means comprising a first antibody that immunoreacts with the
MIER polynucleotide and/or polypeptide. The first antibody is present in an amount sufficient to
perform at least one assay.
Still further, the present invention provides a process of detecting a DNA molecule or RNA
transcript that encodes a MIER polypeptide by hybridizing the DNA or RNA transcript with a
15 polynucleotide that encodes the polypeptide to form a duplex, and then detecting the duplex.
Still further, the present invention provides a process of screening a substance for its ability to
interact with members of the MIER family of proteins.
It is a further object of the present invention to provide a diagnostic marker for rapidly
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proliferating cells. A further aspect ofthe invention is concerned with a diagnostic kit col~L;.il~ g
antibodies to the nucleic acid of the invention. Yet a further aspect of the invention is concerned
with a diagnostic kit Co~ g antibodies to the protein encoded by the nucleic acid of the
instant invention.
5 DESCRIPTION OF THE FIGURES
The drawings form part of the present specification and are included to demonstrate certain
aspects of the present invention. The invention may be better understood by reference to one or
more of these drawings in combination with the detailed description of specific embodiments
presented herein:
10 Figure 1. Nucleotide and predicted amino acid sequence of erl. The nucleotide sequence
numbers of the erl cDNA are shown on the left and the amino acid sequence numbers of the
predicted ERl protein are shown on the right. The TAA termin~tion codon is indicated by an
asterisk. Four stretches of predomin~ntly acidic residues are underlined, the proline-rich region
is in bold and two putative nuclear localization signals (NLS) are indicated by double underlines;
15 the second NLS conforms to the consensus for a bipartite NLS.
Figure 2. Amino acid comparison of ERl to the rat and human MTAl and the C. elegans
CA 02212991 1997-10-10
similar-to-MTAl protein. A, Schematic illustrating alignment of the predicted Xenopus ERl
protein sequence with the rat and human MTAl and the similar-to-MTAl protein from C.
elegans. The N-termini were aligned and gaps (black lines) were introduced in the C. elegans
and Xenopus proteins in order to align the regions of similarity (hatched ) identified by the
BLAST program. White boxes indicate unique regions. B, Alignment of the predicted ERl
amino acid sequence with the MTAl amino acid sequences in the regions of similarity illustrated
in A. Identities are indicated by the one-letter amino acid code, conservative changes are
indicated by a plus sign (+) and dashes (-) indicate non-conservative changes. The amino acid
sequence numbers of the ERl protein are shown on the right.
Figure 3. erl is an FGF immerli~te-early response gene. A, FGF-stim~ ted increase in
steady-state levels of erl . Explants (5 per sample) from stage 8 Xenopus blastulae were treated
for 30 min in the presence (lane 2) or absence (lane 1) of 100 ng/ml XbFGF. Total RNA was
extracted and RT-PCR analysis was performed as described under "Experimental Procedures".
B, FGF-stimlll~ted increase of erl in the absence of protein synthesis. Explants were
pre-incubated for 30 min with (lanes 3, 4) or without (lanes 1, 2) 5ug/ml cycloheximide;
lOOng/ml XbFGF was added to the samples in lanes 2 and 4 and all samples were incubated for
an additional 30 min. Extraction and analysis were performed as described in A.
Figure 4. Expression of erl is restricted to early developmental stages in Xenopus. A,
Northern blot analysis of erl expression. Total RNA was isolated from the following
developmental stages: stage 2 (2-cell; lane 1), stage 6 (64-cell; lane 2), stage 7 (early blastula;
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CA 02212991 1997-10-10
lane 3), stage 8 (mid-blastula; lane 4), stage 12 (mid-gastrula; lane 5), stage 17 (neurula; lane 6),
stage 22 (tailbud; lane 7), stage 30 (lane 8) and stage 41 (tadpole; lane 9). Northern analysis was
performed as in Sambrook et al. (20) using 32P-labeled 2.3-kb erl cDNA as a probe. The blot
was stripped and re-probed with 32P-labeled histone H4 cDNA. B, Qualllil~live PCR analysis of
S erl levels during blastula and gastrula stages of development. Total RNA was isolated at lh
intervals during blastula stages, beginning at stage 7 (lane 1) and ending with stage 9 (lane 4).
For gastrula stages in lanes 5-7, RNA was isolated at stages 10, 10.5 and 12, respectively,
according to morphological criteria (29). RT-PCR and analysis were performed as described in
the legend to Fig. 3.
10 Figure 5. Nuclear locq~ qffon of ERl. A, Immunoprecipitation of in vitro translation
products with anti-ERl. Rabbit reticulocyte lysates programmed with erl cDNA in pcDNA3
were immuno~recil~ilated with either pre-immune (lane 2) or anti-ERl (lane 3) serum prepared
in our laboratory. Total translation products representing one half of the input into each
immunopreciptation are shown in lane 1. B, ERl is localized within the nucleus in transfected
NIH 3T3 cells. NIH 3T3 cells were transfected with either the pcDNA3 vector alone (top) or erl
-pcDNA3 (bottom). After 48h, cells were fixed and stained with anti-ERl, as described in
"Experimental Procedures".
Figure 6. The N-terminus of ERl functions as a transcriptional activator. NIH 3T3 cells
were transiently transfected with various GAL4-ERl fusion constructs along with a CAT
20 reporter plasmid. After 48h, CAT enzyme levels were measured as described in "Experimental
12
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Procedures". Vector denotes the control pM plasmid, cont~ining only the GAL4 DNA binding
domain, while the numbers indicate the amino acids of ERl encoded by each construct. The
value for each construct represents the fold activation relative to the pM plasmid, averaged from
3-12 independent transfections.
5 Figure 7. The partial sequences of some members of the MIER gene family.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to a family of invertebrate genes that are transcribed in the immediate early
phase following exposure to FGF during mesoderm induction, termed Mesoderm Induction Early
Response (MIER) genes. Defining features of the members of this family include that these
10 genes are a) transcribed in response to FGF; b) are expressed within 40 minutes of FGF
treatment; and c) do not require protein synthesis for transcription. There are at least eleven
members within this family.
The unique polynucleotide sequences of the subject invention include MIER gene sequences
which encode the MIER proteins, as well as sequences which drive the ~ures~ion of these
1 5 proteins.
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As an exemplary member of the MIER gene family, erl is an early response gene that encodes a
transcription factor found in the cell nucleus and is activated in response to FGF. The gene is
overexpressed in breast carcinoma and cervical carcinoma cell lines and possibly in general in all
cancer cell lines. Erl is also overexpressed in an abnormal T-cell subset (CD28-) whose
5 numbers increase with disease progression in AIDS patients. This CD28- subset also increases
in chronic infl~mm~tory disorders. Therefore this gene and its product are potential targets for
diagnosis and treatment of various cancers as well as immune disorders such as AIDS.
The ultim~te targets of these signal transduction pathways are the immediate-early genes. To
date, very few FGF immediate-early genes have been identified (11, 12). Accordingly, we have
10 utilized the differential display methodology (13) to isolate cDNAs representing such genes
Definitions and Abbreviations
The term "MIER" refers to Mesoderm Induction Immediate Early Response genes, their nucleic
acid transcription products and translated protein products. Defining features of the members of
this family include that the genes are a) transcribed in response to fibroblast growth factors
15 (FGF); b) are expressed within 40 minutes of FGF treatment; and c) do not require protein
synthesis for transcription. There are at least eleven members within this family; one member is
erl.
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The MIER genes and polypeptides of the present invention are not limited to a particular
invertebrate source. As disclosed herein, the techniques and compositions of the present
invention provide, for example, the identification and isolation of sources from invertebrate
cancerous cell lines. Thus, the invention provides for the general detection and isolation of the
5 genus of MIER genes and polypeptides from a variety of sources. It is believed that a number of
species of the family of MIER genes and polypeptides are amenable to detection and isolation
using the compositions and methods of the present invention.
Polynucleotides and polypeptides of the present invention are prepared by standard techniques
well known to those skilled in the art. Such techniques include, but are not limited to, isolation
10 and purification from tissues known to contain these genes and polypeptides, and expression
from cloned DNA that encodes such polypeptides using kansformed cells.
In one embodiment of the invention, the biological activity of the MIER proteins of the subject
invention can be reduced or elimin~ted by ~f~mini~tering an effective amount of an antibody to
each of the MIER proteins. Alternatively, the activity of the MIER proteins can be controlled by
15 modulation of e~lession of the MIER protein. This can be accomplished by, for example, the
a-~mini~kation of antisense DNA.
As used herein, the terms "nucleic acid" and "polynucleotide sequence" refer to a
deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and
CA 02212991 1997-10-10
unless otherwise limited, would encompass known analogs of natural nucleotides that can
function in a similar manner as naturally-occurring nucleotides. The polynucleotide sequences
include both the DNA strand sequence that is transcribed into RNA and the RNA
sequence that is translated into protein. The polynucleotide sequences include both full-length
S sequences as well as shorter sequences derived from the full-length sequences. It is understood
that a particular polynucleotide sequence includes the degenerate codons of the native
sequence or sequences which may be introduced to provide codon preference in a specific host
cell. Allelic variations of the exemplified sequences also come within the scope of the subject
invention. The polynucleotide sequences falling within the scope of the subject invention further
10 include sequences which specifically hybridize with the exemplified sequences under stringent
conditions. The nucleic acid includes both the sense and antisense strands as either individual
strands or in the duplex.
The terms "hybridize" or "hybridizing" refer to the binding of two single-stranded nucleic acids
via complementary base pairing.
15 The phrase "hybridizing specifically to" refers to binding, duplexing, or hybridizing of a
molecule to a nucleotide sequence under stringent conditions when that sequence is present in a
preparation of total cellular DNA or RNA.
The term "stringent conditions" refers to conditions under which a probe will hybridize to its
target sub-sequence, but not to sequences having little or no homology to the target sequence.
16
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Generally, stringent conditions are selected to be about 5° C. lower than the thermal
melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a complementary probe. Typically, stringent conditions will be those in which the
salt concentration is at least about 0.1 to 1 .ON Na ion concentration at a pH of about 7.0 to 7.5
and the temperature is at least about 60° C. for long sequences (e.g., greater than about 50
nucleotides) and at least about 42° C. for shorter sequences (e.g., about 10 to 50
nucleotides).
The terms "isolated" or "substantially pure" when referring to polynucleotide sequences encoding
the MIER proteins or fragments thereof refers to nucleic acids which encode MIER proteins or
peptides and which are no longer in the presence of sequences with which they are associated in
nature.
The terms "isolated" or "substantially purified" when referring to the proteins of the subject
invention means a chemical composition which is essentially free of other cellular components. It
is preferably in a homogenous state and can be in either a dry or aqueous solution. Purity
and homogeneity are typically determined using analytical chemistry techniques such as
polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which
is the predominant species present in a preparation is substantially purified. Generally, a
substantially purified or isolated protein will comprise more than 80% of all
macromolecular species present in the preparation. Preferably, the protein is purified to represent
17
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greater than 90% of all macromolecular species present. More preferably, the protein is purified
to greater than 95%, and most preferably the protein is purified to essential homogeneity,
wherein other macromolecular species are not detected by conventional techniques.
The phrase "specifically binds to an antibody" or "specifically immunoreactive with," when
S referring to a protein or peptide, refers to a binding reaction which is determin~tive of the
presence of the protein in a heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies bound to a
particular protein do not bind in a significant amount to other proteins present in the sample.
Specific binding to an antibody under such conditions may require an antibody that is selected
1~ for its specificity for a particular protein. A variety of immunoassay formats may be used to
select antibodies specifically immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a
protein. See Harlow and Lan (1988) for a description of immunoassay formats and conditions
that could be used to determine specific immunoreactivity. The subject invention further
15 concerns antibodies raised against the purified MIER molecules or their fragments.
The term "biological sample" as used herein refers to any sample obtained from a living
organism or from an organism that has died. Examples of biological samples include body fluids,
tissue specimens, and tissue cultures lines taken from patients.
The term "recombinant DNA" or "recombinantly-produced DNA" refers to DNA which has been
18
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isolated from its native or endogenous source and modified either chemically or enzymatically to
delete naturally-occurring fl~nking nucleotides or provide fl~nking nucleotides that do not
naturally occur.
Flanking nucleotides are those nucleotides which are either upstream or downstream from the
5 described sequence or sub-sequence of nucleotides.
The term "recombinant protein" or "recombinantly-produced protein" refers to a peptide or
protein produced using cells that do not have an endogenous copy of DNA able to express the
protein. The cells produce the protein because they have been genetically altered by the
introduction of an applopl;ate nucleic acid sequence. The recombinant protein will not be
10 found in association with proteins and other subcellular components normally associated with the
cells producing the protein.
It is well known that DNA possesses a fundamental property called base complementarity. In
nature, DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each
strand projecting from that strand toward the opposite strand. The base adenine (A) on one strand
15 will always be opposed to the base thymine (T) on the other strand, and the base guanine (G) will
be opposed to the base cytosine (C). The bases are held in apposition by their ability to hydrogen
bond in this specific way. Though each individual bond is relatively weak, the net effect of many
adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining of the
two complementary strands. These bonds can be broken by treatrnent~ such as high pH or high
19
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temperature, and these conditions result in the dissociation, or "denaturation," of the two strands.
If the DNA is then placed in conditions which make hydrogen bonding of the bases
thermodynamically favorable, the DNA strands will anneal, or "hybridize," and reform the
original double stranded DNA. If carried out under a~l;ate conditions, this hybridization can
5 be highly specific. That is, only strands with a high degree of base complementarity will be able
to form stable double stranded structures. The relationship of the specificity of hybridization to
reaction conditions is well known. Thus, hybridization may be used to test whether two pieces of
DNA are complementary in their base sequences. It is this hybridization mech~ni~m which
facilitates the use of probes of the subject invention to readily detect and characterize DNA
10 sequences of interest.
As those of ordinary skill in the art will appreciate, any of a number of different nucleotide
sequences can be used, based on the degeneracy of the genetic code, to produce the MIER
proteins described herein. Accordingly, any nucleotide sequence which encodes the MIER
proteins described herein comes within the scope of this invention and the claims appended
15 hereto. Also, as described herein, fragments of the MIER proteins are an aspect of the subject
invention so long as such fragments retain the biological activity so that such fragments are
useful in therapeutic and/or diagnostic procedures as described herein. Such fragments can easily
and routinely be produced by techniques well known in the art. For example, time-controlled
Bal3 1 exonuclease digestion of the full-length DNA followed by expression of the resulting
20 fragments and routine screening can be used to readily identify expression products having the
desired activity.
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Polynucleotide Probes
In addition, PCR-amplified DNA may serve as a hybridization probe. In order to analyze DNA
using the nucleotide sequences of the subject invention as probes, the DNA can first be obtained
in its native, double-stranded form. A number of procedures are currently used to isolate
5 DNA and are well known to those skilled in this art.
One approach for the use of the subject invention as probes entails first identifying by Southern
blot analysis of a DNA library all DNA segments homologous with the disclosed nucleotide
sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the
presence of genes homologous with the polynucleotide sequences described herein. Such
10 a probe analysis provides a rapid diagnostic method.
One hybridization procedure useful according to the subject invention typically includes the
initial steps of isolating the DNA sample of interest and purifying it chemically. For example,
total fractionated nucleic acid isolated from a biological sample can be used. Cells can be
treated using known techniques to liberate their DNA (and/or RNA). The DNA sample can be
15 cut into pieces with an app~opliate restriction enzyme. The pieces can be separated by size
through electrophoresis in a gel, usually agarose or acrylamide. The pieces of interest can be
transferred to an immobilizing membrane in a manner that retains the geometry of the pieces.
The membrane can then be dried and prehybridized to equilibrate it for later immersion in a
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hybridization solution. The manner in which the nucleic acid is affixed to a solid support may
vary. This fixing of the DNA for later processing has great value for the use of this technique in
field studies, remote from laboratory facilities.
The particular hybridization technique is not essential to the subject invention. As improvements
5 are made in hybridization techniques, they can be readily applied.
As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming
a strong non-covalent bond between the two molecules, it can be reasonably assumed that the
probe and sample are essentially identical. The probe's detectable label provides a means for
determining in a known manner whether hybridization has occurred.
10 The nucleotide segments of the subject invention which are used as probes can be synthesized by
use of DNA synthesizers using standard procedures. In the use of the nucleotide segments as
probes, the particular probe is labeled with any suitable label known to those skilled in the art,
including radioactive and non-radioactive labels. Typical radioactive labels include 32 P,
35 S, or the like. A probe labeled with a radioactive isotope can be constructed from a
15 nucleotide sequence complementary to the DNA sample by a conventional nick translation
reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a
hybridization buffer solution and held at an appropl;ate temperature until annealing occurs.
Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound
probe molecules typically detected and quantified by autoradiography and/or liquid
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scintillation counting. For synthetic probes, it may be most desirable to use enzymes such as
polynucleotide kinase or terminal transferase to end-label the DNA for use as probes.
Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as
enzymes such as hydrolases or perixodases, or the various chemiluminescers such as luciferin, or
5 fluorescent compounds like fluorescein and its derivatives. The probes may be made inherently
fluorescent as described in International Application No. W093/16094. The probe may also be
labeled at both ends with different types of labels for ease of separation, as, for example, by
using an isotopic label at the end mentioned above and a biotin label at the other end.
The amount of labeled probe which is present in the hybridization solution will vary widely,
10 depending upon the nature of the label, the amount of the labeled probe which can reasonably
bind to the filter, and the stringency of the hybridization. Generally, substantial excesses of the
probe will be employed to enhance the rate of binding of the probe to the fixed DNA.
Various degrees of stringency of hybridization can be employed. The more severe the conditions,
the greater the complementarity that is required for duplex formation. Severity can be controlled
15 by temperature, probe concentration, probe length, ionic strength, time, and the like.
Preferably, hybridization is conducted under stringent conditions by techniques well known in
the art, as described, for example, in Keller and Manak, 1987.
Duplex formation and stability depend on substantial complementarity between the two strands
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of a hybrid, and, as noted above, a certain degree of mi~m~tch can be tolerated. Therefore, the
nucleotide sequences ofthe subject invention include mutations (both single and multiple),
deletions, insertions of the described sequences, and combinations thereof, wherein
said mutations, insertions and deletions permit formation of stable hybrids with the target
5 polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given
polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled
artisan. Other methods may become known in the future.
The known methods include, but are not limited to:
(1) synthesizing chemically or otherwise an artificial sequence which is a mutation, insertion or
10 deletion of the known sequence;
(2) using a nucleotide sequence of the present invention as a probe to obtain via hybridization a
new sequence or a mutation, insertion or deletion of the probe sequence; and
(3) mllt~ting, inserting or deleting a test sequence in vitro or in vivo.
It is important to note that the mutational, insertional, and deletional variants generated from a
15 given probe may be more or less efficient than the original probe. Notwith~t~n~ling such
differences in efficiency, these variants are within the scope of the present invention.
24
CA 02212991 1997-10-10
Thus, mutational, insertional, and deletional variants of the disclosed nucleotide sequences can
be readily prepared by methods which are well known to those skilled in the art. These variants
can be used in the same manner as the instant probe sequences so long as the variants have
substantial sequence homology with the probes. As used herein, substantial sequence homology
5 refers to homology which is sufficient to enable the variant to function in the same capacity as
the original probe. Preferably, this homology is greater than 50%; more preferably, this
homology is greater than 75%; and most preferably, this homology is greater than 90%. The
degree of homology needed for the variant to function in its intended capacity will depend upon
the intended use of the sequence. It is well within the skill of a person trained in this art to
10 make mutational, insertional, and deletional mutations which are designed to improve the
function of the sequence or otherwise provide a methodological advantage.
It is well known in the art that the amino acid sequence of a protein is determined by the
nucleotide sequence of the DNA. Because of the re~ n~ncy of the genetic code, i.e., more than
one coding nucleotide triplet (codon) can be used for most of the amino acids used to make
15 proteins, different nucleotide sequences can code for a particular amino acid.
The amino acid sequence of the proteins of the subject invention can be encoded by equivalent
nucleotide sequences encoding the same amino acid sequence of the protein. Accordingly, the
subject invention includes probes which would hybridize with various polynucleotide sequences
which would all code for a given protein or variations of a given protein. In addition, it has been
20 shown that proteins of identified structure and function may be constructed by ch~n~;ing the
CA 02212991 1997-10-10
amino acid sequence if such changes do not alter the protein secondary structure (Kaiser and
Kezdy, 1984).
In one aspect, the present invention provides an isolated and purified polynucleotide that encodes
a MIER polypeptide. In a preferred embodiment, a polynucleotide of the present invention is a
5 DNA molecule. Even more preferably, a polynucleotide of the present invention encodes a
polypeptide comprising the amino acid residue sequence of Er-l, a member of the MIER family
(FIG. 1). Most preferably, an isolated and purified polynucleotide of the invention comprises the
nucleotide base sequence of FIG. 1.
As used herein, the term "polynucleotide" means a sequence of nucleotides connected by
10 phosphodiester linkages. Polynucleotides are presented herein in a 5' to 3' direction. A
polynucleotide of the present invention may comprise about several thousand base pairs.
Preferably, a polynucleotide comprises from about 100 to about 10,000 base pairs. Preferred
lengths of particular polynucleotides are set forth hereinafter.
A polynucleotide of the present invention may be a deoxyribonucleic acid (DNA) molecule or
15 ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, that molecule
may be a gene or a cDNA molecule. Nucleotide bases are indi cated herein by a single letter
code: adenine (A), guanine (G), thymine (T) and cytosine (C).
A polynucleotide of the present invention may be prepared using standard techniques
26
CA 02212991 1997-10-10
well-known to one of skill in the art. The preparation of a cDNA molecule encoding an erl
polypeptide of the present invention is described hereinafter in the examples. A polynucleotide
may also be prepared from genomic DNA libraries using, for example, lambda phage
technologies
5 In another aspect, the present invention provides an isolated and purified polynucleotide that
encodes a MIER polypeptide, where the polynucleotide is preparable by a process comprising the
steps of constructing a library of cDNA clones ~om a cell that expresses the polypeptide;
screening the library with a labelled cDNA probe prepared from RNA that encodes the
polypeptide; and selecting a clone that hybridizes to the probe.
10 A further aspect of the claimed invention are antibodies that are raised by immunization of an
animal with a purified protein or polynucleotides of the subject invention. Both polyclonal and
monoclonal antibodies can be produced using standard procedures well known to those skilled in
the art using the proteins of the subject invention as an immunogen (see, for example,
Monoclonal Antibodies: Principles and Practice, 1983; Monoclonal Hybridoma Antibodies:
Techniques and Applications, 1982; Selected Methods in Cellular Immunology, 1980;
Immunological Methods, Vol. II, 1981; Practical Immunology, and Kohler et al., 1975).
The proteins of the subject invention include those which are specifically exemplified herein as
well as related proteins which, for example, are immunoreactive with antibodies which are
27
CA 02212991 1997-10-10
produced by, or are immunologically reactive with, the proteins specifically exemplified
herein.
The proteins described herein can be used in therapeutic or diagnostic procedures.
Probes
5 In another aspect, DNA sequence information provided by the present invention allows for the
preparation of relatively short DNA (or RNA) sequences having the ability to specifically
hybridize to gene sequences of the selected polynucleotide disclosed herein. In these aspects,
nucleic acid probes of an al)plopl;ate length are prepared based on a consideration of a selected
nucleotide sequence, e.g., a sequence such as that shown in FIG. 1. The ability of such nucleic
10 acid probes to specifically hybridize to a polynucleotide encoding a MIER lends them particular
utility in a variety of embodiments. Most importantly, the probes may be used in a variety of
assays for detecting the presence of complementary sequences in a given sample.
In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such
primers is designed using a polynucleotide of the present invention for use in detecting,
15 amplifying or mutating a defined segment of a gene or polynucleotide that encodes a MIER
polypeptide from invertebrate cells using PCR.TM. technology.
To provide certain of the advantages in accordance with the present invention, a preferred nucleic
28
CA 02212991 1997-10-10
acid sequence employed for hybridization studies or assays includes probe molecules that are
complementary to at least an about (14) to an about (70) nucleotide long stretch of a
polynucleotide that encodes a MIER polypeptide, such as the nucleotide base sequences shown
in FIG. 1. A size of at least 14 nucleotides in length helps to ensure that the fragment is of
5 sufficient length to form a duplex molecule that is both stable and selective. Molecules having
complementary sequences over stretches greater than 14 bases in length are generally preferred,
though, in order to increase stability and selectivity of the hybrid, and thereby improve the
quality and degree of specific hybrid molecules obtained, one will generally prefer to design
nucleic acid molecules having gene-complementary stretches of 25 to 40 nucleotides, 55 to 70
10 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for
example, directly synthesizing the fragment by chemical means, by application of nucleic acid
reproduction technology, such as the PCR.TM. technology of U.S. Pat. No. 4,603,102, or by
excising selected DNA fragments from recombinant plasmids cotl~it,itlg al~pl.,pliate inserts and
suitable restriction enzyme sites.
15 In another aspect, the present invention contemplates an isolated and purified polynucleotide
comprising a base sequence that is identical or complementary to a segment of at least 14
contiguous bases of FIG. 1, wherein the polynucleotide hybridizes to a polynucleotide that
encodes a MIER polypeptide. Preferably, the isolated and purified polynucleotide comprises a
base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous
20 bases of FIG. 1. For example, the polynucleotide of the invention may comprise a segment of
bases identical or complementary to 40 or 55 contiguous bases of the disclosed nucleotide
29
CA 02212991 1997-10-10
sequences.
Accordingly, a polynucleotide probe molecule of the invention may be used for its ability to
selectively form duplex molecules with complementary stretches of the gene. Depending on the
application envisioned, one employs varying conditions of hybridization to achieve varying
5 degree of selectivity of the probe toward the target sequence. For applications requiring a high
degree of selectivity, one typically employs relatively stringent conditions to form the hybrids.
For example, one selects relatively low salt and/or high temperature conditions, such as provided
by about 0.02M to about 0.1 5M NaCl at temperatures of about 50~C to about 70~C. Those
conditions are particularly selective, and tolerate little, if any, mi~m~tch between the probe and
10 the template or target strand.
In some applications where it is the intention to prepare mul~ll, employing a mutant primer
strand hybridized to an underlying template or where one seeks to isolate a MIER polypeptide
coding sequence from other cells, functional equivalents, or the like, less stringent hybridization
conditions are typically needed to allow formation of the heteroduplex. In these circumstances,
15 one employs conditions such as about 0.15M to about O.9M salt, at temperatures ranging from
about 20~C C. to about 55~C . Cross-hybridizing species may thereby be readily identified
as positively hybridizing signals with respect to control hybridizations. In any case, it is
generally appreciated that conditions may be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same
20 manner as increased temperature. Thus, hybridization conditions may be readily manipulated,
CA 02212991 1997-10-10
and thus will generally be a method of choice depending on the desired results.
In still another embodiment of the present invention, there is provided a isolated and purified
polynucleotide comprising a base sequence that is identical or complementary to a segment of at
least about 14 contiguous bases of rMIER The polynucleotide of the invention hybridizes to
5 rMIER, or a complement of rMIER. Preferably, the isolated and purified polynucleotide
comprises a base sequence that is identical or complementary to a segment of at least 25 to 70
contiguous bases of rMIER. For example, the polynucleotide of the invention may comprise a
segment of bases identical or complementary to 40 or 55 contiguous bases of rMIER.
Alternatively, the present invention contemplates an isolated and purified polynucleotide that
10 comprises a base sequence that is identical or complementary to a segment of at least about 14
contiguous bases of MIER.
The polynucleotide of the invention hybridizes to MIER, or a complement of MIER. Preferably,
the polynucleotide comprises a base sequence that is identical or complementary to a segment of
at least 25 to 70 contiguous bases of MIER. For example, the polynucleotide may comprise a
15 segment of bases identical or complementary to 40 or 55 contiguous bases of MIER.
In certain embodiments, it is advantageous to employ a polynucleotide of the present invention in
combination with an appropl;ate label for detecting hybrid formation. A wide variety of
a~pl~,pliate labels are known in the art, including radioactive, enzymatic or other ligands, such as
31
CA 02212991 1997-10-10
avidin/biotin, which are capable of giving a detectable signal.
In general, it is envisioned that a hybridization probe described herein is useful both as a reagent
in solution hybridization as well as in embodiments employing a solid phase. In embodiments
involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected
5 matrix or surface. This fixed nucleic acid is then subjected to specific hybridization with selected
probes under desired conditions. The selected conditions will depend on the particular
circumstances and criteria required (e.g., the G~C content, type of target nucleic acid, source of
nucleic acid, size of hybridization probe, etc.). Following washing of the matrix to remove
nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by
lO means ofthe label.
Polynucleotide Primers
Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid
sequence. This procedure is well known and commonly used by those skilled in this art (see
Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., 1985). PCR is based on
15 the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide
primers that hybridize to opposite strands of the target sequence. The primers are oriented with
the 3' ends pointing towards each other. Repeated cycles of heat denaturation of the template,
:~nne~ling of the primers to their complementary sequences, and extension of the annealed
32
CA 02212991 1997-10-10
primers with a DNA polymerase result in the amplification of the segment defined by the S' ends
of the PCR primers. Since the extension product of each primer can serve as a template for the
other primer, each cycle essentially doubles the amount of DNA fragment produced
in the previous cycle. This results in the exponential accumulation of the specific target fi~gment
S up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq
polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the
amplification process can be completely automated.
The DNA sequences of the subject invention can be used as primers for PCR amplification. In
p~lrolllling PCR amplification, a certain degree of mi~m~tch can be tolerated between primer and
10 template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides
to the 5' end) of the exemplified primers fall within the scope of the subject invention. Mutations,
insertions and deletions can be produced in a given primer by methods known to an ordinarily
skilled artisan. It is important to note that the mutational, insertional, and deletional
variants generated from a given primer sequence may be more or less efficient than the original
15 sequences. Notwithstanding such differences in efficiency, these variants are within the scope of
the present invention.
CA 02212991 1997-10-10
DNA Vaccines and Immunotherapy
Tumor Associated Antigens
Certain members of the MIER family of proteins are normally expressed during embryogenesis.
Thus, the proteins should not be present in mature or adult cells. Of these proteins that are not
5 present in adult cells, those that do appear can form the basis of a cancer-antigen indicating a cell
that has turned cancerous. This can be determined, for example, by screening using a labelled
nucleic acid probe indicating the presence of mRNA for the MIER proteins, that is not present at
the same level in normal, healthy cells. In the alternative, labelled antibodies can be used to
detect MIER protein as an antigenic det~rmin~nt of cancerous growth. These types of results are
10 presented in Figures 2-5.
Vaccines
In a plerelled embodiment, the invention relates to specific DNA vaccines and methods of
treating cancer using the immune system and/or providing protective immunity to vertebrates
and/or invertebrates. "Protective i~ lllily" conferred by the method of the invention can elicit
15 humoral and/or cell-mediated immune responses to cancerous growth, but more importantly
interferes with the activity, spread, or growth of a cell that has become cancerous and has begun
to express MIER nucleic acids and/or proteins following a subsequent challenge after
vaccination.
34
CA 02212991 1997-10-10
The DNA vaccines of the invention are transcription units cont~ining DNA encoding a MIER
polypeptide or protein. In the method of the present invention, a DNA vaccine is atlmini~tered to
a vertebrates and/or invertebrates as a mode of therapy, and/or in whom protective immunization
is desired. An object of the invention is to provide an immune response and protective immunity
5 to a vertebrates and/or invertebrates using a DNA vaccine encoding a MIER protein as it has the
potential of achieving high levels of protection in the virtual absence of side effects. Such DNA
vaccines are also stable, easy to ~11mini~ter, and sufficiently cost-effective for widespread
distribution.
An object of the invention is to provide protective immunity to an inoculated host. If the
10 inoculated host is a female vertebrates and/or invertebrates, an object ofthe invention is to
provide protection in the offspring of that female.
The invention features a DNA vaccine co~ inil~g a MIER DNA transcription unit (i.e., an
isolated nucleotide sequence encoding a MIER-encoded protein or polypeptide). The nucleotide
sequence is operably linked to transcriptional and translational regulatory sequences for
15 e~lession of the MIER-coded polypeptide in a cell of a vertebrates and/or invertebrates.
Preferably the polypeptide encoded by the DNA vaccine of the invention is a sequence belonging
to MIER. Preferably, the nucleotide sequence encoding the polypeptide is contained in a plasmid
vector.
The DNA vaccines can be ~11mini~tered to vertebrates (and/or invertebrates) such as humans
CA 02212991 1997-10-10
expressing tumor associated antigens, such as the erl protein.
The DNA vaccines of the invention are preferably contained in a physiologically acceptable
carrier for in vivo ~(1mini~tration to a cell of a vertebrate and/or invertebrate. Administration of
the DNA vaccines of the invention provide an immune response or protective immunity.
5 The invention also features a method of providing an immune response and protective i~ lily
to a vertebrate and/or invertebrate against cancerous growth of cells ~lessing such a tumor
associated antigen. The method includes a(lmini~tering to a cell of a vertebrate and/or
invertebrate, a DNA transcription unit encoding a desired MIER-encoded antigen operably
linked to a promoter sequence. Expression of the DNA transcription unit in the cell elicits a
10 humoral immune response, a cell-mediated immune response, or both against the cell ~ essillg
the protein product of the DNA transcription unit, the tumor associated antigen, which in this
invention would be a MIER-encoded antigen.
The promoter operably linked to the DNA transcription unit is of nonretroviral or retroviral
origin. Preferably the promoter is the cytomegalovirus immediate-early enhancer promoter. The
15 desired MIER-encoded antigen encoded by the DNA transcription unit is one of the members of
the MIER family, demonstrated to be expressed at significantly high levels only in cancerous
cells in the mature organism.
The DNA transcription unit of the method of the invention is preferably contained in a
36
CA 02212991 1997-10-10
physiologically acceptable carrier and is a-lmini~tered to the vertebrate and/or invertebrate by
routes including, but not limited to, inhalation, intravenous, h~ uscular, intraperitoneal,
intradermal, and subcutaneous al1mini.~tration. The DNA transcription unit in a physiologically
acceptable carrier can also be ~lmini~tered by being contacted with a mucosal surface of the
5 vertebrates and/or invertebrates.
Preferably, a-lmini~tration is performed by particle bombardment using gold beads coated with
the DNA transcription units of the invention. Preferably, the gold beads are 1 µm to 2 µm
in diameter. The coated beads are preferably a(lmini~tered intra~erm~lly, inll~lluscularly~ by
organ transfection, or by other routes useful in particle bombardment and known to those of
10 ordinary skill in the art.
The term "immune response" refers herein to a cytotoxic T cells response or increased serum
levels of antibodies to an antigen, or to the presence of neutralizing antibodies to an antigen, such
as a MIER-encoded protein. The term "protection" or "protective immunity" refers herein to the
ability of the serum antibodies and cytotoxic T cell response induced during; ~ 7ation to
15 protect (partially or totally) against cells expressing such tumor associated antigen. That is, a
vertebrate andlor invertebrate immunized by the DNA vaccines of the invention will experience
an immune attack on cancerous cells expressing such tumor associated antigen.
The term "promoter sequence" herein refers to a minim~l sequence sufficient to direct
transcription. Also included in the invention is an enhancer sequence which may or may not be
37
CA 02212991 1997-10-10
contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene
expression and may be located in the 5' or 3' regions of the native gene. Expression is
constitutive or inducible by external signals or agents. Optionally, expression is cell-type
specific, tissue-specific, or species specific.
5 By the term "transcriptional and translational regulatory sequences" is meant nucleotide
sequences positioned adjacent to a DNA coding sequence which direct transcription or
translation of a coding sequence. The regulatory nucleotide sequences include any sequences
which promote sufficient expression of a desired coding sequence and presentation of the
protein product to the inoculated vertebrate's (and/or invertebrate's') immune system such that
10 protective immunity is provided.
By the term "operably linked to transcriptional and translational regulatory sequences" is meant
that a polypeptide coding sequence and minim~l transcriptional and translational controlling
sequences are connected in such a way as to permit polypeptide expression when the applopliate
molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). In the
15 present invention, polypeptide expression in a target vertebrate and/or invertebrate cell is
particularly L~er~ d.
The term "isolated DNA" means DNA that is free of the genes and other nucleotide sequences
that flank the gene in the naturally-occurring genome of the organism from which the isolated
DNA of the invention is derived. The term therefore includes, for example, a recombinant DNA
20 which is incorporated into a vector; into an autonomously replicating plasmid or into the
38
CA 02212991 1997-10-10
genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a
cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion)
independent of other sequences. It also includes a recombinant DNA which is part of a hybrid
gene encoding additional polypeptide sequences.
S A preferred embodiment of this invention relates to a method of providing protective immunity
to vertebrates and/or invertebrates. Protective h~ y of the invention elicits humoral and/or
cell-mediated immune responses. According to the present invention, a DNA transcription unit is
~-lmini~tered to a vertebrate and/or invertebrate in whom imm~mi7~tion and protection is desired.
DNA Transcription Units
10 A DNA transcription unit is a polynucleotide sequence, bounded by an initiation site and a
tennin~lion site, that is transcribed to produce a primary transcript. As used herein, a "DNA
transcription unit" includes at least two components: (1) antigen-encoding DNA, and (2) a
transcriptional promoter element or elements operatively linked for expression of the
antigen-encoding DNA. Antigen-encoding DNA can encode one or multiple antigens, such as
15 antigens from two or more different proteins. The DNA transcription unit can additionally be
inserted into a vector which includes sequences for expression of the DNA transcription unit.
A DNA transcription unit can optionally include additional sequences such as enhancer elements,
splicing signals, termin~tion and polyadenylation signals, viral replicons, and bacterial plasmid
39
CA 02212991 1997-10-10
sequences. In the present method, a DNA transcription unit (i.e., one type oftranscription unit)
can be ~(lministered individually or in combination with one or more other types of DNA
transcription units.
DNA transcription units can be produced by a number of known methods. For example, DNA
5 encoding the desired antigen can be inserted into an expression vector (see, for example,
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory
Press (1989)). With the availability of automated nucleic acid synthesis equipment, DNA can be
synthesized directly when the nucleotide sequence is known, or by a combination of polymerase
chain reaction (PCR), cloning, and fermentation. Moreover, when the sequence of the desired
10 polypeptide is known, a suitable coding sequence for the polynucleotide can be inferred.
The DNA transcription unit can be ~rlministered to an individual, or inoculated, in the presence
of adjuvants or other substances that have the capability of promoting DNA uptake or recruiting
immune system cells to the site of the inoculation. It should be understood that the DNA
transcription unit itself is expressed in the host cell by transcription factors provided by the host
15 cell, or provided by a DNA transcription unit.
The "desired antigen" can be any antigen or combination of antigens from encoded by a MIER
gene. The antigen or antigens can be naturally occurring, or can be mutated or specially
modified. The antigen or antigens can represent different forms, such as subgroups (clades), or
subtypes. These antigens may or may not be structural components of a protein encoded by a
CA 02212991 1997-10-10
MIER gene. The encoded antigens can be translation products or polypeptides. The polypeptides
can be of various lengths, and can undergo normal host cell modifications such as glycosylation,
myristoylation, or phosphorylation. In addition, they can be designated to undergo intracellular,
extracellular, or cell-surface ~les~ion. Furthermore, they can be designed to undergo assembly
5 and release from cells.
Administration of DNA Transcription Units
A vertebrate can be inoculated through any parenteral route. For example, an individual can be
inoculated by intravenous, intraperitoneal, intra~erm~l, subcutaneous, inhalation, or
luscular routes, or by particle bombardment using a gene gun. Muscle is a useful site for
10 the delivery and expression of DNA transcription unit-encoded polynucleotides, because ~nim~
have a proportionately large muscle mass which is conveniently accessed by direct injection
through the skin. A comparatively large dose of polynucleotides can be deposited into muscle
by multiple and/or repetitive injections, for example, to extend therapy over long periods of time.
Muscle cells are injected with polynucleotides encoding immunogenic polypeptides, and these
15 polypeptides are presented by muscle cells in the context of antigens of the major
histocompatibility complex to provoke a selected immlme response against the immunogen (see,
e.g., Felgner, et al. WO90/11092, herein incorporated by reference).
The epidermis is another useful site for the delivery and ~ ssion of polynucleotides, because it
is conveniently accessed by direct injection or particle bombardment. A comparatively large dose
41
CA 02212991 1997-10-10
of polynucleotides can be deposited in the epidermis by multiple injections or bombardments to
extend therapy over long periods of time. In immunization strategies of the invention, skin cells
are injected with polynucleotides coding for immunogenic polypeptides, and these polypeptides
are presented by skin cells in the context of antigens of the major histocompatibility complex
S to provoke a selected immune response against the immunogen.
In addition, an individual can be inoculated by a mucosal route. The DNA transcription unit can
be ~11mini~tered to a mucosal surface by a variety of methods including DNA-col-L ~ g
nose-drops, inh~l~nt~, suppositories, microsphere encapsulated DNA, or by bombardment with
DNA coated gold particles. For example, the DNA transcription unit can be ~-lmini~tered to
10 a respiratory mucosal surface, such as the nares or the trachea.
Any al~plu~liate physiologically compatible medium, such as saline for injection, or gold
particles for particle bombardment, is suitable for introducing the DNA transcription unit into an
individual.
MIER Polypeptides
15 In one embodiment, the present invention contemplates an isolated and purified MIER
polypeptides such as Er- 1 polypeptide. Preferably, a MIER Polypeptide of the invention is a
recombinant polypeptide. Preferably, an exemplary MIER polypeptide of the present invention
comprises an amino acid sequence of FIG. 1.
42
CA 02212991 1997-10-10
Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written
left to right in the direction from the amino to the carboxy terminus. In accordance with standard
nomenclature, amino acid residue sequences are denomin~te~l by either a single letter or a three
letter code.
Modifications and changes may be made in the structure of a polypeptide of the present
invention and still obtain a molecule having MIER-like characteristics. For example, certain
amino acids may be substituted for other amino acids in a sequence without appreciable loss of
activity. Because it is the interactive capacity and nature of a polypeptide that defmes that
polypeptide's biological functional activity, certain amino acid sequence substitutions may be
made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and
nevertheless obtain a polypeptide with like properties.
The importance of the hydropathic amino acid index in conferring interactive biologic function
on a polypeptide is generally understood in the art (Kyte and Doolittle, 1982). It is known that
certain amino acids may be substituted for other amino acids having a similar hydropathic index
or score and still result in a polypeptide with similar biological activity. Each amino acid has
been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine
(-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glut~rn~te (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
43
CA 02212991 1997-10-10
It is believed that the relative hydropathic character of the amino acid determines the secondary
structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide
with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It
is known in the art that an arnino acid may be substituted by another a~nino acid having a similar
S hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the
substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which
are within ±1 are particularly pler~lled, and those within ±0.5 are even more particularly
preferred.
Substitution of like amino acids may also be made on the basis of hydrophilicity, particularly
10 where the biological functional equivalent polypeptide or peptide thereby created is intended for
use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of a polypeptide, as governed by
the hydrophilicity of its adjacent arnino acids, correlates with its immllnogenicity and
antigenicity, i.e., with a biological property of the polypeptide.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to
arnino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glllt~m~te (+3.0±1);
serine (+0.3); asparagine (+0.2); glut~mine (+0.2); glycine (0); proline (-0.5.~-.1); threonine
(-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine
(-1.8); isoleucine (-1.8); tyrosine (-2.3); phenyl~l~nine (-2.5); tryptophan (-3.4). It is understood
20 that an amino acid may be substituted for another having a similar hydrophilicity value and still
44
CA 02212991 1997-10-10
obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In
such changes, the substitution of amino acids whose hydrophilicity values are within ~+-.2 is
preferred, those which are within ±1 are particularly pler~lled, and those within ±0.5 are
even more particularly preferred
5 As outlined above, amino acid substitutions are generally therefore based on the relative
similarity of the amino acid side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the
foregoing characteristics into consideration are well known to those of skill in the art and
include: arginine and lysine; glul~ te and aspartate; serine and threonine; glutamine and
10 asparagine; and valine, leucine and isoleucine (See Table 1, below). The present invention thus
contemplates functional or biological equivalents of a MIER polypeptide as set forth above.
Biological or functional equivalents of a polypeptide may also be prepared using site-specific
mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of second
generation polypeptides, or biologically functional equivalent polypeptides or peptides, derived
15 from the sequences thereof, through specific mutagenesis of the underlying DNA. As noted
above, such changes may be desirable where amino acid substitutions are desirable. The
technique further provides a ready ability to prepare and test sequence variants, for example,
incorporating one or more of the foregoing considerations, by introducing one or more nucleotide
sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants
20 through the use of specific oligonucleotide sequences which encode the DNA sequence of the
CA 02212991 1997-10-10
desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer
sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of about 14 to 25 nucleotides in length is
pleft;lled, with about 5 to 10 residues on both sides of the junction of the sequence being
5 altered.
TABLE 1
Original Exemplary
Residue Substitutions
Ala Gly; Ser
Arg Lys
Asn Gln; His
Asp Glu
Cys Ser
Gln Asn
Glu Asp
Gly Ala
His Asn; Gln
Ile Leu; Val
Leu Ile; Val
46
CA 02212991 1997-10-10
Lys Arg
Met Leu; Tyr
Ser Thr
Thr Ser
Trp Tyr
Tyr Trp; Phe
Val Ile; Leu
The technique of site-specific mutagenesis is generally well-known in the art (Adelman et al.,
1983). As will be appreciated, the technique typically employs a phage vector which may exist in
10 both a single stranded and double stranded form. Typical vectors useful in site-directed
mutagenesis include vectors such as the M13 phage (Messing et al., 1981). These phage are
commercially available and their use is generally known to those of skill in the art.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a
single-stranded vector which includes within its sequence a DNA sequence which encodes all or
15 a portion of the MIER polypeptide sequence selected. An oligonucleotide primer bearing the
desired mutated sequence is prepared, generally synthetically, for exarnple, by the method of
Crea, et al., (1978). This primer is then annealed to the singled-stranded vector, and extended by
the use of enzymes such as the Klenow fragment of E. coli polymerase I, to complete the
synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand
47
CA 02212991 1997-10-10
encodes the original non-ml1t;~ted sequence and the second strand bears the desired mutation.
This heteroduplex vector is then used to transform appropriate cells such as E. coli cells and
clones are selected which include recombinant vectors bearing the mutation. Commercially
available kits come with all the reagents necessary, except the oligonucleotide primers.
5 Expression Vectors
In an alternate embodiment, the present invention provides expression vectors comprising a
polynucleotide that encodes a MIER polypeptide. Preferably, an expression vector of the present
invention comprises a polynucleotide that encodes a polypeptide comprising an amino acid
residue sequence of one of the members of the MIER gene family, eg. erl as in FIG. 1. More
10 preferably, an ~ression vector of the present invention comprises a polynucleotide comprising
a nucleotide base sequence of FIG. 1. Even more preferably, an ~lession vector of the
invention comprises a polynucleotide operatively linked to an enhancer-promoter. More
preferably still, an expression vector of the invention comprises a polynucleotide operatively
linked to a prokaryotic promoter. Alternatively, an ~ples~ion vector of the present invention
15 comprises a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic
promoter, and the expression vector further comprises a polyadenylation signal that is positioned
3' ofthe carboxy-termin~l amino acid and within a transcriptional unit ofthe encoded
polypeptide.
48
CA 02212991 1997-10-10
A promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of
(upstream of) the point at which transcription begins (i.e., a transcription start site). That region
typically contains several types of DNA sequence elements that are located in similar relative
positions in different genes. As used herein, the terrn "promoter" includes what is referred to in
5 the art as an upstream promoter region, a promoter region or a promoter of a generalized
eukaryotic RNA Polymerase II transcription unit.
Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer
provides specificity of time, location and expression level for a particular encoding region (e.g.,
gene). A major function of an enhancer is to increase the level of transcription of a coding
10 sequence in a cell that contains one or more transcription factors that bind to that enhancer.
Unlike a promoter, an enhancer may function when located at variable distances from
transcription start sites so long as a promoter is present.
As used herein, the phrase "enhancer-promoter" means a composite unit that contains both
enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding
15 sequence that encodes at least one gene product. As used herein, the phrase "operatively linked"
means that an enhancer-promoter is connected to a coding sequence in such a way that the
transcription of that coding sequence is controlled and regulated by that enhancer-promoter.
Means for operatively linking an enhancer-promoter to a coding sequence are well known in the
art. As is also well known in the art, the precise orientation and location relative to a coding
20 sequence whose transcription is controlled, is dependent inter alia upon the specific nature of the
49
CA 02212991 1997-10-10
enhancer-promoter. Thus, a TATA box minim~l promoter is typically located from about 25 to
about 30 base pairs upstream of a transcription initiation site and an upstream promoter
element is typically located from about 100 to about 200 base pairs upstream of a transcription
initiation site. In contrast, an enhancer may be located downstream from the initiation site and
5 may be at a considerable distance from that site.
An enhancer-promoter used in a vector construct of the present invention may be any
enhancer-promoter that drives expression in a cell to be transfected. By employing an
enhancer-promoter with well-known properties, the level and pattern of gene product expression
may be optimized.
10 A coding sequence of an expression vector is operatively linked to a transcription termin~ting
region. RNA polymerase transcribes an encoding DNA sequence through a site where
polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream
of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to
herein as transcription-termination regions. Those regions are required for efficient
15 polyadenylation of transcribed messenger RNA (RNA). Transcription-termin~tin~ regions are
well-known in the art. A preferred transcription-termin~ting region used in an adenovirus vector
construct of the present invention comprises a polyadenylation signal of SV40 or the protamine
gene.
An ~res~ion vector comprises a polynucleotide that encodes a MIER polypeptide. Such a
CA 02212991 1997-10-10
polynucleotide is meant to include a sequence of nucleotide bases encoding a MIER polypeptide
sufficient in length to distinguish said segment from a polynucleotide segment encoding a non-
M-erl polypeptide. A polypeptide of the invention may also encode biologically functional
polypeptides or peptides which have variant amino acid sequences, such as with changes selected
S based on considerations such as the relative hydropathic score of the amino acids being
exchanged. These variant sequences are those isolated from natural sources or induced in the
sequences disclosed herein using a mutagenic procedure such as site-directed mutagenesis.
An expression vector of the present invention comprises a polynucleotide that encodes a
polypeptide comprising an amino acid residue sequence of FIG. 1. An expression vector may
10 include a MIER polypeptide-coding region itself or any of the MIER polypeptides noted above
or it may contain coding regions bearing selected alterations or modifications in the basic coding
region of such a MIER polypeptide.
Alternatively, such vectors or fragments may code larger polypeptides or polypeptides which
15 nevertheless include the basic coding region. In any event, it should be appreciated that due to
codon redundancy as well as biological functional equivalence, this aspect of the invention is not
limited to the particular DNA molecules corresponding to the polypeptide sequences noted
above.
Exemplary vectors include the invertebrate expression vectors of the pCMV family including
20 pCMV6b and pCMV6c (Chiron Corp., Emeryville, Calif.). In certain cases, and specifically in
51
CA 02212991 1997-10-10
the case of these individual invertebrate expression vectors, the resulting constructs may require
co-transfection with a vector conlai~ g a selectable marker such as pSV2neo. Via
co-transfection into a dihydrofolate reductase-deficient Chinese hamster ovary cell line, such as
DG44, clones expressing opioid polypeptides by virtue of DNA incorporated into such
S expression vectors may be detected.
A DNA molecule of the present invention may be incorporated into a vector using standard
techniques well known in the art. For instance, the vector pUC18 has been demonstrated to be of
particular value. Likewise, the related vectors M13mpl8 and M13mpl9 may be used in certain
embodiments of the invention, in particular, in performing dideoxy sequencing.
10 An expression vector of the present invention is useful both as a means for preparing quantities
of the MIER polypeptide-encoding DNA itself, and as a means for preparing the encoded
polypeptide and peptides. It is contemplated that where MIER polypeptides of the invention are
made by recombinant means, one may employ either prokaryotic or eukaryotic expression
vectors as shuttle systems. However, in that prokaryotic systems are usually incapable of
15 correctly processing precursor polypeptides and, in particular, such systems are incapable of
correctly processing membrane associated eukaryotic polypeptides, and since eukaryotic MIER
polypeptides are aIlticipated using the teaching of the disclosed invention, one likely expresses
such sequences in eukaryotic hosts. However, even where the DNA segment encodes a
eukaryotic MIER polypeptide, it is contemplated that prokaryotic expression may have some
20 additional applicability. Therefore, the invention may be used in combination with vectors which
CA 02212991 1997-10-10
may shuttle between the eukaryotic and prokaryotic cells. Such a system is described herein
which allows the use of bacterial host cells as well as eukaryotic host cells.
Where ~ es~ion of recombinant MIER polypeptides is desired and a eukaryotic host is
contemplated, it is most desirable to employ a vector such as a plasmid, that incorporates a
5 eukaryotic origin of replication.
Additionally, for the purposes of expression in eukaryotic systems, one desires to position the
MIER encoding sequence adjacent to and under the control of an effective eukaryotic promoter
such as promoters used in combination with Chinese hamster ovary cells. To bring a coding
sequence under control of a promoter, whether it is eukaryotic or prokaryotic, what is generally
10 needed is to position the 5' end of the translation initiation side of the proper translational reading
frame of the polypeptide between about 1 and about 50 nucleotides 3' of or downstream with
respect to the promoter chosen. Furthermore, where eukaryotic ~ression is anticipated, one
would typically desire to incorporate into the transcriptional unit which includes the MIER
polypeptide, an ~propriate polyadenylation side.
15 The pCMV plasmids are a series of ~1 es~ion vectors of particular utility in the present
invention. The vectors are designed for use in essentially all cultured cells and work extremely
well in SV40-transformed simian COS cell lines. The pCMVl, pCMV2, pCMV3, and pCMV5
vectors differ from each other in certain unique restriction sites in the polylinker region of each
plasmid. pCMV4 differs from the other four plasmids in cont~ining a translation enhancer in the
53
CA 02212991 1997-10-10
sequence prior to the polylinker. While they are not directly derived from the pCMVl-pCMV5
series of vectors, the functionally similar pCMV6b and pCMV6c vectors are
commercially available (Chiron Corp., Emeryville, Calif.) and are identical except for the
orientation of the polylinker region which is reversed in one relative to the other.
5 The universal components of the pCMV vectors are as follows: The vector backbone is pTZ18R
(Pharmacia, Piscataway, N.J.), and contains a bacteriophage fl origin of replication for
production of single stranded DNA and an ampicillin (amp~-resistance gene. The CMV region
consists of nucleotides -760 to +3 of the powerful promotor-regulatory region of the
human cytomegalovirus (Towne stain) major immediate early gene (Thomsen et al., 1984;
10 Boshart et al., 1985). The human growth hormone fragment (hGH) contains transcription
termination and poly-adenylation signals representing sequences 1533 to 2157 of this gene
(Seeber-lg, 1982). There is an Alu middle repetitive DNA sequence in this fragment. Finally, the
SV40 origin of replication and early region promoter-enhancer derived from the
pcD-X plasmid (HindIII to PstI fragment) described in (Okayama et al., 1983). The promoter in
15 this fragment is oriented such that transcription proceeds away from the CMV/hGH expression
cassette.
The pCMV plasmids are distinguishable from each other by differences in the polylinker region
and by the presence or absence of the translation enhancer. The starting pCMVl plasmid has
been progressively modified to render an increasing number of unique restriction sites in the
20 polylinker region. To create pCMV2, one of two EcoRI sites in pCMVl were destroyed.
54
CA 02212991 1997-10-10
To create pCMV3, pCMV1 was modified by deleting a short segment from the SV40 region
(StuI to EcoRI), and in so doing made unique the PstI, SalI, and BamHI sites in the polylinker.
To create pCMV4, a synthetic fragment of DNA corresponding to the 5'- untr~n~l~ted region of a
rnRNA transcribed from the CMV promoter was added C'. The sequence acts as a translational
5 enhancer by decreasing the requirements for initiation factors in polypeptide synthesis (Jobling et
al., 1987; Bro~,vning et al., 1988). To create pCMV5, a segment of DNA (HpaI to EcoRI) was
deleted from the SV40 origin region of pCMV1 to render unique all sites in the starting
polylinker.
The pCMV vectors have been successfully expressed in simian COS cells, mouse L cells, CHO
10 cells, and HeLa cells. In several side by side comparisons they have yielded 5- to 10-fold higher
expression levels in COS cells than SV40-based vectors. The pCMV vectors have been used to
express the LDL receptor, nuclear factor 1, Gs .alpha. polypeptide, polypeptide phosphatase,
synaptophysin, synapsin, insulin receptor, influenza hemagglutinin, androgen receptor, sterol
26-hydroxylase, steroid 17- and 21-hydroxylase, cvtochrome P-450 oxidoreductase,
15 .beta.-adrenergic receptor, folate receptor, cholesterol side chain cleavage enzyme, and a
host of other cDNAs. It should be noted that the SV40 promoter in these plasmids may be used
to express other genes such as dominant selectable markers. Finally, there is an ATG sequence in
the polylinker between the HindIII and PstI sites in pCMU that may cause sper-lious translation
initiation. This codon should be avoided if possible in expression plasmids. A paper describing
20 the construction and use of the parenteral pCMV1 and pCMV4 vectors has been published
(Anderson et al., 1989b).
CA 02212991 1997-10-10
Transfected Cells
In yet another embodiment, the present invention provides recombinant host cells transformed or
transfected with a polynucleotide that encodes an MIER polypeptide, as well as transgenic cells
derived from those transformed or transfected cells. Preferably, a recombinant host cell of the
5 present invention is transfected with a polynucleotide of FIG. lC or FIG. lD. Means of
transforming or transfecting cells with exogenous polynucleotide such as DNA molecules are
well known in the art and include techniques such as calcium-phosphate- or DEAE-dextran-
mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct
microinjection and adenovirus infection (Sambrook et al., 1989).
10 The most widely used method is transfection mediated by either calcium phosphate or
DEAE-dextran. Although the mech~ni~m remains obscure, it is believed that the transfected
DNA enters the cytoplasm of the cell by endocytosis and is kansported to the nucleus.
Depending on the cell type, up to 90% of a population of culter-led cells may be transfected at
any one time. Because of its high efficiency, transfection mediated by calcium phosphate or
15 DEAE-dextran is the method of choice for studies requiring transient expression of the foreign
DNA in large numbers of cells. Calcium phosphate-mediated transfection is also used to
establish cell lines that integrate copies of the foreign DNA, which are usually arranged in
head-to-tail tandem arrays into the host cell genome.
In the protoplast fusion method, protoplasts derived from bacteria carrying high numbers of
56
CA 02212991 1997-10-10
copies of a plasmid of interest are mixed directly with cultured cells. After fusion of the cell
membranes (usually with polyethylene glycol), the contents of the bacterium are
delivered into the cytoplasm of the cells and the plasmid DNA is transported to the nucleus.
Protoplast fusion is not as efficient as transfection for many of the cell lines that are commonly
5 used for transient expression assays, but it is useful for cell lines in which
endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies of
the plasmid DNA tandomly integrated into the host chromosome.
The application of brief, high-voltage electric pulses to a variety of m~mm~ n and plant cells
leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly
10 into the cell cytoplasm either through these pores or as a consequence of the redistribution of
membrane components that accompanies closer- 1 e of the pores. Electroporation may be
extremely efficient and may be used both for transient expression of cloned genes and for
establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in
contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives
15 rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by
fusion ofthe liposomes with the cell membrane. The mechanism of how DNA is delivered into
the cell is unclear but transfection efficiencies may be as high as 90%.
Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to
57
CA 02212991 1997-10-10
cellular compartments such as low-pH endosomes. Microinjection is therefore used primarily as
a method to establish lines of cells that carry integrated copies of the DNA of interest.
The use of adenovirus as a vector for cell transfection is well known in the art. Adenovirus
vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet et al.,
1992).
A transfected cell may be prokaryotic or eukaryotic. Preferably, the host cells of the invention are
eukaryotic host cells. More preferably, the recombinant host cells of the invention are COS-l
cells. Where it is of interest to produce MIER polypeptides, cultured or human cells are of
particular interest.
10 In another aspect, the recombinant host cells of the present invention are prokaryotic host cells.
Preferably, the recombinant host cells of the invention are bacterial cells of the DH5.alpha..TM.
(GelBCa BRL, Gaithersber-lg, Md.) strain of E. coli. In general, prokaryotes are preferred for
the initial cloning of DNA sequences and constructing the vectors useful in the invention. For
example, E. coli K12 strains may be particularly useful. Other microbial strains which may be
used include E. coli B, and E. coli X1776 (ATCC No. 31537). These examples are, of coer-lse,
intended to be illustrative rather than limiting.
In general, plasmid vectors corlL~ g replicon and control sequences which are derived from
species compatible with the host cell are used in connection with these hosts. The vector
58
CA 02212991 1997-10-10
ordinarily carries a replication site, as well as m~rking sequences which are capable of providing
phenotypic selection in transformed cells. For example, E. coli may be kansformed using
pBR322, a plasmid derived from an E. coli species (Bolivar et al., 1977). pBR322 contains genes
for amp and tetracycline resistance and thus provides easy means for identifying transformed
5 cells.
The pBR322 or other microbial plasmid or phage must also contain, or be modified to contain,
promoters which may be used by the microbial organism for expression of its own polypeptides.
Those promoters most commonly used in recombinant DNA construction include the
.beta.-lactamase (penicillinase) and .beta.-galactosidase (.beta.-Gal) promoter systems (Chang et
al., 1978; Itaker-la et al., 1977; Goeddel et al., 1979; Goeddel et al., 1980) and a tryptophan
(TRP) promoter system (EPO Appl. Publ. No. 0036776; Siebwenlist et al., 1980). While these
are the most commonly used, other microbial promoters have been discovered and utilized, and
details concerning their nucleotide sequences have been published, enabling a skilled worker to
introduce promoters functional into plasmid vectors (Siebwenlist et al., 1980).
15 In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used. Saccharomyces
cerevisiae or common baker's yeast is the most commonly used among eukaryotic
microorg~ni cm.c, although a number of other strains are commonly available. For expression in
Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979,
Kin~m~n et al., 1979; Tschemper et al., 1980). This plasmid already contains the trpL gene
59
CA 02212991 1997-10-10
which provides a selection marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example ATCC No. 44076 or PEP4- 1 (Jones, 1977). The presence of the trpL
lesion as a characteristic of the yeast host cell genome then provides an effective environment for
detecting transformation by growth in the absence of tryptophan.
5 Suitable promotor sequences in yeast vectors include the promoters for 3-phosphoglycerate
kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al.,
1978) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate
mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and
10 glucokinase. In constructing suitable expression plasmids, the termin~tion sequences associated
with these genes are also introduced into the expression vector downstream from the
sequences to be expressed to provide polyadenylation of the mRNA and termin~tion. Other
promoters, which have the additional advantage of transcription controlled by growth conditions
are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,
15 degradative enzymes associated with nitrogen metabolism, and the aforementioned
glyceraldehyde-3-phosphate dehydrogenase, and enzyrnes responsible for maltose and galactose
utilization. Any plasmid vector Col~f ~ g a yeast-compatible promoter, origin or replication and
termin~tion sequences is suitable.
In addition to microorg~ni~m~, cultures of cells derived from multicellular org~nism.~ may also
20 be used as hosts. In principle, any such cell culture may be employed, whether from vertebrate or
CA 02212991 1997-10-10
invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of
vertebrate cells in tissue culture has become a routine procedure in recent years (Kruse and
Peterson, 1973). Examples of such useful host cell lines are AtT-20, VERO and HeLa cells,
Chinese hamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 and MDCK cell
5 lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a
promoter located upstream of the gene to be expressed, along with any necessary ribosome
binding sites, RNA splice sites, polyadenylation site, and transcriptional termin~tor sequences.
For use in m~mm~ n cells, the control functions on the expression vectors are often derived
from viral material. For example, commonly used promoters are derived from polyoma,
Adenovirus 2, Cytomegalovirus (CMV) and most frequently Simian Virus 40 (SV40). The early
and late promoters of SV40 virus are particularly useful because both are obtained easily
from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al.,
1978). Smaller or larger SV40 fragments may also be used, provided there is included the
ap~ llately 250 bp sequence extending from the HindIII site toward the BglI site located in
15 the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter
or control sequences normally associated with the desired gene sequence, provided such control
sequences are compatible with the host cell systems.
An origin of replication may be provided with by construction of the vector to include an
exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV,
20 BPV, CMV) source, or may be provided by the host cell chromosomal replication mech~ni~m. If
61
CA 02212991 1997-10-10
the vector is integrated into the host cell chromosome, the latter is often sufficient.
Preparing a Recombinant MIER Polypeptide
In yet another embodiment, the present invention describes a process of preparing an MIER
polypeptide comprising transfecting cells with a polynucleotide that encodes an MIER
5 polypeptide to produce a transformed host cell; and m;.i~ g the transformed host cell under
biological conditions sufficient for expression of the polypeptide. Preferably, the transformed
host cell is a eukaryotic cell. Even more preferably, the polynucleotide transfected into the
transformed cells comprises a nucleotide base sequence of FIGl . Most preferably transfection is
accomplished using a hereinbefore disclosed ~x~lession vector.
10 A host cell used in the process is capable of ~ ressillg a functional, recombinant MIER
polypeptide. A variety of cells are amenable to a process of the invention, for instance, yeasts
cells, human cell lines, and other eukaryotic cell lines known well to those of the art.
Following transfection, the cell is m~int~ined under culture conditions for a period of time
sufficient for expression of an MIER polypeptide. Culture conditions are well known in the art
15 and include ionic composition and concentration, temperature, pH and the like. Typically,
transfected cells are m~int~ined under culture conditions in a culture medium. Suitable medium
for various cell types are well-known in the art. In a plefelled embodiment, temperature is from
about 20~C. to about 50~C, more preferably from about 30~C. to about 40~C, and even more
62
CA 02212991 1997-10-10
preferably, about 37~C.
pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a
value of about 6.8 to a value of about 7.8, and most preferably, about 7.4. Osmolality is
preferably from about 200 milliosmols per liter (mosm/L) to about 400 mosm/l and, more
preferably from about 290 mosm/L to about 310 mosm/L. Other biological conditions needed for
transfection and ~pression of an encoded protein are well-known in the art.
Transfected cells are m~int~ined for a period of time sufficient for expression of an MIER
polypeptide. A suitable time depends inter alia upon the cell type used and is readily
determinable by a skilled artisan. Typically, maintenance time is from about 2 to about 14 days.
Recombinant MIER polypeptide is recovered or collected either from the transfected cells or the
medium in which those cells are cultured. Recovery comprises isolating and purifying the MIER
polypeptide. Isolation and purification techniques for polypeptides are well-known in the art and
include such procedures as pLecipilalion, filtration, chromatography, electrophoresis and the like.
Antibodies
In still another embodiment, the present invention provides an antibody immunoreactive with an
MIER polypeptide (e.g., one which is specific for MIER polypeptide). Preferably, an antibody of
the invention is a monoclonal antibody. Preferably, an MIER polypeptide comprises an amino
63
CA 02212991 1997-10-10
acid residue sequence of FIG. . Means for preparing and characterizing antibodies are
well-known in the art (See, e.g., "Antibodies: A Laboratory Manual", E. Howell and D. Lane,
Cold Spring Harbor Laboratory, 1988).
Briefly, a polyclonal antibody is prepared by immuni~ing an animal with an immunogen
5 comprising a polypeptide or polynucleotide of the present invention, and collecting antisera from
that immunized animal. A wide range of animal species may be used for the production of
antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a
h~m~ter or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a
preferred choice for production of polyclonal antibodies.
10 As is well-known in the art, a given polypeptide or polynucleotide may vary in its
immunogenicity. It is often necessary therefore to couple the immunogen (e.g., a polypeptide or
polynucleotide) of the present invention) with a carrier. Exemplary and preferred carriers are
keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as
ovalbumin, mouse serum albumin or rabbit serum albumin may also be used as carriers.
15 Means for conjugating a polypeptide or a polynucleotide to a carrier protein are well-known in
the art and include glutaraldehyde, m-maleimidobencoyl-N- hydroxysuccinimide ester,
carbodiimide and bis-biazotized benzidine.
As is also well-known in the art, immunogencity to a particular immunogen may be enhanced by
64
CA 02212991 1997-10-10
the use of non-specific stimulators of the immune response known as adjuvants. Exemplary and
preferred adjuvants include complete Freund's adjuvant, incomplete Freund's adj uva~ and
alulnillulll hydroxide adjuvant.
The amount of immunogen used of the production of polyclonal antibodies varies inter alia, upon
5 the nature of the immlln~gen as well as the animal used for immunization. A variety of routes
may be used to a-lmini~ter the immunogen (subcutaneous, i~ lluscular, intr~derm~l,
intravenous and intraperitoneal. The production of polyclonal antibodies is monitored by
sampling blood of the inllllullized animal at various points following ill~llunization. When a
desired level of immunogenicity is obtained, the immunized animal may be bled and the serum
10 isolated and stored.
In another aspect, the present invention contemplates a process of producing an antibody
immunoreactive with an MIER polypeptide comprising the steps of (a) transfecting a
recombinant host cell with a polynucleotide that encodes an MIER polypeptide; (b) culturing the
host cell under conditions sufficient for expression of the polypeptide; (c) recovering the
15 polypeptide; and (d) preparing an antibody to the polypeptide. Preferably, the host cell is
transfected with a polynucleotide of FIG 1. The present invention also provides an antibody
prepared according to the process described above.
A monoclonal antibody of the present invention may be readily prepared through use of
well-known techniques such as those exemplified in U.S. Pat. No. 4,196,265. Typically, a
CA 02212991 1997-10-10
technique involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide
or polynucleotide of the present invention) in a manner sufficient to provide an immune
response. Rodents such as mice and rats are pler~lled ~im~31.c. Spleen cells from the i"~"~ ed
animal are then fused with cells of an immortal myeloma cell. Where the h~ lullized animal is a
5 mouse, a preferred myeloma cell is a murine NS-l myeloma cell.
The fused spleen/myeloma cells are cultured in a selective medium to select fused
spleen/myeloma cells from the parental cells. Fused cells are separated from the llli~lul e of
non-fused parental cells, for example, by the addition of agents that block the de novo synthesis
of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin,
10 methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both
pMIERines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin
or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a
soMIERce of nucleotides. Where azaserine is used, the media is supplemented with
hypoxanthine.
15 This culturing provides a population of hybridomas from which specific hybridomas are selected.
Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in
microtiter plates, followed by testing the individual clonal supern~t~tlt~ for reactivity with an
antigen-polypeptides. The selected clones may then be propagated indefinitely to provide the
monoclonal antibody.
66
CA 02212991 1997-10-10
By way of specific example, to produce an antibody of the present invention, mice are injected
intraperitoneally with between about 1 to about 200 llg of an antigen comprising a polypeptide of
the present invention. B lymphocyte cells are stimulated to grow by injecting the antigen in
association with an adjuvant such as complete Freund's adjuvant (a non-specific stim~ tor of the
5 immune response cont~ining killed Mycobacterium tuberculosis). At some time (e.g., at least two
weeks) after the first injection, mice are boosted by injection with a second dose of the antigen
mixed with incomplete Freund's adjuvant.
A few weeks after the second injection, mice are tail bled and the sera tirered by
immunoprecipitation against radiolabeled antigen. Preferably, the process of boosting and
10 titering is repeated until a suitable titer is achieved. The spleen of the mouse with the highest titer
is removed and the spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
Typically, a spleen from an i~ unized mouse contains approximately S x 107 to 2 x 108
lymphocytes.
Mutant lymphocyte cells known as myeloma cells are obtained from laboratory ~nim~l~ in which
15 such cells have been induced to grow by a variety of well-known methods. Myeloma cells lack
the salvage pathway of nucleotide biosynthesis. Because myeloma cells are tumor cells, they
may be propagated indefinitely in tissue culture, and are thus denomin:-ted immortal. Numerous
cultured cell lines of myeloma cells from mice and rats, such as murine NS-l myeloma cells,
have been established.
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CA 02212991 1997-10-10
Myeloma cells are combined under conditions appro~l iate to foster fusion with the normal
antibody-producing cells from the spleen of the mouse or rat injected with the
antigen/polypeptide of the present invention. Fusion conditions include, for example, the
presence of polyethylene glycol. The resulting fused cells are hybridoma cells. Like myeloma
5 cells, hybridoma cells grow indefinitely in culture.
Hybridoma cells are separated from unfused myeloma cells by culturing in a selection medium
such as hypoxanthine-aminopterin-thymidine (HAT) medium. Unfused myeloma cells lack the
enzymes necessary to synthesize nucleotides from the salvage pathway because they are killed in
the presence of aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not
10 continue to grow in tissue culture. Thus, only cells that have successfully fused (hybridoma cells)
may grow in the selection media.
Each of the surviving hybridoma cells produces a single antibody. These cells are then screened
for the production of the specific antibody immunoreactive with an antigen/polypeptide of the
present invention. Single cell hybridomas are isolated by limiting dilutions of the hybridomas.
1~ The hybridomas are serially diluted many times and, after the dilutions are allowed to grow, the
supern~t~nt is tested for the presence of the monoclonal antibody. The clones producing that
antibody are then cultured in large amounts to produce an antibody of the present invention
in convenient quantity.
By use of a monoclonal antibody of the present invention, specific polypeptides and
68
CA 02212991 1997-10-10
polynucleotide of the invention may be recognized as antigens, and thus identified. Once
identified, those polypeptides and polynucleotide may be isolated and purified by techniques
such as antibody-affinity chromatography. In antibody-affinity chromatography, a monoclonal
antibody is bound to a solid substrate and exposed to a solution cu.~ g the desired antigen.
5 The antigen is removed from the solution through an immunospecific reaction with the bound
antibody. The polypeptide or polynucleotide is then easily removed from the substrate and
purified.
Pharmaceutical Compositions
In a preferred embodiment, the present invention provides a pharmaceutical composition
10 comprising an MIER polypeptide and a physiologically acceptable carrier. More preferably, a
pharmaceutical composition comprises an MIER polypeptide comprising an amino acid residue
sequence of FIG. . Alternatively, ph~rm~ceutical compositions include a polynucleotide that
encodes an MIER polypeptide and a physiologically acceptable carrier. An example of a useful
pharmaceutical composition includes a polynucleotide that has the nucleotide sequence of FIG.
15 A composition of the present invention is typically ~(lministered parenterally in dosage unit
formulations cont~ining standard, well-known nontoxic physiologically acceptable carriers,
adjuvants, and vehicles as desired. The term parenteral as used herein includes intravenous,
illll~lluscular, intraarterial injection, or infusion techniques.
69
CA 02212991 1997-10-10
Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, are
formulated according to the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a sterile injectable solution or
suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in
5 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution,
and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed
as a solvent or suspending medium. For this purpose any bland fixed oil may be employed
including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in
10 the preparation of injectables.
Preferred carriers include neutral saline solutions buffered with phosphate, lactate, Tris, and the
like. Of course, one purifies the vector sufficiently to render it essentially free of undesirable
col-t~ nt such as defective interfering adenovirus particles or endotoxins and other
pyrogens such that it does not cause any untoward reactions in the individual receiving the vector
15 construct. Means of purifying the vector may involve the use of buoyant density gradients, such
as cesium chloride gradient centrifugation.
A carrier may also be a liposome. Means for using liposomes as delivery vehicles are
well-known in the art (See, e.g., Gabizon et al., 1990; Ferruti and Tanzi, 1986; Ranade, 1989).
CA 02212991 1997-10-10
A transfected cell may also serve as a carrier. By way of example, a liver cell may be removed
from an org~ni~m, transfected with a polynucleotide of the present invention using methods set
forth above and then the transfected cell returned to the organism (e.g., injected intravascularly).
Detecting MIER Encoding Polynucleotides and MIER Polypeptides
S Alternatively, the present invention provides a process of detecting an MIER polypeptide,
wherein the process comprises immunoreacting the polypeptide with an antibody prepared
according to a process described above to form an antibody-polypeptide conjugate, and detecting
the conjugate.
In yet another embodiment, the present invention contemplates a process of detecting a
10 messenger RNA transcript that encodes an MIER polypeptide, wherein the process comprises (a)
hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes an MIER
polypeptide to form a duplex; and (b) detecting the duplex. Alternatively, the present invention
provides a process of detecting a DNA molecule that encodes an MIER polypeptide, wherein the
process comprises (a) hybridizing a DNA molecule with a polynucleotide that encodes an MIER
15 polypeptide to form a duplex; and (b) detecting the duplex.
Screening Assays
In yet another aspect, the present invention contemplates a process of screening substances for
71
CA 02212991 1997-10-10
their ability to interact with an MIER polypeptide comprising the steps of providing an MIER
polypeptide, and testing the ability of selected substances to interact with the MIER polypeptide.
Utilizing the methods and compositions of the present invention, screening assays for the testing
of candidate substances such as agonists and antagonists of MIERs may be derived. A candidate
5 substance is a substance which potentially may interact with or modulate, by binding or other
intramolecular interaction, an MIER polypeptide. In some instances, such a c~n(lid~te substance
will be an agonist of the polypeptide and in other instances may exhibit antagonistic attributes
when interacting with the polypeptide. In other in~t~n~es, such substances may have mixed
agonistic and antagonistic properties or may modulate the MIER in other ways.
10 Recombinant polypeptide ~les~ion systems of the present invention possess definite
advantages over tissue-based systems. Such a method of the present invention makes it possible
to produce large quantities of MIERs for use in screening assays. More important, however, is
the relative purity of the polypeptides provided by the present invention. A relatively pure
polypeptide preparation for assaying a protein-protein interaction makes it possible to use elutive
15 methods without invoking competing, and unwanted, side-reactions.
Cloned expression systems such as those of the present invention are also usefill where there is
difficulty in obtaining tissue that satisfactorily expresses a particular polypeptide. Cost is another
very real advantage, at least with regard to the microbial expression systems of the present
invention. For antagonists in a primary screen, microorganism ~plession systems of the present
72
CA 02212991 1997-10-10
invention are inexpensive in comparison to prior art tissue-screening methods.
Traditionally, screening assays employed the use of crude polypeptide preparations. Typically,
animal tissue slices thought to be rich in the polypeptide of interest was the source of the
polypeptide. Alternatively, investigators homogenized the tissue and used the crude homogenate
5 as a polypeptide source. A major difficulty with this approach is the provision that the tissue
contain only a single polypeptide type being expressed. The data obtained therefore could not be
definitively correlated with a particular polypeptide. With the recent cloning of polypeptide
sub-types and sub-sub-types, this difficulty is highlighted. A second fundamental difficulty with
the traditional approach is the unavailability of human tissue for screening potential drugs. The
10 traditional approach almost invariably utilized animal polypeptides. With the cloning of human
polypeptides, there is a need for screening assays which utilize human polypeptides.
With the availability of cloned polypeptides, recombinant polypeptide screening systems have
several advantages over tissue based systems. A major advantage is that the investigator may
now control the type of polypeptide that is utilized in a screening assay. Specific polypeptide
15 sub-types and sub-sub-types may be preferentially expressed and its interaction with a ligand
may be identified. Other advantages include the availability of large amounts of polypeptide, the
availability of rare polypeptides previously unavailable in tissue samples, and the lack of
expenses associated with the maintenance of live ~nim~l~
Screening assays of the present invention generally involve determining the ability of a candidate
73
CA 02212991 1997-10-10
substance to bind to the polypeptide and to affect the activity of the polypeptide, such as the
screening of candidate substances to identify those that inhibit or otherwise modify the
polypeptide's function. Typically, this method includes p~a hlg recombinant polypeptide
polypeptide, followed by testing the recombinant polypeptide or cells expressing the polypeptide
with a candidate substance to determine the ability of the substance to affect its physiological
function. In ple~el.~d embodiments, the invention relates to the screening of candidate
substances to identify those that affect the enzymatic activity of the human polypeptide, and thus
can be suitable for use in hllm~n~
A screening assay provides a polypeptide under conditions suitable for the binding of an agent to
the polypeptide. These conditions include but are not limited to pH, temperature, tonicity, the
presence of relevant cofactors, and relevant modifications to the polypeptide such as
glycosylation or prenylation. It is contemplated that the polypeptide can be expressed and
utilized in a prokaryotic or eukaryotic cell. The host cell e~res~ g the polypeptide can be used
whole or the polypeptide can be isolated from the host cell. The polypeptide can be membrane
bound in the membrane of the host cell or it can be free in the cytosol of the host cell. The host
cell can also be fractionated into sub-cellular fractions where the polypeptide can be found. For
example, cells ~ressillg the polypeptide can be fractionated into the nuclei, the endoplasmic
reticulum, vesicles, or the membrane surfaces of the cell.
pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a
value of about 6.8 to a value of about 7.8, and most preferably, about 7.4. In a preferred
74
CA 02212991 1997-10-10
embodiment, temperature is from about 20~C to about 50~C, more preferably, from about 30~C to
about 40~C, and even more preferably about 37~C. Osmolality is preferably from about S
milliosmols per liter (mosm/L) to about 400 mosm/l, and more preferably, from about 200
milliosmols per liter to about 400 mosm/1 and, even more preferably from about 290 mosm/L to
5 about 310 mosm/L. The presence of cofactors can be required for the proper functioning of the
polypeptide. Typical cofactors include sodium, potassium, calcium, magnesium, and chloride. In
addition, small, non-peptide molecules, known as prosthetic groups may also be required. Other
biological conditions needed for polypeptide function are well-known in the art.
It is well-known in the art that proteins can be reconstituted in artificial membranes, vesicles or
10 liposomes. (Danboldt et al., 1990). The present invention contemplates that the polypeptide can
be incorporated into artificial membranes, vesicles or liposomes. The reconstituted polypeptide
can be utilized in screening assays.
It is further contemplated that a polypeptide of the present invention can be coupled to a solid
support, e.g., to agarose beads, polyacrylamide beads, polyacrylic beads or other solid matrices
15 capable of being coupled to polypeptides. Well-known coupling agents include cyanogen
bromide (CNBr), carbonyllliimid~7ole, tosyl chloride, and glutaraldehyde.
In a typical screening assay for identifying candidate substances, one employs the same
recombinant expression host as the starting source for obtaining the polypeptide, generally
prepared in the form of a crude homogenate. Recombinant cells expressing the polypeptide are
CA 02212991 1997-10-10
washed and homogenized to prepare a crude polypeptide homogenate in a desirable buffer such
as disclosed herein. In a typical assay, an amount of polypeptide from the cell homogenate, is
placed into a small volume of an applo~liate assay buffer at an appro~iate pH. Candidate
substances, such as agonists and antagonists, are added to the ad~ e in convenient
5 concentrations and the interaction between the candidate substance and the polypeptide is
monitored.
Where one uses an appr~liate known substrate for the polypeptide, one can, in the foregoing
manner, obtain a baseline activity for the recombinantly produced polypeptide. Then, to test for
inhibitors or modifiers of the polypeptide function, one can incorporate into the ad~ e a
10 candidate substance whose effect on the polypeptide is unknown. By comparing reactions which
are carried out in the presence or absence of the candidate substance, one can then obtain
information regarding the effect of the candidate substance on the normal function of the
polypeptide.
Accordingly, this aspect of the present invention will provide those of skill in the art with
15 methodology that allows for the identification of candidate substances having the ability to
modify the action of MIER polypeptides in one or more manner.
Additionally, screening assays for the testing of candidate substances are designed to allow the
determination of structure-activity relationships of agonists or antagonists with the polypeptides,
e.g., comparisons of binding between naturally-occurring hormones or other substances capable
76
CA 02212991 1997-10-10
of interacting or otherwise mod~ ing with the polypeptide; or comparison of the activity caused
by the binding of such molecules to the polypeptide.
In certain aspects, the polypeptides of the invention are crystallized in order to carry out x-ray
crystallographic studies as a means of evaluating interactions with candidate substances or other
5 molecules with the MIER polypeptide. For instance, the purified recombinant polypeptides of the
invention, when crystallized in a suitable form, are amenable to detection of intra-molecular
interactions by x-ray crystallography.
The recombinantly-produced MIER polypeptide may be used in screening assays for the
identification of substances which may inhibit or otherwise modify or alter the function of the
10 polypeptide. The use of recombinantly-produced polypeptide is of particular benefit because the
naturally-occurring polypeptide is present in only small quantities and has proven difficult to
purify. Moreover, this provides a ready source of polypeptide, which has heretofore been
unavailable.
A screening assay of the invention, in preferred embodiments, conveniently employs an MIER
15 polypeptide directly from the recombinant host in which it is produced. This is achieved most
preferably by simply expressing the selected polypeptide within the recombinant host, typically a
eukaryotic host, followed by preparing a crude homogenate which includes the enzyme. A
portion of the crude homogenate is then admixed with an a~lol~;ate effector of the polypeptide
along with the candidate substance to be tested. By comparing the binding of the selected
77
CA 02212991 1997-10-10
effector to the polypeptide in the presence or absence of the candidate substance, one may obtain
information regarding the physiological properties of the candidate substance.
There are believed to be a wide variety of embodiments which may be employed to determine
the effect of the candidate substance on the polypeptides of the invention, and the invention is
5 not intended to be limited to any one such method. However, it is generally desirable
to employ a system wherein one may measure the ability of the polypeptide to bind to and or be
modified by the effector employed in the presence of a particular substance.
The detection of an interaction between an agent and a polypeptide may be accomplished
through techniques well-known in the art. These techniques include but are not limited to
10 centrifugation, chromatography, electrophoresis and spectroscopy. The use of isotopically
labeled reagents in conjunction with these techniques or alone is also contemplated. Commonly
used radioactive isotopes include 3H, 14C, 22Na, 32p, 35S, 45Ca, 60Co, l25I, and l3lI. Commonly used
stable isotopes include 2H, 13C, 15N, and 130.
For example, if an agent binds to the polypeptide of the present invention, the binding may be
15 detected by using radiolabeled agent or radiolabeled polypeptide. Briefly, if radiolabeled agent or
radiolabeled polypeptide is utilized, the agent-polypeptide complex may be detected by liquid
scintillation or by exposure to x-ray film.
CA 02212991 1997-10-10
When an agent modifies the polypeptide, the modified polypeptide may be detected by
differences in mobility between the modified polypeptide and the unmodified polypeptide
through the use of chromatography, electrophoresis or centrifugation. When the technique
utilized is centrifugation, the differences in mobility is known as the sedimentation coefficient.
5 The modification may also be detected by differences between the spectroscopic properties of the
modified and unmodified polypeptide. As a specific example, if an agent covalently modifies a
polypeptide, the difference in retention times between modified and unmodified polypeptide on a
high pl'eS~;Ul'e liquid chromatography (HPLC) column may easily be detected. Alternatively, the
spectroscopic differences between modified and unmodified polypeptide in the nuclear magnetic
10 resonance (NMR) spectra may be detected. Or, one may focus on the agent and detect the
differences in the spectroscopic properties or the difference in mobility between the free agent
and the agent after modification of the polypeptide.
When a secondary polypeptide is provided, the agent-polypeptide-secondary polypeptide
complex or the polypeptide-secondary polypeptide complex may be detected by differences in
15 mobility or differences in spectroscopic properties as described above. The interaction of an
agent and a polypeptide may also be detected by providing a reporter gene. Well-known reporter
genes include ~-Gal, chloramphenicol (Cml) transferase (CAT) and luciferase. The reporter gene
is expressed by the host and the enzymatic reaction of the reporter gene product may be detected.
In one example, a lni2~1Ult~ co~ g the polypeptide, effector and candidate substance is
20 allowed to incubate. The unbound effector is separable from any effector/polypeptide complex so
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CA 02212991 1997-10-10
formed. One then simply measures the amount of each (e.g., versus a control to which no
candidate substance has been added). This measurement may be made at various time points
where velocity data is desired. From this, one determines the ability of the candidate substance to
alter or modify the function of the polypeptide.
5 Numerous techniques are known for separating the effector from effector/polypeptide complex,
and all such methods are intended to fall within the scope of the invention. Use of thin layer
chromatographic methods (TLC), HPLC, spectrophotometric, gas chromatographic/mass
spectrophotometric or NMR analyses. It is contemplated that any such technique may be
employed so long as it is capable of differentiating between the effector and complex, and may
10 be used to determine enzymatic function such as by identifying or quantifying the substrate and
product.
Screening Assays for MIER Polypeptides
The present invention provides a process of screening a biological sample for the presence of an
MIER polypeptide. A biological sample to be screened may be a biological fluid such as
15 extracellular or intracellular fluid, a cell, a tissue extract, a tissue homogenate or a histological
section. A biological sample may also be an isolated cell (e.g., in culture) or a collection of cells
such as in a tissue sample or histology sample. A tissue sample may be suspended in a liquid
medium or fixed onto a solid support such as a microscope slide.
CA 02212991 1997-10-10
In accordance with a screening assay process, a biological sample is contacted with an antibody
specific for a MIER polypeptide whose presence is being assayed. Typically, one mixes the
antibody and the MIER polypeptide, and either the antibody or the sample with the MIER
polypeptide may be affixed to a solid support (e.g., a column or a microtiter plate). Optimal
5 conditions for the reaction may be accomplished by adjusting temperature, pH, ionic
concentration, etc.
Ionic composition and concentration may range from that of distilled water to a 2 molar solution
of NaCl. Preferably, osmolality is from about 100 mosmols/l to about 400 mosmols/l, and more
preferably, from about 200 mosmols/l to about 300 mosmols/l. Temperature preferably is from
about 4~C. to about 100~C, more preferably from about 15~C to about 50~C, and even more
preferably from about 25~C to about 40~C. pH is preferably from about a value of 4.0 to a value
of about 9.0, more preferably from about a value of 6.5 to a value of about 8.5, and even more
preferably, from about a value of 7.0 to a value of about 7.5. The only limit on biological
reaction conditions is that the conditions selected allow for antibody-polypeptide conjugate
15 formation and that the conditions do not adversely affect either the antibody or the MIER
polypeptide.
Incubation time varies with the biological conditions used, the concentration of antibody and
polypeptide and the nature of the sample (e.g., fluid or tissue sample). Means for deterrnining
exposure time are well-known to one of ordinary skill in the art. Typically, where the sample is
20 fluid and the concentration of polypeptide in that sample is about 10-l~ M, exposure time is from
81
CA 02212991 1997-10-10
about 10 min to about 200 min.
MIER polypeptide in the sample is determined by detecting the formation and presence of
antibody-MIER polypeptide conjugates. Means for detecting such antibody-antigen (e.g.,
polypeptide polypeptide) conjugates or complexes are well-known in the art and include such
5 procedures as centrifugation, affinity chromatography and the like, binding of a secondary
antibody to the antibody-candidate polypeptide complex. Detection may be accomplished by
measuring an indicator affixed to the antibody. Exemplary and well-known such indicators
include radioactive labels (e.g., 32p, ]251, 14C), a second antibody or an enzyme such as horse
radish peroxidase. Methods for ~fflxing indicators to antibodies are well-known in the art.
10 Commercial kits are available.
Screening Assay for MIER Antibody
The present invention provides a process of screening a biological sample for the presence of
antibodies immunoreactive with a MIER polypeptide (i.e., MIER antibody). In accordance with
such a process, a biological sample is exposed to an MIER polypeptide under biological
15 conditions and for a period of time sufficient for antibody-polypeptide conjugate formation and
the formed conjugates are detected.
Screening Assay for a Polynucleotide Encoding A MIER Polypeptide
CA 02212991 1997-10-10
A DNA molecule and, particularly a probe molecule, may be used for hybridizing as
oligonucleotide probes to a DNA source suspected of possessing an MIER polypeptide encoding
polynucleotide or gene. The probing is usually accomplished by hybridizing the oligonucleotide
to a DNA source suspected of po~se~.~ing such a polypeptide gene. In some cases, the probes
5 constitute only a single probe, and in others, the probes constitute a collection of probes based on
a certain amino acid sequence or sequences of the MIER polypeptide and account in their
diversity for the redlm(l~ncy inherent in the genetic code.
A suitable source of DNA for probing in this manner is capable of ~lessillg MIER
polypeptides and may be a genomic library of a cell line of interest. Alternatively, a soer-lce of
10 DNA may include total DNA from the cell line of interest. Once the hybridization process of the
invention has identified a candidate DNA segment, one confirms that a positive clone has been
obtained by fer-lther hybridization, restriction enzyme mapping, sequencing and/or expression
and testing.
Alternatively, such DNA molecules may be used in a number of techniques including their use
15 as: (1) diagnostic tools to detect normal and abnormal DNA sequences in DNA derived from
patient's cells; (2) means for detecting and isolating other members of the MER family and
related polypeptides from a DNA library potentially co~ g such sequences; (3) primers for
hybridizing to related sequences for the per- lpose of amplifying those sequences; and (4) primers
for altering the native MIERDNA sequences; as well as other techniques which rely on the
~0 similarity of the DNA sequences to those of the MIER DNA segments herein disclosed.
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CA 02212991 1997-10-10
As set forth above, in certain aspects, DNA sequence information provided by the invention
allows for the preparation of relatively short DNA (or RNA) sequences (e.g., probes) that
specifically hybridize to encoding sequences of the selected MIER gene. In these aspects, nucleic
acid probes of an ~prupliate length are prepared based on a consideration of the selected MIER
encoding sequence (e.g., a nucleic acid sequence such as shown in FIG. . The ability of such
nucleic acid probes to specifically hybridize to MIER encoding sequences lend them particular
utility in a variety of embodiments.
Most importantly, the probes are useful in a variety of assays for detecting the presence of
complementary sequences in a given sample. These probes are useful in the preparation of
10 mutant species primers and primers for preparing other genetic constructions.
To provide certain of the advantages in accordance with the invention, a preferred nucleic acid
sequence employed for hybridization studies or assays includes probe sequences that are
complementary to at least an about 14 to about 40 or so long nucleotide stretch of the MIER
encoding sequence, such as shown in FIG. A size of at least 14 nucleotides in length helps to
15 ensMIERe that the fragment is of sufficient length to form a duplex molecule that is both stable
and selective. Molecules having complementary sequences over stretches greater than 14 bases in
length are generally preferred, though, to increase stability and selectivity of the hybrid, and
thereby improve the quality and degree of specific hybrid molecules obtained. One will generally
prefer to design nucleic acid molecules having gene-complementary stretches of about 14 to
20 about 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by,
84
CA 02212991 1997-10-10
for example, directly synthesizing the fragment by chemical means, by application of nucleic
acid reproduction technology, such as the PCR.TM. technology of U.S. Pat. No. 4,603,102" or
by introducing selected sequences into recombinant vectors for recombinant production.
Accordingly, a nucleotide sequence of the present invention may be used for its ability to
5 selectively form duplex molecules with complementary stretches of the gene. Depending on the
application envisioned, one employs varying conditions of hybridization to achieve varying
degrees of selectivity of the probe toward the target sequence. For applications requiring a high
degree of selectivity, one typically employs relatively stringent conditions to form the hybrids.
For example, one selects relatively low salt and/or high temperature conditions, such as provided
by about 0.02M to about 0.1 SM NaCl at temperatures of about 50~C to about 70~C. Such
conditions are particularly selective, and tolerate little, if any, mi~m~tch between the probe and
the template or target strand.
Of course, for some applications, for example, where one desires to prepare mutants employing a
mutant primer strand hybridized to an underlying template or where one seeks to isolate MIER
15 coding sequences from related species, functional equivalents, or the like, less stringent
hybridization conditions are typically needed to allow formation of the heteroduplex. Under such
circumstances, one employs conditions such as from about 0.15M to about O.9M salt, at
temperatures ranging from about 20~C to about 55~C. Cross-hybridizing species may thereby
be readily identified as positively hybridizing signals with respect to control hybridizations. In
20 any case, it is generally appreciated that conditions may be rendered more stringent by the
CA 02212991 1997-10-10
addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in
the same manner as increased temperature. Thus, hybridization conditions may be readily
manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it is advantageous to employ a nucleic acid sequence of the present
5 invention in combination with an appropriate means, such as a label, for det~rmining
hybridization. A wide variety of appropl;ate indicator means are known in the art, including
radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a
detectable signal. In preferred embodiments, one likely employs an enzyme tag such a urease,
alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable
10 reagents. In the case of enzyme tags, calorimetric indicator substrates are known which may be
employed to provide a means visible to the human eye or spectrophotometrically, to identify
specific hybridization with complement:~ry nucleic acid-cont~ining samples.
In general, it is envisioned that the hybridization probes described herein are useful both as
reagents in solution hybridization as well as in embodiments employing a solid phase. In
15 embodiments involving a solid phase, the sample co~ g test DNA (or RNA) is adsorbed or
otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then
subjected to specific hybridization with selected probes under desired conditions. The selected
conditions depend inter alia on the particular circumstances based on the particular criteria
required (depending, for example, on the G~C content, type of target nucleic acid, source of
20 nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as
86
CA 02212991 1997-10-10
to remove nonspecifically bound probe molecules, specific hybridization is detected, or even
quantified, by means of the label.
Assay Kits
In another aspect, the present invention contemplates a diagnostic assay kit for detecting the
5 presence of MIER polypeptide in a biological sample, where the kit comprises a first container
cont~ining a first antibody capable of immunoreacting with MIER polypeptide, with the first
antibody present in an amount sufficient to perform at least one assay. An assay kit of the
invention further optionally includes a second container cont~ining a second antibody that
immunoreacts with the first antibody. The antibodies used in the assay kits of the present
10 invention may be monoclonal or polyclonal antibodies. For convenience, one may also provide
the first antibody affixed to a solid support. Additionally, the first and second antibodies may be
combined with an indicator, (e.g., a radioactive label or an enzyme).
The present invention also contemplates a diagnostic kit for screening agents for their ability to
interact with an MIER. Such a kit will contain an MIER of the present invention. The kit may
15 further contain reagents for detecting an interaction between an agent and a polypeptide of the
present
invention. The provided reagent may be radiolabeled. The kit may also contain a known
radiolabeled agent that binds or interacts with a polypeptide of the present invention.
CA 02212991 1997-10-10
The present invention provides a diagnostic assay kit for detecting the presence, in a biological
sample, of a polynucleotide that encodes an MIER polypeptide, the kits comprising a first
container that contains a second polynucleotide identical or complementary to a segment of at
least about 14 contiguous nucleotide bases of a polynucleotide of FIG.
5 In another embodiment, the present invention contemplates a diagnostic assay kit for detecting
the presence, in a biological sample, of an antibody immunoreactive with an MIER polypeptide,
the kits comprising a first container cont~ining an MIER polypeptide that immunoreacts with the
antibody, with the polypeptide present in an amount sufficient to perform at least one assay. The
reagents of the kit may be provided as a liquid solution, attached to a solid support or as a dried
10 powder. When the reagent is provided in a liquid solution, the liquid solution is an aqueous
solution. When the reagent provided is attached to a solid support, the solid support may be
chromatograph media or a microscope slide. When the reagent provided is a dry powder, the
powder may be reconstituted by the addition of a suitable solvent. The solvent may also
be included in the kit.
15 Process of Modifying the Function of a Nuclear Polypeptide using MIER
In another aspect, the present invention provides a process of altering the function of a nuclear
polypeptide. In accordance with that process, a nuclear polypeptide is exposed to an MIER of the
present invention. A preferred nuclear polypeptide used in such a process is the same as set forth
above and includes nuclear polypeptides for thyroid hormone, vitamin D, retinoic acid and the
88
CA 02212991 1997-10-10
like. Preferred MIERs and their corresponding DNA sequences are shown in FIG.
The present invention provides DNA segments, purified polypeptides, methods for obtaining
antibodies, methods of cloning and using recombinant host cells necessary to obtain and use
MIERs. Accordingly, the present invention concerns generally compositions and methods for the
5 preparation and use of MIERs.
MIER Genes and Isoforms in Other Organisms
Er-1 may be considered as a member of a subfamily of early response polypeptides that may
include Mtal. It is probable that Er-1 isoforms are also encoded by multiple genes. Since nuclear
polypeptides usually have a high homology, the sequences of MIER may be used as probes to
10 screen cDNA libraries. Considering the fact that different isoforms of nuclear polypeptides may
have
different tissue distribution patterns and may be expressed to different extents in different tissues,
the MIER may used as a probe to screen genomic libraries for genes encoding MIER isoforms.
The present invention also provides cDNA libraries which are useful for screening of additional
15 MIER isoforms. Using the nucleotide sequences of the present invention, it is possible to
determine structural and genetic information (including restriction enzyrne analysis and DNA
sequencing) concerning these positive clones. Such information will provide important
information concerning the role of these isoforms in vivo and in vitro. MIER sequence
89
CA 02212991 1997-10-10
information may be used to analyze MIER cDNAs and MIER-like gene sequences in other
org~ni~m~. Using PCR.TM. techniques, restriction enzyme analysis, and DNA sequencing, the
structure of these MIER-like isoform genes may be determined with relative facility.
The following examples illustrate preferred embodiments of the invention. Certain aspects of the
5 following examples are described in terms of techniques and procedures found or contemplated
by the present inventors to work well in the practice of the invention. These examples are
exemplified through the use of standard laboratory practices of the inventor.
It should be appreciated by those of skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to function well in the practice of
10 the invention, and thus may be considered to constitute pler~lled modes for its practice.
However, those of skill in the art should, in light of the present disclosure, appreciate that many
changes may be made in the specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the invention.
EXAMPLES
15 As an example of one embodiment of a member of the MIER family, we have utilized the
polymerase chain reaction (PCR)-based differential display methodology (Liang and Pardee,
Science 257, 967-969, 1992) to identify a novel transcript whose expression levels increased in
Xenopus embryo explants during mesoderm induction by fibroblast growth factor (FGF). The
CA 02212991 1997-10-10
PCR product was used to clone a 2.3-kb cDNA representing this transcript, which we have
named erl (_arly response 1). The erl cDNA contained a single open reading frame (ORF)
predicted to encode a protein of 493 amino acid residues. A database homology search
revealed that the predicted ERl amino acid sequence contains three regions of similarity to the
S rat and human proteins encoded by the metastasis-associated gene, mtal, and two regions of
similarity to the C. elegans similar-to-mtal sequence. The FGF-induced increase in erl
steady-state levels was not dependent on de novo protein synthesis, demonstrating that erl is an
immediate-early gene. Northern blot analysis revealed a single 2.8-kb mRNA that was observed
predominantly during the initial cleavage and blastula stages of Xenoplls development, with little
10 or no detectable mRNA during subsequent development. Quan~ilalive PCR analysis of early
developmental stages showed that erl peaked during late blastula. Computer-assisted analysis of
the predicted ERl amino acid sequence revealed two putative nuclear localization signals, four
highly acidic regions clustered at the N-terminus and a proline-rich region located near the
C-tçnninll~. Subcellular localization by immunocytochemistry revealed that the ERl protein was
15 targeted exclusively to the nucleus. Transactivation assays, using various regions of ERl fused
to the DNA binding domain of GAL4, demonstrated that the N-tçnnin:~l acidic region is a potent
transactivator. These data suggest that ERl may function as a transcription factor.
EXAl~IPLE 1
Embryos and mesoderm induction
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CA 02212991 1997-10-10
Xenopus laevis were purchased from Nasco. Embryos were obtained and cultured as in (14). The
recombinant Xenopus bFGF (XbFGF) used for induction was prepared as in (15). Animal pole
explants (animal caps) were induced to form mesoderm as described (9), and animal caps were
treated for 30 min prior to RNA extraction. For inhibition of protein synthesis during induction,
5 animal caps were pre-treated for 30 min with Sug/ml cycloheximide (Sigma), cultured with or
without FGF for an additional 30 min, then processed for PCR analysis, as described below.
Protein synthesis was measured in parallel samples by including 2uCi/ul of 35S-methionine in the
culture medium and 35S-incorporation into TCA precipitable material was determined according
to Clemens (16).
EXAMPLE 2
Differential display
RNA was extracted from induced or uninduced animal caps using the NETS protocol (17).
Reverse transcription (RT) and polymerase chain reaction (PCR) were performed as in (13) with
the following primers: 5'-T"AC-3' and 5'-CTGATCCATG-3'. PCR products were separated on
15 a 6% polyacrylamide/6M urea gel; the gel was dried and the products visualized by
autoradiography. Differentially expressed bands were excised and the PCR products eluted from
the gel in 1 OOul of H20.
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CA 02212991 1997-10-10
E~AMPLE 3
cDNA cloning and sequencing of erl from Xenopus embryos
Eluted PCR products were cloned into the pCRn vector using the TA cloning kit (Invitrogen).
The sequence for both strands of the initial erl PCR product and all subsequent cDNA inserts
S was determined as in (14). A 2.3-kb erl cDNA was isolated from a stage 8 Xenopus (IZAPII)
cDNA library (14), using primers designed according to the erl sequence
(5'-TCCGTTACACCAGGATGTAG-3'; 5'-GGCTGAAATTCCAGTT GGTA-3';
5'-GCATCAGCTGCAGATCAAGG-3';5'-GTTTAAGAAAGGGC-AGTTCG-3') and the IZAP
vector sequence (5'-GCTCGAAATTAACCCTCACTAAAG-3';5'-GGTACCTAATA
10 CGACTCACTATAGGG-3'). The cDNA was cloned into pCRll and the sequence determined
and verified by sequencing several clones on both strands.
EXAMPLE 4
Quantitative PCR and Northern analysis
Quantitative PCR analysis was performed as described in (18) with the following modifications:
RNA was prepared as in (19); 1/8th of the RT sample was added to a 50ul PCl~ reaction and the
annealing temperature was 56~C. Histone H4 was used as a control with forward (F) and reverse
(R) primers as described (18) and the primer sequences for erl were as above. The PCR
93
CA 02212991 1997-10-10
products were analyzed in the linear range for amplification, determined empirically (18) to be
19 cycles for histone H4 and 24 cycles for erl. Quantitation by densitometry was performed as
described in (19) with norm~ tion to histone H4. Northern analysis was carried out as
described in (20), using the 2.3-kb erl or histone H4 cDNA as a probe.
EXAMPLE 5
Immunocytochemistry and protein analysis
Anti-Xenopus ERl antiserum was prepared by immunizing rabbits as (9) with a C-terminal
synthetic peptide (CIKRQRMDSPGKEST) of the predicted ERl protein sequence. Coupled in
vitro transcription-translation, immunoprecipitation and SDS-PAGE were performed as in (9).
10 For immunocytochemistry, NIH 3T3 cells were transfected with either pcDNA3 (Invitrogen) or
erl-pcDNA3. After 48h, the cells were processed for immunocytochemistry as in (19), using a
1:50 dilution of the anti-ERl antiserum.
EXAMPLE 6
Plasmid construction and transient transactivation assays
15 NIH 3T3 cells (ATCC) were m~int~ined in Dulbecco's modified Eagle's medium plus 10% calf
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CA 02212991 1997-10-10
serum and transfected with Lipofectamine according to the m~nllf~cturer's directions (Life
Technologies, Inc.). The ~l es~ion vectors used in this assay were engineered to contain
various portions of ERl fused to the GAL4 DNA binding domain of the pM plasmid (Clontech)
and are named according to the amino acids of ER1 that each encodes. Specific primers
incorporating 5' and 3' BglII sites (ER 1-493, ER 176-493) or a 5'EcoRIand a 3' BamHl site
(ER 1-175, ER 1-25) were used to amplify PCR fragments encoding the ~prop~;ate amino acids.
The digested PCR fragments were inserted into the complementary sites of the pM plasmid and
all plasmids were sequenced to verify the junctions and the erl sequence and to ensure the proper
reading frame. ER 1-98 and ER1-57 were generated by digesting the ER 1-175 construct with
PstI or PvuII, respectively, and re-ligating the cut vector.
0.5ug of a CAT reporter plasmid (pG5CAT, Clontech) was cotransfected into 3X105 cells with
l .Oug of either the pM vector alone, or one of the pM-erl fusion constructs. After 48h, cell
extracts were prepared and assayed for CAT enzyme using a CAT Elisa kit (Boehringer
Mannheim) according to the manufacturer's directions.
Results and Conclusions
In our efforts to elucidate the molecular mech~nism.s of FGF-induced mesoderm differentiation
inXenopus, we employed the PCR-based differential display method (13) to identify and
characterize genes that are expressed early during the cellular response to FGF. RNA was
isolated and reverse-transcribed from five individual sets of 30 min FGF-treated or control
CA 02212991 1997-10-10
animal pole explants (animal caps) from Xenopus blastulae. PCR products from the five sets
were separated on a 6% polyacrylamide/urea gel. Only those bands that were differentially
expressed in all five sets were chosen for further analysis. A total of eleven differentially
expressed bands were identified and one of these was eluted from the gel, cloned and sequenced.
5 A search of the database for similarity to known sequences revealed that this cDNA represented a
novel Xenopus gene, which we have named erl (_arly response 1).
The sequence of the erl PCR product was used to obtain a 2.3-kb cDNA from aXenopus blastula
library (14). This cDNA consisted of a single 1497-bp open reading frame (ORF), bracketed by
a 214-bp 5'-untr~n~l~ted region which contained several stop codons in all three frames and a
626-bp 3'-untr~n~ e~1 region (Fig. 1). The ATG initiation codon is predicted to be at
nucleotides 233-235, as this site is positioned within a Kozak consensus sequence for the start of
translation (21), with a purine in the -3 position and a G in the +4 position. The ORF is predicted
to encode a protein of 493 amino acids, beginning at nucleotide 233 and ending with an in-frame
TAA stop codon at position 1712 (Fig. 1).
15 Computer-assisted analysis of the deduced amino acid sequence using MOTIFS and PSORT
software programs predicts that ERl does not contain an N-terminal signal sequence for transfer
into the endoplasmic reticulum or a hydrophobic domain characteristic of transmembrane
proteins. However, ERl does contain two potential nuclear localization signals (NLS): RRPR
and KKSERYDFFAQQTRFGKKK (Fig. l); the latter conforms to the consensus sequence for a
20 bipartite NLS (22). ERl also contains a proline-rich sequence near the C-terminus which
96
CA 02212991 1997-10-10
corresponds to the PXXP motif found in all high affinity SH3-domain binding ligands (23). The
N-terminus of ER1 includes several highly acidic stretches (Fig. 1), characteristic of the acidic
activation domains of many transcription factors (24).
A database homology search using the National Center for Biotechnology Information BLAST
S Network Service revealed that ER1 contains three regions of similarity to the product of the rat
metastasis-associated gene, mtal (25) (Fig. 2), a gene that was isolated by differential cDNA
library screening and whose expression was associated with a metastatic phenotype. mtal
encodes a 703 amino acid, 79kDa polypeptide of unknown function that contains a putative SH3
binding domain near the C-terrninus. ER1 also displays similarity to the human MTA1
(accession no. U35113) and to the C. elegans MTA1-like sequence (accession no. U41264) (Fig
2). Within the regions of similarity, the percent amino acid similarity ranged from 46% to 64%,
however, the overall percent similarity was only 13.0%, 14.0% and 15.6% to the rat, human and
C. elegans sequences, respectively. To investigate whether erl represents the Xenopus homolog
of mtal or simply a related protein, we screened by RT-PCR a human breast carcinoma cell line,
MDA-468 (26), for erl-related sequences. We obtained a partial human cDNA clone spanning
sequence inside and outside the regions of similarity shown in Fig. 2; this sequence displays 91%
overall similarity to erl at the amino acid level (data not shown). The existence of a human gene
product that is distinct from human mtal and that shows a high degree of similarity to erl
suggests that erl and mtal are not homologs, but possibly related members of a family of
20 proteins or simply proteins co~ g some of the same functional domains.
97
CA 02212991 1997-10-10
Verification that the steady-state levels of erl were increased in response to FGF during
mesoderm induction in vitro was performed by qu~~ ive PCR after a 30 min treatment with
FGF, using histone H4 as an intern~l standard (Fig. 3A). In several independent experiments,
densitometric analysis revealed that erl levels ranged from three- to four-fold higher in
5 FGF-treated samples, after norm~liz~tion to histone H4. These data confirm that erl levels were
increased by treatment with FGF and demonstrate that the increase in erl occurs early during the
cellular response to FGF.
The possibility that erl is an immediate-early gene was investigated further. By definition,
transcription of immediate-early genes is a rapid response and is not dependent on de novo
10 protein synthesis. The FGF-induced increase in erl levels was measured in the presence or
absence of 5ug/ml cycloheximide. Cycloheximide inhibited 90% of 35S-methionine
incorporation into TCA-precipitable material (data not shown) but did not prevent the
FGF-induced increase in erl levels (Fig. 3B), demonstrating that erl is an immediate-early
gene.
15 Northern analysis of the temporal pattern of erl ~lession during embryonic development
revealed a single erl mRNA (Fig. 4A). The estim~te~l 2.8-kb size of the message was slightly
larger than that of the cDNA clone, but this is probably due to the presence of a poly-A tail. erl
was detectable during initial cleavage stages, prior to the start of zygotic transcription which
occurs at mid-blastula transition (27), indicating that erl is a maternally derived mRNA.
20 Densitometric analysis revealed that steady-state levels of erl were relatively constant during
98
CA 02212991 1997-10-10
early cleavage stages (stages 2, 6, 7; Fig. 4A, lanes 1-3), increased slightly at blastula stage (Fig.
4A, lane 4), then decreased sixfold during gastrula, neurula and tailbud stages (stages 12, 17, 22;
Fig. 4A, lane 5-7) and remained below detectable levels during subsequent development (stages
30 and 41; Fig. 4A, lanes 8 and 9). Mesoderm induction, a process in which FGF is known to
5 play a pivotal role, takes place during blastula stages. Therefore, we examined erl levels at lh
time intervals during blastula and gastrula stages, using a quantitative PCR assay (18). erl
expression levels were shown to increase twofold from early blastula (stages 7-8; Fig. 4B, lanes
1-2) to late blastula stages (stages 8-9; Fig. 4B, lanes 3-4), followed by a fivefold decrease at
gastrulation (stage 10; Fig. 4B, lane 5).
10 Our sequence analysis revealed two putative nuclear localization signals, suggesting that ERl is
targeted to the nucleus. We investigated the subcellular localization of the ERl protein using a
polyclonal anti-ERl antibody to stain transfected NIH 3T3 cells ~ples~ g ERl. This antibody,
directed against a synthetic C-terminal peptide, recognizes full-length ERl protein synthesized in
vitro (Fig. 5A, lane 3) and specifically stains the nuclei of cells expressing ERl (Fig. 5B). Cells
15 transfected with the pcDNA3 vector alone (Fig. 5B) as well as pcDNA3-erl transfected cells
stained with pre-immune serum (not shown) gave similar patterns and showed no specific
nuclear st~ining
The fact that ERl is targeted to the nucleus and that its N-terminlls contains stretches of acidic
residues characteristic of acidic activation domains (25), suggests that ERl may function as a
20 transcription factor. We investigated this possibility by testing the transactivation potential of
99
CA 02212991 1997-10-10
various regions of the ERl protein. Constructs, cont~inin~ different portions of erl fused to the
GAL4 DNA binding domain, were used along with a CAT reporter plasmid in transient
transfections. Assays of CAT enzyme levels revealed that, although full-length ERl did not
activate transcription, the N-t~rmin~l region (ER 1175), con~ai~ g all four acidic stretches (Fig.
5 1), stimulated transcription 10-fold (Fig. 6). The complementary C-terminal portion, ER
176-493, on the hand, had no transactivational activity. It is unclear why full-length ERl was
unable to stimulate transcription, but one possible explanation is that fusion of ERl to GAL4
may alter the tertiary structure of the ERl protein, affecting its activity. A similar observation
was made with the ETS transcriptlon factor ER8 1 which, when fused to the GAL4 DNA binding
10 domain, lost its ability to activate transcription (28).
Interestingly, deletion of the N-tçnnin~l region to produce a construct co~ irlg only the first
three acidic stretches (ER 1-98), resulted in a much more potent transactivator that stimulated
transcription 80-fold (Fig. 6). This suggests that a negatively acting domain is located between
amino acids 99-176. Further truncation of the N-terminlls to generate ER 1-57 and ER 1-25
15 completely abolished transactivation. These results demonstrate that the ERl protein contains
regions with transcription transactivating activity and that ERl has the potential to function as a
transcription factor.
An important aspect of the present invention is the use of Er- 1 as a means of detecting and
diagnosing early response polypeptide mutations. The present work suggests that Er-l plays a
20 regulatory role in FGF interaction with mesodermal cells, and as such, Er- 1 may play a critical
100
CA 02212991 1997-10-10
role in regulating growth and differentiation signal transduction. Therefore, by determinin~ the
function and ~plession of Er-l in subjects, it is possible to detect abnormal early response
hormone function in these patients.
By studying Er-l mRNA and Er-l levels in cells from normal individuals and patients with
5 cancer of AIDS, one may determine the effect(s) of Er-l on these cells. DNA isolated from
blood cells or fibroblasts of these patients may also be screened for possible mutations in the Er-
1 gene. The present invention has determined the DNA sequence of the human and rat Er-l
genes. Exons of Er- 1 have been amplified by PCR.TM. techniques and analyzed by nucleotide
sequencing, restriction fragment length polymorphism (RFLP) and single stranded
10 conformational polymorphisms.
The inventors have detected Er- 1 in Xenopus embryos by immunocytochemical staining, and
found that Er- 1 ex~lt;;ssion varies with tissue and stages of development. Thus, levels of Er- 1
may be related to developmental or cellular processes.
Although the make-up of the natural response elements for Er-1, in the control regions of various
15 genes is undoubtedly more complex than the synthetic er-l sequences used in this study, the
interaction of Er-l with FGF and EGF well as Er-l modulation of gene transactivation suggest a
mechanism in which a number of nuclear polypeptides of this subfamily, possibly including
some yet to be discovered, interact in a composite fashion to yield a net transcriptional activity in
the cell nucleus for a given response element. This net transcriptional activity is also dependent
101
CA 02212991 1997-10-10
upon the presence of polypeptide ligands and the particular structure of the response element.
Because numerous modifications and variations in the practice of the present invention are
expected to occur to those skilled in the art, only such limitations as appear in the appended
claims should be placed thereon.
5 All of the compositions and methods disclosed and claimed herein may be made and executed
without undue experimentation in light of the present disclosure. While the compositions and
methods of this invention have been described in terms of pl~r~ d embodiments, it will be
apparent to those of skill in the art that variations may be applied to the composition, methods
and in the steps or in the sequence of steps of the method described herein without departing
10 from the concept, spirit and scope of the invention. More specifically, it will be apparent that
certain agents which are both chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the appended claims.
15 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the a~t to which this invention belongs.
Although methods and materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, the preferred methods and materials are described
below. All publications, patent applications, patents, and other references mentioned herein are
102
CA 02212991 1997-10-10
incorporated by reference. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting. Other features and advantages of the invention
will be apparent from the detailed description, and from the claims.
EXAMPLE 7
5 Sequence Listing
The following is a copy of the genbank listing. The accession no. is AF0 15454.
LOCUS AF015454 2339 bp mRNA VRT 07-SEP-1997
DEFINITION Xenopus laevis ER1 mRNA, complete cds
ACCESSION AF015454
KEYWORDS
SOURCE African clawed frog.
ORGANISM Xenopus laevis
Eukaryotae; mitochondrial eukaryotes; Metazoa; Chordata;
Vertebrata; Amphibia; Batrachia; Anura; Mesobatrachia; Pipoidea;
Pipidae; Xenopodinae; Xenopus.
REFERENCE 1 (bases 1 to 2339)
103
CA 02212991 1997-10-10
FEATURES Location/Qualifiers
source 1..2339
/organism="Xenopus laevis"
/dev_stage="blastula stage embryo"
gene 1... 2339
/note="immediate early gene"
/gene="erl "
CDS 233..1714
/gene="erl"
/fi~ction="potential transcription factor"
/note="nuclear protein; some similarity to mtal"
/codon_start=l
/product="ERl "
/translation=
15 "MAEPSLRTASPGGSAASDDHEFEPSADMLVHEFDDEQTT,F,F,F,F,MLEGEVNFTSF,TF,T~T,F,
RESEMPIDELLRLYGYGSTVPLPGEEDEEDMDNDCNSGCSGEIKDEAIKDSSGQEDETQS
SNDDPTPSFTCRDVREVIRPRRCKYFDTN H l~ EDDEDYVPSEDWKKEIMVGSMFQA
EIPVGICKYRETEKVYENDDQLLWNPEYVMEERVIDFLNEASRRTCEERGLDAIPEGSHI
KDNEQALYEHVKCNFDTEEALRRLRFNVKAAREELSVWTEEECRNFEQGLKAYGKDFH
20 LIQANKVRTRSVGECVAFYYMWKKSERYDFFAQQTRFGKKKYNLHPGVTDYMDRLLD
ESESATSSRAPSPPPTTSNSNTSQSEKEDCTASNNTQNGVSVNGPCAITAYKDEAKQGVH
LNGPTISSSDPSSNETDTNGYNRENVTDDSRFSHTSGKTDTNPDDTNERPIKRQRMDSPG
104
CA 02212991 1997-10-10
KESTGSSEFSQEVFSHGEV"
BASE COUNT 806 a 383 c 513 g 637 t
ORIGIN
1 ttgcatcagc tgcag~tca~ ggttaaaata tatat;~tcag ~g~tac~c ~taatta~
561 atta~tgtc tc,~ca~,t ccttccatat gaaggcctct ctgtacctgt gcagcgtttt
121 tca~caga gcaaggaatt cat~c~tt~c, ~tat~ttt gttgtgtcat aagctacaga
181 gaaagttata gtga~cca~ ca~c~t~a atgacccgtc agtacggcaa acatggcgga
241 gccttcactc aggaccgcaa gcccaggtgg ctcggctgca tc~g~tgacc atgagtttga
301 gccatcagct gacatgcttg ttc~tg~:~tt tgatg~tg~ caaacgttgg ~g~g~gga
10361 gatgctggag gg~g~gtca acttcacttc ~g~tag~g caccttgaaa g~g~agtga
421 aatgccaatt gatg~tt~t tgcgactcta tggttatggc agtacagtgc cactaccagg
481 ~gaag~gat gaggaggata tgg~t~t~a ttgtaacagt ggctgcagtg g~g~t~a~
541 ggatg;~gct attaaggact cttcaggaca gg~g~tg~ acacagtctt c~atg~tga
601 tcctactcca tcttttacat gtag~gatgt acg~g~gt~ atccgtccac gtcggtgcaa
15661 gt~ttttgat aca~tc~tg a~atag~g;~ ggagtctgag ,~tg;~tgagg attatgtacc
721 ttcag~g~t tgga~ g a~ttatggt gggatccatg ttccaggctg aaattccagt
781 tggtatttgc a~tac,ag~g a~cag~g~ agtat~tg~ tg~tgatc, agctcctctg
841 gaatccagaa tatgtaatgg ~g;~ag;~gt aatagacttc tt~tg~gg catccagaag
901 gacttgtgaa g~g~g~gggc tagatgctat tcctgaagga tccc~c~t~ aggac~tga
20961 gcaggcccta tatg~:~catg taaaatgcaa ttttg~caca gaagaggcat tg~g;~g~ct
1021 a~g~ttt~t gtcaaagccg cc~g~ga~ga actttccgtt tggactgaag aagaatgtag
1081 ~attttgag caaggtctaa aagcttatgg c~gatttc cacttgattc aggctaacaa
105
CA 02212991 1997-10-10
1141 ggtaaggaca aggtctgttg gagaatgtgt ggcattctac tacatgtgga a~ tcaga
1201 acgttatgac ttcmgccc aacaaacacg atttggaaaa aagaagtata atctacatcc
1261 tggtgtaacg gattacatgg atcgtctttt ggatg~agc gaaagtgcta cctccagcag
1321 ggcgccatct cccccaccaa ctacctccaa cagcaatact agtcaatccg a~ggagga
1381 ctgtacagcc agt~aca~ca ctc,~ tgg agtttctgtg aatggcccat gtgcaataac
1441 tgcatacaa~ gatgaagcca aacaaggggt gc~ttt:~a~t ggacctacta taagtagcag
1501 tgatccctct tcg~atg~a ccg;~cacca~3 tgggtataat cgtg~ tg ttacggacga
1561 ttccagattt agtcat~c~ gtggaa~a~c tg~cac~at ccagatg~t~ caaacgaaag
1621 gcc~ta~ aggcaacgta tggacagccc tgggaaggaa agtacaggat catctgaatt
1681 ctctcaggaa gtgmtcac atggagaggt tt~a~cattt tgcag~tttg aggg~aaaca
1741 cattttgggg gg~tg~ag~ atttctgggg atcttggaac ttcagggttt ~tt~attat
1801 ttagcaagtt attmttgt attatttttc tatttgtccc atgcacat,tt gagccccaca
1861 gaagagtgaa atattttgtg tagttgaata gtg~atttt tgaagccctc tgg~a:~agt~
1921 agcagccttg ca,~:~tattca gcctatgcct gaatgcagtt tggctttacg tt~tcattcg
1981 ttacatg~g aaggatcttt ~t:~ga~a agaattgttc cag~tatgt ctgcagtgtt
2041 gttgcagtgg ~ t~tt~ ccctgaaagt tgttggtatg ~tttttttta ggtaggtgtt
2101 ~g~t~ , c~tg~ggt ttgtgtatgt ~tttattg~ catca~tg~t gtctttccta
2161 ttcttatctg ggctgaaaaa gat~l~attct gtatttttcc agatctcm gtagcctttg
2221 a~g~ttttt acattatcta tgttttg~tc gaactgcctt tctt~c~ gcttgtataa
2281 ttttcttaac ttgtacagtt gataaacttt tattatg~aa agg~ a ~a~a~a~
106
CA 02212991 1997-10-10
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: SEABRIGHT COORPORATION LTD
(B) STREET: MEMORIAL UNIVERSITY OF NEWFOUNDLAND
(C) CITY: ST. JOHN'S UNIVERSITY
(D) STATE: NEWFOUNDLAND
(E) COUNTRY: CANADA
(F) POSTAL CODE (ZIP): AlC 5S7
(G) TELEPHONE: -(709)-737-4527
(H) TELEFAX: -(709)-737-4029
(ii) TITLE OF lNVENTION: MAMMALIAN MESODERM INDUCTION EARLY
RESPONSE
(MIER) GENE FAMILY
(iii) NUMBER OF SEQUENCES: 8
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2339 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: XENOPUS LAEVIS
CA 02212991 1997-10-10
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
TTGCATCAGC TGCAGATCAA GGTTAAAATA TATATATCAG AAGAATACAC
AAATAATTAA 60
ATTAAATGTC TCAAACAACT CCTTCCATAT GAAGGCCTCT CTGTACCTGT
GCAGCGTTTT 120
TCAAAACAGA GCAAGGAATT CATACATTAC AAATATATTT GTTGTGTCAT
AAGCTACAGA 180
GAAAGTTATA GTGAAACCAA CAAAACATAA ATGACCCGTC AGTACGGCAA
ACATGGCGGA 240
GCCTTCACTC AGGACCGCAA GCCCAGGTGG CTCGGCTGCA TCAGATGACC
ATGAGTTTGA 300
GCCATCAGCT GACATGCTTG TTCATGAATT TGATGATGAA CAAACGTTGG
AAGAAGAGGA 360
GATGCTGGAG GGAGAAGTCA ACTTCACTTC AGAAATAGAG CACCTTGAAA
GAGAAAGTGA 420
AATGCCAATT GATGAATTAT TGCGACTCTA TGGTTATGGC AGTACAGTGC
CACTACCAGG 480
AGAAGAAGAT GAGGAGGATA TGGATAATGA TTGTAACAGT GGCTGCAGTG
GAGAAATAAA 540
GGATGAAGCT ATTAAGGACT CTTCAGGACA GGAAGATGAA ACACAGTCTT
CAAATGATGA 600
TCCTACTCCA TCTTTTACAT GTAGAGATGT ACGAGAAGTA ATCCGTCCAC
GTCGGTGCAA 660
GTATTTTGAT ACAAATCATG AAATAGAAGA GGAGTCTGAG GATGATGAGG
ATTATGTACC 720
TTCAGAAGAT TGGAAAAAGG AAATTATGGT GGGATCCATG TTCCAGGCTG
AAATTCCAGT 780
TGGTATTTGC AAATACAGAG AAACAGAGAA AGTATATGAA AATGATGATC
AGCTCCTCTG 840
CA 02212991 1997-10-10
GAATCCAGAA TATGTAATGG AAGAAAGAGT AATAGACTTC TTAAATGAGG
CATCCAGAAG 900
GACTTGTGAA GAGAGAGGGC TAGATGCTAT TCCTGAAGGA TCCCACATAA
AGGACAATGA 960
GCAGGCCCTA TATGAACATG TAAAATGCAA TTTTGACACA GAAGAGGCAT
TGAGAAGACT 1020
AAGATTTAAT GTCAAAGCCG CCAGAGAAGA ACTTTCCGTT TGGACTGAAG
AAGAATGTAG 1080
AAATTTTGAG CAAGGTCTAA AAGCTTATGG CAAAGATTTC CACTTGATTC
AGGCTAACAA 1 140
GGTAAGGACA AGGTCTGTTG GAGAATGTGT GGCATTCTAC TACATGTGGA
AAAAATCAGA 1200
ACGTTATGAC TTCTTTGCCC AACAAACACG ATTTGGAAAA AAGAAGTATA
ATCTACATCC 1260
TGGTGTAACG GATTACATGG ATCGTCTTTT GGATGAAAGC GAAAGTGCTA
CCTCCAGCAG 1 320
GGCGCCATCT CCCCCACCAA CTACCTCCAA CAGCAATACT AGTCAATCCG
AAAAGGAGGA 1380
CTGTACAGCC AGTAACAACA CTCAGAATGG AGTTTCTGTG AATGGCCCAT
GTGCAATAAC 1440
TGCATACAAA GATGAAGCCA AACAAGGGGT GCATTTAAAT GGACCTACTA
TAAGTAGCAG 1500
TGATCCCTCT TCGAATGAAA CCGACACCAA TGGGTATAAT CGTGAAAATG
TTACGGACGA 1 560
TTCCAGATTT AGTCATACAA GTGGAAAAAC TGACACAAAT CCAGATGATA
CAAACGAAAG 1620
GCCAATAAAA AGGCAACGTA TGGACAGCCC TGGGAAGGAA AGTACAGGAT
CATCTGAATT 1680
CTCTCAGGAA GTGTTTTCAC ATGGAGAGGT TTAAACATTT TGCAGATTTG
AGGGAAAACA 1740
CA 02212991 1997-10-10
CATTTTGGGG GGATGAAGAA ATTTCTGGGG ATCTTGGAAC TTCAGGGTTT
ATTAAATTAT 1800
TTAGCAAGTT A'l"l"l"l"l"l"l'GT ATTATTTTTC TATTTGTCCC ATGCACATTT
GAGCCCCACA 1860
GAAGAGTGAA ATATTTTGTG TAGTTGAATA GTGAAATTTT TGAAGCCCTC
TGGAAAAGTA 1920
AGCAGCCTTG CAGATATTCA GCCTATGCCT GAATGCAGTT TGGCTTTACG
TTATCATTCG 1980
TTACATGAAG AAGGATCTTT AAATAGAAAA AGAATTGTTC CAGAATATGT
CTGCAGTGTT 2040
GTTGCAGTGG AAAATATTAA CCCTGAAAGT TGTTGGTATG A'l"l"l"l''l"l"l"l'A
GGTAGGTGTT 2100
AAGAATAAAC CAAATGAGGT TTGTGTATGT AATTTATTGA CATCAATGAT
GTCTTTCCTA 2160
TTCTTATCTG GGCTGAAAAA GATACATTCT GTATTTTTCC AGATCTCTTT
GTAGCCTTTG 2220
AAAGA'l' l l l l' ACATTATCTA TGTTTTGATC GAACTGCCTT TCTTAACAAA
GCTTGTATAA 2280
TTTTCTTAAC TTGTACAGTT GATAAACTTT TATTATGAAA AGGAAAAAAA
AAAAAAAAA 2339
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 501 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: xenopus laevis
CA 02212991 1997-10-10
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CTGCTCTCAG ATGCAATGAC AACACTATCT CTATTCCAGG ATGACTTCAA
GTCAAATGTT 60
GATGTTGTTT AGTTGCTAAG TTATGTCTGA CTCTTTTGCA ACCCCATGGA
CTATAGCCCA 120
CCTCTGTCCA TAGGATTTCC CAGGCAAGAA TACTGGATGG TTTGCCATTT
CTCTAGGAAA 1 80
TCTTTCCAAC CCAGGGACTG AACCCACATC TTGTGCTTGG CAACCGATTC
TTTACCACTG 240
AGCCACTAGG GAAGCCCTTA AAGTCATATA AAGTAATGTT AATTTCAGAA
TGCTTTCATA 300
TCAAAGTTAA GAGCCCAGAT AAATTTTAAA TGGCTGTGAA TCCATTGCAG
CTATTCCCAC 360
CAAGAGTTGG AGTCTATTTT CAACACTCTC CCCTTACTCT GGGCTGATGG
ATCTATGACT 420
TTCTTTGGCC AACAGACTGT GCTACTTCAA TACTTACCTT CTTACCAGAC
ACTTCTATCT 480
TGTGAAGGAG CCTGAGAGCA G 501
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: xenopus laevis
CA 02212991 1997-10-10
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CTGATCCATG CCTCAAGTAA AATACAAAAT ATAGAAGATG CCCAGCAGTA
ACGTTCAATG 60
TAATGATTCA AGAGATTGTC AGAAAA~AAT ACATGTTAGA TATGGCTCTG
ATAAGGAATG 120
GGAGTCAAGT GTGATAACAG GAATGGCACA CACTTCTTAT AGTTAAGCAA
GCTCTTTGCC 1 80
ACTTTATATC AGCTCATTGC CCATGGATCA G 2 l l
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 190 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Xenopus laevis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CTGATCCATG CGTATAGCCT TGAATAATAA AGCTTTGCTC CCTCTAAATG
ACAAATACCA 60
CATCCACTAC TACCACCTAT GACTGCACTT GAACTTACAA GTAACTAAGG
GAACAAGAGG 120
GGGATAAGAA AACAGAAGTA CAGAACTATC GCAATGACTG CTTTGTGATC
TTATTTCCTA 1 80
CATGGATCAG l 90
(2) INFORMATION FOR SEQ ID NO: 5:
CA 02212991 1997-10-10
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 633 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: XENOPUS LAEVIS
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CTGATCCATG GTTTAAGTAT AAATAATTGT TCACTTATAT CTGTTTCAAT
CACCTTTCAT 60
TGTAGTTCCC AAAATCTCGC CTAAATCATA CATCTGCCCA ACCAACCTTC
TAACAGCAAT 120
GTTAGGGATG GATTCAAAAA GATCTTTGAG GAAATTGGGT GGCAGATACG
CGCTAACAAA 1 80
GATGAGTGAT AGAAACACAA TGGTGATTAC TCCCAATCAG TATAATTCAA
ATAGTATAAT 240
GGGTATAACA GTAATAGAGT ACATGACATG TTAGGCACTT ACTTTGCTGT
GCCAAAGTAT 300
TCCCATCACT TTGTCTCTCA GAGACACCAA CAGTAGAAGC TGTGGCCTAA
TCCCTATCTG 360
TGTACCCTGC TTAACCCAAA CTAATTGACA AACTCGAAAT CGATGGTGCT
AATTCACCAC 420
CCCCATCTAT TGAGAGACAT GCTCTCCAGT TATGTTAGCA ATAGGATAAA
TCCTTATTTT 480
~ l l l l l CCTA TCTCCCTCTG GACTCCCCAT GATCTCTATT TTCCCAATCG
TCGGTTTCTT 540
GCATCCTAAG TAATATCCTC TTCAGGATAC ACTCATGCCT GCTAGAAGGT
CA 02212991 1997-10-10
TAACAAATGA 600
ATTAGGCATG ATAACGATTA TTGCATGGAT CAG 633
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 449 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: XENOPUS LAEVIS
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CTGCTCTCAC AAAAATGCTA TAGAGACGTA TATGACATAA ATAATCTGTG
ATGAAACAAT 60
TTAGGTTTCA TTAGCTTTTA CAAAAATGGA AAAAGTATGA CCATGGTTGC
ACAGTTTGGC 120
AAACCATTTT TTCTATCATT CCTACAAAAT ACTGAGTGTT ACTGGACACT
GATATGATTA 1 80
TTAAAGATAT TTCTTTATAT AAATTGTATA TCAATAAATT ATAATATGCA
GAGTAGGTTG 240
CAGTTACCTA CTTACCTACT TACAGAAGCA ATTATCACTA AACTGCTGAC
ATGCCAGTTT 300
GGTTGTTCAG CATACTTCAG TACAAACAAG AAGCTTCTGG AGTTTCCAGT
ACACTGCATT 360
TTATACAAAT GTAACGTATA GGCTCATAAA CCTAAAGCAC ACTAGTTATT
TATATTTACT 420
ACATACATAA AGATACACAG CTGAGCAGA 449
CA 02212991 1997-10-10
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 211 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ I~D NO: 7:
CTGATCCATG CCCCAAGTAA AATACAAGAT ATAGAAGATG CCCAGCAGTA
ACGTTCAATG 60
TAATGATTCA AGAGATTGTC AGAAAAAAAT ACATATTAGA TATGGCTCTG
ATAAGGAATG 120
GGAGTCAAGT GTGATAACAG GAATGGCACA CACTTCTTAT AGTTAAGCAA
GCTCTTTGCC 1 80
ACTTTATATC AGCTCATTGC CCATGGATCA G 211
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 516 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
TGATCCATGA AAAGTGTTAG TGACCAACCT TCTGGAAATC TTCCATTCCC
GAAACCTGAT 60
CA 02212991 1997-10-10
GATACTCAGT ACTTTGACAA ATTATTGGTT GATGTTGATG AATCTACGCT
AAGTCCAGAA 120
GAACAGAAAG AAAGAAAAAT AATGAAATTA TTGTTAAAAA TAAAAGATGG
CACACCTCCA 1 80
ATGAGGAAGG TCTGCCTTCG ACAAATAACT GATAAAGCTC GTGAGTTTGG
AGCCGGTCCA 240
CTATTCAATC AGATCCTGCC TCTGCTGATG TCGCCAACAC TTGAAGATCA
AGAAAGACAC 300
TTGCTTGTTA AAGTTATTGA TAGAATTTTG TATAAATTGG ATGACTTGGT
CCGCCCATAT 360
GTACATAAGA TTCTTGTCGT TATTGAACCA CTTCTGATTG ATGAAGACTA
TTATGCCAGA 420
GTGGAAGGCA GAGAAATCAT ATCTAATTTA GCCAAGGCTG CTGGTTTAGC
TACAATGATT 480
TCAACTATGC GACCAGATAT TGATAACATG GATCAG 516
