Extracellular signal-regulated kinase, sequences, and methods of production and use

The present invention relates, in general, to an extracellular signal regulated kinase, ERK-5. In particular, the present invention relates to nucleic acid molecules coding for ERK-5; ERK-5 polypeptides; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antisense ERK-5 nucleic acid constructs; antibodies having binding affinity to an ERK-5 polypeptide; hybridomas containing the antibodies; nucleic acid probes for the detection of ERK-5 nucleic acid; a method of detecting ERK-5 nucleic acid or polypeptide in a sample; kits containing nucleic acid probes or antibodies; a method of detecting a compound capable of binding to ERK-5 or a fragment thereof; a method of detecting an agonist or antagonist of ERK-5 activity; a method of agonizing or antagonizing ERK-5 associated activity in a mammal; and a pharmaceutical composition comprising an ERK-5 agonist or antagonist.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates, in general, to an extracellular signal 
regulated kinase, ERK-5. In particular, the present invention relates to 
nucleic acid molecules coding for ERK-5; ERK-5 polypeptides; recombinant 
nucleic acid molecules; cells containing the recombinant nucleic acid 
molecules; antisense ERK-5 nucleic acid constructs; antibodies having 
binding affinity to an ERK-5 polypeptide; hybridomas containing the 
antibodies; nucleic acid probes for the detection of ERK-5 nucleic acid; a 
method of detecting ERK-5 nucleic acid or polypeptide in a sample; kits 
containing nucleic acid probes or antibodies; a method of detecting a 
compound capable of binding to ERK-5 or a fragment thereof; a method of 
detecting an agonist or antagonist of ERK-5 activity; a method of 
agonizing or antagonizing ERK-5 associated activity in a mammal; a method 
of treating diabetes mellitus, skeletal muscle diseases, Alzheimer's 
disease, or peripheral neuropathies in a mammal with an agonist or 
antagonist of ERK-5 activity; and a pharmaceutical composition comprising 
an ERK-5 agonist or antagonist. 
2. Background Information 
Phosphorylation of serine/threonine residues on ribosomal protein S6 
kinases (Ballou et al., J. Biol. Chem. 263:1188-1194 (1988)), phosphatase 
1 G binding protein (Dent et al., Nature 348:302-308 (1990)), and acetyl 
coA-carboxylase (Borthwick et al., Biochem J. 270:795-801 (1990)) occur in 
response to insulin and other extracellular cues. Ray and Sturgill, Proc. 
Natl. Acad. Sci. USA 84:1502-1506 (1987), Cicirelli et al., J. Biol. Chem. 
263:2009-2019 (1988), and Hoshi et al., J. Biol. Chem. 263:5396-5401 
(1988) have identified a micro tubule-associated protein 2 (MAP2)/myelin 
basic protein (MBP) kinase that in response to insulin contains phosphate 
on serine/threonine residues (Ray and Sturgill, Proc. Natl. Acad. Sci. USA 
85:3753-3757 (1988); Boulton et al., Cell 65:663-675 (1991)). Ribosomal 
protein S6 kinase has been identified as one potential target for this 
kinase (Sturgill et al., Nature 334:715-718 (1988); Gregory et al., J. 
Biol. Chem. 264:18397-18401 (1989); Ahn and Krebs, J. Biol. Chem. 
265:11495-11591 (1990)). Boulton et al., Biochemistry 30:278-286 (1991) 
and Boulton et al., Science 249:64-65 (1990) describe the purification and 
cloning of a MAP2/MBP kinase which they named extracellular 
signal-regulated kinase 1 (ERK-1). Using probes derived from ERK-1, two 
novel kinases were identified, ERK-2 and ERK-3 (Boulton and Cobb, Cell 
Regulation 2:357-371 (May 1991); Boulton et al., Cell 65:663-675 (May 17, 
1991)). A fourth ERK has been briefly described (Cobb et al., Cell 
Regulation 2:965-978 (December 1991) and WO 91/19008 published Dec. 12, 
1991). 
The present invention provides a novel ERK, ERK-5. ERK-5 shows 61% 
similarity (38% identity) to the human ERK1 peptide sequence, 64% 
similarity to the rat ERK1 and ERK2 (39% and 37% identity, respectively) 
and 55 % similarity to the rat ERK3 (30% identity). 
SUMMARY OF THE INVENTION 
The invention provides ERK-5. 
The invention also provides an isolated nucleic acid molecule coding for a 
polypeptide comprising an amino acid sequence corresponding to ERK-5, or 
at least 9 contiguous amino acids thereof. 
The invention further provides a substantially pure polypeptide comprising 
an amino acid sequence corresponding to ERK-5, or at least 9 contiguous 
amino acids thereof. 
The invention also provides a nucleic acid probe for the detection of the 
presence of ERK-5 in a sample. 
The invention further provides a method of detecting ERK-5 RNA in a sample. 
The invention also provides a kit for detecting the presence of ERK-5 RNA 
in a sample. 
The invention further provides a recombinant nucleic acid molecule 
comprising, 5' to 3', a promoter effective to initiate transcription in a 
host cell and the above-described isolated nucleic acid molecule. 
The invention also provides a recombinant nucleic acid molecule comprising 
a vector and the above-described isolated nucleic acid molecule. 
The invention further provides a recombinant nucleic acid molecule 
comprising a transcriptional region functional in a cell, a sequence 
complimentary to an RNA sequence encoding an amino acid sequence 
corresponding to the above-described polypeptide, and a transcriptional 
termination region functional in said cell. 
The invention also provides a cell that contains the above-described 
recombinant nucleic acid molecule. 
The invention further provides an organism that contains the 
above-described recombinant nucleic acid molecule. 
The invention also provides an antibody having binding affinity to an ERK-5 
polypeptide, or a binding fragment thereof. 
The invention further provides a method of detecting an ERK-5 polypeptide 
in a sample. 
The invention also provides a method of measuring the amount of ERK-5 in a 
sample. 
The invention further provides a diagnostic kit comprising a first 
container means containing the above-described antibody, and a second 
container means containing a conjugate comprising a binding partner of 
said monoclonal antibody and a label. 
The invention also provides a hybridoma which produces the above-described 
monoclonal antibody. 
The invention further provides a method of detecting a compound capable of 
binding to ERK-5 or a fragment thereof. 
The invention also provides a method of detecting an agonist or antagonist 
of ERK-5 activity. 
The invention further provides a method of agonizing or antagonizing ERK-5 
associated activity in a mammal. 
The invention also provides a method of treating diabetes mellitus, 
skeletal muscle diseases, Alzheimer's disease, or peripheral neuropathies 
in a mammal with an agonist or antagonist of ERK-5 activity. 
The invention further provides a pharmaceutical composition comprising an 
ERK-5 agonist or antagonist. 
Further objects and advantages of the present invention will be clear from 
the description that follows.

DEFINITIONS 
In the description that follows, a number of terms used in recombinant DNA 
(rDNA) technology are extensively utilized. In order to provide a clear 
and consistent understanding of the specification and claims, including 
the scope to be given such terms, the following definitions are provided. 
Isolated Nucleic Acid Molecule. 
An "isolated nucleic acid molecule", as is generally understood and used 
herein, refers to a polymer of nucleotides, and includes but should not be 
limited to DNA and RNA. 
DNA Segment. 
A DNA segment, as is generally understood and used herein, refers to a 
molecule comprising a linear stretch of nucleotides wherein the 
nucleotides are present in a sequence that may encode, through the genetic 
code, a molecule comprising a linear sequence of amino acid residues that 
is referred to as a protein, a protein fragment or a polypeptide. 
Gene. 
A DNA sequence related to a single polypeptide chain or protein, and as 
used herein includes the 5' and 3' untranslated ends. The polypeptide can 
be encoded by a full-length sequence or any portion of the coding 
sequence, so long as the functional activity of the protein is retained. 
Complementary DNA (cDNA). 
Recombinant nucleic acid molecules synthesized by reverse transcription of 
messenger RNA ("mRNA"). 
Structural Gene. 
A DNA sequence that is transcribed into mRNA that is then translated into a 
sequence of amino acids characteristic of a specific polypeptide. 
Restriction Endonuclease. 
A restriction endonuclease (also restriction enzyme) is an enzyme that has 
the capacity to recognize a specific base sequence (usually 4, 5, or 6 
base pairs in length) in a DNA molecule, and to cleave the DNA molecule at 
every place where this sequence appears. For example, EcoRI recognizes the 
base sequence GAATTC/CTTAAG. 
Restriction Fragment. 
The DNA molecules produced by digestion with a restriction endonuclease are 
referred to as restriction fragments. Any given genome may be digested by 
a particular restriction endonuclease into a discrete set of restriction 
fragments. 
Agarose Gel Electrophoresis. 
To detect a polymorphism in the length of restriction fragments, an 
analytical method for fractionating double-stranded DNA molecules on the 
basis of size is required. The most commonly used technique (though not 
the only one) for achieving such a fractionation is agarose gel 
electrophoresis. The principle of this method is that DNA molecules 
migrate through the gel as though it were a sieve that retards the 
movement of the largest molecules to the greatest extent and the movement 
of the smallest molecules to the least extent. Note that the smaller the 
DNA fragment, the greater the mobility under electrophoresis in the 
agarose gel. 
The DNA fragments fractionated by agarose gel electrophoresis can be 
visualized directly by a staining procedure if the number of fragments 
included in the pattern is small. The DNA fragments of genomes can be 
visualized successfully. However, most genomes, including the human 
genome, contain far too many DNA sequences to produce a simple pattern of 
restriction fragments. For example, the human genome is digested into 
approximately 1,000,000 different DNA fragments by EcoRI. In order to 
visualize a small subset of these fragments, a methodology referred to as 
the Southern hybridization procedure can be applied. 
Southern Transfer Procedure. 
The purpose of the Southern transfer procedure (also referred to as 
blotting) is to physically transfer DNA fractionated by agarose gel 
electrophoresis onto a nitrocellulose filter paper or another appropriate 
surface or method, while retaining the relative positions of DNA fragments 
resulting from the fractionation procedure. The methodology used to 
accomplish the transfer from agarose gel to nitrocellulose involves 
drawing the DNA from the gel into the nitrocellulose paper by capillary 
action. 
Nucleic Acid Hybridization. 
Nucleic acid hybridization depends on the principle that two 
single-stranded nucleic acid molecules that have complementary base 
sequences will reform the thermodynamically favored double-stranded 
structure if they are mixed under the proper conditions. The 
double-stranded structure will be formed between two complementary 
single-stranded nucleic acids even if one is immobilized on a 
nitrocellulose filter. In the Southern hybridization procedure, the latter 
situation occurs. As noted previously, the DNA of the individual to be 
tested is digested with a restriction endonuclease, fractionated by 
agarose gel electrophoresis, converted to the single-stranded form, and 
transferred to nitrocellulose paper, making it available for reannealing 
to the hybridization probe. 
Hybridization Probe. 
To visualize a particular DNA sequence in the Southern hybridization 
procedure, a labeled DNA molecule or hybridization probe is reacted to the 
fractionated DNA bound to the nitrocellulose filter. The areas on the 
filter that carry DNA sequences complementary to the labeled DNA probe 
become labeled themselves as a consequence of the reannealing reaction. 
The areas of the filter that exhibit such labeling are visualized. The 
hybridization probe is generally produced by molecular cloning of a 
specific DNA sequence. 
Oligonucleotide or Oligomer. 
A molecule comprised of two or more deoxyribonucleotides or 
ribonucleotides, preferably more than three. Its exact size will depend on 
many factors, which in turn depend on the ultimate function or use of the 
oligonucleotide. An oligonucleotide may be derived synthetically or by 
cloning. 
Sequence Amplification. 
A method for generating large amounts of a target sequence. In general, one 
or more amplification primers are annealed to a nucleic acid sequence. 
Using appropriate enzymes, sequences found adjacent to, or in between the 
primers are amplified. 
Amplification Primer. 
An oligonucleotide which is capable of annealing adjacent to a target 
sequence and serving as an initiation point for DNA synthesis when placed 
under conditions in which synthesis of a primer extension product which is 
complementary to a nucleic acid strand is initiated. 
Vector. 
A plasmid or phage DNA or other DNA sequence into which DNA may be inserted 
to be cloned. The vector may replicate autonomously in a host cell, and 
may be further characterized by one or a small number of endonuclease 
recognition sites at which such DNA sequences may be cut in a determinable 
fashion and into which DNA may be inserted. The vector may further contain 
a marker suitable for use in the identification of cells transformed with 
the vector. Markers, for example, are tetracycline resistance or 
ampicillin resistance. The words "cloning vehicle" are sometimes used for 
"vector." 
Expression. 
Expression is the process by which a structural gene produces a 
polypeptide. It involves transcription of the gene into mRNA, and the 
translation of such mRNA into polypeptide(s). 
Expression Vector. 
A vector or vehicle similar to a cloning vector but which is capable of 
expressing a gene which has been cloned into it, after transformation into 
a host. The cloned gene is usually placed under the control of (i.e., 
operably linked to) certain control sequences such as promoter sequences. 
Expression control sequences will vary depending on whether the vector is 
designed to express the operably linked gene in a prokaryotic or 
eukaryotic host and may additionally contain transcriptional elements such 
as enhancer elements, termination sequences, tissue-specificity elements, 
and/or translational initiation and termination sites. 
Functional Derivative. 
A "functional derivative" of a sequence, either protein or nucleic acid, is 
a molecule that possesses a biological activity (either functional or 
structural) that is substantially similar to a biological activity of the 
protein or nucleic acid sequence. A functional derivative of a protein may 
or may not contain post-translational modifications such as covalently 
linked carbohydrate, depending on the necessity of such modifications for 
the performance of a specific function. The term "functional derivative" 
is intended to include the "fragments," "segments," "variants," "analogs," 
or "chemical derivatives" of a molecule. 
As used herein, a molecule is said to be a "chemical derivative" of another 
molecule when it contains additional chemical moieties not normally a part 
of the molecule. Such moieties may improve the molecule's solubility, 
absorption, biological half life, and the like. The moieties may 
alternatively decrease the toxicity of the molecule, eliminate or 
attenuate any undesirable side effect of the molecule, and the like. 
Moieties capable of mediating such effects are disclosed in Remington's 
Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a 
molecule are well known in the art. 
Fragment. 
A "fragment" of a molecule such as a protein or nucleic acid is meant to 
refer to any portion of the amino acid or nucleotide genetic sequence. 
Variant. 
A "variant" of a protein or nucleic acid is meant to refer to a molecule 
substantially similar in structure and biological activity to either the 
protein or nucleic acid, or to a fragment thereof. Thus, provided that two 
molecules possess a common activity and may substitute for each other, 
they are considered variants as that term is used herein even if the 
composition or secondary, tertiary, or quaternary structure of one of the 
molecules is not identical to that found in the other, or if the amino 
acid or nucleotide sequence is not identical. 
Analog. 
An "analog" of a protein or genetic sequence is meant to refer to a protein 
or genetic sequence substantially similar in function to a protein or 
genetic sequence described herein. 
Allele. 
An "allele" is an alternative form of a gene occupying a given locus on the 
chromosome. 
Mutation. 
A "mutation" is any detectable change in the genetic material which may be 
transmitted to daughter cells and possibly even to succeeding generations 
giving rise to mutant cells or mutant individuals. If the descendants of a 
mutant cell give rise only to somatic cells in multicellular organisms, a 
mutant spot or area of cells arises. Mutations in the germ line of 
sexually reproducing organisms may be transmitted by the gametes to the 
next generation resulting in an individual with the new mutant condition 
in both its somatic and germ cells. A mutation may be any (or a 
combination of) detectable, unnatural change affecting the chemical or 
physical constitution, mutability, replication, phenotypic function, or 
recombination of one or more deoxyribonucleotides; nucleotides may be 
added, deleted, substituted for, inverted, or transposed to new positions 
with and without inversion. Mutations may occur spontaneously and can be 
induced experimentally by application of mutagens. A mutant variation of a 
nucleic acid molecule results from a mutation. A mutant polypeptide may 
result from a mutant nucleic acid molecule. 
Species. 
A "species" is a group of actually or potentially interbreeding natural 
populations. A species variation within a nucleic acid molecule or protein 
is a change in the nucleic acid or amino acid sequence that occurs among 
species and may be determined by DNA sequencing of the molecule in 
question. 
Substantially Pure. 
A "substantially pure" protein or nucleic acid is a protein or nucleic acid 
preparation that is generally lacking in other cellular components. 
DETAILED DESCRIPTION OF THE INVENTION 
Isolated Nucleic Acid Molecules Coding for ERK-5 Polypeptides, and 
Fragments Thereof. 
In one embodiment, the present invention relates to an isolated nucleic 
acid molecule coding for a polypeptide having an amino acid sequence 
corresponding to ERK-5, or at least 9 contiguous amino acids thereof 
(preferably, at least 10, 15, 20, or 30 contiguous amino acids thereof). 
In one preferred embodiment, the isolated nucleic acid molecule comprises 
the sequences set forth in SEQ ID NO:1; allelic, mutant or species 
variation thereof, or at least 27 contiguous nucleotides thereof 
(preferably at least 30, 35, 40, or 50 contiguous nucleotides thereof). In 
another preferred embodiment, the isolated nucleic acid molecule encodes 
the amino acid sequence set forth in SEQ ID NO:2, or mutant or species 
variation thereof, or at least 9 contiguous amino acids thereof 
(preferably, at least 10, 15, 20, or 30 contiguous amino acids thereof). 
Also included within the scope of this invention are the functional 
equivalents of the herein-described isolated nucleic acid molecules. The 
degeneracy of the genetic code permits substitution of certain codons by 
other codons which specify the same amino acid and hence would give rise 
to the same protein. The nucleic acid sequence can vary substantially 
since, with the exception of methionine and tryptophan, the known amino 
acids can be coded for by more than one codon. Thus, portions or all of 
the ERK-5 gene could be synthesized to give a nucleic acid sequence 
significantly different from that shown in SEQ ID NO:1. The encoded amino 
acid sequence thereof would, however, be preserved. 
In addition, the nucleic acid sequence may comprise a nucleotide sequence 
which results from the addition, deletion or substitution of at least one 
nucleotide to the 5'-end and/or the 3'-end of the nucleic acid formula 
shown in SEQ ID NO:1 or a derivative thereof. Any nucleotide or 
polynucleotide may be used in this regard, provided that its addition, 
deletion or substitution does not alter the amino acid sequence of SEQ ID 
NO:2 which is encoded by the nucleotide sequence. For example, the present 
invention is intended to include any nucleic acid sequence resulting from 
the addition of ATG as an initiation codon at the 5'-end of the inventive 
nucleic acid sequence or its derivative, or from the addition of TTA, TAG 
or TGA as a termination codon at the 3'-end of the inventive nucleotide 
sequence or its derivative. Moreover, the nucleic acid molecule of the 
present invention may, as necessary, have restriction endonuclease 
recognition sites added to its 5'-end and/or 3'-end. 
Such functional alterations of a given nucleic acid sequence afford an 
opportunity to promote secretion and/or processing of heterologous 
proteins encoded by foreign nucleic acid sequences fused thereto. All 
variations of the nucleotide sequence of the ERK-5 genes and fragments 
thereof permitted by the genetic code are, therefore, included in this 
invention. 
Further, it is possible to delete codons or to substitute one or more 
codons by codons other than degenerate codons to produce a structurally 
modified polypeptide, but one which has substantially the same utility or 
activity of the polypeptide produced by the unmodified nucleic acid 
molecule. As recognized in the art, the two polypeptides are functionally 
equivalent, as are the two nucleic acid molecules which give rise to their 
production, even though the differences between the nucleic acid molecules 
are not related to degeneracy of the genetic code. 
A. Isolation of Nucleic Acid. 
In one aspect of the present invention, isolated nucleic acid molecules 
coding for polypeptides having amino acid sequences corresponding to ERK-5 
are provided. In particular, the nucleic acid molecule may be isolated 
from a biological sample containing RNA or DNA. 
The nucleic acid molecule may be isolated from a biological sample 
containing RNA using the techniques of cDNA cloning and subtractive 
hybridization as previously described (Birkenbach et al., J. of Virology 
63:9:4079-4084). The nucleic acid molecule may also be isolated from a 
cDNA library using a homologous probe. 
The nucleic acid molecule may be isolated from a biological sample 
containing genomic DNA or from a genomic library using techniques well 
known in the art. Suitable biological samples include, but are not limited 
to, blood, semen and tissue. The method of obtaining the biological sample 
will vary depending upon the nature of the sample. 
One skilled in the art will realize that the human genome may be subject to 
slight allelic variations between individuals. Therefore, the isolated 
nucleic acid molecule is also intended to include allelic variations, so 
long as the sequence is a functional derivative of the ERK-5 gene. 
One skilled in the art will realize that organisms other than humans may 
also contain ERK-5 genes (for example, eukaryotes; more specifically, 
mammals, birds, fish, and plants; more specifically, gorillas, rhesus 
monkeys, and chimpanzees). The invention is intended to include, but not 
be limited to, ERK-5 nucleic acid molecules isolated from the 
above-described organisms. 
B. Synthesis of Nucleic Acid. 
Isolated nucleic acid molecules of the present invention are also meant to 
include those chemically synthesized. For example, a nucleic acid molecule 
with the nucleotide sequence which codes for the expression product of an 
ERK-5 gene may be designed and, if necessary, divided into appropriate 
smaller fragments. Then an oligomer which corresponds to the nucleic acid 
molecule, or to each of the divided fragments, may be synthesized. Such 
synthetic oligonucleotides may be prepared, for example, by the triester 
method of Matteucci et al., J. Am. Chem. Soc. 103:3185-3191 (1981) or by 
using an automated DNA synthesizer. 
An oligonucleotide may be derived synthetically or by cloning. If 
necessary, the 5'-ends of the oligomers may be phosphorylated using T4 
polynucleotide kinase. Kinasing of single strands prior to annealing or 
for labeling may be achieved using an excess of the enzyme. If kinasing is 
for the labeling of probe, the ATP may contain high specific activity 
radioisotopes. Then, the DNA oligomer may be subjected to annealing and 
ligation with T4 ligase or the like. 
II. Substantially Pure ERK-5 Polypeptides. 
In another embodiment, the present invention relates to a substantially 
pure polypeptide having an amino acid sequence corresponding to ERK-5, or 
at least 9 contiguous amino acids thereof (preferably, at least 10, 15, 
20, or 30 contiguous amino acids thereof). In a preferred embodiment, the 
polypeptide has the amino acid sequence set forth in SEQ ID NO:2, or 
mutant or species variation thereof, or at least 9 contiguous amino acids 
thereof (preferably, at least 10, 15, 20, or 30 contiguous amino acids 
thereof). 
A variety of methodologies known in the art can be utilized to obtain the 
peptide of the present invention. In one embodiment, the peptide is 
purified from tissues or cells which naturally produce the peptide. 
Alternatively, the above-described isolated nucleic acid fragments could 
be used to expressed the ERK-5 protein in any organism. The samples of the 
present invention include cells, protein extracts or membrane extracts of 
cells, or biological fluids. The sample will vary based on the assay 
format, the detection method and the nature of the tissues, cells or 
extracts used as the sample. 
Any eukaryotic organism can be used as a source for the peptide of the 
invention, as long as the source organism naturally contains such a 
peptide. As used herein, "source organism" refers to the original organism 
from which the amino acid sequence of the subunit is derived, regardless 
of the organism the subunit is expressed in and ultimately isolated from. 
One skilled in the art can readily follow known methods for isolating 
proteins in order to obtain the peptide free of natural contaminants. 
These include, but are not limited to: size-exclusion chromatography, 
HPLC, ion-exchange chromatography, and immuno-affinity chromatography. 
III. A Nucleic Acid Probe for the Detection of ERK-5. 
In another embodiment, the present invention relates to a nucleic acid 
probe for the detection of the presence of ERK-5 in a sample comprising 
the above-described nucleic acid molecules or at least 27 contiguous 
nucleotides thereof (preferably at least 30, 35, 40, or 50 thereof). In 
another preferred embodiment, the nucleic acid probe has the nucleic acid 
sequence set forth in SEQ ID NO:1 or at least 27 contiguous nucleotides 
thereof (preferably at least 30, 35, 40, or 50 thereof). In another 
preferred embodiment, the nucleic acid probe encodes the amino acid 
sequence set forth in SEQ ID NO:2 or at least 9 contiguous amino acids 
thereof. 
The nucleic acid probe may be used to probe an appropriate chromosomal or 
cDNA library by usual hybridization methods to obtain another nucleic acid 
molecule of the present invention. A chromosomal DNA or cDNA library may 
be prepared from appropriate cells according to recognized methods in the 
art (cf. Molecular Cloning: A Laboratory Manual second edition, edited by 
Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989). 
In the alternative, chemical synthesis is carried out in order to obtain 
nucleic acid probes having nucleotide sequences which correspond to 
N-terminal and C-terminal portions of the amino acid sequence of the 
polypeptide of interest. Thus, the synthesized nucleic acid probes may be 
used as primers in a polymerase chain reaction (PCR) carried out in 
accordance with recognized PCR techniques, essentially according to PCR 
Protocols, A Guide to Methods and Applications, edited by Michael et al., 
Academic Press, 1990, utilizing the appropriate chromosomal or cDNA 
library to obtain the fragment of the present invention. 
One skilled in the art can readily design such probes based on the sequence 
disclosed herein using methods of computer alignment and sequence analysis 
known in the art (cf. Molecular Cloning: A Laboratory Manual second 
edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor 
Laboratory, 1989). 
The hybridization probes of the present invention can be labeled by 
standard labeling techniques such as with a radiolabel, enzyme label, 
fluorescent label, biotin-avidin label, chemiluminescence, and the like. 
After hybridization, the probes may be visualized using known methods. 
The nucleic acid probes of the present invention include RNA, as well as 
DNA probes, such probes being generated using techniques known in the art. 
In one embodiment of the above described method, a nucleic acid probe is 
immobilized on a solid support. Examples of such solid supports include, 
but are not limited to, plastics such as polycarbonate, complex 
carbohydrates such as agarose and sepharose, and acrylic resins, such as 
polyacrylamide and latex beads. Techniques for coupling nucleic acid 
probes to such solid supports are well known in the art. 
The test samples suitable for nucleic acid probing methods of the present 
invention include, for example, cells or nucleic acid extracts of cells, 
or biological fluids. The sample used in the above-described methods will 
vary based on the assay format, the detection method and the nature of the 
tissues, cells or extracts to be assayed. Methods for preparing nucleic 
acid extracts of cells are well known in the art and can be readily 
adapted in order to obtain a sample which is compatible with the method 
utilized. 
IV. A Method of Detecting The Presence of ERK-5 in a Sample. 
In another embodiment, the present invention relates to a method of 
detecting the presence of ERK-5 in a sample comprising a) contacting said 
sample with the above-described nucleic acid probe, under conditions such 
that hybridization occurs, and b) detecting the presence of said probe 
bound to said nucleic acid molecule. One skilled in the art would select 
the nucleic acid probe according to techniques known in the art as 
described above. Samples to be tested include but should not be limited to 
RNA samples of human tissue. 
ERK-5 has been found to be predominantly expressed in muscle. Accordingly, 
ERK-5 probes may be used detect the presence of RNA from muscle in a 
sample. Further, altered expression levels of ERK-5 RNA in an individual, 
as compared to normal levels, may indicate the presence of muscular 
disease or diabetes mellitus. The ERK-5 probes may further be used to 
assay cellular factor activity in general and specifically in muscle 
tissue. 
V. A Kit for Detecting the Presence of ERK-5 in a Sample. 
In another embodiment, the present invention relates to a kit for detecting 
the presence of ERK-5 in a sample comprising at least one container means 
having disposed therein the above-described nucleic acid probe. In a 
preferred embodiment, the kit further comprises other containers 
comprising one or more of the following: wash reagents and reagents 
capable of detecting the presence of bound nucleic acid probe. Examples of 
detection reagents include, but are not limited to radiolabelled probes, 
enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), 
and affinity labeled probes (biotin, avidin, or steptavidin). 
In detail, a compartmentalized kit includes any kit in which reagents are 
contained in separate containers. Such containers include small glass 
containers, plastic containers or strips of plastic or paper. Such 
containers allow the efficient transfer of reagents from one compartment 
to another compartment such that the samples and reagents are not 
cross-contaminated and the agents or solutions of each container can be 
added in a quantitative fashion from one compartment to another. Such 
containers will include a container which will accept the test sample, a 
container which contains the probe or primers used in the assay, 
containers which contain wash reagents (such as phosphate buffered saline, 
Tris-buffers, and the like), and containers which contain the reagents 
used to detect the hybridized probe, bound antibody, amplified product, or 
the like. 
One skilled in the art will readily recognize that the nucleic acid probes 
described in the present invention can readily be incorporated into one of 
the established kit formats which are well known in the art. 
VI. DNA Constructs Comprising a ERK-5 Nucleic Acid Molecule and Cells 
Containing These Constructs. 
In another embodiment, the present invention relates to a recombinant DNA 
molecule comprising, 5' to 3', a promoter effective to initiate 
transcription in a host cell and the above-described nucleic acid 
molecules. In another embodiment, the present invention relates to a 
recombinant DNA molecule comprising a vector and an above-described 
nucleic acid molecules. 
In another embodiment, the present invention relates to a nucleic acid 
molecule comprising a transcriptional region functional in a cell, a 
sequence complimentary to an RNA sequence encoding an amino acid sequence 
corresponding to the above-described polypeptide, and a transcriptional 
termination region functional in said cell. 
Preferably, the above-described molecules are isolated and/or purified DNA 
molecules. 
In another embodiment, the present invention relates to a cell or organism 
that contains an above-described nucleic acid molecule. 
In another embodiment, the peptide is purified from cells which have been 
altered to express the peptide. 
As used herein, a cell is said to be "altered to express a desired peptide" 
when the cell, through genetic manipulation, is made to produce a protein 
which it normally does not produce or which the cell normally produces at 
lower levels. One skilled in the art can readily adapt procedures for 
introducing and expressing either genomic, cDNA, or synthetic sequences 
into either eukaryotic or prokaryotic cells. 
A nucleic acid molecule, such as DNA, is said to be "capable of expressing" 
a polypeptide if it contains nucleotide sequences which contain 
transcriptional and translational regulatory information and such 
sequences are "operably linked" to nucleotide sequences which encode the 
polypeptide. An operable linkage is a linkage in which the regulatory DNA 
sequences and the DNA sequence sought to be expressed are connected in 
such a way as to permit gene sequence expression. The precise nature of 
the regulatory regions needed for gene sequence expression may vary from 
organism to organism, but shall in general include a promoter region 
which, in prokaryotes, contains both the promoter (which directs the 
initiation of RNA transcription) as well as the DNA sequences which, when 
transcribed into RNA, will signal synthesis initiation. Such regions will 
normally include those 5'-non-coding sequences involved with initiation of 
transcription and translation, such as the TATA box, capping sequence, 
CAAT sequence, and the like. 
If desired, the non-coding region 3' to the sequence encoding an ERK-5 gene 
may be obtained by the above-described methods. This region may be 
retained for its transcriptional termination regulatory sequences, such as 
termination and polyadenylation. Thus, by retaining the 3'-region 
naturally contiguous to the DNA sequence encoding an ERK-5 gene, the 
transcriptional termination signals may be provided. Where the 
transcriptional termination signals are not satisfactorily functional in 
the expression host cell, then a 3' region functional in the host cell may 
be substituted. 
Two DNA sequences (such as a promoter region sequence and an ERK-5 
sequence) are said to be operably linked if the nature of the linkage 
between the two DNA sequences does not (1) result in the introduction of a 
frame-shift mutation, (2) interfere with the ability of the promoter 
region sequence to direct the transcription of an ERK-5 gene sequence, or 
(3) interfere with the ability of the an ERK-5 gene sequence to be 
transcribed by the promoter region sequence. Thus, a promoter region would 
be operably linked to a DNA sequence if the promoter were capable of 
effecting transcription of that DNA sequence. 
Thus, to express an ERK-5 gene, transcriptional and translational signals 
recognized by an appropriate host are necessary. 
The present invention encompasses the expression of the ERK-5 gene (or a 
functional derivative thereof) in either prokaryotic or eukaryotic cells. 
Prokaryotic hosts are, generally, very efficient and convenient for the 
production of recombinant proteins and are, therefore, one type of 
preferred expression system for the ERK-5 gene. 
Prokaryotes most frequently are represented by various strains of E. coli. 
However, other microbial strains may also be used, including other 
bacterial strains. 
In prokaryotic systems, plasmid vectors that contain replication sites and 
control sequences derived from a species compatible with the host may be 
used. Examples of suitable plasmid vectors may include pBR322, pUC118, 
pUC119 and the like; suitable phage or bacteriophage vectors may include 
.lambda.gt10, .lambda.gt11 and the like; and suitable virus vectors may 
include pMAM-neo, pKRC and the like. Preferably, the selected vector of 
the present invention has the capacity to replicate in the selected host 
cell. 
Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, 
Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, 
under such conditions, the peptide will not be glycosylated. The 
prokaryotic host must be compatible with the replicon and control 
sequences in the expression plasmid. 
To express ERK-5 (or a functional derivative thereof) in a prokaryotic 
cell, it is necessary to operably link the ERK-5 sequence to a functional 
prokaryotic promoter. Such promoters may be either constitutive or, more 
preferably, regulatable (i.e., inducible or derepressible). Examples of 
constitutive promoters include the int promoter of bacteriophage .lambda., 
the bla promoter of the .beta.-lactamase gene sequence of pBR322, and the 
CAT promoter of the chloramphenicol acetyl transferase gene sequence of 
pPR325, and the like. Examples of inducible prokaryotic promoters include 
the major right and left promoters of bacteriophage .lambda. (P.sub.L and 
P.sub.R), the trp, recA, lacZ, lacI, and gal promoters of E. coli, the 
.alpha.-amylase (Ulmanen et al., J. Bacteriol. 162:176-182 (1985)) and the 
.delta.-28-specific promoters of B. subtills (Gilman et al., Gene sequence 
32:11-20 (1984)), the promoters of the bacteriophages of Bacillus 
(Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., 
New York (1982)), and Streptomyces promoters (Ward et al., Mol. Gen. 
Genet. 203:468-478 (1986)). 
Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 1:277-282 
(1987); Cenatiempo (Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev. 
Genet. 18:415-442 (1984)). 
Proper expression in a prokaryotic cell also requires the presence of a 
ribosome binding site upstream of the gene sequence-encoding sequence. 
Such ribosome binding sites are disclosed, for example, by Gold et al. 
(Ann. Rev. Microbiol. 35:365-404 (1981)). 
The selection of control sequences, expression vectors, transformation 
methods, and the like, are dependent on the type of host cell used to 
express the gene. As used herein, "cell", "cell line", and "cell culture" 
may be used interchangeably and all such designations include progeny. 
Thus, the words "transformants" or "transformed cells" include the primary 
subject cell and cultures derived therefrom, without regard to the number 
of transfers. It is also understood that all progeny may not be precisely 
identical in DNA content, due to deliberate or inadvertent mutations. 
However, as defined, mutant progeny have the same functionality as that of 
the originally transformed cell. 
Host cells which may be used in the expression systems of the present 
invention are not strictly limited, provided that they are suitable for 
use in the expression of the ERK-5 peptide of interest. Suitable hosts may 
often include eukaryotic cells. 
Preferred eukaryotic hosts include, for example, yeast, fungi, insect 
cells, mammalian cells either in vivo, or in tissue culture. Mammalian 
cells which may be useful as hosts include HeLa cells, cells of fibroblast 
origin such as VERO or CHO-K 1, or cells of lymphoid origin and their 
derivatives. Preferred mammalian host cells include SP2/0 and J558L, as 
well as neuroblastoma cell lines such as IMR 332 which may provide better 
capacities for correct post-translational processing. 
In addition, plant cells are also available as hosts, and control sequences 
compatible with plant cells are available, such as the cauliflower mosaic 
virus 35S and 19S, and nopaline synthase promoter and polyadenylation 
signal sequences. 
Another preferred host is an insect cell, for example the Drosophila 
larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase 
promoter can be used. Rubin, Science 240:1453-1459 (1988). Alternatively, 
baculovirus vectors can be engineered to express large amounts of ERK-5 in 
insects cells (Jasny, Science 238:1653 (1987); Miller et al., In: Genetic 
Engineering (1986), Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 
277-297). 
Any of a series of yeast gene sequence expression systems can be utilized 
which incorporate promoter and termination elements from the actively 
expressed gene sequences coding for glycolytic enzymes are produced in 
large quantities when yeast are grown in mediums rich in glucose. Known 
glycolytic gene sequences can also provide very efficient transcriptional 
control signals. 
Yeast provides substantial advantages in that it can also carry out 
posttranslational peptide modifications. A number of recombinant DNA 
strategies exist which utilize strong promoter sequences and high copy 
number of plasmids which can be utilized for production of the desired 
proteins in yeast. Yeast recognizes leader sequences on cloned mammalian 
gene sequence products and secretes peptides bearing leader sequences 
(i.e., pre-peptides). For a mammalian host, several possible vector 
systems are available for the expression of ERK-5. 
A wide variety of transcriptional and translational regulatory sequences 
may be employed, depending upon the nature of the host. The 
transcriptional and translational regulatory signals may be derived from 
viral sources, such as adenovirus, bovine papilloma virus, 
cytomegalovirus, simian virus, or the like, where the regulatory signals 
are associated with a particular gene sequence which has a high level of 
expression. Alternatively, promoters from mammalian expression products, 
such as actin, collagen, myosin, and the like, may be employed. 
Transcriptional initiation regulatory signals may be selected which allow 
for repression or activation, so that expression of the gene sequences can 
be modulated. Of interest are regulatory signals which are 
temperature-sensitive so that by varying the temperature, expression can 
be repressed or initiated, or are subject to chemical (such as metabolite) 
regulation. 
As discussed above, expression of ERK-5 in eukaryotic hosts requires the 
use of eukaryotic regulatory regions. Such regions will, in general, 
include a promoter region sufficient to direct the initiation of RNA 
synthesis. Preferred eukaryotic promoters include, for example, the 
promoter of the mouse metallothionein I gene sequence (Hamer et al., J. 
Mol. Appl. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus 
(McKnight, Cell 31:355-365 (1982)); the SV40 early promoter (Benoist et 
al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence 
promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 
(1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)). 
As is widely known, translation of eukaryotic mRNA is initiated at the 
codon which encodes the first methionine. For this reason, it is 
preferable to ensure that the linkage between a eukaryotic promoter and a 
DNA sequence which encodes ERK-5 (or a functional derivative thereof) does 
not contain any intervening codons which are capable of encoding a 
methionine (i.e., AUG). The presence of such codons results either in a 
formation of a fusion protein (if the AUG codon is in the same reading 
frame as the ERK-5 coding sequence) or a frame-shift mutation (if the AUG 
codon is not in the same reading frame as the ERK-5 coding sequence). 
An ERK-5 nucleic acid molecule and an operably linked promoter may be 
introduced into a recipient prokaryotic or eukaryotic cell either as a 
non-replicating DNA (or RNA) molecule, which may either be a linear 
molecule or, more preferably, a closed covalent circular molecule. Since 
such molecules are incapable of autonomous replication, the expression of 
the gene may occur through the transient expression of the introduced 
sequence. Alternatively, permanent expression may occur through the 
integration of the introduced DNA sequence into the host chromosome. 
In one embodiment, a vector is employed which is capable of integrating the 
desired gene sequences into the host cell chromosome. Cells which have 
stably integrated the introduced DNA into their chromosomes can be 
selected by also introducing one or more markers which allow for selection 
of host cells which contain the expression vector. The marker may provide 
for prototrophy to an auxotrophic host, biocide resistance, e.g., 
antibiotics, or heavy metals, such as copper, or the like. The selectable 
marker gene sequence can either be directly linked to the DNA gene 
sequences to be expressed, or introduced into the same cell by 
co-transfection. Additional elements may also be needed for optimal 
synthesis of single chain binding protein mRNA. These elements may include 
splice signals, as well as transcription promoters, enhancers, and 
termination signals. cDNA expression vectors incorporating such elements 
include those described by Okayama, Molec. Cell. Biol. 3:280 (1983). 
In a preferred embodiment, the introduced nucleic acid molecule will be 
incorporated into a plasmid or viral vector capable of autonomous 
replication in the recipient host. Any of a wide variety of vectors may be 
employed for this purpose. Factors of importance in selecting a particular 
plasmid or viral vector include: the ease with which recipient cells that 
contain the vector may be recognized and selected from those recipient 
cells which do not contain the vector; the number of copies of the vector 
which are desired in a particular host; and whether it is desirable to be 
able to "shuttle" the vector between host cells of different species. 
Preferred prokaryotic vectors include plasmids such as those capable of 
replication in E. coli (such as, for example, pBR322, ColE1, pSC101, pACYC 
184, .pi.VX. Such plasmids are, for example, disclosed by Sambrook (cf. 
Molecular Cloning: A Laboratory Manual, second edition, edited by 
Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)). 
Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids 
are disclosed by Gryczan . (In: The Molecular Biology of the Bacilli, 
Academic Press, New York (1982), pp. 307-329). Suitable Streptomyces 
plasmids include pIJ101 (Kendall et al., J. Bacteriol. 169:4177-4183 
(1987)), and streptomyces bacteriophages such as .phi.C31 (Chater et al., 
In: Sixth International Symposium on Actinomycetales Biology, Akademiai 
Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are 
reviewed by John et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki 
(Jpn. J. Bacteriol. 33:729-742 (1978)). 
Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 
2-micron circle, and the like, or their derivatives. Such plasmids are 
well known in the art (Botstein et al., Miami Wntr. Symp. 9:265-274 
(1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life 
Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y., p. 445-470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al., J. 
Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology: A 
Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, 
New York, pp. 563-608 (1980)). 
Once the vector or nucleic acid molecule containing the construct(s) has 
been prepared for expression, the DNA construct(s) may be introduced into 
an appropriate host cell by any of a variety of suitable means, i.e., 
transformation, transfection, conjugation, protoplast fusion, 
electroporation, particle gun technology, calcium phosphate-precipitation, 
direct microinjection, and the like. After the introduction of the vector, 
recipient cells are grown in a selective medium, which selects for the 
growth of vector-containing cells. Expression of the cloned gene 
molecule(s) results in the production of ERK-5 or fragments thereof. This 
can take place in the transformed cells as such, or following the 
induction of these cells to differentiate (for example, by administration 
of bromodeoxyuracil to neuroblastoma cells or the like). 
A variety of incubation conditions can be used to form the peptide of the 
present invention. The most preferred conditions are those which mimic 
physiological conditions. 
VII. An Antibody Having Binding Affinity to an ERK-5 Polypeptide, or a 
Binding Fragment Thereof and a Hybridoma Containing the Antibody. 
In another embodiment, the present invention relates to an antibody having 
binding affinity to an ERK-5 polypeptide, or a binding fragment thereof. 
In a preferred embodiment, the polypeptide has the amino acid sequence set 
forth in SEQ ID NO:2, or mutant or species variation thereof, or at least 
9 contiguous amino acids thereof (preferably, at least 10, 15, 20, or 30 
contiguous amino acids thereof). 
In another preferred embodiment, the present invention relates to an 
antibody having binding affinity to an ERK-5 polypeptide, or a binding 
fragment thereof and not to ERK-1, ERK-2, ERK-3, or ERK-4. Such an 
antibody may be isolated by comparing its binding affinity to ERK-5 with 
its binding affinity to ERK-1, ERK-2, ERK-3, or ERK-4. Those which bind 
selectively to ERK-5 would be chosen for use in methods requiring a 
distinction between ERK-5 and ERK-1, ERK-2, ERK-3, or ERK-4 polypeptides. 
Such methods could include, but should not be limited to, the analysis of 
altered ERK-5 expression in tissue containing ERK-1, ERK-2, ERK-3, or 
ERK-4. 
The ERK-5 proteins of the present invention can be used in a variety of 
procedures and methods, such as for the generation of antibodies, for use 
in identifying pharmaceutical compositions, and for studying DNA/protein 
interaction. 
The ERK-5 peptide of the present invention can be used to produce 
antibodies or hybridomas. One skilled in the art will recognize that if an 
antibody is desired, such a peptide would be generated as described herein 
and used as an immunogen. 
The antibodies of the present invention include monoclonal and polyclonal 
antibodies, as well fragments of these antibodies, and humanized forms. 
Humanized forms of the antibodies of the present invention may be 
generated using one of the procedures known in the art such as 
chimerization or CDR grafting. 
In another embodiment, the present invention relates to a hybridoma which 
produces the above-described monoclonal antibody, or binding fragment 
thereof. A hybridoma is an immortalized cell line which is capable of 
secreting a specific monoclonal antibody. 
In general, techniques for preparing monoclonal antibodies and hybridomas 
are well known in the art (Campbell, "Monoclonal Antibody Technology: 
Laboratory Techniques in Biochemistry and Molecular Biology," Elsevier 
Science Publishers, Ansterdam, The Netherlands (1984); St. Groth et at., 
J. Immunol. Methods 35:1-21 (1980)). 
Any animal (mouse, rabbit, and the like) which is known to produce 
antibodies can be immunized with the selected polypeptide. Methods for 
immunization are well known in the art. Such methods include subcutaneous 
or intraperitoneal injection of the polypeptide. One skilled in the art 
will recognize that the amount of polypeptide used for immunization will 
vary based on the animal which is immunized, the antigenicity of the 
polypeptide and the site of injection. 
The polypeptide may be modified or administered in an adjuvant in order to 
increase the peptide antigenicity. Methods of increasing the antigenicity 
of a polypeptide are well known in the art. Such procedures include 
coupling the antigen with a heterologous protein (such as globulin or 
.beta.-galactosidase) or through the inclusion of an adjuvant during 
immunization. 
For monoclonal antibodies, spleen cells from the immunized animals are 
removed, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and 
allowed to become monoclonal antibody producing hybridoma cells. 
Any one of a number of methods well known in the art can be used to 
identify the hybridoma cell which produces an antibody with the desired 
characteristics. These include screening the hybridomas with an ELISA 
assay, western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell 
Res. 175:109-124 (1988)). 
Hybridomas secreting the desired antibodies are cloned and the class and 
subclass is determined using procedures known in the art (Campbell, 
Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and 
Molecular Biology, supra (1984)). 
For polyclonal antibodies, antibody containing antisera is isolated from 
the immunized animal and is screened for the presence of antibodies with 
the desired specificity using one of the above-described procedures. 
In another embodiment of the present invention, the above-described 
antibodies are detectably labeled. Antibodies can be delectably labeled 
through the use of radioisotopes, affinity labels (such as biotin, avidin, 
and the like), enzymatic labels (such as horse radish peroxidase, alkaline 
phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, 
and the like), paramagnetic atoms, and the like. Procedures for 
accomplishing such labeling are well-known in the art, for example, see 
(Sternberger et al., J. Histochem. Cytochem. 18:315 (1970); Bayer et al., 
Meth. Enzym. 62:308 (1979); Engval et al., Immunol. 109:129 (1972); 
Goding, J. Immunol. Meth. 13:215 (1976)). The labeled antibodies of the 
present invention can be used for in vitro, in vivo, and in situ assays to 
identify cells or tissues which express a specific peptide. 
In another embodiment of the present invention the above-described 
antibodies are immobilized on a solid support. Examples of such solid 
supports include plastics such as polycarbonate, complex carbohydrates 
such as agarose and sepharose, acrylic resins and such as polyacrylamide 
and latex beads. Techniques for coupling antibodies to such solid supports 
are well known in the art (Weir et al., "Handbook of Experimental 
Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, 
Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, New York 
(1974)). The immobilized antibodies of the present invention can be used 
for in vitro, in vivo, and in situ assays as well as in 
immunochromotography. 
Furthermore, one skilled in the art can readily adapt currently available 
procedures, as well as the techniques, methods and kits disclosed above 
with regard to antibodies, to generate peptides capable of binding to a 
specific peptide sequence in order to generate rationally designed 
antipeptide peptides, for example see Hurby et al., "Application of 
Synthetic Peptides: Antisense Peptides", In Synthetic Peptides, A User's 
Guide, W. H. Freeman, New York, pp. 289-307 (1992), and Kaspczak et al., 
Biochemistry 28:9230-8 (1989). 
Anti-peptide peptides can be generated in one of two fashions. First, the 
anti-peptide peptides can be generated by replacing the basic amino acid 
residues found in the ERK-5 peptide sequence with acidic residues, while 
maintaining hydrophobic and uncharged polar groups. For example, lysine, 
arginine, and/or histidine residues are replaced with aspartic acid or 
glutamic acid and glutamic acid residues are replaced by lysine, arginine 
or histidine. 
VIII. A Method of Detecting an ERK-5 Polypeptide in a Sample. 
In another embodiment, the present invention relates to a method of 
detecting an ERK-5 polypeptide in a sample, comprising: a) contacting the 
sample with an above-described antibody, under conditions such that 
immunocomplexes form, and b) detecting the presence of said antibody bound 
to the polypeptide. In detail, the methods comprise incubating a test 
sample with one or more of the antibodies of the present invention and 
assaying whether the antibody binds to the test sample. Altered levels of 
ERK-5 in a sample as compared to normal levels may indicate muscular 
disease. 
Conditions for incubating an antibody with a test sample vary. Incubation 
conditions depend on the format employed in the assay, the detection 
methods employed, and the type and nature of the antibody used in the 
assay. One skilled in the art will recognize that any one of the commonly 
available immunological assay formats (such as radioimmunoassays, 
enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket 
immunofluorescent assays) can readily be adapted to employ the antibodies 
of the present invention. Examples of such assays can be found in Chard, 
"An Introduction to Radioimmunoassay and Related Techniques" Elsevier 
Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al., 
"Techniques in Immunocytochemistry," Academic Press, Orlando, Fla. Vol. 1 
(1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, "Practice and Theory of 
Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular 
Biology," Elsevier Science Publishers, Amsterdam, The Netherlands (1985). 
The immunological assay test samples of the present invention include 
cells, protein or membrane extracts of cells, or biological fluids such as 
blood, serum, plasma, or urine. The test sample used in the 
above-described method will vary based on the assay format, nature of the 
detection method and the tissues, cells or extracts used as the sample to 
be assayed. Methods for preparing protein extracts or membrane extracts of 
cells are well known in the art and can be readily be adapted in order to 
obtain a sample which is capable with the system utilized. 
IX. A Diagnostic Kit Comprising Antibodies to ERK-5. 
In another embodiment of the present invention, a kit is provided which 
contains all the necessary reagents to carry out the previously described 
methods of detection. The kit may comprise: i) a first container means 
containing an above-described antibody, and ii) second container means 
containing a conjugate comprising a binding partner of the antibody and a 
label. In another preferred embodiment, the kit further comprises one or 
more other containers comprising one or more of the following: wash 
reagents and reagents capable of detecting the presence of bound 
antibodies. Examples of detection reagents include, but are not limited 
to, labeled secondary antibodies, or in the alternative, if the primary 
antibody is labeled, the chromophoric, enzymatic, or antibody binding 
reagents which are capable of reacting with the antibody. The 
compartmentalized kit may be as described above for nucleic acid probe 
kits. 
One skilled in the art will readily recognize that the antibodies described 
in the present invention can readily be incorporated into one of the 
established kit formats which are well known in the art. 
X. Isolation of Compounds Which Interact With ERK-5 
In another embodiment, the present invention relates to a method of 
detecting a compound capable of binding to ERK-5 or a fragment thereof 
comprising incubating the compound with ERK-5 or fragment thereof and 
detecting the presence of the compound bound to ERK-5 or fragment thereof. 
In a preferred embodiment, the compound is present within a complex 
mixture, for example, serum, body fluid, or cell extracts. 
In another embodiment, the present invention relates to a method of 
detecting an agonist or antagonist of ERK-5 activity comprising incubating 
cells that produce ERK-5 in the presence of a compound and detecting 
changes in the level of ERK-5 activity. The compounds thus identified 
would produce a change in activity indicative of the presence of the 
compound. In a preferred embodiment, the compound is present within a 
complex mixture, for example, serum, body fluid, or cell extracts. Once 
the compound is identified it can be isolated using techniques well known 
in the art. 
In a further embodiment, the present invention relates to a method of 
agonizing (stimulating) or antagonizing ERK-5 associated activity in a 
mammal comprising administering to said mammal an agonist or antagonist to 
ERK-5 in an amount sufficient to effect said agonism or antagonism. In a 
preferred embodiment, the present invention relates to a possible method 
of treating diabetes mellitus, skeletal muscle diseases, Alzheimer's 
disease, or peripheral neuropathies in a mammal with an agonist or 
antagonist of ERK-5 activity comprising administering the agonist or 
antagonist to a mammal in an amount sufficient to agonize or antagonize 
ERK-5 associated functions. Further, since ERK-5 is preferentially 
expressed in skeletal muscle, the agonist or antagonist might be used in 
normal individuals. 
One skilled in the art will appreciate that the amounts to be administered 
for any particular treatment protocol can readily be determined. The 
dosage should not be so large as to cause adverse side effects, such as 
unwanted cross-reactions, anaphylactic reactions, and the like. Generally, 
the dosage will vary with the age, condition, sex and extent of disease in 
the patient, counter indications, if any, and other such variables, to be 
adjusted by the individual physician. Dosage can vary from 0.001 mg/kg to 
50 mg/kg, preferably 0.1 mg/kg to 1.0 mg/kg, of the agonist or antagonist 
of the invention, in one or more administrations daily, for one or several 
days. The agonist or antagonist can be administered parenterally by 
injection or by gradual perfusion over time. They can be administered 
intravenously, intraperitoneally, intramuscularly, or subcutaneously. 
Preparations for parenteral administration include sterile or aqueous or 
non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous 
solvents are propylene glycol, polyethylene glycol, vegetable oils such as 
olive oil, and injectable organic esters such as ethyl oleate. Aqueous 
carriers include water, alcoholic/aqueous solutions, emulsions or 
suspensions, including saline and buffered media. Parenteral vehicles 
include sodium chloride solution, Ringer's dextrose and sodium chloride, 
lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and 
nutrient replenishers, electrolyte replenishers, such as those based on 
Ringer's dextrose, and the like. Preservatives and other additives may 
also be present, such as, for example, antimicrobials, antioxidants, 
chelating agents, inert gases and the like. See, generally, Remington's 
Pharmaceutical Science, 16th Ed., Mack Eds. (1980). 
In another embodiment, the present invention relates to a pharmaceutical 
composition comprising the above described ERK-5 agonist or antagonist in 
an amount sufficient to alter ERK-5 associated activity, and a 
pharmaceutically acceptable diluent, carrier, or excipient. Appropriate 
concentrations and dosage unit sizes can be readily determined by one 
skilled in the art as described above (See, for example, Remington's 
Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton Pa. (1980) 
and WO 91/19008). 
The present invention is described in further detail in the following 
non-limiting examples. 
Example 1 
Screening of a Human Skeletal Muscle cDNA Library with Radiolabelled 
Oligonucleotides 
Total RNA was isolated from human skeletal muscle by the acid guanidinium 
thiocyanate-phenol-chloroform extraction procedure as described by 
Puissant et al., Bio Techniques 8:148-149 (1990). Poly (A+) RNA was 
isolated on an oligo(dT) column (Avid et al., Proc. Natl. Acad. Sci. USA 
69:1408-1412 (1972)). A cDNA library was constructed using the methods 
described by Okayama and Berg, Mol. Cell. Biol. 2:161-170 (1982); Okayama 
and Berg, Mol. Cell. Biol. 3:280-289 (1983). The pCDVI-PL vector was used 
for preparation of the primer fragment (Noma et al., Nature 319:640-646 
(1986)). A short synthetic adapter was used as second strand primer as 
recently described (Boel, E. et al., Bio Techniques 11 (1):26-28 (July, 
1991)). E. coli DH5.alpha. (Gibco BRL, Gaithersburg, Md. 20877, USA) was 
used for transformation according to the protocols by H. Inuoue et al., 
Gene 96:23-28 (1990). After transformation, the bacteria were plated on LB 
plates containing 50 .mu.g/ml ampicillin at a density of about 8000 
colonies per 15 cm plate. Nitrocellulose replica filters (Schleicher & 
Schuell, BA85) were screened with standard colony hybridization technique 
(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor, N.Y. 1989). An equimolar mixture of the following three 
oligonucleotides which were labelled at the 5' end using T4 polynucleotide 
kinase and [.gamma.-.sup.32 P] ATP (Amersham, Braunschweig) (Sambrook et 
al., 1989) was used for hybridization. 
__________________________________________________________________________ 
E10 
##STR1## SEQ ID NO:3 
E11 
5' TTA ACT TGT CGA CTA CGT CAG CAG 3' 
SEQ ID NO:5 
E13 
5' A(CT) AT(GT) TGG (GT)CT G(CT) (AG) GGC TGC ATC 3' 
SEQ ID NO:6 
__________________________________________________________________________ 
The nucleotide sequences of the oligonucleotides correspond to nucleotides 
(nt) 378-401 (E10, including the first in frame methioninecodon) and nt 
2033-2010 (E11, reverse primer, including the first in frame stop codon), 
respectively, of the rat ERK-3 sequence published by Boulton et al., Cell 
65:663-675 (1991) with single modifications outside the coding region to 
introduce new restriction sites (NcoI in E10 and SalI in E11, 
respectively). E13 was designed as 32 fold degenerate oligonucleotide 
based on the amino acid sequence of rat ERK1, ERK2 and ERK3 corresponding 
to nt 1030-1052 of rat erk3 (Boulton et al., Cell 65:663-675 (1991)). 
A total of 10 pmoles of the labelled oligonucleotides E10, E11 and E13 in 
50 ml hybridization mixture (6.times.SSC, 5.times. Denhardt's solution, 
0.05% SDS (Current Protocols in Molecular Biology, M. Ausubel et al., 
eds., John Wiley & Sons, New York (1988)) were added to replica 
nitrocellulose filters and allowed to hybridize at 42.degree. C. for 2 h. 
The filters were washed in 6.times.SSC, 0.05% SDS three times 10 min first 
at room temperature, then at 42.degree., 46.degree., 48.degree. and 
50.degree. C., respectively. 
Two positive clones were identified by autoradiography and isolated 
following the procedure described in Sambrook et al., 1989. Partial 
dideoxy sequencing (Sanger etal., Proc. Natl. Acad. Sci. USA 74:5463-5467 
(1977)) of the two positive clones using oligonucleotide E13 as primer 
revealed identical nucleotide sequence. The larger clone with an insert of 
about 1900 bp was then fully sequenced. The first methionine precedes an 
open reading frame of 1179 bp encoding a protein of 393 amino acids (FIG. 
1, SEQ ID NO:1 and SEQ ID NO:2) or 43.2 kD molecular weight which shows 
61% similarity (38% identity) to the human ERK1 peptide sequence, 64% 
similarity to the rat ERK1 and ERK2 (39% and 37% identity, respectively) 
and 55% similarity to the rat ERK3 (30% identity) (the comparison program 
uses the algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443-453 
(1970)). The new clone was termed human ERK5 (hERK5). 
Example 2 
Northern Blot Analysis of Human ERK5 
Total RNA from human tissue was isolated by the acid guanidinium 
thiocyanate-phenol-chloroform extraction procedure (Puissant and 
Houdebine, BioTechniques 8:148-149 (1990)). The preparation of poly(A+) 
RNA was performed as described by Aviv and Leder, Proc. Natl. Acad. Sci. 
USA 69:1408-1412 (1972). Five .mu.g of poly(A+) RNA per lane were loaded 
on a 1.2% agarose-2.2M formaldehyde gel and after separation blotted onto 
a nitrocellulose filter using standard techniques (Sambrook et al., 1989). 
A .sup.32 P labelled 1200 bp BamHI fragment of hERK5 in 30 ml of 
hybridization solution (5.times. Denhardt's solution, 5.times.SSC, 5 
.mu.g/ml salmon sperm DNA, 50 mM Na.sub.2 HPO.sub.4 pH 6.8, 1 mM NaH.sub.2 
PO.sub.4 /Na.sub.4 P.sub.2 O.sub.4, 50% formamide) was used for 
hybridization. The .sup.32 P labelling was done using the Random primed 
DNA labelling kit (Bat No. 1004760, Boehringer Mannheim Biochemica) 
according to the manufacturers instruction. Fifty ng of denatured DNA were 
labelled with 50 .mu.Ci .sup.32 PdATP with an average incorporation of 
2.times.10.sup.8 -10.sup.9 cpm/.mu.g DNA. After the labelling reaction 
unincorporated .sup.32 PdATP was removed using a Sephadex G50 column. The 
washed filter was exposed to an X-ray film. Analysis of the Northern Blot 
showed that there is a major transcript of hERK5 of about 1.9 kb (FIG. 2). 
Further, it appears that hERK5 is preferentially expressed in skeletal 
muscle. 
Example 3 
Production of Polyclonal Antibodies Against hERK5 
Antibodies were raised against an E. coli fusion protein composed of the 
carboxy-terminal part of glutathione S-transferase and the last 264 amino 
acids of hERK5 protein. The vector encoding this construct was generated 
by cloning a 1200 bp BamHI-fragment of hERK5 into the pGEX3X plasmid 
(Pharmacia, Uppsala) which upstream of the multiple cloning site carries 
the cDNA for about 250 amino acids (27.5 kD) of glutathione S-transferase 
under the control of the lac promoter. The construct was transformed in E. 
coli 298F' cells (R. du Bridge, Genentech, San Francisco). After induction 
with IPTG (1 mM final concentration) the expressed soluble fusion protein 
was purified on Glutathione-Sepharose 4B (Pharmacia, Uppsala) according to 
the manufacturer's instruction. The apparent molecular weight in SDS-PAGE 
is 56 kD (FIG. 3). The purified fusion protein (100 .mu.g) was 
subcutaneously injected into a female rabbit using Freund's adjuvant as 
described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring 
Harbor, N.Y. (1988). After the second boost, antiserum was collected and 
used in Western blotting. 
For Western blotting total lysate of E. coli clones expressing either the 
pGEX encoded GST protein portion or the hERK-5-GST fusion protein before 
and after induction with IPTG was separated on SDS-PAGE. The bacteria were 
harvested by centrifugation and resuspended in Laemmli sample buffer (4% 
SDS, 125 mM Tris pH 6.8, 10% .beta.-mercapto ethanol, 10% glycerol, 0.02% 
bromphenol blue) to a concentration of 2.times.10.sup.7 cells/.mu.l of 
sample buffer. After boiling for 5 min, 10 .mu.l pd of this SDS lysate 
were applied to SDS-PAGE (10% polyacrylamide), transferred to 
nitrocellulose using standard techniques (Sambrook et al., (1989), 200 mA, 
40 min), blocked with PBS containing 2% nonfat dry milk, 0.02% Tween20, 
washed (PBS 0.02% Tween 20, 0.2% gelatine) and detected with the 
polyclonal antiserum raised against the hERK-GST fusion protein in 
comparison to preimmune serum (both sera diluted 1:5000 in PBS containing 
0.05% Tween 20 and 0.2% gelatine). The second antibody (horseradish 
peroxidase coupled goat anti-rabbit IgG (BioRad, MCtnchen)) was diluted 
1:20000 fold in PBS 0.02% Tween 20, 0.2% gelatine. Peroxidase reaction was 
performed using the ECL kit (Amersham, Braunschweig). The antiserum 
strongly recognizes a band at 56 kD corresponding to the molecular weight 
of the hERK-GST fusion protein which is not detected by the preimmune 
serum. There is no cross reactivity of the anti-hERK-5-GST antiserum with 
the recombinant GST protein portion (FIG. 4). 
Example 4 
Expression of hERK5 in Eukaryotic Cells 
The herk5 cDNA was cloned in the eukaryotic expression vector pcDNAI 
(Invitrogen, San Diego) and hERK-5 was transiently expressed in human 
embryonal kidney fibroblasts (293 cells:ATCC CRL 1573). The 293 cells were 
grown in DMEM with 4.5 mg/ml glucose and 10% FCS. 5.times.10.sup.4 cells 
per 3.5 cm dish were transfected with 10 .mu.g DNA using the 
calcium-phosphate precipitation method described by Chen and Okayama (Mol. 
Cell. Biol. 7:2745-2752 (1987)). After 16 h at 35.degree. C. and 3% 
CO.sub.2 the medium was changed. The cells were transferred to 37.degree. 
C. and 5% CO.sub.2 for additional 16 h, washed off the cultured dish, 
collected by centrifugation, and resuspended in Laemmli sample buffer 
(composition see Example 3, 40 .mu.l per 3.5 cm dish). 
The Western blot of 293 cell SDS-lysate after pcDNAI-herk5 transfection 
shows major bands at 44 kD (which corresponds to the predicted molecular 
weight of hERK5) and 46 kD, probably representing a different 
phosphorylation state of hERK5 protein (FIG. 5). There is no cross 
reactivity with proteins expressed in mock transfected 293 cells. 
Example 5 
Generation of Stable Cell Lines Expressing hERK5 
NIH3T3 cells, immortalized mouse fibroblasts (Jainchill et al., J. Virol. 
4:549-553 (1969)) were grown in DMEM with 4.5 mg/ml glucose and 10% FCS to 
subconfluency and transfected with 20 .mu.g/1.times.10.sup.7 cells of a 
cvn-construct containing the complete hERK-5 cDNA. The cvn vector carries 
the SV40 early promoter, HBV poly A signal as well as a neomycin 
resistance gene which allows selection of transfected cells on G418 
resistance, and the gene for the DHFR which can be used to increase the 
expression of the integrated cDNA by addition of methotrexate at 
concentrations of 100-1000 nM to the culture medium (Rosenthal et al., 
Cell 46:155-169 (1986)). Transfection was performed as described in 
Example 4. After 16 h at 35.degree. C. and 3% CO.sub.2, the medium was 
changed and the cells were grown at 37.degree. C., 5% CO.sub.2 for 
additional 24 h with one medium change after 8 h. The cells were then 
split to different dilutions and grown in 1 mg/ml G418 containing medium 
until cell colonies appeared which were isolated and selected on 
methotrexate growth. 
The expression of hERE was tested in the Western blot of total cell lysate 
using the antibody raised against the hERK-5-GST fusion protein as 
described in Example 3. The blot shows a double band at 44/46 kD in three 
of four cell clones tested corresponding to the stably expressed hERK5 
protein whose expression is increased when the cells are grown on 200 and 
500 nM methotrexate, respectively (FIG. 6). 
All publications mentioned hereinabove are hereby incorporated in their 
entirety by reference. 
While the foregoing invention has been described in some detail for 
purposes of clarity and understanding, it will be appreciated by one 
skilled in the art from a reading of this disclosure that various changes 
in form and detail can be made without departing from the true scope of 
the invention and appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1260 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 34..1215 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GGCTCTGCGGGGTGGGCAGCTCCCGGGCCTGCCATGAGCTCTCCGCCGCCCGGN54 
MetSerSerProProProGly 
15 
GGCAGTGGCTTTTACCGCCAGGAGGTGACCAAGACGGCCTGGGAGGTG102 
GlySerGlyPheTyrArgGlnGluValThrLysThrAlaTrpGluVal 
10 1520 
CGCGCCGTGTACCGGGACCTGCAGCCCGTGGGCTCGGGCGCCTACGGC150 
ArgAlaValTyrArgAspLeuGlnProValGlySerGlyAlaTyrGly 
25 3035 
GCGGTGTGCTCGGCCGTGGACGGCCGCACCGGCGCTAAGGTTGCCATC198 
AlaValCysSerAlaValAspGlyArgThrGlyAlaLysValAlaIle 
4045 5055 
AAGAAGCTGTATCGGCCCTTCCAGTCCGAGCTGTTCGCCAAGCTCGCC246 
LysLysLeuTyrArgProPheGlnSerGluLeuPheAlaLysLeuAla 
60 6570 
TACCGCGAGCTGCGCCTGCTCAAGCACATGCGCCACGAGAACGTGATC294 
TyrArgGluLeuArgLeuLeuLysHisMetArgHisGluAsnValIle 
75 8085 
GGGCTGCTGGACGTATTCACTCCTGATGAGACCCTGGATGACTTCACG342 
GlyLeuLeuAspValPheThrProAspGluThrLeuAspAspPheThr 
90 95100 
GACTTTTACCTGGTGATGCCGTTCATGGGCACCGACCTGGGCAAGCTC390 
AspPheTyrLeuValMetProPheMetGlyThrAspLeuGlyLysLeu 
105 110115 
ATGAAACATGAGAAGCTAGGCGAGGACCGGATCCAGTTCCTCGTGTAC438 
MetLysHisGluLysLeuGlyGluAspArgIleGlnPheLeuValTyr 
120125 130135 
CAGATGATGAAGGGGCTGAGGTATATCCACGCTGCCGGCATCATCCAC486 
GlnMetMetLysGlyLeuArgTyrIleHisAlaAlaGlyIleIleHis 
140 145150 
AGAGACCTGAAGCCCGGCAACCTGGCTGTGAACGAAGACTGTGAGCTG534 
ArgAspLeuLysProGlyAsnLeuAlaValAsnGluAspCysGluLeu 
155 160165 
AAGATCCTGGACTTCGGCCTGGCCAGGCAGGCAGACAGTGAGATGACT582 
LysIleLeuAspPheGlyLeuAlaArgGlnAlaAspSerGluMetThr 
170 175180 
GGGTACGTGGTGACCCGGTGGTACCGGGCTCCCGAGGTCATCTTGAAT630 
GlyTyrValValThrArgTrpTyrArgAlaProGluValIleLeuAsn 
185 190195 
TGGATCGCGTACACGCAGACGGTGGACATCTGGTCTGTGGGCTGCATC678 
TrpIleAlaTyrThrGlnThrValAspIleTrpSerValGlyCysIle 
200205 210215 
ATGGCGGAGATGATCACAGGCAAGACGCTGTTCAAGGGCAGCGACCAC726 
MetAlaGluMetIleThrGlyLysThrLeuPheLysGlySerAspHis 
220 225230 
CTGGACCAGCTGAAGGAGATCATGAAGGTGACGGGGACGCCTCCGGCT774 
LeuAspGlnLeuLysGluIleMetLysValThrGlyThrProProAla 
235 240245 
GAGTTTGTGCAGCGGCTGCAGAGCGATGAGGCCAAGAACTACATGAAG822 
GluPheValGlnArgLeuGlnSerAspGluAlaLysAsnTyrMetLys 
250 255260 
GGCCTCCCCGAATTGGAGAAGAAGGATTTTGCCTCTATCCTGACCAAT870 
GlyLeuProGluLeuGluLysLysAspPheAlaSerIleLeuThrAsn 
265 270275 
GCAAGCCCTCTGGCTGTGAACCTCCTGGAGAAGATGCTGGTGCTGGAC918 
AlaSerProLeuAlaValAsnLeuLeuGluLysMetLeuValLeuAsp 
280285 290295 
GCGGACATCAGGTTGACTGCAGGCGAGTTTCTTTCCCATCCCTACTTC966 
AlaAspIleArgLeuThrAlaGlyGluPheLeuSerHisProTyrPhe 
300 305310 
GAGTCCCTGCACGACACGGAAGATGAGCCCCAGGTCCAGAAGTATGAT1014 
GluSerLeuHisAspThrGluAspGluProGlnValGlnLysTyrAsp 
315 320325 
GACTCCTTTGACTACTTTGACCGCACACTGGATGAATGGAAGCCGTGT1062 
AspSerPheAspTyrPheAspArgThrLeuAspGluTrpLysProCys 
330 335340 
TACTTACAAAGAGGTGCTCAGCTTCAAGCCTCCCCGGCAGCTGGGGGC1110 
TyrLeuGlnArgGlyAlaGlnLeuGlnAlaSerProAlaAlaGlyGly 
345 350355 
CAGGGTCTCCAAGGAGACGCCTCTGTGAAGATCTCTGGGCTCCGGGGT1158 
GlnGlyLeuGlnGlyAspAlaSerValLysIleSerGlyLeuArgGly 
360365 370375 
GGCAGTGAGGACCACCTTCACCTTCCACCTGAGAGGGGACTCTCGTTG1206 
GlySerGluAspHisLeuHisLeuProProGluArgGlyLeuSerLeu 
380 385390 
CCACCTTGACCTTGGCTGGGGCTTGCATCCCAAGGCATCCATCAGAGCAGACGC1260 
ProPro 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 393 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetSerSerProProProGlyGlySerGlyPheTyrArgGlnGluVal 
151015 
ThrLysThrAlaTrpGluValAr gAlaValTyrArgAspLeuGlnPro 
202530 
ValGlySerGlyAlaTyrGlyAlaValCysSerAlaValAspGlyArg 
3540 45 
ThrGlyAlaLysValAlaIleLysLysLeuTyrArgProPheGlnSer 
505560 
GluLeuPheAlaLysLeuAlaTyrArgGluLeuArgLeuLeuLysHis 
65707580 
MetArgHisGluAsnValIleGlyLeuLeuAspValPheThrProAsp 
859095 
GluT hrLeuAspAspPheThrAspPheTyrLeuValMetProPheMet 
100105110 
GlyThrAspLeuGlyLysLeuMetLysHisGluLysLeuGlyGluAsp 
115 120125 
ArgIleGlnPheLeuValTyrGlnMetMetLysGlyLeuArgTyrIle 
130135140 
HisAlaAlaGlyIleIleHisArgAspLeuLy sProGlyAsnLeuAla 
145150155160 
ValAsnGluAspCysGluLeuLysIleLeuAspPheGlyLeuAlaArg 
165170 175 
GlnAlaAspSerGluMetThrGlyTyrValValThrArgTrpTyrArg 
180185190 
AlaProGluValIleLeuAsnTrpIleAlaTyrThrGlnThr ValAsp 
195200205 
IleTrpSerValGlyCysIleMetAlaGluMetIleThrGlyLysThr 
210215220 
LeuPheLysGlyS erAspHisLeuAspGlnLeuLysGluIleMetLys 
225230235240 
ValThrGlyThrProProAlaGluPheValGlnArgLeuGlnSerAsp 
24 5250255 
GluAlaLysAsnTyrMetLysGlyLeuProGluLeuGluLysLysAsp 
260265270 
PheAlaSerIleLeuThrAsnAl aSerProLeuAlaValAsnLeuLeu 
275280285 
GluLysMetLeuValLeuAspAlaAspIleArgLeuThrAlaGlyGlu 
290295 300 
PheLeuSerHisProTyrPheGluSerLeuHisAspThrGluAspGlu 
305310315320 
ProGlnValGlnLysTyrAspAspSerPheAspTyrPheAspArg Thr 
325330335 
LeuAspGluTrpLysProCysTyrLeuGlnArgGlyAlaGlnLeuGln 
340345350 
AlaS erProAlaAlaGlyGlyGlnGlyLeuGlnGlyAspAlaSerVal 
355360365 
LysIleSerGlyLeuArgGlyGlySerGluAspHisLeuHisLeuPro 
370 375380 
ProGluArgGlyLeuSerLeuProPro 
385390 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AAGGGTTTTACCATGGCAGAGAAA24 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
M etAlaGluLys 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TTAACTTGTCGACTACGTCAGCAG 24 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ACTATGTTGGGTCTGCTAGGGCTGCATC 28