WO1992020365A1 - Therapeutic and diagnostic methods based on neurotrophin-4 expression - Google Patents

Therapeutic and diagnostic methods based on neurotrophin-4 expression Download PDF

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WO1992020365A1
WO1992020365A1 PCT/US1992/004266 US9204266W WO9220365A1 WO 1992020365 A1 WO1992020365 A1 WO 1992020365A1 US 9204266 W US9204266 W US 9204266W WO 9220365 A1 WO9220365 A1 WO 9220365A1
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arg
ser
seq
thr
gly
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PCT/US1992/004266
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French (fr)
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Finn Hallbook
Carlos Fernando Ibanez Moliner
Hakan Bengt Persson
Nancy Ip
George D. Yancopoulos
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Regeneron Pharmaceuticals, Inc.
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Priority to JP5500288A priority Critical patent/JPH08510987A/en
Priority to AU23021/92A priority patent/AU674659B2/en
Publication of WO1992020365A1 publication Critical patent/WO1992020365A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to
  • neurotrophin-4 (NT-4), a newly characterized member of the BDNF/NGF/NT-3 gene family and the therapeutic and diagnostic methods of utilizing neurotrophin-4 in the treatment of neurological disorders.
  • the nerve growth factor family includes ⁇ -nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), also known as hippocampus-derived neurotrophic factor (HDNF).
  • NGF ⁇ -nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • HDNF hippocampus-derived neurotrophic factor
  • BDNF Brain-derived neurotrophic factor
  • NT-3 has been isolated from mouse (Hohn et al., 1990, Nature, 344: 339-341), rat (Maisonpierre et al., 1990, Science 247: 1446- 1451; Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5454-5458), and human (Rosenthal et al., 1990, Neuron 4: 767-773), using degenerate oligonucleotides based on the sequence similarity between the other two factors. The three factors show approximately 55% amino acid similarity to each other, and most sequence differences are present in five regions that contain amino acid motifs characteristic of each protein. The neurotrophic activity in vitro of two of these
  • NGF supports the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons (reviewed in Thoenen and Barde, 1980, Physiol. Rev., 60: 1284-1325; Levi-Montalcini, 1987, Science, 237: 1154-1162). No activity has been seen for BDNF in peripheral sympathetic neurons, but this factor supports in vivo the survival of both placode and neural crest-derived sensory neurons (Hofer and Barde, 1988, Nature, 331: 261-262). The neurons sensitive to NT-3 in vivo remain to be identified.
  • NT-3 exerts both specific and overlapping neurotrophic activities also in vivo (Hohn et al., 1990, Nature, 344: 339-341; Maisonpeirre et al., 1990, Science, 247: 1446-1451; Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458;
  • NGF Basal forebrain cholinergic neurons
  • NGF-R NGF receptor
  • the NGF-R shows a low but significant sequence similarity to the
  • NGF-R can occur in two apparent states, known as the low and high affinity states (Sutter, et al., 1979, J. Biol. Chem., 254: 5972-5982; Landreth and Shooter, 1980, Proc. Natl. Acad. Sci.
  • the gene for the NGF-R appears to encode a protein that forms part of both the low and the high affinity states of the receptor (Hempstead et al., 1989, Science, 243: 373-375), though only the high affinity receptor has been proposed to mediate the biological activity of NGF.
  • Both BDNF Rosriguez-Tebar et al., 1990, Neuron, 4: 487-492
  • NT-3 Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458
  • the low affinity NGF-R may be, in an as yet unknown way, involved in mediating the biological effects of all three factors.
  • NGF and its receptor have been shown to be synthesized in the target area and in the responsive neurons
  • NGF-R mRNA is maximally expressed at early embryonic stages prior to neuronal innervation (Ernfors et al., 1988, Neuron, 1, 983-996), and in the E8 chick embryo high levels of NGF-R mRNA have been detected in the mesenchyme, somites, and neural tube cells (Hallbook et al., 1990, Development, 108: 693-704; Heuer et al., 1990, Dev. Biol., 137: 287-304; Heuer 1990, Neuron, 5: 283-296).
  • NGF neurotrophic factor
  • BDNF and NT-3 mRNA are maximally expressed at E4,5, and BDNF has been shown to control the differentiation of avian neural crest cells in vitro (Kalcheim and Gendreau, 1988, Dev. Brain Res., 41: 79-86).
  • NGF neurotrophic factor
  • NGF mRNA is expressed in spermatocytes and early spermatids in the adult rat testis (Ayer-LeLievre et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2628-2632), and the NGF protein is present in germ cells of all stages from spermatocytes to spermatozoa (Olson et al., 1987, Cell Tissue Res., 248: 275-286; Ayer-LeLievre et al., 1988a, Proc. Natl. Acad. Sci. USA 85: 2628-2632).
  • NGF-R mRNA has also been detected in the adult rat testis, where it is expressed in Sertoli cells under negative control of testosterone, and in the testis NGF has been suggested to control meiosis and spermiation (Persson et al., 1990, Science, 247: 704-707).
  • the present invention relates to
  • NT-4 neurotrophin-4
  • the present invention provides for nucleic acid molecules encoding NT-4.
  • Such molecules may comprise a sequence substantially as set forth for
  • the present invention also provides for protein or peptide molecules which comprise a sequence substantially as set forth for NT-4 in Figure 2 [SEQ ID NO:22 (NT-4, viper), SEQ ID NO:23 (NT-4, Xenopus)], Figure 4 (SEQ ID NO:44), Figure 8 (SEQ ID NO:50),
  • Figure 20 (SEQ ID NO:94), or Figure 21 (SEQ ID NO:117) or may comprise a sequence that is at least about seventy percent homologous to such sequence.
  • the present invention further provides for expression of biologically active NT-4 molecules in prokaryotic and eukaryotic systems.
  • the present invention further provides for the production of NT-4 in quantities sufficient for therapeutic and diagnostic applications.
  • anti-NT-4 antibodies may be utilized in therapeutic and diagnostic applications.
  • the present invention further provides for therapeutic and diagnostic applications based on NT-4 expression by disclosing detectable levels of NT-4 expression in human skeletal muscle, prostate, thymus and testes.
  • FIGURE 1 Alignments of DNA sequences of the isolated fragments coding for NGF, BDNF, NT-3 and the novel neurotrophic factor NT-4 from different species.
  • preproNGF molecule preproNGF molecule.
  • the hatched box indicates the signal sequence (SS), black bars denote proteolytic cleavage sites and the shaded box represents the mature NGF.
  • Regions used for the degenerate primers are indicated by arrows.
  • the upstream primer was from the region coding for lysine 50 to threonine 56 and the downstream primer includes tryptophan 99 to aspartic acid 105.
  • the amplified region comprises DNA sequences from base pair (bp) 168 to 294 in the mature NGF molecules and in all members of the NGF family described so far, this region is located in one exon.
  • SEQ ID NO: 3 (NGF, human), SEQ ID NO:4 (NGF, rat), SEQ ID NO: 5 (NGF, chicken), SEQ ID NO: 6 (NGF, viper), SEQ ID NO:7 (NGF, Xenopus).
  • SEQ ID NO: 8 (NGF, salmon), SEQ ID NO: 9 (BDNF, human), SEQ ID NO: 10 (BDNF, rat), SEQ ID NO: 11 (BDNF, chicken), SEQ ID NO:12 (BDNF, viper), SEQ ID NO: 13 (BDNF, Xenopus..
  • SEQ ID NO: 14 (BDNF, salmon), SEQ ID NO:15 (BDNF, ray), SEQ ID NO: 16 (NT-3, human), SEQ ID NO: 17 (NT-3, rat), SEQ ID NO: 18 (NT-3, chicken), SEQ ID NO:19 (NT-3,
  • FIGURE 2 Alignment of amino acid sequences deduced for NGF, BDNF, NT-3 and NT-4 from different species . The numbering of the amino acids
  • SEQ ID NO:22 (NT-4, viper), SEQ ID NO:23 (NT-4, Xenopus), SEQ ID NO:24 (NGF, human), SEQ ID NO:25 (NGF, rat), SEQ ID NO:26 (NGF, chicken), SEQ ID NO:27 (NGF, viper), SEQ ID NO:28 (NGF, Xenopus) .
  • SEQ ID NO:29 (NGF, salmon), SEQ ID NO:30 (BDNF, human), SEQ ID NO:31 (BDNF, rat), SEQ ID NO:32 (BDNF, chicken), SEQ ID NO:33 (BDNF, viper), SEQ ID NO:34 (BDNF, Xenopus), SEQ ID NO:35 (BDNF, salmon), SEQ ID NO:36 (BDNF, ray), SEQ ID NO:37 (NT-3, human), SEQ ID NO: 38 (NT-3, rat), SEQ ID NO:39 (NT-3, chicken), SEQ ID NO:40 (NT-3,
  • FIGURE 3 Deduced phylogeny of members of the NGF
  • Phylogenetic trees showing speciation of NGF (A), BDNF (B), and NT-3 (C) were constructed using analysis of nucleotide sequences. Human NT-3 was used as a reference point in (A) and (B), human NGF and human BDNF were used in (C).
  • the scale bar in (A) represents a branch length corresponding to a relative difference score of 20. The same scale was used in (B) and (C).
  • the data were compiled from deduced amino acid sequences.
  • the scale bar represents a branch length of 20. All trees shown are
  • FIGURE 4 Sequence of Xenopus NT-4 and Comparison to
  • NGF NGF, BDNF, and NT-3.
  • a potential translation start site is boxed.
  • a putative signal cleavage site is indicated by the arrow labeled SC. Amino acids within the signal sequence that are identical between
  • Xenopus NT-4 and pig and rat BDNF are indicated with stars.
  • a consensus sequence for N- glycosylation is underlined, and the arrow indicates the presumptive start of the mature NT- 4 protein. (SEQ ID NO: 43 and SEQ ID NO: 44)
  • FIGURE 5 Transient expression of the Xenopus NT-4
  • NT-4 gene Shown is an autoradiograph of the dried gel after an overnight exposure to X-ray film.
  • FIGURE 6 Stimulation of neurite outgrowth from
  • A, B, and C Neurite outgrowth elicited in dorsal root ganglia with recombinant NT-4 protein (A), recombinant NGF (B), and BDNF protein (C) .
  • D The response of dorsal root ganglia to conditioned medium from mock-transfected cells.
  • E and F Stimulation of neurite outgrowth from sympathetic ganglia in response to NT-4 (E) or NGF (F).
  • FIGURE 7 Detection of NT-4 mRNA in different Xenopus tissues.
  • the lane labeled CNS includes brain and spinal cord.
  • FIGURE 8 Nucleotide sequence of Xenopus NT-4 with
  • restriction endonuclease cleavage sites SEQ ID NO:49 and SEQ ID NO:50.
  • FIGURE 9 NT-4 mRNA expression in the Xenopus laevis
  • FIGURE 10 Bright-field illumination of emulsion
  • FIGURE 11 Levels of NT-4 mRNA in oocytes at
  • autoradiographs (shown in figure 10) of sections hybridized with the Xenopus NT-4 mRNA specific probe were used to count the number of grains over an area unit.
  • the area unit chosen was about one hundredth of a stage I oocyte. Fifteen area units were analyzed in 10 different oocytes of the indicated stages. Error bars show S.D.
  • FIGURE 12 Northern blot analysis of NT-4 mRNA
  • the oocytes were then grouped in the indicated groups following the stages described by Dumont,
  • HincII fragment from the 3•exon of the Xenopus NT- 4 gene.
  • the filter was washed at high stringency and exposed for five days to a X-ray film. Note the marked decreased in the level of NT-4 mRNA in stages V and VI oocytes.
  • FIGURE 13 (A) The xNT-4 partial amino acid sequence
  • oligonucleotides were synthesized and utilized to prime the amplification of human and rat genomic DNA via the polymerase chain reaction.
  • a set of degenerate oligonucleotides to primer 2Z represent amino acids 184-189 of rBDNF (SEQ ID NO:52).
  • the partial Xenopus NT-4 amino acid sequence represented is from amino acid 167 - amino acid 223 , as described in Figure 4, supra.
  • Oligonucleotide 3Z in Figure 13 is comprised of a mixture of 3Z and 3Z' in order to allow for the degeneracy of the serine codon.
  • 2Y SEQ ID NO:53
  • 2Z SEQ ID NO:54
  • FIGURE 14 DNA sequence of the isolated fragment
  • FIGURE 15 DNA sequence of the isolated fragment
  • FIGURE 16 Alignment of amino acid sequences deduced from representative neurotrophins. Amino acids are indicated using the single letter code.
  • FIGURE 17 DNA sequence of an isolated fragment encoding a portion of human NT-4 (SEQ ID NO: 69). The predicted peptide encoded by the 192 bp hNT-4 nucleic acid fragment is represented by the single letter code (SEQ ID NO: 70). Sequence inside brackets is part of PCR primer.
  • the primer termed 11NT4-5''' [containing a sequence (SEQ ID NO:73) encoding DNAEEG (SEQ ID NO:74)] was utilized with the 3' primer, 4Z (SEQ ID NO:58), to obtain a fragment of 162 bp (plus bp of cloning tail).
  • the 162 bp PCR fragment was then utilized in a patch PCR reaction using our previously utilized upstream PCR
  • FIGURE 18 DNA sequence of the portion of the isolated human genomic phage clone 7-2 encoding human NT-4 (SEQ ID NO:75).
  • the predicted hNT-4 protein encoded by the genomic clone 7-2 is represented by the one-letter symbols for amino acids (SEQ ID NO:76).
  • the boxed region represents the predicted cleavage site of the hNT-4 preprotein. Arrows indicate conserved residues in the presequence.
  • the underlined region (N-R-S) represents a
  • the circled region represents the initiating
  • the splice acceptor site is located at base pair 461-462 (AG) of SEQ ID NO:75,
  • FIGURE 19 Alignment of amino acid sequences deduced from representative neurotrophins (SEQ ID NOS.
  • Amino acids are indicated using the single letter code. Amino acids identical to those encoded by the human genomic phage clone 7-2 (SEQ ID NO: 77) are indicated with an asterik. Dashed lines represent breaks in homologous amino acids as compared to the protein encoded by SEQ ID NO: 77.
  • FIGURE 20 DNA sequence of the isolated fragment
  • FIGURE 21 DNA sequence of the isolated fragment
  • FIGURE 22 Northern blot analysis of human NT-4 mRNA expression.
  • Tissue specific mRNA from human was purchased from Clontech.
  • RNA's (10 ⁇ g) were fractionated by electrophoresis through a 1% agarose-formaldehyde gel followed by capillary transfer to a nylon membrane (MagnaGraph, Micron Separations Inc.) with 10X SSC (pH 7).
  • RNAs were UV-cross-linked to the membranes by exposure to ultraviolet light (Stratlinker, stratagene, Inc.) and hybridized at 65oC with the radiolabeled probe (a 680bp Xhol-Notl fragment containing the radiolabeled probe (a 680bp Xhol-Notl fragment containing the
  • Lane 1 fetal liver poly(A) + mRNA
  • Lane 2 fetal brain poly(A) + mRNA
  • Lane 3 prostate poly A + mRNA
  • Lane 4 muscle poly(A) + mRNA
  • Lane 5 fetal liver poly(A) + mRNA
  • FIGURE 23 COS supernatants from transfected cell
  • FIGURE 24 COS supernatants from Q1 (pCMX-HG7-2Q), and M (pCMX-HG7-2M) cell lines were tested for their survival-promoting activity on DRG
  • volumes tested ranged from 5 ⁇ l to 250 ⁇ l in a total volume of 2 ml.
  • FIGURE 25 Motor neuron enriched cultures isolated
  • the present invention provides for NT-4 genes and proteins. It is based, at least in part, on the cloning, characterization, and expression of the NT-4 gene.
  • Such molecules comprise a sequence substantially as set forth in Figure 1 (SEQ ID NO:l) for viper, Figure 1 (SEQ ID NO: 2), Figure 4 (SEQ ID NO:43) or Figure 8 (SEQ ID NO:49) for Xenopus NT-4, Figure 14 (SEQ ID NO: 61) for rat NT-4, Figure 15 (SEQ ID NO:63), Figure 17 (SEQ ID NO:69) or Figure 18 (SEQ ID NO: 75) for human NT-4, Figure 20 (SEQ ID NO: 93) and Figure 21 (SEQ ID NO: 116) for a human NT-4 like sequence, or a sequence that is at least about seventy percent homologous to any such sequence, in which homology refers to sequence identity (e.g. a sequence that is 70 percent homologous to a second sequence shares 70 percent of the same nucleotide residues with the second sequence).
  • sequence identity e.g. a sequence that is 70 percent homologous to a second sequence shares 70 percent of the same nucleo
  • Example Section 8 and Figure 15 SEQ ID NO: 63, SEQ ID NO: 64
  • the nucleotide and amino acid sequence for a portion of a human neurotrophin molecule is determined.
  • Example Section 9 and Figure 17 SEQ ID NO: 69, SEQ ID NO:70
  • Figure 18 SEQ ID NO: 75, SEQ ID NO:76
  • neurotrophin molecule is determined.
  • SEQ ID NO: 93, SEQ ID NO: 94 and Figure 21 SEQ ID NO: 116, SEQ ID NO: 117 herein, the nucleotide and amino acid sequence for a portion of a human genomic phage clones, 2-1 and 4-2,
  • human neurotrophin-4 is referred to herein as human neurotrophin-4, it should be understood that such a molecule may be the human homologue of the
  • Xenopus neurotrophin-4 described herein, or
  • rat NT-4 may be the rat homologue of NT-4, or alternatively, a distinct yet homologous neurotrophin molecule.
  • the methods and compositions of the present invention do not depend on any single nomenclature.
  • the present invention also provides for substantially purified NT-4 protein or peptide
  • Such molecules may comprise a sequence substantially as set forth in Figure 2, (SEQ ID NO:1 and SEQ ID NO: 2), Figure 4 (SEQ ID NO:44) Figure 8 (SEQ ID NO:50), Figure 14 (SEQ ID NO:62), Figure 15 (SEQ ID NO:64) Figure 17 (SEQ ID NO:70), Figure 18
  • a substantially purified protein or peptide comprises the sequence KCNPSGSTTR (SEQ ID NO: 96).
  • a substantially purified peptide or protein comprises the sequence RGCRGVD (SEQ ID NO: 97). In yet another embodiment of the invention, a substantially purified peptide or protein comprises the sequence KQWIS (SEQ ID NO: 98). In a further embodiment of the invention, a substantially purified peptide or protein comprises the sequence KQSYVR (SEQ ID NO: 99). In yet another embodiment of the invention, a substantially purified peptide or protein comprises the sequence GPGXGGG (SEQ ID NO: 100), where X represents one of the set of 20 amino acids. In a related embodiment of the invention, a substantially purified peptide or protein comprises the sequence GPGVGGG (SEQ ID NO: 101) or GPGAGGG (SEQ ID NO: 102).
  • a substantially purified peptide or protein comprises the sequence ESAGE (SEQ ID NO: 103). In yet a further embodiment of the invention, a substantially purified peptide or protein comprises the sequence DNAEE (SEQ ID NO: 104).
  • proteins and peptides of the invention may be produced by chemical synthesis using standard techniques or may be produced using the NT-4-encoding nucleic acid molecules of the invention, using
  • prokaryotic or eukaryotic expression systems known to one skilled in the art, such as those described in PCT application PCT/US90/04916, filed August 29, 1990, published as WO 91/03569, which is incorporated by reference in its entirety herein, or as exemplified infra (see Section 6.2.4., infra, and Figure 5) for transient expression in COS cells.
  • the present invention also provides for the use of NT-4 in promoting the growth and/or survival of cells of the nervous system, in particular, but not limited to, cells of dorsal root ganglion or neural placode derivatives (see Section 6.2.4., infra, and Figure 6, for example).
  • the present invention also provides for portions of NT-4 nucleic acid or amino acid sequence, substantially as set forth for NT-4 in Figure 1, 2, 4, 8, 14, 15, 17, 18, 20 or 21 (SEQ ID NO's listed,
  • the present invention further provides for a eukaryotic or prokaryotic cell that contains
  • the present invention also provides for recombinant NT-4 protein or peptide that is produced by inserting recombinant nucleic acid encoding NT-4 into a cell (e.g., by transfection, transduction, electroporation, microinjection, etc.) under conditions which permit expression of NT-4 and then isolating NT-4 from the cell.
  • the present invention provides for molecules produced by PCR using, for example, the following oligonucleotides as primers:
  • 3'CGATTGTTTGGCTTTACA (SEQ ID NO: 108) for rat NT-4, and using any suitable genomic or cDNA as template.
  • these primers may be used in conjunction with human cDNA as template to produce fragments of the human NT-4 gene that are suitable for cloning.
  • immunogenicity or antigenicity can be used, for example
  • peptides related to NT-4 can be tested for the desired activity by procedures known in the art.
  • NT-4 related derivatives, analogues, and peptides of the invention can be produced by various methods known in the art.
  • the manipulations which result in their production can occur at the gene or protein level.
  • the cloned NT-4 gene can be modified by any of numerous strategies known in the art (Maniatis, T., 1982, Molecular Cloning, A
  • NT-4 sequence can be cleaved at appropriate sites with restriction
  • the NT-4 gene can be mutated in vitro or in vivo, to create and/or destroy
  • mutagenesis Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551), use of TAB ® linkers (Pharmacia), etc.
  • the prepro or mature coding region of NT-4 may be utilized to construct neurotrophin based chimeric genes.
  • neurotrophin genes including but not limited to NGF, BDNF and NT-3, can provide the prepro region for construction of neurotrophin prepro/NT-4 mature coding region chimeric genes.
  • NT-4 sequence may also be made at the protein level. Any of numerous
  • analogues and peptides related to NT-4 can be chemically synthesized.
  • a peptide corresponding to a portion of NT-4 which mediates the desired neurotrophic activity can be synthesized by use of a peptide synthesizer.
  • the present invention further provides for a method of treating fertility disorders related to ovarian/oocyte dysfunction.
  • NT-4 is involved in the maturation of oocytes.
  • the discussion of Section 7.3 demonstrates that NT-4 is produced by oocytes, is concentrated in immature rather than mature oocytes, and appears to play a role in oogenesis.
  • the putative function of NT-4 protein in the ovary appears to be coupled to events occurring in the pre-vitellogenic and early/mid vitellogenic oocyte.
  • NT-4 has been established that other members of the BDNF/NGF/NT-3 gene family, in particular NGF, are involved in meiotic maturation (Nebrada et al., 1991, Science, 252: 558-563). Because NT-4 has been established that other members of the BDNF/NGF/NT-3 gene family, in particular NGF, are involved in meiotic maturation (Nebrada et al., 1991, Science, 252: 558-563). Because NT-4 has been established that other members of the BDNF/NGF/NT-3 gene family, in particular NGF, are involved in meiotic maturation (Nebrada et al., 1991, Science, 252: 558-563). Because NT-4 has been established that other members of the BDNF/NGF/NT-3 gene family, in particular NGF, are involved in meiotic maturation (Nebrada et al., 1991, Science, 252: 558-563). Because NT-4 has been established that other members of the BDNF
  • NT-4 can be exploited to provide a method for treating infertility disorders and/or other ovarian
  • a method of treating infertility disorders and/or other ovarian dysfunctions comprising administering a therapeutically effective amount of NT-4 or an NT-4 related peptide in a pharmaceutically effective carrier is provided.
  • a therapeutically effective amount is one which induces proper maturation of an oocyte and/or ovulation.
  • a therapeutically effective amount is one which induces proper maturation of an oocyte and/or ovulation.
  • therapeutically effective dose may be one sufficient to maintain circulating serum levels of NT-4 at a concentration of from about 1 to 100 ⁇ 10 -10 M.
  • carriers may be administered by any appropriate route including but not limited to injection (e.g.,
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • NT-4 or NT-4 peptide may be used in any suitable pharmacological carrier, linked to a carrier or targeting molecule (e.g., antibody, hormone, growth factor, etc.) and/or incorporated into liposomes, microcapsules, and controlled release preparation prior to administration in vivo.
  • a carrier or targeting molecule e.g., antibody, hormone, growth factor, etc.
  • Each respective mammalian NT-4 DNA sequence can be utilized as a 32 P-labelled probe to isolate a respective genomic and cDNA clone via the procedures outlined in the Materials and Methods portion Section 8, infra.
  • the rat NT-4 and human NT-4 gene fragments may be utilized directly (as 32 P-labelled probes) or indirectly (to deduce a PCR strategy as described infra) to isolate other mammalian NT-4 genomic and cDNA clones, based on the unique nature of the 7 amino acid insertion in the rNT-4 and hNT-4 coding region, or other unique aspects of the rat or human NT-4 coding region.
  • any mammalian NT-4 gene isolated via the information disclosed by the rat and human NT-4 sequence may be utilized in, although is not limited to, the various manipulations discussed for Xenopus NT-4.
  • the proteins and peptides of mammalian NT-4, subsequent to characterization of the full length gene as discussed in Example Section 9, may be produced using the respective mammalian NT-4 molecules in a prokaryotic or a eukaryotic expression system known to one skilled in the art, such as those described in PCT application PCT/US90/04916, filed August 29, 1990, published as WO91/03569, or as exemplified infra (see Section 6.2.4., supra, and Figure 5) for transient expression in COS cells.
  • Additional functions for mammalian NT-4 include, but are not limited to: the promotion of growth and/or survival of cells of the nervous system, in particular, but not limited to, cells of dorsal root ganglion or neural placode derivatives (see Section 6.2.4., and Figure 6, for example), treating fertility disorders related to ovarian/oocyte dysfunction (see Section 7), the
  • ovarian dysfunction associated with oogenesis see Section 6
  • the treatment of motor neuron diseases see Section 10
  • the treatment of an epitheliac hyperplasia such as benign prostatic hypertrophy
  • the treatment of impotence as related to prostate gland function see Section 10.
  • the therapeutically effective amounts of mammalian NT-4 for the treatment of said disorders as formulated in suitable pharmacological carriers to provide a pharmaceutical composition may be
  • injection e.g., intravenous,
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • rat, human or other mammalian NT-4 or NT-4 peptide may be used in any suitable pharmacological carrier, linked to a carrier or targeting molecule (e.g., antibody, hormone, growth factor, etc.) and/or incorporated into liposomes, microcapsules, and controlled release preparation prior to administration in vivo.
  • a carrier or targeting molecule e.g., antibody, hormone, growth factor, etc.
  • proteins, peptide fragments, or derivatives produced therefrom, as well as antibodies directed against NT-4 protein, peptides, or derivatives, may be utilized to diagnose or monitor the progression of diseases and disorders of the nervous system which are associated with alterations in the pattern of NT-4 expression. Such alterations can be a decrease or increase
  • NT-4 genes and related nucleic acid sequences and subsequences, including complementary sequences may be used in diagnostic hybridization assays.
  • the NT-4 nucleic acid sequences, or subsequences thereof comprising about 15 nucleotides, can be used as hybridization probes.
  • Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in NT-4 levels.
  • the data presented in Example Section 10 discloses tissue specific expression of human NT-4 in skeletal muscle as well as the prostate gland, thymus and testes. The level of expression of human NT-4 in the muscle tissue may be indicative of the presence or absence of neuronal degradation. Therefore, poly(A) + mRNA or total RNA from a tissue sample of a patient could be assayed for the presence of human NT-4 mRNA in
  • Example Section 10 discloses tissue specific expression of NT-4 in the human prostate gland. DNA sequences encoding NT-4 or a portion thereof, as well as NT-4 protein or a peptide may be useful as a therapeutic agent to treat prostate disease.
  • diagnostic assays can be immunoassays.
  • antibodies can be used in immunoassays to quantitate the level of NT-4 in a sample from a patient, in order to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in NT-4 levels.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as
  • radioimmunoassays ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and
  • immunoelectrophoresis assays to name but a few.
  • Anti NT-4 antibody fragments or derivatives containing the binding domain may also be used in such assays.
  • idiotype of the molecule can be generated by known techniques.
  • fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • kits are also provided.
  • a kit can comprise in a suitable
  • the probe is an antibody specific for NT-4. In another embodiment, the probe is a nucleic acid
  • the probe can be detectably labeled; alternatively, the kit can further comprise a labeled specific binding partner for the probe.
  • hybridization assays and immunoassays can also be used to quantitate NT-4 levels as an indication of therapeutic efficacy, by comparing the levels in patient samples before and after treatment of a disorder, particularly, a motor neuron disease.
  • NT-4 mRNA in muscle tissue leads to potential methods of treating motor neuron disorders comprising administering to a patient in need of such treatment an effective amount of an NT-4 factor to support the survival, growth, and/or differentiation of motor neurons.
  • NT-4 mRNA in human muscle suggests further avenues for diagnosing and treating neuron disorders.
  • Retrograde axonal transport may be characteristic of NT-4. The specific retrograde transport of NT-4 can be used to indicate whether neurons are responsive to NT-4 in normal or diseased states.
  • the present invention provides for a method of diagnosing NT-4 related peripheral nervous system disorders comprising injecting a detectably labeled NT-4 protein or peptide into a peripheral nerve and determining whether the labeled NT-4 protein or peptide is retrogradely transported, in which a failure to be retrogradely transported positively correlates with lack of responsiveness to NT-4 and indicates the presence of a peripheral nervous system disorder that is NT-4 related.
  • Analogous methods may be used to diagnose a central nervous system disorder. Evaluation of retrograde transport may be performed by any method known in the art, including but not limited to MRI, CAT, or scintillation scanning. Such methods may be used to identify the location of a nervous system lesion, as retrograde transport should
  • kits for such retrograde evaluation comprising in a container a detectably labeled NT-4 protein, derivative or
  • Such a label can be a radioactive isotope, or other label known in the art.
  • the present invention may be utilized to treat diseases and disorders of the nervous system which may be associated with alterations in the pattern of NT-4 expression or which may benefit from exposure to NT-4 or anti-NT-4 antibodies (or fragments thereof containing the binding domain).
  • diseases and disorders of the nervous system which may be associated with alterations in the pattern of NT-4 expression or which may benefit from exposure to NT-4 or anti-NT-4 antibodies (or fragments thereof containing the binding domain).
  • human NT-4 is expressed in skeletal muscle (See Example Section 10, infra).
  • the invention provides for the treatment of motor neuron diseases.
  • a wide array of neurological disorders may affect motor neurons.
  • Upper motor neurons for example, are predominantly affected by cerebrovascular accidents, neoplasms, infections and trauma.
  • Lower motor neurons, or anterior horn cells are secondarily affected by these processes, but in addition are subject to a number of disorders in which anterior horn cell loss is the primary feature, including amyotrophic lateral sclerosis, infantile and juvenile spinal muscular atrophy, poliomyelitis and the post-polio syndrome, hereditary motor and sensory neuropathies, and toxic motor neuropathies (e.g.
  • NT-4 protein, derivatives, fragments, or antibodies thereto which can be used include but are not limited to those disclosed supra or known in the art.
  • the invention may also be utilized to treat benign prostatic hypertrophy (BPH), a common yet poorly understood condition ocurring mostly in males over 50 years of age.
  • BPH benign prostatic hypertrophy
  • the proliferation of the prostrate during BPH may be induced by a growth factor such as NT-4 through an autocrine loop phenomenon. Synthesis and excretion of NT-4 would be followed by transport of NT-4 back into the prostate cell via a specific receptor on the prostate cell membrane.
  • Autocrine loops have been defined for various growth factor molecules and tumor cell lines. In some cases, these autocrine loops have been experimentally defined by the use of antisense approaches for the disruption of the autocrine loop.
  • a therapeutic application of the present invention includes the use of a nucleic acid anti-sense to human NT-4 or a portion thereof to inhibit translation of NT-4 mRNA in the prostate, (for procedures which can be used, see copending U.S. Application Serial No. 07/728,784 filed July 3, 1991 and incorporated by reference herein in its entirety).
  • a patient suffering from a prostate localized disease characterized by
  • the oligonucleotide should be at least 6 nucleotides in length, complementary to a least a portion of the RNA transcript of the NT-4 gene and, hence, being capable of hybridizing to the NT-4 transcript. Additionally, anti-NT-4 antibodies may be utilized to inhibit binding of NT-4 to its specific receptor on the prostate cell membrane.
  • NT-4 antisense nucleic acid or an anti-NT-4 antibody may be delivered in any fashion described supra.
  • the invention may also be utilized to treat other prostate related dysfunctions, specifically impotence.
  • a malady may be the direct or
  • NT-4 protein or a functional fragment or derivative of NT-4 may be delivered by any method described supra.
  • the present invention discloses the detection of NT-4 expression in human thymus tissue. Therefore, the invention may also be utilized to treat immunological disorders affecting neuromuscular transmission, including but not limited to myasthenia gravis, an acquired autoimmune disorder associated with the acetylcholine receptor (AChR) within the postsynaptic folds at the neuromuscular junction.
  • the disease manifests itself as weakness and muscular fatigue due to blockage of post-synaptic AChR or muscle membranes by binding of antibodies specific to the AChR. (See, e.g., Drachman, 1983, Trends
  • Treatment of such immunological mediated neurological disorders may include
  • NT-4 protein or a functional fragment or derivative of NT-4, delivered by any of the methods described supra.
  • the present invention provides for a method of treating motor neuron disorders comprising
  • an effective amount of an NT-4 protein, derivative or peptide fragment capable of supporting the survival, growth and/or differentiation of motor neurons as demonstrated in an in vitro culture system is administered, to a patient in need of such treatment.
  • effective amounts of neurotrophic factor may desirably be determined on a case by case basis, as motor neurons from different tissue sources or from different species may exhibit different sensitivities to neurotrophic factor.
  • motor neurons from different tissue sources or from different species may exhibit different sensitivities to neurotrophic factor.
  • CAT activity may be measured, for example, by harvesting and lysing treated and untreated motor neurons in a 20 mM
  • neostigmine in 50 mM NaH 2 PO 4 (pH 7.4) buffer, using the micro-Fonnum procedure as described in Fonnum, 1975, J. Neurochem. 24:407-409, incorporated by reference in its entirety herein.
  • motor neurons may be prepared, and cultured in vitro, as follows. At least a portion of a spinal cord, preferably obtained from an embryonic organism such as a rat, may be aseptically obtained and separated from the bulb, sensory ganglia, and adhering meninges. The ventral segments of the cord may then be isolated, as motor neurons are localized in the ventral (anterior) horns of the spinal cord. Ventral cord segments may be diced into small pieces and incubated in about 0.1% trypsin and 0.01%
  • deoxyribonuclease type 1 in calcium and magnesium-free phosphate buffered saline (PBS) at 37°C for about 20 minutes.
  • PBS calcium and magnesium-free phosphate buffered saline
  • the trypsin solution may then be removed, and the cells may be rinsed and placed in fresh medium, such as 45% Eagle's minimum essential (MEM),
  • the tissue may be
  • a nylon filter e.g. Nitex, Tetko; 40 ⁇ m.
  • the filtered cell suspension may then be fractioned using a modification of the method set forth in Schnaar and Schaffner (1981, J. Neurosci. 1:204-217). All steps are desirably carried out at 4°C.
  • Metrizamide may be dissolved in F12:MEM medium (1:1) and a discontinuous gradient may be established that consists of a 18% metrizamide cushion (e.g.
  • the filtered cell suspension (e.g. 2.5 ml) may be layered over the step gradient and the tube may be centrifuged at
  • Fraction I enriched for motor neurons, may be removed in a small volume (e.g. about 1 ml) and rinsed twice with a serum-free defined medium such as 50% F12 and 50% MEM supplemented with glutamine (2 mM), insulin (5 ⁇ g/ml), transferrin (100 ⁇ g/ml), progesterone (20 nM),
  • a serum-free defined medium such as 50% F12 and 50% MEM supplemented with glutamine (2 mM), insulin (5 ⁇ g/ml), transferrin (100 ⁇ g/ml), progesterone (20 nM),
  • Viable cell count may then be obtained by hemocytometer counting in the presence of trypan blue.
  • suspension may then be plated at a density of about 100,000 cells/cm 2 in tissue culture wells (preferably 6 mm) precoated with poly L-ornithine (e.g. 10 ⁇ g/ml) and laminin (e.g. 10 ⁇ g/ml).
  • poly L-ornithine e.g. 10 ⁇ g/ml
  • laminin e.g. 10 ⁇ g/ml
  • An NT-4 protein, derivative or peptide factor may then be added.
  • NT-4 may be added to achieve a final concentration of between about 0.01 and 100 ng/ml, and preferably about 50 ng/ml.
  • the motor neuron cultures may then be maintained in serum- free defined medium at 37°C in a 95% air/5% CO 2 atmosphere at nearly 100% relative humidity.
  • the NT-4 related recombinant nucleic acid sequence such as contained in bacteriophage HG7-2, HG4-2, and/or HG2-1, may be utilized to construct chimeric prepro/mature NT-4 genes.
  • a mature NT-4 protein, derivative or peptide fragment in vivo or in vitro, one can fuse the pre-pro region of a distinct neurotrophic gene to the mature coding region of the NT-4 related sequence.
  • the neurotrophic genes which can provide the prepro region include but are not limited to NGF, BDNF, and NT-3.
  • Such a chimeric construct may promote increased stability of the chimeric mRNA transcript in relation to a wild type NT-4 mRNA transcript, may increase translational efficiency or may generate a more suitable template for proteolytic processing to a mature, biologically active neurotrophin protein or peptide fragment, thus increasing expression.
  • One of ordinary skill in the art possesses the requisite knowledge to construct such chimeric nucleic acid sequences, given the published DNA sequences of other neurotrophin genes such as NGF (Scott et al., 1983, Nature 302: 538-540; Ullrich et al., 1983, Nature
  • chimeric constructions fusing the pre-pro region of an NT-4 related recombinant nucleic acid, such as
  • HG7-2, HG4-2 and HG2-1 to the mature regions of other neurotrophins, can also be used to promote efficient expression of such other neurotrophins, as discussed supra.
  • the present invention also provides methods of detecting or measuring NT-4 activity. As described in Example 12, we have discovered that trkB is a functional receptor for NT-4. Based on this
  • the invention provides methods for
  • detecting or measuring NT-4 activity comprising exposing a cell that expresses trkB to a test agent, and detecting or measuring binding of the test agent to trkB, in which specific binding to trkB positively correlates with NT-4 activity in the test agent.
  • the cell that expresses trkB is a transfected cell such as a 3T3 fibroblast, which expresses recombinant trkB, such that the survival of the cell is dependent upon exposure to neurotrophin-4 or BDNF.
  • detecting of binding of the test agent can be carried out by observing the survival of such transfected cells.
  • Genomic DNA was isolated by standard procedures (Davis et al., 1986, "Basic Methods In Molecular Biology", Elsevier, New York)) from human leukocytes and from liver of Sprague-Dawley rat, frog (Xenopus laevis) and ray (Raja, clavata). Genomic DNA was also obtained from salmon (Salmon) and from the elephant snake (Vipera lebetina). The DNA was precipitated with ethanol, collected using a glass hook, washed in 80% ethanol, dried and dissolved in water to a final concentration of 1 mg/ml. Salmon DNA (Sigma, St. Louis, MO) was dissolved in water, extracted twice with phenol and once with chloroform, and precipitated with ethanol.
  • oligonucleotides representing all possible codons corresponding to the amino acid sequence KQYFYET (SEQ ID NO:110) (5'-oligonucleotide) and WRFIRID (SEQ ID NO:111) (3'-oligonucleotide) (Fig. 1A) were
  • the 5'oligonucleotide contained a synthetic EcoRI site and the 3'-oligonucleotide contained a synthetic HindIII site (Knoth et al., 1988, Nucl. Acids Res. 16: 1093; Nunberg et al., 1989, J. Virology 63: 3240-3249).
  • PCR polymerase chain reaction
  • Xenopus genomic library prepared by insertion of Mboldigested genomic DNA in the BamHI site of phase ⁇ EMBL-3 were screened using conventional procedures with a 182 bp PCR fragment of Xenopus NT-4 labeled with [ ⁇ - 32 P]dCTP by nick translation to a specific activity of approximately 5 ⁇ 10 8 cpm/ ⁇ g.
  • Hybridization was carried out in 4 ⁇ SSC (1 ⁇ SSC is 150 mM NaCl, 15 mM sodium citrate (pH 7.0)), 40% formamide, 1 ⁇ Denhardts solution, 10% dextran sulfate at 42°C.
  • the filters were washed at 55°C in 0.1 ⁇ SSC, 0.1% SDS and exposed to Kodak XAR-5 films at - 70°C. Eight phage clones were isolated, and a
  • BDNF protein a PCR-amplified fragment containing the prepro-BDNF coding sequence from the mouse BDNF gene (Hofer et al., 1990, EMBO J. 9:2459-2464) was also subcloned in pXM.
  • pXM a 1020 bp rat cDNA clone was inserted in pXM (Ernfors et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:5454- 5458).
  • COS cells (Gluzman, 1981, Cell 3:175-182) grown to about 70% confluency were transfected with 25 ⁇ g of plasmid DNA per 100 mm dish using the DEAE-dextran-chloroquine protocol (Luthman and Magnusson, 1983, Nucl. Acids Res. 17:1295-1305). Transfected cells were then grown in complete medium (DMEM plus 10% FCS), and conditioned medium was collected 3 days after transfection. Dishes (35 mm) transfected in parallel were grown over the third night after
  • rat NGF protein was estimated by calculating the area corresponding to each protein relative to that obtained with rat NGF.
  • the absolute amount of rat NGF protein was assessed by quantitative immunoblotting of conditioned media using standards of purified mouse NGF and was used to determine the protein concentration in the samples containing the other recombinant proteins.
  • mouse NGF was labeled with 125 I by the chloramine-T method to an average activity of 7 ⁇ 10 7 cpm/ ⁇ g.
  • Steady-sate binding was measured in competition assays performed at 37°C or 0°C using 1 ⁇ 10 4 cells per ml, 1.5 ⁇ 10 -9 M 125 I-NGF; and serial dilutions of conditioned media containing equivalent amounts of NGF or NT-4. All components were added at the same time, and cells were collected by centrifugation after equilibrium was reached (1-3 hr incubation).
  • the biological activities of the different proteins were measured by the ability of transfected COS cell conditioned media, containing equal amounts of recombinant protein, to stimulate neurite outgrowth from explanted sympathetic, nodose, and dorsal root ganglia from E9 chicken embryos (Ebendal, 1984,
  • Xenopus were dissected and frozen in liquid nitrogen.
  • the brain and spinal cord were pooled. Several lobes of the ovary were dissected out, including oocytes of different stages.
  • the frozen tissue samples were homogenized in 4 M guanidine isothiocyanate, 0.1 M ⁇ -mercaptoethanol, 0.025 M sodium citrate (pH 7.0) and homogenized three times for 15 s with a Polytron.
  • RNA Poly(A) + RNA (10 ⁇ g) from each sample was electrophoresed in a 1% agarose gel containing 0.7% formaldehyde. UV-transillumination of the stained gel was used to confirm that all samples contained similar amounts of intact RNA. The gel was then transferred to a nitrocellulose filter. The filter was hybridized to the indicated DNA probes. The probes were labeled with [ ⁇ - 32 P]dCTP by nick translation to a specific activity of around 5 ⁇ 10 5 cpm/ ⁇ g, and the
  • Filters were washed at high stringency (0.1 ⁇ SSC, 0.1% SDS, 54oC) and exposed to Kodak XAR-5 films.
  • DNA fragments coding for NGF, BDNF and NT-3 from human, rat, snake, frog, and fish were isolated using the PCR technique with degenerate primers from conserved regions in these three proteins located between lysine 50 and threonine 56 for the upstream primer and between tryptophan 99 to aspartic acid 105 for the downstream primer (Fig. 1A).
  • the amplified region contains three of the six cysteine residues and covers approximately one third of the mature molecules.
  • a comparison of the amplified region in already characterized NGF molecules from different species shows that it contains two variable regions, arginine 59 to serine 67 and aspartic acid 93 to alanine 98.
  • a hydrophilic stretch believed to be exposed on the surface of the molecules (Bradshaw, 1978, Ann. Rev. Biochem. 47:191-216), as well as the highly conserved regions glycine 68 to tryptophan 76 and threonine 85 to threonine 91 are also included in the amplified region.
  • the BDNF and NT-3 molecules have an extra amino acid between positions 94 and 95 of the mouse NGF protein which is also included in the amplified region.
  • the sequences of the entire mature molecule of mouse NGF, BDNF and NT-3 proteins were compared in order to calculate how representative the amplified region is of the complete molecule.
  • the entire mature molecules show 65/57% similarity (amino acid sequence similarity/nucleotide sequence identity) between NGF and BDNF, 70/61% similarity between NGF and NT-3 and 68/58% similarity between BDNF and NT-3.
  • NERVE GROWTH FACTOR The nucleotide sequence (Fig. 1B [human (SEQ ID NO:3), rat (SEQ ID NO:4), chicken (SEQ ID NO:5), viper (SEQ ID NO:6), Xenopus (SEQ ID NO:7), salmon (SEQ ID NO: 8)] and the predicted amino acid sequence of the isolated fragments coding for NGF are highly conserved from fish to human (Fig. 2 [human (SEQ ID NO:24), rat (SEQ ID NO:25), chicken (SEQ ID NO:26), viper (SEQ ID NO:27), Xenopus (SEQ ID NO:28), salmon (SEQ ID NO:29]). Most of the non-conservative amino acid changes were found in the variable regions arginine 59 to serine 67 and aspartic acid 93 to alanine 98 (Fig. 2). The similarity between the
  • Xenopus and human NGF sequences is 93/79% (Table I). Xenopus and chicken NGF are identical except for one conservative change from lysine 62 to arginine 62 (Fig. 2). The sequences of viper and salmon NGF contain 11 and 19 amino acid differences (out of 42), respectively, compared with human NGF while all other species only showed four differences. None of the NGF amino acid sequences isolated contained the extra amino acid residue present in BDNF and NT-3 between glutamic acid 94 and lysine 95 of the human NGF sequences.
  • the nucleotide and predicted amino acid sequences for human (SEQ ID NO:16 and 37), rat (SEQ ID NO: 17 and 38), chicken (SEQ ID NO: 18 and 39), Xenopus (SEQ ID NO:19 and 40), salmon (SEQ ID NO:20 and 41), and ray NT-3 are highly similar (Figs. 1B, Figure 2). Most of the changes are silent mutations resulting from changes in the third position of the codons, usually transitions that preserve the pyrimidine or purine feature of the base pair. Only non-conservative amino acid changes were found within the two variable regions and no amino acid replacements were seen outside the two variable regions.
  • the salmon sequence lacks Asp-94 which is present in all other NT-3 molecules (Fig. 2) and has a longer
  • NT-3 indicated that this new sequence is related, but not identical, to the sequences of the other members of the NGF family.
  • the gene including this sequence was therefore named neurotrophin-4 (NT-4).
  • Comparison of the nucleotide and amino acid sequences show that
  • Xenopus and viper NT-4 are 91/73% similar. This
  • NGF, BDNF, and NT-3 showed longer branches in the NGF tree, indicating a higher rate of evolutionary change
  • NGF family to the other members was studied by the construction of a phylogram comparing the deduced amino acid sequences for the four members of the family.
  • the phylogram showed that NGF is more closely related to NT- 3 than to BDNF and NT-4 ( Figure 3D).
  • NT-3 is as related to NGF as to BDNF.
  • NT-4 is clearly more related to BDNF than to the other two members.
  • Xenopus genomic library with the NT-4 PCR fragment and isolated a phage clone containing a 16 kb insert. From this insert, a 1.5 kb Pstl fragment was subcloned and sequenced Figure 4A (SEQ ID NO:43).
  • the nucleotide sequence contained an open reading frame encoding a 236 amino acid protein (SEQ ID NO:44) that showed several structural features characteristic of the other members of the NGF family. The amino terminus of the predicted
  • NT-4 protein contains an 18 amino acid putative signal sequence in which a region of 4 amino acids is identical to the corresponding regions in pig and rat BDNF
  • a potential signal cleavage site which is also identical to the one proposed for BDNF ( Figure 4A), follows.
  • a potential cleavage site for a 123 amino acid mature NT-4 protein is found after amino acid 113 in the prepro-NT-4 protein.
  • a single predicted N-glycosylation site (Asn- Lys-Thr) is located 8 amino acids before the putative cleavage site.
  • NT-4 protein includes all 6 cysteine residues involved in the formation of disulphide bridges [Figure 4B (SEQ ID NO:45-48)].
  • Figure 4B SEQ ID NO:45-48
  • the regions that are identical between NGF, BDNF, and NT-3 are also similar in the NT-4 protein. Most sequence differences between the NT-4 protein and the other three proteins were found within the same variable regions previously identified in the other members of the family.
  • the 1.5 kb Xenopus Pstl fragment was cloned in the expression vector pXM (Yang et al., 1986, Cell
  • the NT-4 protein was less efficient in displacing 125 I-NGF, suggesting that in this case the remaining 125 I-NGF associated with the cells was bound to high affinity or internalized receptors.
  • the fact that this difference could not be seen in a parallel assay performed at 0°C in which no membrane mobilization or internalization occurs suggests that the NT-4 protein is not able to compete with NGF for internalization, a process known to be mediated exclusively through the high affinity receptors (Olender and Stach, 1980, J. Biol. Chem. 255, 9338-9343; Bernd and Greene, 1984; J. Biol. Chem. 259, 15509-15516;
  • NT-4 protein transiently expressed in COS cells was tested for its ability to promote neurite growth from explanted embryonic chick ganglia. A clear stimulation of neurite outgrowth from explanted chicken dorsal root ganglia was seen ( Figure 6A). Comparison of dose-response curves using equal amounts of NT-4 and NGF protein revealed that the activity obtained with NT-4 was lower than that seen with NGF ( Figures 5A and 5B). Recombinant NT-4 and BDNF proteins stimulated neurite outgrowth in the dorsal root ganglia to a similar extent ( Figures 6A and 6C).
  • NT-4 protein elicited a weak, but consistent, neurite outgrowth from the nodose ganglia (Figure 6G), whereas no activity could be detected in sympathetic ganglia (Figure 6E). This is in contrast to NGF, which markedly stimulates neurite outgrowth from sympathetic ganglia ( Figure 6F), and
  • Polyadenylated RNA was prepared from 11 different Xenopus tissues and used for Northern blot analysis.
  • Hybridization with the Xenopus NT-4 probe revealed high levels of two NT-4 transcripts of 2.3 kb and 6.0 kb in the ovary ( Figure 7A).
  • the level of NT-4 mRNA was below the detection limit in all other tissues analyzed.
  • Hybridization with the Xenopus NGF probe showed a 1.3 kb NGF mRNA in the heart ( Figure 7A) and brain. However, the amount of NGF mRNA in these tissues was on the order of 100 times lower than the level of NT-4 mRNA in the ovary.
  • NGF mRNA was also detected in the ovary, though the amount of NGF mRNA was approximately 100 times lower than the level of NT-4 mRNA in this tissue ( Figure 7B) .
  • the levels of BDNF and NT-3 mRNAs in ovary were both below the detection limit ( Figure 7B).
  • PCR polymerase chain reaction
  • NGF, BDNF and NT-3 genes from different species include regions which show complete identity between fish and mammals, as well as regions with lower similarity. A comparison of NGF sequences from
  • NGF neurotrophic factor
  • Salmon NGF and NT-3 are notably more different when compared with these molecules in other species. Some amino acids including the threonine 82 and the histidine-threonine-phenylalanine at position 85 to 87 in NGF, as well as the absence of the amino acid between positions 94 and 95 (compared to the two other proteins), are consistent features of the NGF protein. The fact that the isolated salmon sequence contains all of these NGF specific motifs argues that it is not an additional member of the family, but rather represents salmon NGF. In contrast to all other NT-3 sequences studied, salmon NT-3 lacks the amino acid in position 95.
  • NT-4 is more closely related to BDNF than to NT-3 or NGF, it appears that NT-4 and BDNF were formed from a common ancestral gene. However, since no progenitor-like molecule for all four factors can be distinguished from the present data, the evolutionary relation of the putative BDNF/NT-4 ancestor to the ancestors of NGF and NT-3 cannot be definitely
  • the hippocampus contains the highest levels of NGF, BDNF, and NT-3, mRNA in the brain (Ernfors et al., 1990 J. Dev. Neurosci. 9, 57-66). It is a highly specialized structure derived from the archipallium, which first appeared in the brains of amphibians and reptiles.
  • the mammalian hippocampus is important for memory, learning and cognitive functions known to be associated with high neuronal plasticity (Crutcher and Collins, 1982, Science 277:67-68). These demands may have generated a selective pressure during phylogeny for plasticity-promoting mechanisms, possibly medicated by neurotrophic factors.
  • the organization of the nervous system of primitive vertebrates i.e., cartilaginous fishes, shows some basic similarities to the nervous system of higher vertebrates.
  • the cranial nerves and the somatic sensory and autonomic nervous systems in cartilaginous fishes are in general similar to those of higher vertebrates (Young, J.Z., 1981, The Life of Vertebrates, New York Oxford University Press). It is therefore likely that the principles of neurotrophic interactions are the same in both primitive and higher vertebrates.
  • NGF-like neurotrophic factors also in pri.mi.tive vertebrates suggests that these factors first evolved in invertebrates and were later adapted to function in the development of the vertebrate nervous system.
  • Xenopus Nucleotide sequence analysis of this clone revealed an open reading frame for a 236 amino acid protein, which showed several structural features resembling those of the three other members of the NGF family. These include the presence of a putative amino- terminal signal sequence and a potential N-glycosylation site close to a proteolytic cleavage site that predicts a 123 amino acid mature NT-4 protein.
  • the size of the mature NT-4 protein is 4 amino acids longer than that of BDNF and NT-3 and 5 amino acids longer than that the mature NGF protein.
  • NT-4 protein differs from the other members of the family in the same regions that vary among the sequences of the three other family members. As for NGF, BDNF, and NT-3, the entire prepro-NT-4 protein is encoded in one single exon.
  • NT-4 gene is an additional member of the NGF family.
  • the protein encoded by the low affinity NGF-R gene appears to form part of both the low and the high affinity receptors (Hempstead et al., 1989, Science 243:373-375).
  • the mechanism by which two kinetically different receptors are formed from the same receptor gene is not known, although it has been proposed that the two states can be generated by the formation of a complex between the cytoplasmic domain of the receptor and an intracellular protein (Radeke et al., 1987, Nature 325:593-597: Meakin and Shooter, 1991, Neuron 6:153-163).
  • a high affinity receptor chain may be encoded by a
  • the two receptor chains may form a dimer that constitutes the high affinity
  • the low affinity NGF-R gene has been shown to be expressed in many tissues of both neuronal and nonneuronal origin not known to respond to NGF. These include mesenchyme, somites and neural tube cells in the early chick embryo (Hallbook et al., 1990, Development 108:693-704; Heuer et al., 1990a, Dev. Biol.
  • NT-4 protein is of functional importance in any of these tissues or neuronal populations.
  • the neurotrophic activity of the NT-4 protein was assayed on explanted chick embryonic ganglia, and as for the other three members of the NGF family, the NT-4 protein showed a clear stimulation of neurite outgrowth from dorsal root ganglia. However, when compared to NGF, the NT-4 protein showed lower activity in dorsal root ganglia. Both BDNF and NT-3 readily elicit neurite outgrowth in explanted nodose ganglia, though the response with NT-3 was consistently stronger than that with BDNF. NGF strongly stimulates neurite outgrowth in sympathetic ganglia, and NT-3 also has activity in this ganglia, though it is much lower than that of NGF
  • NT-4 showed weaker activity in nodose ganglia compared with NT-3 and no activity in the sympathetic ganglia.
  • Hybridization to a Xenopus NGF probe revealed NGF mRNA in the Xenopus heart, most likely as a result of NGF mRNA expression in target tissues for neuronal
  • NGF mRNA was, however, more than 100-fold lower than the level of NT-4 mRNA in the ovary. Since the high level of NT-4 mRNA in the ovary does not correlate with neuronal innervation, it appears unlikely that the NT-4 protein has only a neurotrophic function in this case. Instead, the abundant expression of NT-4 mRNA in Xenopus ovary implies an additional and important nonneurotrophic function for the NT-4 protein. NGF mRNA was also detected in Xenopus ovary though at almost 100 times lower levels than those of NT-4 mRNA; BDNF and NT-3 mRNAs were not detected in this tissue.
  • mRNAs for two growth factors have been described as maternal mRNAs in Xenopus oocytes.
  • One of these mRNAs encodes a protein with strong similarity to basic fibroblast growth factor (Kimelman and Kirschner, 1987, Cell 51:869-877); the other mRNA encodes a protein homologous to transforming growth factor ⁇ (Weeks and Melton, 1987, Cell 51:861-867).
  • These factors have been suggested to function as morphogens for the formation of mesoderm and the subsequent induction of this tissue into the neural tube.
  • Fresh-frozen ovaries from adult Xenopus laevis frogs were sectioned (14 ⁇ ) in a cryostat (Leitz, Germany) and the sections were thawed onto poly-L-lysine (50 ⁇ g/ml) pretreated slides after which they were fixed in 10% formalin for 30 min and rinsed twice in PBS.
  • oligonucleotides one specific for Xenopus NT-4 mRNA (5'CCCACAAGCTTGTTGGCATCTATGGTCAGAGCCCTCACATAAGACTGTTTTGC 3' [SEQ ID NO: 109]) and another one, as a control, specific for chicken BDNF mRNA (corresponding to amino acids 61 to 77 of the mature chicken BDNF protein
  • the indicated samples were homogenized in 4M guanidine isothiocyanate, 0.1M ⁇ -mercaptoethanol, 0.025M sodium citrate pH 7.0 and homogenized 3 times for 15 seconds with a Polytrone. Each homogenate was layered over a 4ml cushion of 5.7M CsCl in 0.025M sodium citrate pH 5.5 and centrifuged at 15°C in a Beckman SW41 rotor at 35,000 rpm for 16 hrs. (Chirgwin et al., 1979,
  • RNA was purified by oligo (dT) cellulose chromatography (Aviv and Leder, 1972, PNAS 69: 1408- 1412) and the recovery of RNA (40 ⁇ g) was quantified spectrophotometrically before use in RNA blot analysis. Total cellular RNA (40 ⁇ g) or where indicated
  • poly(A)+RNA (5 ⁇ g) from each sample was electrophoresed in a 1% agarose gel containing 0.7% formaldehyde. UV- transillumination of the stained gel was used to confirm that all samples contained similar amounts of intact RNA. The gel was then transferred to a nitrocellulose filter. The filter was then hybridized to a 350bp
  • HincII fragment from the 3' exon of the Xenopus NT-4 gene (Hallbook et al., 1991, Neuron 6 : 845-858). The fragment was labeled with ⁇ -( 32 p)-dCTP by nick
  • Tissue sections through the adult Xenopus laevis ovary were hybridized to a 35 S-dATP labeled oligonucleotide probe specific for Xenopus NT-4 mRNA.
  • adjacent sections were hybridized to an oligonucleotide probe of the same length and GC-content complementary to mRNA for chicken brain-derived neurotrophic factor
  • BDNF NT-4 mRNA specific probe revealed an
  • NT-4 mRNA corresponding to oocytes in early stages of oogenesis (Fig. 9A)
  • No NT-4 mRNA could be detected over mature, post-vitellogenic stage VI oocytes (arrows in Fig. 9A).
  • the chicken BDNF mRNA specific control probe did not label any cells in the Xenspus ovary.
  • Analysis of emulsion autoradiographs from the hybridized sections revealed an intense labeling over the cytoplasm of oocytes with a diameter of 50-200 ⁇ m (Fig. 10A and 10B) corresponding to stage I oocytes according to Dumont, 1972, supra.
  • stage I oocytes The area unit chosen corresponded to approximately one hundredth of the cross section area of a stage I oocyte.
  • the result of this analysis showed that the intensity of labeling over stage I oocytes was 1.7 and 4.3 times higher than over stage II/III and IV oocytes respectively (Fig. 11) .
  • the number of grains per area unit over stage V and VI oocytes was not significantly above the level of the background labeling.
  • NT-4 mRNA was assessed in developing Xenopus embryos by Northern blots of
  • NT-4 mRNA polyadenylated RNA.
  • a low level of NT-4 mRNA was found in Xenopus somatic Ag cultured kidney cells which were also included in the analysis. However, no NT-4 mRNA could be detected in early embryos from the onset of cleavage divisions to the neurula stage.
  • NT-4 mRNA The abundant expression of NT-4 mRNA in the Xenopus ovary (Hallbook et al., 1991 supra) indicates that this member of the NGF family plays a role in oogenesis and/or early embryogenesis. Localization of cells expressing NT-4 mRNA in the ovary provided insights into the putative function of the NT-4 protein in the ovary. In amphibians, as in all other vertebrates, fertilization of the egg triggers a period of rapid cell cleavage. This event is controlled by a class of soluble maternal mRNAs expressed during oogenesis and stored in the unfertilized egg for subsequent development
  • This class of maternal mRNAs includes two growth factors, basic fibroblast growth factor (Kimelman and Kirschner, 1987, Cell, 51 : 869-77) and transforming growth factor- ⁇ (Weeks and Melton, 1987, Cell, 51: 861-67), as well as several protooncogenes such as c-myc (Godeau et al., 1986, EMBO J., 5 : 3571-77);
  • Immature stage VI Xenopus oocytes are arrested in prophase of meiosis I and both c-mos (Sagata et al., 1988) and ets-2 (Chen et al., 1990) have been shown to function during reinitiation of meiotic division.
  • stage I and II oocytes show that the NT-4 mRNA does not belong to the class of maternal mRNAs. This result also argues against a role of the NT-4 protein in the reinitiation of meiotic division or in early embryogenesis. In agreement with this, addition of recombinant NT-4 protein to immature stage VI oocytes failed to induce germinal vesicle breakdown in vitro and no NT-4 mRNA was detected in
  • NGF Basal-LeLievre et al., 1988, PNAS 85: 2628-2632
  • the 75kD low-affinity NGF receptor Persson et al., 1990, Science, 247: 704-707
  • the trkA high-affinity component of the NGF receptor J.P. Merlo and H. Persson, unpublished
  • neurotrophins do not only function as neurotrophic factors but also play an important role in reproductive tissues.
  • SEQ ID NO:51 QYFYET (SEQ ID NO:52) (5'-oligonucleotide) and, WISECK, CKAKQS and WIRIDT (each contained within SEQ ID NO:52)
  • PCR products from the reamplification procedure were purified as follows: bands of prospective size were gel purified, reamplified, and column purified using Stratagene "primerase” columns.
  • a human ovary cDNA library in ⁇ GT-10 was obtained from Clontech.
  • a human hippocampus cDNA library in ⁇ :ZAPII was obtained from Stratagene.
  • a human genomic DNA library in EMBL3/SP6/T7 was obtained from Clontech.
  • a rat brain cDNA library in ⁇ -ZAP was obtained from
  • a cloned insert encoding the rNT-4 fragment (Fig. 14 [SEQ ID NO:61]) or the hNT-4 fragment (Fig. 15 [SEQ ID NO: 63]) are labeled by PCR to a specific activity of approximately 5 ⁇ 10 8 cpm/ng.
  • Hybridization is carried out in hybridization solution consisting of 0.5 mg/ml salmon sperm DNA at 60°C. The filters are washed at 60°C in 2 ⁇ SSC, 0.1 % SDS and exposed to Kodak XAR-5 film at -70°C.
  • oligonucleotides whose sequence corresponds exactly to the desired mammalian neurotrophin can be used to generate probes (e.g. kinase labelling) and can be used to screen the same libraries by
  • TM buffer (10mM Tris-HCl, pH 7.5; 10mM MgCl 2 ) , layered upon a CsCl step gradient and centrifuged at appropriate speed and length of time to band the bacteriophage.
  • EMBL-3 DNA is precipitated by the addition of 2 volumes of 100% ethanol.
  • the EMBL-3 DNA is recovered by microcentrifugation, washed in 70% ethanol and
  • For a negative control add no phage stock. Incubate 15 minutes at 37°C. Add 5 ml of 2XYT media, shake for 3 hours at 37°C. At the end of the 3 hours, the negative control should be cloudy and the samples clear. Samples are heated at 65°C for 30 minutes, spun at 4000g for 5 minutes. Supernatant contains phagemid stock. To rescue the phagemid, add
  • Lambda phage cDNA libraries are screened according to standard methods (Maniatis, et al., supra) as described supra.
  • Positive plaques are purified, reisolated and subjected to DNA sequence analysis as described supra.
  • a region of the Xenopus NT-4 coding sequence was used as a model for synthesis of degenerate
  • oligonucleotide primers denotes that the 5'-oligonucleotide primer 2Y [SEQ ID NO:53] (QYFFET) and 3'-oligonucleotide primers, 3Y [SEQ ID NO: 55] (WISECK), 3Z [SEQ ID NO: 56] (CKAKQS) and 4Z [SEQ ID NO: 58] (WIRIDT) were derived from the xNT-4 amino acid sequence.
  • the 5'-oligonucleotide primer 2Z [SEQ ID NO: 54] (QYFYET) is derived from the homologous region of rBDNF. All
  • oligonucleotides were utilized to amplify DNA from both rat and human genomic DNA libraries. Since the primers represented by 3Y [SEQ ID NO: 55] and 3Z [SEQ ID NO: 56] of xNT-4 are not conserved in the NGF/BDNF/NT-3 gene family, and therefore were not likely to amplify NGF, BDNF or NT-3, these two primers were utilized in the
  • DNA fragments of the approximate expected sizes were obtained from PCR amplification and
  • the secondary PCR products of the approximate expected size were electrophoresed through a 2% agarose gel, eluted by standard techniques, digested with EcoRI and Sail and ligated in EcoRI-Xhol digested pBS-KS DNA. Positive transformants were selected, and inserted fragments were subjected to DNA sequencing by the dideoxy chain termination method (Sanger, et al., supra).
  • FIG. 14 An open reading frame has been deduced for a portion of the rat NT-4 (Fig. 14 [SEQ ID NO: 62]) and human NT-4 (Fig. 15 [SEQ ID NO: 64]) amino acid coding sequence.
  • Figure 16 illustrates the homologous region of the rNT-4 (SEQ ID NO: 62) and hNT-4 (SEQ ID NO: 64)
  • FIG. 17A An open reading frame encoding a larger portion of human NT-4 than that disclosed in Figure 15 is shown in Figure 17A (SEQ ID NO: 69 and SEQ ID NO: 70).
  • Figure 17A presents additional 3' sequence information for the 3' human NT-4 coding region.
  • the 192 bp nucleic acid fragment was isolated as described supra in the
  • NT-4 (GPGVGGG) [SEQ ID No: 101] and human NT-4 (GPGAGGG)
  • mammalian NT-4 proteins is not presently known, nor whether the 7bp insertion itself will be characteristic of other mammalian NT-4 genes. It is interesting to note that fish NGF has a 22 amino acid insertion in the same region as disclosed in the present invention.
  • Duplicate lifts were made using Schleicher & Schuell nitrocellulose filters, and Were hybridized to a 120 bp probe (from hNT-4 clone 17B, which was obtained from human genomic DNA using primers 2Z4Z followed by 2Z3Z), labelled by PCR using oligonucleotide primers 2Z/3Z.
  • the filters were hybridized at 60oC with the radiolabelled probe (10 6 cpm/ ml) under the following hybridization onditions: 0.5 M NaPO 4 , 1% BSA, 7% SDS, 1 mM EDTA, and 100 ⁇ g/ml salmon sperm DNA.
  • the filters were then washed at 60°C with 2 ⁇ SSC and 0.1% SDS, and subjected to
  • HG4-2, HG7-2 and HG2-1 Phage DNA was made, an aliquot of which was run on agarose gel and subjected to Southern analysis. HG4-2, HG7-2 and HG2-1 were found to hybridize to the rNT-4 180 bp probe (NaPO 4
  • phage DNA for the genomic clones was digested with several restriction enzymes and subjected to Southern analysis.
  • DNA fragments to be subcloned can be subcloned into Bluescript vector.
  • the size of DNA fragments to be subcloned are as follows: clone 2-1 (1.0 kb XhoI fragment), clone 4-2 (4.0 kb XhoI fragment) and clone 7-2 (5.0 kb BamHI fragment).
  • Complete coding sequence can be obtained and this information can be used to identify the exon boundaries to allow subcloning of this gene into an appropriate expression vector.
  • nucleotide sequence analysis was performed on human genomic phage clone 7-2, which had been obtained by screening a human genomic library with a PCR fragment derived from human genomic DNA using
  • this prepro region is preceded by a splice acceptor site localized in the same region as in other neurotrophin genes.
  • the prepro region also contains a consensus glycosylation site at the appropriate position, and terminates at a cleavage site which was very similar to the cleavage sites found in the other neurotrophins ( Figure 18).
  • the prepro region of 7-2 is unusual, however, due to its short length as compared to the prepro region of known neurotrophins. The decrease in length occurs in the N-terminal portion of the prepro region, which is the least conserved portion of prepros between family members.
  • the mature region retains all 6 cysteines found in all previously identified
  • neurotrophins Many of the residues shared between different members of the neurotrophin family are also conserved. Excluding the extensive sequence similarity shared by a PCR fragment derived from rat genomic DNA which may correspond to the rat equivalent of the protein encoded by the human 7-2 clone, computer alignments revealed that the neurotrophin encoded by the 7-2 phage clone was most similar to that of Xenopus NT-4. This was true for both the prepro and mature regions. The protein encoded by the 7-2 clone is unusual, as compared to the known neurotrophins, due to the presence of an insertion situated between the second and third cysteines in the mature region.
  • HG7-2 was radiolabeled and utilized in Northern analysis of various human tissue specific PolyA+ RNAs.
  • the human tissue specific mRNAs were fractionated by
  • RNAs were cross-linked to the membranes by exposure to ultraviolet light and hybridized at 65oC to the 680 bp Xhol-Notl radiolabeled NT-4 probe in the presence of 0.5M NaPO 4 (pH 7), 1% bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA and 100 ng/ml sonicated, denatured salmon sperm DNA.
  • the filter was washed at 65°C with 2X SSC, 0.1% SDS and subjected to autoradiography overnight with one intensifying screen and X-ray film at -70°C. Ethidium bromide staining of the gel demonstrated that equivalent levels of total RNA were being assayed for the different samples.
  • the human NT-4 probe hybridized strongly to mRNA from skeletal muscle, prostate, thymus, testes and placenta (Figure 22).
  • the NT-4 probe hybridized to a larger transcript in skeletal muscle than prostate mRNA. This data suggests that a small human NT-4 multigene family, possessing different expression levels as well as transcript sizes, may be present.
  • the first construction utilized the normal translation initiation site of pCMX (pCMX-HG7-2Q) , while the other utilized the Kozak consensus translation initiation site (pCMX-HG7-2M).
  • pCMX-HG7-2Q the normal translation initiation site of pCMX
  • pCMX-HG7-2M the Kozak consensus translation initiation site
  • hNT4-5'XhoM CGGTACCCTCGAGCCACCATGCTCCCTCTCCCCTCA
  • Oligonucleotide primer combinations of hNT4-5'XhoM andhNT4-3'Not were used to construct pCMX-HG7-2M, while oligos hNT4-5'XhoQ and hNT4-3'Not were used to construct pCMX-HG7-2Q.
  • the PCR fragment was digested with
  • 5'CDM8 GAGACCGGAAGCTTCTAGAGATC [SEQ ID NO: 121]
  • the hNT-3 containing plasmid vector (pC8-hNT3) was amplified by PCR with the 5'CDM8 and hNT3/hNT4 fusion oligonucleotides as primers.
  • the hNT-4 containing plasmid (pCMX-HG7-2Q) was amplified by PCR with the hNT3/hNT4 fusion "DS" oligonucleotide and the hNT4-3'Notl oligonucleotide.
  • the PCR fragment obtained was excised from the gel, and reamplified by PCR with the 5'CDM8 and hNT4-3'Notl oligonucleotides.
  • the product was then digested with HindIII and Pstl and subcloned into
  • the expression plasmid pCMX-hNT3/hNT4 contained the hNT3 prepro region fused to the mature coding region of human NT-4.
  • pCMX-HG7-2Q was amplified by PCR with the 5-CDM8 and xNT4/hNT4/fusion "US" oligonucleotides as primers, while pCMX-HG7-2Q was amplified with the xNT4/hNT4-fusion "DS" oligonucleotide and the hNT4-3'Notl oligonucleotide.
  • the PCR fragment was excised from the gel, and reamplified with the 5'CDM8 and the hNT4-3'Not oligonucleotides.
  • the product was then digested with HindIII and Pstl and subcloned into HindIII/Pstl digested pCMX-HG7-2Q.
  • pCMX-xNT4/hNT4 contains the Xenopus NT-4 prepro region fused to the mature coding region of human NT-4.
  • COS M5 cells were set up at a density of
  • pCMX-xNT4 containing the prepro precursor of Xenopus NT-4; and two human NT-4
  • DMSO/PBS was aspirated and wells washed once with 10% FBS DMEM, then refed with 10% FBS DMEM. The next morning, plates to be bioassayed were washed once with Defined Media (DM) and refed 2 ml/well of DM. Three days post-transfection, supernatants were removed from cells and debris pelleted by microcentrifugation. Supernatants were transferred to fresh tubes and assayed for
  • Embryos from Sprague-Dawley rats (HSD or Zivic-Miller) were used for all experiments. Pregnantrats were sacrificed by carbon dioxide asphyxiation, and embryos were rapidly removed and placed in ice-cold medium for further dissection. Spinal cords were removed aseptically from rat embryos of 14 days gestation. The spinal cord was severed caudal to the bulb (at the level of the first dorsal root ganglion), freed of sensory ganglia and adhering meninges. The cord was then
  • ventral spinal cord tissues were diced into small pieces and incubated in 0.1% trypsin (GIBCO) and 0.01% deoxyribonuclease type 1 (Sigma) in PBS at 37oC for 20 minutes. Trypsin solution was then removed, rinsed and replaced with medium consisting of 45% Eagle's minimum essential medium (MEM), 45% Ham's nutrient mixture F12 (F12), 5% heat inactivated fetal calf serum (GIBCO), 5% heat inactivated horse serum
  • the filtered cell suspension were then subjected to a modification of the fraction procedure described by
  • metrizamide cushion (0.5 ml), 3 ml of 17% metrizamide, 3 ml of 12% metrizamide, and 3 ml of a 8% metrizamide was prepared.
  • fraction I at 0-8% interface
  • fraction II at 8-12% interface
  • fraction III at 12-17% interface
  • the cells from each interface were removed in a small volume (about 1 ml), rinsed twice with serum-free defined medium consisting of 50% F12 and 50% MEM, supplemented with glutamine (2 mM), insulin (5 ⁇ g/ml), transferrin (100 ⁇ g/ml), progesterone (20 nM), putrescine (100 ⁇ M), and sodium selenite (30 nM) (Bottenstein and Sato, 1979, Proc. Natl. Acad. Sci. 76:514-517).
  • Viable cell count was obtained by hemocytometer counting in the presence of trypan blue. Fractionated ventral spinal cord cells (enriched with motor neurons) were then plated at a density of 100,000 cells/cm 2 in 6 mm wells precoated with poly-L-ornithine (Sigma: 10 ⁇ g/ml) and laminin (GIBCO: 10 ⁇ g/ml). Treatment with COS cell supernatants
  • COS cell was given on the day of plating. Cultures were maintained in serum-free defined medium at 37°C in 95% air/ 5% CO 2 atmosphere at nearly 100% relative humidity. On day 2 (48 hours), cells were harvested for measurements of choline acetyltransferase (CAT) as described in Fonnum, 1975, J. Neurochem. 24:407-409.
  • CAT choline acetyltransferase
  • the present invention provides for the
  • the DNA sequence encoding NT-4 prepro precursor was amplified utilizing two PCR amplification strategies to yield pCMX-based expression plasmids containing either the pCMX translation initiation site (pCMX-HG7-2M) or a Kozak consensus translation site (PCMX-HG7-2Q).
  • pCMX-HG7-2M pCMX translation initiation site
  • PCMX-HG7-2Q Kozak consensus translation site
  • neurotrophin genes fusing either the prepro region of Xenopus NT-4 (pCMX-xNT4/hNT4) or the prepro region of human NT-3 (pCMX-hNT3/hNT4) to the mature coding region of NT-4 were constructed for expression in COS cells (see Section 5, supra, for a discussion of the use of chimeric constructions to express NT-4 in vitro).
  • NT-4 a biologically active form of human NT-4 in an in vitro eukaryotic expression system substantially increases the ease at which the production of human recombinant NT-4, peptides or derivatives thereof may be scaled up for both therapeutic and
  • a plasmid containing an identical DNA sequence as disclosed or a similar DNA sequence encoding a homologous yet distinct NT-4 like protein or derivative thereof can readily construct a plasmid containing an identical DNA sequence as disclosed or a similar DNA sequence encoding a homologous yet distinct NT-4 like protein or derivative thereof.
  • the skilled artisan can also pick and choose between numerous DNA plasmid vectors known in the art to construct an expression plasmid for use in a eukaryotic expression system.
  • recombinant human NT-4 whether produced as a full prepro precursor or via a neurotrophin-based chimeric construction, is biologically active as demonstrated by the stimulating effect of recombinant NT-4 COS supernatants on neurite outgrowth in DRG explants and the bioactivity of cultured motor neurons. 12.
  • TRKB IS A RECEPTOR FOR NEUROTROPHIN-4 COS cell supernatants were also examined in a survival assay utilizing 3T3 fibroblasts.
  • 3T3 fibroblasts which do not express
  • neurotrophin receptor proteins are transfected with mammalian expression vectors encoding either trkA or trkB. 3T3 fibroblast survival is dependent on the addition and receptor specific binding of the respective neurotrophic factor.
  • COS-M5 cells were cultured and transfected
  • rat trkA cDNA clone was obtained from Dr. Eric Shooter of Stanford University.
  • the rat trkA cDNA was subcloned into the mammalian expression vector, pCMX, to generate pCMX-trkA.
  • rat trkB cDNA clone was obtained by screening a rat brain cDNA library in the lambda ZAP2 vector (Stratagene) with rat trkB-specific
  • oligonucleotides corresponding to the most 5' and 3' coding regions of trkB The rat trkB cDNA was subcloned into pCMX to generate pCMX-trkB.
  • 3T3 fibroblasts were cultured and transfected as described in Glass, et al., 1991, Cell 66:405-413.
  • 3T3 fibroblasts which do not express neurotrophin receptor proteins, have been transfected with trkA, a protooncogene encoding a tyrosine kinase receptor for NGF, or with trkB, a tyrosine kinase which serves as a functional binding protein for BDNF and NT-3.
  • the transfected cells are dependent upon the addition of the corresponding neurotrophin for survival, and thus may be used to assay for biological activity of neurotrophins. Addition of
  • NT-4 protein has biological activity in this system, and suggests that trkB, but not trkA, serves as a functional binding protein for NT-4.
  • MOLECULE TYPE DNA (genomic)
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  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 2:
  • MOLECULE TYPE DNA (genomic)
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  • CAAGTGCAAA AATCCAAGTC CAGTATCAGG TGGGTGCAGG GGCATTGATG CCAAGCATTG 60 GAATTCGTAT TGCACCACAA CAGACACATT TGTCAGGGCA TTAACCATGG AAGGCAATCA 120 GGCATCT 127
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
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  • MOLECULE TYPE DNA (genomic)
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Abstract

The present invention relates to neurotrophin-4 (NT-4), a newly characterized member of the BDNF/NGF/NT-3 gene family. The present invention also provides for nucleic acid molecules encoding NT-4.

Description

THERAPEUTIC AND DIAGNOSTIC METHODS
BASED ON NEUROTROPHIN-4 EXPRESSION
1. INTRODUCTION
The present invention relates to
neurotrophin-4 (NT-4), a newly characterized member of the BDNF/NGF/NT-3 gene family and the therapeutic and diagnostic methods of utilizing neurotrophin-4 in the treatment of neurological disorders.
2. BACKGROUND OF THE INVENTION
The nerve growth factor family includes β-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), also known as hippocampus-derived neurotrophic factor (HDNF). This family of proteins plays an important role in both the developing and the adult vertebrate nervous system, where they support neuronal survival.
Based on the amino acid sequence of the mouse NGF protein (Angeletti, et al., 1973,
Biochemistry 12:100-115) DNA sequences coding for mouse and human NGF have been isolated (Scott et al., 1983, Nature 302:538-540; Ullrich et al., 1983, Nature 303:821-8251. Comparison of mouse and human NGF showed that the protein is conserved within mammals and in support of this, NGF-like activities have been isolated from several species (Harper and Thoenen, 1981, Ann. Rev. Pharmacol. Toxicol. 21:205-229).
Subsequently, DNA sequences from bull (Meier et al., 1986, EMBO J. 5:1489-1493); chick (Meier et al., 1986, EMBO J. 5:1489-1493; Ebendal et al., 1986, EMBO J.
5:1483-1487; Wion et al., 1986, FEBS Letters 203:82-86; cobra (Selby et al., 1987, J. Neurosci. Res.
18:293-298); rat (Whittemore et al., 1988, J.
Neurosci . Res . 20 : 403-410) ; and guinea pig (Schwarz et al., 1989, Neurochem. 52: 1203-1209) NGFs were also determined. Brain-derived neurotrophic factor (BDNF) was first isolated from pig brain (Barde et al., 1982, EMBO J. 1:549-553) and subsequently cloned as a cDNA from this tissue (Leibrock et al., 1989 Nature
341:149-152). The gene for NT-3 has been isolated from mouse (Hohn et al., 1990, Nature, 344: 339-341), rat (Maisonpierre et al., 1990, Science 247: 1446- 1451; Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5454-5458), and human (Rosenthal et al., 1990, Neuron 4: 767-773), using degenerate oligonucleotides based on the sequence similarity between the other two factors. The three factors show approximately 55% amino acid similarity to each other, and most sequence differences are present in five regions that contain amino acid motifs characteristic of each protein. The neurotrophic activity in vitro of two of these
proteins have recently been shown to be acquired by specific combinations of these variable regions.
NGF supports the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons (reviewed in Thoenen and Barde, 1980, Physiol. Rev., 60: 1284-1325; Levi-Montalcini, 1987, Science, 237: 1154-1162). No activity has been seen for BDNF in peripheral sympathetic neurons, but this factor supports in vivo the survival of both placode and neural crest-derived sensory neurons (Hofer and Barde, 1988, Nature, 331: 261-262). The neurons sensitive to NT-3 in vivo remain to be identified.
However, in explanted chick ganglia or dissociated neuronal cultures in vitro, the three factors support both overlapping and unique sets of neuronal
populations, suggesting that NT-3 exerts both specific and overlapping neurotrophic activities also in vivo (Hohn et al., 1990, Nature, 344: 339-341; Maisonpeirre et al., 1990, Science, 247: 1446-1451; Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458;
Rosenthal et al., 1990, Neuron 4: 767-773). All three factors are expressed in specific sets of neurons in the brain, with the highest levels of mRNA for all three factors in the hippocampus (Ayer-LeLievre et al., 1988, Science 240: 1339-1341; Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458;
Ernfors et al., 1990, Neuron 5 : 511-526; Watmore et al., 1991, Neurol. 109: 141-152; Hofer et al., 1990, EMBO J., 9: 2459-2464; Phillips et al., 1990, Science, 250: 290-294). In the brain, NGF has been shown to support basal forebrain cholinergic neurons (reviewed in Whittemore and Seiger, 1987, Brain Res., 434: 439-464; Thoenen et al., 1987, Rev. Physiol. Biochem.
Pharmacol., 105: 145-178; Ebendal, 1989, Prog. Growth Factor Res. 1: 143-159) and BDNF has been shown to stimulate the survival of these neurons in vitro
(Alderson et al., 1990, Neuron 5 : 297-306).
The effects of the three proteins are mediated by their interaction with specific receptors present on sensitive cells. Molecular clones have been isolated for the rat, human, and chicken NGF receptor (NGF-R), and nucleotide sequence analysis of these clones has shown that the NGF-R contains one plasma membrane-spanning domain, a cytoplasmic region, and an extracellular cysteine-rich amino-terminal domain (Johnson et al., 1986, Cell, 47: 545-554;
Radeke et al., 1987, Nature, 325: 593-597; Large et al., 1989, Neuron 2: 1123-1134). The NGF-R shows a low but significant sequence similarity to the
receptor for a tumor necrosis factor (Schall et al., 1990, Cell, 61 : 361-370) as well as to the lymphocyte surface antigens CD40 (Stamenkovic et al., 1989, EMBO J., 8 : 1403-1410) and OX40 (Mallett et al., 1990, EMBO J., 9: 1063-1068). The NGF-R can occur in two apparent states, known as the low and high affinity states (Sutter, et al., 1979, J. Biol. Chem., 254: 5972-5982; Landreth and Shooter, 1980, Proc. Natl. Acad. Sci. USA, 77: 4751-4755; Schechter and Bothwell, 1991, Cell 24 : 867-874). The gene for the NGF-R appears to encode a protein that forms part of both the low and the high affinity states of the receptor (Hempstead et al., 1989, Science, 243: 373-375), though only the high affinity receptor has been proposed to mediate the biological activity of NGF. Both BDNF (Rodriguez-Tebar et al., 1990, Neuron, 4: 487-492) and NT-3 (Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458) can interact with the low affinity NGF-R, suggesting that the low affinity NGF-R may be, in an as yet unknown way, involved in mediating the biological effects of all three factors.
In the developing nervous system, NGF and its receptor have been shown to be synthesized in the target area and in the responsive neurons,
respectively, at the time when the growing axon reaches its target (Davies et al., 1987, Nature, 326: 353-358). In agreement with this, the level of NGF mRNA in the developing chick embryo reaches a maximum at embryonic day 8 (E8) (Ebendal and Persson, 1988, Development, 102: 101-106), which coincides with the time of sensory innervation. However, in the chick, NGF-R mRNA is maximally expressed at early embryonic stages prior to neuronal innervation (Ernfors et al., 1988, Neuron, 1, 983-996), and in the E8 chick embryo high levels of NGF-R mRNA have been detected in the mesenchyme, somites, and neural tube cells (Hallbook et al., 1990, Development, 108: 693-704; Heuer et al., 1990, Dev. Biol., 137: 287-304; Heuer 1990, Neuron, 5: 283-296). This observation, together with the fact that NGF mRNA is expressed in the E3 chick embryo at relatively high levels (Ebendal and Persson, 1988, Development, 102: 101-106), indicates that NGF may play a role in early development that is distinct from its function as a neurotrophic factor. In agreement with this possibility, NGF has recently been shown to control proliferation and differentiation of E14 rat embryonic striatal precursor cells in culture
(Cattaneo and McKay, 1990, Nature, 347: 762-765). In the chick embryo BDNF and NT-3 mRNA are maximally expressed at E4,5, and BDNF has been shown to control the differentiation of avian neural crest cells in vitro (Kalcheim and Gendreau, 1988, Dev. Brain Res., 41: 79-86).
Moreover, evidence for a non-neuronal function of NGF has also been presented. The still unexplained high levels of NGF found in the male mouse submandibular gland may indicate other functions for NGF (Levi-Montalcini, 1987, Science, 237: 1154-1162). In the adult rat, NGF has been shown to induce DNA synthesis and to stimulate IgM secretion in B-cells (Otten et al., 1989, Proc. Natl. Acad. Sci. USA 86: 10059-10063). Additionally, NGF is present in
sufficient quantity in guinea pig prostate such that Rubin and Bradshaw (1981, J. Neur. Res. 6: 451-464) were successful in isolating and characterizing substantially pure NGF from this exocrine tissue. The high level of NGF in pig prostate support the
hypothesis that this neurotrophic factor functions in a non-neuronal capacity not yet understood (Bradshaw,
1978, Ann. Rev. Biochem. 47:191-216; Harper, et al.,
1979, Nature 279:160-162; Harper and Thoenen, 1980, J. Neurochem. 34:893-903).
Furthermore, NGF mRNA is expressed in spermatocytes and early spermatids in the adult rat testis (Ayer-LeLievre et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 2628-2632), and the NGF protein is present in germ cells of all stages from spermatocytes to spermatozoa (Olson et al., 1987, Cell Tissue Res., 248: 275-286; Ayer-LeLievre et al., 1988a, Proc. Natl. Acad. Sci. USA 85: 2628-2632). NGF-R mRNA has also been detected in the adult rat testis, where it is expressed in Sertoli cells under negative control of testosterone, and in the testis NGF has been suggested to control meiosis and spermiation (Persson et al., 1990, Science, 247: 704-707).
3. SUMMARY OF THE INVENTION
The present invention relates to
neurotrophin-4 (NT-4), a newly characterized member of the BDNF/NGF/NT-3 gene family.
The present invention provides for nucleic acid molecules encoding NT-4. Such molecules may comprise a sequence substantially as set forth for
NT-4 in Figure 1 [SEQ ID NO:1 (NT-4, viper), SEQ ID NO:2 (NT-4, Xenopus.1. Figure 4 (SEQ ID NO:43), Figure
8 (SEQ ID NO:49), Figure 14 (SEQ ID NO: 61), Figure 15
(SEQ ID NO:63), Figure 17 (SEQ ID NO:69), Figure 18
(SEQ ID NO:75), Figure 20 (SEQ ID NO:93) and Figure 21
(SEQ ID NO: 116) or may comprise a sequence that is at least about seventy percent homologous to such
sequence.
The present invention also provides for protein or peptide molecules which comprise a sequence substantially as set forth for NT-4 in Figure 2 [SEQ ID NO:22 (NT-4, viper), SEQ ID NO:23 (NT-4, Xenopus)], Figure 4 (SEQ ID NO:44), Figure 8 (SEQ ID NO:50),
Figure 14 (SEQ ID NO: 62), Figure 15 (SEQ ID NO: 64),
Figure 17 (SEQ ID NO:70), Figure 18 (SEQ ID NO:76),
Figure 20 (SEQ ID NO:94), or Figure 21 (SEQ ID NO:117) or may comprise a sequence that is at least about seventy percent homologous to such sequence.
The present invention further provides for expression of biologically active NT-4 molecules in prokaryotic and eukaryotic systems.
The present invention further provides for the production of NT-4 in quantities sufficient for therapeutic and diagnostic applications. Likewise, anti-NT-4 antibodies may be utilized in therapeutic and diagnostic applications. For most purposes, it is preferable to use NT-4 genes or gene products from the same species for therapeutic or diagnostic purposes, although cross-species utility of NT-4 may be useful in specific embodiments of the invention.
The present invention further provides for therapeutic and diagnostic applications based on NT-4 expression by disclosing detectable levels of NT-4 expression in human skeletal muscle, prostate, thymus and testes.
4. DESCRIPTION OF THE FIGURES FIGURE 1. Alignments of DNA sequences of the isolated fragments coding for NGF, BDNF, NT-3 and the novel neurotrophic factor NT-4 from different species.
(A) Schematic representation of the mouse
preproNGF molecule. The hatched box indicates the signal sequence (SS), black bars denote proteolytic cleavage sites and the shaded box represents the mature NGF. Regions used for the degenerate primers are indicated by arrows. The upstream primer was from the region coding for lysine 50 to threonine 56 and the downstream primer includes tryptophan 99 to aspartic acid 105. The amplified region comprises DNA sequences from base pair (bp) 168 to 294 in the mature NGF molecules and in all members of the NGF family described so far, this region is located in one exon.
(B) Alignment of nucleotide sequences for NGF, BDNF, NT-3 and NT-4 isolated from different species. The fragments correspond to amino acids 57 to 98 in the mature mouse NGF. Identical bases are indicated by dots. The numbering refers to nucleotides in the sequences of mouse mature NGF (Scott et al., 1983, Nature 300:538- 540). SEQ ID NO:l (NT-4, viper), SEQ ID NO: 2 (NT-4, Xenopus.. SEQ ID NO: 3 (NGF, human), SEQ ID NO:4 (NGF, rat), SEQ ID NO: 5 (NGF, chicken), SEQ ID NO: 6 (NGF, viper), SEQ ID NO:7 (NGF, Xenopus). SEQ ID NO: 8 (NGF, salmon), SEQ ID NO: 9 (BDNF, human), SEQ ID NO: 10 (BDNF, rat), SEQ ID NO: 11 (BDNF, chicken), SEQ ID NO:12 (BDNF, viper), SEQ ID NO: 13 (BDNF, Xenopus.. SEQ ID NO: 14 (BDNF, salmon), SEQ ID NO:15 (BDNF, ray), SEQ ID NO: 16 (NT-3, human), SEQ ID NO: 17 (NT-3, rat), SEQ ID NO: 18 (NT-3, chicken), SEQ ID NO:19 (NT-3,
Xenopus), SEQ ID NO:20 (NT-3, salmon), SEQ ID NO:21 (NT-3, ray).
FIGURE 2. Alignment of amino acid sequences deduced for NGF, BDNF, NT-3 and NT-4 from different species . The numbering of the amino acids
(single letter code) is taken from the mature mouse NGF (Scott et al., 1983, Nature 300:538- 540). Identical amino acids are indicated with dots. Positions that show conservative amino acid replacements in all species variants of the same factor are underlined. The broken line indicates that the corresponding sequence was not isolated. Bars represent variable regions in the different molecules (R59 to S67 and D93 to A98). SEQ ID NO:22 (NT-4, viper), SEQ ID NO:23 (NT-4, Xenopus), SEQ ID NO:24 (NGF, human), SEQ ID NO:25 (NGF, rat), SEQ ID NO:26 (NGF, chicken), SEQ ID NO:27 (NGF, viper), SEQ ID NO:28 (NGF, Xenopus) . SEQ ID NO:29 (NGF, salmon), SEQ ID NO:30 (BDNF, human), SEQ ID NO:31 (BDNF, rat), SEQ ID NO:32 (BDNF, chicken), SEQ ID NO:33 (BDNF, viper), SEQ ID NO:34 (BDNF, Xenopus), SEQ ID NO:35 (BDNF, salmon), SEQ ID NO:36 (BDNF, ray), SEQ ID NO:37 (NT-3, human), SEQ ID NO: 38 (NT-3, rat), SEQ ID NO:39 (NT-3, chicken), SEQ ID NO:40 (NT-3,
Xenopus), SEQ ID NO: 41 (NT-3, salmon), SEQ ID NO: 42 (NT-3, ray).
FIGURE 3. Deduced phylogeny of members of the NGF
family. Phylogenetic trees showing speciation of NGF (A), BDNF (B), and NT-3 (C) were constructed using analysis of nucleotide sequences. Human NT-3 was used as a reference point in (A) and (B), human NGF and human BDNF were used in (C). The scale bar in (A) represents a branch length corresponding to a relative difference score of 20. The same scale was used in (B) and (C).
(D) shows a phylogram of the evolutionary
relationship between the different members of the NGF family. The data were compiled from deduced amino acid sequences. The scale bar represents a branch length of 20. All trees shown are
unrooted so that the branches are measured relative to one another with no outside
reference. Abbreviations: chi, chicken; hum, human; sal, salmon; vip, viper; xen, Xenopus.
FIGURE 4. Sequence of Xenopus NT-4 and Comparison to
NGF, BDNF, and NT-3. (A) A potential translation start site is boxed. A putative signal cleavage site is indicated by the arrow labeled SC. Amino acids within the signal sequence that are identical between
Xenopus NT-4 and pig and rat BDNF are indicated with stars. A consensus sequence for N- glycosylation is underlined, and the arrow indicates the presumptive start of the mature NT- 4 protein. (SEQ ID NO: 43 and SEQ ID NO: 44)
(B) Amino acid (single-letter code) sequence comparison of Xenopus NT-4 (SEQ ID NO: 45) with mouse NGF (Scott et al., 1983, Nature 300: 538- 540) (SEQ ID NO:46), mouse BDNF (Hofer et al., 1990, EMBO J. 9: 2459-2464) (SEQ ID NO:47), and mouse NT-3 (Hohn et al., 1990, Nature 344: 339- 341) (SEQ ID NO: 48). Identical amino acid replacements compared with the NT-4 amino acid sequence are shown by dots. Sequences that differ between NGF, BDNF, and NT-3 also differ in the sequence of the NT-4 protein.
FIGURE 5. Transient expression of the Xenopus NT-4
protein in COS cells and its interaction with NGF-Rs on PC12 cells.
(A) SDS-PAGE of conditioned media from in vivo labeled COS cell cultures (3 × 104 cpm loaded in each lane) transfected with the rat NGF gene, a control plasmid without insert, or the Xenopus
NT-4 gene. Shown is an autoradiograph of the dried gel after an overnight exposure to X-ray film.
(B) Serial dilutions of transfected COS cell medium containing equal amounts of NT-4 (open circles) or NGF (closed circles) protein were assayed for their ability to displace 125I-NGF from its receptor on PC12 cells. Binding assays were performed at 37°C using 1.5 × 109 M 125I-NGF and 1 × 104 cells per ml. Medium from mock-transfected cells failed to displace binding of 125I-NGF from PC12 cells. Each point represents the mean + SD of triplicate determinations.
FIGURE 6. Stimulation of neurite outgrowth from
chicken embryonic ganglia.
(A, B, and C) Neurite outgrowth elicited in dorsal root ganglia with recombinant NT-4 protein (A), recombinant NGF (B), and BDNF protein (C) . (D) The response of dorsal root ganglia to conditioned medium from mock-transfected cells. (E and F) Stimulation of neurite outgrowth from sympathetic ganglia in response to NT-4 (E) or NGF (F).
(G, H, and I) Nodose ganglia stimulated with recombinant NT-4 (G), NT-3 (H), and BDNF (I) proteins. All figures are bright-field
micrographs of ganglia after 1.5 days in culture. FIGURE 7. Detection of NT-4 mRNA in different Xenopus tissues.
(A) Poly(A)+ RNA (10 μg per slot) from the indicated tissues of adult female Xenopus was electrophoresed in a formaldehyde-containing agarose gel, blotted onto a nitrocellulose filter, and hybridized to a 500 bp Hindi fragment from the 3' exon of the Xenopus NT-4 gene. For comparison, the filter was also hybridized to a 180 bp PCR fragment from the Xenopus NGF gene (lane marked heart, NGF). The filter hybridized to the NT-4 probe was exposed for 2 days; the filter hybridized to the NGF probe was exposed for 2 weeks. A prolonged 2 week exposure of the filter hybridized to the NT-4 probe did not reveal NT-4 mRNA in any tissues other than the ovary, which includes oocytes of different
stages. The lane labeled CNS includes brain and spinal cord.
(B) Poly(A)+ RNA (10 μg) from Xenopus ovary was analyzed for the expression of the four members of the NGF family. Each filter was hybridized with the indicated probes obtained by labeling of PCR fragments from their respective Xenopus
genes. The location of the labeled PCR fragments in the 3' exon of their genes is shown in Figure 1A. The filters were washed at high stringency and exposed to X-ray films for 5 days.
FIGURE 8. Nucleotide sequence of Xenopus NT-4 with
restriction endonuclease cleavage sites (SEQ ID NO:49 and SEQ ID NO:50).
FIGURE 9. NT-4 mRNA expression in the Xenopus laevis
ovary. Ovary from adult Xenopus laevis was
sectioned in a cryostat (14 μm thick sections) and the sections were then hybridized to the
indicated 48-mer oligonucleotides labeled with
35S-dATP using terminal deoxynucleotidyl
transferase.
(A) Hybridization using a Xenopus NT-4 mRNA
specific oligonucleotide with the sequence
5'CCCACAAGCTTGTTGGCATCTATGGTCAGAGCCCTCACATAAGACTGTTTTGC3 ' (SEQ ID NO:95)
(B) Hybridization using a control
oligonucleotide of similar length and G+C content. After hybridization, sections were washed in
iχ SSC at 55°C followed by exposure to X-ray film for 10 days. Shown in the figure are photographs of the developed X-ray films. Note the intense labeling over many small cells with the NT-4 probe and the absence of labeling with the control
probe. Arrows point at large (stage VI) oocytes which are not labeled with either of the two probes. Scale bar, 2 mm.
FIGURE 10. Bright-field illumination of emulsion
autoradiographs showing NT-4 mRNA expressing oocytes in the Xenopus ovary. Sections hybridized to the Xenopus NT-4 mRNA specific (A,B) or control (C) probe as described in FIG. 9 were coated with Kodak NTB2 emulsion, exposed for 5 weeks,
developed and lightly counterstained with cresyl violet This figure shows bright-field
photomicrographs of the developed sections. Note in panel A the intense NT-4 mRNA labeling over small size oocytes (stages I and II) and the absence of labeling over large size (stages V and VI) oocytes. Panel B shows a higher magnification of the boxed in area in panel A. Note the intense labeling of the cytoplasm of the stage II oocytes shown in the picture.
(C) No labeling can be seen using the control probe. Abbreviations: n, nucleus; fc, follicle cells; pi, pigmented layer. Scale bar in A, 50 μm; in B and C, 15 μm.
FIGURE 11. Levels of NT-4 mRNA in oocytes at
different stages of oogenesis. Emulsion
autoradiographs (shown in figure 10) of sections hybridized with the Xenopus NT-4 mRNA specific probe were used to count the number of grains over an area unit. The area unit chosen was about one hundredth of a stage I oocyte. Fifteen area units were analyzed in 10 different oocytes of the indicated stages. Error bars show S.D.
FIGURE 12. Northern blot analysis of NT-4 mRNA
expression during oogenesis in Xenopus laevis.
Ovaries from two adult Xenopus laevis were
dissected out and treated with collagenase to remove follicle cells and release the oocytes.
The oocytes were then grouped in the indicated groups following the stages described by Dumont,
1972, J. Morphol. 136: 153-180. Total ovary and the released follicle cells were also included in the analysis. Total cellular RNA was then prepared and a 40 μg/slot of RNA was
electrophoresed in a formaldehyde-containing 1% agarose gel. This was blotted onto a
nitrocellulose filter and hybridized to a 600bp
HincII fragment from the 3•exon of the Xenopus NT- 4 gene. The filter was washed at high stringency and exposed for five days to a X-ray film. Note the marked decreased in the level of NT-4 mRNA in stages V and VI oocytes.
FIGURE 13. (A) The xNT-4 partial amino acid sequence
(SEQ ID NO: 51) indicating positions where
degenerate oligonucleotides were synthesized and utilized to prime the amplification of human and rat genomic DNA via the polymerase chain reaction.
Arrows indicate oligonucleotides representing sense and antisense degenerate oligonucleotides.
A set of degenerate oligonucleotides to primer 2Z represent amino acids 184-189 of rBDNF (SEQ ID NO:52). The partial Xenopus NT-4 amino acid sequence represented is from amino acid 167 - amino acid 223 , as described in Figure 4, supra.
(B) Degenerate oligonucleotides used for cloning of human and rat NT-4. Oligonucleotide 3Z in Figure 13 is comprised of a mixture of 3Z and 3Z' in order to allow for the degeneracy of the serine codon. 2Y (SEQ ID NO:53), 2Z (SEQ ID NO:54),
3Y (SEQ ID NO: 55), 3Z (SEQ ID NO: 56), (SEQ ID
NO:57) and 4Z (SEQ ID NO:58). (C) Cloning tails for degenerate oligonucleotides 3' (SEQ ID NO: 59) and 5' (SEQ ID NO: 60).
FIGURE 14. DNA sequence of the isolated fragment
encoding a portion of rat NT-4 (SEQ ID NO: 61). The predicted open reading frame for the peptide encoded by the rNT-4 nucleic acid fragment is represented by the single letter code (SEQ ID NO: 62). Sequence inside brackets is part of PCR primer.
FIGURE 15. DNA sequence of the isolated fragment
encoding a portion of human NT-4 (SEQ ID NO: 63). The predicted open reading frame for the peptide encoded by the hNT-4 nucleic acid fragment is represented by the single letter code. (SEQ ID NO: 64) Sequence inside brackets is part of PCR primer.
FIGURE 16. Alignment of amino acid sequences deduced from representative neurotrophins. Amino acids are indicated using the single letter code.
Identical amino acids are indicated with dots. Dashed lines indicate a 7 amino acid insertion within the conserved region of both rNT-4 (SEQ ID NO:62) and hNT-4 (SEQ ID NO:64). XNT-4 (SEQ ID NO:65), rNGF (SEQ ID NO:66), rBDNF (SEQ ID NO:67), rNT-3 (SEQ ID NO: 68). x=Xenopus, r=rat, h=human. Sequence inside brackets is part of PCR primer. FIGURE 17. (A) DNA sequence of an isolated fragment encoding a portion of human NT-4 (SEQ ID NO: 69). The predicted peptide encoded by the 192 bp hNT-4 nucleic acid fragment is represented by the single letter code (SEQ ID NO: 70). Sequence inside brackets is part of PCR primer.
(B) Oligonucleotide sequence of the 5' -end primer, termed hNT4-5'' [containing a sequence (SEQ ID NO: 71) encoding ETRCKA (SEQ ID NO:72)], used in the primary amplification of human genomic DNA along with the 3' -end primer, termed 4Z (SEQ ID NO:58) [containing a nucleotide sequence encoding WIRIDT]. (C) Oligonucleotide sequence of the 5'-end primer used to amplify the primary PCR reaction product. The primer, termed 11NT4-5''' [containing a sequence (SEQ ID NO:73) encoding DNAEEG (SEQ ID NO:74)] was utilized with the 3' primer, 4Z (SEQ ID NO:58), to obtain a fragment of 162 bp (plus bp of cloning tail). The 162 bp PCR fragment was then utilized in a patch PCR reaction using our previously utilized upstream PCR
fragment (termed 2YZ3Z) to generate the single fragment of 192 bp plus cloning tail shown in (A). Additional 3' extended nucleic acid sequence information was obtained following the subcloning and sequencing of this fragment.
FIGURE 18. DNA sequence of the portion of the isolated human genomic phage clone 7-2 encoding human NT-4 (SEQ ID NO:75). The predicted hNT-4 protein encoded by the genomic clone 7-2 is represented by the one-letter symbols for amino acids (SEQ ID NO:76). The boxed region represents the predicted cleavage site of the hNT-4 preprotein. Arrows indicate conserved residues in the presequence. The underlined region (N-R-S) represents a
consensus sequence for n-glycosylation. The circled region represents the initiating
methionine. The splice acceptor site is located at base pair 461-462 (AG) of SEQ ID NO:75,
representing the 3'-end of the intron.
FIGURE 19. Alignment of amino acid sequences deduced from representative neurotrophins (SEQ ID NOS.
77-92) Amino acids are indicated using the single letter code. Amino acids identical to those encoded by the human genomic phage clone 7-2 (SEQ ID NO: 77) are indicated with an asterik. Dashed lines represent breaks in homologous amino acids as compared to the protein encoded by SEQ ID NO: 77.
FIGURE 20. DNA sequence of the isolated fragment
encoding a portion of the human genomic phage clone, 2-1 (SEQ ID NO:93). The predicted open reading frame for the peptide encoded by the isolated nucleic acid fragment is represented by the single letter code (SEQ ID NO: 94).
FIGURE 21. DNA sequence of the isolated fragment
encoding a portion of the human genomic phage clone, 4-2 (SEQ ID NO: 116). The predicted open reading frame for the peptide encoded by the isolated nucleic acid fragment is represented by the single letter code (SEQ ID NO: 117).
FIGURE 22. Northern blot analysis of human NT-4 mRNA expression. Tissue specific mRNA from human was purchased from Clontech. RNA's (10 μg) were fractionated by electrophoresis through a 1% agarose-formaldehyde gel followed by capillary transfer to a nylon membrane (MagnaGraph, Micron Separations Inc.) with 10X SSC (pH 7). RNAs were UV-cross-linked to the membranes by exposure to ultraviolet light (Stratlinker, stratagene, Inc.) and hybridized at 65ºC with the radiolabeled probe (a 680bp Xhol-Notl fragment containing the
complete coding region of HG7-2 NT-4 (see Example Section 9, infra) in the presence of 0.5 M NaPO4 (pH 7), 1% bovine serum albumin (Fraction V,
Sigma), 7% SDS, 1 mM EDTA (Mahmoudi and Lin, 1989, Biotechniques 7:331-333), and 100 μg/ml sonicated, denatured salmon sperm DNA. The filter was washed at 65°C with 2X SSC, 0.1% SDS and subjected to autoradiography overnight with one intensifying screen (Cronex, DuPont) and X-ray film (XAR-5, Kodak) at -70°C. Ethidium bromide staining of the gel demonstrated that equivalent levels of total RNA were being assayed for the different samples (as in Maisonpierre et al., 1990, Science
247:1446-1451).
Lane 1: fetal liver poly(A)+ mRNA; Lane 2: fetal brain poly(A)+ mRNA; Lane 3: prostate poly A+ mRNA; Lane 4: muscle poly(A)+ mRNA; Lane 5:
intestine poly(A)+ mRNA; Lane 6: kidney poly(A)+ mRNA; Lane 7: liver poly(A)+ mRNA; Lane 8:
spleen poly(A)+ mRNA; Lane 9: thymus poly(A)+ mRNA; Lane 10: ovary poly(A)+ mRNA; Lane 11:
testes poly(A)+ mRNA; Lane 12: placenta poly(A)+ mRNA; Lane 13: brain poly(A)+ mRNA; Lane 14:
brain total RNA.
FIGURE 23. COS supernatants from transfected cell
lines; Ql (pCMX-HG7-2Q), N7 (pCMX-hNT3/hNT4) and χι (pCMX-χNT4/hNT4) were tested in volumes of 10 μl, 50 μl and 250 μl for neurite promoting activity in DRG explants. A supernatant from a mock transfected COS cell line was utilized as a control.
FIGURE 24. COS supernatants from Q1 (pCMX-HG7-2Q), and M (pCMX-HG7-2M) cell lines were tested for their survival-promoting activity on DRG
associated cells. Volumes tested ranged from 5 μl to 250 μl in a total volume of 2 ml.
FIGURE 25. Motor neuron enriched cultures isolated
from E14 rat embryos were treated with two
dilutions of COS cell supernatants from the M cell line (pCMX-HG7-2M). Biological activity was measured by choline acetyltransferase (CAT)
activity as described in Fonnum, 1975, J. Neurochem. 24:407-409. Both a mock transfected COS cell line (MOC COS) and an untreated motor neuron (C-NT) are presented as controls.
5. DETAILED DESCRIPTION OF THE INVENTION The present invention provides for NT-4 genes and proteins. It is based, at least in part, on the cloning, characterization, and expression of the NT-4 gene.
In particular, the present invention
provides for recombinant nucleic acid molecules that encode NT-4. Such molecules comprise a sequence substantially as set forth in Figure 1 (SEQ ID NO:l) for viper, Figure 1 (SEQ ID NO: 2), Figure 4 (SEQ ID NO:43) or Figure 8 (SEQ ID NO:49) for Xenopus NT-4, Figure 14 (SEQ ID NO: 61) for rat NT-4, Figure 15 (SEQ ID NO:63), Figure 17 (SEQ ID NO:69) or Figure 18 (SEQ ID NO: 75) for human NT-4, Figure 20 (SEQ ID NO: 93) and Figure 21 (SEQ ID NO: 116) for a human NT-4 like sequence, or a sequence that is at least about seventy percent homologous to any such sequence, in which homology refers to sequence identity (e.g. a sequence that is 70 percent homologous to a second sequence shares 70 percent of the same nucleotide residues with the second sequence).
In a particular aspect the present invention detailed in Example Section 8 and Figure 15 (SEQ ID NO: 63, SEQ ID NO: 64) herein, the nucleotide and amino acid sequence for a portion of a human neurotrophin molecule is determined. In another aspect of the present invention detailed in Example Section 9 and Figure 17 (SEQ ID NO: 69, SEQ ID NO:70) and Figure 18 (SEQ ID NO: 75, SEQ ID NO:76) herein, the nucleotide and amino acid sequence for the entire human
neurotrophin molecule is determined. In another aspect of the present invention detailed in Example Section 9 and Figure 20 (SEQ ID NO: 93, SEQ ID NO: 94) and Figure 21 (SEQ ID NO: 116, SEQ ID NO: 117) herein, the nucleotide and amino acid sequence for a portion of a human genomic phage clones, 2-1 and 4-2,
respectively, which are similar but not identical to the nucleotide and amino acid sequence described in Figure 18 (SEQ ID NO: 75), are detailed. While such human neurotrophin molecule is referred to herein as human neurotrophin-4, it should be understood that such a molecule may be the human homologue of the
Xenopus neurotrophin-4 described herein, or
alternatively, a distinct yet homologous neurotrophin molecule. Similarly, the molecule referred to herein as rat NT-4 may be the rat homologue of NT-4, or alternatively, a distinct yet homologous neurotrophin molecule. The methods and compositions of the present invention do not depend on any single nomenclature.
The present invention also provides for substantially purified NT-4 protein or peptide
molecules. Such molecules may comprise a sequence substantially as set forth in Figure 2, (SEQ ID NO:1 and SEQ ID NO: 2), Figure 4 (SEQ ID NO:44) Figure 8 (SEQ ID NO:50), Figure 14 (SEQ ID NO:62), Figure 15 (SEQ ID NO:64) Figure 17 (SEQ ID NO:70), Figure 18
(SEQ ID NO:76), Figure 20 (SEQ ID NO:94) and Figure 21 (SEQ ID NO:117) for NT-4, or a sequence that is at least about seventy percent homologous to any such sequence. In additional nonlimiting specific
embodiments of the invention, a substantially purified protein or peptide comprises the sequence KCNPSGSTTR (SEQ ID NO: 96). In another embodiment of the
invention, a substantially purified peptide or protein comprises the sequence RGCRGVD (SEQ ID NO: 97). In yet another embodiment of the invention, a substantially purified peptide or protein comprises the sequence KQWIS (SEQ ID NO: 98). In a further embodiment of the invention, a substantially purified peptide or protein comprises the sequence KQSYVR (SEQ ID NO: 99). In yet another embodiment of the invention, a substantially purified peptide or protein comprises the sequence GPGXGGG (SEQ ID NO: 100), where X represents one of the set of 20 amino acids. In a related embodiment of the invention, a substantially purified peptide or protein comprises the sequence GPGVGGG (SEQ ID NO: 101) or GPGAGGG (SEQ ID NO: 102). In a further embodiment of the invention, a substantially purified peptide or protein comprises the sequence ESAGE (SEQ ID NO: 103). In yet a further embodiment of the invention, a substantially purified peptide or protein comprises the sequence DNAEE (SEQ ID NO: 104).
The proteins and peptides of the invention may be produced by chemical synthesis using standard techniques or may be produced using the NT-4-encoding nucleic acid molecules of the invention, using
prokaryotic or eukaryotic expression systems known to one skilled in the art, such as those described in PCT application PCT/US90/04916, filed August 29, 1990, published as WO 91/03569, which is incorporated by reference in its entirety herein, or as exemplified infra (see Section 6.2.4., infra, and Figure 5) for transient expression in COS cells.
The present invention also provides for the use of NT-4 in promoting the growth and/or survival of cells of the nervous system, in particular, but not limited to, cells of dorsal root ganglion or neural placode derivatives (see Section 6.2.4., infra, and Figure 6, for example).
The present invention also provides for portions of NT-4 nucleic acid or amino acid sequence, substantially as set forth for NT-4 in Figure 1, 2, 4, 8, 14, 15, 17, 18, 20 or 21 (SEQ ID NO's listed,
supra) that are not identical to portions of BDNF, NGF, or NT-3 of substantially the same size.
The present invention further provides for a eukaryotic or prokaryotic cell that contains
recombinant nucleic acid that encodes NT-4 and that expresses recombinant NT-4 protein. In a specific embodiment the cell is a eukaryotic cell, such as a COS cell. Accordingly, the present invention also provides for recombinant NT-4 protein or peptide that is produced by inserting recombinant nucleic acid encoding NT-4 into a cell (e.g., by transfection, transduction, electroporation, microinjection, etc.) under conditions which permit expression of NT-4 and then isolating NT-4 from the cell.
In addition, the present invention provides for molecules produced by PCR using, for example, the following oligonucleotides as primers:
5'CAGTATTTTTACGAAACC (SEQ ID NO: 105) and
3'GTCTTGTTTGGCTTTACA (SEQ ID NO: 106) for human NT-4 and 5'CAGTATTTTTACGAGACG (SEQ ID NO: 107) and
3'CGATTGTTTGGCTTTACA (SEQ ID NO: 108) for rat NT-4, and using any suitable genomic or cDNA as template. In a specific embodiment of the invention, these primers may be used in conjunction with human cDNA as template to produce fragments of the human NT-4 gene that are suitable for cloning.
The production and use of derivatives, analogues, and peptides related to NT-4 are also envisioned, and within the scope of the present
invention. Such derivatives, analogues, or peptides which have the desired neurotrophic activity,
immunogenicity or antigenicity can be used, for
example therapeutically, or in immunoassays, for immunization, etc. Derivatives, analogues, or
peptides related to NT-4 can be tested for the desired activity by procedures known in the art.
The NT-4 related derivatives, analogues, and peptides of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned NT-4 gene can be modified by any of numerous strategies known in the art (Maniatis, T., 1982, Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The NT-4 sequence can be cleaved at appropriate sites with restriction
endonuclease(s), followed by further enzymatic
modification if desired, isolated, and ligated
in vitro. In the production of the gene encoding a derivative, analogue, or peptide related to NT-4, care should be taken to ensure that the modified gene remains within the same translational reading frame as NT-4, uninterrupted by translational stop signals, in the gene region where the desired NT-4-specific activity is encoded.
Additionally, the NT-4 gene can be mutated in vitro or in vivo, to create and/or destroy
translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy
preexisting ones, to facilitate further in vitro
modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem. 253:6551), use of TAB® linkers (Pharmacia), etc.
As discussed infra. the prepro or mature coding region of NT-4 may be utilized to construct neurotrophin based chimeric genes. For example, neurotrophin genes, including but not limited to NGF, BDNF and NT-3, can provide the prepro region for construction of neurotrophin prepro/NT-4 mature coding region chimeric genes.
Manipulations of the NT-4 sequence may also be made at the protein level. Any of numerous
chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
In addition, analogues and peptides related to NT-4 can be chemically synthesized. For example, a peptide corresponding to a portion of NT-4 which mediates the desired neurotrophic activity can be synthesized by use of a peptide synthesizer.
The present invention further provides for a method of treating fertility disorders related to ovarian/oocyte dysfunction. As shown in the examples infra, in particular, Section 7, NT-4 is involved in the maturation of oocytes. The discussion of Section 7.3 demonstrates that NT-4 is produced by oocytes, is concentrated in immature rather than mature oocytes, and appears to play a role in oogenesis. The putative function of NT-4 protein in the ovary appears to be coupled to events occurring in the pre-vitellogenic and early/mid vitellogenic oocyte.
it has been established that other members of the BDNF/NGF/NT-3 gene family, in particular NGF, are involved in meiotic maturation (Nebrada et al., 1991, Science, 252: 558-563). Because NT-4 has
exhibited properties similar to NGF (see Section 6, infra) it may be used as a factor involved in the regulation of oocyte development. These properties of NT-4 can be exploited to provide a method for treating infertility disorders and/or other ovarian
dysfunctions associated with oogenesis.
Therefore, in accordance with the invention a method of treating infertility disorders and/or other ovarian dysfunctions comprising administering a therapeutically effective amount of NT-4 or an NT-4 related peptide in a pharmaceutically effective carrier is provided. A therapeutically effective amount is one which induces proper maturation of an oocyte and/or ovulation. For example, a
therapeutically effective dose may be one sufficient to maintain circulating serum levels of NT-4 at a concentration of from about 1 to 100 × 10-10M.
Establishing additional effective doses is within the purview of one skilled in the art.
Effective doses of NT-4 or an NT-4 related peptide formulated in suitable pharmacological
carriers may be administered by any appropriate route including but not limited to injection (e.g.,
intravenous, intraperitoneal, intramuscular,
subcutaneous, etc.), by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.); etc.
In addition, NT-4 or NT-4 peptide may be used in any suitable pharmacological carrier, linked to a carrier or targeting molecule (e.g., antibody, hormone, growth factor, etc.) and/or incorporated into liposomes, microcapsules, and controlled release preparation prior to administration in vivo.
Each respective mammalian NT-4 DNA sequence can be utilized as a 32P-labelled probe to isolate a respective genomic and cDNA clone via the procedures outlined in the Materials and Methods portion Section 8, infra. The rat NT-4 and human NT-4 gene fragments may be utilized directly (as 32P-labelled probes) or indirectly (to deduce a PCR strategy as described infra) to isolate other mammalian NT-4 genomic and cDNA clones, based on the unique nature of the 7 amino acid insertion in the rNT-4 and hNT-4 coding region, or other unique aspects of the rat or human NT-4 coding region.
Any mammalian NT-4 gene isolated via the information disclosed by the rat and human NT-4 sequence may be utilized in, although is not limited to, the various manipulations discussed for Xenopus NT-4. For example, the proteins and peptides of mammalian NT-4, subsequent to characterization of the full length gene as discussed in Example Section 9, may be produced using the respective mammalian NT-4 molecules in a prokaryotic or a eukaryotic expression system known to one skilled in the art, such as those described in PCT application PCT/US90/04916, filed August 29, 1990, published as WO91/03569, or as exemplified infra (see Section 6.2.4., supra, and Figure 5) for transient expression in COS cells.
Additional functions for mammalian NT-4, as described infra for Xenopus NT-4, include, but are not limited to: the promotion of growth and/or survival of cells of the nervous system, in particular, but not limited to, cells of dorsal root ganglion or neural placode derivatives (see Section 6.2.4., and Figure 6, for example), treating fertility disorders related to ovarian/oocyte dysfunction (see Section 7), the
treatment of infertility disorders and/or other
ovarian dysfunction associated with oogenesis (see Section 6), the treatment of motor neuron diseases (see Section 10), the treatment of an epitheliac hyperplasia such as benign prostatic hypertrophy (see Section 10), the treatment of impotence as related to prostate gland function (see Section 10) and,
therefore, the therapeutically effective amounts of mammalian NT-4 for the treatment of said disorders as formulated in suitable pharmacological carriers to provide a pharmaceutical composition may be
administered by any appropriate route including but not limited to injection (e.g., intravenous,
intraperitoneal, intramuscular, subcutaneous, etc.), by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.); etc.
In addition, rat, human or other mammalian NT-4 or NT-4 peptide may be used in any suitable pharmacological carrier, linked to a carrier or targeting molecule (e.g., antibody, hormone, growth factor, etc.) and/or incorporated into liposomes, microcapsules, and controlled release preparation prior to administration in vivo.
In addition, the present invention, which relates to nucleic acids encoding NT-4 and to
proteins, peptide fragments, or derivatives produced therefrom, as well as antibodies directed against NT-4 protein, peptides, or derivatives, may be utilized to diagnose or monitor the progression of diseases and disorders of the nervous system which are associated with alterations in the pattern of NT-4 expression. Such alterations can be a decrease or increase
relative to that in normal patients, preferably, or in other samples taken from the patient, or in samples from the same patient taken at an earlier time.
In various embodiments of the invention, NT-4 genes and related nucleic acid sequences and subsequences, including complementary sequences, may be used in diagnostic hybridization assays. The NT-4 nucleic acid sequences, or subsequences thereof comprising about 15 nucleotides, can be used as hybridization probes. Hybridization assays can be used to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in NT-4 levels. For example, the data presented in Example Section 10 discloses tissue specific expression of human NT-4 in skeletal muscle as well as the prostate gland, thymus and testes. The level of expression of human NT-4 in the muscle tissue may be indicative of the presence or absence of neuronal degradation. Therefore, poly(A)+ mRNA or total RNA from a tissue sample of a patient could be assayed for the presence of human NT-4 mRNA in
skeletal muscle tissue. Additionally, the data presented in Example Section 10 discloses tissue specific expression of NT-4 in the human prostate gland. DNA sequences encoding NT-4 or a portion thereof, as well as NT-4 protein or a peptide may be useful as a therapeutic agent to treat prostate disease.
In a similar method, diagnostic assays can be immunoassays. Thus, antibodies can be used in immunoassays to quantitate the level of NT-4 in a sample from a patient, in order to detect, prognose, diagnose, or monitor conditions, disorders, or disease states associated with changes in NT-4 levels.
The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and
immunoelectrophoresis assays, to name but a few.
Anti NT-4 antibody fragments or derivatives containing the binding domain may also be used in such assays.
Antibody fragments which contain the
idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
Diagnostic kits are also provided. For example, such a kit can comprise in a suitable
container an NT-4 specific probe. In one embodiment, the probe is an antibody specific for NT-4. In another embodiment, the probe is a nucleic acid
(molecular probe) capable of hybridizing to an NT-4 nucleic acid sequence. The probe can be detectably labeled; alternatively, the kit can further comprise a labeled specific binding partner for the probe.
The above-described hybridization assays and immunoassays can also be used to quantitate NT-4 levels as an indication of therapeutic efficacy, by comparing the levels in patient samples before and after treatment of a disorder, particularly, a motor neuron disease.
In an analogous fashion, the expression of human NT-4 mRNA in muscle tissue leads to potential methods of treating motor neuron disorders comprising administering to a patient in need of such treatment an effective amount of an NT-4 factor to support the survival, growth, and/or differentiation of motor neurons. Expression of NT-4 mRNA in human muscle suggests further avenues for diagnosing and treating neuron disorders. Retrograde axonal transport may be characteristic of NT-4. The specific retrograde transport of NT-4 can be used to indicate whether neurons are responsive to NT-4 in normal or diseased states. Therefore, the present invention provides for a method of diagnosing NT-4 related peripheral nervous system disorders comprising injecting a detectably labeled NT-4 protein or peptide into a peripheral nerve and determining whether the labeled NT-4 protein or peptide is retrogradely transported, in which a failure to be retrogradely transported positively correlates with lack of responsiveness to NT-4 and indicates the presence of a peripheral nervous system disorder that is NT-4 related. Analogous methods may be used to diagnose a central nervous system disorder. Evaluation of retrograde transport may be performed by any method known in the art, including but not limited to MRI, CAT, or scintillation scanning. Such methods may be used to identify the location of a nervous system lesion, as retrograde transport should
substantially diminish upon reaching the lesion.
The invention further provides kits for such retrograde evaluation comprising in a container a detectably labeled NT-4 protein, derivative or
fragment. Such a label can be a radioactive isotope, or other label known in the art.
The present invention may be utilized to treat diseases and disorders of the nervous system which may be associated with alterations in the pattern of NT-4 expression or which may benefit from exposure to NT-4 or anti-NT-4 antibodies (or fragments thereof containing the binding domain). We show that human NT-4 is expressed in skeletal muscle (See Example Section 10, infra). Based on this discovery, the invention provides for the treatment of motor neuron diseases. A wide array of neurological disorders may affect motor neurons. Upper motor neurons, for example, are predominantly affected by cerebrovascular accidents, neoplasms, infections and trauma. Lower motor neurons, or anterior horn cells, are secondarily affected by these processes, but in addition are subject to a number of disorders in which anterior horn cell loss is the primary feature, including amyotrophic lateral sclerosis, infantile and juvenile spinal muscular atrophy, poliomyelitis and the post-polio syndrome, hereditary motor and sensory neuropathies, and toxic motor neuropathies (e.g.
vincristine). The disorders of motor neurons which can be treated according to the present invention include but are not limited to the foregoing. Methods of formulation and administration of NT-4 protein, derivatives, fragments, or antibodies thereto which can be used include but are not limited to those disclosed supra or known in the art.
The invention may also be utilized to treat benign prostatic hypertrophy (BPH), a common yet poorly understood condition ocurring mostly in males over 50 years of age. The proliferation of the prostrate during BPH may be induced by a growth factor such as NT-4 through an autocrine loop phenomenon. Synthesis and excretion of NT-4 would be followed by transport of NT-4 back into the prostate cell via a specific receptor on the prostate cell membrane. Autocrine loops have been defined for various growth factor molecules and tumor cell lines. In some cases, these autocrine loops have been experimentally defined by the use of antisense approaches for the disruption of the autocrine loop. Therefore, a therapeutic application of the present invention includes the use of a nucleic acid anti-sense to human NT-4 or a portion thereof to inhibit translation of NT-4 mRNA in the prostate, (for procedures which can be used, see copending U.S. Application Serial No. 07/728,784 filed July 3, 1991 and incorporated by reference herein in its entirety). For example, a patient suffering from a prostate localized disease characterized by
increased transcription in prostate tissue of an NT-4 gene relative to that of transcription levels of the NT-4 gene in the prostate of normal patients could be administered an effective amount of an oligonucleotide to treat a prostate disease, preferably benign
prostatic hypertrophy. The oligonucleotide should be at least 6 nucleotides in length, complementary to a least a portion of the RNA transcript of the NT-4 gene and, hence, being capable of hybridizing to the NT-4 transcript. Additionally, anti-NT-4 antibodies may be utilized to inhibit binding of NT-4 to its specific receptor on the prostate cell membrane. A
therapeutically effective amount of either an NT-4 antisense nucleic acid or an anti-NT-4 antibody may be delivered in any fashion described supra.
The invention may also be utilized to treat other prostate related dysfunctions, specifically impotence. Such a malady may be the direct or
indirect result of inadequate levels of NT-4 in the prostate. Therefore, both the detection of the dysfunction as well as treating the patient for
impotence via application of a therapeutically
effective amount of NT-4 protein or a functional fragment or derivative of NT-4 may be delivered by any method described supra.
The present invention discloses the detection of NT-4 expression in human thymus tissue. Therefore, the invention may also be utilized to treat immunological disorders affecting neuromuscular transmission, including but not limited to myasthenia gravis, an acquired autoimmune disorder associated with the acetylcholine receptor (AChR) within the postsynaptic folds at the neuromuscular junction. The disease manifests itself as weakness and muscular fatigue due to blockage of post-synaptic AChR or muscle membranes by binding of antibodies specific to the AChR. (See, e.g., Drachman, 1983, Trends
Neurosci. 6:446-451). Treatment of such immunological mediated neurological disorders may include
therapeutic applications of the NT-4 protein or a functional fragment or derivative of NT-4, delivered by any of the methods described supra.
The present invention provides for a method of treating motor neuron disorders comprising
administering, to a patient in need of such treatment, an effective amount of an NT-4 protein, derivative or peptide fragment capable of supporting the survival, growth and/or differentiation of motor neurons as demonstrated in an in vitro culture system.
In in vitro embodiments, effective amounts of neurotrophic factor may desirably be determined on a case by case basis, as motor neurons from different tissue sources or from different species may exhibit different sensitivities to neurotrophic factor. For any particular culture, it may be desirable to
construct a dose response curve that correlates neurotrophic factor concentration and motor neuron response. To evaluate motor neuron survival, growth, and/or differentiation, one can compare motor neurons exposed to an NT-4 protein, derivative or peptide fragment to motor neurons not exposed to an NT-4 protein, derivative or peptide fragments, using, for example, vital dyes to evaluate survival, phase- contrast microscopy and/or neurofilament stain to measure neurite sprouting, or techniques that measure the bioactivity of motor neuron-associated compounds, such as choline acetyltransferase (CAT), or any other methods known in the art. CAT activity may be measured, for example, by harvesting and lysing treated and untreated motor neurons in a 20 mM
Tris-HCl (pH 8.6) solution containing about 0.1% Triton X-100, removing an aliquot of several
microliters, and measuring for CAT activity using, as a substrate, 0.2 ml [1 - γC] acetyl-CoA, 300 mM NaCl, 8 mM choline bromide, 20 mM EDTA, and 0.1 mM
neostigmine in 50 mM NaH2PO4 (pH 7.4) buffer, using the micro-Fonnum procedure as described in Fonnum, 1975, J. Neurochem. 24:407-409, incorporated by reference in its entirety herein.
In a specific, non-limiting embodiment of the invention, motor neurons may be prepared, and cultured in vitro, as follows. At least a portion of a spinal cord, preferably obtained from an embryonic organism such as a rat, may be aseptically obtained and separated from the bulb, sensory ganglia, and adhering meninges. The ventral segments of the cord may then be isolated, as motor neurons are localized in the ventral (anterior) horns of the spinal cord. Ventral cord segments may be diced into small pieces and incubated in about 0.1% trypsin and 0.01%
deoxyribonuclease type 1 in calcium and magnesium-free phosphate buffered saline (PBS) at 37°C for about 20 minutes. The trypsin solution may then be removed, and the cells may be rinsed and placed in fresh medium, such as 45% Eagle's minimum essential (MEM),
45% Ham's nutrient mixture F12, 5% heat inactivated fetal calf serum, 5% heat inactivated horse serum, glutamine (2 mM), penicillin G (0.5 U/ml), and
streptomycin (0.5 μg/ml). The tissue may be
mechanically dissociated by gentle trituration through a Pasteur pipet, and the supernatants pooled and filtered through a nylon filter (e.g. Nitex, Tetko; 40 μm). The filtered cell suspension may then be fractioned using a modification of the method set forth in Schnaar and Schaffner (1981, J. Neurosci. 1:204-217). All steps are desirably carried out at 4°C. Metrizamide may be dissolved in F12:MEM medium (1:1) and a discontinuous gradient may be established that consists of a 18% metrizamide cushion (e.g.
0.5 ml), 3 ml of 17% metrizamide, 3 ml of 12%
metrizamide, and 3 ml of 8% metrizamide. The filtered cell suspension (e.g. 2.5 ml) may be layered over the step gradient and the tube may be centrifuged at
2500 g for about 15 minutes using a swing-out rotor (e.g. Sorvall HB4). Centrifugation may be expected to result in three layers of cells: fraction I (at 0-8% interface), fraction II (at 8-12% interface) and fraction III (at 12-17% interface). Fraction I, enriched for motor neurons, may be removed in a small volume (e.g. about 1 ml) and rinsed twice with a serum-free defined medium such as 50% F12 and 50% MEM supplemented with glutamine (2 mM), insulin (5 μg/ml), transferrin (100 μg/ml), progesterone (20 nM),
putrescine (100 μM), and sodium selenite (30 nM, see Bottenstein and Sato, 1979, Proc. Natl. Acad. Sci.
U.S.A. 76:514-517). Viable cell count may then be obtained by hemocytometer counting in the presence of trypan blue. The motor neuron enriched cell
suspension may then be plated at a density of about 100,000 cells/cm2 in tissue culture wells (preferably 6 mm) precoated with poly L-ornithine (e.g. 10 μg/ml) and laminin (e.g. 10 μg/ml). An NT-4 protein, derivative or peptide factor may then be added. For example, in specific embodiments, NT-4 may be added to achieve a final concentration of between about 0.01 and 100 ng/ml, and preferably about 50 ng/ml. The motor neuron cultures may then be maintained in serum- free defined medium at 37°C in a 95% air/5% CO2 atmosphere at nearly 100% relative humidity.
In a further embodiment of the invention, the NT-4 related recombinant nucleic acid sequence, such as contained in bacteriophage HG7-2, HG4-2, and/or HG2-1, may be utilized to construct chimeric prepro/mature NT-4 genes. For example, when it is desired to express a mature NT-4 protein, derivative or peptide fragment in vivo or in vitro, one can fuse the pre-pro region of a distinct neurotrophic gene to the mature coding region of the NT-4 related sequence. The neurotrophic genes which can provide the prepro region include but are not limited to NGF, BDNF, and NT-3. Such a chimeric construct may promote increased stability of the chimeric mRNA transcript in relation to a wild type NT-4 mRNA transcript, may increase translational efficiency or may generate a more suitable template for proteolytic processing to a mature, biologically active neurotrophin protein or peptide fragment, thus increasing expression. One of ordinary skill in the art possesses the requisite knowledge to construct such chimeric nucleic acid sequences, given the published DNA sequences of other neurotrophin genes such as NGF (Scott et al., 1983, Nature 302: 538-540; Ullrich et al., 1983, Nature
303:821-825). BDNF (Leibrock et al., 1989, Nature
341:149-152) and NT-3 (Hohn et al., 1990, Nature
344:339-341: Maisonpierre et al., 1990 Science
247:1446-1451: Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA 87:5454-5458; Rosenthal et al., 1990, Neuron 4:767-773), as well as guidance as to
strategies for generating a fusion junction (for example, see Darling et al . , 1983 , Cold Spring Harbor Symposium Quantative Biology 48:427-434; Edwards et al., 1988, J. Biol. Chem. 263:6810-6815; Suter et al., 1991, EMBO J. 10:2395-2400). In another embodiment, chimeric constructions fusing the pre-pro region of an NT-4 related recombinant nucleic acid, such as
contained in bacteriophage HG7-2, HG4-2 and HG2-1, to the mature regions of other neurotrophins, can also be used to promote efficient expression of such other neurotrophins, as discussed supra.
The present invention also provides methods of detecting or measuring NT-4 activity. As described in Example 12, we have discovered that trkB is a functional receptor for NT-4. Based on this
discovery, the invention provides methods for
detecting or measuring NT-4 activity comprising exposing a cell that expresses trkB to a test agent, and detecting or measuring binding of the test agent to trkB, in which specific binding to trkB positively correlates with NT-4 activity in the test agent. In a specific embodiment, the cell that expresses trkB is a transfected cell such as a 3T3 fibroblast, which expresses recombinant trkB, such that the survival of the cell is dependent upon exposure to neurotrophin-4 or BDNF. Thus detecting of binding of the test agent can be carried out by observing the survival of such transfected cells. 6. EXAMPLE: EVOLUTIONARY STUDIES OF THE
NERVE GROWTH FACTOR FAMILY REVEAL A NOVEL MEMBER ABUNDANTLY EXPRESSED IN XENOPUS OVARY
6. 1. MATERIALS AND METHODS
6. 1. 1. DNA PREPARATION
Genomic DNA was isolated by standard procedures (Davis et al., 1986, "Basic Methods In Molecular Biology", Elsevier, New York)) from human leukocytes and from liver of Sprague-Dawley rat, frog (Xenopus laevis) and ray (Raja, clavata). Genomic DNA was also obtained from salmon (Salmon) and from the elephant snake (Vipera lebetina). The DNA was precipitated with ethanol, collected using a glass hook, washed in 80% ethanol, dried and dissolved in water to a final concentration of 1 mg/ml. Salmon DNA (Sigma, St. Louis, MO) was dissolved in water, extracted twice with phenol and once with chloroform, and precipitated with ethanol.
6. 1. 2. POLYMERASE CHAIN REACTIONS, MOLECULAR
CLONING AND DNA SEQUENCING
Six separate mixtures of 28-mer
oligonucleotides representing all possible codons corresponding to the amino acid sequence KQYFYET (SEQ ID NO:110) (5'-oligonucleotide) and WRFIRID (SEQ ID NO:111) (3'-oligonucleotide) (Fig. 1A) were
synthesized on an Applied Biosystem A381 DNA
synthesizer. The 5'oligonucleotide contained a synthetic EcoRI site and the 3'-oligonucleotide contained a synthetic HindIII site (Knoth et al., 1988, Nucl. Acids Res. 16: 1093; Nunberg et al., 1989, J. Virology 63: 3240-3249). Each mixture of
oligonucleotides was then used to prime the
amplification of 0.8 μg of genomic DNA using the polymerase chain reaction (PCR) (Taq DNA polymerase, Promega) (Saiki et al., 1985, Science 230:1350-1354). The PCR products were restricted with HindIII and
EcoRI, analyzed on a 2% agarose gel and cloned into plasmid Bluescript KS+ (Stratagene, La Jolla,CA). The size of the amplified region plus primers is 179 base pairs (bp) for NGF and 182 bp for BDNF and NT-3. As a result of internal EcoRI sites in some cases, shorter fragments of 144 bp and 95 bp were also isolated. The cloned DNA fragments were sequenced using the dideoxy nucleotide chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467) with T7 DNA polymerase (Pharmacia, Uppsala). Between 2 and 20 independent clones were sequenced for each gene and species, and altogether more than 200 independent clones were sequenced.
Approximately 2,000,000 clones from a
Xenopus genomic library prepared by insertion of Mboldigested genomic DNA in the BamHI site of phase λEMBL-3 were screened using conventional procedures with a 182 bp PCR fragment of Xenopus NT-4 labeled with [α-32P]dCTP by nick translation to a specific activity of approximately 5 × 108 cpm/μg.
Hybridization was carried out in 4 × SSC (1 × SSC is 150 mM NaCl, 15 mM sodium citrate (pH 7.0)), 40% formamide, 1 × Denhardts solution, 10% dextran sulfate at 42°C. The filters were washed at 55°C in 0.1 × SSC, 0.1% SDS and exposed to Kodak XAR-5 films at - 70°C. Eight phage clones were isolated, and a
hybridizing 1.5 kb PstI fragment from one of these clones was subcloned in the plasmid pBS-KS
(Stratagene). The nucleotide sequence of the
subcloned fragment was determined by the dideoxy chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467). 6. 1. 3. COMPUTER ANALYSIS OF THE SEQUENCE DATA DNA and amino acid sequence comparisons and alignments shown in Table I were performed on a VAX computer using UWGCG software (Devereux et al., 1984, Nucl. Acids Res. 72:387-395). The results of
comparing amino acid sequences using the UWGCG programs are presented as percent amino acid
similarity or nucleotide identity between the
sequences, taking conservative amino acid changes into consideration (Gribskov and Burgess, 1986, Nucl. Acids Res. 14:6745-6763; Schwartz and Dayhoff, 1979, "An Atlas of Protein Sequence and Structure", ed., Natl. Biomed. Res. Found., Washington D. C., pp. 353-358). Phylogenetic Analysis Using Parsimony (PAUP version 3. Of) was used for the construction of the phylograms (Felsenstein, 1988; Annu. Rev. Gene. 22:521-555:
Swofford and Olsen, 1990, in "Molecular Systematics," Hills and Moritz, Eds., Sunderland, MA., Sinaver
Assoc., Inc. pp. 441-501). Searches for the most probable trees were run using both exhaustive and heuristic (branch swapping) algorithms.
6. 1. 4. PRODUCTION OF RECOMBINANT PROTEIN,
BINDING ASSAY TO PC12 CELLS, AND
ASSAYS OF NEUROTROPHIC ACTIVITIES
For transient expression of recombinant proteins in COS cells, appropriate DNA fragments were cloned in the vector pXM (Yang et al., 1986, Cell
47:3-10). For NT-4 the sequenced 1.5 kb PstI fragment from Xenopus was cloned in pXM, and for NGF a 771 bp BstEII-PstI fragment from the 3' exon of the rat NGF gene was used (Halbook et al., 1988, Development
108:693-704). To express BDNF protein, a PCR-amplified fragment containing the prepro-BDNF coding sequence from the mouse BDNF gene (Hofer et al., 1990, EMBO J. 9:2459-2464) was also subcloned in pXM. For NT-3, a 1020 bp rat cDNA clone was inserted in pXM (Ernfors et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:5454- 5458).
COS cells (Gluzman, 1981, Cell 3:175-182) grown to about 70% confluency were transfected with 25 μg of plasmid DNA per 100 mm dish using the DEAE-dextran-chloroquine protocol (Luthman and Magnusson, 1983, Nucl. Acids Res. 17:1295-1305). Transfected cells were then grown in complete medium (DMEM plus 10% FCS), and conditioned medium was collected 3 days after transfection. Dishes (35 mm) transfected in parallel were grown over the third night after
transfection in the presence of 200 μCi/ml
[35S]cysteine (Amersham, UK). Aliquots (10-20 μl each) of the in vivo labeled conditioned media were analyzed by SDS-PAGE in 13% polyacrylamide gels. The gels were treated with EnHance (New England Nuclear, Boston, MA), dried, and exposed to Kodak XAR5 films with intensifying screens for 24-48 hr at 80ºC.
Autoradiographs were scanned in a Shimadzu
densitometer, and the relative amounts of the
different recombinant proteins were estimated by calculating the area corresponding to each protein relative to that obtained with rat NGF. The absolute amount of rat NGF protein was assessed by quantitative immunoblotting of conditioned media using standards of purified mouse NGF and was used to determine the protein concentration in the samples containing the other recombinant proteins.
For binding assay of recombinant proteins to PC12 cells (Greene and Tischler, 1976, Proc. Natl.
Acad. Sci. U.S.A. 73:2424-2428), mouse NGF was labeled with 125I by the chloramine-T method to an average activity of 7 × 107 cpm/μg. Steady-sate binding was measured in competition assays performed at 37°C or 0°C using 1 × 104 cells per ml, 1.5 × 10-9 M 125I-NGF; and serial dilutions of conditioned media containing equivalent amounts of NGF or NT-4. All components were added at the same time, and cells were collected by centrifugation after equilibrium was reached (1-3 hr incubation). Control experiments using medium from mock-transfected COS cells showed that other proteins present in the conditioned medium had no effect on the binding of I25I-NGF to PC12 cells. Nonspecific binding was measured in a parallel incubation to which at least a 1000-fold excess of unlabeled NGF was added. All results were corrected for this nonspecific binding which was always less than 10% of the total binding.
The biological activities of the different proteins were measured by the ability of transfected COS cell conditioned media, containing equal amounts of recombinant protein, to stimulate neurite outgrowth from explanted sympathetic, nodose, and dorsal root ganglia from E9 chicken embryos (Ebendal, 1984,
"Organizing Principles of Neural Development, S.
Sharms, ed., New York: Plenum Publishing Corp., pp. 93-107; Ebendal, 1989, "Use of Collagen Gels to
Bioassay Nerve Growth Factor Activity In Nerve Growth Factors", R. A. Rush, ed. (Chichester: John Wiley & Sons, pp. 81-93). Serial dilutions of conditioned medium were assayed and the fiber outgrowth was scored. 6. 1. 5. RNA PREPARATIONS AND BLOT ANALYSIS
The indicated tissues from adult female
Xenopus were dissected and frozen in liquid nitrogen. The brain and spinal cord were pooled. Several lobes of the ovary were dissected out, including oocytes of different stages. The frozen tissue samples were homogenized in 4 M guanidine isothiocyanate, 0.1 M β-mercaptoethanol, 0.025 M sodium citrate (pH 7.0) and homogenized three times for 15 s with a Polytron.
Each homogenate was layered over a 4 ml cushion of 5.7 M CsCl in 0.025 M sodium citrate (pH 5.5) and
centrifuged at 15°C in a Beckman SW41 rotor at 35,000 rpm for 76 hr (Chirgwin et al., 1979, Biochemistry
78:5294-5299). Poly(A)+ RNA was purified by
oligo(dT)-cellulose chromatography (Aviv and Leder, 1972, Proc. Natl. Acad. Sci. U.S.A. 69:1408-1412), and the recovery of RNA was quantified
spectrophotometrically before use in RNA blot
analysis. Poly(A)+ RNA (10 μg) from each sample was electrophoresed in a 1% agarose gel containing 0.7% formaldehyde. UV-transillumination of the stained gel was used to confirm that all samples contained similar amounts of intact RNA. The gel was then transferred to a nitrocellulose filter. The filter was hybridized to the indicated DNA probes. The probes were labeled with [α-32P]dCTP by nick translation to a specific activity of around 5 × 105 cpm/μg, and the
hybridization was carried out as described above.
Filters were washed at high stringency (0.1 × SSC, 0.1% SDS, 54ºC) and exposed to Kodak XAR-5 films.
6. 2. RESULTS
DNA fragments coding for NGF, BDNF and NT-3 from human, rat, snake, frog, and fish were isolated using the PCR technique with degenerate primers from conserved regions in these three proteins located between lysine 50 and threonine 56 for the upstream primer and between tryptophan 99 to aspartic acid 105 for the downstream primer (Fig. 1A). The amplified region contains three of the six cysteine residues and covers approximately one third of the mature molecules. A comparison of the amplified region in already characterized NGF molecules from different species shows that it contains two variable regions, arginine 59 to serine 67 and aspartic acid 93 to alanine 98. A hydrophilic stretch believed to be exposed on the surface of the molecules (Bradshaw, 1978, Ann. Rev. Biochem. 47:191-216), as well as the highly conserved regions glycine 68 to tryptophan 76 and threonine 85 to threonine 91 are also included in the amplified region. The BDNF and NT-3 molecules have an extra amino acid between positions 94 and 95 of the mouse NGF protein which is also included in the amplified region.
The sequences of the entire mature molecule of mouse NGF, BDNF and NT-3 proteins were compared in order to calculate how representative the amplified region is of the complete molecule. The entire mature molecules show 65/57% similarity (amino acid sequence similarity/nucleotide sequence identity) between NGF and BDNF, 70/61% similarity between NGF and NT-3 and 68/58% similarity between BDNF and NT-3. When
comparing the region isolated in this study, the similarity between NGF and BDNF is 62/53%, that between NGF and NT-3 is 67/58%, and that between BDNF and NT-3 69/60%. This strongly suggests that the region isolated in this study is representative for the entire molecule and that it can be used to monitor the evolutionary relationships among the different factors. Pairwise sequence comparison were performed (Table I) taking conservative amino acid replacements into consideration, using the comparison matrix of Schwartz and Dayhoff (1979, "In Atlas of Protein
Sequence and Structure, M.O. Dayhoff, ed., Washington, D.C., Natl. Biomed. Res. Found., pp. 353-358).
Therefore, comparisons of amino acid sequences given below and shown in Table I indicate percent
similarity, not identity. Phylogenetic trees were constructed using parsimony analysis (Felsenstein, 1988, Ann. Rev. Genet. 22:521-565; Swofford and Olsen, 1990, "In Molecular Systematics, D. M. Hills and C. Moritz, eds., Sunderland, MA:Sinauer Assoc, Inc., pp. 411-501). As shown below, all isolated DNA fragments with predicted amino acid sequences related to those of NGF, BDNF, and NT-3 contained conserved cysteine residues at the correct positions. This was used as an initial criterion for a sequence to be considered as a member of the nerve growth factor gene family.
6. 2. 1. NGF, BDNF AND HDNF/NT-3 ARE HIGHLY
CONSERVED DURING EVOLUTION
6. 2. 1. 1. NERVE GROWTH FACTOR The nucleotide sequence (Fig. 1B [human (SEQ ID NO:3), rat (SEQ ID NO:4), chicken (SEQ ID NO:5), viper (SEQ ID NO:6), Xenopus (SEQ ID NO:7), salmon (SEQ ID NO: 8)] and the predicted amino acid sequence of the isolated fragments coding for NGF are highly conserved from fish to human (Fig. 2 [human (SEQ ID NO:24), rat (SEQ ID NO:25), chicken (SEQ ID NO:26), viper (SEQ ID NO:27), Xenopus (SEQ ID NO:28), salmon (SEQ ID NO:29]). Most of the non-conservative amino acid changes were found in the variable regions arginine 59 to serine 67 and aspartic acid 93 to alanine 98 (Fig. 2). The similarity between the
Xenopus and human NGF sequences is 93/79% (Table I). Xenopus and chicken NGF are identical except for one conservative change from lysine 62 to arginine 62 (Fig. 2). The sequences of viper and salmon NGF contain 11 and 19 amino acid differences (out of 42), respectively, compared with human NGF while all other species only showed four differences. None of the NGF amino acid sequences isolated contained the extra amino acid residue present in BDNF and NT-3 between glutamic acid 94 and lysine 95 of the human NGF sequences.
The interspecies relationships of the different NGF sequences were analyzed by the
construction of a phylogenetic tree (Figure 3A). The salmon NGF sequence appears to have diverged more than the NGF sequences isolated from other species. No NGF sequence could be isolated from ray using the
described PCR technique, suggesting that ray NGF sequences may be above the mismatch tolerance of the primers used in our PCR protocol. Alternatively, the absence of NGF in cartilaginous fishes would imply that NGF appeared after the splitting of the branch leading to the evolution of the bony fishes (some 450 million years ago) but before amphibians and higher vertebrates evolved from this branch (about 400 million years ago).
Figure imgf000049_0001
6. 2. 1. 2. BRAIN-DERIVED NEUROTROPHIC FACTOR
DNA sequences similar to that of human BDNF were found in all species investigated (Fig. 1B [SEQ ID NOS: 1-21, listed supra). The similarity in amino acid and nucleotide sequences between ray, the most primitive species investigated, and human are 93/77% (Table I). Only two non-conservative changes were seen outside the variable regions, whereas ten similar changes were found in the two variable regions (Fig. 2). In Xenopus, (SEQ ID NO:34) leucine 90 is replaced by a phenylalanine as a result of a single base pair mutation, C to T in the first position of the codon, and in salmon (SEQ ID NO:35), tryptophan 77 is
replaced by tyrosine as a result of a double mutation, changing the codon from TGG to TAT (Fig. IB [SEQ ID NO: 14]). All isolated sequences contained an extra amino acid residue at position 96, compared with NGF (Fig. 2 [SEQ ID NO:24-29]). The BDNF sequences from different species appeared as a homogenous group of sequences when analyzed by the parsimony method
(Figure 3B).
6. 2. 1. 3. NEUROTROPHIN-3
The nucleotide and predicted amino acid sequences for human (SEQ ID NO:16 and 37), rat (SEQ ID NO: 17 and 38), chicken (SEQ ID NO: 18 and 39), Xenopus (SEQ ID NO:19 and 40), salmon (SEQ ID NO:20 and 41), and ray NT-3 are highly similar (Figs. 1B, Figure 2). Most of the changes are silent mutations resulting from changes in the third position of the codons, usually transitions that preserve the pyrimidine or purine feature of the base pair. Only non-conservative amino acid changes were found within the two variable regions and no amino acid replacements were seen outside the two variable regions. The salmon sequence lacks Asp-94 which is present in all other NT-3 molecules (Fig. 2) and has a longer
distance from the branching point in the phylogenetic tree than NT-3 sequences from other species (Figure 3C).
6. 2. 1. 4. A NOVEL MEMBER OF THE NERVE
GROWTH FACTOR GENE FAMILY
Additional DNA fragments were isolated from viper (SEQ ID NO:1) and Xenopus (SEQ ID NO:2), and the predicted ammo acid sequences (SEQ ID NO: 22 and SEQ ID
NO:23, respectively) revealed that these fragments contained all three cysteine residues in the same positions as in NGF, BDNF and NT-3 (Fig. 1B, Fig. 2). A comparison with the sequences of Xenopus NGF, BDNF and
NT-3 indicated that this new sequence is related, but not identical, to the sequences of the other members of the NGF family. The gene including this sequence was therefore named neurotrophin-4 (NT-4). Comparison of the nucleotide and amino acid sequences show that
Xenopus and viper NT-4 are 91/73% similar. This
similarity is in the same range as the one seen between Xenopus and viper NGF and BDNF (Table I). As for the other members of the NGF family, non-conservative amino acid changes were only seen in the two variable regions (Fig. 2).
6. 2. 1. 5. COMPARISON AND PHYLOGENY OF THE MEMBERS
IN THE NERVE GROWTH FACTOR GENE FAMILY
A comparison of the phylogenetic trees for
NGF, BDNF, and NT-3 showed longer branches in the NGF tree, indicating a higher rate of evolutionary change
(Figures 3A-3C). The relationship of each member of the
NGF family to the other members was studied by the construction of a phylogram comparing the deduced amino acid sequences for the four members of the family. The phylogram showed that NGF is more closely related to NT- 3 than to BDNF and NT-4 (Figure 3D). NT-3 is as related to NGF as to BDNF. NT-4 is clearly more related to BDNF than to the other two members.
6. 2. 2. STRUCTURAL FEATURES OF THE NT-4 PROTEIN
To enable a more detailed characterization of the NT-4 gene and its gene product, we screened a
Xenopus genomic library with the NT-4 PCR fragment and isolated a phage clone containing a 16 kb insert. From this insert, a 1.5 kb Pstl fragment was subcloned and sequenced Figure 4A (SEQ ID NO:43). The nucleotide sequence contained an open reading frame encoding a 236 amino acid protein (SEQ ID NO:44) that showed several structural features characteristic of the other members of the NGF family. The amino terminus of the predicted
NT-4 protein contains an 18 amino acid putative signal sequence in which a region of 4 amino acids is identical to the corresponding regions in pig and rat BDNF
(Leibrock et al. 1989, Nature Ml, 149-152;
Maisonpierre, et al., 1990, Science, 247, 1446-1451). A potential signal cleavage site, which is also identical to the one proposed for BDNF (Figure 4A), follows. A potential cleavage site for a 123 amino acid mature NT-4 protein is found after amino acid 113 in the prepro-NT-4 protein. A single predicted N-glycosylation site (Asn- Lys-Thr) is located 8 amino acids before the putative cleavage site.
A comparison of the mature NT-4 protein to the mature BDNF, NT-3, and NGF proteins from mouse revealed 60%, 58% and 51% amino acid identity,
respectively. Included in the mature NT-4 protein are all 6 cysteine residues involved in the formation of disulphide bridges [Figure 4B (SEQ ID NO:45-48)]. The regions that are identical between NGF, BDNF, and NT-3 are also similar in the NT-4 protein. Most sequence differences between the NT-4 protein and the other three proteins were found within the same variable regions previously identified in the other members of the family.
6. 2. 3. BINDING TO THE NGF-R AND
NEUROTROPHIC ACTIVITY OF NT-4
The 1.5 kb Xenopus Pstl fragment was cloned in the expression vector pXM (Yang et al., 1986, Cell
47: 3-10) and transiently expressed in COS cells. SDS- PAGE of conditioned media from transfected cells labeled with [35 -S] cysteine showed an NT-4 protein with an Mr of 14K (Figure 5A). NGF protein produced and labeled in parallel dishes migrated somewhat faster than the NT-4 protein. This difference in mobility is most likely due to variations in the charge of the two proteins.
Similar mobility differences have also been observed for NGF proteins with identical sizes from different
species.
Conditioned media from transfected COS cells containing equal amounts of rat NGF and Xenopus NT-4 protein were tested for their ability to compete for binding of 125I-labeled NGF to its receptor on PC12 cells. Binding assays were done at 37ºC and under conditions in which 80% of the 125I-NGF associated to the cells is bound to the low affinity NGF-R (Sutter et al., 1979, J. Biol. Chem. 254, 3972). Similar concentrations of NGF and NT-4 (6×10-10M) were required to displace 50% of the 125I-NGF from the PC12 cells, indicating that the two proteins bind to the low affinity NGF-R with a similar affinity (Figure 5B). At higher concentrations, the NT-4 protein was less efficient in displacing 125I-NGF, suggesting that in this case the remaining 125I-NGF associated with the cells was bound to high affinity or internalized receptors. The fact that this difference could not be seen in a parallel assay performed at 0°C in which no membrane mobilization or internalization occurs suggests that the NT-4 protein is not able to compete with NGF for internalization, a process known to be mediated exclusively through the high affinity receptors (Olender and Stach, 1980, J. Biol. Chem. 255, 9338-9343; Bernd and Greene, 1984; J. Biol. Chem. 259, 15509-15516;
Hosang and Shooter, 1987, EMBO J. 6, 1197-1202).
The NT-4 protein transiently expressed in COS cells was tested for its ability to promote neurite growth from explanted embryonic chick ganglia. A clear stimulation of neurite outgrowth from explanted chicken dorsal root ganglia was seen (Figure 6A). Comparison of dose-response curves using equal amounts of NT-4 and NGF protein revealed that the activity obtained with NT-4 was lower than that seen with NGF (Figures 5A and 5B). Recombinant NT-4 and BDNF proteins stimulated neurite outgrowth in the dorsal root ganglia to a similar extent (Figures 6A and 6C). The NT-4 protein elicited a weak, but consistent, neurite outgrowth from the nodose ganglia (Figure 6G), whereas no activity could be detected in sympathetic ganglia (Figure 6E). This is in contrast to NGF, which markedly stimulates neurite outgrowth from sympathetic ganglia (Figure 6F), and
NT-3, which showed a clear activity in the nodose
ganglia (Figure 6H). As for NT-4, the neurite
outgrowth-promoting activity of BDNF in the nodose ganglia (Figure 6I) was lower than the activity seen with NT-3.
6. 2. 4. EXPRESSION OF NT-4 mRNA IN
DIFFERENT XENOPUS TISSUES
Polyadenylated RNA was prepared from 11 different Xenopus tissues and used for Northern blot analysis. Hybridization with the Xenopus NT-4 probe revealed high levels of two NT-4 transcripts of 2.3 kb and 6.0 kb in the ovary (Figure 7A). In contrast, the level of NT-4 mRNA was below the detection limit in all other tissues analyzed. Hybridization with the Xenopus NGF probe showed a 1.3 kb NGF mRNA in the heart (Figure 7A) and brain. However, the amount of NGF mRNA in these tissues was on the order of 100 times lower than the level of NT-4 mRNA in the ovary. NGF mRNA was also detected in the ovary, though the amount of NGF mRNA was approximately 100 times lower than the level of NT-4 mRNA in this tissue (Figure 7B) . The levels of BDNF and NT-3 mRNAs in ovary were both below the detection limit (Figure 7B). 6. 3. DISCUSSION
We have used the polymerase chain reaction (PCR) in combination with degenerate oligonucleotide primers to isolate the genes for different members in the NGF family from different species. A comparison of the nucleotide and amino acid sequences of the entire mature NGF, BDNF and NT-3 proteins revealed similarities that are the same as those obtained by comparing the region of the genes analyzed in this study. Hence, this region appears to be representative for the rest of the gene and can therefore be used to study the evolutionary conservation of the entire mature protein.
The NGF, BDNF and NT-3 genes from different species include regions which show complete identity between fish and mammals, as well as regions with lower similarity. A comparison of NGF sequences from
different species with the corresponding sequences of BDNF or NT-3 showed that the NGF gene is less conserved in vertebrates than both BDNF and HDNF/NT-3. The two latter genes appear to be equally conserved in all species studied, except in salmon, in which NT-3 is less conserved than BDNF. In this context, it is interesting to speculate about the fact that the molecular clock seems sped up in some branches, notably NGF, and not in others. It is generally believed that there is a selective force that preserves the correct tertiary structure of a protein (Dickerson, 1971, J. Mol.
Evol. 1, 26-45; Kimura & Ohta, 1974, Proc. Natl. Acad. Sci. USA, 71: 2848-2852). The difference in the
evolutionary conservation of the three factors suggests that there has been a higher selective pressure on BDNF and NT-3 than on the NGF gene. Environmental changes have been proposed to lead to changes in the selective pressure altering the performance optimum of a specific gene product (Kimura 1983, in "Evolution of Genes and Proteins", pp. 208-233). In this context, it is
possible that the more extensive evolutionary changes seen in NGF compared to BDNF and NT-3 reflect the fact that the function of NGF has changed more during
evolution. Structure-function studies of NGF have shown that this molecule can tolerate considerable structural changes without loss or modification of its activity profile, suggesting that the lower degree of
evolutionary conservation of NGF could be due to a more stable structure of this protein, which is therefore less easily perturbed by substitutions. Another
possible explanation is that the regions of the genome where the genes for the different factors are located have different general mutation rates. Different
mutation rates have been shown for non-coding regions of the genome (Wolfe, et al., 1989, Nature, 337: 283-285) but it is less clear if this can lead to an increased number of changes in coding regions.
Salmon NGF and NT-3 are notably more different when compared with these molecules in other species. Some amino acids including the threonine 82 and the histidine-threonine-phenylalanine at position 85 to 87 in NGF, as well as the absence of the amino acid between positions 94 and 95 (compared to the two other proteins), are consistent features of the NGF protein. The fact that the isolated salmon sequence contains all of these NGF specific motifs argues that it is not an additional member of the family, but rather represents salmon NGF. In contrast to all other NT-3 sequences studied, salmon NT-3 lacks the amino acid in position 95. Since the extra amino acid is present in ray NT-3, it is likely that the common ancestor of ray and salmon had an ancestral NT-3 sequence which included the extra amino acid in position 95. Therefore, the changes in the salmon NT-3 molecule must have occurred after this gene split from the common ancestor. Most of the changes in the amino acids of the salmon sequence are in the same regions that vary, to a lesser degree, also in the other species, strongly suggesting that the isolated salmon NGF or NT-3 sequences are not pseudogenes. The greater divergence of salmon NGF and NT-3, compared with the other species, probably reflects the high degree of evolutionary expansion of the bony fishes.
The results in this study indicate that the NGF family probably existed 500 million years ago in the primitive fishes, which were the ancestors of todays higher vertebrates. The gene family could have been formed by gene duplication, which is believed to be the most common mechanism whereby new genes evolve (Li, W., 1983, in "Evolution of Genes and Proteins, pp. 14-37). Duplications of functional genes could have been
facilitated, since all information required for the synthesis of a biologically active protein is contained within a 3' exon (Hallbook, et al., 1988, Mol. Cell.
Biol. 8: 452-456; Leibrock, et al., 1989, Nature, 341: 149-152; Hohn, et al., 1990, Nature, 344: 339-341). The formation of the family has involved several gene duplications (Figure 3D).
Since NT-4 is more closely related to BDNF than to NT-3 or NGF, it appears that NT-4 and BDNF were formed from a common ancestral gene. However, since no progenitor-like molecule for all four factors can be distinguished from the present data, the evolutionary relation of the putative BDNF/NT-4 ancestor to the ancestors of NGF and NT-3 cannot be definitely
established. The topology of the phylograms using data from different species is in general agreement with the consensus evolutionary relationship among different species. However, for both NGF and BDNF, the chicken sequences show an earlier branching in the phylogram than expected. Comparison of NT-4, NGF, and BDNF from viper and Xenopus revealed that the NT-4 sequences in these species have 11 amino acid replacements, compared with 9 and 8 replacements in NGF and BDNF, respectively. This suggests that in these species, NT-4 has diverged with a rate that is comparable to, or faster than, the rate of NGF or BDNF divergence.
Replacements of highly conserved amino acids in the NGF molecule do not abolish the biological activity, but in many cases these affect the amount of protein produced, indicating that there are constraints other than the biological activity, such as protein stability, which may be important for the conservation of the NGF protein. In addition, the fact that all members of the NGF family can interact with the low affinity NGF-R suggests that the complete conservation of certain regions in these factors may be due to constraints on these genes to retain proteins that can interact with the NGF-R. The basic mechanisms and strategies for the early ontogeny of the embryo are similar in all vertebrates and presumably involve genes that are conserved in all vertebrates. The evolutionary conservation of the neurotrophic factors is therefore consistent with the notion that they are important in early embryonic development in many different species,
The hippocampus contains the highest levels of NGF, BDNF, and NT-3, mRNA in the brain (Ernfors et al., 1990 J. Dev. Neurosci. 9, 57-66). It is a highly specialized structure derived from the archipallium, which first appeared in the brains of amphibians and reptiles. The mammalian hippocampus is important for memory, learning and cognitive functions known to be associated with high neuronal plasticity (Crutcher and Collins, 1982, Science 277:67-68). These demands may have generated a selective pressure during phylogeny for plasticity-promoting mechanisms, possibly medicated by neurotrophic factors. However, the results in this study clearly show that the duplication event of the genes for the neurotrophic factors preceded by far the formation of the hippocampus. This finding indicates that the neurotrophic factors did not evolve as a consequence of the formation of the hippocampus and supports the notion that the neuronal plasticity in this brain region is at least in part due to these molecules.
The organization of the nervous system of primitive vertebrates, i.e., cartilaginous fishes, shows some basic similarities to the nervous system of higher vertebrates. The cranial nerves and the somatic sensory and autonomic nervous systems in cartilaginous fishes are in general similar to those of higher vertebrates (Young, J.Z., 1981, The Life of Vertebrates, New York Oxford University Press). It is therefore likely that the principles of neurotrophic interactions are the same in both primitive and higher vertebrates. The
evolutionary conservation of the NGF-like neurotrophic factors also in pri.mi.tive vertebrates suggests that these factors first evolved in invertebrates and were later adapted to function in the development of the vertebrate nervous system.
Our study of the evolutionary conservation of the NGF family led to the isolation of a novel member of this family, named neurotrophin-4 or NT-4, PCR fragments from the NT-4 gene were isolated from Xenopus and viper, and a genomic clone was subsequently isolated from
Xenopus. Nucleotide sequence analysis of this clone revealed an open reading frame for a 236 amino acid protein, which showed several structural features resembling those of the three other members of the NGF family. These include the presence of a putative amino- terminal signal sequence and a potential N-glycosylation site close to a proteolytic cleavage site that predicts a 123 amino acid mature NT-4 protein. The size of the mature NT-4 protein is 4 amino acids longer than that of BDNF and NT-3 and 5 amino acids longer than that the mature NGF protein. Within the mature NT-4 protein, all 6 cystein residues involved in the formation of
disulphide bridges are conserved. The NT-4 protein differs from the other members of the family in the same regions that vary among the sequences of the three other family members. As for NGF, BDNF, and NT-3, the entire prepro-NT-4 protein is encoded in one single exon.
Hence, both the gene organization and the structural features of the predicted protein indicate that the NT-4 gene is an additional member of the NGF family. The fact that the NT-4 gene was isolated from both reptiles and amphibians suggests that it is present in several different species.
Both BDNF and NT-3 have been shown to
interact with the low affinity NGF-R (Rodriguez-Tebar et al., 1990, Nueron 4:487-492; Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA 87:5454-5458). The Xenopus NT-4 protein displaced 125-NGF from its low affinity receptor on PC12 cells, indicating that the fourth member of this family can also interact with the low affinity NGF-R. The comparison of displacement curves obtained at 37ºC and 0°C suggests that the NT-4 protein cannot compete for binding to the high affinity NGF-R. The protein encoded by the low affinity NGF-R gene appears to form part of both the low and the high affinity receptors (Hempstead et al., 1989, Science 243:373-375). The mechanism by which two kinetically different receptors are formed from the same receptor gene is not known, although it has been proposed that the two states can be generated by the formation of a complex between the cytoplasmic domain of the receptor and an intracellular protein (Radeke et al., 1987, Nature 325:593-597: Meakin and Shooter, 1991, Neuron 6:153-163). Alternatively, a high affinity receptor chain may be encoded by a
separate gene and, similar to the interleukin-2 receptor (Hatakeyama et al., 1989, Science 744:551-556) and the platelet-derived growth factor receptor (Matsui et al., 1989, Science 243:800-804), the two receptor chains may form a dimer that constitutes the high affinity
receptor. The fact that all four members of the NGF family can interact with the low affinity NGF-R suggests that the low affinity state of the NGF-R may be, in an as yet unknown way, involved in mediating the biological effects of all these factors. In this context, it is interesting to note that the low affinity NGF-R gene has been shown to be expressed in many tissues of both neuronal and nonneuronal origin not known to respond to NGF. These include mesenchyme, somites and neural tube cells in the early chick embryo (Hallbook et al., 1990, Development 108:693-704; Heuer et al., 1990a, Dev. Biol.
137:287-304; Heuer et al., 1990b, Neuron 5:283-296), as well as developing and regenerating spinal cord motorneurons (Ernfors et al., 1989, Neuron 2:1605-1613; Ernfors et al., 1991, J. Dev. Neurosci. 9:57-66). It would therefore be of interest to investigate whether the NT-4 protein is of functional importance in any of these tissues or neuronal populations.
The neurotrophic activity of the NT-4 protein was assayed on explanted chick embryonic ganglia, and as for the other three members of the NGF family, the NT-4 protein showed a clear stimulation of neurite outgrowth from dorsal root ganglia. However, when compared to NGF, the NT-4 protein showed lower activity in dorsal root ganglia. Both BDNF and NT-3 readily elicit neurite outgrowth in explanted nodose ganglia, though the response with NT-3 was consistently stronger than that with BDNF. NGF strongly stimulates neurite outgrowth in sympathetic ganglia, and NT-3 also has activity in this ganglia, though it is much lower than that of NGF
(Maisonpierre et al., 1990, Science 247: 1446-1451;
Ernfors et al., 1990, Proc. Natl. Acad. Sci. USA
87:5454-5458). NT-4 showed weaker activity in nodose ganglia compared with NT-3 and no activity in the sympathetic ganglia. The spectrum of the biological activity of NT-4 on peripheral explanted ganglia
resembles that of BDNF, which is in agreement with the fact that NT-4 is structurally similar to BDNF.
Northern blot analysis of 11 different tissues from Xenopus showed high levels of NT-4 in the ovary, whereas the level of NT-4 mRNA was below the detection limit in all other tissues examined. Two NT-4 mRNAs of 2.3 kb and 6.0 kb were seen in the oocytes.
The presence of two transcripts from the same gene has previously been observed for BNDF, in which case two mRNAs of 1.4 kb and 4.0 kb are present in the rat brain (Leibrock et al., 1989, Nature 341:149-152; Maisonpierre et al., 1990, Science 247:1446-1451: Ernfors et al., 1990a, Proc. Natl. Acad. Sci. USA 87:5454-5458).
Hybridization to a Xenopus NGF probe revealed NGF mRNA in the Xenopus heart, most likely as a result of NGF mRNA expression in target tissues for neuronal
innervation. The level of NGF mRNA in the heart was, however, more than 100-fold lower than the level of NT-4 mRNA in the ovary. Since the high level of NT-4 mRNA in the ovary does not correlate with neuronal innervation, it appears unlikely that the NT-4 protein has only a neurotrophic function in this case. Instead, the abundant expression of NT-4 mRNA in Xenopus ovary implies an additional and important nonneurotrophic function for the NT-4 protein. NGF mRNA was also detected in Xenopus ovary though at almost 100 times lower levels than those of NT-4 mRNA; BDNF and NT-3 mRNAs were not detected in this tissue.
mRNAs for two growth factors have been described as maternal mRNAs in Xenopus oocytes. One of these mRNAs encodes a protein with strong similarity to basic fibroblast growth factor (Kimelman and Kirschner, 1987, Cell 51:869-877); the other mRNA encodes a protein homologous to transforming growth factor α (Weeks and Melton, 1987, Cell 51:861-867). These factors have been suggested to function as morphogens for the formation of mesoderm and the subsequent induction of this tissue into the neural tube. In the rat, in situ hybridization studies have revealed NT-3 mRNA in the epithelium of secondary and tertiary follicles, and a role for NT-3 in oogenesis has been suggested (Ernfors et al., 1990, Neuron 5:511-526). 7. EXAMPLE: IDENTIFICATION OF CELLS
EXPRESSING NT-4 mRNA IN THE XENOPUS LAEVIS OVARY BY IN SITU HYBRIDIZATION
7.1. MATERIALS AND METHODS
7.1.1. ISOLATION, HANDLING AND CULTURE OF
XENOPUS OOCYTES, EMBRYOS AND CELLS
Male and female X. laevis frogs were
maintained in the laboratory at 19°C. After immersionanesthesia of the animals in 0.25% tricaine methane sulfonate (Sandoz, Switzerland), ovarian lobes were surgically removed, washed with modified Barth's saline
Hepes (MBSH) (Gurdon and Wickens, 1983, Methods Enzymol,
101: 370-86) and dissociated by overnight incubation at
20°C in calcium-free MBSH containing 2 mg/ml
collagenase. Crude separation of pre-vitellogenic and vitelloenic oocytes was obtained by differential
sedimentation, and oocytes were further sorted manually under a dissecting microscope into the developmental classes described by Dumont (1972, supra).
Synchronously cleaving embryos were obtained by in vitro fertilization essentially as described by Newport and Kirschner (1982).
A6 Xenopus kidney cells were cultured in
Leibowitz L15 medium diluted with distilled water 60:40 (v/v) and supplemented with 10 mM Hepes pH 7.35, 10 μM hypoxanthine (Sigma), 4 mM glutamine and 10% fetal bovine serum (Gibco) at 20°C. Cultures were
equilibrated with air and kept in the dark. 7.1.2. IN SITU HYBRIDIZATION
Fresh-frozen ovaries from adult Xenopus laevis frogs were sectioned (14 μ) in a cryostat (Leitz, Germany) and the sections were thawed onto poly-L-lysine (50 μg/ml) pretreated slides after which they were fixed in 10% formalin for 30 min and rinsed twice in PBS.
Dehydration was carried out in a graded series of ethanol including a 5 min incubation in chloroform after which the slides were air dried. Two 53-mer
oligonucleotides, one specific for Xenopus NT-4 mRNA (5'CCCACAAGCTTGTTGGCATCTATGGTCAGAGCCCTCACATAAGACTGTTTTGC 3' [SEQ ID NO: 109]) and another one, as a control, specific for chicken BDNF mRNA (corresponding to amino acids 61 to 77 of the mature chicken BDNF protein
(Hallbook et al., 1991, Neuron 6: 845-58 [contained within SEQ ID NOS.: 11 and 32]), were labeled at the 3' end with α35S-dATP using terminal deoxyribonucleotidyl transferase (IBI, New Haven) to a specific activity of approximately 1×109 cpm/μ. Hybridization was performed at 42°C for 16 hours in 50% formamide, 4x SSC, lx
Denhardts solution, 1% Sarcosyl, 0.02M NaPO4 (pH 7.0), 10% dextransulphate, 0.5 mg/ml yeast tRNA, 0.06M DDT,0.1 mg/ml sheared salmon sperm DNA and 1×107 cpm/ml of 35S-labeled oligonucleotide probe. Sections were
subsequently rinsed, washed 4 times (15 min. each) at 55°C in 1 × SSC, rinsed in water, dehydrated in a graded series of ethanol and air-dried. The sections were exposed to X-ray film followed by coating in Kodak NTB-3 photo emulsion (diluted 1:1 in water), exposed for 5-6 weeks at -20°C, developed, fixed and counterstained with cresyl violet.
7.1.3 RNA BLOT ANALYSIS
The indicated samples were homogenized in 4M guanidine isothiocyanate, 0.1M β-mercaptoethanol, 0.025M sodium citrate pH 7.0 and homogenized 3 times for 15 seconds with a Polytrone. Each homogenate was layered over a 4ml cushion of 5.7M CsCl in 0.025M sodium citrate pH 5.5 and centrifuged at 15°C in a Beckman SW41 rotor at 35,000 rpm for 16 hrs. (Chirgwin et al., 1979,
Biochemistry 78: 5294-5299). Polyadenylated RNA
(Poly(A) + RNA) was purified by oligo (dT) cellulose chromatography (Aviv and Leder, 1972, PNAS 69: 1408- 1412) and the recovery of RNA (40 μg) was quantified spectrophotometrically before use in RNA blot analysis. Total cellular RNA (40 μg) or where indicated
poly(A)+RNA (5 μg) from each sample was electrophoresed in a 1% agarose gel containing 0.7% formaldehyde. UV- transillumination of the stained gel was used to confirm that all samples contained similar amounts of intact RNA. The gel was then transferred to a nitrocellulose filter. The filter was then hybridized to a 350bp
HincII fragment from the 3' exon of the Xenopus NT-4 gene (Hallbook et al., 1991, Neuron 6 : 845-858). The fragment was labeled with α-(32p)-dCTP by nick
translation to a specific activity of around 5×108 cpm/μg and the hybridization was carried out as described
(Ernfors et al., 1988, Neuron 1: 983-96). Filters were washed at high stringency (0.1×SSC, 0.1% SDS, 54ºC) and exposed to Kodak AR-5 films at -70ºC. 7.2 RESULTS
Tissue sections through the adult Xenopus laevis ovary were hybridized to a 35S-dATP labeled oligonucleotide probe specific for Xenopus NT-4 mRNA. As a control for the specificity of the hybridization, adjacent sections were hybridized to an oligonucleotide probe of the same length and GC-content complementary to mRNA for chicken brain-derived neurotrophic factor
(BDNF). The NT-4 mRNA specific probe revealed an
intense labeling over many cells scattered throughout the ovary with a size (50-400 μm in diameter)
corresponding to oocytes in early stages of oogenesis (Fig. 9A), No NT-4 mRNA could be detected over mature, post-vitellogenic stage VI oocytes (arrows in Fig. 9A). The chicken BDNF mRNA specific control probe did not label any cells in the Xenspus ovary. Analysis of emulsion autoradiographs from the hybridized sections revealed an intense labeling over the cytoplasm of oocytes with a diameter of 50-200 μm (Fig. 10A and 10B) corresponding to stage I oocytes according to Dumont, 1972, supra. The NT-4 mRNA
specific probe also labeled oocytes with a larger diameter corresponding to stages II to IV, though the intensity of labeling over these cells was lower than that seen over stage I oocytes. In agreement with the analysis of low magnification dark-field illuminations (Fig. 9), the emulsion autoradiographs did not show any labeling over more mature oocytes of stages V and VI. No labeling was seen over any cells after hybridization with the control BDNF probe (Fig. 10C) . To enable a more detailed determination of the level of NT-4 mRNA during oogenesis, the number of grains per an
arbitrarily chosen area unit was counted. The area unit chosen corresponded to approximately one hundredth of the cross section area of a stage I oocyte. The result of this analysis showed that the intensity of labeling over stage I oocytes was 1.7 and 4.3 times higher than over stage II/III and IV oocytes respectively (Fig. 11) . The number of grains per area unit over stage V and VI oocytes was not significantly above the level of the background labeling.
7.2.1 NORTHERN BLOT ANALYSIS OF NT-4 mRNA EXPRESSION
DURING XENOPUS OOGENESIS AND EARLY DEVELOPMENT
A fixed amount of total cellular RNA (40 μg) prepared from different stages of oocytes as well as from a fraction enriched from follicle cells was
analyzed by Northern blots using a Xenopus NT-4 specific probe (Hallbook et al., 1991 supra). In agreement with the results of the in situ hybridization, the highest levels of NT-4 transcripts with sizes of 2.3 kb and 6.0 kb was present in the smallest oocytes (stages I and II) (Fig. 12). The level of NT-4 mRNA declined abruptly in more mature stage V and VI oocytes. A weak
hybridization signal was seen in the follicle cell preparation which was probably due to a contamination with a small number of stage I and II oocytes. The same result was obtained when a fixed amount (5 μg) of polyadenylated RNA was analyzed from the difference samples shown in Fig. 12.
The results of the analysis of the NT-4 mRNA expression in the ovary showed that NT-4 mRNA is
restricted to immature oocytes. To test the possibility that expression of NT-4 mRNA is induced after
fertilization, the level of NT-4 mRNA was assessed in developing Xenopus embryos by Northern blots of
polyadenylated RNA. A low level of NT-4 mRNA was found in Xenopus somatic Ag cultured kidney cells which were also included in the analysis. However, no NT-4 mRNA could be detected in early embryos from the onset of cleavage divisions to the neurula stage.
7.3 DISCUSSION
The abundant expression of NT-4 mRNA in the Xenopus ovary (Hallbook et al., 1991 supra) indicates that this member of the NGF family plays a role in oogenesis and/or early embryogenesis. Localization of cells expressing NT-4 mRNA in the ovary provided insights into the putative function of the NT-4 protein in the ovary. In amphibians, as in all other vertebrates, fertilization of the egg triggers a period of rapid cell cleavage. This event is controlled by a class of soluble maternal mRNAs expressed during oogenesis and stored in the unfertilized egg for subsequent development
(Davidson, 1986, Gene Activity in Early Development (New
York, Academic Press). This class of maternal mRNAs includes two growth factors, basic fibroblast growth factor (Kimelman and Kirschner, 1987, Cell, 51 : 869-77) and transforming growth factor-β (Weeks and Melton, 1987, Cell, 51: 861-67), as well as several protooncogenes such as c-myc (Godeau et al., 1986, EMBO J., 5 : 3571-77);
(Vriz et al., 1989, EMBO J. 8: 4091-97), c-fos (Mohun et al., 1989, Development, 107: 835-46), ras (Andeol et al.,
1990, Dev. Biol., 139: 24-34), ets-2 (Chen et al., 1990, Science, 250: 1416-18) and c-mos (Sagata et al., 1988, Nature, 335: 519-25). Immature stage VI Xenopus oocytes are arrested in prophase of meiosis I and both c-mos (Sagata et al., 1988) and ets-2 (Chen et al., 1990) have been shown to function during reinitiation of meiotic division. The finding of high levels of NT-4 mRNA in stage I and II oocytes but a decreased level below the detection limit of both Northern blots and in situ hybridization in stage V and VI oocytes strongly suggests that the NT-4 mRNA does not belong to the class of maternal mRNAs. This result also argues against a role of the NT-4 protein in the reinitiation of meiotic division or in early embryogenesis. In agreement with this, addition of recombinant NT-4 protein to immature stage VI oocytes failed to induce germinal vesicle breakdown in vitro and no NT-4 mRNA was detected in
Xenopus early embryos. Instead, the putative function of the NT-4 protein in the ovary appears to be coupled to events occurring in the pre-vitellogenic and early mid vitellogenic oocyte. Both NGF (Ayer-LeLievre et al., 1988, PNAS 85: 2628-2632) the 75kD low-affinity NGF receptor (Persson et al., 1990, Science, 247: 704-707) and the trkA high-affinity component of the NGF receptor (J.P. Merlo and H. Persson, unpublished) are expressed in the testes where NGF has recently been shown to stimulate DNA synthesis at the onset of meiosis (Parvinen et al.,
1991, submitted). Hence, it appears that the
neurotrophins do not only function as neurotrophic factors but also play an important role in reproductive tissues.
8. EXAMPLE: ISOLATION AND CHARACTERIZATION OF NUCLEIC ACID FRAGMENTS ENCODING MAMMALIAN NT-4
8.1. MATERIALS AND METHODS
8.1.1. DNA PREPARATION
Genomic DNA was isolated as described in
6.1.1, supra.
8.1.2. POLYMERASE CHAIN REACTIONS,
MOLECULAR CLONING AND DNA SEQUENCING
Mixtures of 34-mer oligonucleotides (including tail) representing all possible codons corresponding to the amino acid sequences QYFFET (contained within SEQ ID
NO:51) and QYFYET (SEQ ID NO:52) (5'-oligonucleotide) and, WISECK, CKAKQS and WIRIDT (each contained within SEQ
ID NO:51) (3'-oligonucleotide) (Fig. 13) were
synthesized, with linkers, as described in 6.1.2., supra.
Together, 2Y (derived from xNT-4 [SEQ ID NO:50]) and 2Z
(derived from BDNF/NT-3 [SEQ ID NO:51]) represent all known sequence for neurotrophins from all species in this region. A primary amplification of both rat and human genomic DNA was carried out with Taq polymerase (Cetus) with cycles of 1 minute at 95°C, 2 minutes at 43°C and 2 minutes at 72ºC. An aliquot from the primary PCR
reactions was then reamplified using either the same primers as in the primary amplification or with new nested degenerate oligonucleotide primers which would result in an expected size shift. PCR products from the reamplification procedure were purified as follows: bands of prospective size were gel purified, reamplified, and column purified using Stratagene "primerase" columns.
These were then digested to completion with EcoRI and Sail, analyzed and re-purified using Primerase columns (Stratagene) and ligated into EcoRI-Xhol digested
Bluescript KS(-). Transformants were screened for pBS-KS containing an insert of the approximate predicted size. The cloned fragments were subjected to DNA sequence analysis as described in 6.1.2, supra.
8.1.3 ISOLATION OF FULL LENGTH GENOMIC AND
CDNA CLONES ENCODING rNT-4 and hNT-4
A human ovary cDNA library in λGT-10 was obtained from Clontech. A human hippocampus cDNA library in λ:ZAPII was obtained from Stratagene. A human genomic DNA library in EMBL3/SP6/T7 was obtained from Clontech. A rat brain cDNA library in λ-ZAP was obtained from
Stratagene.
Isolation of NT-4 clones can be carried out as follows:
A cloned insert encoding the rNT-4 fragment (Fig. 14 [SEQ ID NO:61]) or the hNT-4 fragment (Fig. 15 [SEQ ID NO: 63]) are labeled by PCR to a specific activity of approximately 5×108 cpm/ng. Hybridization is carried out in hybridization solution consisting of 0.5 mg/ml salmon sperm DNA at 60°C. The filters are washed at 60°C in 2 × SSC, 0.1 % SDS and exposed to Kodak XAR-5 film at -70°C. Alternatively, oligonucleotides whose sequence corresponds exactly to the desired mammalian neurotrophin can be used to generate probes (e.g. kinase labelling) and can be used to screen the same libraries by
conventional methods. Positive phage are plaque purified and infected at low multiplicity in an appropriate
E. coli strain in liquid broth as described by Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). GT-10 and EMBL3/SP6/T7 phage are prepared as follows:
Cultures are incubated overnight at 37° with constant shaking. The overnight suspension is brought to 1M NaCl and 8% PEG, mixed well and incubated overnight at 4°C to precipitate the bacteriophage. The bacteriophage are pelleted via centrifucation, resuspended in TM buffer (10mM Tris-HCl, pH 7.5; 10mM MgCl2) , layered upon a CsCl step gradient and centrifuged at appropriate speed and length of time to band the bacteriophage. The
bacteriophage are removed, transferred to a fresh
Eppendorf tube and lysed by the addition of 1 volume of formamide. EMBL-3 DNA is precipitated by the addition of 2 volumes of 100% ethanol. The EMBL-3 DNA is recovered by microcentrifugation, washed in 70% ethanol and
resuspended in TE buffer (10mM Tris-HCl, pH 7.5; 1mM Na2- EDTA). The DNA is extracted several times with
phenol:chloroform:isomyl alcohol (24:24:1), ethanol precipitated, resuspended in TE buffer, digested with various restriction enzymes and electrophoresed through a 1% agarose gel. Subsequent to electrophoresis, the restricted DNA is transferred to nitrocellulose and hybridized to the 32P-labelled rNT-4 or hNT-4 probe, under conditions described supra. The hybridizing band is subcloned into pBS-KS plasmid vector and subjected to DNA sequence analysis by the dideoxy chain termination method (Sanger, et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74: 5463-5467).
The λ-ZAP plasmid preparations are peformed as follows: 200λ of OD600=1.0 XLl-Blue cells, 200λ of the hititer phase stock, and 1λ of R408 helper phage (1×10 minutes pfu/ml) are combined. For a negative control, add no phage stock. Incubate 15 minutes at 37°C. Add 5 ml of 2XYT media, shake for 3 hours at 37°C. At the end of the 3 hours, the negative control should be cloudy and the samples clear. Samples are heated at 65°C for 30 minutes, spun at 4000g for 5 minutes. Supernatant contains phagemid stock. To rescue the phagemid, add
0.5λ of the stock to 200 of XLl-Blue cells (OD600=1) . Incubate 37°C for 15 minutes. Plate 1-100λ (preferably 10λ) on LB ampicillin plates. Incubate 37ºC × overnight and large colonies are picked. After plasmid DNA is purified, it is sequenced as above.
Lambda phage cDNA libraries are screened according to standard methods (Maniatis, et al., supra) as described supra.
Positive plaques are purified, reisolated and subjected to DNA sequence analysis as described supra.
8.2. RESULTS AND DISCUSSION
A region of the Xenopus NT-4 coding sequence was used as a model for synthesis of degenerate
oligonucleotide primers. Figure 13 denotes that the 5'-oligonucleotide primer 2Y [SEQ ID NO:53] (QYFFET) and 3'-oligonucleotide primers, 3Y [SEQ ID NO: 55] (WISECK), 3Z [SEQ ID NO: 56] (CKAKQS) and 4Z [SEQ ID NO: 58] (WIRIDT) were derived from the xNT-4 amino acid sequence. The 5'-oligonucleotide primer 2Z [SEQ ID NO: 54] (QYFYET) is derived from the homologous region of rBDNF. All
possible combinations of these degenerate
oligonucleotides were utilized to amplify DNA from both rat and human genomic DNA libraries. Since the primers represented by 3Y [SEQ ID NO: 55] and 3Z [SEQ ID NO: 56] of xNT-4 are not conserved in the NGF/BDNF/NT-3 gene family, and therefore were not likely to amplify NGF, BDNF or NT-3, these two primers were utilized in the
reamplification, or secondary PCR.
DNA fragments of the approximate expected sizes were obtained from PCR amplification and
reamplification of both the rat and human genomic
libraries when the following primer combinations were utilized:
(1) 2Y/3Z (primary PCR) : 2Y, 2Z/3Y (secondary PCR)
(2) 2Y/3Z (primary PCR) : 2Y, 2Z/32 (secondary PCR) (3) 2Y/4Z (primary PCR) : 2Y, 2Z/32 (secondary PCR)
(4) 2Z/4Z (primary PCR) : 2Y, 2Z/3Z (secondary PCR)
The secondary PCR products of the approximate expected size were electrophoresed through a 2% agarose gel, eluted by standard techniques, digested with EcoRI and Sail and ligated in EcoRI-Xhol digested pBS-KS DNA. Positive transformants were selected, and inserted fragments were subjected to DNA sequencing by the dideoxy chain termination method (Sanger, et al., supra).
An open reading frame has been deduced for a portion of the rat NT-4 (Fig. 14 [SEQ ID NO: 62]) and human NT-4 (Fig. 15 [SEQ ID NO: 64]) amino acid coding sequence. Figure 16 illustrates the homologous region of the rNT-4 (SEQ ID NO: 62) and hNT-4 (SEQ ID NO: 64)
fragment to representative members of the NGF/BDNF/NT-3 gene family.
An open reading frame encoding a larger portion of human NT-4 than that disclosed in Figure 15 is shown in Figure 17A (SEQ ID NO: 69 and SEQ ID NO: 70).
Figure 17A presents additional 3' sequence information for the 3' human NT-4 coding region. The 192 bp nucleic acid fragment was isolated as described supra in the
Description of the Figures.
The actual size of the PCR products recovered from the reamplification procedure was larger than predicted due to the additional 7 amino acids in the rat
NT-4 (GPGVGGG) [SEQ ID No: 101] and human NT-4 (GPGAGGG)
[SEQ ID NO: 102] DNA fragment.
The 7 amino acid insertions of rNT-4 and hNT-4 are described as 'GPGXGGG' [SEQ ID NO:100], where X=V for rNT-4 and X=A for hNT-4. Valine and alanine possess nonpolar R group. Whether position four is conserved to contain a nonpolar R group at position 4 in other
mammalian NT-4 proteins is not presently known, nor whether the 7bp insertion itself will be characteristic of other mammalian NT-4 genes. It is interesting to note that fish NGF has a 22 amino acid insertion in the same region as disclosed in the present invention.
9. EXAMPLE: ISOLATION AND CHARACTERIZATION OF AN NT-4 HUMAN GENOMIC CLONE
We have screened a human placenta genomic library in EMBL3 SP6/T7 (Clontech, K802 as host). A total of 1.25 × 106 pfu were plated on large NZY plates.
Duplicate lifts were made using Schleicher & Schuell nitrocellulose filters, and Were hybridized to a 120 bp probe (from hNT-4 clone 17B, which was obtained from human genomic DNA using primers 2Z4Z followed by 2Z3Z), labelled by PCR using oligonucleotide primers 2Z/3Z. The filters were hybridized at 60ºC with the radiolabelled probe (106 cpm/ ml) under the following hybridization onditions: 0.5 M NaPO4, 1% BSA, 7% SDS, 1 mM EDTA, and 100 μg/ml salmon sperm DNA. The filters were then washed at 60°C with 2×SSC and 0.1% SDS, and subjected to
autoradiography. Following four days of exposure , positive signals were identified on the duplicate lifts. A total of seven plugs were picked, put into 1 ml SM buffer, shaken for 2 hr, and replated as follows: 1) 100 μl of 10-3 dilution (1 μl in 1 ml), mixed with 100 μl cells, and plated; an almost confluent plate was
obtained; 2) 200 μl of 10-5 dilution, which gave isolated plaques. Duplicate lifts were made, and screened as described above with the hNT-4 120 bp probe. Following a 2 day exposure, many positives were identified on the confluent plate for plugs HG2, 4, and 7. A well-isolated positive was identified on both HG4-2 and HG7-2 plates. A single plaque for HG4-2 and HG7-2 was picked, put into 500 μl of SM buffer, and shaken for 2 hr, following which 100 μl of eluant was mixed with 100 μl cells and plated. The plate was then flooded with 3 ml SM buffer, and supernatant collected as the first high titer stock.
Three plates were then plated using 100 μl of this first stock mixed with 100 μl cells. The plates were flooded with 3 ml SM and shaken on rotator for 3 hr at room temperature. Supernatant was removed, spun to remove debris, following which chloroform was added, and this used as the second high titer stock. Two μl of HG4-2 and HG7-2 high titer stock was spotted onto Schleicher & Schuell nitrocellulose filter, and was found to hybridize to the rNT-4 180 bp probe [isolated from the plasmid containing an insert obtained by PCR from rat genomic DNA using primers designed based on our rat NT-4 clone sequence coding for the amino acid GELSVCD (SEQ ID
NO: 112) (degenerate primer) and KAESAG (SEQ ID NO: 113) (exact primer)]. Plate lysates and liquid lysates were prepared for HG4-2, HG7-2 and HG2-1. Phage DNA was made, an aliquot of which was run on agarose gel and subjected to Southern analysis. HG4-2, HG7-2 and HG2-1 were found to hybridize to the rNT-4 180 bp probe (NaPO4
hybridization as above, 65ºC), and a 45mer
oligonucleotide probe
(GGAGGGGGCTGCCGGGGAGTGGACAGGAGGCACTGGGTATCTGAG) [SEQ ID NO:114] corresponding to amino acid GGGCRGVDRRHWVSE [SEQ ID NO: 115] coded for by human PCR fragment clone 17B (6×SSC, 45ºC hybridization). The size of the insert for these three genomic clones is approximately 9-23 kb.
They both contain the coding exon of the gene(s) that is closely related to the probes used for the screening, hNT4 (120 bp) and rNT4 (180 bp). The phage DNA for the genomic clones was digested with several restriction enzymes and subjected to Southern analysis. The
appropriate fragment that hybridizes to the probe rNT4
(180 bp) can be subcloned into Bluescript vector. The size of DNA fragments to be subcloned are as follows: clone 2-1 (1.0 kb XhoI fragment), clone 4-2 (4.0 kb XhoI fragment) and clone 7-2 (5.0 kb BamHI fragment).
Complete coding sequence can be obtained and this information can be used to identify the exon boundaries to allow subcloning of this gene into an appropriate expression vector.
To this end, nucleotide sequence analysis was performed on human genomic phage clone 7-2, which had been obtained by screening a human genomic library with a PCR fragment derived from human genomic DNA using
degenerate oligonucleotides to the DNA sequence of
Xenopus NT-4 (see discussion, supra). Sequence analysis revealed that human phage clone 7-2 contains a sequence identical to the sequence of the PCR fragment used as a probe to screen the genomic library. This sequence is contained within what appears to be an exon encoding a novel neurotrophic factor (Figure 18, SEQ ID NO:75 and SEQ ID NO:76).
Alignment of the protein encoded by this exon (Figure 19, SEQ ID NO:77) with the known neurotrophins revealed that it shares features found in all the known neurotrophins (Figure 19, SEQ ID NOS:78-92). It contains a prepro region in which are conserved many of the identical amino acids conserved between the prepro regions of previously defined neurotrophins.
Furthermore, this prepro region is preceded by a splice acceptor site localized in the same region as in other neurotrophin genes. The prepro region also contains a consensus glycosylation site at the appropriate position, and terminates at a cleavage site which was very similar to the cleavage sites found in the other neurotrophins (Figure 18). The prepro region of 7-2 is unusual, however, due to its short length as compared to the prepro region of known neurotrophins. The decrease in length occurs in the N-terminal portion of the prepro region, which is the least conserved portion of prepros between family members. The mature region retains all 6 cysteines found in all previously identified
neurotrophins. Many of the residues shared between different members of the neurotrophin family are also conserved. Excluding the extensive sequence similarity shared by a PCR fragment derived from rat genomic DNA which may correspond to the rat equivalent of the protein encoded by the human 7-2 clone, computer alignments revealed that the neurotrophin encoded by the 7-2 phage clone was most similar to that of Xenopus NT-4. This was true for both the prepro and mature regions. The protein encoded by the 7-2 clone is unusual, as compared to the known neurotrophins, due to the presence of an insertion situated between the second and third cysteines in the mature region.
Sequence analysis was also performed on two additional human clones isolated in the same screening procedure that yielded clone 7-2 (see discussion, supra). The sequence of these clones was similar to, but not identical to, that obtained from clone 7-2, raising the possibility that they encode novel neurotrophins more closely related to 7-2 than to the other known
neurotrophins. The partial sequence of one of these clones, clone 2-1, is presented in Figure 20 (SEQ ID NO:93 and SEQ ID NO:94). The sequence disclosed starts at a position corresponding to amino acid number 50 in the alignments depicted in Figure 19. Partial sequence of the other clone, clone 4-2, is presented in Figure 21 (SEQ ID NO:116 and SEQ ID NO:117).
10. EXAMPLE: TISSUE SPECIFIC EXPRESSION OF HUMAN NT-4
A 680 bp Xhol-Notl fragment, containing the entire coding region of the human genomic NT-4 clone,
HG7-2, was radiolabeled and utilized in Northern analysis of various human tissue specific PolyA+ RNAs. The human tissue specific mRNAs were fractionated by
electrophoresis through a 1% agarose-formaldehyde gel followed by capillary transfer to a nylon membrane with 10X SSC. The RNAs were cross-linked to the membranes by exposure to ultraviolet light and hybridized at 65ºC to the 680 bp Xhol-Notl radiolabeled NT-4 probe in the presence of 0.5M NaPO4 (pH 7), 1% bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA and 100 ng/ml sonicated, denatured salmon sperm DNA. The filter was washed at 65°C with 2X SSC, 0.1% SDS and subjected to autoradiography overnight with one intensifying screen and X-ray film at -70°C. Ethidium bromide staining of the gel demonstrated that equivalent levels of total RNA were being assayed for the different samples.
The human NT-4 probe hybridized strongly to mRNA from skeletal muscle, prostate, thymus, testes and placenta (Figure 22). The NT-4 probe hybridized to a larger transcript in skeletal muscle than prostate mRNA. This data suggests that a small human NT-4 multigene family, possessing different expression levels as well as transcript sizes, may be present.
The high expression of human NT-4 in muscle tissue suggests that the present invention may be
utilized to treat disorders of the nervous system, specifically the wide array of neurological disorders affecting motor neurons (see discussion, supra).
Additionally, high expression of human NT-4 in prostate tissue suggests that the present invention may be
utilized to treat prostate disease, preferably BPH and impotency (see discussion, supra). Finally, expression of human NT-4 in thymus tissue suggests that the present invention may be utilized to treat immunological related disorders of nerve and muscle tissue, including but not limited to myasthenia gravis (see discussion, supra). 11. EXAMPLE: CONSTRUCTION OF HUMAN NT-4 IN
EUKARYOTIC EXPRESSION VECTORS AND THE MEASUREMENT OF BIOLOGICAL ACTIVITY OF RECOMBINANT HUMAN NT-4
11.1 MATERIALS AND METHODS
11.1.1. CONSTRUCTION OF EUKARYOTIC EXPRESSION
VECTORS ENCODING HUMAN NT-4
Two eukaryotic expression vectors containing the prepro precursor coding region of the human genomic clone HG7-2 were constructed in pCMX (NRRL Accession No.
B-18790). The first construction utilized the normal translation initiation site of pCMX (pCMX-HG7-2Q) , while the other utilized the Kozak consensus translation initiation site (pCMX-HG7-2M). A 5 kb genomic fragment of HG7-2, containing the entire coding region cloned in the BamHI site of Bluescript, was amplified by PCR utilizing the following oligonucleotides:
hNT4-5'XhoM:CGGTACCCTCGAGCCACCATGCTCCCTCTCCCCTCA
[SEQ ID NO: 118]
hNT4-3'Not:CGGTACAAGCGGCCGCTTCTTGGGCATGGGTCTCAG
[SEQ ID NO: 119]
hNT4-5'XhoQ:CGGTACCCTCGAGCCACCCAGGTGCTCCGAGAGATG
[SEQ ID NO: 120]
Oligonucleotide primer combinations of hNT4-5'XhoM andhNT4-3'Not were used to construct pCMX-HG7-2M, while oligos hNT4-5'XhoQ and hNT4-3'Not were used to construct pCMX-HG7-2Q. The PCR fragment was digested with
Xhol/Notl and subcloned into Xhol/Notl digested pCMX. 11.1.2. CONSTRUCTION OF CHIMERIC GENES FUSING A
NEUROTROPHIN PREPRO REGION TO THE MATURE CODING REGION OF HUMAN NT-4
Two additional eukaryotic expression vectors encoding the mature portion of human NT-4 were
constructed. First, the prepro region of human NT-4 was replaced with the prepro region of Xenopus NT-4 (pCMX-xNT4/hNT4). Second, the prepro region of human NT-4 was replaced with the prepro region of human NT-3 (pCMX-hNT3/hNT4). The following oligonucleotides were utilized in the construction of pCMX-xNT4/hNT4 andpCMX-hNT3/hNT4:
(1) 5'CDM8: GAGACCGGAAGCTTCTAGAGATC [SEQ ID NO: 121]
(2) hNT3/hNT4 fusion ("US" oligonucleotide):
TGCAGTTTCGCTCACCCCCCGTTTCCGCCGTGATGT [SEQ ID NO: 122] (3) hNT3/hNT4 fusion ("DS" oligonucleotide):
ACATCACGGCGGAAACGGGGGGTGAGCGAAACTGCA [SEQ ID NO: 123]
(4) xNT4/hNT3 fusion ("DS" oligonucleotide):
ACTTCCCGGCTAAAACGGGGGGTGAGCGAAACTGCA [SEQ ID NO: 124]
(5) xNT4/hNT4 fusion ("US" oligonucleotide):
TGCAGTTTCGCTCACCCCCCGTTTTAGCCGGGAAGT [SEQ ID NO: 109]
The hNT-3 containing plasmid vector (pC8-hNT3) was amplified by PCR with the 5'CDM8 and hNT3/hNT4 fusion oligonucleotides as primers. The hNT-4 containing plasmid (pCMX-HG7-2Q) was amplified by PCR with the hNT3/hNT4 fusion "DS" oligonucleotide and the hNT4-3'Notl oligonucleotide. The PCR fragment obtained was excised from the gel, and reamplified by PCR with the 5'CDM8 and hNT4-3'Notl oligonucleotides. The product was then digested with HindIII and Pstl and subcloned into
Hindlll/Pstl digested pCMX-HG7-2Q. Therefore, the expression plasmid pCMX-hNT3/hNT4 contained the hNT3 prepro region fused to the mature coding region of human NT-4. Similarly, the human NT-4 expression plasmid
(pCMX-HG7-2Q) was amplified by PCR with the 5-CDM8 and xNT4/hNT4/fusion "US" oligonucleotides as primers, while pCMX-HG7-2Q was amplified with the xNT4/hNT4-fusion "DS" oligonucleotide and the hNT4-3'Notl oligonucleotide. The PCR fragment was excised from the gel, and reamplified with the 5'CDM8 and the hNT4-3'Not oligonucleotides. The product was then digested with HindIII and Pstl and subcloned into HindIII/Pstl digested pCMX-HG7-2Q.
Therefore, the resulting eukaryotic expression plasmid, pCMX-xNT4/hNT4 contains the Xenopus NT-4 prepro region fused to the mature coding region of human NT-4.
11.1.3. EXPRESSION OF RECOMBINANT
HUMAN NT-4 IN COS CELLS
COS M5 cells were set up at a density of
1.5 × 105 cells/well of a Costar 6 well dish in DMEM media supplemented with 10% FBS, glutamine and Na pyruvate (all from Irvine Scientific except FBS).
The next day the cells were aspirated and refed with 2 ml/well of RPMI media containing 400 μg/ml
DEAE-Dextran (Pharmacia), 400 μM chloroquine (Sigma),
4 mM glutamine (Irvine), 1 × ITS (insulin, transferrin, selenium, Sigma). To each well 2 μg of the appropriate DNA was added and mixed by swirling. Three separate constructs were used: pCMX-xNT4, containing the prepro precursor of Xenopus NT-4; and two human NT-4
constructions, pCMX-HG7-2M and pCMX-HG7-2Q. After the addition of the DNA the plates were returned to 37°C, 5% CO2 incubator for 3 hours 15 minutes. The media/DNA mixture was then aspirated and 2 ml/well of 10% DMSO in PBS without Ca2+, Mg2+ was added for 2 minutes. The
DMSO/PBS was aspirated and wells washed once with 10% FBS DMEM, then refed with 10% FBS DMEM. The next morning, plates to be bioassayed were washed once with Defined Media (DM) and refed 2 ml/well of DM. Three days post-transfection, supernatants were removed from cells and debris pelleted by microcentrifugation. Supernatants were transferred to fresh tubes and assayed for
bioactivity. 11.1.4. PREPARATION OF ENRICHED MOTOR
NEURON CULTURES
Embryos (E14) from Sprague-Dawley rats (HSD or Zivic-Miller) were used for all experiments. Pregnantrats were sacrificed by carbon dioxide asphyxiation, and embryos were rapidly removed and placed in ice-cold medium for further dissection. Spinal cords were removed aseptically from rat embryos of 14 days gestation. The spinal cord was severed caudal to the bulb (at the level of the first dorsal root ganglion), freed of sensory ganglia and adhering meninges. The cord was then
subdivided into ventral and mediodorsal segments for separate cultures. The ventral spinal cord tissues were diced into small pieces and incubated in 0.1% trypsin (GIBCO) and 0.01% deoxyribonuclease type 1 (Sigma) in PBS at 37ºC for 20 minutes. Trypsin solution was then removed, rinsed and replaced with medium consisting of 45% Eagle's minimum essential medium (MEM), 45% Ham's nutrient mixture F12 (F12), 5% heat inactivated fetal calf serum (GIBCO), 5% heat inactivated horse serum
(GIBCO), glutamine (2 mM), penicillin G (0.5 U/ml), and streptomycin (0.5 μg/ml). The tissue was then
mechanically dissociated by gentle trituration through a Pasteur pipet, and the supernatants were pooled and filtered through a nylon fiber (Nitex, Tetko; 40 μm).
The filtered cell suspension were then subjected to a modification of the fraction procedure described by
Schnaar and Schaffner (1981, J. Neurosci, 1:204-217).
All steps were carried out at 4ºC. Metrizamide was dissolved in F12:MEM (1:1) medium, and a discontinuous gradient was established which consisted of a 18%
metrizamide cushion (0.5 ml), 3 ml of 17% metrizamide, 3 ml of 12% metrizamide, and 3 ml of a 8% metrizamide was prepared. The filtered ventral spinal cord cell
suspension (2.5 ml) obtained as described above was layered over the step gradient, the tube was centrifuged at 2500 × g for 15 minutes using a swing-out rotor
(Sorvall HB4). Centrifugation resulted in three layers of cells: fraction I (at 0-8% interface), fraction II (at 8-12% interface), and fraction III (at 12-17% interface). The cells from each interface were removed in a small volume (about 1 ml), rinsed twice with serum-free defined medium consisting of 50% F12 and 50% MEM, supplemented with glutamine (2 mM), insulin (5 μg/ml), transferrin (100 μg/ml), progesterone (20 nM), putrescine (100 μM), and sodium selenite (30 nM) (Bottenstein and Sato, 1979, Proc. Natl. Acad. Sci. 76:514-517). Viable cell count was obtained by hemocytometer counting in the presence of trypan blue. Fractionated ventral spinal cord cells (enriched with motor neurons) were then plated at a density of 100,000 cells/cm2 in 6 mm wells precoated with poly-L-ornithine (Sigma: 10 μg/ml) and laminin (GIBCO: 10 μg/ml). Treatment with COS cell supernatants
containing NT-4 was COS cell was given on the day of plating. Cultures were maintained in serum-free defined medium at 37°C in 95% air/ 5% CO2 atmosphere at nearly 100% relative humidity. On day 2 (48 hours), cells were harvested for measurements of choline acetyltransferase (CAT) as described in Fonnum, 1975, J. Neurochem. 24:407-409.
11.2 RESULTS
11.2.1. EUKARYOTIC EXPRESSION OF
BIOLOGICALLY ACTIVE RECOMBINANAT
HUMAN NT-4
Plasmid DNA from each of the pCMX-based
constructions (pCMX-HG7-2Q, pCMX-HG7-2M, pCMX-hNT3/hNT4 and pCMX-xNT4/hNT4) was prepared and individually
transfected into COS cells. COS supernatants from each transfected cell line were utilized in order to assess the biological activity of each respective recombinant form of NT-4. The volumes of COS supernatants tested were 10, 50 and 250 μl in a total volume of 2 ml. Q1 (PCMX-HG7-2Q), N7 (pCMX-hNT3/hNT4 fusion), and X1 (pCMXxNT4/hNT4) possessed neurite-promoting activity on DRG explants (Figure 23). In addition, both Q (pCMX-HG7-2Q) and M (pCMX-HG7-2M) were examined for their survival-promoting activity on DRG dissociated cells. Volumes tested were 5-250 μl in a total volume of 2 ml. When added to cultures of dissociated DRG neurons, COS supernatant containing hNT4 promoted 30% neuronal survival compared to 10% survival with mock transfected COS supernatants (Figure 24).
The biological effect of human recombinant protein from supernatants of COS cells transfected with pCMX-HG7-2M was tested on motor neuron enriched cultures prepared as described supra in Example Section 11.1.4. Treatment of motor neuron enriched cultures with
pCMX-HG7-2M derived human NT-4 diluted to 1:5 resulted in a 2.9 fold increase in choline acetyltransferase (CAT) activity after 48 hours as compared to untreated (C-NT) and mock transfected (MOC COS) controls (Figure 25). The increase in CAT activity dropped to 1.7 fold when a 1:50 dilution was tested, suggesting that it was a dose dependent response (Figure 25).
11.3 DISCUSSION
The present invention provides for the
utilization of an in vitro eukaryotic expression system to express recombinant human NT-4. The present invention discloses several strategies to express a biologically active form of recombinant human NT-4 in COS cells. In one example, the DNA sequence encoding NT-4 prepro precursor was amplified utilizing two PCR amplification strategies to yield pCMX-based expression plasmids containing either the pCMX translation initiation site (pCMX-HG7-2M) or a Kozak consensus translation site (PCMX-HG7-2Q). In another example, two chimeric
neurotrophin genes fusing either the prepro region of Xenopus NT-4 (pCMX-xNT4/hNT4) or the prepro region of human NT-3 (pCMX-hNT3/hNT4) to the mature coding region of NT-4 were constructed for expression in COS cells (see Section 5, supra, for a discussion of the use of chimeric constructions to express NT-4 in vitro).
Expression of a biologically active form of human NT-4 in an in vitro eukaryotic expression system substantially increases the ease at which the production of human recombinant NT-4, peptides or derivatives thereof may be scaled up for both therapeutic and
diagnostic applications discussed supra. In view of the instant invention, one of ordinary skill in the art can readily construct a plasmid containing an identical DNA sequence as disclosed or a similar DNA sequence encoding a homologous yet distinct NT-4 like protein or derivative thereof. The skilled artisan can also pick and choose between numerous DNA plasmid vectors known in the art to construct an expression plasmid for use in a eukaryotic expression system. We have demonstrated that recombinant human NT-4, whether produced as a full prepro precursor or via a neurotrophin-based chimeric construction, is biologically active as demonstrated by the stimulating effect of recombinant NT-4 COS supernatants on neurite outgrowth in DRG explants and the bioactivity of cultured motor neurons. 12. EXAMPLE: TRKB IS A RECEPTOR FOR NEUROTROPHIN-4 COS cell supernatants were also examined in a survival assay utilizing 3T3 fibroblasts. In this assay system, 3T3 fibroblasts, which do not express
neurotrophin receptor proteins, are transfected with mammalian expression vectors encoding either trkA or trkB. 3T3 fibroblast survival is dependent on the addition and receptor specific binding of the respective neurotrophic factor.
COS-M5 cells were cultured and transfected
With either pCMX-HG7-2Q, pCMX-HG7-2M or pCMX-HG7-2Q as described in Example Section 11.1.3.
A full-length rat trkA cDNA clone was obtained from Dr. Eric Shooter of Stanford University. The rat trkA cDNA was subcloned into the mammalian expression vector, pCMX, to generate pCMX-trkA.
A full-length rat trkB cDNA clone was obtained by screening a rat brain cDNA library in the lambda ZAP2 vector (Stratagene) with rat trkB-specific
oligonucleotides corresponding to the most 5' and 3' coding regions of trkB. The rat trkB cDNA was subcloned into pCMX to generate pCMX-trkB.
3T3 fibroblasts were cultured and transfected as described in Glass, et al., 1991, Cell 66:405-413.
in this survival assay system, 3T3 fibroblasts, which do not express neurotrophin receptor proteins, have been transfected with trkA, a protooncogene encoding a tyrosine kinase receptor for NGF, or with trkB, a tyrosine kinase which serves as a functional binding protein for BDNF and NT-3. The transfected cells are dependent upon the addition of the corresponding neurotrophin for survival, and thus may be used to assay for biological activity of neurotrophins. Addition of
NT-4 containing COS cell supernatants in this bioassay indicated that viable cells remain after 48 hours only in 3T3 trkB cultures (Table 2). Thus, these results
demonstrate that NT-4 protein has biological activity in this system, and suggests that trkB, but not trkA, serves as a functional binding protein for NT-4.
TABLE 2
Assay of COS Supernatants on 3T3 Cell Lines Expressing TrkA and TrkB
Dilutions MOCk HG7- HG7- hNT3/
2Q 2M hNT4
1:5 - - - -
1:10 - - - - 3T3 1:20 - - - -
1:50 - - - -
1:5 - - - -
1:10 - - - - 3T3-trkA 1:20 - - -
1:50 - - - -
1:5 - + + +
1:10 - + + + 3T3-trkB 1:20 - + + +
1:50 - + + +
13. DEPOSIT OF MICROORGANISMS
The following recombinant bacteriophage, containing a human genomic sequence related to
neurotrophin-4, were deposited on August 22, 1991 (HG4-2 and HG7-2) and September 11, 1991 (HG2-1) with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, and assigned the indicated accession number. Additionally, the chimeric gene construction, pCMX-hNT3/hNT4, was deposited on
October 30, 1991 with the American Type Culture
Collection and assigned the indicated accession number.
Bacteriophage ATCC Accession Number
HG4-2 75069
HG7-2 75070
HG2-1 75098
pCMX-hNT3/hNT4 75133
The present invention is not to be limited in scope by the deposited microorganisms or the specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various publications have been cited herein which are incorporated by reference in their entireties. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Ip and Yancopoulos
(ii) TITLE OF INVENTION: Neurotrophin-4
(iii) NUMBER OF SEQUENCES: 124
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Pennie & Edmonds
(B) STREET: 1155 Avenue of the Americas
(C) CITY: New York
(D) STATE: New York
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(F) ZIP: 10036-2711
(v) 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 #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Misrock, S. LesIle
(B) REGISTRATION NUMBER: 18,872
(C) REFERENCE/DOCKET NUMBER: 6526-079
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 212 790-9090
(B) TELEFAX: 212 8698864/9741
(C) TELEX: 66141 PENNIE
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CAAATGTAAT CCCGCTGGTG GAACTGTGGG TGGCTGCCGG GGTGTTGATC GACGCCATTG 60 GATCTCTGAG TGCAAGGCTA AACAGTCTTA CGTGAGGGCT CTGACTATGG ATTCTGACAA 120 GATTGTTGGC 130
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CAAGTGCAAT CCATCAGGCA GCACCACTAG AGGATGCCGA GGTGTAGACA AAAAGCAATG 60 GATATCTGAG TGCAAAGCAA AACAGTCTTA TGTGAGGGCT CTGACCATAG ATGCCAACAA 120 GCTTGTGGGT 130
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CAAGTGCCGG GACCCAAATC CCGTTGACAG CGGGTGCCGG GGCATTGACT CAAAGCACTG 60 GAACTCATAT TGTACCACGA CTCACACCTT TGTCAAGGCG CTGACCATGG ATGGCAAGCA 120 GGCTGCC 127
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CAAGTGCCGG GCCCCAAATC CTGTAGAGAG TGGATGCCGG GGCATTGACT CCAAGCACTG 60 GAACTCATAC TGCACCACGA CTCACACCTT TGTCAAGGCG TTGACAACAG ACGACAAACA 120 GGCTGCC 127
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CAAGTGCAGG GACCCTAGGC CGGTGTCCAG CGGGTGCCGA GGGATCGATG CGAAGCATTG 60
GAACTCTTAC TGCACCACGA CACACACCTT CGTCAAAGCA CTGACCATGG AGGGCAAGCA 120
AGCAGCC 127
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CAAGTGCAAA AATCCAAGTC CAGTATCAGG TGGGTGCAGG GGCATTGATG CCAAGCATTG 60 GAATTCGTAT TGCACCACAA CAGACACATT TGTCAGGGCA TTAACCATGG AAGGCAATCA 120 GGCATCT 127
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CAAATGCAGG GACCCAAAGC TAGTTTCAAG CGGATGCCGT GGGATTGATG CAAAGCATTG 60 GAACTCTTAT TGTACCACCA CGCACACCTT TGTCAAAGCA TTAACAATGG AAGGGAAGCA 120 AGCAGCA 127
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
CACGTGCCGT GGCGCCCGGG CGGGCAGCTC TGGCTGCCTG GGCATCGACG GGCGACACTG 60 GAACTCCTAC TGCACCAACT CGCACACCTT CGTGCGGGCG CTGACTTCCT TTAAGGACCT 120 GGTGGCC 127
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CAAGTGCAAT CCCATGGGTT ACACAAAAGA AGGCTGCAGG GGCATAGACA AAAGGCATTG 60
GAACTCCCAG TGCCGAACTA CCCAGTCGTA CGTGCGGGCC CTTACCATGG ATAGCAAAAA 120
GAGAATTGGC 130 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CAAGTGTAAT CCCATGGGTT ACACGAAGGA AGGCTGCAGG GGCATAGACA AAAGGCACTG 60 GAACTCGCAA TGCCGAACTA CCCAATCGTA TGTTCGGGCC CTTACTATGG ATAGCAAAAA 120 GAGAATTGGC 130
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CAAATGCAAC CCCAAGGGGT ACACAAAGGA AGGCTGCAGG GGCATAGACA AGAGGCACTG 60 GAACTCACAG TGCCGAACTA CCCAGTCTTA CGTGAGAGCT CTCACCATGG ATAACAAAAA 120 GAGAGTTGGC 130
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
CAAGTGCAGC ACGAAGGGTT ATGCAAAAGA AGGCTGTAGA GGCATAGACA AGAGGTACTG 60
GAATTCCCAG TGCCGAACTA CTCAGTCTTA CGTCCGCGCT CTCACCATGG ATAACAAAAA 120
GAGGATTGGA 130
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CAAATGCAAC CCTATGGGTT ACATGAAAGA AGGCTGCAGA GGCATAGACA AAAGGTACTG 60 GAACTCTCAG TGCCGAACTA CTCAGTCTTA CGTGCGGGCT TTCACCATGG ATAGCAGAAA 120 AAAAGTTGGT 130
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CAAATGTAAC CCTATGGGGT ACACAAAGGA GGGCTGCCGT GGAATAGACA AGAGGCATTA 60 TAACTCCCAA TGCAGGACAA CCCAGTCCTA CGTGCGAGCG CTCACCATGG ATAGCAAAAA 120 GAAGATTGGC 130
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CAAATGTAAC CCCAAGGGTT TCACCAACGA AGGCTGCAGA GGGATAGACA AGAAACATTG 60 GAATTCGCAG TGTAGAACCA GCCAATCCTA TGTGCGAGCT CTAACCATGG ATAGTAGGAA 120 GAAGATTGGG 130
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGATGTAAG GAAGCCAGGC CGGTCAAAAA CGGTTGCAGG GGTATTGATG ATAAACACTG 60
GAACTCTCAG TGCAAAACAT CCCAAACCTA CGTCCGAGCA CTGACTTCAG AGAACAATAA 120
ACTCGTGGGC 130 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GAGGTGTAAA GAAGCCAGGC CAGTCAAAAA CGGTTGCAGG GGGATTGATG ACAAACACTG 60 GAACTCTCAG TGCAAAACGT CGCAAACCTA CGTCCGAGCA CTGACTTCAG AAAACAACAA 120 ACTCGTAGGC 130
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
AAGGTGTAAA GAAGCCAAAC CTGTTAAAAA TGGCTGCCGA GGCATTGACG ACAAGCACTG 60 GAACTCCCAG TGCAAGACAT CCCAAACTTA CGTTAGAGCA TTGACTTCAG AAAACAATAA 120 ACTTGTAGGC 130
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
AAGGTGTAAA GAGGCAAGAC CTGTCAAAAA TGGCTGTCGA GGCATAGACG ACAAACACTG 60 GAATTC 66
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CAAGTGTCGG ACTGCCAAAC CTTTTAAGAG CGGCTGTCGC GGCATCGATG ACAAACACTG 60 GAACTCGCAG TGTAAGACCT CTCAGACGTA CGTCAGAGTC TCTGACGCAG GACCGTACCT 120 CTGTGGGC 128
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CCGCTGCAAG GAGTCGAAGC CGGGCAAGAA CGGGTGCCGG GGCATCGACG ACAAACACTG 60 GAACTCGCAG TGCAAGACCA GCCAGACCTA TGTCCGAGCG CTGAGCAAGG AGAACAATAA 120 ATATGTGGGC 130
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Lys Cys Asn Pro Ala Gly Gly Thr Val Gly Gly Cys Arg Gly Val Asp 1 5 10 15
Arg Arg His Trp Ile Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Met Asp Ser Asp Lys Ile Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Lye Cys Asn Pro Ser Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp 1 5 10 15
Lys Lys Gln Trp Ile Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Ile Asp Ala Asn Lys Leu Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile Asp 1 5 10 15
Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys
20 25 30
Ala Leu Thr Met Asp Gly Lys Gln Ala Ala
35 40
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Lys Cys Arg Ala Pro Asn Pro Val Glu Ser Gly Cys Arg Gly Ile Asp 1 5 10 15
Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys
20 25 30
Ala Leu Thr Thr Asp Asp Lys Gln Ala Ala
35 40
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Lys Cys Arg Asp Pro Arg Pro Val Ser Ser Gly Cys Arg Gly Ile Asp 1 5 10 15 Ala Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys 20 25 30
Ala Leu Thr Met Glu Gly Lys Gln Ala Ala
35 40
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
( ii) MOLECULE TYPE : peptide
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 27 :
Lys Cys Lys Asn Pro Ser Pro Val Ser Gly Gly Cys Arg Gly Ile Asp
1 5 10 15
Ala Lys His Trp Asn Ser Tyr Cys Thr Thr Thr Asp Thr Phe Val Arg
20 25 30
Ala Leu Thr Met Glu Gly Asn Gln Ala Ser
35 40
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Lys Cys Arg Asp Pro Lys Pro Val Ser Ser Gly Cys Arg Gly Ile Asp 1 5 10 15
Ala Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys
20 25 30
Ala Leu Thr Met Glu Gly Lys Gln Ala Ala
35 40
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Thr Cys Arg Gly Ala Arg Ala Gly Ser Ser Gly Cys Leu Gly Ile Asp 1 5 10 15
Gly Arg His Trp Asn Ser Tyr Cys Thr Asn Ser His Thr Phe Val Arg
20 25 30
Ala Leu Thr Ser Phe Lys Asp Leu Val Ala
35 40
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile Asp 1 5 10 15
Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Met Asp Ser Lys Lys Arg Ile Gly
35 40
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile Asp 1 5 10 15
Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Met Asp Ser Lys Lys Arg Ile Gly
35 40
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Lys Cys Asn Pro Lys Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile Asp 1 5 10 15
Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Met Asp Asn Lys Lys Arg Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Lys Cys Ser Thr Lys Gly Tyr Ala Lys Glu Gly Cys Arg Gly Ile Asp 1 5 10 15
Lys Arg Tyr Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Met Asp Asn Lys Lys Arg Ile Gly
35 40
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Lys Cys Asn Pro Met Gly Tyr Met Lys Glu Gly Cys Arg Gly Ile Asp 1 5 10 15
Lye Arg Tyr Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg
20 25 30
Ala Phe Thr Met Asp Ser Arg Lys Lys Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Cys Arg Gly Ile Asp 1 5 10 15
Lys Arg His Tyr Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Met Asp Ser Lys Lys Lys Ile Gly
35 40
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Lys Cys Asn Pro Lys Gly Phe Thr Asn Glu Gly Cys Arg Gly Ile Asp 1 5 10 15
Lys Lys His Trp Asn Ser Gln Cys Arg Thr Ser Gln Ser Tyr Val Arg
20 25 30
Ala Leu Thr Met Asp Ser Arg Lys Lys Ile Gly
35 40
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Arg Cys Lys Glu Ala Arg Pro Val Lys Asn Gly Cys Arg Gly Ile Asp 1 5 10 15
Asp Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg
20 25 30
Ala Leu Thr Ser Glu Asn Asn Lys Leu Val Gly
35 40 (2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Arg Cys Lys Glu Ala Arg Pro Val Lys Asn Gly Cys Arg Gly Ile Asp 1 5 10 15
Asp Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg
20 25 30
Ala Leu Thr Ser Glu Asn Asn Lys Leu Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Arg Cys Lys Glu Ala Lys Pro Val Lys Asn Gly Cys Arg Gly Ile Asp 1 5 10 15
Asp Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg
20 25 30
Ala Leu Thr Ser Glu Asn Asn Lys Leu Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
Arg Cys Lys Glu Ala Arg Pro Val Lys Asn Gly Cys Arg Gly Ile Asp 1 5 10 15
Asp Lye His Trp Asn
20 ( 2 ) INFORMATION FOR SEQ ID NO : 41 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
Lys Cys Arg Thr Ala Lys Pro Phe Lys Ser Gly Cys Arg Gly Ile Asp 1 5 10 15
Asp Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg
20 25 30
Ala Leu Thr Gln Asp Arg Thr Ser Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42 :
Arg Cys Lys Glu Ser Lys Pro Gly Lys Asn Gly Cys Arg Gly Ile Asp 1 5 10 15
Asp Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg
20 25 30
Ala Leu Ser Lys Glu Asn Asn Lys Tyr Val Gly
35 40
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1302 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(529..534, 538..1248)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
CAATCATACT TATGAACAGC AGGGGGAGCC CTCGCCTTAC TTCCCAGCCA TGCAGAACTC 60 AAGCAGCTTT GTTTATGCCG ATCCCTAAGC AGCCCAGACC ACACTGAGCA TGTGCACAGT 120
CTTAGTCTTG CAAAGATGTT TAACAAAGTT ACAAGATGGT GACCCCCTGT AGCCAACTTT 180
GAAAGCATAA ATCATTTGTT TGATTAGGCT TGTGGTGCAG TAAGTTCATG TTTATATTTA 240
GCATACAAAA TACAGCATTT CTAGCCTTAT TCTATTTTAG ACTTTACCCT TTAATGCCCA 300
GTTCTGCCCA TTGCCTTATA GATGTTAAAG TCCCAATATC ACATTGGCAT CCTCGGCTGT 360
TTACAACAAA CATTAAAACT TGTACTTATA TTTAACATTC TGTTGTTCTT CCAATATTCC 420
ATCACACTTA GACCCTAAAA GAATTATATG TATATAATTT GCATAAATTA TATAATGGCA 480
GCCGTATTCT AATTCTGTTT TTTTTTTTTT TTTTTGCAGT GGTCTGAG GTG GAT 534
Val Asp
1
TAA GTA ATG ATC CTC CGC CTT TAT GCC ATG GTG ATC TCA TAC TGT TGT 582 Val Met Ile Leu Arg Leu Tyr Ala Met Val Ile Ser Tyr Cys Cys
5 10 15
GCC ATC TGC GCT GCC CCC TTC CAG AGC CGG ACC ACA GAT TTG GAT TAT 630 Ala Ile Cys Ala Ala Pro Phe Gln Ser Arg Thr Thr Asp Leu Asp Tyr
20 25 30
GGC CCC GAT AAA ACA TCA GAA GCC TCA GAC CGG CAA TCA GTT CCC AAC 678 Gly Pro Asp Lys Thr Ser Glu Ala Ser Asp Arg Gln Ser Val Pro Asn
35 40 45
AAC TTC AGT CAT GTC CTG CAA AAT GGG TTC TTT CCA GAT TTG TCA TCC 726
Asn Phe Ser His Val Leu Gln Asn Gly Phe Phe Pro Asp Leu Ser Ser
50 55 60 65
ACC TAT TCC AGC ATG GCT GGT AAG GAC TGG AAC CTA TAC TCA CCT AGA 774 Thr Tyr Ser Ser Met Ala Gly Lys Asp Trp Asn Leu Tyr Ser Pro Arg
70 75 80
GTG ACT CTT TCA AGT GAG GAG CCT TCT GGA CCT CCA CTA CTT TTC TTG 822 Val Thr Leu Ser Ser Glu Glu Pro Ser Gly Pro Pro Leu Leu Phe Leu
85 90 95
TCA GAG GAG ACA GTG GTA CAT CCA GAA CCA GCC AAC AAG ACT TCC CGG 870 Ser Glu Glu Thr Val Val His Pro Glu Pro Ala Asn Lys Thr Ser Arg
100 105 110
CTA AAA CGG GCA TCA GGA TCT GAT TCG GTC AGC TTG TCC CGT CGG GGA 918 Leu Lys Arg Ala Ser Gly Ser Asp Ser Val Ser Leu Ser Arg Arg Gly
115 120 125
GAG CTC TCT GTG TGT GAC AGT GTC AAC GTC TGG GTT ACC GAT AAA CGT 966 Glu Leu Ser Val Cys Asp Ser Val Asn Val Trp Val Thr Asp Lys Arg
130 135 140 145
ACA GCC GTG GAT GAT CGG GGT AAA ATA GTG ACT GTC ATG TCT GAG ATT 1014 Thr Ala Val Asp Asp Arg Gly Lys Ile Val Thr Val Met Ser Glu Ile
150 155 160
CAG ACT CTA ACA GGA CCA CTG AAG CAA TAC TTC TTT GAG ACC AAG TGC 1062 Gln Thr Leu Thr Gly Pro Leu Lys Gln Tyr Phe Phe Glu Thr Lys Cys
165 170 175
AAT CCA TCA GGC AGC ACC ACT AGA GGA TGC CGA GGT GTA GAC AAA AAG 1110 Asn Pro Ser Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp Lys Lys
180 185 190
CAA TGG ATA TCT GAG TGC AAA GCA AAA CAG TCT TAT GTG AGG GCT CTG 1158 Gln Trp Ile Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg Ala Leu
195 200 205 ACC ATA GAT GCC AAC AAG CTT GTG GGT TGG CGT TGG ATC CGT ATT GAC 1206 Thr Ile Asp Ala Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile Asp
210 215 220 225
ACA GCG TGT GTC TGT ACC TTG TTG AGT CGG ACA GGA AGG ACG 1248
Thr Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Thr
230 235
TAAAAGACGA GGGTTAGCAA AATAGAGAGA AGAGGTTGAT CCGTTGACCT GCAG 1302
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 239 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
Val Asp Val Met Ile Leu Arg Leu Tyr Ala Met Val Ile Ser Tyr Cys
1 5 10 15
Cys Ala Ile Cys Ala Ala Pro Phe Gln Ser Arg Thr Thr Asp Leu Asp
20 25 30
Tyr Gly Pro Asp Lys Thr Ser Glu Ala Ser Asp Arg Gln Ser Val Pro
35 40 45
Asn Asn Phe Ser His Val Leu Gln Asn Gly Phe Phe Pro Asp Leu Ser
50 55 60
Ser Thr Tyr Ser Ser Met Ala Gly Lys Asp Trp Asn Leu Tyr Ser Pro
65 70 75 80
Arg Val Thr Leu Ser Ser Glu Glu Pro Ser Gly Pro Pro Leu Leu Phe
85 90 95
Leu Ser Glu Glu Thr Val Val His Pro Glu Pro Ala Asn Lys Thr Ser
100 105 110
Arg Leu Lys Arg Ala Ser Gly Ser Asp Ser Val Ser Leu Ser Arg Arg
115 120 125
Gly Glu Leu Ser Val Cys Asp Ser Val Asn Val Trp Val Thr Asp Lys
130 135 140
Arg Thr Ala Val Asp Asp Arg Gly Lys Ile Val Thr Val Met Ser Glu
145 150 155 160
Ile Gln Thr Leu Thr Gly Pro Leu Lys Gln Tyr Phe Phe Glu Thr Lys
165 170 175
Cys Asn Pro Ser Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp Lys
180 185 190
Lys Gln Trp Ile Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg Ala
195 200 205
Leu Thr Ile Asp Ala Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile
210 215 220
Asp Thr Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Thr
225 230 235
(2) INFORMATION FOR SEQ ID NO: 45: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 123 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Ala Ser Gly Ser Asp Ser Val Ser Leu Ser Arg Arg Gly Glu Leu Ser
1 5 10 15
Val Cys Asp Ser Val Asn Val Trp Val Thr Asp Lys Arg Thr Ala Val
20 25 30
Asp Asp Arg Gly Lys Ile Val Thr Val Met Ser Glu Ile Gln Thr Leu
35 40 45
Thr Gly Pro Leu Lys Gln Tyr Phe Phe Glu Thr Lys Cys Asn Pro Ser 50 55 60
Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp Lys Lys Gln Trp Ile 65 70 75 80
Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg Ala Leu Thr Ile Asp
85 90 95
Ala Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile Asp Thr Ala Cys
100 105 110
Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Thr
115 120
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Ser Ser Thr His Pro Val Phe His Met Gly Glu Phe Ser Val Cys Asp 1 5 10 15
Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys
20 25 30
Gly Lys Glu Val Thr Val Leu Ala Glu Val Asn Ile Asn Asn Ser Val
35 40 45
Phe Arg Gln Tyr Phe Phe Glu Thr Lys Cys Arg Ala Ser Asn Pro Val 50 55 60
Glu Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys 65 70 75 80
Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Thr Asp Glu Lys Gln
85 90 95
Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu 100 105 110
Ser Arg Lys Ala Thr Arg
115
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Ser Ile 1 5 10 15
Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Met Ser
20 25 30
Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys Gly Gln
35 40 45
Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr Thr 50 55 60
Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Gln Cys 65 70 75 80
Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys Lys
85 90 95
Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Thr
100 105 110
Leu Thr Ile Lys Arg Gly Arg
115
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48 :
Tyr Ala Glu His Lys Ser His Arg Gly Glu Tyr Ser Val Cys Asp Ser 1 5 10 15
Glu Ser Leu Trp Val Thr Asp Lys Ser Ser Ala Ile Asp Ile Arg Gly
20 25 30 His Gln Val Thr Val Leu Gly Glu Ile Lys Thr Gly Asn Ser Pro Val 35 40 45
Lys Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Lys 50 55 60
Asn Gly Cys Arg Gly Ile Asp Asp Lys His Trp Asn Ser Gln Cys Lys 65 70 75 80
Thr Ser Gln Thr Tyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu
85 90 95
Val Gly Trp Arg Trp Ile Arg Ile Asp Thr Ser Cys Val Cys Ala Leu
100 105 110
Ser Arg Lys Ile Gly Arg Thr
115
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1313 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 552..1259
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
CTGCAGGGAA ACAATCATAC TTATGAACAG CAGGGGGAGC CCTCGCCTTA CTTCCCAGCC 60
ATGCAGAACT CAAGCAGCTT TGTTTATGCC GATCCCTAAG CAGCCCAGAC CACACTGAGC 120
ATGTGCACAG TCTTAGTCTT GCAAAGATGT TTAACAAAGT TACAAGATGG TGACCCCCTG 180
TAGCCAACTT TGAAAGCATA AATCATTTGT TTGATTAGGC TTGTGGTGCA GTAAGTTCAT 240
GTTTATATTT AGCATACAAA ATACAGCATT TCTAGCCTTA TTCTATTTTA GACTTTACCC 300
TTTAATGCCC AGTTCTGCCC ATTGCCTTAT AGATGTTAAA GTCCCAATAT CACATTGGCA 360
TCCTCGGCTG TTTACAACAA ACATTAAAAC TTGTACTTAT ATTTAACATT CTGTTGTTCT 420
TCCAATATTC CATCACACTT AGACCCTAAA AGAATTATAT GTATATAATT TGCATAAATT 480
ATATAATGGC AGCCGTATTC TAATTCTGTT TTTTTTTTTT TTTTTTGCAG TGGTCTGAGG 540
TGGATTAAGT A ATG ATC CTC CGC CTT TAT GCC ATG GTG ATC TCA TAC TGT 590
Met Ile Leu Arg Leu Tyr Ala Met Val Ile Ser Tyr Cys
1 5 10
TGT GCC ATC TGC GCT GCC CCC TTC CAG AGC CGG ACC ACA GAT TTG GAT 638 Cys Ala Ile Cys Ala Ala Pro Phe Gln Ser Arg Thr Thr Asp Leu Asp
15 20 25
TAT GGC CCC GAT AAA ACA TCA GAA GCC TCA GAC CGG CAA TCA GTT CCC 686 Tyr Gly Pro Asp Lys Thr Ser Glu Ala Ser Asp Arg Gln Ser Val Pro
30 35 40 45 AAC AAC TTC AGT CAT GTC CTG CAA AAT GGG TTC TTT CCA GAT TTG TCA 734 Asn Asn Phe Ser His Val Leu Gln Asn Gly Phe Phe Pro Asp Leu Ser
50 55 60
TCC ACC TAT TCC AGC ATG GCT GGT AAG GAC TGG AAC CTA TAC TCA CCT 782 Ser Thr Tyr Ser Ser Met Ala Gly Lys Asp Trp Asn Leu Tyr Ser Pro
65 70 75
AGA GTG ACT CTT TCA AGT GAG GAG CCT TCT GGA CCT CCA CTA CTT TTC 830 Arg Val Thr Leu Ser Ser Glu Glu Pro Ser Gly Pro Pro Leu Leu Phe
80 85 90
TTG TCA GAG GAG ACA GTG GTA CAT CCA GAA CCA GCC AAC AAG ACT TCC 878 Leu Ser Glu Glu Thr Val Val His Pro Glu Pro Ala Asn Lys Thr Ser
95 100 105
CGG CTA AAA CGG GCA TCA GGA TCT GAT TCG GTC AGC TTG TCC CGT CGG 926 Arg Leu Lys Arg Ala Ser Gly Ser Asp Ser Val Ser Leu Ser Arg Arg
110 115 120 125
GGA GAG CTC TCT GTG TGT GAC AGT GTC AAC GTC TGG GTT ACC GAT AAA 974 Gly Glu Leu Ser Val Cys Asp Ser Val Asn Val Trp Val Thr Asp Lys
130 135 140
CGT ACA GCC GTG GAT GAT CGG GGT AAA ATA GTG ACT GTC ATG TCT GAG 1022 Arg Thr Ala Val Asp Asp Arg Gly Lys Ile Val Thr Val Met Ser Glu
145 150 155
ATT CAG ACT CTA ACA GGA CCA CTG AAG CAA TAC TTC TTT GAG ACC AAG 1070 Ile Gln Thr Leu Thr Gly Pro Leu Lys Gln Tyr Phe Phe Glu Thr Lys
160 165 170
TGC AAT CCA TCA GGC AGC ACC ACT AGA GGA TGC CGA GGT GTA GAC AAA 1118 Cys Asn Pro Ser Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp Lys
175 180 185
AAG CAA TGG ATA TCT GAG TGC AAA GCA AAA CAG TCT TAT GTG AGG GCT 1166 Lys Gln Trp Ile Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg Ala
190 195 200 205
CTG ACC ATA GAT GCC AAC AAG CTT GTG GGT TGG CGT TGG ATC CGT ATT 1214 Leu Thr Ile Asp Ala Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile
210 215 220
GAC ACA GCG TGT GTC TGT ACC TTG TTG AGT CGG ACA GGA AGG ACG 1259 Asp Thr Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Thr
225 230 235
TAAAAGACGA GGGTTAGCAA AATAGAGAGA AGAGGTTGAT CCGTTGACCT GCAG 1313
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 236 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Met Ile Leu Arg Leu Tyr Ala Met Val Ile Ser Tyr Cys Cys Ala Ile
1 5 10 15
Cys Ala Ala Pro Phe Gin Ser Arg Thr Thr Asp Leu Asp Tyr Gly Pro
20 25 30
Asp Lys Thr Ser Glu Ala Ser Asp Arg Gin Ser Val Pro Asn Asn Phe
35 40 45 Ser His Val Leu Gln Asn Gly Phe Phe Pro Asp Leu Ser Ser Thr Tyr 50 55 60
Ser Ser Met Ala Gly Lys Asp Trp Asn Leu Tyr Ser Pro Arg Val Thr 65 70 75 80
Leu Ser Ser Glu Glu Pro Ser Gly Pro Pro Leu Leu Phe Leu Ser Glu
85 90 95
Glu Thr Val Val His Pro Glu Pro Ala Asn Lys Thr Ser Arg Leu Lys
100 105 110
Arg Ala Ser Gly Ser Asp Ser Val Ser Leu Ser Arg Arg Gly Glu Leu
115 120 125
Ser Val Cys Asp Ser Val Asn Val Trp Val Thr Asp Lys Arg Thr Ala
130 135 140
Val Asp Asp Arg Gly Lys Ile Val Thr Val Met Ser Glu Ile Gln Thr 145 150 155 160
Leu Thr Gly Pro Leu Lys Gln Tyr Phe Phe Glu Thr Lys Cys Asn Pro
165 170 175
Ser Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp Lys Lys Gln Trp
180 185 190 Ile Ser Glu Cys Lye Ala Lys Gln Ser Tyr Val Arg Ala Leu Thr Ile
195 200 205
Asp Ala Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile Asp Thr Ala
210 215 220
Cys Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Thr
225 230 235
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Gln Tyr Phe Phe Glu Thr Lys Cys Asn Pro Ser Gly Ser Thr Thr Arg
1 5 10 15
Gly Cys Arg Gly Val Asp Lys Lys Gln Trp Ile Ser Glu Cys Lys Ala
20 25 30
Lys Gln Ser Tyr Val Arg Ala Leu Thr Ile Asp Ala Asn Lys Leu Val
35 40 45
Gly Trp Arg Trp Ile Arg Ile Asp Thr
50 55
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Gln Tyr Phe Tyr Glu Thr
1 5
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
CARTAYTTYT TYGARAC
17
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
CARTAYTTYT AYGARAC 17 (2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 9
(D) OTHER INFORMATION: /label= N
/note= "N = I"
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 12
(D) OTHER INFORMATION: /label= N
/note= "N = I"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
TTRCAYTCNS WNATCCA 17 ( 2 ) INFORMATION FOR SEQ ID NO : 56 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 17 base pairs
( B ) TYPE : nucleic acid
( C ) STRANDEDNESS : single
(D ) TOPOLOGY : unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 9
(D) OTHER INFORMATION: /label= N
/note= "N = I"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
GAYTGYTTNG CYTTRCA 17
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 9
(D) OTHER INFORMATION: /label= N
/note= "N = I"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
CTYTGYTTNG CYTTRCA 17
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 6
(D) OTHER INFORMATION: /label= n
/note= "N = I"
(ix) FEATURE:
(A) NAME/KEY: modified base
(B) LOCATION: 9
(D) OTHER INFORMATION: /label= N
/note= "N = I"
(ix) FEATURE:
(A) NAME/KEY: modified base
(B) LOCATION: 12 (D) OTHER INFORMATION: /label= N
/note= "N = I"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
GTRTCNATNC KNATCCA 17 (2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
CCAAGCTTCT AGAATTC 17 (2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
GACTCGAGTC GACATCG 17 (2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 126 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
( ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..126
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
CAG TAT TTT TAC GAG ACG CGC TGC AAG GCC GAA AGC GCT GGG GAA GGT 48 Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Ala Glu Ser Ala Gly Glu Gly
1 5 10 15
GGC CCA GGT GTG GGC GGA GGG GGC TGT CGC GGC GTG GAT CGG AGG CAC 96 Gly Pro Gly Val Gly Gly Gly Gly Cys Arg Gly Val Asp Arg Arg His
20 25 30
TGG CTC TCA GAA TGT AAA GCC AAA CAA TCG 126
Trp Leu Ser Glu Cys Lys Ala Lys Gln Ser
35 40
(2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Ala Glu Ser Ala Gly Glu Gly
1 5 10 15
Gly Pro Gly Val Gly Gly Gly Gly Cys Arg Gly Val Asp Arg Arg His
20 25 30
Trp Leu Ser Glu Cys Lys Ala Lys Gln Ser
35 40
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 126 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..126
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
CAG TAT TTT TAC GAA ACC CGC TGC AAG GCT GAT AAC GCT GAG GAA GGT 48 Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Ala Asp Asn Ala Glu Glu Gly
1 5 10 15
GGC CCG GGG GCA GGT GGA GGG GGC TGC CGG GGA GTG GAC AGG AGG CAC 96 Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg Gly Val Asp Arg Arg His
20 25 30
TGG GTA TCT GAG TGT AAA GCC AAA CAA TCG 126
Trp Val Ser Glu Cys Lys Ala Lys Gln Ser
35 40
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Ala Asp Asn Ala Glu Glu Gly
1 5 10 15
Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg Gly Val Asp Arg Arg His
20 25 30
Trp Val Ser Glu Cys Lys Ala Lys Gln Ser
35 40
(2) INFORMATION FOR SEQ ID NO:65: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Gln Tyr Phe Phe Glu Thr Lys Cys Asn Pro Ser Gly Ser Thr Thr Arg 1 5 10 15
Gly Cys Arg Gly Val Asp Lys Lys Gln Trp Ile Ser Glu Cys Lys Ala
20 25 30
Lys Gln Ser
35
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Gln Tyr Phe Phe Glu Thr Lys Cys Arg Ala Pro Asn Pro Val Glu Ser 1 5 10 15
Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr
20 25 30
Thr His Thr
35
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu 1 5 10 15
Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Gln Cys Arg Thr
20 25 30
Thr Gln Ser
35
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Gln Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Lys Asn 1 5 10 15
Gly Cys Arg Gly Ile Asp Asp Lys His Trp Asn Ser Gln Cys Lys Thr
20 25 30
Ser Gln Thr
35
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..192
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
CAA TAT TTT TTC GAG ACC CGC TGC AAG GCT GAT AAC GCT GAG GAA GGT 48 Gln Tyr Phe Phe Glu Thr Arg Cys Lys Ala Asp Asn Ala Glu Glu Gly
1 5 10 15
GGT CCG GGG GCA GGT GGA GGG GGC TGC CGG GGA GTG GAC AGG AGG CAC 96 Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg Gly Val Asp Arg Arg His
20 25 30
TGG GTA TCT GAG TGC AAG GCC AAG CAG TCC TAT GTG CGG GCA TTG ACC 144 Trp Val Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg Ala Leu Thr
35 40 45
GCT GAT GCC CAG GGC CGT GTG GGC TGG CGA TGG ATC CGC ATC GAT ACG 192 Ala Asp Ala Gln Gly Arg Val Gly Trp Arg Trp Ile Arg Ile Asp Thr
50 55 60
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
Gln Tyr Phe Phe Glu Thr Arg Cys Lys Ala Asp Asn Ala Glu Glu Gly
1 5 10 15
Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg Gly Val Asp Arg Arg His
20 25 30 Trp Val Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg Ala Leu Thr
35 40 45
Ala Asp Ala Gln Gly Arg Val Gly Trp Arg Trp Ile Arg Ile Asp Thr
50 55 60
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 18..35
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
GACTCGAGTC GACATCG GAA ACC CGC TGC AAG GCT 35
Glu Thr Arg Cys Lys Ala
1 5
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
Glu Thr Arg Cys Lys Ala
1 5
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 18..35
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
GACTCGAGTC GACATCG GAT AAC GCT GAG GAA GGT 35
Asp Asn Ala Glu Glu Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
Asp Asn Ala Glu Glu Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1404 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 460..1104
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
CTTGTCACCC AGGTGGCACC CGAGTGGTGC ACTCTCTGCT CACTGCAACC TCGGCCTCCT 60
GGGTTCGAGT GATTCTCCTA CCTCAGCCTA CTGAGTAGCT GGGATTACAG GCGTGCAGCA 120
CTATGCCCGG TTAATTTTGG TATTTTTGGT AGAGATGAGG TTTCACAATG TTGACCAGCT 180
GCTCTGGAAC TCCTGACCTC AAGTCATCCA CCTGCCTCAG CCTCCCAGAG TGCTGGGATT 240
AGAGGTGTGG GGCACAGTGC CTGGCCTGTA GTAGTTGAAT ATTTATTATT AATCTACAAG 300
TTGCGCATTA CGCAAGCCCT AGATATAGGG TCCCCCAAAC TTCTAGAACA AGGGCTTCCC 360
CACAATCCTG GCAGGCAAGC CTCCCCTGGG GTTCCCAACT TCTTTCCCCA CTGAAGTTTT 420
TACCCCCTTC TCTAATCCCA GCCTCCCTCT TTCTGTCTC CAG GTG CTC CGA GAG 474
Gln Val Leu Arg Glu
1 5
ATG CTC CCT CTC CCC TCA TGC TCC CTC CCC ATC CTC CTC CTT TTC CTC 522 Met Leu Pro Leu Pro Ser Cys Ser Leu Pro Ile Leu Leu Leu Phe Leu
10 15 20
CTC CCC AGT GTG CCA ATT GAG TCC CAA CCC CCA CCC TCA ACA TTG CCC 570 Leu Pro Ser Val Pro Ile Glu Ser Gln Pro Pro Pro Ser Thr Leu Pro
25 30 35
CCT TTT CTG GCC CCT GAG TGG GAC CTT CTC TCC CCC CGA GTA GTC CTG 618 Pro Phe Leu Ala Pro Glu Trp Asp Leu Leu Ser Pro Arg Val Val Leu
40 45 50
TCT AGG GGT GCC CCT GCT GGG CCC CCT CTG CTC TTC CTG CTG GAG GCT 666 Ser Arg Gly Ala Pro Ala Gly Pro Pro Leu Leu Phe Leu Leu Glu Ala
55 60 65
GGG GCC TTT CGG GAG TCA GCA GGT GCC CCG GCC AAC CGC AGC CGG CGT 714 Gly Ala Phe Arg Glu Ser Ala Gly Ala Pro Ala Asn Arg Ser Arg Arg
70 75 80 85
GGG GTG AGC GAA ACT GCA CCA GCG AGT CGT CGG GGT GAG CTG GCT GTG 762 Gly Val Ser Glu Thr Ala Pro Ala Ser Arg Arg Gly Glu Leu Ala Val 90 95 100
TGC GAT GCA GTC AGT GGC TGG GTG ACA GAC CGC CGG ACC GCT GTG GAC 810 Cys Asp Ala Val Ser Gly Trp Val Thr Asp Arg Arg Thr Ala Val Asp
105 110 115
TTG CGT GGG CGC GAG GTG GAG GTG TTG GGC GAG GTG CCT GCA GCT GGC 858 Leu Arg Gly Arg Glu Val Glu Val Leu Gly Glu Val Pro Ala Ala Gly
120 125 130
GGC AGT CCC CTC CGC CAG TAC TTC TTT GAA ACC CGC TGC AAG GCT GAT 906 Gly Ser Pro Leu Arg Gln Tyr Phe Phe Glu Thr Arg Cys Lys Ala Asp
135 140 145
AAC GCT GAG GAA GGT GGT CCG GGG GCA GGT GGA GGG GGC TGC CGG GGA 954 Asn Ala Glu Glu Gly Gly Pro Gly Ala Gly Gly Gly Gly Cys Arg Gly
150 155 160 165
GTG GAC AGG AGG CAC TGG GTA TCT GAG TGC AAG GCC AAG CAG TCC TAT 1002 Val Asp Arg Arg His Trp Val Ser Glu Cys Lys Ala Lys Gln Ser Tyr
170 175 180
GTG CGG GCA TTG ACC GCT GAT GCC CAG GGC CGT GTG GGC TGG CGA TGG 1050 Val Arg Ala Leu Thr Ala Asp Ala Gln Gly Arg Val Gly Trp Arg Trp
185 190 195
ATT CGA ATT GAC ACT GCC TGC GTC TGC ACA CTC CTC AGC CGG ACT GGC 1098 Ile Arg Ile Asp Thr Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly
200 205 210
CGG GCC TGAGACCCAT GCCCAGGAAA ATAACAGAGC TGGATGCTGA GAGACCTCAG 1154 Arg Ala
215
GGATGGCCCA GCTGATCTAA GGACCCCAGT TTGGGAACTC ATCAAATAAT CACAAAATCA 1214
CAATTCTCTG ATTTGGAGCT CAATCTCTGC AGGATGGGTG AAACCACATG GGGTTTTGGA 1274
GGTTGAATAG GAGTTCTCCT GGAGCAACTT GAGGGTAATA ATGATGATGA TATAATAATA 1334
ATAGCCACTA TTTACTGAGT GTTTACTGTT TCTTATCCCT AATACATAAC TCCTCAGATC 1394
AACTCTCATG 1404
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
Gln Val Leu Arg Glu Met Leu Pro Leu Pro Ser Cys Ser Leu Pro Ile
1 5 10 15
Leu Leu Leu Phe Leu Leu Pro Ser Val Pro Ile Glu Ser Gln Pro Pro
20 25 30
Pro Ser Thr Leu Pro Pro Phe Leu Ala Pro Glu Trp Asp Leu Leu Ser
35 40 45
Pro Arg Val Val Leu Ser Arg Gly Ala Pro Ala Gly Pro Pro Leu Leu
50 55 60
Phe Leu Leu Glu Ala Gly Ala Phe Arg Glu Ser Ala Gly Ala Pro Ala
65 70 75 80 Asn Arg Ser Arg Arg Gly Val Ser Glu Thr Ala Pro Ala Ser Arg Arg 85 90 95
Gly Glu Leu Ala Val Cys Asp Ala Val Ser Gly Trp Val Thr Asp Arg
100 105 110
Arg Thr Ala Val Asp Leu Arg Gly Arg Glu Val Glu Val Leu Gly Glu
115 120 125
Val Pro Ala Ala Gly Gly Ser Pro Leu Arg Gln Tyr Phe Phe Glu Thr
130 135 140
Arg Cys Lys Ala Asp Asn Ala Glu Glu Gly Gly Pro Gly Ala Gly Gly 145 150 155 160
Gly Gly Cys Arg Gly Val Asp Arg Arg His Trp Val Ser Glu Cys Lys
165 170 175
Ala Lys Gln Ser Tyr Val Arg Ala Leu Thr Ala Asp Ala Gln Gly Arg
180 185 190
Val Gly Trp Arg Trp Ile Arg Ile Asp Thr Ala Cys Val Cys Thr Leu
195 200 205
Leu Ser Arg Thr Gly Arg Ala
210 215
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Val Leu Arg Glu Met Leu Pro Leu Pro Ser Cys Ser Leu Pro Ile Leu 1 5 10 15
Leu Leu Phe Leu Leu Pro Ser Val Pro Ile Glu Ser Gln Pro Pro Pro
20 25 30
Ser Thr Leu Pro Pro Phe Leu Ala Pro Glu Trp Asp Leu Leu Ser Pro
35 40 45
Arg Val Val Leu Ser Arg Gly Ala Pro Ala Gly Pro Pro Leu Leu Phe 50 55 60
Leu Leu Glu Ala Gly Ala Phe Arg Glu Ser Ala Gly Ala Pro Ala Asn 65 70 75 80
Arg Ser Arg Arg Gly Val Ser Glu Thr Ala Pro Ala Ser Arg Arg Gly
85 90 95
Glu Leu Ala Val Cys Asp Ala Val Ser Gly Trp Val Thr Asp Arg Arg
100 105 110
Thr Ala Val Asp Leu Arg Gly Arg Glu Val Glu Val Leu Gly Glu Val
115 120 125
Pro Ala Ala Gly Gly Ser Pro Leu Arg Gln Tyr Phe Phe Glu Thr Arg 130 135 140
Cys Lys Ala Asp Asn Ala Glu Glu Gly Gly Pro Gly Ala Gly Gly Gly 145 150 155 160
Gly Cys Arg Gly Val Asp Arg Arg His Trp Val Ser Glu Cys Lys Ala
165 170 175
Lys Gln Ser Tyr Val Arg Ala Leu Thr Ala Asp Ala Gln Gly Arg Val
180 185 190
Gly Trp Arg Trp Ile Arg Ile Asp Thr Ala Cys Val Cys Thr Leu Leu
195 200 205
Ser Thr Arg Gly Arg Ala
210
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 176 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Ser Ser Thr Tyr Ser Ser Met Ala Gly Lys Asp Trp Asn Leu Tyr Ser 1 5 10 15
Pro Arg Val Thr Leu Ser Ser Glu Glu Pro Ser Gly Pro Pro Leu Leu
20 25 30
Phe Leu Ser Glu Glu Thr Val Val His Pro Glu Pro Ala Asn Lys Thr
35 40 45
Ser Arg Leu Lys Arg Ala Ser Gly Ser Asp Ser Val Ser Leu Ser Arg 50 55 60
Arg Gly Glu Leu Ser Val Cys Asp Ser Val Asn Val Trp Val Thr Asp 65 70 75 80
Lys Arg Thr Ala Val Asp Asp Arg Gly Lys Ile Val Thr Val Met Ser
85 90 95
Glu Ile Gln Thr Leu Thr Gly Pro Leu Lys Gln Tyr Phe Phe Glu Thr
100 105 110
Lys Cys Asn Pro Ser Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp
115 120 125
Lys Lys Gln Trp Ile Ser Glu Cys Lys Ala Lys Gln Ser Tyr Val Arg 130 135 140
Ala Leu Thr Ile Asp Ala Asn Lys Leu Val Gly Trp Arg Trp Ile Arg 145 150 155 160 Ile Asp Thr Ala Cys Val Cys Thr Leu Leu Ser Arg Thr Gly Arg Thr
165 170 175
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 177 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D ) TOPOLOGY : unknown
( ii ) MOLECULE TYPE : peptide
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 79 :
Glu Phe Gln Pro Met Ile Ala Thr Asp Thr Glu Leu Leu Arg Gln Gln 1 5 10 15
Arg Arg Tyr Asn Ser Pro Arg Val Leu Leu Ser Asp Ser Thr Pro Leu
20 25 30
Glu Pro Pro Pro Leu Tyr Leu Met Glu Asp Tyr Val Gly Asn Pro Val
35 40 45
Val Ala Asn Arg Thr Ser Pro Arg Arg Lys Tyr Ala Glu His Lys Ser 50 55 60
His Arg Gly Glu Tyr Ser Val Cys Asp Ser Glu Ser Leu Trp Val Thr 65 70 75 80
Asp Lys Ser Ser Ala Ile Asp Ile Arg Gly His Gln Val Thr Val Leu
85 90 95
Gly Glu Ile Lys Thr Gly Asn Ser Pro Val Lys Gln Tyr Phe Tyr Glu
100 105 110
Thr Arg Cys Lys Glu Ala Arg Pro Val Lys Asn Gly Cys Arg Gly Ile
115 120 125
Asp Asp Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val 130 135 140
Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp Ile 145 150 155 160
Arg Ile Asp Thr Ser Cys Val Cys Ala Leu Ser Arg Lys Ile Gly Arg
165 170 175
Thr
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 175 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
Gln Pro Val Ile Ala Met Asp Thr Glu Leu Leu Arg Gln Gln Arg Arg 1 5 10 15
Tyr Asn Ser Pro Arg Val Leu Leu Ser Asp Thr Thr Pro Leu Glu Pro
20 25 30
Pro Pro Leu Tyr Leu Met Glu Asp Tyr Val Gly Ser Pro Val Val Ala
35 40 45
Asn Arg Thr Ser Arg Arg Lys Arg Tyr Ala Glu His Lys Ser His Arg 50 55 60
Gly Glu Tyr Ser Val Cys Asp Ser Glu Ser Leu Trp Val Thr Asp Lys 65 70 75 80
Ser Ser Ala Ile Asp Ile Arg Gly His Gln Val Thr Val Leu Gly Glu
85 90 95 Ile Lys Thr Gly Asn Ser Pro Val Lys Gln Tyr Phe Tyr Glu Thr Arg
100 105 110
Cys Lys Glu Ala Arg Pro Val Lys Asn Gly Gly Arg Gly Ile Asp Asp
115 120 125
Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg Ala 130 135 140
Leu Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile 145 150 155 160
Asp Thr Ser Cys Val Cys Ala Leu Ser Arg Lys Ile Gly Arg Thr
165 170 175
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
Glu Phe Gln Pro Met Ile Ala Thr Asp Thr Glu Leu Leu Arg Gln Gln 1 5 10 15
Arg Arg Tyr Asn Ser Pro Arg Val Leu Leu Ser Asp Ser Thr Pro Leu
20 25 30
Glu Pro Pro Pro Leu Tyr Leu Met Glu Asp Tyr Val Gly Asn Pro Val
35 40 45
Val Thr Asn Arg Thr Ser Pro Arg Arg Lys Arg Tyr Ala Glu His Lys 50 55 60
Ser His Arg Gly Glu Tyr Ser Val Cys Asp Ser Glu Ser Leu Trp Val 65 70 75 80
Thr Asp Lys Ser Ser Ala Ile Asp Ile Arg Gly His Gln Val Thr Val
85 90 95
Leu Gly Glu Ile Lys Thr Gly Asn Ser Pro Val Lys Gln Tyr Phe Tyr
100 105 110
Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Lys Asn Gly Gly Arg Gly
115 120 125 Ile Asp Asp Lys His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr 130 135 140
Val Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp 145 150 155 160 Ile Arg Ile Asp Thr Ser Cys Val Cys Ala Leu Ser Arg Lys Ile Gly
165 170 175
Arg Thr (2) INFORMATION FOR SEQ ID NO:82: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
Asp Leu Tyr Thr Ser Arg Val Met Leu Ser Ser Gln Val Pro Leu Glu 1 5 10 15
Pro Pro Leu Leu Phe Leu Leu Glu Glu Tyr Lys Asn Tyr Leu Asp Ala
20 25 30
Ala Asn Met Ser Met Arg Val Arg Arg His Ser Asp Pro Ala Arg Arg
35 40 45
Gly Glu Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala 50 55 60
Asp Lys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu 65 70 75 80
Glu Lys Val Pro Val Ser Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu
85 90 95
Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Gly Ile
100 105 110
Asp Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val
115 120 125
Arg Ala Leu Thr Met Asp Ser Lys Lys Arg Ile Gly Trp Arg Phe Ile 130 135 140
Arg Ile Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg Gly Arg 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Asp Leu Tyr Thr Ser Arg Val Met Leu Ser Ser Gln Val Pro Leu Glu 1 5 10 15
Pro Pro Leu Leu Phe Leu Leu Glu Glu Tyr Lys Asn Tyr Leu Asp Ala
20 25 30
Ala Asn Met Ser Met Arg Val Arg Arg His Ser Asp Pro Ala Arg Arg
35 40 45
Gly Glu Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala 50 55 60
Asp Lys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu 65 70 75 80
Glu Lys Val Pro Val Ser Lys Gly Gln Leu Lys Gln Phe Phe Tyr Glu 85 90 95
Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Asp Ile
100 105 110
Asp Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val
115 120 125
Arg Ala Leu Thr Met Asp Ser Lys Lys Arg Ile Gly Trp Arg Phe Ile 130 135 140
Arg Ile Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg Gly Arg 145 150 155 160
(2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
Asp Leu Tyr Thr Ser Arg Val Met Leu Ser Ser Gln Val Pro Leu Glu 1 5 10 15
Pro Pro Leu Leu Phe Leu Leu Glu Glu Tyr Lys Asn Tyr Leu Asp Ala
20 25 30
Ala Asn Met Ser Met Arg Val Arg Arg His Ser Asp Pro Ala Arg Arg
35 40 45
Gly Glu Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala 50 55 60
Asp Lys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu 65 70 75 80
Glu Lys Val Pro Val Ser Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu
85 90 95
Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Asp Ile
100 105 110
Asp Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val
115 120 125
Arg Ala Leu Thr Met Asp Ser Lys Lys Arg Ile Gly Trp Arg Phe Ile 130 135 140
Arg Ile Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg Gly Arg 145 150 155 160
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 160 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: Asp Met Tyr Thr Ser Arg Val Met Leu Ser Ser Gln Val Pro Leu Glu 1 5 10 15
Pro Pro Leu Leu Phe Leu Leu Glu Glu Tyr Lys Asn Tyr Leu Asp Ala
20 25 30
Ala Asn Met Ser Met Arg Val Arg Arg His Ser Asp Pro Ala Arg Arg
35 40 45
Gly Glu Leu Ser Val Cys Asp Ser Ile Ser Glu Trp Val Thr Ala Ala 50 55 60
Asp Lys Lys Thr Ala Val Asp Met Ser Gly Gly Thr Val Thr Val Leu 65 70 75 80
Glu Lys Val Pro Val Ser Lys Gly Gln Leu Lys Gln Tyr Phe Tyr Glu
85 90 95
Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Asp Ile
100 105 110
Asp Lys Arg His Trp Asn Ser Gln Cys Arg Thr Thr Gln Ser Tyr Val
115 120 125
Arg Ala Leu Thr Met Asp ser Lys Lys Arg Ile Gly Trp Arg Phe Ile 130 135 140
Arg Ile Asp Thr Ser Cys Val Cys Thr Leu Thr Ile Lys Arg Gly Arg 145 150 155
60
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
Ala Ala Arg Val Thr Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg 1 5 10 15
Leu Phe Lys Lys Arg Arg Leu His Ser Pro Arg Val Leu Phe Ser Thr
20 25 30
Gln Pro Pro Pro Thr Ser Ser Asp Thr Leu Asp Leu Asp Phe Gln Ala
35 40 45
His Gly Thr Ile Pro Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser 50 55 60
Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp Ser Val 65 70 75 80
Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys
85 90 95
Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys
100 105 110
Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser
115 120 125 Gly Gly Arg Asp Ile Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr 130 135 140
Thr His Thr Phe Val Lys Ala Leu Thr Thr Asp Glu Lys Gln Ala Ala 145 150 155 160
Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Val Leu Ser Arg
165 170 175
Lys Ala Thr Arg
180
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
Ala Ala Arg Val Thr Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Lys 1 5 10 15
Leu Phe Lys Lys Arg Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr
20 25 30
Gln Pro Pro Pro Thr Ser Ser Asp Thr Leu Asp Leu Asp Phe Gln Ala
35 40 45
His Gly Thr Ile Ser Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser 50 55 60
Thr His Pro Val Phe His Met Gly Glu Phe Ser Val Cys Asp Ser Val 65 70 75 80
Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys
85 90 95
Glu Val Thr Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys
100 105 110
Gln Tyr Phe Phe Glu Thr Lys Cys Arg Ala Pro Asn Pro Val Glu Ser
115 120 125
Gly Gly Arg Asp Ile Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr 130 135 140
Thr His Thr Phe Val Lys Ala Leu Thr Thr Asp Asp Lys Gln Ala Ala 145 150 155 160
Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Val Leu Ser Arg
165 170 175
Lys Ala Ala Arg
180
(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 179 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
Ala Ala Arg Val Ala Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg 1 5 10 15
Phe Lys Lys Arg Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln
20 25 30
Pro Pro Arg Glu Ala Asp Thr Thr Gln Asp Leu Asp Phe Glu Val Gly
35 40 45
Gly Ala Ala Pro Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser Ser 50 55 60
His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser 65 70 75 80
Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu
85 90 95
Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln
100 105 110
Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly
115 120 125
Gly Arg Asp Ile Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr 130 135 140
His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp 145 150 155 160
Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Val Leu Ser Arg Lys
165 170 175
Ala Val Arg
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
Phe Phe Lys Lys Lys Arg Phe Arg Ser Ser Arg Val Leu Phe Ser Thr 1 5 10 15 Gln Pro Pro Pro Glu Ser Arg Lys Gly Gln Ser Thr Gly Phe Leu Ser
20 25 30
Ser Ala Val Ser Leu Asn Arg Thr Ala Arg Thr Lys Arg Thr Ala His
35 40 45
Pro Val Leu His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Met 50 55 60
Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val 65 70 75 80 Thr Val Leu Gly Glu Val Asn Ile Asn Asn Asn Val Phe Lys Gln Tyr
85 90 95
Phe Phe Glu Thr Lys Cys Arg Asp Pro Arg Pro Val Ser Ser Gly Gly
100 105 110
Arg Asp Ile Asp Ala Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His
115 120 125
Thr Phe Val Lys Ala Leu Thr Met Glu Gly Lys Gln Ala Ala Trp Arg 130 135 140
Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Val Leu Ser Arg Lys Ser 145 150 155 160
Gly Arg Pro
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 124 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
His Arg Ser Lys Arg Ser Ser Glu Ser His Pro Val Phe His Arg Gly 1 5 10 15
Glu Phe Ser Val Cys Asp Ser Ile Ser Val Trp Val Gly Asp Lys Thr
20 25 30
Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu Gly Glu Val
35 40 45
Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys 50 55 60
Arg Asp Pro Asn Pro Val Asp Ser Gly Gly Arg Asp Ile Asp Ala Lys 65 70 75 80
His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val Lys Ala Leu
85 90 95
Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr
100 105 110
Ser Cys Val Cys Val Leu Ser Arg Lys Thr Gly Gln
115 120
(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
Val Asp Pro Lys Leu Phe Gln Lys Arg Gln Phe Gln Ser Pro Arg Val 1 5 10 15
Leu Phe Ser Thr Gln Pro Pro Leu Leu Ser Arg Asp Glu Glu Ser Val
20 25 30
Glu Phe Leu Asp Asn Glu Asp Ser Leu Asn Arg Asn Ile Arg Ala Lys
35 40 45
Arg Glu Asp His Pro Val His Asn Leu Gly Glu His Ser Val Cys Asp 50 55 60
Ser Val Ser Ala Trp Val Gly Lys Thr Thr Ala Thr Asp Ile Lys Gly 65 70 75 80
Asn Thr Val Thr Val Met Glu Asn Val Asn Leu Asp Asn Lys Val Tyr
85 90 95
Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asn Pro Asn Pro Glu Pro
100 105 110
Ser Gly Gly Arg Asp Ile Asp Ser Ser His Trp Asn Ser Tyr Cys Thr
115 120 125
Glu Thr Asp Gly Phe Ile Lys Ala Leu Thr Met Glu Gly Asn Gln Ala 130 135 140
Ser Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys Val Ile Thr 145 150 155 160
Lys Lys Lys Gly
(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
Pro Ala Gly Ser Ser Pro Asp Pro Ser Ser Pro Val Val Asp Pro Lys 1 5 10 15
Leu Phe Ser Lys Arg His Tyr Pro Ser Pro Arg Val Val Phe Ser Glu
20 25 30
Val Ile Pro Ser His Asp Val Leu Asp Gly Glu Gly Tyr Asp Phe Glu
35 40 45
Arg Val Arg Gly Leu Arg Val Arg Arg Lys Ala Val Ser His Thr Met 50 55 60
His Arg Gly Glu Tyr Ser Val Cys Asp Ser Ile Asn Thr Trp Val Asn 65 70 75 80
Thr Lys Arg Ala Thr Asp Met Ser Gly Asn Glu Val Thr Val Leu Ser
85 90 95
His Val Thr Val Asn Asn Lys Val Lys Lys Gln Leu Phe Tyr Glu Thr
100 105 110
Thr Cys Arg Ser Pro Thr His Arg Ser Ser Gly Ile Val Ile Gly Gly
115 120 125 Arg Ser Gly Gly Arg Gly Gly Ser Gln Gly Ser Lys Thr Gly Asn Ser 130 135 140
Gly Gly Arg Asp Ile Asp Ser Arg Tyr Trp Asn Ser His Cys Thr Asn 145 150 155 160
Thr Asp Ile Tyr Val Ser Ala Leu Thr Val Phe Lys Glu Gln Thr Ala
165 170 175
Trp Arg Phe Ile Arg Ile Asn Ala Ser Cys Val Cys Val Ser Arg Thr
180 185 190
Asn Ser Trp Ser
195
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..225
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
CGA GTG GTC CTG TCT AGG GGT GCC GCT GCC GGG CCC CCT CTG GTC TTC 48 Arg Val Val Leu Ser Arg Gly Ala Ala Ala Gly Pro Pro Leu Val Phe
1 5 10 15
CTG CTG GAG ACT GGA GCC TTT CGG GAG TCA GCA GGC GCC CGG GCC AAC 96 Leu Leu Glu Thr Gly Ala Phe Arg Glu Ser Ala Gly Ala Arg Ala Asn
20 25 30
CGC AGC CAG CGA GGG GTG AGC GAT ACT TCA CCG GCG AGT CAT CAG GGT 144 Arg Ser Gln Arg Gly Val Ser Asp Thr Ser Pro Ala Ser His Gln Gly
35 40 45
GAG CTG GCC CTG TGC GAT GCA GTC AGT GTC TGG GTG ACA GAC CCC TGG 192 Glu Leu Ala Leu Cys Asp Ala Val Ser Val Trp Val Thr Asp Pro Trp
50 55 60
ACT GCT GTG GAC TTG GGT GTG CTC GAG GTC GAG 225
Thr Ala Val Asp Leu Gly Val Leu Glu Val Glu
65 70 75
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
Arg Val Val Leu Ser Arg Gly Ala Ala Ala Gly Pro Pro Leu Val Phe
1 5 10 15
Leu Leu Glu Thr Gly Ala Phe Arg Glu Ser Ala Gly Ala Arg Ala Asn
20 25 30 Arg Ser Gln Arg Gly Val Ser Asp Thr Ser Pro Ala Ser His Gln Gly 35 40 45
Glu Leu Ala Leu Cys Asp Ala Val Ser Val Trp Val Thr Asp Pro Trp 50 55 60
Thr Ala Val Asp Leu Gly Val Leu Glu Val Glu
65 70 75
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
CCCACAAGCT TGTTGGCATC TATGGTCAGA GCCCTCACAT AAGACTGTTT TGC
53
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
Lys Cys Asn Pro Ser Gly Ser Thr Thr Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
Arg Gly Cys Arg Gly Val Asp
1 5
(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
Lys Gln Trp Ile Ser
1 5
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
Lys Gln Ser Tyr Val Arg
1 5
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
Gly Pro Gly Xaa Gly Gly Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
Gly Pro Gly Val Gly Gly Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
Gly Pro Gly Ala Gly Gly Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 103: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
Glu Ser Ala Gly Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
Asp Asn Ala Glu Glu
1 5
(2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
CAGTATTTTT ACGAAACC 18
(2) INFORMATION FOR SEQ ID NO: 106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
ACATTTCGGT TTGTTCTG 18
(2) INFORMATION FOR SEQ ID NO: 107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: CAGTATTTTT ACGAGACG 18
(2) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:
ACATTTCGGT TTGTTAGC 18 (2) INFORMATION FOR SEQ ID NO: 109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:
TGCAGTTTCG CTCACCCCCC GTTTTAGCCG GGAAGT 36 (2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
Lys Gln Tyr Phe Tyr Glu Thr
1 5
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:
Trp Arg Phe Ile Arg Ile Asp
1 5
(2) INFORMATION FOR SEQ ID NO:112: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
Gly Glu Leu Ser Val Cys Asp
1 5
(2) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
Lys Ala Glu Ser Ala Gly
1 5
(2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:
GGAGGGGGCT GCCGGGGAGT GGACAGGAGG CACTGGGTAT CTGAG
45
(2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
Gly Gly Gly Cys Arg Gly Val Asp Arg Arg His Trp Val Ser Glu 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 582 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..396
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116:
GTA GTT TGT CCA ATT ATG TCA CAC CAC AGA AGT AAG GTT CCT TCA CAA
48
Val Val Cys Pro Ile Met Ser His His Arg Ser Lys Val Pro Ser Gln
1 5 10 15
AGA TCC TCT AGA GTC GCG CCC GCG ACC TGC AGG CGC AGA ACT GGT AGG
96
Arg Ser Ser Arg Val Ala Pro Ala Thr Cys Arg Arg Arg Thr Gly Arg
20 25 30
TAT GGA AGA TCC CTC GAG GTG GAG GTG TTG GGC GAG GTG CCT CCA GCT
144
Tyr Gly Arg Ser Leu Glu Val Glu Val Leu Gly Glu Val Pro Pro Ala
35 40 45
GTC GGC AGT TCC CTC CGC CAG CAC TTC TTT GTT GCC CGC TTC GAG GCC
192
Val Gly Ser Ser Leu Arg Gln His Phe Phe Val Ala Arg Phe Glu Ala
50 55 60
GAT AAA TCT GAG GAA GGT GGC CCG GGG GTA GGT GGA GGG GCT GCC GCC
240
Asp Lys Ser Glu Glu Gly Gly Pro Gly Val Gly Gly Gly Ala Ala Ala
65 70 75 80
GGG GTG TGG ACC GGG GGG CAC TGG GTG TCT GAG TGC AAG GCC AAG CAG
288
Gly Val Trp Thr Gly Gly His Trp Val Ser Glu Cys Lys Ala Lys Gln
85 90 95
TCC TAT GTG CGG GCA TTG ACC GCT GAT GCC CAG GGC CGT GTG GAC TGG
336
Ser Tyr Val Arg Ala Leu Thr Ala Asp Ala Gln Gly Arg Val Asp Trp
100 105 110
CGA TGG ATT CAA ACT GGC ACA GCC TGT GTC TGC ACA CTC CTC AGC CGG
384
Arg Trp Ile Gln Thr Gly Thr Ala Cys Val Cys Thr Leu Leu Ser Arg
115 120 125
ACT GGC TGG GCC TGAGACTTAT ACCCAGGAAC TGGTCAGGCA GAAAAAGAAC 436
Thr Gly Trp Ala
130
AGAGCTGGAT GCTGAGAGAC CTCAGGGTTG GCCCAGCTGC TCTACGGACG GACCCCAGTT 496
GGGGAACTCA TGAAATCATC ACAAAATCAC AACTCTCTGA ATTTGAGCTC AATCTCTGCA 556
GGATGGGTGC CACCACATGT GGTTTT 582
(2) INFORMATION FOR SEQ ID NO: 117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:
Val Val Cys Pro Ile Met Ser His His Arg Ser Lys Val Pro Ser Gln
1 5 10 15
Arg Ser Ser Arg Val Ala Pro Ala Thr Cys Arg Arg Arg Thr Gly Arg
20 25 30
Tyr Gly Arg Ser Leu Glu Val Glu Val Leu Gly Glu Val Pro Pro Ala
35 40 45
Val Gly Ser Ser Leu Arg Gln His Phe Phe Val Ala Arg Phe Glu Ala
50 55 60
Asp Lys Ser Glu Glu Gly Gly Pro Gly Val Gly Gly Gly Ala Ala Ala
65 70 75 80
Gly Val Trp Thr Gly Gly His Trp Val Ser Glu Cys Lys Ala Lys Gln
85 90 95
Ser Tyr Val Arg Ala Leu Thr Ala Asp Ala Gln Gly Arg Val Asp Trp
100 105 110
Arg Trp Ile Gln Thr Gly Thr Ala Cys Val Cys Thr Leu Leu Ser Arg
115 120 125
Thr Gly Trp Ala
130
(2) INFORMATION FOR SEQ ID NO: 118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:
CGGTACCCTC GAGCCACCAT GCTCCCTCTC CCCTCA 36
(2) INFORMATION FOR SEQ ID NO: 119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:
CGGTACAAGC GGCCGCTTCT TGGGCATGGG TCTCAG 36
(2) INFORMATION FOR SEQ ID NO: 120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
CGGTACCCTC GAGCCACCCA GGTGCTCCGA GAGATG 36 (2) INFORMATION FOR SEQ ID NO: 121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:
GAGACCGGAA GCTTCTAGAG ATC 23 (2) INFORMATION FOR SEQ ID NO: 122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:
TGCAGTTTCG CTCACCCCCC GTTTCCGCCG TGATGT 36 (2) INFORMATION FOR SEQ ID NO: 123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123:
ACATCACGGC GGAAACGGGG GGTGAGCGAA ACTGCA 36 (2) INFORMATION FOR SEQ ID NO: 124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:
ACTTCCCGGC TAAAACGGGG GGTGAGCGAA ACTGCA 36 -
Figure imgf000141_0001
Figure imgf000142_0001

Claims

WE CLAIM:
1. A method of treating an infertility disorder involving oocytes comprising administering to a patient a therapeutically effective amount of NT-4 or an NT-4 related peptide in a pharmaceutically
acceptable carrier.
2. The method of claim 1 wherein the protein or peptide is derived from human NT-4.
3. A method of treating an NT-4 related motor neuron disorder comprising administering, to a patient in need of such treatment, a therapeutically effective amount of an NT-4 protein, NT-4 peptide or derivative, capable of supporting the survival, growth and/or differentiation of motor neurons.
4. The method of claim 3 in which the disorder is selected from the group consisting of amyotrophic lateral sclerosis, progressive spinal muscular
atrophy, infantile muscular atrophy, poliomyelitis, post-polio syndrome, Chariot-Marie Tooth disease, nerve trauma or nerve injury.
5. The method of claim 3 wherein the NT-4 protein is encoded by a recombinant nucleic acid sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
6. A method of diagnosing an NT-4-related motor neuron disorder comprising injecting a detectably labeled NT-4 protein, NT-4 peptide or derivative, in a manner such that the labeled NT-4 protein, NT-4 peptide or derivative enters a motor nerve, and determining whether the labeled NT-4 protein, NT-4 peptide or derivative is retrogradely transported, in which a failure to be retrogradely transported correlates with dysfunction of motor neurons.
7. The method of claim 6 in which the disorder is selected from the group consisting of amyotrophic lateral sclerosis, progressive spinal muscular atrophy, infantile muscular atrophy, poliomyelitis, post-polio syndrome, Charcot-Marie tooth disease, nerve trauma or nerve injury.
8. The method of claim 6 wherein the NT-4 protein is encoded by a recombinant nucleic acid sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
9. The method of claim 6 wherein the NT-4 protein is encoded by a recombinant nucleic acid molecule which is at least about 70% homologous to the corresponding DNA sequence as contained in
bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
10. A method of promoting motor neuron survival, growth, and/or differentiation comprising exposing motor neurons to a therapeutically effective
concentration of an NT-4 protein, NT-4 peptide or derivative that is capable of promoting the survival, growth, and/or differentiation of motor neurons.
11. The method of claim 10 that is carried out in vivo.
12. The method of claim 10 wherein the NT-4 protein is encoded by a recombinant nucleic acid molecule comprising the DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
13. The method of claim 10 wherein the NT-4 protein, NT-4 peptide or derivative is encoded by a recombinant nucleic acid molecule which is at least about 70% homologous to the corresponding DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
14. A method of diagnosing a motor neuron disorder in a patient comprising the steps of:
(a) determining a concentration of NT-4 in a patient sample by means of an
immunometric assay utilizing an antiNT-4 antibody;
(b) calculating a difference in
concentration of NT-4 in the patient sample from an average concentration of NT-4 in analogous samples from normal individuals; and
(d) diagnosing a motor neuron disorder when the difference between the
concentration of NT-4 in the patient sample and the concentration of NT-4 in an analogous sample from at least one normal individual positively correlates with the motor neuron disorder.
15. The method of claim 14 wherein the anti-NT-4 antibody is capable of binding to an NT-4 protein encoded by a recombinant nucleic acid molecule comprising the NT-4 related DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
16. The method of claim 14 wherein the anti-NT-4 antibody is capable of binding to an NT-4 protein encoded by a recombinant nucleic acid molecule at least 70% homologous to the corresponding DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
17. A method of treating a patient with a prostate localized disease characterized by increased transcription in prostate tissue of an NT-4 gene, relative to transcription levels in prostate tissue of normal patients, comprising administering to the patient an effective amount of an oligonucleotide, said oligonucleotide
(a) consisting of at least six nucleotides;
(b) comprising a sequence complementary to at least a portion of an RNA transcript of the NT-4 gene; and
(c) is hybridizable to the RNA transcript.
18. The method of claim 17 wherein the prostate localized disease is benign prostatic hypertrophy.
19. The method of claim 18 wherein the
oligonucleotide comprises a sequence which is 100% complementary to the corresponding portion of the RNA transcript encoded by the DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
20. The method of claim 19 wherein the
oligonucleotide comprises a sequence which is at least 70% complementary to the corresponding portion of the RNA transcript encoded by the DNA sequence as
contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
21. A method of treating an impotence disorder involving the human prostate comprising administering to a patient a therapeutically effective amount of an NT-4 protein in a pharmaceutically acceptable carrier.
22. The method of claim 21 wherein the NT-4 protein is encoded by a recombinant nucleic acid molecule comprising the NT-4 related DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
23. The method of claim 21 wherein the NT-4 protein is encoded by a recombinant nucleic acid molecule which is at least about 70% homologous to the corresponding DNA sequence as contained in
bacteriophage HG7-2 as deposited with the ATCC and designated accession number 75070.
24. A diagnostic kit for detecting retrograde transport in a motor neuron comprising in a suitable container a detectably labeled NT-4 protein,
derivative or peptide fragment.
25. The diagnostic kit of claim 24 wherein the detectably labeled NT-4 protein, derivative or peptide fragment is encoded by a recombinant nucleic acid molecule comprising the NT-4 related DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
26. The diagnostic kit of claim 24 wherein the detectably labeled NT-4 protein, derivative or peptide fragment is encoded by a nucleic acid fragment at least about 70% homologous to the corresponding DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
27. A chimeric prepro protein comprising
(a) an amino acid sequence of a prepro
region of a neurotrophin other than NT- 4;
(b) an amino acid sequence of a prepro
region of a mature form of an NT-4 related protein.
28. The chimeric prepro protein of claim 27 wherein the amino acid sequence of the mature form of an NT-4 related protein is encoded by a recombinant nucleic acid molecule comprising at least a portion of the DNA sequence encoding NT-4 as contained in
bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
29. The chimeric prepro protein of claim 27 in which the neurotrophin other than NT-4 is nerve growth factor, brain-derived neurotrophic factor, or
neurotrophin-3.
30. The chimeric prepro protein of claim 27 wherein the prepro region of the other than NT-4 neurotrophin is derived from Xenopus NT-4.
31. The chimeric prepro protein of claim 27 wherein the prepro region of the other than NT-4 neurotrophin is derived from human NT-3.
32. A nucleic acid molecule comprising a nucleotide sequence which encodes the chimeric prepro protein of claim 27.
33. A DNA vector that is pCMX-hNT3/hNT4 as deposited with the ATCC, having accession number 75133.
34. A DNA vector that is pCMX-xNT4/hNT4.
35. The substantially pure recombinant NT-4 protein encoded by the DNA vector of claim 33.
36. The substantially pure recombinant NT-4 protein encoded by the DNA vector of claim 34.
37. A method of treating an NT-4 related
immunological disorder affecting neuromuscular
transmission comprising administering to a patient a therapeutically effective amount of an NT-4 protein in a pharmaceutically acceptable carrier.
38. The method of claim 37 in which the disorder is myasthenia gravis.
39. The method of claim 37 wherein the NT-4 protein is encoded by a recombinant nucleic acid molecule comprising the NT-4 related DNA sequence as contained in bacteriophage HG7-2 as deposited with the ATCC and assigned accession number 75070.
40. The method of claim 37 wherein the NT-4 protein is encoded by a recombinant nucleic acid molecule which is at least about 70% homologous to the corresponding DNA sequence as contained in
bacteriophage HG7-2 as deposited with the ATCC and designated accession number 75070.
41. A method of producing NT-4 comprising growing a recombinant cell containing the nucleic acid molecule of claim 31, under conditions such that the chimeric prepro protein is expressed and processed by the cell to produce the mature form of NT-4.
42. A method of detecting or measuring NT-4 activity comprising (a) exposing a cell that expresses trkB to a test agent; and (b) detecting or measuring the specific binding of the test agent to trkB, in which specific binding to trkB positively correlates with NT-4 activity in the test agent.
PCT/US1992/004266 1991-05-21 1992-05-20 Therapeutic and diagnostic methods based on neurotrophin-4 expression WO1992020365A1 (en)

Priority Applications (2)

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JP5500288A JPH08510987A (en) 1991-05-21 1992-05-20 Therapeutic and diagnostic methods based on neurotrophin-4 expression
AU23021/92A AU674659B2 (en) 1991-05-21 1992-05-20 Therapeutic and diagnostic methods based on neurotrophin-4 expression

Applications Claiming Priority (12)

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US762,674 1985-08-05
US70345091A 1991-05-21 1991-05-21
US703,450 1991-05-21
US72925391A 1991-07-12 1991-07-12
US729,253 1991-07-12
US73442291A 1991-07-23 1991-07-23
US734,422 1991-07-23
US751,356 1991-08-08
US75135691A 1991-08-28 1991-08-28
US76267491A 1991-09-20 1991-09-20
US79192491A 1991-11-14 1991-11-14
US791,924 1991-11-14

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AU (1) AU674659B2 (en)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025684A1 (en) * 1992-06-12 1993-12-23 Regeneron Pharmaceuticals, Inc. Therapeutic and diagnostic methods based on neurotrophin-4 expression
WO2000072871A1 (en) * 1999-05-27 2000-12-07 Licentia Ltd. Neurotrophic factors in the treatment of peripheral nerve dysfunction of pelvic area

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU654302B2 (en) * 1990-09-25 1994-11-03 Genentech Inc. Novel neurothrophic factor

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5169764A (en) * 1990-08-08 1992-12-08 Regeneron Pharmaceuticals, Inc. Multitrophic and multifunctional chimeric neurotrophic factors, and nucleic acids and plasmids encoding the chimeras
AU654302B2 (en) * 1990-09-25 1994-11-03 Genentech Inc. Novel neurothrophic factor
IL101661A (en) * 1991-04-23 1998-02-08 Regeneron Pharma Method for detecting, identifying or measuring agents having neurotrophin activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NATURE, Vol. 331, issued 21 January 1988, HOFER M.M. et al., "Brain-Derived Neurotrophic Factor Prevents Neuronal Death In Vivo", pages 261-262. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 87, issued October 1990, JONES K.R. et al., "Molecular Cloning of a Human Gene that is a Member of the Nerve Growth Factor Family", pages 8060-8064. *
See also references of EP0587806A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025684A1 (en) * 1992-06-12 1993-12-23 Regeneron Pharmaceuticals, Inc. Therapeutic and diagnostic methods based on neurotrophin-4 expression
WO2000072871A1 (en) * 1999-05-27 2000-12-07 Licentia Ltd. Neurotrophic factors in the treatment of peripheral nerve dysfunction of pelvic area

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PT100511A (en) 1993-09-30
EP0587806A1 (en) 1994-03-23
CA2109598A1 (en) 1992-11-26
AU2302192A (en) 1992-12-30
AU674659B2 (en) 1997-01-09
EP0587806A4 (en) 1995-04-26
NZ242813A (en) 1996-09-25
IE921636A1 (en) 1992-12-02

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