Abstract:
The present invention relates to a transgenic  C. elegans  which expresses an amyloid precursor protein (APP) or a part thereof, to the transgene itself, to the protein encoded by the transgene, and also to a process for preparing the transgenic  C. elegans  and to its use.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Field of the Invention  
           [0002]    The present invention relates to a transgenic  C. elegans  which expresses an amyloid precursor protein (APP) or a part thereof, to the transgene itself, to the protein encoded by the transgene, and also to a process for preparing the transgenic  C. elegans  and to its use.  
           [0003]    2. Description of Related Art  
           [0004]    Several publications are referenced in the application. These references describe the state of the art to which this invention pertains, and are incorporated herein by reference.  
           [0005]    Alzheimer&#39;s disease (morbus Alzheimer) is a neurodegenerative disorder of the brain which, at the cellular level, is accompanied by a massive loss of neurons in the limbic system and in the cerebral cortex. At the molecular level, it is possible to detect protein depositions, so-called plaques, in the affected areas of the brain, which depositions constitute an important feature of Alzheimer&#39;s disease. The protein which most frequently occurs in these plaques is a peptide of from 40 to 42 amino acids in size which is termed the Aβ peptide. This peptide is a cleavage product of a substantially larger protein of from 695 to 751 amino acids, the so-called amyloid precursor protein (APP).  
           [0006]    APP is an integral transmembrane protein which traverses the lipid double layer once. By far the largest part of the protein is located extracellularly, while the shorter C-terminal domain is directed into the cytosol (FIG. 1). The Aβ peptide is shown in dark gray in FIG. 1. About two thirds of the Aβ peptide are derived from the extracellular domain of APP and about one third from the transmembrane domain.  
           [0007]    In addition to the APP which is located in the membrane, it is also possible to detect a secreted form of the amyloid precursor protein, which form comprises the large ectodomain of the APP and is termed APPsec (“secreted APP”). APPsec is formed from APP by proteolytic cleavage which is effected by α-secretase. The proteolytic cleavage takes place at a site in the amino acid sequence of APP which lies within the amino acid sequence of the Aβ peptide (after amino acid residue 16 of the Aβ peptide). Proteolysis of APP by the α-secretase consequently rules out the possibility of the Aβ peptide being formed.  
           [0008]    The Aβ peptide can consequently only be formed from APP by an alternative processing route. It is postulated that two further proteases are involved in this processing route, with one of the proteases, which is termed β-secretase, cutting the APP at the N terminus of the Aβ peptide and the second protease, which is termed γ-secretase, releasing the C terminus of the Aβ peptide (Kang, J. et al., Nature, 325, 733) (FIG. 1).  
           [0009]    It has not as yet been possible to identify any of the three secretases or proteases (α-secretase, β-secretase and γ-secretase). However, knowledge of the secretases is of great interest, in particular within the context of investigations with regard to Alzheimer&#39;s disease and with regard to identifying the proteins involved, which proteins can then in turn be employed as targets in follow-up studies since, on the one hand, inhibition of the β-secretase, and in particular of the γ-secretase, could lead to a decrease in Aβ production and, on the other hand, activation of the α-secretase would increase the processing of APP into APPsec and thereby simultaneously reduce formation of the Aβ peptide.  
           [0010]    There is a large amount of evidence that the Aβ peptide is a crucial factor in the development of Alzheimer&#39;s disease. Inter alia, Aβ fibrils are postulated to be neurotoxic in cell culture (Yankner, B. A. et al., (1990) Proc Natl Acad Sci USA,87, 9020). Furthermore, the neuropathology which is characteristic of Alzheimer&#39;s disease already appears at the age of 30 in Down&#39;s syndrome patients, who have an additional copy of APP. In this case, it is assumed that overexpression of APP is followed by an increased conversion into the Aβ peptide (Rumble, B. et al., (1989), N. Engl. J. Med., 320,1446).  
           [0011]    The familial forms of Alzheimer&#39;s disease constitute what is probably the most powerful evidence of the central role of the Aβ peptide. In these forms, there are mutations in the APP gene around the region of the β-secretase and γ-secretase cleavage sites or in two further AD-associated genes (presenilins) which, in cell culture, lead to a substantial increase in Aβ production (Scheuner, D. et al., (1996), Nature Medicine, 2, 864).  
           [0012]    While  C. elegans  has already been used as a model organism in Alzheimer&#39;s disease, these studies do not relate to the processing of APP into the Aβ peptide. Some of the studies are concerned with two other Alzheimer-associated proteins, i.e. the presenilins. The presenilins are transmembrane proteins which traverse the membrane 6-8 times. They are of great importance in familial cases of Alzheimers since specific mutations in the presenilin genes lead to Alzheimer&#39;s disease. In this connection, it was shown that homologs to the human presenilins (sel-12, spe-4 and hop-1) are present in  C. elegans , with the function of the presenilins being conserved in humans and worm (Levitan D, Greenwald I (1995) Nature 377, 351; Levitan et al.(1996) Proc Natl Acad Sci USA, 93, 14940; Baumeister R (1997) Genes &amp; Function 1, 149; Xiajun Li and Iva Greenwald (1997) Proc Natl Acad Sci USA, 94, 12204).  
           [0013]    Other studies deal with the APP homolog in  C. elegans,  which is termed Apl-1, and with expression of the Aβ peptide in  C. elegans.  However, Apl-1 does not possess any region which is homologous with the amino acid sequence of the Aβ peptide;  C. elegans  does not therefore possess any endogenous Aβ peptide (Daigle I, Li C (1993) Proc Natl Acad Sci USA, 90 (24), 12045).  
           [0014]    C. D. Link, Proc Natl Acad Sci USA (1995) 92, 9368 described the expression of Aβ peptide (but not that of an Aβ precursor protein) in  C. elegans.  These studies involve preparing transgenic worms which express an Aβ1-42 peptide (i.e. the Aβ peptide which consists of 42 amino acids) as a fusion protein together with a synthetic signal peptide and under the control of the muscle-specific promoter unc 54. Muscle-specific protein depositions which reacted with anti-β-amyloid antibodies were detected in the studies.  
           [0015]    Other studies (e.g. C. Link et al. personal communication) relate to investigations of the aggregation and toxicity of the Aβ peptide in the  C. elegans  model system.  
           [0016]    Transgenic  C. elegans  lines were established in the present study in order to investigate the existence of a processing machinery in  C. elegans  which is involved in the formation of Aβ peptide and to identify potential secretases in this worm.  
         SUMMARY OF THE INVENTION  
         [0017]    In this invention, APP genes have been transferred into  C. elegans  to create a transgenic  C. elegans  organism. This transgenic  C. elegans  can then be used to investigate the processing machinery involved in the formation of the Aβ peptide and to identify potential secretases.  
           [0018]    The present invention relates to a transgene (a gene that has been transferred from one species to another by genetic engineering) which contains  
           [0019]    a) a nucleotide sequence encoding an amyloid precursor protein (APP) or a part thereof, wherein the nucleotide sequence comprising the APP peptide or part thereof, contains, as part of the sequence, a nucleotide sequence comprising a complete Aβ peptide or a part of the Aβ peptide, and  
           [0020]    b) where appropriate, one or more further coding and/or non-coding nucleotide sequences, and  
           [0021]    c) a promoter for expression in a cell of the nematode  Caenorhabditis elegans  ( C. elegans ).  
           [0022]    The nucleotide sequence preferably encodes the 100 carboxyterminal amino acids of APP, beginning with the sequence of the Aβ peptide and ending with the carboxyterminal amino acid of APP (C100 fragment). The APP is preferably one of the isoforms APP695 (695 amino acids), APP751 (751 amino acids), APP770 (770 amino acids) and L-APP. All the isoforms are formed from the same APP gene by means of alternative splicing. In APP695, exons 7 and 8 were removed by splicing, whereas only exon 8 is lacking in APP751 and exon 7 and 8 are present in APP770. In addition to this, other splicing forms of APP exist in which exon 15 has been removed by splicing. These forms are termed L-APP and are likewise present in the forms which are spliced with regard to exons 7 and 8.  
           [0023]    In one particular embodiment of the invention, the transgene contains the nucleotide sequence SEQ ID NO.: 1 or a part thereof or a sequence homologous to SEQ ID No. 1.  
           [0024]    The transgene can preferably contain an additional coding nucleotide sequence which is located at the 5′ end of the nucleotide sequence encoding APP or a part thereof. In one particular embodiment of the invention, the additional nucleotide sequence encodes a signal peptide or a part thereof, for example encodes the APP signal peptide (SP) having the amino acid sequence SEQ ID NO.:9 or a part thereof. The sequence from the N terminus of the Aβ peptide to the C terminus of APP consists of 99 amino acids. The APP signal peptide consists of 17 amino acids. When a fusion product comprising the N terminus of the Aβ peptide to the C terminus of APP and the APP signal peptide is cloned, one or more spacer amino acids is/are preferably inserted between these two parts of the fusion product, with preference being given to inserting one amino acid, for example leucine. The C-terminal fragment is therefore given different designations, e.g. C100 (C=C terminus), LC99 (L=leucine), LC1-99, C99 or SPA4CT (SP=signal peptide, A4=Aβ peptide and CT=C terminus).  
           [0025]    In one particular embodiment of the invention, the transgene contains the nucleotide sequence SEQ ID NO.: 2 or a part thereof and/or the nucleotide sequence SEQ ID NO.: 3 or a part thereof.  
           [0026]    In addition to this, the transgene can also contain one or more additional non-coding and/or one or more additional coding nucleotide sequences.  
           [0027]    For example, the transgene can contain, as an additional non-coding nucleotide sequence, a sequence from an intron of the APP gene, e.g. a sequence which is derived from the 42 bp intron of the APP gene and exhibits the sequence SEQ ID NO.: 4. A transgene which contains the nucleotide sequence SEQ ID NO.: 5 is part of the subject-matter of the invention.  
           [0028]    The transgene also preferably contains one or more gene-regulating sequences for regulating expression of the encoded protein, preferably a constitutive promoter or a promoter which can be regulated. For example, the promoter can be active in the neuronal, muscular or dermal tissue of  C. elegans  or be ubiquitously active in  C. elegans.  A promoter can, for example, be selected from the group of the  C. elegans  promoters unc-54, hsp 16-2, unc-119, goa-1 and sel-12. In one particular embodiment of the invention, the transgene contains a promoter having the nucleotide sequence SEQ ID NO.: 6. In one particular embodiment, the transgene contains the nucleotide sequence SEQ ID NO.: 7.  
           [0029]    The transgene can be present in a vector, for example in an expression vector. For example, a recombinant expression vector can contain the nucleotide sequence SEQ ID NO.: 8.  
           [0030]    The invention also relates to the preparation of an expression vector, with a transgene being integrated into a vector in accordance with known methods. In particular, the invention relates to the use of an expression vector for preparing a transgenic cell, with it being possible for this cell to be part of a non-human organism, e.g.  C. elegans.    
           [0031]    The invention also relates to the preparation of the transgene, with suitable part sequences being ligated in the appropriate order and in the correct reading frame, where appropriate while inserting linkers. In particular, the invention relates to the use of the transgene, for example for preparing a transgenic cell, with it being possible for this cell to be part of a non-human organism. For example, the cell can be a  C. elegans  cell.  
           [0032]    One particular embodiment of the invention relates to a transgenic  C. elegans  which contains the transgene. The transgene can also be present in the  C. elegans  in an expression vector. The transgene can be present in the  C. elegans  intrachromosomally and/or extrachromosomally. One or more transgenes or expression vectors which contain the transgene can be present intrachromosomally and/or extrachromosomally as long tandem arrays. A transgenic cell or a transgenic organism preferably contains another expression vector as well, which vector contains a nucleotide sequence which encodes a marker, with the marker either being a temperature-sensitive marker or a phenotypic marker. For example, the marker can be a visual marker or a behaviorally phenotypic marker. Examples are fluorescent markers, e.g. GFP (green fluorescent protein) or EGFP (enhanced green fluorescent protein), marker genes which encode a dominant, mutated form of a particular protein, e.g. a dominant Rol6 mutation, or marker sequences which encode antisense RNA, e.g. the antisense RNA of Unc-22.  
           [0033]    One or more copies of the transgene and/or of the expression vector and, where appropriate, of an additional expression vector are preferably present in the germ cells and/or the somatic cells of the transgenic  C. elegans.    
           [0034]    The invention also relates to a process for preparing a transgenic  C. elegans,  with a transgene and/or an expression vector, where appropriate in the presence of an additional expression vector which contains a nucleotide sequence which encodes a marker, being microinjected into the germ cells of a  C. elegans.  A DNA construct which expresses SP-C100 (SP=signal peptide) under the control of a neuron-specific promoter can, for example, be used for preparing the transgenic  C. elegans  lines (FIG. 2). Since C100 is composed of the Aβ sequence and the C terminus of APP, only the γ-secretase cleavage is required in order to release the Aβ peptide from C100. C100 is also a substrate for the γ-secretase.  
           [0035]    The invention also relates to the use of a transgenic  C. elegans,  for example for expressing an SP-C100 fusion protein. An SP-C100 fusion protein having the amino acid sequence SEQ ID NO.: 10 is part of the subject-matter of the invention.  
           [0036]    In particular, the invention relates to the use of a transgenic  C. elegans  for identifying a γ-secretase activity and/or an α-secretase activity in  C. elegans,  to its use in methods for identifying and/or characterizing substances which inhibit the γ-secretase activity, to its use in methods for identifying and/or characterizing substances which increase the α-secretase activity, and to its use in methods for identifying and/or characterizing substances which can be used as active compounds for treating and/or preventing Alzheimer&#39;s disease.  
         DESCRIPTION OF THE PREFERRED EMBODIMENTS  
         [0037]    In the present study, the nematode  Caenorhabditis elegans  ( C. elegans ) was chosen as the model organism for identifying secretases which are involved in processing APP into the Aβ peptide. This worm is outstandingly suitable for genetic studies and has therefore in the past been employed on many occasions for investigating universally important processes such as programmed cell death, neuronal guidance and RAS/MAP kinase signaling (Riddle, D. L. et al. (1997) ).  
           [0038]    The important points which make  C. elegans  especially appropriate for such studies include the following (C. Kenyon, Science (1988) 240, 1448; P. E. Kuwabara (1997), TIG, 13, 454):  
           [0039]    Its small genome, which is composed of about 19,000 genes or 97 Mb and which was sequenced completely in December 1998. (The  C. elegans  Sequencing Consortium, Science (1998), 282, 2012).  
           [0040]    Its reproduction by self fertilization. In the case of the two sexes of  C. elegans,  a distinction is made between males and hermaphrodites, i.e. hermaphroditic animals which fertilize their eggs themselves before laying. A crucial advantage of this type of reproduction is that, after a transgene has been introduced into the germ line, a hermaphrodite can automatically generate homozygous transgenic descendants. There is therefore no need for any further crossing steps, as in the case of Drosophila, for example, for preparing transgenic lines.  
           [0041]    Its easy handling in the laboratory due to its small size (about 1 mm in length) and its relatively undemanding growth conditions. As a result, a large number of worms can be handled routinely in the laboratory.  
           [0042]    Its short generation time of 3 days, which makes it possible to obtain large quantities of biological material for analysis within a very short time.  
           [0043]    A complete cell description for the development and anatomy of  C. elegans  is available.  
           [0044]    Detailed genetic maps and methods for genetic analysis in  C. elegans  are available.  
           [0045]    Technologies for preparing knock-out animals are available. In the same way, technologies exist for mutagenizing the  C. elegans  genome (transposon mutagenesis and ethyl methanesulfonate (EMS) mutagenesis).  
           [0046]    The following are possible uses of the transgenic  C. elegans  lines:  
           [0047]    1. Identification of a γ-secretase-like activity in  C. elegans  using mutagenesis approaches. It is planned that a transposon mutagenesis, which destroys the γ-secretase-like activity, should be carried out and that the corresponding gene should be sought by detecting the worms which no longer possess this activity. Such a screening method is described in the literature: Korswagen H. C. et al., (1996), 93, 14680 Proc Natl Acad Sci USA.  
           [0048]    Alternative approaches would be mutagenesis using ethyl methanesulfonate (EMS) or else anti-sense RNA approaches. In the latter case, an attempt could be made to find motifs which were common to all  C. elegans  proteases and to downregulate these proteases specifically using anti-sense RNAs which were directed against these motifs. Screening for the Aβ peptide could then show whether one of the proteases was involved in Aβ peptide production.  
           [0049]    2. Identification of a γ-secretase-like activity in  C. elegans,  perhaps by a similar route to that described in item 1.  
           [0050]    3. Armed with knowledge of a γ-secretase or γ-secretase-like activity in  C. elegans,  it is possible to search for human γ-secretase or γ-secretase-like activity by means of a homology comparison.  
           [0051]    4. Identification of drugs which  
           [0052]    inhibit the activity of γ-secretase, in order to inhibit Aβ production from the amyloid precursor protein directly.  
           [0053]    activate γ-secretase and thereby indirectly inhibit formation of the Aβ peptide by increasing APPsec production.  
           [0054]    This approach could take place in a 96-well format since  C. elegans  can be maintained in suspension in 96-well plates.  
           [0055]    Since the screening is carried out on a whole organism, it is possible, to a large extent, to exclude drugs which have an unspecific toxic effect.  
           [0056]    5. Investigation of the aggregation behavior, and of a possible neurotoxic effect, of the Aβ peptide in  C. elegans.  Screening for drugs which inhibit aggregation of the Aβ peptide.  
           [0057]    6. Investigation of the modulation of APP processing by other proteins (e.g. presenilins or ApoE) as a result of their overexpression or knock-out. Since the presenilins are Alzheimer-associated proteins and ApoE constitutes a risk factor in Alzheimer&#39;s disease, these proteins could have an effect on formation of the Aβ peptide and, as a consequence, their role in the APP processing pathway could be investigated.  
           [0058]    7. Where appropriate, validation of an α-secretase and/or γ-secretase activity which has been found using other experimental approaches known to the skilled person. 
       
    
    
       [0059]    [0059]FIG. 1: FIG. 1 shows the amyloid precursor protein (APP695 isoform and APP770 and APP751 isoforms) and secretase cleavage products.  
         [0060]    [0060]FIG. 2: FIG. 2 describes the construction of the transgenic vector “Unc-119-SP-C100”, which contains an unc-119 promoter, an APP signal peptide and the C100 fragment from APP, with “unc-119” being a neuron-specific  C. elegans  promoter, the APP signal peptide corresponding to amino acids 1 to 24 of APP and C100 corresponding to the 100 C-terminal amino acids of APP (=C100). C100 is composed of the Aβ sequence and the C terminus of APP (Shoji, M et al., (1992) Science 258, 126). The vector Unc-119-SP-C100 possesses 5112 base pairs. 
     
    
     EXAMPLES  
       [0061]    The following examples are illustrative of some of the products and compositions and methods of making and using the same falling within the scope of the present invention.  
         [0062]    Example 1  
       Preparing an Expression Vector Which Contains the Transgene  
       [0063]    Two vectors, i.e. pSKLC1-99, which encodes SP-C100, and pBY103, which contains the unc-119 promoter, were used for the cloning, with the SP-C100-encoding DNA being cloned into the pBY103 vector behind the unc-119 promoter. The basic vector pBY103 is composed of the vector backbone pPD49.26, which is described in “ Caenorhabditis elegans:  Modern Biological Analysis of an Organism” (1995) Ed. Epstein et al., Vol 48, pp. 473, into which the unc-119 promoter (Maduro et al. Genetics (1995) , 141, p. 977) has been cloned by way of the HindIII/BamHI sites. The plasmid unc-119-SP-C100 was prepared by KpnI/SacI digestion of pSKLC1-99 and cloning of the LC99 fragment into pBY103 (Shoji et al. (1992).  
       Example 2  
     Preparing the Transgenic  C. elegans  Lines  
       [0064]    The method of microinjection was used for preparing the transgenic  C. elegans  lines (Mello et al., (1991) EMBO J. 10 (12) 3959; C. Mello and A. Fire, Methods in Cell Biology, Academic Press Vol. 48, pp. 451, 1995; C. D. Link, Proc Natl Acad Sci USA (1995) 92, 9368).  
         [0065]    Two different  C. elegans  strains, i.e. wild-type N2 and him-8 (high incidence of males), were used. The unc-119-SP-C100 construct was microinjected into the gonads of young adult hermaphrodites using a microinjection appliance. The DNA concentration was about 20 ng/μl.  
         [0066]    A marker plasmid was injected together with the unc-119-SP-C100 construct. This marker plasmid is the plasmid ttx3-GFP, which encodes the green fluorescent protein under the control of the ttx3 promoter. The activity of the ttx3 promoter is specific for particular neurons of the  C. elegans  head, the so-called AIY neurons, which play a role in the thermotaxis of the worm.  
         [0067]    When plasmid DNA is microinjected, it is assumed that long tandem arrays, which are composed of many copies of plasmid DNA (in our case, of the ttx3-GFP plasmid and the unc-119-SP-C100 plasmid), are formed by recombination. A certain percentage of these arrays integrate into the  C. elegans  genome. However, the arrays are more likely to be present extrachromosomally.  
         [0068]    Worms which had been injected successfully exhibit a green fluorescence in the AIY neurons of the head region when stimulated with light of a wavelength of about 480 nm. It was possible to detect such nematodes.  
       Example 3  
     Describing the C100 Transgenic  C. elegans  Lines  
       [0069]    1. Phenotypic Features  
         [0070]    Following stimulation with light of a wavelength of 480 nm, C100-transgenic worms exhibit a green fluorescence in the AIY neurons of the head region. Since it was also possible to detect green fluorescence in the head neurons once again in the descendants of the worms, it can be assumed that the plasmids are able to pass down through the germ line. However, the penetrance is not 100%, which makes it possible to conclude that the long tandem arrays composed of ttx3-GFP marker DNA and unc-119-SP-C100 are present extrachromosomally rather than being integrated into the genome.  
       Example 4  
     Detecting C100 Expression in a Blot  
       [0071]    Six different transgenic C100  C. elegans  lines (three in an N2 wt background and three in a him 8 background) were examined in a Western blot for expression of the C100 fragment using a polyclonal antiserum directed against the C terminus of APP. A band having the appropriate molecular weight of about 10 kDa was detectable in all the six lines.  
       Example 5  
     Detecting the C100 in an ELISA  
       [0072]    In an Aβ Sandwich ELISA, signals which were above the background level, and which were statistically significant in two cases, were detected in cell extracts from transgenic animals. This indicates that  C. elegans  could possess a γ-secretase-like activity.  
         [0073]    In the Aβ Sandwich ELISA assay, 96-well plates are first of all incubated with the monoclonal antibody clone 6E10 (SENETEK PLC., MO, USA), which reacts specifically with the Aβ peptide (amino acids 1-17), and then coated with worm extracts from transgenic worms or control worms. The Aβ peptide is detected using the monoclonal Aβ antibody 4G8 (SENETEK PLC., MO, USA), which recognizes amino acids 17-24 in the Aβ peptide and is labeled with biotin. The detection is effected by way of the alkaline phosphatase reaction using an appropriate antibody which is directed against biotin. Disruption of the worms involves detergent treatment, nitrogen shock freezing, sonication and rupture of the cells using glass beads.  
         [0074]    The ELISA signal from the above-described experiment can be based either on weak expression of the Aβ peptide or on expression of the C100 precursor protein, since the appropriate epitopes are present in both proteins.  
         [0075]    Expression of the Aβ peptide could, for example, also be specifically detected in an analogous manner: for this, Aβ-specific antibodies which do not react with the C100 precursor would have to be employed in an Aβ Sandwich ELISA. An Aβ-specific antibody could, for example, be a monoclonal antibody which specifically recognizes the C-terminal end of the Aβ form, which is composed of 40 or 42 amino acids. In parallel, the Aβ peptide could be detected in a Western blot using the monoclonal antibodies 4G8 and 6E10 and then be distinguished from the larger C100 precursor by its molecular weight of 4 kD.  
         [0076]    The vectors can be obtained from Andrew Fire (Department of Embryology, Carnegie Institution of Washington, Baltimore, Md. 21210, USA) in the case of pPD49.26 and LC99 (amyloid precursor protein), which is deposited under ATCC number 106372. The unc-119 promoter can be obtained from Maduro, M. (Department of Biological Science, Universitiy of Alberta Edmonton, Canada), while unc-54 and unc-16.2 can be obtained from Andrew Fire.  
         [0077]    The above description of the invention is intended to be illustrative and not limiting. Various changes or modifications in the embodiments described may occur to those skilled in the art. These can be made without departing from the spirit or scope of the invention.  
                           SEQ ID NO.1: Nucleotide sequence of C100           CTGGATGC AGAATTCCGA CATGACTCAG GATATGAAGT TCATCATCAAAAATTGGTGT               TCTTTGCAGA AGATGTGGGT TCAAACAAAG GTGCAATCAT TGGACTCATGGTGGGCGGTG               TTGTCATAGC GACAGTGATC GTCATCACCT TGGTGATGCT GAAGAAGAAACAGTACACAT               CCATTCATCA TGGTGTGGTG GAGGTTGACG CCGCTGTCAC CCCAGAGGAGCGCCACCTGT               CCAAGATGCA GCAGAACGGC TACGAAAATC CAACCTACAA GTTCTTTGAGCAGATGCAGA ACTAG               SEQ ID NO.2: Nucleotide sequence of SP       ATG CTGCCCGGTT TGGCACTGTT CCTGCTGGCC GCCTGGACGG CTCGGGCG               SEQ ID NO.3: Nucleotide sequence of SP+C100       ATG CTGCCCGGTT TGGCACTGTT CCTGCTGGCC GCCTGGACGG CTCGGGCGCT G               GATGC AGAATTCCGA CATGACTCAG GATATGAAGT TCATCATCAA AAATTGGTGT               TCTTTGCAGA AGATGTGGGT TCAAACAAAG GTGCAATCAT TGGACTCATG               GTGGGCGGTG TTGTCATAGC GACAGTGATC GTCATCACCT TGGTGATGCT GAAGAAGAAA               CAGTACACAT CCATTCATCA TGGTGTGGTG GAGGTTGACG CCGCTGTCAC CCCAGAGGAG               CGCCACCTGT CCAAGATGCA GCAGAACGGC TACGAAAATC CAACCTACAA GTTCTTTGAG               CAGATGCAGA ACTAG               SEQ ID NO.4: Nucleotide sequence of the 42bp intron       GTATGTTTCGAATGATACTAACATAACATAGAACATTTTCAG               SEQ ID NO.5: Nucleotide sequence of intron+SP+C100       GTATGTTTCGAATGATACTAACATAACATAGAACATTTTCAGGAGGACCCTTGGCTAGCGTCGACGGT               ACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAACCTTCACAGCAGCGCACTCGGTGCCCCGCG               CAGGGTCGCGATG CTGCCCGGTT TGGCACTGTT CCTGCTGGCCGCCTGGACGG CTCGGGCGCT               GGATGC AGAATTCCGAATGACTCAGGATATGAAGTCATCATCAAAAATTGGTGT TCTTTGCAGA               AGATGTGGGT TCAAACAAAG GTGCAATCAT TGGACTCATG GTGGGCGGTG TTGTCATAGC               GACAGTGATC GTCATCACCT TGGTGATGCT GAAGAAGAAA CAGTACACAT CCATTCATCA               TGGTGTGGTG GAGGTTGACG CCGCTGTCAC CCCAGAGGAGCGCCACCTGT CCAAGATGCA               GCAGAACGGC TACGAAAATC CAACCTACAA GTTCTTTGAG CAGATGCAGA ACTAG               SEQ ID NO.6: Nucleotide sequence of unc-119       AAGCTTCAGTAAAAGAAGTAGAATTTTATAGTTTTTTTTCTGTTTGAAAAATTCTCCCCATCAATGTTCT               TTCAAATAAATACATCACTAATGCAAAGTATTCTATAACCTCATATCTAAATTCTTCAAAATCTTAACAT               ATC               TTATCATTGCTTTAAGTCAACGTAACATTAAAAAAAATGTTTTGGAAAATGTGTCAAGTCTCTCAAAATT               CAGTTTTTTAAACCACTCCTATAGTCCTATAGTCCTATAGTTACCCATGAAATCCTTATATATTACTGTA               AAATGTTTCAAAAACCATTGGCAAATTGCCAGAACTGAAAATTTCCGGCAAATTGGGGAACCGGCAA               ATTGCCAATTTGCTGAATTTGCCGGAAACGGTAATTGCCGAAAGTTTTTGACACGAAAATGGCAAATT               GTGGTTTTAAAATTTTTTTTTTTGGAAATTTCAGAATTTCAATTTTAATCGGCAAAACTGTAGGCATCCT               AAGAATGTTCCTACATCTATTTTGAAAAGTAAGCGAATTAATTCTATGAAAATGTCTAAAGAAAATGGG               GAAACAATTTCAAAAAGGCACAGTTTCAATGGTTTCCGAATTATACTAAATCCCTCTAAAAACTTCCGG               CAAATTGATATCCGTAAAAGAGCAAATCCGCATTTTTGCCGAAAATTAAAATTTCCGACAAATCGGCA               AACCGGCAATTTGGCGAAATTTGCCGGAACGATTGCCGCCCACCCCTGTTCCAGAGGTTCAAACTG               GTAGCAAAGCTCAAAATTTCTCAAATTCTCCAATTTTTTTTTGAATTTTGGCAGTGTACCAAAATGACA               TTCAGTCATATTGGTTTATTATAGATTTATTTAGATAAAATCCTAAATGATTCTACCTTTAAAGATGCCC               ACTTTAAAAGTAATGACTCAAACTTCAAATTGCTCTAAGATTCTATTGAATTACCATCTTTTCCTCTCAT               TTTCTCTCACTGTCTATTTCATCACAAATTCATCCCTCTCTCCTCTCTTCTCTCTCCCTCTCTCTCTCTT               TCTCTTTGCTCATCATCTGTCATTTTGTCCGTTCCTCTCTCTGCGCCCTCAGCGTTCCCCACACTCTC               TCGCTTCTCTTTTCCTAGACGTCTTCTTTTTTCATCTTCTTCAGCCTTTTTCGCCATTTTCCATCTCTGT               CAATCATTACGGACGACCCCCATTATCGAT               SEQ ID NO.7: Nucleotide sequence of unc-119+intron+SP+C100       AAGCTTCAGTAAAAGAAGTAGAATTTTATAGTTTTTTTTCTGTTTGAAAAATTCTCCCCATCA               ATGTTCTTTCAAATAAATACATCACTAATGCAAAGTATTCTATAACCTCATATCTAAATTCTTCAAAATC               TTAACATATCTTATCATTGCTTTAAGTCAACGTAACATTAAAAAAAATGTTTTGGAAAATGTGTCAAGTC               TCTCAAAATTCAGTTTTTTAAACCACTCCTATAGTCCTATAGTCCTATAGTTACCCATGAAATCCTTATA               TATTACTGTAAAATGTTTCAAAAACCATTGGCAAATTGCCAGAACTGAAAATTTCCGGCAAATTGGGG               AACCGGCAAATTGCCAATTTGCTGAATTTGCCGGAAACGGTAATTGCCGAAAGTTTTTGACACGAAAA               TGGCAAATTGTGGTTTTAAAATTTTTTTTTTTGGAAATTTCAGAATTTCAATTTTAATCGGCAAAACTGT               AGGCATCCTAAGAATGTTCCTACATCTATTTTGAAAAGTAAGCGAATTAATTCTATGAAAATGTCTAAA               GAAAATGGGGAAACAATTTCAAAAAGGCACAGTTTCAATGGTTTCCGAATTATACTAAATCCCTCTAA               AAACTTCCGGCAAATTGATATCCGTAAAAGAGCAAATCCGCATTTTTGCCGAAAATTAAAATTTCCGA               CAAATCGGCAAACCGGCAATTTGGCGAAATTTGCCGGAACGATTGCCGCCCACCCCTGTTCCAGAG               GTTCAAACTGGTAGCAAAGCTCAAAATTTCTCAAATTCTCCAATTTTTTTTTGAATTTTGGCAGTGTAC               CAAAATGACATTCAGTCATATTGGTTTATTATAGATTTATTTAGATAAAATCCTAAATGATTCTACCTTT               AAAGATGCCCACTTTAAAAGTAATGACTCAAACTTCAAATTGCTCTAAGATTCTATTGAATTACCATCT               TTTCCTCTCATTTTCTCTCACTGTCTATTTCATCACAAATTCATCCCTCTCTCCTCTCTTCTCTCTCCCT               CTCTCTCTCTTTCTCTTTGCTCATCATCTGTCAT               TTTGTCCGTTCCTCTCTCTGCGCCCTCAGCGTTCCCCACACTCTCTCGCTTCTCTTTTCCTAGACGTC               TTCTTTTTTCATCTTCTTCAGCCTTTTTCGCCATTTTCCATCTCTGTCAATCATTACGGACGACCCCCA               TTATCGATAAGATCTCCACGGTGGCCGCGAATTCCTGCAGCCCGGGGGATCCCCGGGATTGGCCAA               AGGACCCAAAGGTATGTTTCGAATGATACTAACATAACATAGAACATTTTCAGGAGGACCCTTGGCTA               GCGTCGACGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAACCTTCACAGCAGCGCACTC               GGTGCCCCGCGCAGGGTCGCGATGCTGCCCGGTT               TGGCACTGTTCCTGCTGGCCGCCTGGACGGCTCGGGCGCTGGATGCAGAATTCCGA               CATGACTCAGGATATGAAGTTCATCATCAAAAATTGGTGTTCTTTGCAGAAGATGTGGGTTCAAACAA               AG GTGCAATCAT TGGACTCATGGTGGGCGGTGTTGTCATAGCGACAGTGATCGTCATCACCT               TGGTGATGCT GAAGAAGAAACAGTACACAT CCATTCATCA TGGTGTGGTG GAGGTTGACG               CCGCTGTCAC CCCAGAGGAGCGCCACCTGT CCAAGATGCA GCAGAACGGC TACGAAAATC               CAACCTACAA GTTCTTTGAGCAGATGCAGA ACTAG               SEQ ID NO.8: Nucleotide sequence of the expression vector       ACCCCCGCCACAGCAGCCTCTGAAGTTGGACACGGATCCACTAGTTCTAGAGCGGCCGCCACCGC               GGTGGAGCTCCGCATCGGCCGCTGTCATCAGATCGCCATCTCGCGCCCGTGCCTCTGACTTCTAAG               TCCAATTACTCTTCAACATCCCTACATGCTCTTTCTCCCTGTGCTCCCACCCCCTATTTTTGTTATTAT               CAAAAAAACTTCTTCTTAATTTCTTTGTTTTTTAGCTTCTTTTAAGTCACCTCTAACAATGAAATTGTGT               AGATTCAAAAATAGAATTAATTCGTAATAAAAAGTCGAAAAAAATTGTGCTCCCTCCCCCCATTAATAA               TAATTCTATCCCAAAATCTACACAATGTTCTGTGTACACTTCTTATGTTTTTTTTACTTCTGATAAATTTT               TTTTGAAACATCATAGAAAAAACCGCACACAAAATACCTTATCATATGTTACGTTTCAGTTTATGACCG               CAATTTTTATTTCTTCGCACGTCTGGGCCTCTCATGACGTCAAATCATGCTCATCGTGAAAAAGTTTT               GGAGTATTTTTGGAATTTTTCAATCAAGTGAAAGTTTATGAAATTAATTTTCCTGCTTTTGCTTTTTGGG               GGTTTCCCCTATTGTTTGTCAAGAGTTTCGAGGACGGCGTTTTTCTTGCTAAAATCACAAGTATTGAT               GAGCACGATGCAAGAAAGATCGGAAGAAGGTTTGGGTTTGAGGCTCAGTGGAAGGTGAGTAGAAGT               TGATAATTTGAAAGTGGAGTAGTGTCTATGGGGTTTTTGCCTTAAATGACAGAATACATTCCCAATATA               CCAAACATAACTGTTTCCTACTAGTCGGCCGTACGGGCCCTTTCGTCTCGCGCGTTTCGGTGATGAC               GGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGG               AGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGC               GGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAG               GAGAAAATACCGCATCAGGCGGCCTTAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCAT               GATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT               TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT               ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTT               GCCTTCCTGTTTTTGCTC               ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCG               AACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAG               CACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT               CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGG               ATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTT               ACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA               ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACG               ATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCC               GGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC               CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG               CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTA               TGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA               CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAG               ATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC               CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA               AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGT               AACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC               TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCA               GTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGT               CGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGA               TACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCG               GTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCT               TTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG               CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTG               CTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT               GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGC               GCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGT               TTCCGGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCAC               CCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC               ACAGGAAACAGCTATGACCATGATTACGCCAAGCTT               SEQ ID NO.9: Amino acid sequence of SP       MLPGLALFLL AAWTARA               SEQ ID NO.10: Amino acid sequence of the fusion protein       MLPGLALFLL AAWTARALDA EFRHDSGYEV HHQKLVFFAE DVGSNKGAII               GLMVGGVVIA TVIVITLVML KKKQYTSIHH GVVEVDAAVT PEERHLSKMQ               QNGYENPTYK FFEQMQN               SEQ ID NO. 11: Nucleotide sequence of the vector unc-119-SP-C100       ATGACCATGATTACGCCAAGCTTCAGTAAAAGAAGTAGAATTTTATAGTTTTTTTTCTGTTTGAAAAAT               TCTCCCCATCAATGTTCTTTCAAATAAATACATCACTAATGCAAAGTATTCTATAACCTCATATCTAAAT               TCTTCAAAATCTTAACATATCTTATCATTGCTTTAAGTCAACGTAACATTAAAAAAAATGTTTTGGAAAA               TGTGTCAAGTCTCTCAAAATTCAGTTTTTTAAACCACTCCTATAGTCCTATAGTCCTATAGTTACCCAT               GAAATCCTTATATATTACTGTAAAATGTTTCAAAAACCATTGGCAAATTGCCAGAACTGAAAATTTCCG               GCAAATTGGGGAACCGGCAAATTGCCAATTTGCTGAATTTGCCGGAAACGGTAATTGCCGAAAGTTT               TTGACACGAAAATGGCAAATTGTGGTTTTAAAATTTTTTTTTTTGGAAATTTCAGAATTTCAATTTTAAT               CGGCAAAACTGTAGGCATCCTAAGAATGTTCCTACATCTATTTTGAAAAGTAAGCGAATTAATTCTAT               GAAAATGTCTAAAGAAAATGGGGAAACAATTTCAAAAAGGCACAGTTTCAATGGTTTCCGAATTATAC               TAAATCCCTCTAAAAACTTCCGGCAAATTGATATCCGTAAAAGAGCAAATCCGCATTTTTGCCGAAAA               TTAAAATTTCCGACAAATCGGCAAACCGGCAATTTGGCGAAATTTGCCGGAACGATTGCCGCCCACC               CCTGTTCCAGAGGTTCAAACTGGTAGCAAAGCTCAAAATTTCTCAAATTCTCCAATTTTTTTTTGAATT               TTGGCAGTGTACCAAAATGACATTCAGTCATATTGGTTTATTATAGATTTATTTAGATAAAATCCTAAAT               GATTCTACCTTTAAAGATGCCCACTTTAAAAGTAATGACTCAAACTTCAAATTGCTCTAAGATTCTATT               GAATTACCATCTTTTCCTCTCATTTTCTCTCACTGTCTATTTCATCACAAATTCATCCCTCTCTCCTCTC               TTCTCTCTCCCTCTCTCTCTCTTTCTCTTTGCTCATCATCTGTCATTTTGTCCGTTCCTCTCTCTGCGC               CCTCAGCGTTCCCCACACTCTCTCGCTTCTCTTTTCCTAGACGTCTTCTTTTTTCATCTTCTTCAGCCT               TTTTCGCCATTTTCCATCTCTGTCAATCATTACGGACGACCCCCATTATCGATAAGATCTCCACGGTG               GCCGCGAATTCCTGCAGCCCGGGGGATCCCCGGGATTGGCCAAAGGACCCAAAGGTATGTTTCGAA               TGATACTAACATAACATAGAACATTTTCAGGAGGACCCTTGGCTAGCGTCGACGGTACCGGGCCCCC               CCTCGAGGTCGACGGTATCGATAACCTTCACAGCAGCGCACTCGGTGCCCCGCGCAGGGTCGCGA               TG CTGCCCGGTT TGGCACTGTT CCTGCTGGCCGCCTGGACGG CTCGGGCGCT GGATGC               AGAATTCCGA CATGACTCAG GATATGAAGT TCATCATCAAAAATTGGTGT TCTTTGCAGA               AGATGTGGGT TCAAACAAAG GTGCAATCAT TGGACTCATGGTGGGCGGTG TTGTCATAGC               GACAGTGATC GTCATCACCT TGGTGATGCT GAAGAAGAAACAGTACACAT CCATTCATCA               TGGTGTGGTG               GAGGTTGACG CCGCTGTCAC CCCAGAGGAGCGCCACCTGT CCAAGATGCA GCAGAACGGC               TACGAAAATCCAACCTACAATTCTTTGAGCAGATGCAGAACTAGACCCCCGCCACAGCAGCCTCTGA               AGTTGGACACGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCGCATCGGCCGCT               GTCATCAGATCGCCATCTCGCGCCCGTGCCTCTGACTTCTAAGTCCAATTACTCTTCAACATCCCTAC               ATGCTCTTTCTCCCTGTGCTCCCACCCCCTATTTTTGTTATTATCAAAAAAACTTCTTCTTAATTTCTTT               GTTTTTTAGCTTCTTTTAAGTCACCTCTAACAATGAAATTGTGTAGATTCAAAAATAGAATTAATTCGTA               ATAAAAAGTCGAAAAAAATTGTGCTCCCTCCCCCCATTAATAATAATTCTATCCCAAAATCTACACAAT               GTTCTGTGTACACTTCTTATGTTTTTTTTACTTCTGATAAATTTTTTTTGAAACATCATAGAAAAAACCG               CACACAAAATACCTTATCATATGTTACGTTTCAGTTTATGACCGCAATTTTTATTTCTTCGCACGTCTG               GGCCTCTCATGACGTCAAATCATGCTCATCGTGAAAAAGTTTTGGAGTATTTTTGGAATTTTTCAATCA               AGTGAAAGTTTATGAAATTAATTTTCCTGCTTTTGCTTTTTGGGGGTTTCCCCTATTGTTTGTCAAGAG               TTTCGAGGACGGCGTTTTTCTTGCTAAAATCACAAGTATTGATGAGCACGATGCAAGAAAGATCGGA               AGAAGGTTTGGGTTTGAGGCTCAGTGGAAGGTGAGTAGAAGTTGATAATTTGAAAGTGGAGTAGTGT               CTATGGGGTTTTTGCCTTAAATGACAGAATACATTCCCAATATACCAAACATAACTGTTTCCTACTAGT               CGGCCGTACGGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAG               CTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC               GTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAG               AGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGGCC               TTAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAG               GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT               ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATT               CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAA               ACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGAT               CTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAA               AGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCAT               ACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA               CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC               AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT               GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTA               GCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAAT               TAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCT               GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGC               CAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAAC               GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTA               CTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTT               GATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA               GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG               CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT               TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGT               TAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGT               GGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA               GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA               CCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCG               GACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAA               ACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC               TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTT               TGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC               CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG               AAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCT               GGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCA               CTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG               ATAACAATTTCACACAGGAAACAGCT          
 
       References  
       [0078]    Baumeister R (1997) Genes &amp; Function 1, 149  
         [0079]    Daigle I, Li C (1993), 90 (24), 12045  
         [0080]    Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum J. M., Masters C. L., Grzeschik, K. H., Multhaupt, G., Beyreuther, K., Mueller-Hill, B. (1987) Nature, 325, 733  
         [0081]    Kenyon, C., Science (1988) 240, 1448  
         [0082]    Korswagen H. C., Durbin, R. M., Smits, M. T., Plasterk, R. H. A. (1996), 93, 14680 Proc Natl Acad Sci USA  
         [0083]    Kuwabara, P. E. (1997), Trends in Genetics, 13, 454  
         [0084]    Levitan D., Doyle T G, Brousseau D., Lee M K. Thinakaran G., Slunt H H., Sisodia S S. Greenwald I. (1996) Proc Natl Acad Sci USA, 93,14940  
         [0085]    Levitan D, Greenwald I (1995) Nature 377, 351  
         [0086]    Link C. D. (1995) Proc Natl Acad Sci USA, 92, 9368  
         [0087]    Mello, C. and Fire, A., Methods in Cell Biology, Academic Press Vol. 48, pp 451, 1995  
         [0088]    Riddle et al. (1997)  C. elegans  II, Cold Spring Harbor Laboratory Press  
         [0089]    Rumble, B., Retallack, R., Hilbich, C., Simms, G., Multhaup, G., Martins, R., Hockey, A., Montgomery, P., Beyreuther, K., Masters, C. L., (1989) , N. Engl. J. Med., 320, 1446  
         [0090]    Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T., Hardy, M., Hutton, W., Kukull, W., Farson, E., Levy-Lahad, E., Vitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfeld, D., Selkoe, D., Younkin, S. G. (1996), Nature Medicine, 2, 864  
         [0091]    Shoji M., Golde T E., Ghiso J., Cheung T T., Estus S., Shaffer L M., Cai X-D., McKay D M., Tintner R., Fraggione B., Younkin S G. (1992) Science 258,126  
         [0092]    Xiajun Li and Iva Greenwald (1997) Proc Natl Acad Sci USA, 94,12204  
         [0093]    Yankner, B. A., Caceres, A., Duffy, L. K. (1990) Proc Natl Acad Sci USA, 87, 9020