NEUROGENIC DIFFERENTIATION (NeuroD) GENES AND PROTEINS

Abstract: Neurogenic differentiation genes and proteins from human, mouse, and frog are identified, isolated, and sequenced. Expression of neuroD1 has been demonstrated in neural, pancreatic, and gastrointestinal cells, and expression of neuroD2 was detected in brain. Ectopic expression of neuroD1 and neuroD2 in non-neuronal cells of Xenopus embryos induced formation of neurons. NeuroD1 knock-out mice had diabetes.

Description: NEUROGENIC DIFFERENTIATION (NeuroD) GENES AND PROTEINS This invention was made with government support under grant CA42506 awarded by the National Institutes of Health The government has certain rights in the invention This application is a continuation-in-part of co-pending U.S application No 08/552,142, filed November 2, 1995, which is a continuation-in-part of PCT application No PCT/US95/05741, which is a continuation-in-part of parent application U S Serial No 08/239,238, filed May 6, 1994 (abandoned) Field ofthe Invention The invention relates to molecular biology and in particular to genes and proteins involved in vertebrate neural development Background ofthe Invention Transcription factors of the basic-helix-loop-helix (bHLH) family are implicated in the regulation of differentiation in a wide variety of cell types, including trophoblast cells (Cross et al , Development 121 2513-2523, 1995), pigment cells (Steingrimsson et al , Nature Gen 8 251-255, 1994), B-cells (Shen, C P and T Kadesch , Molec. & Cell. Biol 15.3813-3822, 1995, Zhuang et al , Cell 79 875-884, 1994), chondrocytes and osteoblasts (Cserjesi et al., Development 121 1099-1110, 1995, Tamura, M and M Noda , J. Cell Biol 126 773-782, 1994), and cardiac muscle (Burgess et al , Develop. Biol 168.296-306, 1995, Hollenberg et al , Molec. & Cell. Biol 15.3813-3822, 1995) bHLH proteins form homodimeric and heterodimeric complexes that bind with DNA m the 5' regulatory regions of genes controlling expression Perhaps the most extensively studied sub-families of bHLH proteins are those that regulate myogenesis and neurogenesis The myogenic bHLH factors, (MyoD, myogenin, Myf5, and MRF4), appear to have unique as well as redundant functions during myogenesis (Weintraub, H , Cell 75 1241-1244, 1993, Weintraub et al , Science 251 761-766, 1991) It is thought that either Myf5 or MyoD is necessary to determine myogenic fate, whereas myogenin is necessary for events involved in terminal differentiation (Hasty et al , Nature 364 501-506, 1993, Nabeshima et al , Nature 364 532-535, 1993, Rudnicki et al , Cell 75 1351-1359, 1993, Venuti et al , J. Cell Biol 128.563-576, 1995) Recent work on neurogenic bHLH proteins suggests parallels between the myogenic and neurogenic sub-families of bHLH proteins Genes of the Drosophila melanogaster achaete-scute complex and the atonal gene have been shown to be involved in neural cell fate determination (Anderson, D J , Cur. Biol 5 1235-1238, 1995, Campuzano, S and j Modolell , Trends in Genetics 8 202-208, 1992, Jaman et al , Cell 73 1307-1321, 1993), and the mammalian homologs, MASH1 and MATH1, are expressed in the neural tube at the time of neurogenesis (Akazawa et al , J. Biol. Chem 270 8730-8738, 1995, Lo et al , Genes & Dev 5 1524-1537, 1991) Two related vertebrate bHLH proteins, neuroD (hereafter referred to as "neuroDl ") and NEX-l/MATH-2, are expressed slightly later in CNS development, predominantly in the marginal layer of the neural tube and persisting in the mature nervous system (Bartholoma, A and K A Nave , Mech. Dev 48 217-228, 1994, Lee et al , Science 268 836-844, 1995, Shimizu et al , Eur. J. Biochem 229.239-248, 1995) NeuroDl was also cloned as a factor that regulates insulin transcription in pancreatic beta cells and named "Beta2" (Naya et al , Genes & Dev 9 1009-1019, 1995) Constitutive expression of neuroDl in developing Xenopus embryos produces ectopic neurogenesis in the ectodermal cells, indicating that neuroD is capable of regulating a neurogenic program A neuroDl homolog having 36,873 nucleotides has been identified in C. elegans (Lee et al , 1995, Genbank Accession No 010402), suggesting that this molecular mechanism of regulating neurogenesis may be conserved between vertebrates and invertebrates Neural tissues and endocrine tissues do not regenerate Damage is permanent Paralysis, loss of vision or hearing, and hormonal insufficiency are also intractable medical conditions Furthermore, tumors in neural and endocrine tissues can be very difficult to treat because of the toxic side effects that conventional chemotherapeutic drugs may have on nervous tissues. The medical community and public would greatly benefit from the availability of agents active in triggering differentiation in neuroectodermal stem cells. Such neuronal differentiating agents could be used for construction of test cell lines, assays for identifying candidate therapeutic agents capable of inducing regeneration of neuronal and endocrine tissues, gene therapy, and differentiation of tumor cells. Summary ofthe Invention The presently disclosed neuroD proteins represent a new sub-family of bHLH proteins and are implicated in vertebrate neuronal, endocrine and gastrointestinal development. Mammalian and amphibian neuroD proteins were identified, and polynucleotide molecules encoding neuroD proteins were isolated and sequenced. NeuroD genes encode proteins that are distinctive members of the bHLH family. In addition, the present invention provides a family of neuroD proteins that share a highly conserved HLH region. Representative polynucleotide molecules encoding members ofthe neuroD family include neuroDl, neuroDl and neuroD3. A representative nucleotide sequence encoding murine neuroDl is shown in SEQ ID NO: l. The HLH coding domain of murine neuroDl resides between nucleotides 577 and 696 in SEQ ID NO: l. The deduced amino acid sequence of murine neuroDl is shown in SEQ ID NO:2. There is a highly conserved region following the helix-2 domain from amino acid 150 through amino acid 199 of SEQ ID NO: 2 that is not shared by other bHLH proteins. A representative nucleotide sequence encoding Xenopus neuroDl is shown in SEQ ID NO:3. The HLH coding domain of Xenopus neuroDl resides between nucleotides 376 and 495 in SEQ ID NO:3. The deduced amino acid sequence of Xenopus neuroDl is shown in SEQ ID NO:4. There is a highly conserved region following the helix-2 domain from amino acid 157 through amino acid 199 of SEQ ID NO:4 that is not shared by other bHLH proteins. Representative nucleotide and deduced amino acid sequences of the human neuroD family are shown in SEQ ID NOS:8-15. Representative nucleotide and deduced amino acid sequences of a human homolog of murine neuroDl are shown in SEQ ID NOS: 8 and 9 (partial genomic sequence) and SEQ ID NOS: 14 and 15 (human neuroDl cDNA). Representative nucleotide and deduced amino acid sequences of the human and murine neuroDl are shown in SEQ ID NOS: 10 and 11, and 16 and 17, respectively. Representative nucleotide and deduced amino acid sequences for human neuroD 3 are shown in SEQ ID NOS: 12 and 13. The disclosed human clones, 9Fl(and its corresponding cDNA HC2A; now referred to as human neuroDl) and 14B1 (now referred to as human neuroDl), have an identical HLH motif: Amino acid residues 117-156 in SEQ ID NO:9 and 15, and residues 137-176 in SEQ ED NO: 11 (corresponding to nucleotides 405-524 of SEQ ID NO: 8 and SEQ ID NO: 14, and nucleotides 463-582 of SEQ ID NO: 10). Comparison ofthe deduced amino acid sequences of these neuroD genes shows that human neuroD3 contains an HLH domain between amino acid residues 108-147 of SEQ ID NO: 13 (corresponding to nucleotides 376-495 of SEQ ID NO: 12) and that murine neuroD2 contains an HLH domain between amino acids residues 138-177 of SEQ ID NO: 17 (corresponding to nucleotides 641-760 of SEQ ID NO: 16) The HLH domain of murine neuroD2 is identical to that of the human neuroDl and human neuroD2 proteins. Similar analyses indicated that mouse neuroD3 contains an HLH domain between amino acid residues 109-148 of SEQ ID NO:22 (corresponding to nucleotides 425-544 of SEQ ID NO:21) Brief Description ofthe Drawings FIGURE 1 schematically depicts the domain structure of the murine and Xenopus neuroD bHLH proteins. Detailed Description ofthe Preferred Embodiment Tissue-specific bHLH proteins that regulate early neuroectodermal differentiation were discovered using expression cloning and screening assays designed to identify possible bHLH proteins capable of interacting with the protein product of the Drosophila daughterless gene. These proteins belong to a family of proteins that share conserved residues in the HLH region. The subject invention provides neuroDl and neuroD3, which are two novel genes related to neuroDl, and which have been isolated and whose nucleotide sequences have been determined. The term "neuroD," as used here, encompasses all members of the neuroD family, and includes neuroDl, neuroDl and neuroD3 coding sequences and proteins. The neuroD family of genes function during the development of the nervous system. Like MATH1 (Lo et al., Genes & Dev. 5:1524-1537, 1991), the expression of neuroD 3 peaks during embryonic development and is not detected in the mature nervous system. NeuroDl shows a high degree of sequence similarity to both neuroDl and NEX-1 MATHl, and is similarly expressed both during embryogenesis and in the mature nervous system, demonstrating an expression pattern that partially overlaps with neuroDl. Like neuroDl, neuroDl when expressed by transfection in Xenopus embryos induces neurogenesis in ectodermal cells. Transfection of expression vectors for neuroDl and neuroDl indicates that these highly similar transcription factors demonstrate some target specificity, with the GAP-43 promoter being activated by neuroDl and not by neuroDl. The partially overlapping expression pattern and target specificity of neuroDl and neuroDl suggests that this group of neurogenic transcription factors may contribute to the establishment of neuronal identity in the nervous system by acting on an overlapping but non- congruent set of target genes. NeuroD proteins are transiently expressed in differentiating neurons during embryogenesis. NeuroD proteins are also detected in adult brain, in the granule layer of the hippocampus and the cerebellum. In addition, murine neuroDl expression has been detected in the pancreas and gastrointestinal tissues of developing embryos and post-natal mice (see, e.g., Example 14). NeuroD proteins contain the basic helix-loop-helix (bHLH) domain structure that has been implicated in the binding of bHLH proteins to upstream recognition sequences and activation of downstream target genes. The present invention provides representative neuroD proteins, which include the murine neuroDl protein of SEQ ID NO:2, the amphibian neuroDl protein of SEQ ID NO:4, murine neuroD2 protein of SEQ ID NO: 17, human neuroDl protein of SEQ ID NOS 9 and 15, human neuroD2 protein of SEQ ID NO: 11, human neuroD3 protein of SEQ ID NO: 13, and mouse neuroD3 protein SEQ ID NO:22. Based on homology with other bHLH proteins, the bHLH domain for murine neuroDl is predicted to reside between amino acids 102 and 155 of SEQ ID NO:2, and between amino acids 101 and 157 of SEQ ID NO:4 for the amphibian neuroDl . As detailed below, the present invention provides the identification of human neuroDl and, in addition, provides unexpected homologous genes of the same family based on highly conserved sequences across the HLH domain shared between the two human genes at the amino acid level (neuroDl and neuroD3, SEQ ID NOS: 10 and 11, and 12 and 13, respectively). NeuroD proteins are transcriptional activators that control transcription of downstream target genes including genes that among other activities cause neuronal progenitors to differentiate into mature neurons. In the neurula stage of the mouse embryo (elO), murine neuroDl is highly expressed in the neurogenic derivatives of neural crest cells, the cranial and dorsal root ganglia, and postmitotic cells in the central nervous system (CNS). During mouse development, neuroDl is expressed transiently and concomitant with neuronal differentiation in differentiating neurons in sensory organs such as in nasal epithelium and retina In Xenopus embryos, ectopic expression of neuroDl in non-neuronal cells induced formation of neurons As discussed in more detail below, neuroD proteins are expressed in differentiating neurons and are capable of causing the conversion of non-neuronal cells into neurons The present invention encompasses variants of neuroD genes that, for example, are modified in a manner that results in a neuroD protein capable of binding to its recognition site, but unable to activate downstream genes The present invention also encompasses fragments of neuroD proteins that, for example, are capable of binding the natural neuroD partner, but that are incapable of activating downstream genes NeuroD proteins encompass proteins retrieved from naturally occurring materials and closely related, functionally similar proteins retrieved by antisera specific to neuroD proteins, and recombinantly expressed proteins encoded by genetic materials (DNA, RNA, cDNA) retrieved on the basis of their similarity to the unique regions in the neuroD family of genes The present invention provides representative isolated and purified polynucleotide molecules encoding proteins of the neuroD family Representative polynucleotide molecules encoding various neuroD proteins include the sequences presented in SEQ ED NOS.1, 3, 8, 10, 12, 14, and 16 Polynucleotide molecules encoding neuroD include those sequences resulting in minor genetic polymoφhisms, differences between species, and those that contain amino acid substitutions, additions, and/or deletions. According to the present invention, polynucleotide molecules encoding neuroD proteins encompass those molecules that encode neuroD proteins or peptides that share identity with the sequences shown in SEQ ID NOS.2, 4, 9, 11, 13, 15, and 17 Such molecules will generally share greater than 35% identity at the amino acid level with the disclosed sequences The neuroD genes of the present invention may share greater identity at the amino acid level across highly conserved regions such as the HLH domain For example, the deduced amino acid sequences of murine and Xenopus neuroDl genes are 96% identical within this domain. In some instances, one may employ such changes in the sequence of a recombinant neuroD polynucleotide molecule to substantially decrease or even increase the biological activity of neuroD protein relative to the wild-type neuroD activity, depending on the intended use ofthe preparation Such changes may also be directed towards endogenous neuroD polynucleotide sequences using, for example, gene therapy methods to alter the gene product. Such changes are envisioned with regard to neuroDl, neuroDl, neuroD3, or other members ofthe neuroD gene family. The neuroDl proteins of the present invention are capable of inducing the expression in a frog embryo of neuron-specific genes, such as N-C AM, β-tubulin, and Xen-1, neurofilament M (NF-M), Xen-2, tanabin-1, shaker- 1, and frog HSCL. As described below in Example 10, neuroDl activity may be detected when neuroD is ectopically expressed in frog oocytes following, for example, injection of Xenopus neuroDl RNA into one of the two cells in a two-cell stage Xenopus embryo, and monitoring expression of neuronal-specific genes in the injected as compared to uninjected side ofthe embryo by immunochemistry or in situ hybridization. "Over-expression" means an increased level of a neuroD protein or oϊ neuroD transcripts in a recombinant transformed host cell relative to the level of protein or transcripts in the parental cell from which the host cell is derived. As noted above, the present invention provides isolated and purified polynucleotide molecules encoding various members of the neuroD family. The disclosed sequences may be used to identify and isolate additional neuroD polynucleotide molecules from suitable mammalian or non-mammalian host cells such as canine, ovine, bovine, caprine, lagomorph, or avian. In particular, the nucleotide sequences encoding the HLH region may be used to identify polynucleotide molecules encoding other proteins of the neuroD family. Complementary DNA molecules encoding neuroD family members may be obtained by constructing a cDNA library mRNA from, for example, fetal brain, newborn brain, and adult brain tissues. DNA molecules encoding neuroD family members may be isolated from such a library using the disclosed sequences to provide probes to be used in standard hybridization methods (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989, which is incorporated herein by reference), and Bothwell, Yancopoulos and Alt, ibid.) or by amplification of sequences using polymerase chain reaction (PCR) amplification (e.g., Loh et al., Science 243:217-222, 1989; Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998-9002, 1988; Erlich (ed.), PCR Technology: Principles and Applications for DNA Amplification, Stockton Press, 1989; and Mullis et al., PCR: The Polymerase Chain Reaction, 1994, which are incorporated by reference herein in their entirety). In a similar manner, genomic DNA encoding neuroD proteins may be obtained using probes designed from the sequences disclosed herein. Suitable probes for use in identifying neuroD genes or transcripts may be obtained from newroZ)-specific sequences that are highly conserved regions between mammalian and amphibian neuroD coding sequences. Nucleotide sequences, for example, from the region encoding the approximately 40 residues following the helix-2 domain are suitable for use in designing PCR primers. Alternatively, oligonucleotides containing specific DNA sequences from a human neuroDl, neuroDl, or neuroD3 coding region may be used within the described methods to identify related human neuroD genomic and cDNA clones. Upstream regulatory regions of neuroD may be obtained using the same methods. Suitable PCR primers are between 7-50 nucleotides in length, more preferably between 15 and 25 nucleotides in length. Alternatively, neuroD polynucleotide molecules may be isolated using standard hybridization techniques with probes of at least about 15 nucleotides in length and up to and including the full coding sequence. Southern analysis of mouse genomic DNA probed with the murine neuroDl cDNA under stringent conditions showed the presence of only one gene, suggesting that under stringent conditions bHLH genes from other protein families will not be identified. Other members of the neuroD family can be identified using degenerate oligonucleotides based on the sequences disclosed herein for PCR amplification or by hybridization at moderate stringency using probes based on the disclosed sequences. The regulatory regions of neuroD may be useful as tissue-specific promoters. Such regulatory regions may find use in, for example, gene therapy to drive the tissue- specific expression of heterologous genes in pancreatic, gastrointestinal, or neural cells, tissues or cell lines. As shown in Example 14, murine neuroDl promoter sequences reside within the 1.4 kb 5' untranslated region. Regulatory sequences within this region are identified by comparison to other promoter sequences and/or deletion analysis ofthe region itself. In other aspects ofthe invention, a DNA molecule coding a neuroD protein is inserted into a suitable expression vector, which is in turn used to transfect or transform a suitable host cell. Suitable expression vectors for use in carrying out the present invention include a promoter capable of directing the transcription of a polynucleotide molecule of interest in a host cell and may also include a transcription termination signal, these elements being operably linked in the vector. Representative expression vectors may include both plasmid and/or viral vector sequences. Suitable vectors include retroviral vectors, vaccinia viral vectors, CMV viral vectors, BLUESCRIPT® vectors, baculovirus vectors, and the like. Promoters capable of directing the transcription of a cloned gene or cDNA may be inducible or constitutive promoters and include viral and cellular promoters. For expression in mammalian host cells, suitable viral promoters include the immediate early cytomegalovirus promoter (Boshart et al., Cell 41:521-530, 1985) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981). Suitable cellular promoters for expression of proteins in mammalian host cells include the mouse metallothionine-1 promoter (Palmiter et al., U.S. Patent No. 4,579,821), a mouse Vk promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983; Grant et al. Nucleic Acid. Res. 15:5496, 1987), and tetracycline-responsive promoter (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992, and Pescini et al., Biochem. Biophys. Res. Comm. 202: 1664-1667, 1994). Also contained in the expression vectors, typically, is a transcription termination signal located downstream of the coding sequence of interest. Suitable transcription termination signals include the early or late polyadenylation signals from SV40 (Kaufman and Shaφ, Mol. Cell. Biol. 2: 1304- 1319, 1982), the polyadenylation signal from the Adenovirus 5 elB region, and the human growth hormone gene terminator (DeNoto et al., Nucleic Acid. Res. 9:3719- 3730, 1981). Mammalian cells, for example, may be transfected by a number of methods including calcium phosphate precipitation (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973), lipofection, microinjection, and electroporation (Neumann et al., EMBO J. 1:8410845, 1982). Mammalian cells can be transduced with viruses such as SV40, CMV, and the like. In the case of viral vectors, cloned DNA molecules may be introduced by infection of susceptible cells with viral particles. Retroviral vectors may be preferred for use in expressing neuroD proteins in mammalian cells particularly if the neuroD genes used for gene therapy (for review, see, Miller et al. Methods in Enzymology 217:581-599, 1994; which is incoφorated herein by reference in its entirety). It may be preferable to use a selectable marker to identify cells that contain the cloned DNA. Selectable markers are generally introduced into the cells along with the cloned DNA molecules and include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. Selectable markers may also complement auxotrophs in the host cell. Yet other selectable markers provide detectable signals, such as β-galactosidase to identify cells containing the cloned DNA molecules. Selectable markers may be amplifiable. Such amplifiable selectable markers may be used to amplify the number of sequences integrated into the host genome. As would be evident to one of ordinary skill in the art, the polynucleotide molecules of the present invention may be expressed in Saccharomyces cerevisiae, filamentous fungi, and E. coli. Methods for expressing cloned genes in Saccharomyces cerevisiae are generally known in the art (see, "Gene Expression Technology," Methods in Enzymology, Vol. 185, Goeddel (ed ), Academic Press, San Diego, CA, 1990; and "Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology, Guthrie and Fink (eds ), Academic Press, San Diego, CA, 1991, which are incoφorated herein by reference). Filamentous fungi may also be used to express the proteins of the present invention; for example, strains of the fungi Aspergillus (McKnight et al., U.S. Patent No. 4,935,349, which is incoφorated herein by reference). Methods for expressing genes and cDNAs in cultured mammalian cells and in E. coli are discussed in detail in Sambrook et al., 1989. As will be evident to one skilled in the art, one can express the protein ofthe instant invention in other host cells such as avian, insect, and plant cells using regulatory sequences, vectors and methods well established in the literature. NeuroD proteins produced according to the present invention may be purified using a number of established methods such as affinity chromatography using anti- neuroD antibodies coupled to a solid support. Fusion proteins of antigenic tag and neuroD can be purified using antibodies to the tag. Additional purification may be achieved using conventional purification means such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others Methods of protein purification are known in the art (see generally, Scopes, R., Protein Purification, Springer- Verlag, NY, 1982, which is incoφorated herein by reference) and may be applied to the purification of recombinant neuroD described herein. The term "capable of hybridizing under stringent conditions" as used herein means that the subject nucleic acid molecules (whether DNA or RNA) anneal under stringent hybridization conditions to an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16. It is generally known that oligonucleotides 15 nucleotides or more in length are extremely unlikely to be represented more than once in a mammalian genome, hence such oligonucleotides can form specific hybrids (see, for example, Sambrook et al, Molecular Cloning, [2d ed ], Cold Spring Harbor Laboratory Press, 1989, at Section 11.7). "Stringent hybridization" is generally understood in the art to mean that the nucleic acid duplexes that form during the hybridization reaction are perfectly matched or nearly perfectly matched Several rules governing nucleic acid hybridization have been well established For example, it is standard practice to achieve stringent hybridization for polynucleotide molecules >200 nucleotides in length by hybridizing at a temperature 15°-25°C below the melting temperature (Tm) of the expected duplex, and 5°-10°C below the Tm for oligonucleotide probes (e.g., Sambrook et al., at Section 11.45). The Tm of a nucleic acid duplex be calculated using a formula based on the % G+C contained in the nucleic acids, and that takes chain length into account, such as the formula Tm = 81.5 - 16.6 (log [Na + ]) + 0.41 (% G+C) - (600/N), where N = chain length (Sambrook et al., 1989, at Section 1 1.46). It is apparent from this formula that the effects of chain length on Tm is significant only when rather short nucleic acids are hybridized, and also that the length effect is negligible for nucleic acids longer than a few hundred bases. The choice of hybridization conditions will be evident to one skilled in the art and will generally be guided by the puφose of the hybridization, the type of hybridization (DNA-DNA or DNA-RNA), and the level of desired relatedness between the sequences. As discussed above, methods for hybridization are well established in the literature. See also, for example: Sambrook et al., ibid.; Hames and Higgins, eds., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington DC, 1985; Berger and Kimmel, eds., Methods in Enzymology, Vol. 51, Guide to Molecular Cloning Techniques, Academic Press Inc., New York, NY, 1987; and Bothwell, Yancopoulos and Alt, eds., Methods for Cloning and Analysis of Eukaryotic Genes, Jones and Bartlett Publishers, Boston, MA 1990; which are incoφorated by reference herein in their entirety. One of ordinary skill in the art realizes that the stability of nucleic acid duplexes will decrease with an increased number and location of mismatched bases; thus, the stringency of hybridization may be used to maximize or minimize the stability of such duplexes. Hybridization stringency can be altered by: adjusting the temperature of hybridization; adjusting the percentage of helix-destabilizing agents, such as formamide, in the hybridization mix; and adjusting the temperature and/or salt concentration of the wash solutions. In general, the stringency of hybridization is adjusted during the post-hybridization washes by varying the salt concentration and/or the temperature. Stringency of hybridization may be reduced by reducing the percentage of formamide in the hybridization solution or by decreasing the temperature of the wash solution. High stringency conditions may involve high temperature hybridization (e.g., 65-68°C in aqueous solution containing 4-6 X SSC (1 X SSC = 0.15 M NaCl, 0.015 M sodium citrate), or 42°C in 50% formamide) combined washes at high temperature (e.g., 5- 25°C below the T m ), in a solution having a low salt concentration (e.g., 0.1 X SSC) Low stringency conditions may involve lower hybridization temperatures (e.g., 35- 42°C in 20-50% formamide) with washes conducted at an intermediate temperature (e.g., 40-60°C) and in a wash solution having a higher salt concentration (e.g., 2-6 X SSC). Moderate stringency conditions, which may involve hybridization in 0.2-0.3M NaCl at a temperature between 50°C and 65°C and washes in 0.1 X SSC, 0.1% SDS at between 50°C and 55°C, may be used in conjunction with the disclosed polynucleotide molecules as probes to identify genomic or cDNA clones encoding members ofthe neuroD family. The invention provides isolated and purified polynucleotide molecules encoding neuroD proteins that are capable of hybridizing under stringent conditions to an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO: l, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and/or SEQ ID NO: 16, and also including the polynucleotide molecules complementary to the coding strands. The subject isolated neuroD polynucleotide molecules preferably encode neuroD proteins that trigger differentiation in ectodermal cells, particularly neuroectodermal stem cells, and in more committed cells of that lineage, for example, epidermal precursor cells, pancreatic and gastrointestinal cells. Such neuroD expression products typically form heterodimeric bHLH protein complexes that bind in the 5 -regulatory regions of target genes and enhance or suppress transcription of the target gene. In some instances, cancer cells may contain a non-functional neuroD protein or may contain no neuroD protein due to genetic mutation or somatic mutations such that these cells fail to differentiate. For cancers of this type, the cancer cells may be treated in a manner to cause the over-expression of wild-type neuroD protein to force differentiation ofthe cancer cells. Antisense neuroD nucleotide sequences, that is, nucleotide sequences complementary to the non-transcribed strand of a neuroD gene, may be used to block expression of mutant neuroD expression in neuronal precursor cells to generate and harvest neuronal stem cells The use of antisense oligonucleotides and their applications have been reviewed in the literature (see, for example, Mol and Van der Krul, eds., Antisense Nucleic Acids and Proteins Fundamentals and Applications, New York, NY, 1992; which is incoφorated by reference herein in its entirety). Suitable antisense oligonucleotides are at least 11 nucleotide in length and may include untranslated (upstream or intron) and associated coding sequences. As will be evident to one skilled in the art, the optimal length of an antisense oligonucleotide depends on the strength of the interaction between the antisense oligonucleotide and the complementary mRNA, the temperature and ionic environment in which translation takes place, the base sequence of the antisense oligonucleotide, and the presence of secondary and tertiary structure in the target mRNA and/or in the antisense oligonucleotide. Suitable target sequences for antisense oligonucleotides include intron-exon junctions (to prevent proper splicing), regions in which DNA RNA hybrids will prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, and sites that interfere with ribosome progression. A particularly preferred target region for antisense oligonucleotide is the 5' untranslated (promoter/enhancer) region ofthe gene of interest. Antisense oligonucleotides may be prepared by the insertion of a DNA molecule containing the target DNA sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself The expression vector may then be transduced, transformed or transfected into a suitable cell resulting in the expression of antisense oligonucleotides. Alternatively, antisense oligonucleotides may be synthesized using standard manual or automated synthesis techniques. Synthesized oligonucleotides may be introduced into suitable cells by a variety of means including electroporation, calcium phosphate precipitation, liposomes, or microinjection. The selection of a suitable antisense oligonucleotide administration method will be evident to one skilled in the art. With respect to synthesized oligonucleotides, the stability of antisense oligonucleotide-mRNA hybrids may be increased by the addition of stabilizing agents to the oligonucleotide. Stabilizing agents include intercalating agents that are covalently attached to either or both ends of the oligonucleotide. Oligonucleotides may be made resistant to nucleases by, for example, modifications to the phosphodiester backbone by the introduction of phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates, or phosphorodithioates. Oligonucleotides may also be made nuclease resistant by synthesis of the oligonucleotides with alpha-anomers ofthe deoxyribonucleotides. NeuroD proteins bind to 5' regulatory regions of neurogenic genes that are involved in neuroectodermal differentiation, including development of neural and endocrine tissues. As described in more detail herein, murine neuroDl has been detected in neuronal, pancreatic and gastrointestinal tissues in embryonic and adult mice suggesting that neuroDl functions in the transcription regulation in these tissues. NeuroD proteins alter the expression of subject genes by, for example, down- regulating or up-regulating transcription, or by inducing a change in transcription to an alternative open reading frame. The subject polynucleotide molecules find a variety of uses, e.g., in preparing oligonucleotide probes, expression vectors, and transformed host cells, as disclosed below in the following Examples. DNA sequences recognized by the various neuroD proteins may be determined using a number of methods known in the literature including immunoprecipitation (Biedenkapp et al, Nature 335:835-837, 1988; Kinzler and Vorgelstein, Nuc. Acids Res. 17:3645-3653, 1989; and Sompayrac and Danna, Proc. Natl. Acad. Sci. USA 87:3274-3278, 1990; which are incoφorated by reference herein), protein affinity columns (Oliphant et al., Mol. Cell. Biol. 9:2944-2949, 1989; which is incoφorated by reference herein), gel mobility shifts (Blackwell and Weintraub, Science 250: 1104-1110, 1990; which is incoφorated by reference herein), and Southwestern blots (Keller and Maniatis, Nuc. Acids Res. 17:4675-4680, 1991; which is incoφorated by reference herein). One embodiment of the present invention involves the construction of inter- species hybrid neuroD proteins and hybrid neuroD proteins containing at least one domain from two or more neuroD family members to facilitate structure-function analyses or to alter neuroD activity by increasing or decreasing the neuroD-mediated transcriptional activation of neurogenic genes relative to the wild-type neuroD(s). Hybrid proteins of the present invention may contain the replacement of one or more contiguous amino acids ofthe native neuroD protein with the analogous amino acid(s) of neuroD from another species or other protein of the neuroD family. Such interspecies or interfamily hybrid proteins include hybrids having whole or partial domain replacements. Such hybrid proteins are obtained using recombinant DNA techniques. Briefly, DNA molecules encoding the hybrid neuroD proteins of interest are prepared using generally available methods such as PCR mutagenesis, site- directed mutagenesis, and or restriction digestion and ligation. The hybrid DNA is then inserted into expression vectors and introduced into suitable host cells. The biological activity may be assessed essentially as described in the assays set forth in more detail in the Examples that follow. The invention also provides synthetic peptides, recombinantly derived peptides, fusion proteins, and the like that include a portion of neuroD or the entire protein. The subject peptides have an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions with an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 8, SEQ ID NO 10, SEQ ED NO 12, SEQ ID NO 14, or SEQ ID NO 16 Representative amino acid sequences ofthe subject peptides are disclosed in SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, and SEQ ID NO 17 The subject peptides find a vanety of uses, including preparation of specific antibodies and preparation of agonists and antagonists of neuroD activity As noted above, the invention provides antibodies that bind to neuroD proteins The production of non-human antisera or monoclonal antibodies (e g , murine, lagormoφh, porcine, equine) is well known and may be accomplished by, for example, immunizing an animal with neuroD protein or peptides For the production of monoclonal antibodies, antibody producing cells are obtained from immunized animals, immortalized and screened, or screened first for the production of the antibody that binds to the neuroD protein or peptides and then immortalized It may be desirable to transfer the antigen binding regions (e g , F(ab')2 or hypervariable regions) of non-human antibodies into the framework of a human antibody by recombinant DNA techniques to produce a substantially human molecule Methods for producing such "humanized" molecules are generally well known and described in, for example, U S Patent No 4,816,397, which is incoφorated by reference herein in its entirety Alternatively, a human monoclonal antibody or portions thereof may be identified by first screening a human B-cell cDNA library for DNA molecules that encode antibodies that specifically bind to the neuroD family member, e g , according to the method generally set forth by Huse et al (Science 246 1275-1281, 1989, which is incoφorated by reference herein in its entirety) The DNA molecule may then be cloned and amplified to obtain sequences that encode the antibody (or binding domain) ofthe desired specificity The invention also provides methods for inducing the expression of genes associated with neuronal phenotype in a cell that does not normally express those genes Examples of neuronal phenotypes that may be modulated by neuroD expression include expression of neurotransmitters or neuromodulatory factors Cells that can be used for the puφose of modulation of gene expression by neuroD include cells of the neuroectodermal lineage, glial cells, neural crest cells, and epidermal epithelial basal stem cells, and all types of both mesodermal and endodermal lineage cells NeuroD expression may also be used within methods that induce expression of genes associated with pancreatic and gastrointestinal phenotype Examples of such gene expression include insulin expression, and gastrointestinal-specific enzyme expression. As illustrated in Example 10, the expression of Xenopus neuroDl protein in stem cells causes redirection of epidermal cell differentiation and induces terminal differentiation into neurons, i.e., instead of epidermal cells. Epithelial basal stem cells (i e , in skin and mucosal tissues) are one of the few continuously regenerating cell types in an adult mammal. Introduction of the subject nucleotide sequences into an epithelial basal stem cell may be accomplished in vitro or in vivo using a suitable gene therapy vector delivery system (e.g., a retroviral vector), a microinjection technique (see, for example, Tarn, Basic Life Sciences 37 187-194, 1986, which is incoφorated by reference herein in its entirety), or a transfection method (e.g , naked or liposome encapsulated DNA or RNA, see, for example, Trends in Genetics 5 138, 1989, Chen and Okayama, Biotechniques 6 632-638, 1988, Mannino and Gould-Fogerite, Biotechmques 6 682-690, 1988, Kojima et al , Biochem. Biophys. Res. Comm. 207:8-12, 1995, which are incoφorated by reference herein in their entirety) The introduction method may be chosen to achieve a transient expression of neuroD in the host cell, or it may be preferable to achieve constitutive or regulated expression in a tissue specific manner Transformed host cells ofthe present invention find a variety of in vitro uses, for example i) as convenient sources of neuronal and other growth factors, ii) in transient and continuous cultures for screening anti-cancer drugs capable of driving terminal differentiation in neural tumors, iii) as sources of recombinantly expressed neuroD protein for use as an antigen in preparing monoclonal and polyclonal antibodies useful in diagnostic assays, and iv) in transient and continuous cultures for screening for compounds capable of increasing or decreasing the activity of neuroD Transformed host cells of the present invention also find a variety of in vivo uses, for example, for transplantation at sites of traumatic neural injury where motor or sensory neural activity has been lost Representative patient populations that may benefit from transplantation include patients with hearing or vision loss due to optical or auditory nerve damage, patients with peripheral nerve damage and loss of motor or sensory neural activity, and patients with brain or spinal cord damage from traumatic injury. For example, donor cells from a patient such as epithelial basal stem cells are cultured in vitro and then transformed or transduced with a neuroD nucleotide sequence The transformed cells are then returned to the patient by microinjection at the site of neural dysfunction In addition, as neuroD appears capable of regulating expression of insulin, transformed host cells of the present invention may be useful for transplantation into patients with diabetes. For example, donor cells from a patient such as fibroblasts, pancreatic islet cells, or other pancreatic cells are harvested and transformed or transfected with a neuroD nucleotide sequence. The genetically engineered cells are then returned to the patient. In another embodiment, such engineered host cells may find use in the treatment of malabsoφtion syndromes. Representative uses of the nucleotide sequences of the invention include the following: 1. Construction of cDNA and oligonucleotide probes useful in Northern or Southern blots, dot-blots, or PCR assays for identifying and quantifying the level of expression of neuroD in a cell. High level expression of neuroD in neuroendocrine tumors and in rapidly proliferating regions of embryonic neural development (see below) indicates that measuring the level of neuroD expression may provide prognostic markers for assessing the growth rate and invasiveness of a neural tumor. In addition, considering the important role of neuroD in embryonic development it is thought highly likely that birth defects and spontaneous abortions may result from expression of an abnormal neuroD protein. In this case, neuroD may prove highly useful in prenatal screening of mothers and/or for in utero testing of fetuses. 2. Construction of recombinant cell lines, ova, and transgenic embryos and animals including dominant-negative and "knock-out" recombinant cell lines in which the transcription regulatory activity of neuroD protein is down-regulated or eliminated. Such cells may contain altered neuroD coding sequences that result in the expression of a neuroD protein that is not capable of enhancing, suppressing or activating transcription ofthe target gene. The subject cell lines and animals find uses in screening for candidate therapeutic agents capable of either substituting for a function performed by neuroD or correcting the cellular defect caused by a defective neuroD. Considering the important regulatory role of neuroD in embryonic development, birth defects may occur from expression of mutant neuroD proteins, and these defects may be correctable in utero or in early post-natal life through the use of compounds identified in screening assays using neuroD. In addition, neuroD polynucleotide molecules may be joined to reporter genes, such as β-galactosidase or luciferase, and inserted into the genome of a suitable embryonic host cell such as a mouse embryonic stem cell by, for example, homologous recombination (for review, see Capecchi, Trends in Genetics 5:70-76, 1989; which is incoφorated by reference). Cells expressing neuroD may then be obtained by subjecting the differentiating embryonic cells to cell sorting, leading to the purification of a population of neuroblasts. Neuroblasts may be useful for studying neuroblast sensitivity to growth factors or chemotherapeutic agents. The neuroblasts may also be used as a source from which to purify specific protein products or gene transcripts. These products may be used for the isolation of growth factors, or for the identification of cell surface markers that can be used to purify stem cell population from a donor for transplantation. As illustrated in Example 14, "knock-out" mice were generated by replacing the murine neuroDl coding region with the β-galactosidase reporter gene and the neomycin resistance gene to assess the consequences of eliminating the murine neuroDl protein and to examine the tissue distribution of neuroDl in fetal and postnatal mice. Mice that were homozygous for the mutation (lacking neuroDl) had diabetes, as demonstrated by high blood glucose levels, and died by day four. Homozygous mutants had blood glucose levels between 2 and 3 times the blood glucose level of wild-type mice. Heterozygous mutants exhibited similar blood glucose levels as wild-type mice. Examination of stained tissue from fetal and postnatal mice heterozygous for the mutation confirmed the neuroDl expression pattern in neuronal cells demonstrated by in situ hybridization (Example 4) and also demonstrated neuroD expression in the pancreas and gastrointestinal tract. "Knock-out" mice may be useful as a model system for diabetes. Such mice may be used to study methods to rescue homozygous mutants and as hosts to test transplant tissue for treating diabetes. 3. Construction of gene transfer vectors (e.g., retroviral vectors, and the like) wherein neuroD is inserted into the coding region ofthe vector under the control of a promoter. NeuroD gene therapy may be used to correct traumatic neural injury that has resulted in loss of motor or sensory neural function, and also for the treatment of diabetes. For these therapies, gene transfer vectors may either be injected directly at the site of the traumatic injury, or the vectors may be used to construct transformed host cells that are then injected at the site of the traumatic injury. The results disclosed in Example 10 indicate that introduction of neuroDl induces a non-neuronal cell to become a neuron. This discovery raises for the first time the possibility of using transplantation and/or gene therapy to repair neural defects resulting from traumatic injury. In addition, the discovery of neuroDl provides the possibility of providing specific gene therapy for the treatment of certain neurological disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease, in which a population of neurons have been damaged. Two basic methods of neuroDl utilization are envisioned in this regard. In one method, neuroDl is expressed in existing populations of neurons to modulate aspects of their neuronal phenotype (e.g., neurotransmitter expression or synapse targeting) to make the neurons express a factor or phenotype to overcome the deficiency that contributes to the disease. In this method, recombinant neuroDl sequences are introduced into existing neurons or endogenous neuroDl expression is induced. In another method, neuroDl is expressed in non-neuronal cells (e.g., glial cells in the brain or another non-neuronal cell type such as basal epithelial cells) to induce expression of genes that confer a complete or partial neuronal phenotype that ameliorates aspects of the disease. As an example, Parkinson's disease is caused, at least in part, by the death of neurons that supply the neurotransmitter dopamine to the basal ganglia. Increasing the levels of neurotransmitter ameliorates the symptoms of Parkinson's disease. Expression of neuroDl in basal ganglia neurons or glial cells may induce aspects of a neuronal phenotype such that the neurotransmitter dopamine is produced directly in these cells. It may also be possible to express neuroDl in donor cells for transplantation into the affected region, either as syngeneic or allogeneic transplantations. Within yet another embodiment, neuroDl is expressed in non- pancreatic cells to induce expression of genes that confer a complete or partial pancreatic phenotype that ameliorates aspects of diabetes. Within yet another embodiment, neuroDl is expressed in pancreatic islet cells to induce expression of genes that induce the expression of insulin. 4. Preparation of transplantable recombinant neuronal precursor cell populations from embryonic ectodermal cells, non-neural basal stem cells, and the like. Establishing cultures of non-malignant neuronal cells for use in therapeutic screening assays has proven to be a difficult task. The isolated polynucleotide molecules encoding neuroD proteins ofthe present invention permit the establishment of primary (or continuous) cultures of proliferating embryonic neuronal stem cells under conditions mimicking those that are active in development and cancer. The resultant cell lines find uses: i) as sources of novel neural growth factors, ii) in screening assays for anti-cancer compounds, and iii) in assays for identifying novel neuronal growth factors. For example, a high level of expression of neuroD was observed in the embryonic optic tectum, indicating that neuroD 1 protein may regulate expression of factors trophic for growing retinal cells. Such cells may be useful sources of growth factors, and may be useful in screening assays for candidate therapeutic compounds. The cell lines and transcription regulatory factors disclosed herein offer the unique advantage that since they are active very early in embryonic differentiation they represent potential switches, e.g , ON→OFF or OFF→ON, controlling subsequent cell fate. If the switch can be shown to be reversible (i.e., ON<-»OFF), the neuroD transcription regulatory factor and neuroD nucleic acids disclosed herein provide exciting opportunities for restoring lost neural and/or endocrine functions in a subject. The following examples are offered by way of illustration and not by way of limitation EXAMPLE 1 Construction ofthe embryonic stem cell "179" cDNA library A continuous murine embryonic stem cell line (i.e., the ES cell line) having mutant E2 A (the putative binding partner of myoD) was used as a cell source to develop a panel of embryonic stem cell tumors Recombinant ES stem cells were constructed (i.e., using homologous recombination) wherein both alleles of the putative myoD binding partner E2A were replaced with drug-selectable marker genes. ES cells do not make functional El 2 or E47 proteins, both of which are E2A gene products. ES cells form subcutaneous tumors in congenic mice (i.e., strain 129J) that appear to contain representatives of many different embryonal cell types as judged histologically and through the use of RT-PCR gene expression assays. Individual embryonic stem cell tumors were induced in male 129J strain mice by subcutaneous injection of 1 x 10' cells/site. Three weeks later each tumor was harvested and used to prepare an individual sample of RNAs Following random priming and second strand synthesis the ds-cDNAs were selected based on their size on 0.7% agarose gels and those cDNAs in the range of 400-800 bp were ligated to either Bam HI or Bgl II linkers. (Linkers were used to minimize the possibility that an internal Bam HI site in a cDNA might inadvertently be cut during cloning, leading to an abnormally sized or out-of-frame expression product.) The resultant individual stem cell tumor DNAs were individually ligated into the Bam HI cloning site in the "fl-VP16" 2μ yeast expression vector. This expression vector, fl-VP16, contains the VPI 6 activation domain of Heφes simplex virus (HSV) located between Hind III (HIII) and Eco RI (RI) sites and under the control of the Saccharomyces cereviseae alcohol dehydrogenase promoter; with LEU1 and Ampicillin-resistance selectable markers. Insertion of a DNA molecule of interest into the Hind III site of the fl-VP16 vector (i.e., 5' to the VP16 nucleotide sequence), or into a Bam HI site (i.e., 3' to the VP16 sequence but 5 * to the Eco RI site), results in expression of a VPI 6 fusion protein having the protein of interest joined in-frame with VPI 6. The resultant cDNA library was termed the " 179-library" . EXAMPLE 2 Identification and cDNA cloning of mouse neuroDl. A two-hybrid yeast screening assay was used essentially as described by Fields and Song (Nature 340:245, 1989) and modified as described herein was used to screen the 179-library described in Example 1. Yeast two-hybrid screens are reviewed as disclosed in Fields and Sternglanz (Trends in Genetics 10:286-292, 1994). The library was screened for cDNAs that interacted with exA-Da, a fusion protein between the Drosophila Da (Daughterless) bHLH domain and the prokaryotic LexA-DNA binding domain. The S. cerevisiae strain L40 contained multimerized LexA binding sites cloned upstream of two reporter genes, namely, the HIS3 gene, and the β-galactosidase gene, each of which was integrated into the L40 genome. The S. cereviseae strain L40 containing a plasmid encoding the LexA-Da fusion protein was transformed with CsCl gradient-purified fl-VP16-179-cDNA library. Transformants were maintained on medium selecting both plasmids (the LexA-Da plasmid and the cDNA library plasmid) for 16 hours before being subjected to histidine selection on plates lacking histidine, leucine, tryptophan, uracil, and lysine. Clones that were HIS + were subsequently assayed for the expression of LacZ. To eliminate possible non-specific cloning artifacts, plasmids from HIS + /LacZ + were isolated and transformed into S. cereviseae strain L40 containing a plasmid encoding a LexA-Lamin fusion. Clones that scored positive in the interaction with lamin were discarded. Approximately 400 cDNA clones, which represented 60 different transcripts, were identified as positive in these assays. Twenty-five percent of the original clones were subsequently shown to be known bHLH genes on the basis of their reactivity with specific cDNA probes. One cDNA clone encoding a VPlό-fusion protein that interacted with Da but not lamin was identified as unique by sequence analysis. This clone, initially termed tango, is now referred to as neuroDl. The unique cDNA identified above, VP\6-neuroD, contained an approximately 450 bp insert that spanned the bHLH region. Sequence analysis showed that the clone contained an insert encoding a complete bHLH amino acid sequence motif that was unique and previously unreported. Further analysis suggested that while the cDNA contained conserved residues common to all members of the bHLH protein family, several residues were unique and made it distinct from previously identified bHLH proteins. The DNA cloned in V?\6-neuroD is referred to as "neuroDl ." The neuroDl cDNA insert was subcloned as a Bam HI-Not I insert into Bam HI-Not I linearized pBluescript SK . The resulting plasmid was designated pSK+1-83. The neuroDl insert contained in the VP\6-neuroD plasmid was used to re- probe a mouse cDNA library prepared from mouse embryos at developmental stage el 0.5. Candidate clones were isolated and sequenced essentially as described above. Several clones were isolated. One clone, designated pKS m7a RX, was deposited at the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD 20852 USA, on May 6, 1994, under accession number 75768. Plasmid pKS m7a RX contains 1646 bp of murine neuroDl cDNA as an EcoRI-XhoI insert. The amino acid sequence encoded by the insert begins at amino acid residue +73 and extends to the carboxy-terminus of the neuroDl protein. The plasmid contains about 855 bp of neuroDl coding sequence (encoding amino acids 73-536). None of the mouse cDNAs contained the complete 5' coding sequence. To obtain the 5' neuroDl coding sequence, a mouse strain 129/Sv genomic DNA library was screened with the VP16-wewraD plasmid insert (450 bp). Genomic clones were isolated and sequenced and the sequences were aligned with the cDNA sequences. Alignment of the sequence and comparison of the genomic 5' coding sequences with the Xenopus neuroDl clone (Example 8) confirmed the 5' neuroDl coding sequence. The complete neuroDl coding sequence and deduced amino acid sequence are shown in SEQ ID NOS: l and 2. EXAMPLE 3 NeuroD/neuroD bHLH proteins share common structural similarities that include a basic region that binds DNA and an HLH region involved in protein-protein interactions required for the formation of homodimers and heterodimeric complexes. A comparison of the amino acid sequence of the basic region of murine neuroDl (amino acids 102 to 113 of SEQ ID NO: 2) with basic regions of other bHLH proteins revealed that murine neuroD contained all of the conserved residues characteristic among this family of proteins. However, in addition, neuroDl contained several unique residues. These unique amino acid residues were not found in any other known HLH, making neuroDl a distinctive new member ofthe bHLH family. The NARERNR basic region motif in neuroD (amino acids 107-1 13 of SEQ ID NO:2) is also found in the Drosophila AS-C protein, a protein thought to be involved in neurogenesis. Similar, but not identical, NARERRR and NERERNR motifs (SEQ ID NOS: 5 and 6, respectively) have been found in the Drosophila Atonal and MASH (mammalian achaete-scute homolog) proteins, respectively, which are also thought to be involved in neurogenesis. The NARER motif (SEQ ID NO:7) of neuroDl is shared by other bHLH proteins, and the Drosophila Daughterless (Da) and Mammalian E proteins. The basic region of bHLH proteins is important for DNA binding site recognition, and there is homology between neuroDl and other neuro-proteins in this functional region. Within the important dimer-determining HLH region of neuroDl, a low level of homology was recorded with mouse twist protein (i.e., 51% homology) and with MASH (i.e., 46% homology). NeuroDl contains several regions of unique peptide sequence within the bHLH domain including the junction sequence (MHG). EXAMPLE 4 Tissue expression patterns of neuroDl, neuroDl, and neuroD 3 NeuroDl expression was analyzed during embryonic development of mouse embryos using in situ hybridization. The probe used was an antisense neuroDl single-stranded riboprobe labeled with digoxigenin (Boehringer Mannheim). Briefly, a riboprobe was prepared from plasmid pSK+1-83 using T7 polymerase and digoxigenin- 11-UTP for labeling. The hybridized probe was detected using anti- digoxigenin antibody conjugated with alkaline phosphatase. Color development was carried out according to the manufacturer's instructions. Stages of development are commonly expressed as days following copulation and where formation ofthe vaginal plug is eθ.5. The results recorded in the in situ hybridization studies were as follows: In the e9.5 mouse embryo, neuroDl expression was observed in the developing trigeminal ganglia. In the el 0.5 mouse embryo, a distinctive pattern of neuroDl expression was observed in all the cranial ganglia (i.e., V-XI) and in dorsal root ganglia (DRG) in the trunk region of the embryo. At this time, neuroDl expression was also observed in the central nervous system in post-mitotic cells in the brain and spinal cord that were undergoing neuronal differentiation. In the spinal cord, the ventral portion ofthe cord from which the motor neurons arise and differentiate was observed to express neuroDl at high levels; and expression in the posterior-ventral spinal cord was higher when compared to more mature anterior- ventral spinal cord. In the el 1.5 mouse embryo, the ganglionic expression pattern of neuroDl observed in el 0.5 persisted. Expression in the spinal cord was increased over the level of expression observed in el 0.5 embryos, which was consistent with the presence of more differentiating neurons at this stage. At this stage neuroDl expression was also observed in other sensory organs in which neuronal differentiation occurs, for example, in the nasal epithelium, otic vesicle, and retina of the eye. In both of these organs neuroDl expression was observed in the region containing differentiating neurons. In the el 4.5 mouse embryo, expression of neuroDl was observed in cranial ganglia and DRG, but expression of neuroDl persisted in the neuronal regions of developing sensory organs and the central nervous system (CNS). Thus, neuroDl expression was observed to be transient during neuronal development. In summary, expression of euroDl in the neurula stage of the embryo (elO), in the neurogenic derivatives of neural crest cells, the cranial and dorsal root ganglia, and post mitotic cells in the CNS suggests an important possible link between expression and generation of sensory and motor nerves. Expression occurring later in embryonic development in differentiating neurons in the CNS and in sensory organs (i.e., nasal epithelium and retina) also supports a role in development of the CNS and sensory nervous tissue. Since neuroDl expression was transient, the results suggest that neuroDl expression is operative as a switch controlling formation of sensory nervous tissue. It is noteworthy that in these studies neuroDl expression was not observed in embryonic sympathetic and enteric ganglia (also derived from migrating neural crest cells). Overall, the results indicate that neuroDl plays an important role in neuronal differentiation. In addition to the in situ studies described above, Northern blot analysis was done to determine in what tissues of the mouse neuroDl, neuroDl, and neuroD3 were expressed. Total RNA was isolated from whole mouse embryos and adult mouse tissues. RNA isolation was performed using RNazol B according to the protocol provided (Cinna/Biotex CS-105B). RNA was size fractionated on 1.5% agarose gels and transferred to Hybond-N membranes. Hybridization was carried out in 7% SDS, 0.25 M Na 2 PO 4 , lOmg/ml BSA, 1 mM EDTA at 65°C for at least 5 hours and then washed in 0.1X SSC and 0.1% SDS at 55°C-60°C. Probes for analyzing mouse mRNA were prepared from fragments representing the divergent carboxy- terminal regions 3 -prime of the bHLH domain to avoid cross-hybridization between genes. Probe for neuroDl was made from a 350 base pair PstI fragment from the mouse neuroDl cDNA (Lee et al., 1995) that encompasses the region coding for amino acids 187-304; probe for neuroDl was made from a 635 base pair PstI fragment from the mouse neuroDl cDNA that encompasses the region from amino acid 210 through to the 3 -prime non-translated region; and probe for neuroD3 was made from a 400 base pair Apal-BamHI fragment from the neuroD3 genomic region that is 3-prime to the region coding the bHLH domain. After labeling with 32 P, the above-described fragments were used to probe Northern blots containing RNAs prepared from various tissues of newborn and adult mice. Both neuroDl and neuroDl were detected in the brain of both newborn and adult mice, whereas, neuroD3 transcripts were not detected in any of the tissues tested. RNA extracted from dissected regions of the adult mouse nervous system demonstrated that neuroDl was more abundant in the cerebellum than the cortex, whereas neuroDl was expressed at relatively equivalent levels in both cerebellum and cortex. To determine when during mouse embryonic development neuroDl and neuroD3 were expressed in comparison to neuroDl, RNA was prepared from whole embryos at various developmental stages. In accord with previous reports (Lee et al., 1995), neuroDl mRNA was first detected at low levels at embryonic day 9.5 and at increasing levels through embryonic day 12.5, the latest embryonic stage tested. NeuroDl mRNA was first detected at embryonic day 11 and also increased in abundance through embryonic day 12.5. Although we did not detect neuroD 3 in the adult tissues, the embryonic expression pattern showed a transient expression between embyronic day 10 and 12 and then declined to undetectable levels by embryonic day 16. Collectively, these data demonstrate that neuroD3 is expressed transiently during embryogenesis, similar to the expression pattern of MATHl (Akazawa et al., 1995), and that the temporal expression of neuroDl and neuroDl partly overlap with neuroD3, but that their expression persists in the adult nervous system. EXAMPLE 5 NeuroDl is expressed in neural and brain tumor cells: murine probes identify human neuroDl. Given the expression pattern in mouse embryo (Example 4), Northern blots of tumor cell line mRNAs were examined using murine neuroDl cDNA (Example 2) as a molecular probe. As a first step, cell lines that have the potential for developing into neurons were screened. The D283 human medullablastoma cell line, which expressed many neuronal markers, expressed high levels of neuroDl by Northern blot analysis. NeuroDl was also transcribed at various levels by different human neuroblastoma cell lines and in certain rhabdomyosarcoma lines that are capable of converting to neurons. EXAMPLE 6 Recombinant cells expressing NeuroDl. Recombinant murine 3T3 fibroblast cells expressing either a myc-tagged murine neuroDl protein or myc-tagged Xenopus neuroDl protein were made. The recombinant cells were used as a test system for identifying antibody to neuroD described below. Xenopus neuroD 1 protein was tagged with the antigenic marker Myc to allow the determination of the specificity of anti-neuroDl antibodies to be determined. Plasmid CS2+MT was used to produce the Myc fusion protein. The CS2+MT vector (Turner and Weintraub, ibid.) contains the simian cytomegalovirus EE94 enhancer/promoter (and an SP6 promoter in the 5' untranslated region of the IE94- driven transcript to allow in vitro RNA synthesis) operatively linked to a DNA sequence encoding six copies of the Myc epitope tag (Roth et al, J. Cell Biol. 115:587-596, 1991; which is incoφorated herein in its entirety), a polylinker for insertion of coding sequences, and an SV40 late polyadenylation site. CS2-MT was digested with Xho I to linearize the plasmid at the polylinker site downstream of the DNA sequence encoding the Myc tag. The linearized plasmid was blunt-ended using Klenow and dNTPs. A full length Xenopus neuroDl cDNA clone was digested with Xho I and Eae I and blunt-ended using Klenow and dNTPs, and the 1.245 kb fragment of the Xenopus neuroDl cDNA was isolated. The neuroDl fragment and the linearized vector were ligated to form plasmid CS2+MT xl-83. CS2+MT was digested with Eco RI to linearize the plasmid at the polylinker site downstream ofthe DNA sequence encoding the Myc tag. The linearized plasmid was blunt-ended using Klenow and dNTPs and digested with Xho I to obtain a linearized plasmid having an Xho I adhesive end and a blunt end. Plasmid pKS+m7a containing a partial murine neuroDl cDNA was digested with Xho I, and the neuroDl containing fragment was blunt-ended and digested with Xba I to obtain the approximately 1.6 kb fragment of the murine neuroDl cDNA. The neuroDl fragment and the linearized vector were ligated to form plasmid CS2+MT Ml- 83(m7a). Plasmids CS2+MT xl-83 and CS2+MT Ml-83(m7a) were each transformed into murine 3T3 fibroblast cells and used as a test system for identifying antibody against neuroDl (Example 7). EXAMPLE 7 Antibodies to NeuroDl . A recombinant fusion protein of maltose binding protein (MBP) and amino acid residues 70-355 of murine neuroDl was used as an antigen to evoke antibodies in rabbits. Specificity of the resultant antisera was confirmed by immunostaining of the recombinant 3T3 cells described above. Double-immunostaining of the recombinant cells was observed with monoclonal antibodies to Myc (i.e., the control antigenic tag on the transfected DNA) and with rabbit anti-murine neuroDl in combination with anti-rabbit IgG. The specificity of the resultant anti-murine neuroDl sera was investigated further by preparing mouse 3T3 fibroblasts cells transfected with different portions of neuroDl DNA. Specificity seemed to map to the glutamic acid-rich domain (i.e., amino acids 66-73 of SEQ ID NO:2). The anti- murine antisera did not react with cells transfected with the myc-tagged Xenopus neuroDl. In a similar manner, Xenopus neuroDl was used to generate rabbit anti- neuroD antisera. The antisera was _Yt?/7θ/n/s-specific and did not cross react with cells transfected with Myc-tagged murine neuroDl. EXAMPLE 8 NeuroDl is a highly evolutionarily conserved protein: sequence of Xenopus neuroDl. Approximately one million clones from a stage 17 Xenopus head cDNA library made by Kintner and Melton (Development 99:311, 1987) were screened with the mouse cDNA insert as a probe at low stringency. The hybridization was performed with 50% formamide/4 X SSC at 33°C and washed with 2 X SSC/0.1% SDS at 40°C. Positive clones were identified and sequenced. Analysis of the Xenopus neuroDl cDNA sequence (SEQ ID NO:3) revealed that neuroDl is a highly conserved protein between frog and mouse. The deduced amino acid sequences of frog and mouse (SEQ ID NOS:2 and 4) show 96% identity in the bHLH domain (50 of 52 amino acids are identical) and 80% identity in the region that is carboxy- terminal to the bHLH domain (159 of 198 amino acids are identical). The domain structures of murine and Xenopus neuroDl are highly homologous with an "acidic" N-terminal domain (i.e., glutamic or aspartic acid rich); a basic region; helix 1, loop, helix 2; and a proline rich C-terminal region. Although the amino terminal regions of murine and Xenopus neuroDl differ in amino acid sequence, both retain a glutamic or aspartic acid rich "acidic domain" (amino acids 102 to 113 of SEQ ID NO: 2 and amino acids 56 to 79 of SEQ ID NO:4) It is highly likely that the acidic domain constitutes an "activation" domain for the neuroDl protein, in a manner analogous to the activation mechanisms currently understood for other known transcription regulatory factors. EXAMPLE 9 Neuronal expression of Xenopus neuroDl. The expression pattern of neuroDl in whole mount Xenopus embryos was determined using in situ hybridization with a single stranded digoxigenin-labeled Xenopus neuroDl antisense cDNA riboprobe. Embryos were examined at several different stages. Consistent with the mouse expression pattern, by late stage, all cranial ganglia showed very strong staining patterns. In Xenopus, as in other vertebrate organisms, neural crest cells give rise to skeletal components of the head, all ganglia of the peripheral nervous system, and pigment cells. Among these derivatives, the cranial sensory ganglia, which are of mixed crest and placode origin, represent the only group of cells that express neuroDl. High levels of neuroDl expression in the eye were also observed, correlating with active neuronal differentiation in the retina at this stage. Expression is observed in the developing olfactory placodes and otic vesicles, as was seen in mice. The pineal gland also expressed neuroDl. All of this expression was transient, suggesting that neuroDl functions during the differentiation process but is not required for maintenance of these differentiated cell types. As early as stage 14 (i.e., the mid-neurula stage) neuroDl expression was observed in the cranial neural crest region where trigeminal ganglia differentiate. Primary mechanosensory neurons in the spinal cord, also referred to as Rohon-Beard cells and primary motor neurons, showed neuroDl expression at this stage. By stage 24, all of the developing cranial ganglia, trigeminal, facio-acoustic, glosso-pharyngeal, and vagal nervous tissues showed a high level of neuroDl expression. High levels of expression of neuroDl were also observed in the eye at this stage. (Note that in Xenopus neuronal differentiation in the retina occurs at a much earlier stage than in mice, and neuroDl expression was correspondingly earlier and stronger in this animal model.) In summary, in Xenopus as in mouse, neuroDl expression was correlated with sites of neuronal differentiation. The remarkable evolutionary conservation of the pattern of neuroDl expression in differentiating neurons supports the notion that neuroDl has been evolutionarily conserved both structurally and functionally in these distant classes, which underscores the critical role performed by this protein in embryonic development. EXAMPLE 10 Expression of neuroDl and neuroDl converts non-neuronal cells into neurons. To further analyze the biological functions of neuroDl, a gain-of-function assay was conducted. In this assay, RNA was microinjected into one of the two cells in a 2-cell stage Xenopus embryo, and the effects on later development of neuronal phenotype were evaluated. For these experiments /wye-tagged Xenopus neuroDl transcripts were synthesized in vitro using SP6 RNA polymerase. The myc-tzggeά- neuroDl transcripts were microinjected into one of the two cells in a Xenopus 2-cell embryo, and the other cell ofthe embryo served as an internal control. Synthesis of capped RNA for the Xenopus laevis injections was done essentially as described (Kreig, P. A. and D. A. Melton., Meth. Enzymol. 155:397-415, 1987) using the SP6 transcription of the pCS2-hND2, pCS2-hNDl, pCS2-mND2, and pCS2MT-mND2. The capped RNA was phenol/chloroform extracted followed by separation of unincoφorated nucleotides using a G-50 spin column. Approximately 350 pg or capped RNA was injected into one cell of 2-cell stage albino Xenopus laevis embryo in a volume of approximately 5 nl, as described previously (Turner and Weintraub, 1994). Embryos were allowed to develop in 0.1X modified Barth's saline (MBS) and staged according to Nieukwoop and Faber (Nieuwkoop, P D. and J. Faber, "Normal Table of Xenopus laevis," North-Holland Publishing Co., Amsterdam, The Netherlands, 1967). Embryos were fixed in MEMFA for 2 hours at room temperature and stored in methanol. Embryos were hydrated through a graded series of methanol/PBS solutions and prepared for immunohistochemistry as described (Turner and Weintraub, 1994). The embryos were stained with an anti-NCAM antibody (Balak et al. Develop. Biol. 119:540-550, 1987) diluted 1:500 (gift of Urs Rutishauser) followed by a goat anti-rabbit alkaline phosphatase conjugated secondary antibody, or stained with the monoclonal anti-myc tag 9el0 antibody. Presence of the antibody was visualized by NBT/BCIP color reaction according to protocol provided (Gibco). Antibodies to Xenopus N-CAM, a neural adhesion molecule, anti-Myc (to detect the exogenous protein tag), and immunostaining techniques were used to evaluate phenotypic expression of the neuronal marker (and control) gene during the subsequent developmental stages of the microinjected embryos. Remarkably, an evaluation of over 130 embryos that were injected with neuroDl RNA showed a striking increase in ectopic expression of N-CAM on the microinjected side of the embryo (i.e., Myc ), as judged by increased immunostaining. The increased staining O 97/16548 PCI7US96/17532 -30- was observed in the region from which neural crest cells normally migrate. It is considered likely that ectopic expression (or over-expression) of neuroDl caused neural crest stem cells to follow a neurogenic cell fate. Outside the neural tube, the ectopic immunostaining was observed in the facio-cranial region and epidermal layer, and in some cases the stained cells were in the ventral region of the embryo far from the neural tube. The immunostained cells not only expressed N-CAM ectopically, but displayed a moφhological phenotype of neuronal cells. At high magnification, the N- CAM expressing cells exhibited typical neuronal processes reminiscent of axonal processes. To confirm that the ectopic N-CAM expression resulted from a direct effect on the presumptive epidermal cells and not from aberrant neural cell migration into the lateral and ventral epidermis, neuroDl RNA was injected into the top tier of 32- cell stage embryos, in order to target the injection into cells destined to become epidermis. N-CAM staining was observed in the lateral and ventral epidermis without any noticeable effect on the endogenous nervous system, indicating that the staining of N-CAM in the epidermis represents the conversion of epidermal cell fate into neuronal cell fate. Ectopic generation of neurons by neuroDl was confirmed with other neural specific markers, such as neural-specific class II β-tubulin (Richter et al., Proc. Natl. Acad. Sci. USA 85:8066, 1988), acetylated I-tubulin (Piperno and Fuller, J. Cell. Biol. 101:2085, 1985), tanabin (Hemmati-Brinvanlou et al., Neuron 9:417, 1992), neurofilament(NF)-M (Szaro et al., J. Comp. Neurol. 273:344, 1988), and Xen-1,2 (Ruiz i Altaba, Development 115:67, 1992). The embryos were subjected to immunochemistry as described by Turner and Weintraub (Genes Dev. 8: 1434, 1994, which is incoφorated by reference herein) using primary antibodies detected with alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit antibodies diluted to 1:2000 (Boehringer-Mannheim). Anti-acetylated alpha-tubulin was diluted 1 :2000. Anti-Xen-1 was diluted 1:1. Anti-NF-M was diluted 1:2000. Embryos stained for NF-M were fixed in Dent's fixative (20% dimethylsulfoxide/80% methanol) and cleared in 2:1 benzyl benzoate/benzyl alcohol as described by Dent et al. (Development 105:61, 1989, which is incoφorated by reference herein). In situ hybridization of embryos was carried out essentially as described by Harland (in Methods in Cell Biology, B K. Kay, H J. Pend, eds., Academic Press, New York, NY, Vol. 36, pp. 675-685, 1991, which is incoφorated by reference herein) as modified by Turner and Weintraub (ibid ). In situ hybridization with β-tubulin without RNase treatment can also detect tubulin expression in the ciliated epidermal cells. All of these markers displayed ectopic staining on the neuroDl RNA injected side. Injection of neuroDl mRNA into vegetal cells led to no ectopic expression of neural markers except in one embryo that showed internal N-CAM staining in the trunk region, suggesting the absence of cofactors or the presence of inhibitors in vegetal cells. However, the one embryo that showed ectopic neurons in the internal organ tissue suggests that it may be possible to convert non-ectodermal lineage cells into neurons under certain conditions. The embryos were also stained with markers that detect Rohon-Beard cells (cells in which neuroDl is normally expressed). Immunostaining using the method described above for Rohon-Beard cell-specific markers such as HNK-1 (Nordlander, Dev. Brain Res. 50: 147, 1989, which is incoφorated by reference herein) at a dilution of 1 : 1, Islet- 1 (Ericson et al., Science 256: 1555, 1992 and Korzh et al., Development 118:417, 1993) at a dilution of 1 :500, and in situ hybridization as described above with shaker-1 (Ribera et al., J. Neurosci. 13:4988, 1993) showed more cells staining on the injected side ofthe embryos. The combined results support the notion that ectopic expression of neuroD 1 induced differentiation of neuronal cells from cells that, without neuroDl microinjection, would have given rise to non-neuronal cells In summary, these experiments support the notion that ectopic neuroDl expression can be used to convert a non-neuronal cell (i.e., uncommitted neural crest cells and epidermal epithelial basal stem cells) into a neuron. These findings offer for the first time the potential for gene therapy to induce neuron formation in injured neural tissues. Interesting moφhological abnormalities were observed in the microinjected embryos. In many cases the eye on the microinjected side of the embryo failed to develop. In other embryos, the spinal cord on the microinjected side of the embryo failed to develop properly, and the tissues were strongly immunopositive when stained with anti-N-CAM. In addition, at the mid-neurula stage many microinjected embryos exhibited an increase in cell mass in the cranial region ofthe embryo from which (in a normal embryo) the neural crest cells and their derivatives (i.e., cranial ganglionic cells) would migrate. The observed cranial bulge exhibited strong immunostaining with antibodies specific for N-CAM. These results were inteφreted to mean that moφhological changes in the eye, neural crest, and spinal cord resulted from premature neural differentiation which altered the migration of neural and neural crest precursor cells. O 97/16548 PCΪ7US96/17532 -32- NewroZ)7-injected embryos were also assayed for alteration in the expression of Xtwist, the Xenopus homolog of Drosophila twist, to determine whether neuroDl converted non-neuronal components of neural crest cells into the neural lineage In wild-type embryos, Xtwist is strongly expressed in the non-neuronal population cephalic neural crest cells that give rise to the connective tissue and skeleton of the head. NewrøZ)V-injected embryos were completely missing Xtwist expression in the migrating cranial neural crest cells on the injected side The failure to generate sufficient cranial mesenchymal neural crest precursors in «e«rσ£ ) /-injected embryos was also observed moφhologically, since many of the injected embryos exhibited poor branchial arch development in the head Furthermore, the increased mass of cells in the cephalic region stained very strongly for Ν-CAM, β-tubulin, and Xen-1, indicating that these cells were neural in character The converse experiment in which frog embryos were injected with Xtwist mRΝA showed that ectopic expression of Xtwist significantly decreased neuroDl expression on the injected side. Thus, two members of the bHLH family, neuroDl and Xtwist, may compete for defining the identity of different cell types derived from the neural crest. In the wewroDZ-injected embryos, exogenous neuroDl may induce premigratory neural crest to differentiate into neurons in situ, and consequently they fail to migrate to their normal positions. The effect of introduction of exogenous neuroDl on the fate of cells that normally express neuroDl, such as cranial ganglia, eye, otic vesicle, olfactory organs, and primary neurons, and on other CΝS cells that normally do not express neuroDl, was determined by staining for differentiation markers When the cranial region ofthe embryo was severely affected by ectopic neuroDl, the injected side of the embryos displayed either small or no eyes in addition to poorly organized brains, otic vesicles, and olfactory organs. Moreover, as the embryos grew, the spinal cord showed retarded growth, remaining thinner and shorter on the weurøZ)/-injected side. Ν-CAM staining in the normal embryo at early stages was not uniform throughout the entire neural plate, but rather was more prominent in the medial region of the neural plate Injected embryos analyzed for Ν-CAM expression showed that the neural plate on the injected side of the early stage embryos was stained more intensely and more laterally The increase in Ν-CAM staining was not associated with any lateral expansion of the neural plate as assayed by visual inspection and staining with the epidermal marker EpA This was in contrast to what has been observed with XASH-3 injection that causes neural plate expansion. These observations suggest that the first effects of neuroDl are to cause neuronal precursors in the neural plate to differentiate prematurely. To determine whether neuroDl caused neuronal precursors to differentiate prematurely, injected embryos were stained using two neuronal markers that are expressed in differentiated neurons, neural specific β-tubulin and tanabin. In situ hybridization for β-tubulin and tanabin was carried out as described above. Over¬ expression of neuroDl dramatically increased the β-tubulin signals in the region ofthe neural plate containing both motor neurons and Rohon-Beard cells at stage 14. The earliest ectopic β-tubulin positive cells on the injected side were observed at the end of gastrulation when the control side did not yet show any β-tubulin positive cells. Tanabin was also expressed in more cells in the spinal cord in the neuroDl injected side of the embryos at stage 14. These results suggest that neuroDl can cause premature differentiation of the neural precursors into differentiated neurons. This is a powerful indication that, when ectopically expressed or over-expressed, neuroDl can differentiate mitotic cells into non-dividing mature neurons. To determine if neuroD2 also was capable of inducing ectopic neuronal development in the frog, mouse neuroDl RNA was injected into one side of a two cell X. laevis embryo, the uninjected side serving as a control. The neuroDl mRNA was made from pCS2-MTmND2, an expression vector that was constructed as follows. Expression vectors were made in the pCS2+ or pCS2+MT (Turner, D.L. and H. Weintraub, Genes & Dev. 8: 1434-1447, 1994), both contain the simian CMV promoter and the MT contains six copies of the myc epitope recognized by the 9el0 monoclonal antibody (ATCC: CRL 1729) cloned in-frame upstream of the insert. The 1.75 kb full length human neuroDl cDNA (Tamimi et al., Genomics 34: 418-421, 1996) from plasmid phcndl-17a was cloned into the EcoRI site to make pCS2-hNDl- 17s (hereafter referred to as pCS2-hNDl). The 1.53 kb genomic region containing the entire coding sequence of the human neuroDl gene (described in Example 11) was cloned into the Stul-Xbal site to make pCS2-hND2-14Bl (hereafter referred to as pCS2-hND2). The mouse 1.95 kb neuroDl cDNA was cloned into the EcoRI- Xhol sites to make pCS2-mND2- 1.1.1 (hereafter referred to as pCS2-mND2). For the myc-tagged construct, a synthetic oligonucleotide mediated mutagenesis was used to introduce an EcoRI site adjacent to the initial ATG codon to result in the /wy -tag and neuroD2 coding regions being in-frame to make pCS2MT-mND2. When injected into Xenopus laevis, mouse neuroDl mRNA was able to induce ectopic neuronal development as determined by immunohistochemistry with an anti-NCAM antibody. An anti-myc tag antibody, 9E10, was used to confirm that most ectodermal cells on the injected side of the frog expressed the myc-tagged mouse neuroD2 and approximately 80-90% of injected embryos stained positively with either the anti-myc or anti-NCAM antibodies. Injection of RNA encoding the human neuroD2 gene resulted in an ectopic neuronal phenotype similar to that seen with Xenopus neuroDl and murine neuroDl. This demonstrates that both neuroDl and neuroD2 can regulate the formation of neurons and that the human and mouse neuroD2 proteins are capable of functioning in the developing Xenopus embryo. Developmental expression patterns suggest two distinct sub-families of neurogenic bHLH genes. MATHl and neuroD3 share similarity in the bHLH region and have similar temporal expression patterns, with RNA expression detected around embryonic day 10, but not persisting in the mature nervous system. MATH-1 RNA was localized to the dorsal neural tube in 10.5-11.5 day embryos, but by birth was present only in the external granule cell layer ofthe cerebellum, the progenitors ofthe cerebellar granule cell layer (Akazawa et al., 1995). In contrast, the neuroDl, neuroDl, and MATHl/NEX-1 genes are expressed in both differentiating and mature neurons. Northern analysis demonstrated that neuroDl expression begins around embryonic day 11 and continues through day 16, the latest embryonic time point tested. NeuroDl was detected in the brain of neonates as well as adult mice, with relatively equal abundance in both the cerebellum and cortex. Similar to neuroDl, the CNS expression of neuroDl persists postnatally, as well as does its expression in the beta cells of the pancreas (Naya et al., 1995). Northern blot analysis indicated that neuroDl expression in the adult mouse brain is most abundant in the cerebellum with lower levels in the cerebral cortex and brain stem. NEX-l/MATH-1 gene expression is reported to occur by embryonic day 11.5 and at embryonic day 15.5 its expression is limited to the intermediate zone adjacent to the mitotically active ventricular zone, suggesting that NEX-1 /MATHl is expressed primarily in the newly differentiating neurons at this stage (Bartholoma, A. and K. A. Nave, 1994; Shimizu et al., 1995). In mature brain, NEX-l/MATH-1 is expressed in neurons comprising the hippocampus, subsets of cortical neurons, and post migratory cerebellar granule cells, but the reports disagree on whether this gene is expressed in the dentate gyrus of the hippocampus. It is interesting to note that the Northern analysis of MATHl expression reported by Shimizu et al. (1995) shows high levels in the cerebral cortex and low levels in the cerebellum, the opposite of the expression pattern seen for neuroDl, suggesting that these genes may also have significant differences in relative abundance in specific regions of the nervous system. Therefore, it appears that MATH-1 and neuroD3 are expressed early in nervous system development and may have a role in either determining or expanding a population of neuronal precursors, whereas the persistent expression of neuroDl, neuroDl and NEX-l/MATH-1 suggest a role in initiating and maintaining expression of genes related to neuronal differentiation. Kume et al. (Biochem. Biophys. Res. Comm. 219:526-530, 1996) have reported the cloning of a helix-loop-helix gene from rat brain using a strategy designed to identify genes that are expressed during tetanic stimulation of hippocampal neurons in a model of long-term-potentiation. The gene they describe, KW8, is the rat homolog of the mouse and human neuroDl gene described here. Kume et al. also describe expression in the adult brain, including the hippocampus. Subsequently, Yasunami et al. (Biophys. Res. Comm. 220:754-758, 1996) reported the mouse NDRF gene, which is nearly identical to neuroDl and demonstrates a similar expression pattem in adult brain by in situ hybridization. While expression of either neuroDl or neuroDl in Xenopus leavis embryos resulted in ectopic neuronal development, it is interesting to note that neither neuroDl nor neuroD2 was capable of converting all cell types in which it was present into neurons. As in the case of neuroDl, the ectopic neurons induced by neuroD2 were confined to a subpopulation of ectodermal cells, as indicated by the spotty NCAM positive staining pattem. The apparent restricted activity of the neuroD proteins to a subset of cells derived from the ectoderm suggests that other factors may regulate their activity, such as the notch pathway that mediates lateral inhibition during Drosophila neurogenesis. While the induction of ectopic neurogenesis by both neuroDl and neuroD2 in Xenopus embryos suggests a similar function, the developmental expression patterns and in vitro transfection experiments indicate that the family members may serve both overlapping and distinct functions. Previous studies have demonstrated that neuroD/beta2 and NEX-1/MATH2 can bind the core CANNTG sequence of an E- box as a heterodimer with an E-protein and activate transcription. In the work presented here, it is shown that both neuroDl and neuroD2 can activate a construct containing multimerized E-boxes. They also activate a construct driven by a genomic fragment from the neuroDl gene that presumably contains regulatory regions for neuroDl, and the temporal expression pattem of neuroD 1 and neuroD2 proteins in embryogenesis and P19 differentiation suggests a model in which neuroDl may activate neuroD2 expression during development. Most important, however, is the demonstration that neuroDl and neuroD2 have different capacities to activate a construct driven by the core regulatory sequences of the GAP-43 gene, demonstrating that the highly related neuroDl and neuroD2 proteins are capable of regulating specific subsets of genes. This promoter contains several E-boxes and it remains to be determined if neuroD2 directly binds to these sites. In the bHLH region, neuroDl and neuroD2 differ by only 2 amino acids and it would be anticipated that they recognize the same core binding sequences. Therefore, the differential regulation of transcriptional activity may be determined independently of DNA binding. The amino acid following the histidine in the junction region of the basic region is a glycine in neuroDl, NEX-1/MATH2, and MATHl, an aspartate in neuroD2, and an asparagine in neuroD3. This residue is positioned at the same site as the lysine residue in the myogenic bHLH proteins that has been shown to be one of the critical for myogenic activity (Davis et al., Cell 60:733-746, 1990; Davis, R. L. and H. Weintraub, Science 256: 1027-1030, 1992; Weintraub et al., Genes & Dev. 5: 1377-1386, 1991). In this case, it has been postulated to be a site of potential interaction with co-activator factors that regulate transcriptional activity If the neuroD proteins have a similar mechanism for exerting their regulatory activities, it is possible that amino acid variability in this amino acid mediates different target specificities. Alternatively, the more divergent amino- and carboxyterminal regions could confer regulation by interaction with other activators or repressors. The different expression patterns in the mature nervous system and the subtle differences in target genes is similar to myogenic bHLH proteins. In mature muscle, MyoD is expressed in fast muscle fibers and myogenin in slow fibers (Asakura et al., Develop. Biol. 171:386-398, 1995; Hughes et al., Development 118:1137-1147, 1993) and transfection studies demonstrate that sequences adjacent to the core E-box sequence can differentially regulate the ability of MyoD and myogenin to function as transcriptional activators (Asakura et al., Molec. & Cell. Biol. 13:7153-7162, 1993), presumably by interaction of other regulatory factors with the non-bHLH regions of MyoD and myogenin. For the wewrøZ)-related genes, the partially overlapping expression patterns and partially overlapping target genes suggest that they may act in a combinatorial fashion to directly regulate overlapping subsets of genes and thereby confer specific neuronal phenotypes. In this model, it is possible that a small family of neuroD-related transcription factors acts to establish the identity of a limited number of neuronal sub-types and that local inductive events influence the generation of a higher complexity. Alternatively, it is possible that many additional members of this sub-family are yet to be identified and they may act to directly determine specific neuronal attributes. EXAMPLE 11 Genomic clones of human neuroDl, neuroDl and neuroD3 and mouse neuroD3. Genomic clones encoding human neuroDl were obtained by probing a human fibroblast genomic library with the mouse neuroDl cDNA. Host E. coli strain LE392 (New England Biolabs) were grown in LB + 10 mM MgSO4 ) 0.2% maltose overnight at 37°C. The cells were harvested and resuspended in 10 mM MgSO4 to a final OD600 of 2. The resuspended cells were used as hosts for phage infection. The optimal volume of phage stock for use in this screening was determined by using serial dilutions of the phage stock of a human fibroblast genomic library in lambda FIX II (Stratagene®) to infect LE392 cells (New England Biolabs). To obtain approximately 50,000 plaques per plate, a 2.5 μl aliquot of the phage stock was used to infect 600 μl of the resuspended LE392 cells. The cells were incubated with the phage for 15 minutes at 37°C, after which the cells were mixed with 6.5 ml of top agar warmed to 50°C. The top agar was plated on solid LB, and incubated overnight at 37°C. A total of 22 15-cm plates were prepared in this manner. Duplicate plaque lifts were prepared. A first set of Hybond membranes (Amersham) were placed onto the plates and allowed to sit for 2 minutes. The initial membranes were removed and the duplicate membranes were laid on the plates for 4 minutes. The membranes were allowed to air dry; then the phage were denatured in 0.5 M NaOH, 1.5 M NaCl for 7 minutes. The membranes were neutralized with two washes in neutralization buffer (1.5M NaCl, 0.5 M Tris, pH 7.2). After neutralization, the membranes were crosslinked by exposure to UV. A 1 kb Eco RI- Hind III fragment containing murine neuroDl coding sequences was random primed using the Random Priming Kit (Boehringer Mannheim) according to the manufacturer's instructions. Membranes were prepared for hybridization by placing six membranes in 10 ml of FBI hybridization buffer [100 g polyethylene glycol 800, 350 ml 20% SDS, 75 ml 20X SSPE; add water to a final volume of one liter] and incubating the membranes at 65°C for 10 minutes. After 10 minutes, denatured salmon sperm DNA was added to a final concentration of 10 μg/ml and denatured 7 probe was added to a final concentration of 0.25-0.5 x 10 cpm/ml. The membranes were hybridized at 65°C for a period of 8 hours to overnight. After incubation, the excess probe was removed, and the membranes were washed first in 2 X SSC, 0.1% SDS for 30 minutes at 50°C. The first wash was followed by a final wash in 0.1 X SSC, 0.1% SDS for 30 minutes at 55°C (moderate stringency). Autoradiographs of the membranes were prepared. The first screen identified 55 putative positive plaques. Thirty-one of the plaques were subjected to a secondary screen using the method essentially set forth above. Ten positive clones were identified and subjected to a tertiary screen as described above. Eight positive clones were identified after the tertiary screen. Of these eight clones, three (14B1, 9F1 and 20A1) were chosen for further analysis. Clones 14B1 and 20A1 were deposited at the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD 20852 USA, on November 1, 1995, under accession numbers 69943 and 69942, respectively. Phage DNA was prepared from clones 14B1, 9F1, and 20A1. The 14B1 and 20A1 phage DNA were digested with Pst I to isolate the 1.2 kb and 1.6 kb fragments, respectively, that hybridized to the mouse neuroDl probe. The 9F1 phage DNA was digested with Eco RI and Sad to obtain an approximately 2.2 kb fragment that hybridizes with the mouse neuroDl probe. The fragments were each subcloned into plasmid BLUESCRIPT SK (Stratagene) that had been linearized with the appropriate restriction enzyme(s). The fragments were sequenced using Sequenase Version 2.0 (US Biochemical) and the following primers: the universal primer Ml 3-21, the T7 primer, and the T3 primer. Sequence analysis of clones 9F1 (SEQ ID NOS: 8 and 9), and 14B1 (SEQ ID NOS: 10 and 11) showed a high similarity between the mouse and human coding sequences at both the amino acid and nucleotide level. In addition, while clones 9F1 and 14B1 shared 100% identity in the HLH region at the amino acid level (i.e., residues 117-156 in SEQ ID NO:9 and residues 137-176 in SEQ ID NO: 11), they diverged in the amino-terminal ofthe bHLH. This finding strongly suggests that 14B1 is a member of the neuroD family of genes. Sequence analysis demonstrates that clone 9F1 has a high degree of homology throughout the sequence region that spans the translation start site to the end of the bHLH region. The 9F1 clone has 100% identity to mouse neuroDl in the HLH region (i.e., residues 117-156 in SEQ ID NO:9 and residues 117-156 in SEQ ID NO:2), and an overall identity of 94%. The 14B1 clone also has 100% identity to the HLH region (i.e., residues 137-176 in SEQ ID NO: 11 and residues 117-156 in SEQ ID NO:2), but only 40% identity to 9F1 and 39% identity to mouse neuroDl in the amino-terminal region. This demonstrates that 9F1 is the human homolog of mouse neuroDl, whereas the strong conservation of the neuroD HLH identifies 14B1 as another member of the neuroD HLH subfamily. Human clone 9F1 (represented by SEQ ID NOS: 8 and 9) is referred to as human neuroDl. Human clone 14B1 is referred to as neuroDl (SEQ ID NOS: 10 and 1 1, and human clone 20A1 is referred to as neuroD3 (SEQ ID NOS: 12 and 13). A fragment of the human neuroDl gene was used to screen both a mouse genomic library and an embryonic day 16 mouse cDNA library. An 800 bp Hind III- Eag I fragment from the neuroDl sequences from clone 14B1 was random primed with 32 P, and used to screen a 16-day mouse embryo cDNA library essentially as described previously. Filters were prehybridized in FBI hybridization buffer (see above) at 50°C for 10 minutes. After prehybridization, denatured salmon sperm DNA was added to a final concentration of 10 μg/ml; denatured probe was added to a final concentration of one million cpm/ml. The filters were hybridized at 50°C overnight. After incubation, excess probe was removed, and the filters was washed first in 2 X SSC, 0.1% SDS for 30 minutes at 60°C. Genome clones were obtained and characterized. Five independent cDNAs were mapped by restriction endonucleases and demonstrated identical restriction sites and sequence. One clone, designated 1.1.1, contained 1.46 kb of murine neuroDl cDNA as an Eco RI-Hind III insert. The nucleotide sequence and deduced amino acid sequences are shown in SEQ ED NOS: 16 and 17, respectively. A comparison with the corresponding mouse genomic sequence demonstrated that the entire coding region of neuroDl is contained in the second exon. The mouse neuroDl cDNA sequence indicated a predicted protein of 382 amino acids that differs from the major open reading frame in the human neuroDl gene at only 9 residues, all in the aminoterminal portion of the protein. The human neuroD2 protein was found to have 98% similarity to neuroDl and MATH2 in the bHLH region and 90% similarity in the 30 amino acids immediately carboxyterminal to the bHLH region. Similar to neuroDl and MATH2, neuroD2 contains an aminoterminal region rich in glutamate residues that may constitute an acidic activation domain, and has other regions of similarity to neuroDl throughout the protein. Mouse neuroD3 was obtained by screening a 129SV mouse genomic library cloned in lambda-Dash II (Stratagene®), using a labeled Pst-Pst genomic fragment containing the human neuroD coding sequence using conditions essentially as described above for selecting mouse neuroDl, with the exception that the prehybridization and hybridization were carried out at 55°C and the final wash was carried out at 50°C Since all identified members of the family of genes related to neuroDl are known to have their entire coding sequence in a single exon, the major open reading frame (ORF) encoded in the genomic DNA from human and mouse neuroD3 were determined (SEQ ID NO:12 and SEQ ID NO:21, respectively). The predicted amino acid sequences of the mouse and human neuroD3 proteins are based on the major ORF in the corresponding genomic DNAs, since cDNAs have not been cloned for these genes. The genomic sequence of mouse neuroD 3 contains a major ORF of 244 amino acids and the human neuroD3 gene an ORF of 237 amino acids that differs from the predicted mouse protein at 26 positions. The entire coding region of other neuroD family members is contained within a single exon, and therefore it is possible that the ORF in the neuroD3 genomic DNA represents the entire coding region, a notion supported by the conservation between mouse and human that extends to the stop codon. The major ORF predicts a smaller protein than related neuroD family members, and lacks the acidic rich aminoterminal region. The bHLH region has some elements ofthe loop that are similar to MATHl, but the overall level of homology in the bHLH region is closer to the neuroD-τέlated genes. In contrast to neuroD2, the neuroD3 protein does not contain significant regions of homology to neuroDl or MATH2/NEX-1 outside of the bHLH region and does not have an aminoterminal region rich in glutamates or acidic amino acids. The Genbank accession numbers are: human neuroD2, U58681; mouse neuroD2, U58471; human neuroD3, U63842; mouse neuroD3, U63841. EXAMPLE 12 Chromosome mapping of human neuroDl clones. FISH karyotyping was performed on fixed metaphase spreads of the microcell hybrids essentially as described (Trask et al., Am. J. Hum. Genet. 48:1-15, 1991; and Brandriff et al., Genomics 10:75-82, 1991; which are incoφorated by reference herein in their entirety). NeuroDl sequences were detected using the 9F1 or 20 A 1 phage DNA as probes labeled using digoxigenin-dUTP (Boehringer Mannheim) according to the manufacturer's instructions. Phage DNA was biotinylated by random priming (Gibco/BRL BioNick Kit) and hybridized in situ to denatured metaphase chromosome spreads for 24-48 hours. Probes were detected with rhodamine-conjugated antibodies to digoxigenin, and chromosomes were counterstained with DAPI (Sigma). Signals were viewed through a fluorescence microscope and photographs were taken with color slide film. FISH analysis indicated clone 9F1 maps to human chromosome 2q, and clone 20 A 1 maps to human chromosome 5. Chromosome mapping was also carried out on a human/rodent somatic cell hybrid panel (National Institute of General Medical Sciences, Camden, NJ). This panel consists of DNA isolated from 24 human/rodent somatic cell hybrids retaining one human chromosome. For one set of experiments, the panel of DNA's were digested with Eco RI and electrophoresed on an agarose gel. The DNA was transferred to Hybond-N membranes (Amersham). A random primed (Boehringer Mannheim) 4 kb Eco RJ-Sac I fragment of clone 9F1 was prepared. The filter was prehybridized in 10 ml of FBI hybridization buffer (see above) at 65°C for 10 minutes. After prehybridization, denatured salmon sperm DNA was added to a final concentration of 10 μg/ml, denatured probe was added to a final concentration of one million cpm/ml. The filter was hybridized at 65°C for a period of 8 hours to overnight. After incubation, excess probe was removed, and the filter was washed first in 2 X SSC, 0.1% SDS for 30 minutes at 65°C. The first wash was followed by a final stringent wash in 0.1 X SSC, 0.1% SDS for 30 minutes at 65°C. An autoradiograph of the filter was prepared. Autoradiographs confirmed the FISH mapping results. In the second experiment, the panel was digested with Pst I, electrophoresed and transferred essentially as described above. A random-primed (Boehringer Mannheim) 1.6 kb Pst I fragment of clone 20 A 1 was prepared. The membrane was prehybridized, hybridized with the 20A1 probe and washed as described above Autoradiographs of the Southern filter showed that 20A1 mapped to human chromosome 5 and confirmed the FISH mapping results. After autoradiography, the 20Al-probed membrane was stripped by a wash in 0.5 M NaOH, 1.5 M NaCl. The membrane was neutralized in 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl. The filter was washed in 0.1 X SSC before prehybridization. A random-primed (Boehringer Mannheim) 1.2 kb Pst I fragment of clone 14B1 was prepared. The washed membrane was prehybridized and hybridized with the 14B1 probe as described above. After washing under the previously described conditions, the membrane was autoradiographed. Autoradiographs demonstrated that clone 14B1 mapped to chromosome 17. EXAMPLE 13 Human neuroDl complementary DNA. To obtain a human neuroDl cDNA, one million plaque forming units (pfu) were plated onto twenty LB + 10 mM MgSO4 ( 50 mm) plates using the Stratagene human cDNA library in Lambda ZAP II in the bacterial strain XL-1 Blue (Stratagene). Plating and membrane lifts were performed using standard methods, as described in Example 11. After UV cross-linking, the membranes were pre-hybridized in an aqueous hybridization solution (1% bovine serum albumin, 1 mM EDTA, 0.5 M Na2HPO4 (pH 7.4), 7% SDS) at 50°C for two hours. The mouse neuroDl cDNA insert was prepared by digesting the pKS+ m7a RX plasmid with Eco RI and Xho I, and isolating the fragment containing the cDNA by electroelution. A probe was made with the cDNA containing fragment by random primed synthesis with random hexanucleotides, dGTP, dATP, dTTP, alpha- 32 P-labeled dCTP, and Klenow in a buffered solution (25 mM Tris (pH6.9), 50mM KCl, 5mM MgCl2, ImM DTT). The probe was purified from the unincoφorated nucleotides on a G-50 Sepharose® column. The purified probe was heat denatured at 90°C for 3 minutes. After prehybridization, the denatured probe was added to the membranes in hybridization solution. The membranes were hybridized for 24 hours at 50°C. Excess probe was removed from the membranes, and the membranes were washed in 0.1 X SSC, 0.1% SDS for 20 minutes at 50°C. The wash solution was changed five times. The membranes were blotted dry and covered with plastic film before being subjected to autoradiography. Autoradiography of the filters identified 68 positive clones. The clones were plaque-purified and rescreened to obtain 40 pure, positive clones. The positive clones were screened with a random-primed Pst I fragment from clone 9F1 (human neuroDl) Twelve positive clones that hybridized with the human neuroDl genomic probe were isolated. The plasmid vector containing cDNA insert was excised in vivo from the lambda phage clone according to the Stratagene methodology. Briefly, eluted phage and XL-1 Blue cells (200 microliters of OD 600=1) were mixed with R408 helper phage provided by Stratagene for 15 minutes at 37°C. Five milliliters of rich bacterial growth media (2 X YT, see Sambrook et al., ibid.) was added, and the cultures were incubated for 3 hours at 37°C. The tubes were heated at 70°C for 20 minutes and spun for 5 minutes at 4,000 X g. After centrifugation, 200 microliters of supernatant were added to the same volume of XL-1 Blue cells (OD=l), and the mixture was incubated for 15 minutes at 37°C, after which the bacterial cells were plated onto LB plates containing 50 mg/ml ampicillin. Each colony was picked and grown for sequencing template preparation. The clones were sequenced and compared to the human genomic sequence. A full length cDNA encoding human neuroDl that was identical to the 9F1 neuroDl genomic sequence was obtained and designated HC2A. The nucleotide and deduced amino acid sequences are shown in SEQ ID NOS: 14 and 15, respectively. Clone HC2A was deposited at the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD 20852 USA, on November 1, 1995, under accession number 69944. Using a random-primed radiolabeled antisense probe to the mouse neuroDl (Boehringer Mannheim), the expression pattem was determined using Northern analysis. Filters containing murine RNA from the brain and spinal cords of embryonic through adult mice were probed at high stringency and washed in 0.1 X SSC, 0.1% SDS at 65°C. Northern analysis showed neuroDl expression in the brain and spinal cords of mice from embryonic day 12.5 through adult. Experiments were conducted also to isolate a cDNA corresponding to mouse neuroD3 mRNA. Using procedures similar to those described above, a random- primed 1.1 kb Pst I fragment from human neuroD 3 clone 20 A 1 was prepared and used to screen mouse embryo and newborn mouse brain libraries. For unknown reasons, no positive clones were obtained. Likewise, attempts to clone human neuroD 3 cDNA have been unsuccessful. The difficulty in obtaining neuroD 3 cDNA may be secondary to instability of the construct in the library, since deletions in the genomic DNA were common during amplification. EXAMPLE 14 Construction of knock-out mice Knock-out mice in which the murine neuroDl coding sequence was replaced with the θ-galactosidase gene and the neomycin resistance gene (neo) were generated i) to assess the consequences of eliminating the murine neuroDl protein during mouse development and ii) to permit examination of the expression pattem of neuroDl in embryonic mice. Genomic neuroDl sequences used for these knock-out mice were obtained from the 129/Sv mice so that the homologous recombination could take place in a congenic background in 129/Sv mouse embryonic stem cells. Several murine neuroDl genomic clones were isolated from a genomic library prepared from 129/Sv mice (Zhuang et al., Cell 79:875-884, 1994; which is incoφorated herein by reference in its entirety) using the Bam HI-Not I neuroDl cDNA containing fragment of pSK+1-83 (Example 2) as a random-primed probe essentially as described in Example 11. Plasmid pPNT (Tybulewicz et al., Cell 65:1153-1163, 1991; which is incoφorated herein by reference in its entirety) containing the neomycin resistance gene (neo; a positive selection marker) and the Herpes simplex virus thymidine kinase gene (hsv-tk, a negative selection marker) under the control of the PGK promoter provided the vector backbone for the targeting construct A 1 4 kb 5' murine neuroDl genomic fragment together with the 3 kb cytoplasmic β-galactosidase gene were inserted between the Eco RI and Xba I sites of the pPNT vector, and an 8 kb fragment containing the genomic 3' untranslated sequence of neuroDl was inserted into the vector backbone between into the Xho I and Not I sites To prepare an Eco Rl-Xba I fragment containing neuroDl promoter sequences joined to the β-galactosidase gene, a 1 4 kb Eco RI(vector- derived)-Asp 718 fragment containing the 5' untranslated murine neuroDl genomic sequence was ligated to a Hind III-Xba I fragment containing the cytoplasmic β-galactosidase gene such that the Asp 718 and Hind III sites were destroyed The resulting approximately 4 4 kb Eco Rl-Xba I fragment, containing the 5' neuroDl genomic sequence (including the neuroDl promoter) and the β-galactosidase gene in the same transcriptional orientation, was inserted into Eco Rl-Xba I linearized pPNT to yield the plasmid pPNT/5'+β-gal A neuroDl fragment containing 3' untranslated DNA was obtained from a murine neuroDl genomic clone that had been digested with Spe I and Not I(vector-derived) to yield an 8 kb fragment To obtain a 5' Xho I site, the 8 kb fragment was inserted into Spe I-Not I linearized pBlueskriptSK+ (Stratagene), and the resulting plasmid digested with Xho I and Not I to obtain the 8 kb neuroDl 3' genomic fragment The Xho I-Not I fragment was inserted into Xho I-Not I linearized pPNT/5'+β-gal to yield the neuroDl targeting vector The final construct contained the 5' neuroDl fragment, the β-galactosidase gene, and the 3' genomic neuroDl fragment in the same orientation, and the hsv-tk and neomycin resistance genes in the opposite orientation The targeting construct was transfected by electroporation into mouse embryonic stem (ES) cells. A 129/Sv derived ES cell line, AK-7 described by Zhuang et al (ibid.) was used for electroporation These ES cells were routinely cultured on mitomycin C-treated (Sigma) SNL 76/7 cells (feeder cells) as described by McMahon and Bradley (Cell 62:1073-1085, 1990; which is incoφorated herein by reference in its entirety) in culture medium containing high glucose DMEM supplemented with 15% fetal bovine serum (Hyclone) and 0 1 μM β-mercaptoethanol To prepare the targeting construct for transfection, 25 μg ofthe targeting construct was linearized by digestion with Not I, phenol-chloroform extracted, and ethanol precipitated The 7 linearized vector was then electroporated into 1-2 x 10 AK-7 (ES) cells The electroporated cells were seeded onto three 10-cm plates, with one plate receiving 50% of the electroporated cells and the remaining two plates each receiving 25% of the electroporated cells After 24 hours, G418 was added to each of the plates to a final concentration of 150 μg/ml After an additional 24 hours, gancyclovir was added to a final concentration of 0.2 μM to the 50% plate and one of the 25% plates The third plate containing 25% ofthe electroporated cells was subjected to only G418 selection to assess the efficiency of gancyclovir selection The culture medium for each plate was changed every day for the first few days, and then changed as needed after selection had occurred After 10 days of selection, a portion of each colony was picked microscopically with a drawn micropipette, and was directly analyzed by PCR as described by Joyner, et al (Nature 338 153-156, 1989, which is incoφorated herein by reference in its entirety) Briefly, PCR amplification was performed as described (Kogan et al , New England J. Med. 317 985-990, 1987, which is incoφorated herein by reference in its entirety) using 40 cycles of 93 °C for 30 seconds, 57°C for 30 seconds, and 65°C for 3 minutes To detect the wild-type allele, primers JL34 and JL36 (SEQ ID NOS 18 and 19, respectively) were used in the PCR reaction, to detect the mutant neuroDl allele, primers JL34 and JL40 (SEQ ID NOS 18 and 20, respectively) were used in the PCR reaction Positive colonies, identified by PCR, were subcloned into 4-well plates, expanded into 60 mm plates and frozen into 2-3 ampules Among the clones that were selected for both G418-resistance (positive selection for neo gene expression) and gancyclovir-resistance (negative selection for hsv-tk gene expression), 10% of the population contained correctly targeted integration of the vector into the murine neuroDl locus (an overall 10% targeting frequency) The negative selection provided 4-8 fold enrichment for homologous recombination events To generate chimeric mice, each positive clone was thawed and passaged once on feeder cells The transfected cells were trypsinized into single cells, and blastocysts obtained from C57BL/6J mice were injected with approximately 15 cells The injected blastocysts were then implanted into pseudopregnant mice (C57BL/6J x CBA) Four male chimeras arose from the injected blastocysts (AK-71, AK-72, AK-74 and AK-75) The male chimeras AK-71 and AK-72 gave germ-line transmission at a high rate as determined by the frequency of agouti coat color transmission to their offspring (Fl) in a cross with C57BL/6J female mice Since 50% of the agouti coat color offspring (Fl) should represent heterozygous mutants, their genotypes were determined by Southern blot analysis Briefly, genomic DNA prepared from tail biopsies was digested with Eco RI and probed with the 1 4 kb 5' genomic sequence used to make the targeting construct. This probe detects a 4 kb Eco RI fragment from the wild-type allele and a 6.3 kb Eco RI fragment from the mutant allele. Therefore, a Southern analysis would show a single 4 kb band for a wild-type mouse, 4 kb and 6.3 kb fragments for a heterozygous mouse, and a single 6.3 kb band for a homozygous mutant mouse. The resulting offspring (Fl) heterozygous (+/-) mice, were mated with sibling heterozygous mice to give rise to the homozygous (-/-) mutant mice. To study neuroDl expression patterns in embryonic mice, chimeric mice or Fl heterozygous progeny from the chimera x C57B/6J mating were crossed with C57B/6J. Litters resulting from these crosses were harvested from pregnant females and stained for β-galactosidase activity The embryos were dissected away from all the extra-embryonic tissue and the yolk sac was reserved for DNA analysis The embryos were fixed for one hour in a fixing solution (0.1 M phosphate buffer containing 0.2% glutaraldehyde, 2% formaldehyde, 5 mM EGTA (pH 7.3), 2 mM MgCl2) The fixing solution was removed by three thirty-minute rinses with rinse solution (0.1 M phosphate buffer (pH 7.3) containing 2 mM MgCl2, 0.1% sodium deoxycholate, 0.2% NP-40). The fixed embryos were stained overnight in the dark in rinse solution containing 1 mg/ml X-gal, 5 mM sodium ferricyanide, 5 mM sodium ferrocyanide. After staining, the embryos were rinsed with PBS and stored in the fixing solution before preparation for examination. Examination of stained tissue from fetal and postnatal mice heterozygous for the mutation confirmed the neuroDl expression pattem in neuronal cells demonstrated previously by in situ hybridization (Example 4), and also demonstrated neuroDl expression in the pancreas and gastrointestinal tract. Blood glucose levels were detected using PRECISION QID blood glucose test strips and a PRECISION QID blood glucose sensor (Medisens Inc., Waltham, MA) according to the manufacturer's instruction. A tissue sample was taken for DNA analysis and the pups were fixed for further histological examination. Blood glucose levels in mice homozygous for the mutation (neuroDl) had blood glucose levels between 2 and 3 times higher than the blood glucose level of wild-type mice. Heterozygous mutants exhibited similar blood glucose levels as wild-type mice. Mice that were homozygous for the mutation (lacking neuroDl) had diabetes as demonstrated by high blood glucose levels and died by day four; some homozygous mice died at birth. EXAMPLE 15 NeuroDl expression and activity in PC 11 and P 19 embryonic carcinoma cells Murine PC 12 pheochromacytoma cells differentiate into neurons in tissue culture in the presence of appropriate inducers, i.e., nerve growth factor. Neither induced nor non-induced murine PC 12 cells expressed neuroDl transcripts, nor did control 3T3 fibroblasts produce detectable levels of neuroDl transcription products. P19 cells are a well characterized mouse embryonic carcinoma cell line with the ability to differentiate into numerous cell types, including skeletal and cardiac muscle, or neurons and glia following treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA) (Jones- Villeneuve et al., Molec. & Cell. Biol. 3:2271-2279, 1983), respectively. To determine whether P19 cells expressed endogenous neuroD genes during neuronal differentiation, RNA expression was analyzed for neuroDl, neuroDl, and neuroD3 in both uninduced and induced P19 cells. To induce the formation of neurons, P19 cells were cultured as aggregates in Petri dishes in the presence of retinoic acid for four days. The aggregates were then plated into tissue culture dishes in the absence of retinoic acid and neuronal differentiation occurred during a five day period, as evidenced by the formation of neurofilament positive process bearing cells. NeuroDl mRNA was most abundant after the cells were aggregated and treated with RA for 4 days, and continued to be expressed at decreased levels during the period of neuronal differentiation. NeuroDl was not detected during the period of RA induction, but became abundant during the period of neuronal differentiation. Both neuroDl and neuroDl signals were modestly enhanced when the differentiated P19 cultures were grown in the presence of Ara-C which eliminates some of the non- neuronal dividing cells, suggesting that the neuroDl and neuroDl genes are preferentially expressed in the post-mitotic cell population but further experiments will be necessary to prove this point. NeuroD 3 was first detected after two days of induction, and was most abundant after 4 days of induction), however, unlike neuroDl, neuroD 3 mRNA was not detected at the later, more differentiated, time points. Therefore, the temporal expression pattem of neuroDl, neuroDl, and neuroD 3 in differentiating P19 cells was similar to that seen during embryonic development: a peak of neuroD expression at the time of neuronal commitment and early neurogenesis, early and persistent expression of neuroDl, and slightly later and persistent expression of neuroDl. Hence, P19 cells are potentially useful in screening assays for identifying inducers of neuroDl expression that may stimulate nerve regeneration and differentiation of neural tumor cells. NeuroDl and neuroDl are both expressed in neurons and both can induce neurogenesis when expressed in frog embryos. To determine if they have the ability to activate similar target genes, expression vectors were constructed driving the human neuroDl or neuroD2 coding regions from a simian cytomegalovirus promoter; these vectors are pCS2-hNDl and pCS2-hND2, whose construction is described in Example 10. The activity of neuroDl and neuroD2 was assayed on reporter constructs co-transfected into P19 cells. Other members of the neuroD family have been shown to bind consensus E-box sequences in vitro. Gel shift assays have demonstrated that MATH-1 and NEX-l/MATH-2 bind the consensus E-box site CAGGTG as a heterodimer with the E47 protein, and activate the transcription of reporter constructs (Akazawa et al., 1995; Bartholoma, A. and K. A. Nave., 1994; Shimizu et al., 1995). In vitro gel shift assays demonstrated that neuroDl and neuroD2 proteins can bind to an oligo containing the core E-box CACCTG as a heterodimer with an E-protein. Therefore, we tested the ability of neuroDl and neuroD2 proteins to activate transcription of a simple reporter construct composed of a multimerized E-box with the same core sequence and the minimal promoter from the thymidine kinase gene driving luciferase, p4RTK-luc. P19 cells to be transfected were cultured in minimal essential medium alpha supplemented with 10% fetal bovine serum. Transfections were performed as previously described (Tamura, M. and M. Noda., 1994), using a BBS calcium chloride precipitation. Forty-eight hours after transfection, the cells were harvested and assayed for luciferase and lacZ. Construction of the expression vectors pCS2- hNDl and pCS2-hND2 were as described in Example 10. The ρGAP43-luc construct, a neuronal specific promoter construct that is upregulated in vivo in post- mitotic, terminally differentiating neurons (Nedivi et al., J. Neurosci. 12:691-704, 1992), contained the GAP 43 760 base pair promoter region driving luciferase in a pGL2 vector modified to contain a poly-A site upstream of the multiple cloning site, and was the generous gift of Pate Skene and Joseph Weber. The pND2-luciderase construct was made by cloning a lkb fragment of mouse neuroDl sequence terminating in the first exon, cloning this fragment into the pGL3 luciferase vector. The p4RTK-luciferase construct was made by placing the 4RTK region from Hindlll to Xhol of the p4RTK-CAT vector (Weintraub et al., Proc. Natl. Acad. Sci. 87:5623-5627, 1990) into the promoterless luciferase vector. Luciferase assays were performed according to Current Protocols in Molecular Biology (Brasier, A. R., John Wiley & Sons, New York, 1989). When P19 cells were transfected as described above, it was observed that cotransfection with either pCS2-NDl or pCS2-ND2 modestly increased the level of activity from p4RTK-luc in P19 cells, increasing the activity between two and four¬ fold. Additional reporter constructs were tested in P19 cells to determine whether the neuroD and neuroD2 proteins had different transcriptional activation potentials. Tests were conducted to determine the ability of pCS2-NDl and pCS2-ND2 to transactivate the luciferase reporter constmct, pGAP43-luciferase. In contrast to the simple E-box driven reporter, pCS2-NDl did not show significant transactivation of the pGAP43 -luciferase, while pCS2-ND2 induced expression from this constmct by approximately 4-fold over the basal activity. The myogenic bHLH proteins show auto- and cross-regulation, and expression of NEX-l/MATH-2 has been shown to activate a reporter driven by the NEX-l/MATH-2 promoter (Bartholoma and Nave, 1994). To determine if neuroDl or neuroD2 could activate a constmct containing the neuroDl promoter, we made a constmct that contained a one kilobase fragment upstream of the mouse neuroDl gene, terminating in the first exon, driving the luciferase reporter gene. P19 cells were co-transfected with this pND2-luc reporter constmct and the neuroD expression vectors. Both pCS2-NDl and pCS2-ND2 transactivated this reporter constmct, suggesting that neuroDl may be auto-regulated and cross-regulated by other members of the neuroD family, in a manner analogous to the regulation of the myogenic bHLH genes. Together these transfection experiments demonstrate that neuroDl and neuroD2 proteins can both activate some target genes, such as a multimerized E-box reporter and the neuroDl promoter; whereas the reporter constmct driven by the GAP 43 promoter seems to be preferentially activated by neuroD2. At this time the amount of protein made from each vector following transfection cannot be quantitated, and inteφretations rely on the relative activity ofthe reporter constructs. Further analysis of the specificity of neuroD and neuroD2 will require identifying specific cis acting sequences in these reporters that mediate activity. EXAMPLE 16 In situ localization of neuroDl and neuroDl RNA in adult mouse brain To address the question of whether neuroDl and neuroDl were expressed in neurons in the adult mouse brain and whether they were expressed in the same cells, in situ hybridizations were performed using 35 S-UTP labeled RNA probes Sections of adult mouse brain were hybridized to anti-sense probes derived from the mouse neuroDl and neuroDl cDNA fragments using T3 and T7 generated transcripts for sense and anti-sense probes, and incoφorating 35 S-UTP label Frozen 4-5 micron sagittal sections of adult mouse brain were cut, placed on Fisher Superfrost slides, and frozen at -80°C Hybridization to 35 SUTP labeled probes and autoradiography was performed according to Masters et al. (J. Neurosci 14 5844-5857, 1994), which is hereby incoφorated by reference After washing to remove unhybridized probe, sections were coated with liquid photographic emulsion After development of the emulsion, dark field optics illuminated the silver grains as white spots at magnification X160 In the cerebellum, neuroDl was easily detected in the granule layer, whereas the neuroDl signal was less intense in this region and was largely restricted to the region of the Purkinje cells In contrast, the neuroDl and neuroDl signals in the pyramidal cells and dentate gyms of the hippocampus were easily detected The neuroDl probe hybridized preferentially to the region of the Purkinje cell layer These results demonstrate that neuroDl and neuroDl are expressed in neuronal populations in the mature nervous system, and that their relative level of expression varies among neuronal populations. From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses of illustration, various modification may be made without deviating from the spirit and scope of the invention SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANTS: Weintraub, Harold M. Lee, Jacqueline E. Tapscott, Stephen J. Hollenberg, Stanley M. (ii) TITLE OF INVENTION: Neurogenic Differentiation (NeuroD) Genes and Proteins (iii) NUMBER OF SEQUENCES: 22 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Christensen O'Connor Johnson Kindness PLLC (B) STREET: 1420 Fifth Avenue, Suite 2800 (C) CITY: Seattle (D) STATE: WA (E) COUNTRY: USA (F) ZIP: 98101-2347 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/239,238 (B) FILING DATE: 06-MAY-1994 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: WO PCT/US95/05741 (B) FILING DATE: 08-MAY-1995 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Sheiness, Diana K. (B) REGISTRATION NUMBER: 35,356 (C) REFERENCE/DOCKET NUMBER: FHCR-1-10112 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 206-224-0735 (B) TELEFAX: 206-225-0779 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2089 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 229..1302 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ACTACGCAGC ACCGAGGTAC AGACACGCCA GCATGAAGCA CTGCGTTTAA CTTTTCCTGG 60 AGGCATCCAT TTTGCAGTGG ACTCCTGTGT ATTTCTATTT GTGTGCATTT CTGTAGGATT 120 AGGGAGAGGG AGCTGAAGGC TTATCCAGCT TTTAAATATA GCGGGTGGAT TTCCCCCCCT 180 TTCTTCTTCT GCTTGCCTCT CTCCCTGTTC AATACAGGAA GTGGAAAC ATG ACC AAA 237 Met Thr Lys 1 TCA TAC AGC GAG AGC GGG CTG ATG GGC GAG CCT CAG CCC CAA GGT CCC 285 Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gin Pro Gin Gly Pro 5 10 15 CCA AGC TGG ACA GAT GAG TGT CTC AGT TCT CAG GAC GAG GAA CAC GAG 333 Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gin Asp Glu Glu His Glu 20 25 30 35 GCA GAC AAG AAA GAG GAC GAG CTT GAA GCC ATG AAT GCA GAG GAG GAC 381 Ala Asp Lys Lys Glu Asp Glu Leu Glu Ala Met Asn Ala Glu Glu Asp 40 45 50 TCT CTG AGA AAC GGG GGA GAG GAG GAG GAG GAA GAT GAG GAT CTA GAG 429 Ser Leu Arg Asn Gly Gly Glu Glu Glu Glu Glu Asp Glu Asp Leu Glu 55 60 65 GAA GAG GAG GAA GAA GAA GAG GAG GAG GAG GAT CAA AAG CCC AAG AGA 477 Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Gin Lys Pro Lys Arg 70 75 80 CGG GGT CCC AAA AAG AAA AAG ATG ACC AAG GCG CGC CTA GAA CGT TTT 525 Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu Glu Arg Phe 85 90 95 AAA TTA AGG CGC ATG AAG GCC AAC GCC CGC GAG CGG AAC CGC ATG CAC 573 Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn Arg Met His 100 105 110 115 GGG CTG AAC GCG GCG CTG GAC AAC CTG CGC AAG GTG GTA CCT TGC TAC 621 Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val Pro Cys Tyr 120 125 130 TCC AAG ACC CAG AAA CTG TCT AAA ATA GAG ACA CTG CGC TTG GCC AAG 669 Ser Lys Thr Gin Lys Leu Ser Lys Ile Glu Thr Leu Arg Leu Ala Lys 135 140 145 AAC TAC ATC TGG GCT CTG TCA GAG ATC CTG CGC TCA GGC AAA AGC CCT 717 Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly Lys Ser Pro 150 155 160 GAT CTG GTC TCC TTC GTA CAG ACG CTC TGC AAA GGT TTG TCC CAG CCC 765 Asp Leu Val Ser Phe Val Gin Thr Leu Cys Lys Gly Leu Ser Gin Pro 165 170 175 ACT ACC AAT TTG GTC GCC GGC TGC CTG CAG CTC AAC CCT CGG ACT TTC 813 Thr Thr Asn Leu Val Ala Gly Cys Leu Gin Leu Asn Pro Arg Thr Phe 180 185 190 195 TTG CCT GAG CAG AAC CCG GAC ATG CCC CCG CAT CTG CCA ACC GCC AGC 861 Leu Pro Glu Gin Asn Pro Asp Met Pro Pro His Leu Pro Thr Ala Ser 200 205 210 GCT TCC TTC CCG GTG CAT CCC TAC TCC TAC CAG TCC CCT GGA CTG CCC 909 Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gin Ser Pro Gly Leu Pro 215 220 225 AGC CCG CCC TAC GGC ACC ATG GAC AGC TCC CAC GTC TTC CAC GTC AAG 957 Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe His Val Lys 230 235 240 CCG CCG CCA CAC GCC TAC AGC GCA GCT CTG GAG CCC TTC TTT GAA AGC 1005 Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe Phe Glu Ser 245 250 255 CCC CTA ACT GAC TGC ACC AGC CCT TCC TTT GAC GGA CCC CTC AGC CCG 1053 Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro Leu Ser Pro 260 265 270 275 CCG CTC AGC ATC AAT GGC AAC TTC TCT TTC AAA CAC GAA CCA TCC GCC 1101 Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu Pro Ser Ala 280 285 290 GAG TTT GAA AAA AAT TAT GCC TTT ACC ATG CAC TAC CCT GCA GCG ACG 1149 Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro Ala Ala Thr 295 300 305 CTG GCA GGG CCC CAA AGC CAC GGA TCA ATC TTC TCT TCC GGT GCC GCT 1197 Leu Ala Gly Pro Gin Ser His Gly Ser Ile Phe Ser Ser Gly Ala Ala 310 315 320 GCC CCT CGC TGC GAG ATC CCC ATA GAC AAC ATT ATG TCT TTC GAT AGC 1245 Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe Asp Ser 325 330 335 CAT TCG CAT CAT GAG CGA GTC ATG AGT GCC CAG CTT AAT GCC ATC TTT 1293 His Ser His His Glu Arg Val Met Ser Ala Gin Leu Asn Ala Ile Phe 340 345 350 355 CAC GAT TAGAGGGCAC GTCAGTTTCA CTATTCCCGG GAAACGAATC CACTGTGCGT 1349 His Asp ACAGTGACTG TCCTGTTTAC AGAAGGCAGC CCTTTTGCTA AGATTGCTGC AAAGTGCAAA 1409 TACTCAAAGC TTCAAGTGAT ATATGTATTT ATTGTCGTTA CTGCCTTTGG AAGAAACAGG 1469 GGATCAAAGT TCCTGTTCAC CTTATGTATT GTTTTCTATA GCTCTTCTAT TTTAAAAATA 1529 ATAATACAGT AAAGTAAAAA AGAAAATGTG TACCACGAAT TTCGTGTAGC TGTATTCAGA 1589 TCGTATTAAT TATCTGATCG GGATAAAAAA AATCACAAGC AATAATTAGG ATCTATGCAA 1649 TTTTTAAACT AGTAATGGGC CAATTAAAAT ATATATAAAT ATATATTTTT CAACCAGCAT 1709 TTTACTACCT GTGACCTTTC CCATGCTGAA TTATTTTGTT GTGATTTTGT ACAGAATTTT 1769 TAATGACTTT TTATAACGTG GATTTCCTAT TTTAAAACCA TGCAGCTTCA TCAATTTTTA 1829 TACATATCAG AAAAGTAGAA TTATATCTAA TTTATACAAA ATAATTTAAC TAATTTAAAC 1889 CAGCAGAAAA GTGCTTAGAA AGTTATTGCG TTGCCTTAGC ACTTCTTTCT TCTCTAATTG 1949 TAAAAAAGAA AAAAAAAAAA AAAAAACTCG AGGGGGGGCC CGGTACCCAG CTTTTGTTCC 2009 CTTTAGTGAG GGTTAATTGC GCGCTTGGCG TAATCATGGT CATAGCTGTT TCCTGTGTGA 2069 ATTGTTATCC GCTCACAATT 2089 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 357 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gin Pro 1 5 10 15 Gin Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gin Asp Glu 20 25 30 Glu His Glu Ala Asp Lys Lys Glu Asp Glu Leu Glu Ala Met Asn Ala 35 40 45 Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Glu Glu Asp Glu 50 55 60 Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Gin Lys 65 70 75 80 Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu 85 90 95 Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110 Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125 Pro Cys Tyr Ser Lys Thr Gin Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140 Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly 145 150 155 160 Lys Ser Pro Asp Leu Val Ser Phe Val Gin Thr Leu Cys Lys Gly Leu 165 170 175 Ser Gin Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gin Leu Asn Pro 180 185 190 Arg Thr Phe Leu Pro Glu Gin Asn Pro Asp Met Pro Pro His Leu Pro 195 200 205 Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gin Ser Pro 210 215 220 Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe 225 230 235 240 His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe 245 250 255 Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro 260 265 270 Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu 275 280 285 Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro 290 295 300 Ala Ala Thr Leu Ala Gly Pro Gin Ser His Gly Ser Ile Phe Ser Ser 305 310 315 320 Gly Ala Ala Ala Pro Arg Cys Glu Ile Pro lie Asp Asn Ile Met Ser 325 330 335 Phe Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gin Leu Asn 340 345 350 Ala Ile Phe His Asp 355 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1275 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Xenopus laevis (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 25..1083 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATTTCCTTTC TCCAGATCTA AAAA ATG ACC AAA TCG TAT GGA GAG AAT GGG 51 Met Thr Lys Ser Tyr Gly Glu Asn Gly 1 5 CTG ATC CTG GCC GAG ACT CCG GGC TGC AGA GGA TGG GTG GAC GAA TGC 99 Leu Ile Leu Ala Glu Thr Pro Gly Cys Arg Gly Trp Val Asp Glu Cys 10 15 20 25 CTG AGT TCT CAG GAT GAA AAC GAT CTG GAG AAA AAG GAG GGA GAG TTG 147 Leu Ser Ser Gin Asp Glu Asn Asp Leu Glu Lys Lys Glu Gly Glu Leu 30 35 40 ATG AAA GAA GAC GAT GAA GAC TCA CTG AAT CAT CAC AAT GGA GAG GAG 195 Met Lys Glu Asp Asp Glu Asp Ser Leu Asn His His Asn Gly Glu Glu 45 50 55 AAC GAG GAA GAG GAT GAA GGG GAT GAG GAG GAG GAG GAC GAT GAA GAT 243 Asn Glu Glu Glu Asp Glu Gly Asp Glu Glu Glu Glu Asp Asp Glu Asp 60 65 70 GAT GAT GAG GAT GAC GAC CAG AAA CCC AAA AGG CGA GGA CCG AAA AAG 291 Asp Asp Glu Asp Asp Asp Gin Lys Pro Lys Arg Arg Gly Pro Lys Lys 75 80 85 AAA AAA ATG ACG AAA GCC CGG GTG GAG CGA TTT AAA GTG AGA CGC ATG 339 Lys Lys Met Thr Lys Ala Arg Val Glu Arg Phe Lys Val Arg Arg Met 90 95 100 105 AAG GCA AAC GCC AGG GAG AGG AAT CGC ATG CAC GGA CTC AAC GAT GCC 387 Lys Ala Asn Ala Arg Glu Arg Asn Arg Met His Gly Leu Asn Asp Ala 110 115 120 CTG GAC AGT CTG CGC AAA GTT GTG CCC TGC TAC TCC AAA ACA CAA AAG 435 Leu Asp Ser Leu Arg Lys Val Val Pro Cys Tyr Ser Lys Thr Gin Lys 125 130 135 TTG TCT AAG ATT GAA ACT CTG CGC CTG GCT AAG AAC TAC ATC TGG GCT 483 Leu Ser Lys Ile Glu Thr Leu Arg Leu Ala Lys Asn Tyr Ile Trp Ala 140 145 150 CTT TCT GAG ATT TTA AGG TCC GGC AAA AGC CCA GAC CTG GTG TCC TTT 531 Leu Ser Glu Ile Leu Arg Ser Gly Lys Ser Pro Asp Leu Val Ser Phe 155 160 165 GTA CAA ACT CTC TGC AAA GGT TTG TCG CAG CCC ACC ACC AAT CTA GTA 579 Val Gin Thr Leu Cys Lys Gly Leu Ser Gin Pro Thr Thr Asn Leu Val 170 175 180 185 GCG GGG TGT CTG CAG CTG AAC CCC AGA ACT TTC CTT CCT GAG CAG AGT 627 Ala Gly Cys Leu Gin Leu Asn Pro Arg Thr Phe Leu Pro Glu Gin Ser 190 195 200 CAG GAC ATC CAG TCG CAC ATG CAA ACA GCG AGC TCT TCC TTC CCT CTG 675 Gin Asp Ile Gin Ser His Met Gin Thr Ala Ser Ser Ser Phe Pro Leu 205 210 215 CAG GGC TAT CCC TAT CAG TCC CCT GGT CTT CCC AGT CCC CCC TAT GGT 723 Gin Gly Tyr Pro Tyr Gin Ser Pro Gly Leu Pro Ser Pro Pro Tyr Gly 220 225 230 ACC ATG GAC AGC TCC CAT GTA TTC CAC GTC AAG CCT CAC TCC TAT GGG 771 Thr Met Asp Ser Ser His Val Phe His Val Lys Pro His Ser Tyr Gly 235 240 245 GCG GCC CTG GAG CCT TTC TTT GAC AGC AGC ACC GTC ACT GAG TGT ACC 819 Ala Ala Leu Glu Pro Phe Phe Asp Ser Ser Thr Val Thr Glu Cys Thr 250 255 260 265 AGC CCG TCA TTC GAT GGT CCC CTG AGC CCA CCC CTT AGT GTT AAT GGG 867 Ser Pro Ser Phe Asp Gly Pro Leu Ser Pro Pro Leu Ser Val Asn Gly 270 275 280 AAC TTT ACT TTT AAA CAC GAG CAT TCG GAG TAT GAT AAA AAT TAC ACG 915 Asn Phe Thr Phe Lys His Glu His Ser Glu Tyr Asp Lys Asn Tyr Thr 285 290 295 TTC ACT ATG CAC TAT CCT GCA GCC ACT ATA TCC CAG GGC CAC GGA CCA 963 Phe Thr Met His Tyr Pro Ala Ala Thr Ile Ser Gin Gly His Gly Pro 300 305 310 TTG TTC TCC ACG GGG GGA CCA CGC TGT GAA ATC CCA ATA GAC ACC ATC 1011 Leu Phe Ser Thr Gly Gly Pro Arg Cys Glu Ile Pro Ile Asp Thr lie 315 320 325 ATG TCC TAT GAC GGT CAC TCC CAC CAT GAA AGA GTC ATG AGT GCC CAG 1059 Met Ser Tyr Asp Gly His Ser His His Glu Arg Val Met Ser Ala Gin 330 335 340 345 CTA AAT GCC ATC TTT CAT GAT TAACCCTTGG AAGATCAAAA CAACTGACTG 1110 Leu Asn Ala Ile Phe His Asp 350 TGCATTGCCA GGACTGTCTT GTTTACCAAG GGCAGACACG TGGGTAGTAA AAGTGCAAAT 1170 GCCCCACTCT GGGGCTGTAA CAAACTTGAT CTTGTCCTGC CTTTAGATAT GGGGAAACCT 1230 AATGTATTAA TTCCCACCTC CTTCCAATCG ACACTCCTTT AAATT 1275 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 352 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Thr Lys Ser Tyr Gly Glu Asn Gly Leu Ile Leu Ala Glu Thr Pro 1 5 10 15 Gly Cys Arg Gly Trp Val Asp Glu Cys Leu Ser Ser Gin Asp Glu Asn 20 25 30 Asp Leu Glu Lys Lys Glu Gly Glu Leu Met Lys Glu Asp Asp Glu Asp 35 40 45 Ser Leu Asn His His Asn Gly Glu Glu Asn Glu Glu Glu Asp Glu Gly 50 55 60 Asp Glu Glu Glu Glu Asp Asp Glu Asp Asp Asp Glu Asp Asp Asp Gin 65 70 75 80 Lys Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg 85 90 95 Val Glu Arg Phe Lys Val Arg Arg Met Lys Ala Asn Ala Arg Glu Arg 100 105 110 Asn Arg Met His Gly Leu Asn Asp Ala Leu Asp Ser Leu Arg Lys Val 115 120 125 Val Pro Cys Tyr Ser Lys Thr Gin Lys Leu Ser Lys Ile Glu Thr Leu 130 135 140 Arg Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser 145 150 155 160 Gly Lys Ser Pro Asp Leu Val Ser Phe Val Gin Thr Leu Cys Lys Gly 165 170 175 Leu Ser Gin Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gin Leu Asn 180 185 190 Pro Arg Thr Phe Leu Pro Glu Gin Ser Gin Asp Ile Gin Ser His Met 195 200 205 Gin Thr Ala Ser Ser Ser Phe Pro Leu Gin Gly Tyr Pro Tyr Gin Ser 210 215 220 Pro Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val 225 230 235 240 Phe His Val Lys Pro His Ser Tyr Gly Ala Ala Leu Glu Pro Phe Phe 245 250 255 Asp Ser Ser Thr Val Thr Glu Cys Thr Ser Pro Ser Phe Asp Gly Pro 260 265 270 Leu Ser Pro Pro Leu Ser Val Asn Gly Asn Phe Thr Phe Lys His Glu 275 280 285 His Ser Glu Tyr Asp Lys Asn Tyr Thr Phe Thr Met His Tyr Pro Ala 290 295 300 Ala Thr Ile Ser Gin Gly His Gly Pro Leu Phe Ser Thr Gly Gly Pro 305 310 315 320 Arg Cys Glu Ile Pro Ile Asp Thr Ile Met Ser Tyr Asp Gly His Ser 325 330 335 His His Glu Arg Val Met Ser Ala Gin Leu Asn Ala Ile Phe His Asp 340 345 350 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Asn Ala Arg Glu Arg Arg Arg 1 5 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (v) FRAGMENT TYPE internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Asn Glu Arg Glu Arg Asn Arg 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asn Ala Arg Glu Arg 1 5 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 524 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: 9F1 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 57..524 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TTTTTCTGCT TTTCTTTCTG TTTGCCTCTC CCTTGTTGAA TGTAGGAAAT CGAAAC 56 ATG ACC AAA TCG TAC AGC GAG AGT GGG CTG ATG GGC GAG CCT CAG CCC 104 Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gin Pro 1 5 10 15 CAA GGT CCT CCA AGC TGG ACA GAC GAG TGT CTC AGT TCT CAG GAC GAG 152 Gin Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gin Asp Glu 20 25 30 GAG CAC GAG GCA GAC AAG AAG GAG GAC GAC CTC GAA GCC ATG AAC GCA 200 Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala 35 40 45 GAG GAG GAC TCA CTG AGG AAC GGG GGA GAG GAG GAG GAC GAA GAT GAG 248 Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu 50 55 60 GAC CTG GAA GAG GAG GAA GAA GAG GAA GAG GAG GAT GAC GAT CAA AAG 296 Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gin Lys 65 70 75 80 CCC AAG AGA CGC GGC CCC AAA AAG AAG AAG ATG ACT AAG GCT CGC CTG 344 Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu 85 90 95 GAG CGT TTT AAA TTG AGA CGC ATG AAG GCT AAC GCC CGG GAG CGG AAC 392 Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110 CGC ATG CAC GGA CTG AAC GCG GCG CTA GAC AAC CTG CGC AAG GTG GTG 440 Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125 CCT TGC TAT TCT AAG ACG CAG AAG CTG TCC AAA ATC GAG ACT CTG CGC 488 Pro Cys Tyr Ser Lys Thr Gin Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140 TTG GCC AAG AAC TAC ATC TGG GCT CTG TCG GAG ATC 524 Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile 145 150 155 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 156 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gin Pro 1 5 10 15 Gin Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gin Asp Glu 20 25 30 Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala 35 40 45 Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu 50 55 60 Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gin Lys 65 70 75 80 Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu 85 90 95 Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110 Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125 Pro Cys Tyr Ser Lys Thr Gin Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140 Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile 145 150 155 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1535 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: 14B1 , neuroD2) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 55..1194 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CCCCTCACTT TGTGCTGTCT GTCTCCCCTT CCCGCCCGGG GNCCCTCAGG CACCATGCTG 60 ACCCGCCTGT TCAGCGAGCC CGGCCTTCTC TCGGACGTGC CCAAGTTCGC CAGCTGGGGC 120 GACGGCGAAG ACGACGAGCC GAGGAGCGAC AAGGGCGACG CGCCGCCACC GCCACCGCCT 180 GCGCCCGGGC CAGGGGCTCC GGGGCCAGCC CGGGCGGCCA AGCCAGTCCC TCTCCGTGGA 240 GAAGAGGGGA CGGAGGCCAC GTTGGCCGAG GTCAAGGAGG AAGGCGAGCT GGGGGGAGAG 300 GAGGAGGAGG AAGAGGAGGA GGAAGAAGGA CTGGACGAGG CGGAGGGCGA GCGGCCCAAG 360 AAGCGCGGGC CCAAGAAGCG CAAGATGACC AAGGCGCGCT TGGAGCGCTC CAAGCTTCGG 420 CGGCAGAAGG CGAACGCGCG GGAGCGCAAC CGCATGCACG ACCTGAACGC AGCCCTGGAC 480 AACCTGCGCA AGGTGGTGCC CTGCTACTCC AAGACGCAGA AGCTGTCCAA GATCGAGACG 540 CTGCGCCTAG CCAAGAACTA TATCTGGGCG CTCTCGGAGA TCCTGCGCTC CGGCAAGCGG 600 CCAGACCTAG TGTCCTACGT GCAGACTCTG TGCAAGGGTC TGTCGCAGCC CACCACCAAT 660 CTGGTGGCCG GCTGTCTGCA GCTCAACTCT CGCAACTTCC TCACGGAGCA AGGCGCCGAC 720 GGTGCCGGCC GCTTCCACGG CTCGGGCGGC CCGTTCGCCA TGCACCCCTA CCCGTACCCG 780 TGCTCGCGCC TGGCGGGCGC ACAGTGCCAG GCGGCCGGCG GCCTGGGCGG CGGCGCGGCG 840 CACGCCCTGC GGACCCACGG CTACTGCGCC GCCTACGAGA CGCTGTATGC GGCGGCAGGC 900 GGTGGCGGCG CGAGCCCGGA CTACAACAGC TCCGAGTACG AGGGCCCGCT CAGCCCCCCG 960 CTCTGTCTCA ATGGCAACTT CTCACTCAAG CAGGACTCCT CGCCCGACCA CGAGAAAAGC 1020 TACCACTACT CTATGCACTA CTCGGCGCTG CCCGGTTCGC GCCACGGCCA CGGGCTAGTC 1080 TTCGGCTCGT CGGCTGTGCG CGGGGGCGTC CACTCGGAGA ATCTCTTGTC TTACGATATG 1140 CACCTTCACC ACGACCGGGG CCCCATGTAC GAGGAGCTCA ATGCGTTTTT TCATAACTGA 1200 GACTTCGCGC CGNCTCCCTN CTTTTTCTTT TGCCTTTGCC CGCCCCCCTG TCCCCAGCCC 1260 CCAGAGCGCA GGGACACCCC CATNCTACCC CGGCNCCGGC GGAGCGGGCC ACCGGTCTGC 1320 CGCTCTCCTG GGGCAGCGCA GTCTGTTACN TGTGGGTGGC TGTCCCAGGG GCCTCGCTTC 1380 CCCCAGGGAC TCGCCTTCTC TCTCCAAGGG GTTCCCTCCT CCTCTCTCCC AAGGAGTGCT 1440 TCTCCAGGGA CCTCTCTCCG GGGGCTCCCT GGAGGCACCC CTCCCCCATT CCCAATATCT 1500 TCGCTGAGGT TTCCTCCTCC CCCTCCTCCC TGCAG 1535 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 381mino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Leu Thr Arg Leu Phe Ser Glu Pro Gly Leu Leu Ser Asp Val Pro 1 5 10 15 Lys Phe Ala Ser Trp Gly Asp Gly Glu Asp Asp Glu Pro Arg Ser Asp 20 25 30 Lys Gly Asp Ala Pro Pro Pro Pro Pro Pro Ala Pro Gly Pro Gly Ala 35 40 45 Pro Gly Pro Ala Arg Ala Ala Lys Pro Val Pro Leu Arg Gly Glu Glu 50 55 60 Gly Thr Glu Ala Thr Leu Ala Glu Val Lys Glu Glu Gly Glu Leu Gly 65 70 75 80 Gly Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Leu Asp Glu Ala 85 90 95 Glu Gly Glu Arg Pro Lys Lys Arg Gly Pro Lys Lys Arg Lys Met Thr 100 105 110 Lys Ala Arg Leu Glu Arg Ser Lys Leu Arg Arg Gin Lys Ala Asn Ala 115 120 125 Arg Glu Arg Asn Arg Met His Asp Leu Asn Ala Ala Leu Asp Asn Leu 130 135 140 Arg Lys Val Val Pro Cys Tyr Ser Lys Thr Gin Lys Leu Ser Lys Ile 145 150 155 160 Glu Thr Leu Arg Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile 165 170 175 Leu Arg Ser Gly Lys Arg Pro Asp Leu Val Ser Tyr Val Gin Thr Leu 180 185 190 Cys Lys Gly Leu Ser Gin Pro Thr Thr Asn Leu Val Ala Gly Cys Leu 195 200 205 Gin Leu Asn Ser Arg Asn Phe Leu Thr Glu Gin Gly Ala Asp Gly Ala 210 215 220 Gly Arg Phe His Gly Ser Gly Gly Pro Phe Ala Met His Pro Tyr Pro 225 230 235 240 Tyr Pro Cys Ser Arg Leu Ala Gly Ala Gin Cys Gin Ala Ala Gly Gly 245 250 255 Leu Gly Gly Gly Ala Ala His Ala Leu Arg Thr His Gly Tyr Cys Ala 260 265 270 Ala Tyr Glu Thr Leu Tyr Ala Ala Ala Gly Gly Gly Gly Ala Ser Pro 275 280 285 Asp Tyr Asn Ser Ser Glu Tyr Glu Gly Pro Leu Ser Pro Pro Leu Cys 290 295 300 Leu Asn Gly Asn Phe Ser Leu Lys Gin Asp Ser Ser Pro Asp His Glu 305 310 315 320 Lys Ser Tyr His Tyr Ser Met His Tyr Ser Ala Leu Pro Gly Ser Arg 325 330 335 His Gly His Gly Leu Val Phe Gly Ser Ser Ala Val Arg Gly Gly Val 340 345 350 His Ser Glu Asn Leu Leu Ser Tyr Asp Met His Leu His His Asp Arg 355 360 365 Gly Pro Met Tyr Glu Glu Leu Asn Ala Phe Phe His Asn 370 375 380 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1268 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: 20A1 (neuroD3) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 55..768 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CTGCAGCGCT CTGAGCCGCT TTCTATCTGT CCGTCGGTCC TGCACAGCGC AACG ATG 57 Met 1 CCA GCC CGC CTT GAG ACC TGC ATC TCC GAC CTC GAC TGC GCC AGC AGC 105 Pro Ala Arg Leu Glu Thr Cys Ile Ser Asp Leu Asp Cys Ala Ser Ser 5 10 15 AGC GGC AGT GAC CTA TCC GGC TTC CTC ACC GAC GAG GAA GAC TGT GCC 153 Ser Gly Ser Asp Leu Ser Gly Phe Leu Thr Asp Glu Glu Asp Cys Ala 20 25 30 AGA CTC CAA CAG GCA GCC TCC GCT TCG GGG CCG CCC GCG CCG GCC CGC 201 Arg Leu Gin Gin Ala Ala Ser Ala Ser Gly Pro Pro Ala Pro Ala Arg 35 40 45 AGG GGC GCG CCC AAT ATC TCC CGG GCG TCT GAG GTT CCA GGG GCA CAG 249 Arg Gly Ala Pro Asn Ile Ser Arg Ala Ser Glu Val Pro Gly Ala Gin 50 55 60 65 GAC GAC GAG CAG GAG AGG CGG CGG CGC CGC GGC CGG ACG CGG GTC CGC 297 Asp Asp Glu Gin Glu Arg Arg Arg Arg Arg Gly Arg Thr Arg Val Arg 70 75 80 TCC GAG GCG CTG CTG CAC TCG CTG CGC AGG AGC CGG CGC GTC AAG GCC 345 Ser Glu Ala Leu Leu His Ser Leu Arg Arg Ser Arg Arg Val Lys Ala 85 90 95 AAC GAT CGC GAG CGC AAC CGC ATG CAC AAC TTG AAC GCG GCC CTG GAC 393 Asn Asp Arg Glu Arg Asn Arg Met His Asn Leu Asn Ala Ala Leu Asp 100 105 110 GCA CTG CGC AGC GTG CTG CCC TCG TTC CCC GAC GAC ACC AAG CTC ACC 441 Ala Leu Arg Ser Val Leu Pro Ser Phe Pro Asp Asp Thr Lys Leu Thr 115 120 125 AAA ATC GAG ACG CTG CGC TTC GCC TAC AAC TAC ATC TGG GCT CTG GCC 489 Lys Ile Glu Thr Leu Arg Phe Ala Tyr Asn Tyr Ile Trp Ala Leu Ala 130 135 140 145 GAG ACA CTG CGC CTG GCG GAT CAA GGG CTG CCC GGA GGC GGT GCC CGG 537 Glu Thr Leu Arg Leu Ala Asp Gin Gly Leu Pro Gly Gly Gly Ala Arg 150 155 160 GAG CGC CTC CTG CCG CCG CAG TGC GTC CCC TGC CTG CCC GGT CCC CCA 585 Glu Arg Leu Leu Pro Pro Gin Cys Val Pro Cys Leu Pro Gly Pro Pro 165 170 175 AGC CCC GCC AGC GAC GCG GAG TCC TGG GGC TCA GGT GCC GCC GCC GCC 633 Ser Pro Ala Ser Asp Ala Glu Ser Trp Gly Ser Gly Ala Ala Ala Ala 180 185 190 TCC CCG CTC TCT GAC CCC AGT AGC CCA GCC GCC TCC GAA GAC TTC ACC 681 Ser Pro Leu Ser Asp Pro Ser Ser Pro Ala Ala Ser Glu Asp Phe Thr 195 200 205 TAC CGC CCC GGC GAC CCT GTT TTC TCC TTC CCA AGC CTG CCC AAA GAC 729 Tyr Arg Pro Gly Asp Pro Val Phe Ser Phe Pro Ser Leu Pro Lys Asp 210 215 220 225 TTG CTC CAC ACA ACG CCC TGT TTC ATT CCT TAC CAC TAGGCCCTTT 775 Leu Leu His Thr Thr Pro Cys Phe Ile Pro Tyr His 230 235 GTAGACACTG TTACTTTCCC CCTCCCCTAG TCAGCAGGCA ATAGATTGGG CCCAGCTGCC 835 GCCTCGGGAC CCCTCTCCAG GCGGAGGGAG GAAGCGGGAG CTTTAAAGCA GTCGGGGATA 895 CCTGAGCCGC TTGTTAGGTC GCCGCACCCT CGCGGCGGAT GTCTCTTGGT CTGTTTCTCC 955 GGCCCTCAGC CCAGCGCCCC TCCTGCCCGC CCCTAGACGG CCTTTCCTTT TGCACTTTCT 1015 GAACTCCACA AAACCTCCTT TGTGACTGGC TCAGAACTGA CCCCAGCCAC CACTTCAGTG 1075 TGATTTAGAA AAGGGACAGA TCAGCCCCTG AAGACGAGGT GAAAAGTCAA TTTTACAATT 1135 TGTAGAACTC TAATGAAGAA AAACGAGCAT GAAAATTCGG TTTGAGCCGG CTGACAATAC 1195 AATGAAAAGG CTTAAAAAGC AGAGACAAGG AGTGGGCTTC ATGCATTATG GATCCCGACC 1255 CCCACCACTG CAG 1268 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 237 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Met Pro Ala Arg Leu Glu Thr Cys Ile Ser Asp Leu Asp Cys Ala Ser 1 5 10 15 Ser Ser Gly Ser Asp Leu Ser Gly Phe Leu Thr Asp Glu Glu Asp Cys 20 25 30 Ala Arg Leu Gin Gin Ala Ala Ser Ala Ser Gly Pro Pro Ala Pro Ala 35 40 45 Arg Arg Gly Ala Pro Asn Ile Ser Arg Ala Ser Glu Val Pro Gly Ala 50 55 60 Gin Asp Asp Glu Gin Glu Arg Arg Arg Arg Arg Gly Arg Thr Arg Val 65 70 75 80 Arg Ser Glu Ala Leu Leu His Ser Leu Arg Arg Ser Arg Arg Val Lys 85 90 95 Ala Asn Asp Arg Glu Arg Asn Arg Met His Asn Leu Asn Ala Ala Leu 100 105 110 Asp Ala Leu Arg Ser Val Leu Pro Ser Phe Pro Asp Asp Thr Lys Leu 115 120 125 Thr Lys Ile Glu Thr Leu Arg Phe Ala Tyr Asn Tyr Ile Trp Ala Leu 130 135 140 Ala Glu Thr Leu Arg Leu Ala Asp Gin Gly Leu Pro Gly Gly Gly Ala 145 150 155 160 Arg Glu Arg Leu Leu Pro Pro Gin Cys Val Pro Cys Leu Pro Gly Pro 165 170 175 Pro Ser Pro Ala Ser Asp Ala Glu Ser Trp Gly Ser Gly Ala Ala Ala 180 185 190 Ala Ser Pro Leu Ser Asp Pro Ser Ser Pro Ala Ala Ser Glu Asp Phe 195 200 205 Thr Tyr Arg Pro Gly Asp Pro Val Phe Ser Phe Pro Ser Leu Pro Lys 210 215 220 Asp Leu Leu His Thr Thr Pro Cys Phe Ile Pro Tyr His 225 230 235 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1560 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: HC2A (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 57..1126 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TTTTTCTGCT TTTCTTTCTG TTTGCCTCTC CCTTGTTGAA TGTAGGAAAT CGAAACATGA 60 CCAAATCGTA CAGCGAGAGT GGGCTGATGG GCGAGCCTCA GCCCCAAGGT CCTCCAAGCT 120 GGACAGACGA GTGTCTCAGT TCTCAGGACG AGGAGCACGA GGCAGACAAG AAGGAGGACG 180 ACCTCGAAGC CATGAACGCA GAGGAGGACT CACTGAGGAA CGGGGGAGAG GAGGAGGACG 240 AAGATGAGGA CCTGGAAGAG GAGGAAGAAG AGGAAGAGGA GGATGACGAT CAAAAGCCCA 300 AGAGACGCGG CCCCAAAAAG AAGAAGATGA CTAAGGCTCG CCTGGAGCGT TTTAAATTGA 360 GACGCATGAA GGCTAACGCC CGGGAGCGGA ACCGCATGCA CGGACTGAAC GCGGCGCTAG 420 ACAACCTGCG CAAGGTGGTG CCTTGCTATT CTAAGACGCA GAAGCTGTCC AAAATCGAGA 480 CTCTGCGCTT GGCCAAGAAC TACATCTGGG CTCTGTCGGA GATCCTGCGC TCAGGCAAAA 540 GCCCAGACCT GGTCTCCTTC GTTCAGACGC TTTGCAAGGG CTTATCCCAA CCCACCACCA 600 ACCTGGTTGC GGGCTGCCTG CAACTCAATC CTCGGACTTT TCTGCCTGAG CAGAACCAGG 660 ACATGCCCCC GCACCTGCCG ACGGCCAGCG CTTCCTTCCC TGTACACCCC TACTCCTACC 720 AGTCGCCTGG GCTGCCCAGT CCGNCTTACG GTACCATGGA CAGCTCCCAT GTCTTCCACG 780 TTAAGCCTCC GCCGCACGCC TACAGCGCAG CGCTGGAGCC CTTCTTTGAA AGCCCTCTGA 840 CTGATTGCAC CAGCCCTTCC TTTGATGGAC CCCTCAGCCC GCCGCTCAGC ATCAATGGCA 900 ACTTCTCTTT CAAACACGAA CCGTCCGCCG AGTTTGAGAA AAATTATGCC TTTACCATGC 960 ACTATCCTGC AGCGACACTG GCAGGGGCCC AAAGCCACGG ATCAATCTTC TCAGGCACCG 1020 CTGCCCCTCG CTGCGAGATC CCCATAGACA ATATTATGTC CTTCGATAGC CATTCACATC 1080 ATGAGCGAGT CATGAGTGCC CAGCTCAATG CCATATTTCA TGATTAGAGG CACGCCAGTT 1140 TCACCATTTC CGGGAAACGA ACCCACTGTG CTTACAGTGA CTGTCGTGTT TACAAAAGGC 1200 AGCCCTTTGG TACTACTGCT GCAAAGTGCA AATACTCCAA GCTTCAAGTG ATATATGTAT 1260 TTATTGTCAT TACTGCCTTT GGAAGAAACA GGGGATCAAA GTTCCTGTTC ACCTTATGTA 1320 TTATTTTCTA TAGACTCTTC TATTTTAAAA AATAAAAAAA TACAGTAAAG TTTAAAAAAT 1380 ACACCACGAA TTTGGTGTGG CTGTATTCAG ATCGTATTAA TTATCTGATC GGGATAACAA 1440 AATCACAAGC AATAATTAGG ATCTATGCAA TTTTTAAACT AGTAATGGGC CAATTAAAAT 1500 ATATATAAAT ATATATTTCA ACCAGCATTT TACTACTTGT TACCTCCCAT GCTGAATTAT 1560 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 356 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gin Pro 1 5 10 15 Gin Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gin Asp Glu 20 25 30 Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala 35 40 45 Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu 50 55 60 Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gin Lys 65 70 75 80 Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu 85 90 95 Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110 Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125 Pro Cys Tyr Ser Lys Thr Gin Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140 Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly 145 150 155 160 Lys Ser Pro Asp Leu Val Ser Phe Val Gin Thr Leu Cys Lys Gly Leu 165 170 175 Ser Gin Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gin Leu Asn Pro 180 185 190 Arg Thr Phe Leu Pro Glu Gin Asn Gin Asp Met Pro Pro His Leu Pro 195 200 205 Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gin Ser Pro 210 215 220 Gly Leu Pro Ser Pro Xaa Tyr Gly Thr Met Asp Ser Ser His Val Phe 225 230 235 240 His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe 245 250 255 Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro 260 265 270 Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu 275 280 285 Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro 290 295 300 Ala Ala Thr Leu Ala Gly Ala Gin Ser His Gly Ser Ile Phe Ser Gly 305 310 315 320 Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe 325 330 335 Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gin Leu Asn Ala 340 345 350 Ile Phe His Asp 355 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1951 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -69- (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (vii) IMMEDIATE SOURCE: (B) CLONE: 1.1.1 (mouse neuroD2) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 230..1378 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GAATTCAAGC TAGAGGCTGG TACCCCGCCT GGTAGAGATG CCACACTCGC TCCGCGGCTC 60 GCATGGCGCT CTGAAGACGC CGGCGCCCGC GCCTTGAGGA ACCGCTGCCC CCGCTCCCTG 120 AAGATGGGGG AACAATGAAA TAAGCGAGAA GATTCCTCTT CTCCCCCCTC TCTCTCTTGC 180 CCCCTCCCCC CTCCCCTCCC CTCTCCCCTT GACTCCTCTC TGAGGCACCA TGCTGACCCG 240 CCTGTTCAGC GAGCCCGGCC TCCTCTCGGA CGTGCCCAAG TTCGCCAGCT GGGGCGACGG 300 CGACGACGAC GAGCCGAGGA GCGACAAGGG CGACGCGCCG CCGCAGCCTT CTCCTGCTCC 360 CGGGTCGGGG GCTCCAGGAC CCGCCCGGGC CGCCAAGCCA GTGTCTCTTC GTGGAGGAGA 420 AGAGATCCCT GAACCCACGT TGGCTGAGGT CAAGGAGGAA GGAGAGCTGG GCGGCGAGGA 480 GGAGGAGGAA GAGGAGGAGG AGGAAGGACT GGACGAGGCG GAAGGCGAGC GGCCCAAGAA 540 GCGCGGGCCG AAGAAACGCA AGATGACCAA GGCGCGTCTG GAGCGCTCCA AGCTGCGGCG 600 ACAGAAGGCC AATGCGCGCG AGCGCAACCG CATGCACGAC CTGAACGCGG CTCTGGACAA 660 CCTGCGCAAG GTGGTCCCCT GCTACTCCAA GACCCAGAAG CTGTCCAAGA TCGAGACCCT 720 GCGCCTGGCC AAGAACTACA TCTGGGCTCT CTCGGAGATC TTGCGCTCCG GGAAGCGGCC 780 GGATCTGGTG TCCTACGTGC AGACTCTGTG CAAGGGGCTG TCACAGCCCA CCACGAATCT 840 GGTGGCCGGC TGCCTGCAGT TAAACTCTCG TAACTTCCTC ACGGAGCAGG GCGCGGACGG 900 CGCCGGCCGC TTTCACGGCT CGGGTGGCCC GTTCGCCATG CATCCGTACC CATACCCGTG 960 CTCCCGCCTG GCAGGCGCAC AGTGTCAGGC GGCTGGCGGC CTGGGCGGAG GCGCGGCGCA 1020 CGCCCTGCGG ACCCACGGCT ACTGCGCCGC CTACGAGACG CTGTACGCGG CGGCCGGTGG 1080 CGGCGGCGCT AGCCCGGACT ACAACAGCTC CGAGTACGAG GGTCCACTCA GTCCCCCGCT 1140 CTGTCTCAAC GGCAACTTCT CGCTCAAGCA GGACTCGTCC CCCGATCACG AGAAGAGCTA 1200 CCACTACTCT ATGCACTACT CGGCGCTGCC CGGCTCACGC CACGGCCACG GGCTGGTCTT 1260 CGGCTCGTCG GCCGTGCGCG GGGGCGTCCA CTCCGAGAAT CTCTTGTCTT ACGATATGCA 1320 CCTTCACCAC GATCGGGGCC CCATGTACGA GGAGCTCAAC GCATTTTTCC ATAACTGAGA 1380 CCTCNCGCCG ACCCCTTCTT TTTCTTTGCC TTTGTCCGGC CCCTTAGCCC CAGCCCCANN 1440 AGCTCAGGGA GCTCCCACCG ACTCCAGAGC CGGGCNCTCG NCNCGCCGCC GGTTCTGCAG 1500 CTCTCCAGAG CGGCGTGCTC TCTTACCTGT GGGTGGCCCG TCCCAGGGGC CTCGCTTGCC 1560 TCTGGGGACT CGCCTTCTCT CTCTCCCCAG CGGCTTCCTC CTCCCTTCTC TCGTGGAGAG 1620 CATCTCTNNN GATCTCCCGC CAGCCCTCCC AAGAGACTTC CTCCACATTC CCAAACTTGG 1680 GTTTTCTCTC CCCACCTCCA ACAGGCCAGA GGAGTTGGTA AGGGGTGCTG AGTCTCGGGA 1740 TAGTGTCTCC CCACTTATAG TTACTTAAAC AAACAAACAG ACACAGAGCT TCCAGCNAAA 1800 AGAGTTGGTA TCTCTTCCTT CTCGAAGANC ACCAGCCAGG AGCCCAACCG CCTTCACCCT 1860 AACACNGAAT CTCCNNGTTT TTTATTTTTT ATTTTGGTGG GAGGGGATGT GGATTGAGAG 1920 GAAAGAGAGA GCCAAGCCAA TTTGTAACTA G 1951 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 382 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (vii) IMMEDIATE SOURCE: (B) CLONE: 1.1.1 (murine neuroD2) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Met Leu Thr Arg Leu Phe Ser Glu Pro Gly Leu Leu Ser Asp Val Pro 1 5 10 15 Lys Phe Ala Ser Trp Gly Asp Gly Asp Asp Asp Glu Pro Arg Ser Asp 20 25 30 Lys Gly Asp Ala Pro Pro Gin Pro Ser Pro Ala Pro Gly Ser Gly Ala 35 40 45 Pro Gly Pro Ala Arg Ala Ala Lys Pro Val Ser Leu Arg Gly Gly Glu 50 55 60 Glu Ile Pro Glu Pro Thr Leu Ala Glu Val Lys Glu Glu Gly Glu Leu 65 70 75 80 Gly Gly Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Leu Asp Glu 85 90 95 Ala Glu Gly Glu Arg Pro Lys Lys Arg Gly Pro Lys Lys Arg Lys Met 100 105 110 Thr Lys Ala Arg Leu Glu Arg Ser Lys Leu Arg Arg Gin Lys Ala Asn 115 120 125 Ala Arg Glu Arg Asn Arg Met His Asp Leu Asn Ala Ala Leu Asp Asn 130 135 140 Leu Arg Lys Val Val Pro Cys Tyr Ser Lys Thr Gin Lys Leu Ser Lys 145 150 155 160 Ile Glu Thr Leu Arg Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu 165 170 175 Ile Leu Arg Ser Gly Lys Arg Pro Asp Leu Val Ser Tyr Val Gin Thr 180 185 190 Leu Cys Lys Gly Leu Ser Gin Pro Thr Thr Asn Leu Val Ala Gly Cys 195 200 205 Leu Gin Leu Asn Ser Arg Asn Phe Leu Thr Glu Gin Gly Ala Asp Gly 210 215 220 Ala Gly Arg Phe His Gly Ser Gly Gly Pro Phe Ala Met His Pro Tyr 225 230 235 240 Pro Tyr Pro Cys Ser Arg Leu Ala Gly Ala Gin Cys Gin Ala Ala Gly 245 250 255 Gly Leu Gly Gly Gly Ala Ala His Ala Leu Arg Thr His Gly Tyr Cys 260 265 270 Ala Ala Tyr Glu Thr Leu Tyr Ala Ala Ala Gly Gly Gly Gly Ala Ser 275 280 285 Pro Asp Tyr Asn Ser Ser Glu Tyr Glu Gly Pro Leu Ser Pro Pro Leu 290 295 300 Cys Leu Asn Gly Asn Phe Ser Leu Lys Gin Asp Ser Ser Pro Asp His 305 310 315 320 Glu Lys Ser Tyr His Tyr Ser Met His Tyr Ser Ala Leu Pro Gly Ser 325 330 335 Arg His Gly His Gly Leu Val Phe Gly Ser Ser Ala Val Arg Gly Gly 340 345 350 Val His Ser Glu Asn Leu Leu Ser Tyr Asp Met His Leu His His Asp 355 360 365 Arg Gly Pro Met Tyr Glu Glu Leu Asn Ala Phe Phe His Asn 370 375 380 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: (B) CLONE: JL34 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CTCAGCATCA GCAACTCGGC 20 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: (B) CLONE: JL36 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TCGGATCCCG TTCTAGGCGC GCCTTGGTC 29 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vii) IMMEDIATE SOURCE: (B) CLONE: JL40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GTTTTCCCAG TCACGACGTT G 21 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1333 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (vii) IMMEDIATE SOURCE: (B) CLONE: neuroD3 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 101..835 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CTGCAGAGGA CAGGTAGCCC CGGGTCGTAC GGACAGTAAG TGCGCTTCGA AGGCCGACCT 60 CCAAACCTCC TGTCCGTCTG TCGGTCCTGC ACACTGCAAG ATG CCT GCC CCT TTG 115 Met Pro Ala Pro Leu 1 5 GAG ACC TGC ATC TCT GAT CTC GAC TGC TCC AGC AGC AAC AGC AGC AGC 163 Glu Thr Cys Ile Ser Asp Leu Asp Cys Ser Ser Ser Asn Ser Ser Ser 10 15 20 GAC CTG TCC AGC TTC CTC ACC GAC GAG GAG GAC TGT GCC AGG CTA CAG 211 Asp Leu Ser Ser Phe Leu Thr Asp Glu Glu Asp Cys Ala Arg Leu Gin 25 30 35 CCC CTA GCC TCC ACC TCG GGG CTG TCC GTG CCA GCC CGG AGG AGC GCT 259 Pro Leu Ala Ser Thr Ser Gly Leu Ser Val Pro Ala Arg Arg Ser Ala 40 45 50 CCC GCC CTC TCC GGG GCA TCG AAT GTT CCC GGT GCC CAG GAC GAA GAG 307 Pro Ala Leu Ser Gly Ala Ser Asn Val Pro Gly Ala Gin Asp Glu Glu 55 60 65 CAG GAA CGG CGG AGG CGG CGA GGT CGC GCT CGG GTG CGG TCC GAG GCT 355 Gin Glu Arg Arg Arg Arg Arg Gly Arg Ala Arg Val Arg Ser Glu Ala 70 75 80 85 CTG CTG CAC TCC CTG CGG AGG AGT CGT CGC GTC AAA GCC AAC GAT CGC 403 Leu Leu His Ser Leu Arg Arg Ser Arg Arg Val Lys Ala Asn Asp Arg 90 95 100 GAG CGC AAC CGC ATG CAC AAC CTC AAC GCT GCG CTG GAC GCC TTG CGC 451 Glu Arg Asn Arg Met His Asn Leu Asn Ala Ala Leu Asp Ala Leu Arg 105 110 115 AGC GTG CTG CCC TCG TTC CCC GAC GAC ACC AAG CTC ACC AAG ATT GAG 499 Ser Val Leu Pro Ser Phe Pro Asp Asp Thr Lys Leu Thr Lys Ile Glu 120 125 130 ACG CTG CGC TTC GCC TAC AAC TAC ATC TGG GCC CTG GCT GAG ACA CTG 547 Thr Leu Arg Phe Ala Tyr Asn Tyr Ile Trp Ala Leu Ala Glu Thr Leu 135 140 145 CGC CTG GCA GAT CAA GGG CTC CCC GGG GGC AGT GCC CGG GAG CGC CTC 595 Arg Leu Ala Asp Gin Gly Leu Pro Gly Gly Ser Ala Arg Glu Arg Leu 150 155 160 165 CTG CCT CCG CAG TGT GTC CCC TGT CTG CCC GGG CCC CCG AGC CCG GCC 643 Leu Pro Pro Gin Cys Val Pro Cys Leu Pro Gly Pro Pro Ser Pro Ala 170 175 180 AGC GAC ACT GAG TCC TGG GGT TCC GGG GCC GCT GCC TCC CCC TGC GCC 691 Ser Asp Thr Glu Ser Trp Gly Ser Gly Ala Ala Ala Ser Pro Cys Ala 185 190 195 ACT GTG GCA TCA CCA CTC TCT GAC CCC AGT AGT CCC TCG GCT TCA GAA 739 Thr Val Ala Ser Pro Leu Ser Asp Pro Ser Ser Pro Ser Ala Ser Glu 200 205 210 GAC TTC ACC TAT GGC CCG GGC GAT CCC CTT TTC TCC TTT CCT GGC CTG 787 Asp Phe Thr Tyr Gly Pro Gly Asp Pro Leu Phe Ser Phe Pro Gly Leu 215 220 225 CCC AAA GAC CTG CTC CAC ACG ACG CCC TGT TTC ATC CCA TAC CAC TAGGCCTTTG 842 Pro Lys Asp Leu Leu His Thr Thr Pro Cys Phe Ile Pro Tyr His 230 235 240 245 TAAGGCAACA TCAATACATT CTTCCTCCCC CAGTCTAAGA GCAATAATAG ATGGGGAACT 902 GGCTGAAGCC TCCGGGGGCC ACACTTACCC CCAAGTGAAT TCTGGGAGCT TTAAAGGGGG 962 GAGGGGGAAT ACCTGACCAC TTGTTAGGTT GCTGCACCCT CGCTGAAGCT GCCCTCGGTC 1022 TATTTCTCCA CCCCCAGCAC GGCCTCCCCC CCCCCCGCCC GCCCCCAGAC GGCCTTTCGT 1082 TTTTGTTGCA CTTTCTGAAC TTCACAAAAC CTTCTTTGTG ACTGGCTCAG AACTGACCCC 1142 AGCCACCACT TCAGTGTGGT TTGGAAAAGG GACAGATGAG CCCCTGAAGA CGAGGTGAAA 1202 AGTCAATTTT ACAATTTGTA GAACTCTAAT GAAGAAAAAC GAGCATGAAA ATTCGGTTTG 1262 AGCCGGCTGA CAATACAATG GCAAGGCTTA AAAAGGAGCC ACAAGGAGTG GGCTTCATGC 1322 ATTATGGATC C 1333 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 244 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Met Pro Ala Pro Leu Glu Thr Cys Ile Ser Asp Leu Asp Cys Ser Ser 1 5 10 15 Ser Asn Ser Ser Ser Asp Leu Ser Ser Phe Leu Thr Asp Glu Glu Asp 20 25 30 Cys Ala Arg Leu Gin Pro Leu Ala Ser Thr Ser Gly Leu Ser Val Pro 35 40 45 Ala Arg Arg Ser Ala Pro Ala Leu Ser Gly Ala Ser Asn Val Pro Gly 50 55 60 Ala Gin Asp Glu Glu Gin Glu Arg Arg Arg Arg Arg Gly Arg Ala Arg 65 70 75 80 Val Arg Ser Glu Ala Leu Leu His Ser Leu Arg Arg Ser Arg Arg Val 85 90 95 Lys Ala Asn Asp Arg Glu Arg Asn Arg Met His Asn Leu Asn Ala Ala 100 105 110 Leu Asp Ala Leu Arg Ser Val Leu Pro Ser Phe Pro Asp Asp Thr Lys 115 120 125 Leu Thr Lys Ile Glu Thr Leu Arg Phe Ala Tyr Asn Tyr Ile Trp Ala 130 135 140 Leu Ala Glu Thr Leu Arg Leu Ala Asp Gin Gly Leu Pro Gly Gly Ser 145 150 155 160 Ala Arg Glu Arg Leu Leu Pro Pro Gin Cys Val Pro Cys Leu Pro Gly 165 170 175 Pro Pro Ser Pro Ala Ser Asp Thr Glu Ser Trp Gly Ser Gly Ala Ala 180 185 190 Ala Ser Pro Cys Ala Thr Val Ala Ser Pro Leu Ser Asp Pro Ser Ser 195 200 205 Pro Ser Ala Ser Glu Asp Phe Thr Tyr Gly Pro Gly Asp Pro Leu Phe 210 215 220 Ser Phe Pro Gly Leu Pro Lys Asp Leu Leu His Thr Thr Pro Cys Phe 225 230 235 240 Ile Pro Tyr His

Claims: The embodiments ofthe invention in which an exclusive property or privilege is claimed are defined as follows: 1. An isolated polynucleotide molecule that encodes a neuroD polypeptide and that hybridizes under stringent conditions with a nucleic acid molecule selected from among SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 8, SEQ ID NO.TO, SEQ ID NO:12, SEQ ID NO 14, SEQ ID NO:16, SEQ ID NO:21, or its complement. 2. The isolated polynucleotide molecule of Claim 1, said polynucleotide molecule encoding a human neuroD2 polypeptide, and further being capable of hybridizing under stringent conditions with the nucleotide sequence of SEQ ID NO: 10, or its complement. 3. The isolated polynucleotide molecule of Claim 1, said polynucleotide molecule encoding a human neuroD3 polypeptide, and further being capable of hybridizing under stringent conditions with the nucleotide sequence of SEQ ID NO: 12, or its complement. 4. An isolated polynucleotide molecule that comprises at least 15 nucleotides and that hybridizes under stringent conditions with a neuroD HLH domain selected from among nucleotides 577-696 of SEQ ID NO:l, nucleotides 376-495 of SEQ ID NO: 3, nucleotides 149-268 of SEQ ID NO: 8, nucleotides 463-582 of SEQ ID NO: 10, nucleotides 368-496 of SEQ ID NO: 12, nucleotides 405-524 of SEQ ID NO: 14, nucleotides 642-761 of SEQ ID NO: 16, nucleotides 425-544 of SEQ ID NO:21, or its complement. 5. A vector comprising the following operably linked elements: a promoter, the polynucleotide molecule of Claim 1, and a transcription termination signal. 6. A cell transformed by the polynucleotide molecule of Claim 1. 7. A recombinant peptide encoded by the polynucleotide molecule of Claim 1. 8. An antibody or antigen-binding fragment thereof that binds to the recombinant peptide of Claim 9. 9. An antibody or antigen-binding fragment thereof that binds to a polypeptide selected from among SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:9, SEQ ED NOT 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO:22. 10. An antibody or antigen-binding fragment thereof that binds to a peptide selected from among amino acid residues 117-156 of SEQ ID NO:2, amino acid residues 118-157 of SEQ ID NO:4, amino acid residues 117-156 of SEQ ID NO:9, amino acid residues of 137-176 of SEQ BD NO: l l, amino acid residues 108- 147 of SEQ ID NO. 13, amino acid residues 117-156 of SEQ ID NO: 15, amino acid residues 138-177 of SEQ ID NO: 17, and amino acid residues 109-148 of SEQ ID NO:22.

Inventors: HOLLENBERG STANLEY M, LEE JACQUELINE E, TAPSCOTT STEPHEN J, WEINTRAUB HAROLD M, HOLLENBERG, STANLEY, M., LEE, JACQUELINE, E., TAPSCOTT, STEPHEN, J., WEINTRAUB, HAROLD, M. +DI
Applicants: HOLLENBERG STANLEY M, HUTCHINSON FRED CANCER RES, LEE JACQUELINE E, TAPSCOTT STEPHEN J, WEINTRAUB NANCY & EF, FRED HUTCHINSON CANCER RESEARCH CENTER, HOLLENBERG, STANLEY, M., LEE, JACQUELINE, E., TAPSCOTT, STEPHEN, J., WEINTRAUB, NANCY +EF
Application year: 1996
Main classification: C12N15
IPC codes: A61K31, A61K38, A61K39, A61K48, A61P25, C07K14, C07K16, C12N5, C12N15, C12R1