EP0644937A1 - Therapeutic and diagnostic methods based on neurotrophin-4 expression - Google Patents

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 provides for nucleic acid molecules encoding NT-4. Such molecules may comprise a sequence substantially as set forth for NT-4 in the figure [SEQ ID NO:1 (NT-4, viper)]. The present invention provides for therapeutic and diagnostic methods based on human NT-4 expression, specifically the potential to treat motor neuron diseases and prostate localized diseases, immunological related neuromuscular disorders, and peripheral and central nervous system disorders including Alzheimer's disease, Parkinson's disease and Huntington's chorea and epilepsy.

Description

THERAPEUTIC AND DIAGNOSTIC METHODS BASED ON NEUROTROPHIN^

EXPRESSION

The present application is a continuation-in-part of copending United States application Serial No. 07/898,194, filed on June 12, 1992 which is a continuation in part of copending United States application Serial No. 791 ,924 filed on November 14, 1991.

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 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 303:821-825). 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 ; 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). 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 , Neural. 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, Cel 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 Sertoii 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)], 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.

Further therapeutic and diagnostic applications are based on the demonstration of binding of NT-4 to brain and retina, as well as retrograde transport of NT-4 and on the ability of NT-4 to support the survival of various neuronal cell populations. 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 t o 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:1 (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), SK3 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 x 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 125|-NGF from its receptor on PC12 cells. Binding assays were performed at 37°C using 1.5 x 109 M i25|-NGF and 1 x 104 cells per ml. Medium from mock-transfected cells failed to displace binding of 12S|-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 g 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' axon 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 g) 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' axon 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 Iaevis ovary. Ovary from adult Xenopus Iaevis was sectioned in a cryostat (14 m 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 m RNA specific oligonucleotide with the sequence δ'CXϊCAC GCTTGTTGGCATCTATGGTCAGAGCCCTCACATAAGACTGTTTTG

C3'. (SEQ ID NO:95)

(B) Hybridization using a control oligonucleotide of similar length and G-t-C content. After hybridization, sections were washed in 1x 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 emulsionautoradiographs 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 m; in B and C, 15 m.

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 Iaevis. Ovaries from two adult Xenopus Iaevis 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 g/slot of RNA was electrophoresed in a formaldehyde-containing 1% agarose gel. This was blotted onto a nitrocellulose filter and hybridized to a 600bp Hindi 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 hNT4-5'" [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 h (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 asterisk. 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). URE 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 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 65°C with the radiolabeled probe (a 680bp Xho1-Not1 fragment containing the complete coding region of HG7-2 NT-4 (see Example Section 9, infra) in the presence of 0.5 M NaPO (pH 7), 1% bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM

EDTA (Mahmoudi and Lin, 1989, Biotechniques 7:331-333), and 100 g/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 h 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 supematants from transfected cell lines; Q1 (pCMX-

HG7-2Q), N7 (pCMX-hNT3 hNT4) and X1 (pCMX-xNT4/hNT4) were tested in volumes of 10 I, 50 I and 250 I 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 I to 250 I 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 acetyltransf erase (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.

FIGURE 26. COS supernatants containing human, rat and xenopus NT-4 were tested for their ability to induce the tyrosine phosphorylation of trkA, trkB and trkC.

FIGURE 27. (A) Tyrosine phosphorylations of trkB induced by varying concentrations of human and xenopus NT-4.

(B) Growth response of NIH3T3 fibroblasts expresssing trkB to various concentrations of COS supernatants containing human or xenopus NT-4. A mock transfected COS cell line is presented as a control.

FIGURE 28. Tyrosine phosphorylation of trk receptors induced by varying concentrations of purified preparations of NGF, BDNF, NT-3 and NT-

4. (A)Phosphorylation of trkA; (B) Phosphorylation of trkB; (C) Phosphorylation of trkC.

The effect of varying concentrations of purified preparations of NGF, BDNF, NT-3 and NT-4 on cell growth of NIH3T3 cells expressing (D) trkA; (E)trkB and (F)trkC.

FIGURE29. The effect of varying concentrations of purified preparations of NGF, BDNF, NT-3 and NT-4 on parental PC12 cells and PC12 cells expressing trkB. (A)Differentiation as demonstrated by neurite extension. (B) Number of surviving cells; (C) Tyrosine phosphorylation assays. FIGURE 30. (A) Crosslinking of iodinated NGF to PC12 cells and rat postnatal day 7 striatal homogenates and competition with cold neurotrophins.

(B) Crosslinking of iodinated BDNF to3T3 trkB cells and rat postnatal day 7 cortex and competition with cold neurotrophins. (C) Crosslinking of iodinated

NT-4 to 3T3 trkB cells and rat postnatal day 7 cortex and hippocampus and competition with cold neurotrophins.

FIGURE 31. Cell survival of embryonic E14 rat DRG explants in response to increasing concentrations of human NT-4. FIGURE 32. Expression of trkB and trkC mRNA in ganglionic neurons surviving in the presence of NT-4.

FIGURE 33. (A) Induction of fos mRNA in hippocampi from D18 rat embryos by purified neurotrophins.

(B) Phosphorylation of trk in hippocampi from D18 rat embryos by purified neurotrophins.

FIGURE 34. (A) The effect of NT-4 on the number of calbindin- immunopositive cells in hippocampal cultures.

(B) The effect of NT-4 and BDNF on the number of acetylcholinesterase-positive cells in hippocampal cultures. FIGURE 35. The dose related effect of purified human NT-4 on choline acetyltransferase activity in cultures of basal forebrain neurons.

FIGURE 36. The effect of increasing concentrations of purified human NT-4 on tyrosine hydroxylase positive dopaminergic neurons of the rat embryonic substantia nigra. (A) NT-4 added on day 1; (B) NT-4 added on days 1 , 4 and 7.

FIGURE 37. Effect of NT-4 treatment on calbindin-immunoreactive neurons in D17 striatal cultures at 8 days in vitro.

FIGURE 38. Effect of NT-4 on high-affinity GABA uptake in E17 striatal cultures at 8 days in vitro. FIGURE 39. Effect of increasing concentrations of purified human NT- 4 on choline acetyltransferase activity in E14 rat motor neurons.

FIGURE 40. Effect of CNTF or NT-3 alone or in combination with NT- 4 on choline acetyltransferase activity in E14 rat motor neurons. FIGURE41. Immunocytochemical staining of GABAergic neurons.

Cells were prepared as described and plated at 50,000 cells/cm2 in 35 mm culture dishes. After 3, 6 and 10 days (41 A, 41 B and 41 C), cultures were processed for immunocytochemical staining using a polyclonal antiserum directed against recombinant feline GAD (courtesty Dr. A. Tobin (UCLA, CA). 41 D: control indicating lack of staining in 7 day cultures in the absence of antibody. Scale bar, 100μM.

FIGURE 42. Effect of neurotrophins on GAD activity in mesencephalic cultures. Cells were prepared as described above and plated at 50,000 cells/cm2 in 35 mm dishes. Upon media change after the initial attachment period, cultures were exposed to increasing concentrations of either BDNF,

NT-3, or NT-4 (n=5 per group). All cultures were maintained for 7 days in vitro, and were than processed for the measurement of GAD as described. GAD activity is expressed as a percentage of the control value determined in untreated cultures. The baseline GAD activity in untreated control samples was calculated as 6.23 +/-0.39 pmol/120 min/μg protein. ^{* *}, p<0.01 ; ^*p<0.05 compared to control using students t test.

FIGURE 43. Effect of neurotrophins on high affinity GABA uptake. Cells were prepared as described above and plated at 50,000 cells/cms in 35 mm dishes. After the initial attachment period, the culture media was changed to a serum-free formulation, and BDNF, NT-3 or NT-4 were added in increasing concentrations. After 7 days, cultures were processed for the measurement of high-affinity GABA uptake as described. 43A: dose response curves for BDNF, NT-3 and NT-4. The GABA uptake activities are expressed as a percentage of the GABA uptake determined in control cultures. 43B: GABA uptake activity determined in cultures which were maintained in the presence of either 25 ng/ml BDNF, 10ng/ml NT-3, 2.5 ng/ml NT-4, 50 ng/ml NGF, or combinations of BDNF and NT-3 or NT-4 at these same concentrations. The baseline GABA uptake activity in untreated control cultures was calculated as 41 ,358+/- 2161 cpm/15 min/dish. FIGURE 44. BDNF, NT-3 and NT-4 increase the GABA content of mesencephalic cultures. Cultures were prepared as described, and plated at 50,000 cells/cm2 into 35 mm dishes. Upon change to serum-free media, BDNF (50 ng/ml), NT-3 (10 ng/ml), NT-4 (2.5 ng/ml) or NGF (50 ng/ml) were added (n=6 per group). Cultures were mainained for 7 days before being processed for GABA determination as described. The GABA content is expressed as pg/μg protein. Data are the mean +/- sem ^*, p<0.05 compared to control in students t test.

RGURE45 Localization of trkB and trkC mRNA in adult rat substantia nigra. Dark field photomicrographs of coronal sections of adult rat brain showing the autoradiographic localization of hybridization signal to mRNA encoding trkB (45B, 45C) and trkC (45D, 45F). 45A: schematic illustration of a cross section of the area of midbrain which is representative of the brain sections in panels B-F. At low magnification (45B), trkB mRNA is detected in the ventral tegmental area (VTA) and medial s. nigra (SN). 45C: higher magnification of the righth hemisphere, ventral aspect of the tissue section shown in B. 45D: corresponding tissue section to that in C, hybridized to trkC cRNA. 45E, 45F: high magnification pair of matching bright field/dark field photomicrographs of the tissue section in D, showing the autoradiographic localization of trkC hybridization to large perikarya, presumed to be neurons. Scale bar=3125 μm for B, 2185 μm for C and D, and 546 μm for E and F.

F 3URE46. Northern blot analysis of TrkB and TrkC mRNA in cultures derived from E14 ventral mesencephalon. Cells were plated at 50,000 eel Is/cm 2 into 60 mm dishes. After maintainence in serum-free conditions for 72 hours, BDNF (50 ng/ml) or NT-3 (25 ng/ml) was added and cultures maintained for 5, 16, 24 or 29 hours. Northern blots prepared from the cultures were probed for TrkB or TrkC mRNA. 46A: autoradiogram of a blot following hybridization with the TrkB probe; C=control, B=BDNF, TB=total adult rat brain RNA; 46B: the corresponding (to 46A) ethidium bromide stained gel; 46C: autoradiogram of a blot folowing hybridization with the TrkC probe; C=control, N=NT-3, TB=total brain RNA; 46D corresponding (to 46C) ethidium bromide stained gel.

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:1) for viper, Figure 1 (SEQ ID N02), 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, dopaminergic neurons, cholinergic neurons, sensory neurons, striatal cells, cells of the cortex, striatum, hippocampus, cerebellum, olfactory bulbs, periaqueductal gray, raphe nude, locus coeruleus, dorsal root ganglion, neural placode derivatives, sympathetic neurons and upper and lower motor neurons. 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, t o 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 x IO-10M. Establishing additional effective doses is within the purview of one skilled in the art. In various embodiments of the invention, NT-4 protein, peptide fragments or derivatives can be administered to patients in whom the nervous system has been damaged by trauma, surgery, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, or toxic agents. The invention in particular can be used to treat conditions in which damage has occurred to neurons in the basal forebrain, hippocampus or striatum. in addition, it can be used to treat conditions in which damage or degeneration has occurred to spinal sensory neurons, cranial sensory neurons involved in hearing, taste, vision, balance, etc., motor neurons or retinal cells, by administering effective therapeutic amounts of NT-4 protein or peptide fragments or derivatives. Such uses include, but are not limited to, treatment of retinal detachment, age related or other maculopathies, photic retinopathy, surgery-induced retinopathy, retinopathy of prematurity, viral retinopathy, uvetis, ischemic retinopathy due to venous or arterial occlusion or other vascular disorders, retinopathy due to trauma or penetrating lesions of the eye, peripheral vitreoretinopathy or inherited retinal degeneration.

In various specific embodiments of the invention, NT-4 can be locally administered to sensory neurons which have been severed, including, but not limited to, neurons in dorsal root ganglia or in the retina. It may be desirable to administer the NT-4-related peptides or NT-4 protein by adsorption onto a membrane, e.g. a silastic membrane, that could be implanted in the proximity of the severed nerve. The present invention can also be used for example in hastening the recovery of patients suffering from peripheral neuropathies. In further embodiments of the invention, NT-4 protein or peptide fragments or derivatives derived therefrom, can be used to treat congenital conditions or neurodegenerative disorders, including, but not limited to, Alzheimer's disease, Parkinson's disease, Parkinson-Plus syndromes (in which Parkinsonian symptoms result from degeneration of dopaminergic neurons), such as Progressive Supranuclear Palsy (Steele-Richardson-Olszewski

Syndrome), Olivoponto- cerebellar Atrophy (OPCA), Shy-Drager Syndrome (multiple systems atrophy), and Guamanian Parkinsonism dementia complex, and Huntington's chorea; in particular, the invention can be used to treat congenital or neurodegenerative disorders associated with sensory nerve dysfunction and degenerative diseases of the retina. For example, the NT-4 protein, or peptide fragments, or derivatives of the invention can be used in the treatment of hereditary spastic paraplegia with retinal degeneration (Kjellin and Barnard-Scholz syndromes), retinitis pigmentosa, Stargardt disease, Usher syndrome (retinitis pigmentosa with congenital hearing loss), and Refsum syndrome (retinitis pigmentosa, hereditary hearing loss, and polyneuropathy), to name but a few. It is possible that a defect in NT-4 synthesis or responsiveness may be the underlying etiology for syndromes characterized by a combination of retinal degeneration and other sensory dysfunction. In a specific embodiment of the invention, administration of NT-4 protein, or peptide fragments or derivatives derived therefrom, can be used in conjunction with surgical implantation of tissue in the treatment of Alzheimer's disease and/or Parkinson's disease. As discussed in Section 18 , infra NT-4 may be used to promote the survival of dopaminergic neurons of the substantia nigra in a dose-dependent manner, supporting the use of

NT-4 in the treatment of disorders of CNS dopaminergic neurons, induding, but not limited to, Parkinson's disease. In addition, NT-4 has been observed to sustain the survival of CNS cholinergic neurons (Section 17) and, in particular, basal forebrain cholinergic neurons, indicating that NT-4 may be useful in the treatment of disorders involving cholinergic neurons, induding, but not limited to Alzheimer's disease. It has been shown that approximately 35% of patients with Parkinson's disease suffer from Alzheimer-type dementia; NT-4 produced according to the invention may prove to be a useful single agent therapy for this disease complex. Similarly, NT-4 produced according to the invention may be used therapeutically to treat Alzheimer's disease in conjunction with Down's

Syndrome. NT-4 produced according to the invention can be used in the treatment of a variety of dementias as well as congenital learning disorders.

In another specific embodiment of the invention, the administration of NT-4 protein or peptide fragments or derivatives derived therefrom can be used for the treatment of diseases or disorders which involve striatal cells, which include, but are not limited to Huntington's chorea, striatonigral degeneration and cerebral palsy. This is based on the disclosure herein (Section 19) indicating the ability of NT-4 to support striatal cultures, as indicated by an increase in calbindin immunoreactivity and a high affinity uptake of GABA. A dramatic decrease in calbindin and calbindin mRNA has been detected in the striata of Huntington's chorea patients [Kiyama et al, Brain Res. 525:209-214 (1990); lacopino et al Proc. Natl. Acad. Sd. 87:4078-4082 (1990)]. In another embodiment of the invention, the administration of NT-4 protein or peptide fragments or derivatives derived therefrom can be used for the treatment of other diseases or disorders which are related to damage or degeneration of striatal or hippocampal cells. Such diseases or disorders may be caused by, for example, stroke, ischemia, hypoglycemia or hypoxia.

In yet another embodiment of the invention, NT-4 may be administered in the treatment of epilepsy-related or other seizures. Reduced levels of the inhibitory transmitter GABA are known to be associated with seizures. For example, high doses of penicillin, which reduce GABA levels, can be used to induce experimental focal epilepsy. Adminisration of the GABA agonist muscimol into the area of the substantia nigra has been shown to markedly suppress motor and iimbic seizures (as measured electrographically) induced by electrical stimulation. McNamara, J. et al., 1984, J. Neurosci. 4:2410-2417. Accordingly, the present invention contemplates use of the neurotrophins, including NT-4, to enhance levels of

GABA and thereby prevent the motor manifestations of seizures.

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, compiement- 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 of NT-4 has been demonstrated in both the central and peripheral nervous system (see Section 14, infra.)

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 motor neuron, central and peripheral nervous system disorders comprising injecting a detectably labeled NT-4 protein or peptide into a 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 nervous system disorder that is NT-4 related. 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 o r 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 occurring 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 t o 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 h 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 t o 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-HCI (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 - C] acetyl-CoA, 300 mM NaCl, 8 mM choline bromide, 20 mM EDTA, and 0J mM neostigmine in 50 mM NaH₂P0₄ (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 h 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 g/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 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. 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 g/ml), transferrin (100 g/ml), progesterone (20 nM), putrescine (100 M), 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 g/ml) and laminin (e.g. 10 g/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 Iaevis) 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:11 1 ) (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 Hindlll 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 g 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 Hindlll 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 Mbol-digested genomic DNA in the Bam HI site of phase μEMBL-3 were screened using conventional procedures with a 182 bp PCR fragment of Xenopus NT-4 labeled with [ -32P]dCTP by nick translation to a specific activity of approximately 5 x 108 cpm/ g. Hybridization was carried out in 4 x SSC (1 x SSC is 150 mM NaCl, 15 mM sodium citrate (pH 7.0)), 40% formamide, 1 x Denhardts solution, 10% dextran sulfate at 42°C.

The filters were washed at 55°C in 0.1 x SSC, 0.1% SDS and exposed t o Kodak XAR-5 films at -70°C. Eight phage clones were isolated, and a hybridizing 1.5 kb Pstl fragment from one of these clones was sub oned in the plasmid pBS-KS (Stratagene). The nucleotide sequence of the subdoned 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 3.0f) 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 Pstl 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 subdoned 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 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 transfection in the presence of 200 Ci/ml [35S]cysteine (Amersham, UK). Aliquots (10-20 I 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 80°C. 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 t o 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 1251 y the chloramine-T method to an average activity of 7 x 107 cpm/ g. Steady-state binding was measured in competition assays performed at 37°C or 0°C using 1 x 104 cells per ml, 1.5 x 10-9 M I2SI-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 125I-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. RNAPREPARATIONSANDBLOTANALYSIS

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 mi cushion of 5.7 M CsCI 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 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 [ - 32p]dCTP by nick translation to a specific activity of around 5 x 105 cpm/ g, and the hybridization was carried out as described above. Filters were washed at high stringency (0.1 x SSC, 0.1% SDS, 54°C) 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 t o 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).

Nucleotide identities & amino acid similarities were calculated with a VAX computer (software package from the UWGCG; Devereux et al., 1984, Nucl. Acids Res. 12: 389-395) according to the comparison matrix of

Schwartz and Dayhoff (1979, Washington, D.C. Nat'l Biomed. Res. Found, pp. 353-358), taking conservative amino acid changes into consideration. The figures below the diagonals show percent nucleotide identity. The figures above the diagonals show the percent amino acid similarity. X indicated that the sequences were not isolated from those species (NGF) from ray and NT-3 from viper). Hum, human; Chi, chicken; Vip, viper; Sal, salmon; Xen, Xenopus.

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. 1 B [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 amino 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 t o NGF as to BDNF. NT-4 is clearly more related to BDNF than to the other two members.

6.2.2. STRUCTURALFEATURESOFTHENT-4PROTEIN

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 subdoned 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 341 , 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 deavage 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-giycosylation 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 disulfide 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 [35S] 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 i25i-|_abeled NGF to its receptor on PC12 cells. Binding assays were done at 37°C and under conditions in which 80% of the 125I- GF 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 (6x10-10M) were required to displace 50% of the 1251-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 1251-NGF, suggesting that in this case the remaining 1 5I-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 61) 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 seledive 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-phenylaianine 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 today's 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 primitive 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 disulfide 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, Neuron 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 37°C 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, Sάence 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 t o 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 BDNF, 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 h 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. 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^ mRNA IN THE XENOPUS LAEVIS OVARY BY IN SITU HYBRIDIZATION

7J. MATERIALS AND METHODS

7.1.1. ISOLATION, HANDLING AND CULTURE OF XENOPUS OOCYTES, EMBRYOS AND CELLS

Male and female X. Iaevis frogs were maintained in the laboratory at 19°C. After immersion-anesthesia 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 M hypoxanthine (Sigma), 4 mM glutamine and 10% fetal bovine serum (Gibco) at 20°C. Cultures were equilibrated with air and kept in the dark.

7J.2. IN SITU HYBRIDIZATION

Fresh-frozen ovaries from adult Xenopus Iaevis 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. 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 (5CCCACAAGCTTGTTGGCATCTATGGTCAGAGCCCT

CACATAAGACTGTTTTGC3' [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 35S-dATP using terminal deoxyribonucleotidyl transferase (IBI, New Haven) to a specific activity of approximately 1x109 cpm/ . Hybridization was performed at 42°C for 16 hours in 50% formamide, 4x SSC, Ix Denhardts solution, 1% Sarcosyl, 0.02M NaP0 (pH 7.0), 10% dextransulphate, 0.5 mg/ml yeast tRNA, 0.06M DDT.0J mg/ml sheared salmon sperm DNA and 1x107 cpm/ml of 35S-labeled oligonucleotide probe. Sections were subsequently rinsed, washed 4 times (15 min. each) at 55°C in 1 x 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, 0J M 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 CsCI 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 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 Hindi fragment from the 3' exon of the Xenopus NT-4 gene (Hallbook et al., 1991 , Neuron 6: 845-858). The fragment was labeled with -(32p)-dCTP by nick translation to a specific activity of around

5x108 cpm/ g and the hybridization was carried out as described (Ernfors et al., 1988, Neuron 1 : 983-96). Filters were washed at high stringency (OJxSSC, 0.1% SDS, 54°C) and exposed to Kodak AR-5 films at -70°C.

7.2 RESULTS

Tissue sections through the adult Xenopus Iaevis 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 m 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 Xenopus 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. 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 ll/lll 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.

7.2J NORTHERN BLOT ANALYSIS OF NT-4 mRNA EXPRESSION DURING XENOPUS OOGENESIS AND EARLY DEVELOPME TT

A fixed amount of total cellular RNA (40 g) 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 g) of poiyadenylated 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 poiyadenylated RNA. A low level of NT-4 mRNA was found in Xenopus somatic A6 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-U (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, ΛBO 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 t o 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 6J J, 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 72°C. 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.

8J .3. ISOLATION OF FULL LENGTH GENOMIC AND cDNA CLONES ENCODING rNT- 4 AND NT-4

A human ovary cDNA library in μGT-10 was obtained from Clontech.

A human hippocampus cDNA library in μ:ZAPII was obtained from Stratagene. A human genomic DNA library in EMBL3/SP6/T7 was obtained from Clontech. A rat brain cDNA library in μ-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 5x108 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 x 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 centrifugation, resuspended in TM buffer (10mM Tris-HCI, pH 7.5; 10mM

MgCI2), layered upon a CsCI 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-HCI, 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 subdoned 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 μ-ZAP plasmid preparations are performed as follows: 200μ of OD600=1.0 XL1 -Blue cells, 200μ of the hi-titer phase stock, and 1μ of R408 helper phage (1x10 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 0.5μ of the stock to 200 of XL1 -Blue cells (OD600=1). Incubate 37°C for 15 minutes. Plate 1 -1 OOμ (preferably 10μ) on LB ampicillin plates. Incubate 37°C x 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.

82. 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 x 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 60°C with the radiolabeled probe (106 cpm/ ml) under the following hybridization conditions: 0.5 M NaP0₄, 1% BSA, 7% SDS, 1 mM EDTA, and 100 g/ml salmon sperm DNA. The filters were then washed at 60°C with 2xSSC 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 mI SM buffer, shaken for 2 hr, and replated as follows: 1) 100 I of 10-3 dilution (1 I in 1 ml), mixed with 100 I cells, and plated; an almost confluent plate was obtained; 2) 200 I 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 I of SM buffer, and shaken for 2 hr, following which 100 I of eluant was mixed with 100 I 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 I of this first stock mixed with 100 I 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 I 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 (NaP0 hybridization as above, 65°C), and a 45mer oligonucleotide probe

(GGAGGGGGCTGCCGGGGAGTGGACAGGAGGCA CTGGGTATCTGAG) [SEQ ID NO:114] corresponding to amino acid GGGCRGVDRR HWVSE [SEQ ID NO:115] coded for by human PCR fragment clone 17B (6xSSC, 45°C 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 subdoned into Biuescript vector. The size of DNA fragments to be subdoned are as follows: clone 2-1 (1.0 kb Xhol fragment), clone 4-2 (4.0 kb Xhol 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 Xho1-Not1 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 eiectrophoresis through a 1 % agarose-formaidehyde 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 65°C to the 680 bp Xho1-

Not1 radiolabeled NT-4 probe in the presence of 0.5M NaP0₄ (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, induding 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.LLCONSTRUCTION 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

Bam HI site of Biuescript, was amplified by PCR utilizing the following oligonucleotides: h π^*4^'XhoM:CX3GTACCCTCGAG<XACCATGCTCCCTCTCCCCTCA [SEQ ID NO:118] hNT4-3"NotCGGTACAAGCGGCCGCTTCTTGGGCATGGGTCTCAG

[SEQ ID NO:119] hNT 5'XhoQ:α3GTACCCTCGAGCCAC∞AGGTGCTCCGAGAGATG [SEQ ID NO:120] Oligonucleotide primer combinations of hNT4-5'XhoM and hNT4-3'Not were used to construct pCMX-HG7-2M, while oiigos hNT4-5'XhoQ and hNT4-3'Not were used to construct pCMX-HG7-2Q. The PCR fragment was digested with Xho1/Not1 and subdoned into Xho1/Not1 digested pCMX.

11.^.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 and pCMX-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'Not1 oligonucleotide.

The PCR fragment obtained was excised from the gel, and reamplified by PCR with the 5'CDM8 and hNT4-3'Not1 oligonucleotides. The product was then digested with Hindlll and Pstl and subdoned into Hindlll/Pst1 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 5ODM8 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'Not1 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 Hindlll and Pstl and subdoned into Hindi Il/Pst1 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 x 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 g/ml DEAE-Dextran (Pharmacia), 400 M chloroquine (Sigma), 4 mM glutamine (Irvine), 1 x ITS (insulin, transferrin, selenium, Sigma). To each well 2 g 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% C0₂ 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. Pregnant rats 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 t o 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 37°C 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 g/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 m). 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 4°C. 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 x 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 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/cm2 in 6 mm wells precoated with poly-L-ornithine (Sigma: 10 g/ml) and laminin (GIBCO: 10 g/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% CO≥ 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.

11.1.5. PREPARATION OF ENRICHED HUMAN MOTOR NEURON CULTURES

Seven to 9 week old human embryonic material was obtained from aspiration abortions at the Geneva Cantonal Hospital. Appropriate consent forms for experimental use of embryonic tissues were obtained from the Ethics Commission for the Department of Gynecology and Obstetrics at the Hospital. The age of the embryo was estimated according to menstrual history, foot size (Streeter, 1920 Contri. Embryol. 11, 143) and external characteristics (Moore, 1982, in The Developing Human", K.L. Moore, ed., pp 70-92, W.B. Saunders, Philadelphia). The material was kept at 4° C for 2 to 6 hours until dissection. The spinal cords were carefully isolated, all the spinal roots were removed, and the meninges and other adhering tissue were discarded. The cords were minced and incubated in 0.12% trypsin in Ca2+, Mg2+- free salt solution for 10 minutes at 37°C. The cells were dispersed into a suspension by repeated trituration through a pipette. Cells were centrifuged and resuspended in a standard culture medium (MEM plus 13% decomplemented human serum). The Petri dishes were covered with a solution of polyornithine at a concentration of 1 mg/ml for 1 hr at 37° C and rinsed three times with phosphate-buffered saline solution (PBS) before plating. Cells were plated at a density of 4 X 104 and 10-15 X 104 cells per 6 and 11 mm tissue culture well, respedively. The cultures were maintained at 37°C in 5% CO₂/O₂ air. The medium was changed every three days and cytosine arabinoside (ara C) (aO-⁶M) was added during the last 4 days of culture. Human neurotrophic fadors CNTF, NT-3 and NT-4 were added from the start of the culture period at a concentration of 10ng/ml and the culture medium was changed every 3 days. Choline acetyltransferase (ChAT) was determined by measuring the synthesis of 3H-acdtylcoenzyme

A. The ChAT measurements were done according to the method of

Fonnum, F. J., 1975, Neurochem. 24:407-409 with the modifications of

Raynaud, et al., 1987, Dev. Biol. 119:305-312 and Martinou et al., 1989, J.

Neurosci. 9:3645-3656 (1989).

11.2 RESULTS

11.2.1. EUKARYOTIC EXPRESSION OF BIOLOGICALLY ACTIVE RECOMBINANT 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 CCS supernatants tested were 10, 50 and 250 I in a total volume of 2 ml. Q1

(pCMX-HG7-2Q), N7 (pCMX-hNT3/hNT4 fusion), and X1 (pCMX-xNT4 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 I 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 CCS 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 J.4. and on human spinal cord neurons as described supra in Example Section 11.1.5. 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).

The biological effect of the neurotrophins on cultured human spinal cord neurons, as measured by ChAT activity, was as follows. Since there was a difference in the ChAT values from one experiment to another, the values in the control wells were normalized to 100% in order to compare results from different experiments. The results have been pooled from 20 different cultures and are expressed as the mean +/- S.E.M. (n=number of wells):

Condition (π) % ChAT Activity

Control (77) 100

NT-3 (24) 231 +/- 23

BDNF (12) 252 +/- 27 NT-4 (25) 318 +/- 38 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 adive 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 Sedion 5, supra, for a discussion of the use of chimeric construdions to express NT-4 in vitro).

Expression of a biologically adive form of human NT-4 in an in vitro eukaryotic expression system substantially increases the ease at which the produdion 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 distind 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 construdion, is biologically adive as demonstrated by the stimulating effed of recombinant NT-4 COS supernatants on neurite outgrowth in DFG explants and the bioactivity on rat and human cultured motor neurons.

12. EXAMPLE: TRKB IS A RECEPTOR FOR NEUROTROPHINS

12J MATERIALS AND METHODS

12.1.1 3T3 FIBROBLAST SURVIVAL ASSAYS

COS cell supernatants were examined in a survival assay utilizing 3T3 fibroblasts. In this assay system, 3T3 fibroblasts, which do not express neurotrophin receptor proteins, are transfeded with mammalian expression vedors encoding either trkA, trkB or trkC. 3T3 fibroblast survival is dependent on the addition and receptor specific binding of the respective neurotrophic fador.

COS-M5 cells were cultured and transfeded with either pCMX-HG7-2Q, pCMX-HG7-2M or pCMX-HG7-2Q as described in Example Sedion 11.1.3.

A full-length rat trkA cDNA clone was obtained from Dr. Eric Shooter of Stanford University. The rat trkA cDNA was subdoned into the mammalian expression vedor, pCMX, to generate pCMX-trkA.

Full-length rat trkB and trkC cDNA clones were obtained by screening a rat brain cDNA library in the lambda ZAP2 vedor (Stratagene) with rat trkB-specific and trkC specific oligonucleotides corresponding to the most 5' and 3' coding regions of trkB and trkC. The rat trkB and trkC cDNAs were subdoned into pCMX to generate pCMX-trkB and pCMX-trkC.

3T3 fibroblasts were cultured and transfeded 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 transfeded with trkA, a proto- oncogene encoding a tyrosine kinase receptor for NGF, with trkB, a tyrosine kinase which serves as a functional binding protein for BDNF and NT-3, or with trkC, a tyrosine kinase which serves as a fundional binding protein for 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 adivity of neurotrophins.

12J.2 TRANSFECTION OF PC12 CELLS

A full length rat trkB cDNA was placed under control of the CMV promoter in the pcDNA I expression vedor (Invitrogen), which also contains an LTR-promoted neo resistance gene. PC12 cells (a gift of Dr. Eric Shooter, Stanford University) (107 cells per 10cm dish) were incubated for 24 hr in 5 ml OptiMEM medium (GIBCO BRL) containing 25 mg DNA and 100 mg Lipofedin (GIBCO BRL), then rinsed and placed in fresh medium. Five days after transfedion cells were seleded in 0.4 mg/ml G418. Resistant colonies were assayed for differentiation in the presence of 100ng/ml BDNF and one clone (PC12 trkB) was seleded for further study.

12.L3 CROSS LINKING STUDIES

Cross-linking studies were performed as follows: Briefly, cell lines and cell suspensions from cortex, hippocampus and striatum were incubated in PBS-glucose with 1nM of ¹ 5| labelled NT-4, in the presence or absence of excess cold neurotrophins for 2 hr at 4°C. The cross-linking agent (6m M EDAC for 12S|-BDNF and i25|-NT-4, 0.2mM DSS for 125|-NGF) was added, and rotated at room temperature for 20 minutes. The mixture was washed 3 times with a solution containing tris/NaCI. The cell pellet was resuspended in complete RIPA lysis buffer. After centrifugation, the supernatant containing cross-linked complexes was immunoprecipitated with trk-antibody (RG22), and subjeded to electrophoresis. The fixed and dried gels were exposed for autoradiography.

12.2 RESULTS

12.2J 3T3 trkB CELLS REMAIN VIABLE IN THE PRESENCE OF NT-4

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; data not shown indicated inability of NT-4 containing COS supernatants to support 3T3-trkC cultures). Thus, these results demonstrate that NT-4 protein has biological adivity in this system, and suggests that trkB, but not trkA or trkC, serves as a fundional 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

5

10

3T3 20 50

5

10

3T3-trkA 20 50

5 + +

10 + +

3T3-trkB 20 + + + 50 + + + 12.2.2 HUMAN AND RAT NT-4 ARE SIMILAR TO XENOPUS NT-4 IN THEIR ABILITY TO SPECIFICALLY ACTIVATE trkB.

To compare human and rat NT-4 to xNT-4 for their abilities t o activate the various trk receptors, we first expressed all three of these proteins transiently in COS cells; metabolic labeling was used to demonstrate that all three proteins were produced in approximately equal amounts by these cells (data not shown). COS cell supernatents containing the three proteins were then assayed for their ability to induce tyrosine phosphorylation of the three known trks expressed in NIH3T3 fibroblasts. Human and rat NT-4 were identical to xNT-4 in that they were specifically able to induce the tyrosine phosphorylation of trkB, but not trkA or trkC (Figure26). Furthermore, human NT-4 and xNT-4 induced trkB tyrosine phosphorylation with very similar dose-dependencies (Figure 27A). Human

NT-4 and xNT-4 also displayed similar dose-dependencies in their ability to elicit growth of NIH3T3 cells expressing trkB receptors (Figure 27B). In addition, both stimulated neurite outgrowth in PC 12 cells expressing introduced trkB receptors; neither human NT-4 nor xNT-4 could elicit phenotypic effects from untransfected versions of these cells, or cells expressing the other trk receptors (not shown). Thus mammalian NT-4 and xNT-4 are very similar in their ability to functionally activate trkB but not trkA or trkC.

122.3 TYROSINE PHOSPHORYLATION OF trkB BY NT-4

The assay described above suggested that not only are mammalian

NT-4/5 and xNT-4 similar to BDNF in being specific ligands for trkB, but they might also be as potent as BDNF in their ability to adivate trkB. To diredly determine the specific adivities of all the known mammalian neurotrophins for each of the known trk receptors, we first obtained highly purified preparations of each of the mammalian neurotrophins. Each of these purified factors was then tested for its ability to induce tyrosine phosphorylation of each of the trk receptors expressed in NIH3T3 fibroblasts. NGF was clearly the preferred ligand for trkA, with very minor indudions by very high concentrations of NT-3 and NT-4, while NT-3 was clearly the preferred ligand for trkC, with minor induction by very high concentrations of BDNF (Figure 28A and 28C). BDNF, NT-3 and NT-4 were all quite effective at inducing tyrosine phosphorylation of trkB, although the NT-3 induction appeared to require higher concentrations to achieve saturation (Figure28B).

To compare specific activities for inducing phosphorylation with those required for function effects, each of the purified neurotrophins was then assayed for its ability to promote cell growth of NIH3T3 cells expressing each of the trk receptors. Strikingly, the dose-responses for cell growth in fibroblasts almost exadly paralleled those for phosphorylation (compare panels A, B and C with D, E and F in Figure 28); by this assay, BDNF and NT- 4 were essentially indistinguishable in their ability to activate trkB, whereas NT-3 was about 50-fold less potent. In all cases, the "preferred" ligands for each trk receptor (i.e. NGF for trkA, BDNF or NT-4 for trkB, and NT-3 for trkC) exhibited 50% of their maximum adivity (EC₅₀'s) of between 1-

10ng/ml.

12.2.4 ACTIVATION OF PC12 trkB CELLS BY NT-4

In addition to the 3T3/trkB system, we have also compared the abilities of human NT-4 to activate trkB in PC12/trkB cells. Of the 4 neurotrophins tested, parental PC12 cells are only responsive to NGF (Figure 29). In PC12 cells stably transfected with trkB, NT-4 increased the survival (Figure 29B) and percentage of neurite-bearing PC12 trkB cells (Figure 29A) in a dose-dependent manner, similar to that seen with BDNF. PC12 trkB cells responded to NT-3 only at high concentrations. Results of tyrosine phosphorylation assays performed with 4 neurotrophins in PC12 or PC12trkB cells showed that NGF induced trkA phosphorylation in PC12 cells, while BDNF and NT-4 and NT-3 (to a lesser extent) induced trkB phosphorylation in PC12/trkB cells (Figure 29C).

12.2.5 NT-4 COMPETES FOR trkB BINDING WITH BDNF and NT-3

Cross-linking experiments were carried out with radiodinated neurotrophins. Iodinated NGF was found to cross-link to a protein

(presumed trkA) immunoprecipitable with trk antibody in PC12 cells and striatal homogenates (rat postnatal day 7) (Figure 30A), and 3T3/trkA cells (data not shown). In all cases, this cross-linked protein could be competed by excess cold NGF, and not by BDNF, NT-3 or NT-4. This data indicates that NT-4 did not interad appreciably with the trkA receptor in cell lines or in vivo. Similarly, iodinated BDNF could be cross-linked to a trk- immunoprecipitable protein (presumed trkB) in 3T3/trkB cells, PC12/trkB cells (data not shown), and cortex (rat postnatal day 7); this cross-linked protein could be competed by BDNF, and NT-4, to a lesser extent by NT-3, but not by NGF (Figure 30B). Iodinated NT-4 cross-linking experiments gave similar results. That is, in 3T3 trkB cells, PC12/trkB cells (data not shown), cortex and hippocampus (postnatal day 7), NT-4 cross-linked protein could be competed by BDNF and NT-4, to a lesser extent by NT-3, and not by NGF (Figure 30C).

13. EXAMPLE: NT-4 EFFECTS NEURITE OUTGROWTH AND CELL SURVIVAL OF DORSAL ROOT GANGLION EXPLANTS

Neurotrophin responses in cultured primary neurons were examined. The survival of sensory neurons derived from embryonic E14 rat dorsal root ganglia were supported by all of the known neurotrophins, albeit to differing extents, which suggest that different neuronal subpopulations in the ganglia are responding to the different neurotrophins (Figure 31 ). RNA was prepared from immediately explanted dorsal root ganglia, as well as ganglia that had been maintained for 24 hours in the presence of individual neurotrophins; after this 24 hour treatment, only neurons responsive to

(and thus presumably expressing the appropriate trk receptor for) the given neurotrophin survive, while non-responding neurons die. Untreated ganglia immediately removed from the animals expressed all of the trks (Figure 32). In contrast, ganglionic neurons surviving in the presence of BDNF or NT-4 showed significant trkB message, but no detectable trkC message (Figure 32). Ganglionic neurons surviving in the presence of NT-3 expressed trkC but not trkB. These data demonstrated that NT-4 ad on trkB and not on trkC. Similar studies were not possible with trkA because, in contrast to the mutually exclusive relationship between trkB and trkC expressing neurons, it appears that substantial numbers of trkB and trkC expressing neurons co-express trkA (data not shown).

14. EXAMPLE: DISTRIBUTION AND RETROGRADE TRANSPORT OF NT-4

14J MATERIALS AND METHODS

NT-4 was iodinated by a modification of the ladoperoxidase method. Briefly, 1 mCi of Nai 5i (NEN) was added with 1.2 μg of lactoperoxidase (Sigma), 85 μM H202 and 10 μg of NT-4 at pH 6.0 for 12 min. The readion was stopped by addition of 0J M Nal, 0J M Na phosphate and

1.0 M NaCl, pH 7.5. The readion was diluted 1:1 with 2% bovine serum albumin (Boehringer Mannheim) in PBS. The solution was dialyzed t o eliminate free (unincorporated) ¹²sι. Percent incorporation (55%) was determined by thin layer chromatography. Specific activity (2047 cpm/fmole) was calculated based on a molecular weight of 26,000 for NT-4. For sciatic nerve studies adult male Sprague-Dawley rats (Zivic Miller; 200-220 g; n=30) were anesthetized with a mixture of pentobarbital (35.2 mg/kg) and chloral hydrate (170 mg/kg), and the right sciatic nerve was exposed. Two μl of 1251-iabeled NT-4, containing PBS or a 50-fold excess of unlabeled neurotrophins, were injected into the nerve at the level of the tendon of the obturator internus muscle with a Hamilton syringe. Wounds were sutured and the animals allowed to recover for 18 hr. Rats were killed, the DRGs were disseded, placed in 4% paraformaldehyde and counted in a gamma counter for 1 min. Differences in mean transport values were analyzed by analysis of variance (ANOVA).

For sympathetic neuron transport studies, i25iJabeled NT-4 was injected into the anterior eye chamber as described (Johnson et al., 1978). After 16 hr SCGs were removed and counted in 4% paraformaldehyde as for DRGs.

Fixed DRGs and spinal cords were equilibrated with buffered sucrose, frozen in methyl butane and sedioned in a cryostat (10 μm for DRG and SCG, and 20 μm for spinal cord) and then mounted onto microscope slides. The brains from perfused animals were removed, equilibrated in buffered sucrose, and 25 μm frozen sedions were cut in the coronal plane and mounted. Slides were then processed for emulsion autoradiography (using Kodak NTB-2 emulsion) following established procedures (Cowan et al., 1972). Exposure times ranged 1-3 weeks; however, comparable exposure times were used for any individual region. After being developed, tissues were counterstained through the emulsion (NTB-2, Kodak) with thionin.

For studies involving transport in the brain, male Sprague-Dawley rats were anesthetized with chloral hydrate-pentobarbital and fixed in a stereotaxic apparatus. Small volumes of [¹²si]-iabeled trophic factors (0.2- 0.5 μl) were injeded slowly into the hippocampus or neostriatum by way of a borosilicate glass micropipette. In other experiments, larger volumes (10 μl) were injeded into the right lateral cerebral ventricle. The wounds were closed and the animals allowed to recover. Approximately 24 hours later, the animals were sacrificed and the brains fixed by transcardial perfusion of buffered paraformaldehyde. The brains were then removed, sedioned and processed for film and emulsion autoradiography. Hippocampal injections were centered in the Dentate Gyrus/CA4 - Hilar region. Striatal injections were located centrally in the rostral caudate-putamen. Intracerebroventricular (ICV) injedions were verified as being made into the ventricular space. Similar amounts of [125I]-NGF, NT-3 and BDNF have been injected at these sites previous experiments, permitting a clear determination as to the specificity of the patterns of distribution and retrograde transport within the CNS.

14.2 RESULTS

14.2.1 TRANSPORT OF NT-4 IN THE BRAIN

Film and emulsion autoradiographic experiments (Table 3) showed that, like NGF labeling associated with retrogradely transported NT-4 was well localized to magnocellular neurons of the medial septum and diagonal band, cells which are known to provide the cholinergic input to hippocampus.

In general, more magnocellular neurons appear to be labeled in NGF-injeded animals compared to animals injeded with NT-4.

Examination of film and emulsion autoradiograms available to date have not provided evidence of transport of NT-4 to any other CNS cell group following intrastriatal or intrahippocampal injedions, in marked contrast to results obtained with BDNF which is widely transported within the CNS (Table 3). Table 3: Retrograde Neuronal Labeling Following Hippocampal Injedions of Radiodinated NGF, BDNF and NT-4

Data reported above is derived from emulsion autoradiographic experiments. Pluses represent relative numbers of cells labeled in a given area, from "++++" indicating many labeled cells to "+" indicating a few. Minus signs indicates that no labeled cells were observed. A question mark indicates areas that were difficult to evaluate given their proximity to the injedion site.

14.2.2 DISTRIBUTION OF NT-4 FOLLOWING ICV ADMINISTRATION

In previous experiments, we have found that NGF diffuses widely into the brain parenchyma following ICV administration, such that the radiolabeled ligand becomes available to and concentrated within NGF- responsive neuronal populations (eg. the cholinergic neurons of the basal forebrain). Diffusion of NT-3, and particularly BDNF into the brain substance is much more limited in the rat, such that at most a few neurons within the neural parenchyma concentrate sufficient amounts of labeled BDNF to be clearly discriminable. Following ICV administration of BDNF (and to a lesser extent NT-3), labeling of the apical surface of the ventricular ependyma is particularly prominent.

Following ICV administration of [i 5i]. NT-4, the pattern of distribution seen is distinctly different from that described above for the other neurotrophins. As for NGF, label associated with NT-4 is distributed for some distance into the brain substance bordering the ventricles, especially at the level of the injedion site. There is likewise some diffusion into neural tissues from the extracerebral subarachnoid CSF space. In contrast to NGF, no concentration of NT-4 associated label was apparent in neurons following ICV administration.

14.2.3 RETROGRADE TRANSPORT OF NT-4 BY L4 AND L5 DRG NEURONS

125I-NT-4 was retrogradely transported by L4 and L5 DRG neurons. This accumulation was specific as assessed by the fad that few counts accumulated in the contralateral (non-injected) L4 or L5 DRGs and that transport was blocked by the co-injedion of a 16-fold excess of NT-4. NT- 4 transport was blocked to varying degrees by all members of the neurotrophin family. BDNF and NGF (57-fold) were approximately equal in blocking transport, while NT-3 was as effedive as NT-4, when injeded at a

57-fold excess. NT-4 was transported to SCG neurons when injected into the anterior eye chamber.

15. EXAMPLE: BINDING OF 125I-NT-4 TO BRAIN AND RETINA

15J MATERIALS AND METHODS

NT-4 was iodinated by the lactoperoxidase method to a specific activity of 2400-3500 cpm/fmol (1211-1789 Ci/mmol of NT-4). 25i]-NT-4 was stored at 4°C and used within 1-3 days after preparation. Biological activity of the radioiiodinated NT-4 was verified by bioassay.

Male Sprague-Dawley rats (200-250 g, Zivic Miller) were maintained on a 12:12 h light:dark cycle and given food and water ad libitum. The brains and eyes of each rat were frozen in isopentane at -15°C within 5 min. of death. Serial, 12 urn thick sedions of these tissues were collected on gelatin coated slides and were used for binding studies.

Sagittal whole body sedions of adult male rat were mounted on a large piece of adhesive tape which was then attached to a plastic frame. This plastic frame created a recess in which each sedion was incubated with the [i2Si]-NT-4.

Binding assays using p25i]-NT-4 were performed as follows: After being thawed, adjacent brain and whole body sedions were preincubated for 0.5 h in phosphate buffered saline, pH 7.4 followed by a 3 h incubation at room temperature in DMEM tissue culture medium containing high glucose, 10% heat- inadivated fetal calf serum (60°C for 0.5 h), 25 mM Hepes buffer, 4 ug/mi leupeptin, 0.5 mM PMSF (BRL, Gaithersburg, MD., dissolved to 0.1 mg/mg ethanol, 0.5 mM MgCI₂ and I nM [i2si]-NT-4 with (non-specific) or without (total) I μM unlabeled NT-3 or NT-4. Following the incubation, the sections were washed for 0.5 h in PBS. Eye sections were not preincubated, and were washed for 0.5 h in binding buffer without labeled or unlabeled neurotrophin, since this procedure maintained the anatomical integrity of the retina during the binding procedure. After washing, sedions were fixed for I0 min in 4% paraformalde-hyde at 22°C, rinsed for 2 seconds in dH₂0 and dried with a stream of room temperature air. The labeled sections and 1251-containing radioactivity standards

(Amersham, Inc.) were exposed at room temperature for 2-5 days to 1251- sensitive film (Hyperfilm, Amersham, Inc.). Slides were then dipped in Kodak NTB-2 photographic emulsion diluted 1 :1 with distilled water and developed 1 to 2 weeks later in Kodak D-19 at 15°C. Slides containing eye sedions were stained with hematoxylin-eosin for histological examination.

15.2 RESULTS

In whole body sections of adult rats, specific binding of [i25i]-NT-4 was restricted to the central nervous system, including the brain, spinal cord and retina as well as to the dorsal root ganglia.

In brain sedions, specific [i2si]-NT-4 binding was found to be widely distributed throughout the brain including the cortex, striatum, hippocampus, cerebellum, olfadory bulbs, periaquedudal gray, and raphe nudei.

Results from dry film exposure of [i25i]-NT-4 labeled rat eye sedions and human eye sedions revealed high levels of displaceable binding in the retina. When examined at the emulsion level, intense, displaceable labeling was found in the inner plexiform and ganglion cell layers of the retina as well as in the human optic nerve.

16. EXAMPLE: THE COMPARATIVE EFFECT OF NEUROTROPHINS IN HIPPOCAMPUS

16J MATERIALS AND METHODS

Hippocampi were disseded from E18 rat embryos of Sprague- Dawley rats, and colleded in F10 medium. The tissues were minced, rinsed twice with F10 medium (Gibco) and trypsinized with 0.25% trypsin (Gibco) for 20 minutes at 37°C. Trypsin was inadivated by the addition of a serum- containing medium composed of minimum essential medium (MEM supplemented with fetal calf serum (FCS, 10%), glutamine (2 mM), penicillin (25 U/ml) and streptomycin (25 μg/ml) in DME plus 10% fetal calf serum.

After 4 hours of culture, the medium was changed to DME plus I mg/ml BSA and N2 media supplement [Bottenstein, et al., Methods Enzymol. 58:94-109] and 1 mM pyruvate, at which time NT-4 was added. The media was changed every three to four days, with re-addition of the fador.

16.2 RESULTS

Purified recombinant human NT-4 produced an increase in fos mRNA in these cells similar to that seen with BDNF or NT-3 (Figure 33A). This increase was followed by an increase in fos protein when examined at 2 hr after treatment. The three neurotrophins (BDNF, NT-3 and NT-4) were found to cause tyrosine phosphorylation of proteins immunoprecipitable by a pan trk-specific antibody (Figure 33B). Two cell populations that were shown to respond to BDNF also responded to NT-4. That is, there was an increase in the number of acetylcholinesterase-positive cells and calbindin- immunopositive cells in hippocampal cultures treated with NT-4 (Figure 34). 17. EXAMPLE: NT-4 INCREASES SURVIVAL AND DIFFERENTIATED FUNCTIONS OF RATSEPTAL CHOLINERGIC NEURONS IN CULTURE

17J MATERIALS AND METHODS

17.1.1. PREPARATION OF DISSOCIATED CELLS AND CELL CULTURE CONDITIONS

The septal region from rats (Sprague-Dawley) after 17 days of gestation was dissected free from the surrounding tissue. Tissue fragments were pooled, washed three times with Ham's F-10, and then transferred to a 35mm tissue culture dish and minced. A single cell suspension was made by incubating the tissue with 0.25% trypsin for 20 minutes at 37^βC. Following the inadivation of the trypsin by a five minute incubation at room temperature in growth medium (infra), containing 100 μg/ml deoxyribonuclease type 1 (Sigma), the cells were dissociated by passing the fragments repeatedly through the constrided tip of a Pasteur pipet. The dissociated cells were then centrifuged at 500xg for 45 seconds. The supernatant was removed and recentrifuged.

The loose cell pellets were resuspended and combined in normal growth medium (5% (v/v) horse serum (Gibco), 1% N3 additives (v/v) (Romijn et al. 1982, Dev. Brain Res. 2: 583-589), 0.5% (v/v) glutamine (200mM, Gibco), and 0.25% (v/v) penicillin-streptomycin (10,000 units/ml, 10,000 mcg/ml respectively, Gibco) in Dulbecco's modified Eagle's medium

(DMEM). Three to four hours after plating, sterile coverslips were placed in each well to cover the cells. Neuronal-enriched cultures were prepared by replacing 2/3 of the growth medium, 24 and 72 hours after plating with N3/DMEM: DMEM containing 1% N3 additives, 0.5% glutamine, and 0.25% penicillin and streptomycin. All subsequent medium changes, carried out every other day, were done by removing 1/2 the volume of medium and replacing it with an equal volume of fresh N3/DMEM. To limit the growth of astrocytes, the cultures were treated for 24 hours with cytosine arabinoside at a concentration of 2μM after 1 week in culture.

17.1.2 ASSAY OF CHOLINE ACETYLTRANSFERASE (ChAT) ACTIVITY

The growth medium was removed from the cultures and 125 μl of the lysis buffer (50 mM KH_aP0₄ pH 6.7 containing 200 mM NaCl and 25% (v/v) Triton x-100) was added. With the tissue culture plates on ice, the cells were scraped from the plates and the wells were rinsed with an additional 125μl of lysis buffer. The two aliquotes were then combined in eppindorf tubes and quick frozen in a dry-ice methanol slurry.

17.2 RESULTS

The culture conditions used in this study limit the proliferation of astrocytes and allow for the long-term maintenance of basal forebrain neurons in vitro. E. coli produced NT-4 was added to the cultures 24 hrs after plating and was replenished with every medium change. At the end of the two week treatment period, the cells were harvested. Figure35 depids the dose related effect of NT-4 on ChAT activity. At a saturating concentration of 25 ng/ml, NT-4 produced an approximate two-fold increase in ChAT adivity. This level of enzyme indudion was maintained up to the highest concentration (100 ng/ml) of NT-4 tested. The level of ChAT indudion observed with NT-4 is approximately equivalent to that observed with BDNF. These data thus demonstrate that the basal forebrain cholinergic neurons are a target for NT-4. 18 EXAMPLE: NT-4 SUSTAINS THE SURVIVAL OF DOPAMINERGIC

NEURONS

18J MATERIALS AND METHODS

18.1.1. METHODS FOR CULTURING DOPAMINERGIC SUBSTANTIA NIGRA NEURONS

Ventral mesencephalon was disseded from brains of rat embryos varying in age from embryonic day 13 to embryonic day 15. Typically, two litters were used in each experiment. The dissection solution had the following composition: NaCl, 136.8 mM, KC1 , 2.7 mM, Na₂HPO₄.7H;>0, 8.0mM, KH₂P0 , 1.5 mM, glucose, 6 mg/ml, and BSA, 0.1 mg/ml, pH 7.4. This solution was prepared and subsequently filter sterilized through a 0.2μM pore filter. The dissection was performed under non-sterile conditions. Once the tissue was disseded from all the brains, the rest of the procedure was carried out under sterile conditions. The tissue fragments were placed in a 35 mm culture dish and minced using a fine scissors. Two ml of F-12 nutrient media containing 0.125% trypsin was then added to the tissue, and incubated at 37°C. At the end of this incubation period, DNAsel was added to the slurry such that the final concentration was 80 ng/ml. Another identical incubation was carried out, and the tissue slurry was subsequently added to 8.0 ml of growth medium consisting of Minimal Essential Medium (MEM) supplemented with 2mM glutamine, 6 mg/ml glucose, 5 units/ml penicillin, 5mg/ml streptomycin, and

7.5% fetal calf serum (FCS). The sample was centrifuged in a tabletop centrifuge at room temperature at 500 rpm for a period of 5 minutes. The medium was aspirated, and 2 ml growth medium was added to the cell pellet. A fire polished pipette with an opening of I mm was used to triturate the cells eight times. The remaining tissue fragments were allowed to settle by gravity, and a small aliquot of the supernatant was taken to assess cell number by counting in a hemocytometer. After cell density was determined, the cells were plated into tissue culture plates at a density of 50,000/cm2.

The culture plates were prepared on the day prior to dissection. Tissue plates (24 well, 2 cm2/well) were precoated with polyornithine (molecular weight 30,000-70,000 g/mol), 0.5 mg/ml, at room temperature for 3 hours. The plates were extensively washed with water, and subsequently treated with mouse laminin, 5 μg/ml, at room temperature for 3 hours. The plates were then washed with water as above, and incubated overnight at 37°C in a humidified atmosphere consisting of 5% C0₂, 95% air, in the presence of growth medium. The medium in the plates was removed the following day and replaced with fresh growth medium.

Once the cells were plated onto the culture plates, the cells were placed in an incubator set at 37°C and 5% CO₂/95% air for a period of 24 hours. The culture medium was changed to a serum-free formulation (SFM) having the following composition: a 1 :1 (vo vol) mixture of Basal Eagle

Medium (BEM) and nutrient mixture F-12 with glucose (33 mM), glutamine (2mM), NaHCO₃ (15 mM)< HEPES (10mM), supplemented with insulin (25 μg/ml), putrescine (60 μM), progesterone (20 nM), sodium selenite (30 nM), penicillin (5 U/ml), streptomycin (5 mg/ml), and T₃ (30 nM). In some experiments, purified BDNF was added to the cultures after the media change to SFM on culture day 2.

The solutions used for culturing dopaminergic neurons were prepared using water taken from a Milli-Q reagent water system. The tissue culture media formulations were obtained through Gibco Laboratories (Santa Clara, California), as was the fetal cal serum (lot number 43N1086) and the mouse laminin. All other media components were purchased from Sigma Chemical (St. Louis, MO), and were cell culture tested grade. The polyornithine and DNAsel were also obtained from Sigma. Trypsin was obtained from Worthington (Freehold, NJ), lot number 3667. Commercial chemicals were of analytical grade, purchased from Baker Chemical (Phillipsburg, NJ).

18.1.2. METHODS FOR IMMUNOCYTOCHEMICAL STAINING OF VENTRAL MESENCEPHALON CULTURES

Fixative solutions were prepared fresh for each experiment. For the staining of tyrosine hydroxylase (TH), the fixative was 4.0% paraformaldehyde in Sorenson's phosphate buffer. The Sorenson buffer was prepared by adding a 0.2 M solution of KH₂P0 to a stock of 0.2 M

NA₂H P 0₄ until the pH reached 7.3. The paraformaldehyde was subsequently added to the solution and briefly heated, to allow it to be dissolved, and cooled to room temperature before use.

To begin the procedure, culture medium was removed from the culture dishes by gentle sudion, and the proper fixative solution was gently added to the dish. A room temperature incubation of 20 minutes was carried out. Three washes in Sorenson's phosphate buffer, for 5 minutes each, with gentle rotation, followed. The cells were then incubated in a quench solution for 30 minutes at room temperature with gentle rotation. The quench solution for the cultures to be stained for TH consisted of

Sorenson's phosphate buffer containing 2% normal horse serum. Next, the cultures were incubated in permeabilization buffer at room temperature for 30 minutes with gentle rotation. The solution consisted of Sorenson's buffer containing 0.2% saponin, and 1.5% of normal horse serum for the cultures to be stained for TH. Following the permeabilization step, the cultures were incubated in the presence of primary antibody overnight at 4°C. The antibody against rat TH was a mouse monoclonal antibody of isotype lgG2a. It was used at 40 μg/ml in a colution of 20 mM NaP0 , 50m M NaCl, 0.2% saponin pH 7.5. Following the primary antibody incubation, the cultures were washed 5 times for 15 minutes each in the appropriate permeabilization buffer. Next, the cultures were incubated with secondary antibody conjugated to biotin, that is biotinylated horse anti-mouse IgG. This incubation was carried out at room temperature for two hours with gentle rotation. Washes identical to those described above followed, and the cultures were then incubated in the presence of a preformed avidin- biotinylated horseradishperoxidase complex (ABC reagent, Vector

Laboratories, Burlingame, CA) prepared according to manufacturer's protocol. After a 30 min. incubation at room temperature with gentle rotation, the cultures were washed as described above. The cultures were subsequently incubated with 55 mM Tris-Cl pH 7.3 containing 0.5 mg/ml diaminobenzidine and 0.02% hydrogen peroxide. The development of reaction product was allowed to proceed for 2-5 min. after which the solution was removed and the cultures were washed several times with the ice cold PBS. The number of positive cells/cm2 was then ascertained.

The paraformaldehyde and the glutaraldehyde were obtained from Fluka Chemical. Vedastain kits containing normal serum (used as a blocking agent), biotinylated, affinity-purified anti-immunoglobulin, avidin DH, and biotinylated HRP-H were purchased from Vector Laboratories. The diaminobenzidine was obtained from BRBDNFL (Gaithersberg, MD).

18.2 RESULTS

Two sets of data from experiments in which 2 lots of human NT- 4(made in E. coli) were used are shown in Figures 36A and 36B. Cultures were prepared as previously described, and were treated with increasing concentrations of NT-4. In the experiment shown in Figure 36A, the treatment of cultures with NT-4 was given as a single addition on the day of plating. In the experiment shown in Figure 36B, the NT-4 treatment was given as multiple additions at the day of plating (culture day 1), and subsequently at culture days 4 and 7 (CD4, CD7). At culture day 8, the cultures were processed for immunocytochemical staining for the dopaminergic marker tyrosine hyroxylase. The number of dopaminergic neurons present in each dish was then determined. Each treatment group represents 5 replicate cultures.

The results from both experiments show that NT-4 treatment leads to an increase in the number of dopaminergic neurons deteded by TH immunocytochemical staining. This increase is dose dependent and saturable as shown in Figure 36B.

19. EXAMPLE: EFFECTS OF NT-4 ON STRIATAL NEURONS IN VITRO

19J METHODS

19.1.1 DISSOCIATED CULTURE PREPARATION

Striatal neuronal cultures were prepared from E17 rat brains as follows: striatal tissue was minced in calcium- and magnesium-free Hank's balanced salt solution and dissociated by enzymatic treatment with 0.25% trypsin and DNAase (0.2 mg/ml) followed by mechanical trituration in medium composed of Dulbecco'sModified Eagle's Medium and 10% fetal calf serum (DME-FCS). Dissociated cells were seeded at a density of 104 cells well in serum-free N2 medium in 96-well tissue culture plates that had been previously coated with polylysine and merosin. Human NT-4 (0.1-50 ng/ml) was added at the time of plating and replenished every other day.

19J 2. IMMUNOHISTOCHEMICAL STAINING FOR CALBINDIN

Striatal cultures at 8 days in vitro (8 DIV) were fixed in 4% paraformaldehyde for 30 minutes, rinsed with PBS, permeabiiized for 15 minutes in 0.1% Triton X-100/PBS, and blocked with 10% horse serum/1% bovine serum albumin/PBS for 90 minutes at room temperature. Cultures were then incubated with the primary antibody (Mouse-anti-calbindin, Sigma, 1 :5000 dilution) plus 5% normal horse serum overnight at 4°C prior to incubation with the secondary antibody (biotinylated horse-anti-mouse (Vedor Labs, 1 :400 dilution) for 90 minutes at room temperature. Calbindin immunoreactivity was visualized by using the Vectastatin

ABC kit (Vector Labs). The number of total neurons as well as the number of neurons immunoreadive for calbindin were counted in approximately 5-10% of the total area of each of four duplicate culture wells.

19J.3 MEASUREMENT OF HIGH-AFFINITY GABA UPTAKE

High-affinity GABA uptake was measured according to a modification of the procedure of Tomozawa and Appel, 1986, Brain Res. 399:111-124. Cells were washed once in buffer containing 140 mM NaCl, 2.6 mM KCI, 0.75 mM MgCI2, 0.75 mM CaCI2, 1 mM KH2P0₄, 1 mM Na2HP0₄, 6 mg/ml glucose,

2 mM β-alanine, and 1 mg/ml BSA. Following one wash, cells were incubated in the uptake buffer for 5 minutes at 37°C. 3H-GABA (NEN, NET-191XJ00 Ci/mmol) was then added at a final concentration of 17 nM, and cells were incubated for 15 minutes at 37°C. In both incubations, neuron-specific uptake was verified by the addition of 1 mM nipecotic acid to some wells.

Following the second incubation, cells were washed four times with uptake buffer at 4°C, and then incubated with 0.5 M NaOH for two hours at room temperature. The cell extrad was then colleded and the 3H-GABA in the extrad counted.

19.2 RESULTS

Striatal cultures were treated with human NT-4 purified from E. coli. In striatal cultures treated with NT-4 for 8 days, the percent of total neurons that were calbindin-immunoreadive increased by approximately 3 to 4 fold compared to untreated control cultures (Figure 37). NT-4 treatment produced an approximately 3 to 4 fold increase h the level of high-affinity uptake of GABA as compared to untreated controls (Figure 38).

20. EXAMPLE: EFFECT OF NT-4 ON NIGRAL GABAergic NEURONS

20J METHODS

20J .1 PREPARATION OF VENTRAL MESENCEPHALON CULTURES

Cultures were prepared from the ventral mesencephalon of embryonic day 14 (E14) rat embryos as before (Hyman et al, 1991 , Nature, 350:230-

232; Spina et al., 1992, J. Neurochem., 59:99-106). The single cell suspension obtained following trypsinization and mechanical dissociation of the brain tissue was seeded at a density of 5 x 10 cell per cm2 onto 35mm dishes which had been precoated with poly-L-lysine and merosin (Collaborative Research). After a 4 hour incubation in MEM supplemented with glutamine

(2mM), glucose (6 mg/ml), penicillin G (0.5U/ml), streptomycin (5 μg/ml), and fetal calf serum (FCS,7.5%) to allow for cell attachment to the substratum, cells were cultured in the presence or absence of trophic factors in a defined medium consisting of F12 and basal Eagle medium (1 :1 , v v) with N₂ supplements as described by Bottenstein and Sato ,1979,

Proc. Natl. Acad. Sci. USA, 64:787-794, except that the insulin concentration was reduced to 20 ng/ml, and giutathione was included at 2.5 μg/ml.

20.1.2 3H-GABA UPTAKE MEASUREMENT

The measurement of high-affinity GABA uptake activity was carried out according to the method set forth in Example 19.

20.L3 GLUTAMIC ACID DECARBOXYLASE (GAD) IMMUNOCYTOCHEMICAL STAINING Cultures to be stained for GAD were prefixed in a 1 :1 mixture of 4% paraformaldehyde and F12/BME (1 :1 , v/v) for 10 minutes, followed by 30 minutes incubation in 4% paraformaldehyde. Cultures were then incubated in the presence of phosphate buffer containing 4% goat serum and 0.02% saponin, after which the cultures were incubated overnight at 4°C with goat anti-feline GAD ₆₇ antiserum (generously supplied by Dr. A. Tobin, UCLA, CA) at a dilution of 1/7500 . Cultures were washed, incubated with goat anti- rabbit IgG at a concentration of 1.5 μg/ml, and specifically bound antibody was detected after binding of avidin-HRP, by development using DAB with NiS0 intensification (Hancock et al, 1982,Neurosci. Lett., 31 :247-252).

20J .4 MEASUREMENT OF GABA CONTENT

Cultures were prepared and maintained in vitro for various times, either in the presence or absence of trophic fadors. At the end of the culture period, cells were harvested and immediately acidified with 0.4 N perchlorate containing 0.1 mM ascorbate and 2.5 ng/ml dihydroxybenzylamine (DHBA, an internal standard). Following homogenization and centrifugation to remove precipitated protein, the catecholamines in the extract were absorbed onto alumina (ICN). The samples were then washed extensively, and the catecholamines recovered by extradion from the alumina with 174nM acetic acid containing 0.05% sodium disulfite and 0.025% EDTA. The protein content of the sample was determined after resuspension of the pelleted precipitate in PBS by the method of Smith et al, 1985, Anal. Biochem. 76-85. GABA determinations were carried out, after derivatization with ophthalaldehyde, by an HPLC separation using a reverse phase C-iβ column in a mobile phase consisting of 25% methanol, 3.1% acetonitrile, 0.1 M Na₂P0₄, pH 6.8. Quantitation of the various amino acids was performed after electrochemical detection (ESA 5500 coulochem eledrode array system detedor) of the eluted peaks from the HPLC, and the data normalized to the protein content of the sample. 20.2 RESULTS 202.1 EFFECT OF NEUROTROPHINS ON GABAERGIC NEURONS

To examine possible effects of the neurotrophins on nigral GABAergic neurons, high-affinity GABA uptake, GABA content, and GAD adivity were measured in control and treated cultures. The presence of GABAergic neurons in the cultures was first deteded by immunocytochemical staining using an antibody specifically direded against the 67kD form of GAD, which is found in terminal processes and neuronal perikarya (Gonzales et al, 1991 , J. Neurocytol., 20: 953-961 ; Kaufman et al, 1991 , J. Neurochem.

56:720-723). Fig. 4 shows representative GAD staining patterns obtained in cultures maintained in the absence of any neurotrophic fadors for 4, 7, o r 11 days (41A-C, respectively). No staining was observed in the absence of primary antibody (41 D). Cell counts indicated that the number of GAD positive neurons in cultures maintained for one week in vitro in the absence of neurotrophins represented 3.3±0.7% of the total cell count at that time. Only NT-3 produced a significant increase (63%) in the number of GAD- positive neurons after 7 days in vitro.

Although BDNF did not increase the number of GAD-positive neurons, BDNF as well as NT-3 produced dose-dependent increases in GAD enzymatic adivity in cultures maintained for 7 days, as shown in Fig. 42A and 42B respedively. NT-3 produced a greater increase than BDNF (3-fold vs 1.8- fold) whereas NT-4/5 was without effed at any concentration tested (Fig. 42C; and up to 200ng/ml, data not shown). As another marker of GABAergic neurons, we explored the effeds of each of the neurotrophins on the high-affinity GABA uptake capacity of cells cultured for 7 days. As shown in Fig. 43A, all three neurotrophins tested (BDNF, NT-3 and NT-4/5) elicited increases of 2- to 3-fold in GABA uptake. The dose responses of BDNF and NT-3 were similar, reaching maxima at approximately 20 ng/ml. NT-4/5 was effedive at lower concentrations than BDNF or NT-3, saturating at 10 ng/ml with decreased effects at higher concentrations. When we assessed possible additive or synergistic effects on GABA uptake, no combinatorial effects of any of the neurotrophins were observed (Fig. 43B). NGF (50 ng/ml) had no effect on GABA uptake activity.

As a further marker of the GABAergic phenotype, we measured GABA content in cultures treated with each of the neurotrophins (Fig. 44). BDNF, NT-3, and NT4/5 all produced modest but significant increases in the GABA content of cultures grown with these factors for 7 days. Again, NGF treatment was without effect. These data are consistent with the increases observed in the high-affinity GABA uptake adivity measurements.

20.2.2 ANALYSIS OF TRK RECEPTOR DISTRIBUTION IN SUBSTANTIA NIGRA To address the question of the site of adion of the neurotrophins h mediating the above effeds on cultured nigral neurons, it was of interest to ascertain the expression pattern of high-affinity BDNF and NT-3 receptors (TrkB and TrkC, respectively) in both embryonic cultures and adult rat brain substantia nigra. As shown in Fig. 45, in situ hybridization clearly indicated high levels of TrkB (45B, 45C) and TrkC (45D-F) mRNA in both the ventral tegmental area (VTA) and substantia nigra of adult rat brain. The distribution of TrkC is more widespread than TrkB. Examination of the TrkC localization pattern at higher magnification (Fig. 45E.45F), demonstrates that Trk C is expressed in large perikarya, indicative of neurons. To ascertain if TrkB and TrkC are expressed in embryonic rat ventral mesencephalic tissue, RNA prepared from E14 mesencephaiic cultures grown in the presence or absence of BDNF or NT-3 for various times was probed by Northern blot analysis. As shown in Fig. 46A, a probe to the kinase domain of TrkB indicated the presence of a 9 kb transcript under all conditions. Exposure of cultures to BDNF or NT-3 for up to 29 hours did not substantially alter the expression levels of TrkB mRNA. The 9 kb transcript deteded is one of the 2 brain-specific transcripts described by Klein et al, 1991, Cell 65:189-197 which was shown to correspond to the full length Trk B tyrosine kinase cell surface receptor. In the blot probed for TrkC (Fig. 46C), a 14kb transcript was detected both in adult brain and

E14VM culture RNA. This species has been identified as the major transcript encoding full length TrkC (Valenzuela et al, 1993, in press). The expression level of this transcript was not strikingly altered in NT-3 treated cultures. An additional 5kb TrkC transcript was detected. Migrating just above the 28S ribosomal RNA band, this is one of two known transcripts which encode truncated forms of TrkC (Valenzuela et al., 1993, in press).

20.2.3 DISCUSSION

The above data provides evidence that the neurotrophins may provide a role in supporting GABAergic neurons in the substantia nigra. In general, altered GABAergic neurotransmission is associated with generalized seizures. In particular, generalized seizures can be prevented by increasing GABAergic neurotransmission within the substantia nigra. Gale, K., 1985, Federation Proceedings 44:2414-2424; Olsen, R.W., et al., 1986, in Neurotransmitters, Seizures and Epilepsy III, Nistico, et al. (eds), Raven

Press, New York. Accordingly, data provided herein suggest that the neurotrophins may have potential utility for the treatment of seizure related disorders.

21. EXAMPLE: NT-4 IN COMBINATION WITH OTHER NEUROTROPHIC

FACTORS SUSTAINS THE SURVIVAL OF MOTOR NEURONS

The effect of NT-4 on motor neurons was measured by monitoring

ChAT adivity as described above. NT-4 stimulated ChATactivity in motor neuron enriched cultures in a dose dependent manner (Figure 39).

Simultaneous treatment of motor neuron enriched cultures with NT-4 and CNTF in combination as well as NT-4 and NT-3 in combination resulted in a more than additive effect on ChAT adivity. NT-4 (100 ng/ml) alone increased ChAT activity 3.8 fold and CNTF (50ng/ml) alone increase ChAT activity 4.5 fold. However, when they were added simultaneously, ChAT activity was elevated 13.4-fold, suggesting synergistic actions of the two factors. (Figure 40).

21. 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 Colledion, 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. A recombinant bacteriophage (hCNTF-G1), containing a human genomic sequence related to ciliary neurotrophic factor (CNTF), was deposited on September 12, 1989, with the American Type

Culture Collection and assigned accession number ATCC 40657. A recombinant baderiophage [phi hN3(G1)], containing a human genomic sequence related to neurotrophin-3 (NT-3), was deposited on February 28, 1990 with the American Type Culture Colledion and assigned accession number ATCC 40763.

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.

International Application No: PCT/

MICROORGANISMS

Optional Sheet in connection with the microorganism referred to on page 1 10. lines 9-30 and page 1 1 1 . line 1 of the description '

A. IDENTIFICATION OF DEPOSIT '

Further deposits are identified on an additional sheet '

Name of depositary institution ' American Type Culture Collection

Address of depositary institution (including postal code and country)

12301 Parklawn Drive Rockville. D 10582 US

Date of deposit ' August 22, 1991 Accession Number * 75069

B. ADDITIONAL INDICATIONS ' (leave blank if Dot spplicabfe). Tha mfoπnalion a continued on a aepanlc suachcd sheet Si

C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' or*..

D. SEPARATE FURNISHING OF INDICATIONS ' ( .« blank if not appli able)

The indications listed below will be submitted to the International Bureau later * fSpecifγ the general nature of the indications e.g., -Accession Number of Deposit")

E Ξ.. W Qj TThhiis sheet was received with the International

CD The date of receipt (from the applicant) by the International Bureau "

(Authorized Officer)

Form PCT/RO/134 (January 1981 ) International Application No: PCT/ /

Form PCT/RO/134 (cont.)

American Type Culture Collection

12301 Parklawn Drive Rockville. MD 10582 US

Accession No. Date of Deposit

75070 August 22, 1991

75098 September 1 1 , 1991

75133 October 30, 1991

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(1) GENERAL INFORMATION:

(i) APPLICANT: Ip and Yancopoulos

(ii) TITLE OF INVENTION: Therapeutic and Diagnostic Methods Based on Neurotrophin-4 Expression

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(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 lib Ser Glu Cys Lys Ala Lys Gin Ser Tyr Val Arg 20 25 30

Ala Leu Thr Met Asp Ser Asp Lys lie 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:

Lys Cys Asn Pro Ser Gly Ser Thr Thr Arg Gly Cys Arg Gly Val Asp 1 5 10 15

Lys Lys Gin Trp lie Ser Glu Cys Lys Ala Lys Gin Ser Tyr Val Arg 20 25 30

Ala Leu Thr lie 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 lie 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 Gin 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 lie 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 Gin 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 lie 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 Gin 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 lie 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 Gin 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 lie 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 Gin 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 lie 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 lie Asp 1 5 10 15

Lys Arg His Trp Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val Arg 20 25 30

Ala Leu Thr Met Asp Ser Lys Lys Arg lie 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 lie Asp 1 5 10 15

Lys Arg His Trp Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val Arg 20 25 30

Ala Leu Thr Met Asp Ser Lys Lys Arg lie 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

( i) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Lys Cys Asn Pro Lys Gly Tyr Thr Lys Glu Gly Cys Arg Gly lie Asp 1 5 10 15

Lys Arg His Trp Asn Ser Gin Cys Arg Thr Thr Gin 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 lie Asp 1 5 10 15

Lys Arg Tyr Trp Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val Arg 20 25 30 Ala Leu Thr Met Aβp Asn Lys Lys Arg lie 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 lie Asp 1 5 10 15

Lys Arg Tyr Trp Asn Ser Gin Cys Arg Thr Thr Gin 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 lie Asp 1 5 10 15

Lys Arg His Tyr Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val Arg 20 25 30

Ala Leu Thr Met Asp Ser Lys Lys Lys lie 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 Cyβ Asn Pro Lye Gly Phe Thr Asn Glu Gly Cys Arg Gly lie Asp 1 5 10 15

Lys Lys His Trp Asn Ser Gin Cys Arg Thr Ser Gin Ser Tyr Val Arg 20 25 30

Ala Leu Thr Met Asp Ser Arg Lys Lys lie 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 lie Asp 1 5 10 15

Asp Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin 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 lie Asp 1 5 10 15

Asp Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin 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 lie Asp 1 5 10 15

Asp Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin 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 lie Asp 1 5 10 15

Asp Lys 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 lie Asp 1 5 10 15

Asp Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin Thr Tyr Val Arg 20 25 30

Ala Leu Thr Gin 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 lie Asp 1 5 10 15

Asp Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin 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 lie Leu Arg Leu Tyr Ala Met Val lie 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 lie Cys Ala Ala Pro Phe Gin 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 Gin 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 Gin 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 He Val Thr Val Met Ser Glu He 150 155 160

CAG ACT CTA ACA GGA CCA CTG AAG CAA TAC TTC TTT GAG ACC AAG TGC 1062 Gin Thr Leu Thr Gly Pro Leu Lye Gin 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 Gin Trp He Ser Glu Cys Lys Ala Lys Gin 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 He Asp Ala Asn Lys Leu Val Gly Trp Arg Trp He Arg He 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 He Leu Arg Leu Tyr Ala Met Val He Ser Tyr Cys 1 5 10 15

Cys Ala He Cys Ala Ala Pro Phe Gin Ser Arg Thr Thr Asp Leu Asp 20 25 30

Tyr Gly Pro Asp Lys Thr Ser Glu Ala Ser Asp Arg Gin Ser Val Pro 35 40 45

Asn Asn Phe Ser His Val Leu Gin 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 He Val Thr Val Met Ser Glu 145 150 155 160

He Gin Thr Leu Thr Gly Pro Leu Lys Gin 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 Gin Trp He Ser Glu Cys Lys Ala Lys Gin Ser Tyr Val Arg Ala 195 200 205

Leu Thr He Asp Ala Asn Lys Leu Val Gly Trp Arg Trp He Arg He 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 He Val Thr Val Met Ser Glu He Gin Thr Leu 35 40 45

Thr Gly Pro Leu Lys Gin 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 Gin Trp He 65 70 75 80

Ser Glu Cys Lys Ala Lys Gin Ser Tyr Val Arg Ala Leu Thr He Asp 85 90 95

Ala Asn Lys Leu Val Gly Trp Arg Trp He Arg He 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 He Lys 20 25 30

Gly Lye Glu Val Thr Val Leu Ala Glu Val Asn He Asn Asn Ser Val 35 40 45

Phe Arg Gin Tyr Phe Phe Glu Thr Lys Cys Arg Ala Ser Asn Pro Val 50 55 60

Glu Ser Gly Cys Arg Gly He 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 Gin 85 90 95

Ala Ala Trp Arg Phe He Arg He 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 He 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 Gin 35 40 45

Leu Lys Gin Tyr Phe Tyr Glu Thr Lye Cys Asn Pro Met Gly Tyr Thr 50 55 60

Lys Glu Gly Cys Arg Gly He Asp Lys Arg His Trp Asn Ser Gin Cys

65 70 75 80

Arg Thr Thr Gin Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys Lys 85 90 95

Arg He Gly Trp Arg Phe He Arg He Asp Thr Ser Cys Val Cys Thr 100 105 110

Leu Thr He 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 He Asp He Arg Gly 20 25 30

His Gin Val Thr Val Leu Gly Glu He Lys Thr Gly Asn Ser Pro Val 35 40 45

Lys Gin Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Lys 50 55 60

Asn Gly Cys Arg Gly He Asp Asp Lys His Trp Asn Ser Gin Cys Lys 65 70 75 80

Thr Ser Gin Thr Tyr Val Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu 85 90 95

Val Gly Trp Arg Trp He Arg He Asp Thr Ser Cys Val Cys Ala Leu 100 105 110

Ser Arg Lys He Gly Arg Thr

115

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1313 baβe 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 He Leu Arg Leu Tyr Ala Met Val He 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 He Cys Ala Ala Pro Phe Gin 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 Gin 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 Gin 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 He 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 He Gin Thr Leu Thr Gly Pro Leu Lys Gin 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 Gin Trp He Ser Glu Cys Lys Ala Lys Gin 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 He Asp Ala Asn Lys Leu Val Gly Trp Arg Trp He Arg He 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 He Leu Arg Leu Tyr Ala Met Val He Ser Tyr Cys Cys Ala He 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 Gin 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 He Val Thr Val Met Ser Glu He Gin Thr 145 150 155 160

Leu Thr Gly Pro Leu Lys Gin 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 Gin Trp 180 185 190

He Ser Glu Cys Lys Ala Lys Gin Ser Tyr Val Arg Ala Leu Thr He 195 200 205

Asp Ala Asn Lys Leu Val Gly Trp Arg Trp He Arg He 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:

Gin 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 Gin Trp He Ser Glu Cys Lys Ala 20 25 30

Lys G n Ser Tyr Val Arg Ala Leu Thr He Asp Ala Asn Lys Leu Val 35 40 45

Gly Trp Arg Trp He Arg He 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:

Gin 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_baβe

(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 Gin 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 Gin 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:

Gin 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 Gin 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 Gin 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 Lye Gin 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:

Gin 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 Lye Ala Lys Gin 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:

Gin 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 Gin Trp He Ser Glu Cys Lys Ala 20 25 30

Lys Gin 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:

Gin Tyr Phe Phe Glu Thr Lys Cys Arg Ala Pro Asn Pro Val Glu Ser 1 5 10 15

Gly Cys Arg Gly He 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:

Gin Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu 1 5 10 15

Gly Cys Arg Gly He Asp Lys Arg His Trp Asn Ser Gin Cys Arg Thr 20 25 30

Thr Gin 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:

Gin Tyr Phe Tyr Glu Thr Arg Cys Lys Glu Ala Arg Pro Val Lys Asn 1 5 10 15

Gly Cys Arg Gly He Asp Asp Lys His Trp Asn Ser Gin Cys Lys Thr 20 25 30

Ser Gin 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 Gin 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 Gin 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 Gin Gly Arg Val Gly Trp Arg Trp He Arg He 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:

Gin 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 Gin Ser Tyr Val Arg Ala Leu Thr 35 40 45

Ala Asp Ala Gin Gly Arg Val Gly Trp Arg Trp He Arg He 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

Gin 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 He 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 He Glu Ser Gin 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 Gin 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 Gin 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 Gin 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 He Arg He 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:

Gin Val Leu Arg Glu Met Leu Pro Leu Pro Ser Cys Ser Leu Pro He 1 5 10 15

Leu Leu Leu Phe Leu Leu Pro Ser Val Pro He Glu Ser Gin 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 Gin 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 Gin Ser Tyr Val Arg Ala Leu Thr Ala Asp Ala Gin Gly Arg 180 185 190

Val Gly Trp Arg Trp He Arg He 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 He Leu

1 5 10 15

Leu Leu Phe Leu Leu Pro Ser Val Pro He Glu Ser Gin 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 Gin 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 Gin Ser Tyr Val Arg Ala Leu Thr Ala Asp Ala Gin Gly Arg Val 180 185 190

Gly Trp Arg Trp He Arg He 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 He Val Thr Val Met Ser 85 90 95

Glu He Gin Thr Leu Thr Gly Pro Leu Lys Gin 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 Gin Trp He Ser Glu Cys Lys Ala Lys Gin Ser Tyr Val Arg 130 135 140

Ala Leu Thr He Asp Ala Asn Lys Leu Val Gly Trp Arg Trp He Arg 145 150 155 160

He 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 Gin Pro Met He Ala Thr Asp Thr Glu Leu Leu Arg Gin Gin 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 He Asp He Arg Gly His Gin Val Thr Val Leu 85 90 95

Gly Glu He Lys Thr Gly Asn Ser Pro Val Lys Gin Tyr Phe Tyr Glu 100 105 110

Thr Arg Cys Lys Glu Ala Arg Pro Val Lye Asn Gly Cys Arg Gly He 115 120 125

Asp Asp Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin Thr Tyr Val 130 135 140

Arg Ala Leu Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp He 145 150 155 160

Arg He Asp Thr Ser Cys Val Cys Ala Leu Ser Arg Lys He 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:

Gin Pro Val He Ala Met Asp Thr Glu Leu Leu Arg Gin Gin 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 He Asp He Arg Gly His Gin Val Thr Val Leu Gly Glu 85 90 95

He Lys Thr Gly Asn Ser Pro Val Lys Gin Tyr Phe Tyr Glu Thr Arg 100 105 110

Cys Lys Glu Ala Arg Pro Val Lys Asn Gly Gly Arg Gly He Asp Asp 115 120 125

Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin Thr Tyr Val Arg Ala 130 135 140

Leu Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp He Arg He 145 150 155 160

Asp Thr Ser Cys Val Cys Ala Leu Ser Arg Lys He 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 Gin Pro Met He Ala Thr Asp Thr Glu Leu Leu Arg Gin Gin 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 He Asp He Arg Gly His Gin Val Thr Val 85 90 95

Leu Gly Glu He Lys Thr Gly Asn Ser Pro Val Lys Gin 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

He Asp Asp Lys His Trp Asn Ser Gin Cys Lys Thr Ser Gin 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

He Arg He Asp Thr Ser Cys Val Cys Ala Leu Ser Arg Lys He 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 Gin 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 He 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 Gin Leu Lys Gin Tyr Phe Tyr Glu 85 90 95 Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Gly He 100 105 110

Asp Lys Arg His Trp Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val 115 120 125

Arg Ala Leu Thr Met Aβp Ser Lys Lys Arg He Gly Trp Arg Phe He 130 135 140

Arg He Asp Thr Ser Cys Val Cys Thr Leu Thr He 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 Gin 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 He 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 Gin Leu Lys Gin Phe Phe Tyr Glu 85 90 95

Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Asp He 100 105 110

Asp Lys Arg His Trp Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val 115 120 125

Arg Ala Leu Thr Met Asp Ser Lys Lys Arg He Gly Trp Arg Phe He 130 135 140

Arg He Asp Thr Ser Cys Val Cys Thr Leu Thr He 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 Gin 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 He 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 Gin Leu Lys Gin Tyr Phe Tyr Glu 85 90 95

Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Asp He 100 105 110

Asp Lys Arg His Trp Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val 115 120 125

Arg Ala Leu Thr Met Asp Ser Lys Lys Arg He Gly Trp Arg Phe He 130 135 140

Arg He Asp Thr Ser Cys Val Cys Thr Leu Thr He 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 Gin 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 He 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 Gin Leu Lys Gin Tyr Phe Tyr Glu 85 90 95

Thr Lys Cys Asn Pro Met Gly Tyr Thr Lys Glu Gly Gly Arg Asp He 100 105 110

Asp Lys Arg His Trp Asn Ser Gin Cys Arg Thr Thr Gin Ser Tyr Val 115 120 125 Arg Ala Leu Thr Met Asp Ser Lys Lys Arg He Gly Trp Arg Phe He 130 135 140

Arg He Asp Thr Ser Cys Val Cys Thr Leu Thr He Lys Arg Gly Arg 145 150 155 160

(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 Gin Thr Arg Asn He 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

Gin Pro Pro Pro Thr Ser Ser Asp Thr Leu Asp Leu Asp Phe Gin Ala 35 40 45

His Gly Thr He Pro Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser 50 55 60

Ser His Pro He 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 He Lys Gly Lys 85 90 95

Glu Val Met Val Leu Gly Glu Val Asn He Asn Asn Ser Val Phe Lys 100 105 110

Gin Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser 115 120 125

Gly Gly Arg Asp He 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 Gin Ala Ala 145 150 155 160

Trp Arg Phe He Arg He 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 Gin Thr Arg Asn He 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

Gin Pro Pro Pro Thr Ser Ser Asp Thr Leu Asp Leu Asp Phe Gin Ala 35 40 45

His Gly Thr He 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 He Lys Gly Lys 85 90 95

Glu Val Thr Val Leu Gly Glu Val Asn He Asn Asn Ser Val Phe Lys 100 105 110

Gin Tyr Phe Phe Glu Thr Lys Cys Arg Ala Pro Asn Pro Val Glu Ser 115 120 125

Gly Gly Arg Asp He 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 Gin Ala Ala 145 150 155 160

Trp Arg Phe He Arg He 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 Gin Thr Arg Asn He Thr Val Asp Pro Arg 1 5 10 15

Phe Lys Lys Arg Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gin 20 25 30

Pro Pro Arg Glu Ala Asp Thr Thr Gin 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 He Phe His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser 65 70 75 80 Val Trp Val Gly Aβp Lys Thr Thr Ala Thr Asp He Lys Gly Lys Glu 85 90 95

Val Met Val Leu Gly Glu Val Asn He Asn Asn Ser Val Phe Lys Gin 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 He 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 Gin Ala Ala Trp 145 150 155 160

Arg Phe He Arg He 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

Gin Pro Pro Pro Glu Ser Arg Lys Gly Gin 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 He Lys Gly Lys Glu Val 65 70 75 80

Thr Val Leu Gly Glu Val Asn He Asn Asn Asn Val Phe Lys Gin 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 He 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 Gin Ala Ala Trp Arg 130 135 140

Phe He Arg He 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 He Ser Val Trp Val Gly Asp Lys Thr 20 25 30

Thr Ala Thr Asp He Lys Gly Lys Glu Val Met Val Leu Gly Glu Val 35 40 45

Asn He Asn Asn Ser Val Phe Lys Gin Tyr Phe Phe Glu Thr Lys Cys 50 55 60

Arg Asp Pro Asn Pro Val Asp Ser Gly Gly Arg Asp He 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 Gin Ala Ala Trp Arg Phe He Arg He Asp Thr 100 105 110

Ser Cys Val Cys Val Leu Ser Arg Lys Thr Gly Gin 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 Gin Lys Arg Gin Phe Gin Ser Pro Arg Val 1 5 10 15

Leu Phe Ser Thr Gin 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 He 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 He 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 Gin Tyr Phe Phe Glu Thr Lys Cys Arg Asn Pro Asn Pro Glu Pro 100 105 110

Ser Gly Gly Arg Asp He Asp Ser Ser His Trp Asn Ser Tyr Cys Thr 115 120 125

Glu Thr Asp Gly Phe He Lys Ala Leu Thr Met Glu Gly Asn Gin Ala 130 135 140

Ser Trp Arg Phe He Arg He Asp Thr Ser Cys Val Cys Val He 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 He 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 He 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 Gin Leu Phe Tyr Glu Thr 100 105 110

Thr Cys Arg Ser Pro Thr His Arg Ser Ser Gly He Val He Gly Gly 115 120 125

Arg Ser Gly Gly Arg Gly Gly Ser Gin Gly Ser Lys Thr Gly Asn Ser 130 135 140

Gly Gly Arg Asp He Asp Ser Arg Tyr Trp Asn Ser His Cys Thr Asn 145 150 155 160

Thr Asp He Tyr Val Ser Ala Leu Thr Val Phe Lys Glu Gin Thr Ala 165 170 175

Trp Arg Phe He Arg He 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 Gin Arg Gly Val Ser Asp Thr Ser Pro Ala Ser His Gin 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 Gin Arg Gly Val Ser Asp Thr Ser Pro Ala Ser His Gin 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 Gin Trp He 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 Gin 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 Gin 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 He Arg He 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 He Met Ser His His Arg Ser Lys Val Pro Ser Gin 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 Gin 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 Gin 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 Gin 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 He Gin 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 He Met Ser His His Arg Ser Lys Val Pro Ser Gin 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 Gin 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 Gin 85 90 95

Ser Tyr Val Arg Ala Leu Thr Ala Asp Ala Gin Gly Arg Val Asp Trp 100 105 110

Arg Trp He Gin 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

Claims

WE CLAIM:

1. A method of treating an NT-4 related motor neuron disorder comprising administering, to a patient in need of such treatment, an effective amount of an NT-4 protein capable of supporting the survival, growth and/or differentiation of motor neurons as demonstrated in an in vitro culture system.

2. The method of claim 1 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.

3. The method of claim 1 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.

4. The method of claim 1 comprising administering, in combination with said NT-4 protein, an effective amount of a second neurotrophic factor capable of supporting the survival, growth and/or differentiation of motor neurons as demonstrated in an in vitro culture system.

5. The method of claim 4 in which the second neurotrophic factor is ciliary neurotrophic factor, neurotrophin-3 or nerve growth factor.

6. A method of promoting dopaminergic neuron survival, growth, and/or differentiation comprising exposing the neurons to an effective concentration of an NT-4 protein that is capable of promoting the survival, growth, and/or differentiation of dopaminergic neurons as demonstrated in an in vitro culture system.

7. A method of treating a dopaminergic neuron disease or disorder comprising administering, to a patient in need of such treatment, an effective amount of an NT-4 protein that is capable of promoting the survival, growth, and/or differentiation of dopaminergic neurons as demonstrated in an in vitro culture system.

8. The method of claim 59 wherein said disease or disorder is Parkinson's disease.

9. A method of promoting cholinergic neuron survival, growth, and/or differentiation comprising exposing the neurons to an effective concentration of an NT-4 protein that is capable of promoting the survival, growth, and/or differentiation of cholinergic neurons as demonstrated in an in vitro culture system.

10. A method of treating a cholinergic neuron disease or disorder comprising administering, to a patient in need of such treatment, an effective amount of an NT-4 protein that is capable of promoting the survival, growth, and/or differentiation of cholinergic neurons as demonstrated in an in vitro culture system.

11. The method according to claim 10 in which the cholinergic neurons are basal forebrain cholinergic neurons.

12. The method according to claim 10 in which the cholinergic neurons are septal cholinergic neurons.

13. The method of claim 10 wherein said disease or disorder is Alzheimer's disease.

14. A method of treating a peripheral neuropathy comprising administering, to a patient in need of such treatment, an effective amount of an NT-4 related protein that is capable of promoting the survival, growth, and/or differentiation of dorsal root ganglia or other sensory neurons as demonstrated in an in vitro culture system.

15. The method of claim 14 wherein said peripheral neuropathy is acute neurapraxia, neurotmesis, axotmesis, diabetic neuropathy, amyotrophic lateral sclerosis and compression.

16. A method of treating a disease or disorder involving cells of the

hippocampus comprising administering, to a patient in need of such treatment, an effective amount of an NT-4 related protein that is capable of promoting the survival, growth, and/or differentiation of hippocampal cells as demonstrated in an in vitro culture system.

17. The method of claim 16 wherein said disease or disorder is related to ischaemia, hypoxia, hypoglycemia or stroke.

18. A method of treating a disease or disorder involving cells of the striatum comprising administering, to a patient in need of such treatment, an effective amount of an NT-4 related protein that is capable of promoting the survival, growth, and/or differentiation of striatal cells as demonstrated in an in vitro culture system

19. The method of claim 18 wherein said disease or disorder is Huntington's chorea, striatonigral degeneration, cerebral palsy, stroke, ischaemia, hypoxia or hypoglycemia.

20. A method of diagnosing an NT-4 related peripheral nervous system disorder comprising injecting detectably labeled NT-4 into a peripheral nerve and determining whether the labeled NT-4 is retrogradely transported, in which a failure to be retrogradely transported positively correlates with lack of responsiveness to NT-4 and indicates that the peripheral nervous system disorder is NT-4 related.

21. The method of claim 20 in which the disease or disorder is selected from the group consisting of acute neurapraxia, neurotmesis, axotmsesis, diabetic neuropathy, amyotrophic lateral sclerosis and compression.

22. A method of diagnosing an NT-4 related central nervous system

disorder comprising injecting detectably labeled NT-4 into a central nerve and determining whether the labeled NT-4 is retrogradely transported, in which a failure to be retrogradely transported positively correlates with lack of responsiveness to NT-4 and indicates that the central nervous system disorder is NT-4 related.

23. The method of claim 22 in which the disease or disorder is selected from the group consisting of tumor, abscess, trauma, Alzheimer's disease, and Parkinson's disease.

24. A method of treating a disease or disorder of the retina comprising administering to a patient in need of such treatment, an effective amount of an NT-4 related protein.

25. The method of claim 24 where said disease or disorder is retinal

detachment, age related or other maculopathies, photic retinopathy, surgery-induced retinopathy, retinopathy of prematurity, viral retinopathy, uvetis, ischemic retinopathy due to venous or arterial occlusion or other vascular disorders, retinopathy due to trauma or penetrating lesions of the eye, peripheral vitreoretinopathy or inherited retinal degeneration.

26. The method of claim 24 wheren said disease or disorder involves the optic nerve.

27. The method of claim 24 wherein said disease or disorder involves degeneration of retinal ganglion cells.

28. A method of treating seizures comprising admistering to a patient in need of such treatment, an effective amount of NT-4.