MXPA97009014A - Transgenic animal models for diabetes mellitus type - Google Patents

Abstract

The generation of transgenic animal models to test various treatments of Type II Diabetes Mellitus is described: the constructed DNA allows the specific expression in pancreatic cells of the polypeptide associated with the human islet (IAPP) under the regulation of the rat insulin II promoter both in lines of cells in transgenic animals, the constructed DNA is introduced into animal embryos by microinjection as one or several copies or in cell lines established by electroporation, the transgenic animals develop amyloid plaque deposits in the islets of Langerhans in the pancreas, fasting hyperglycemia , glycosuria and glomerulosclerosis at 3 to 5 months of age, cell lines can be traced for treatments that inhibit the expression of human IAPP, transgenic animals can be traced for treatments, either by potentiating or inhibiting the progression of this phenotype. sick

Description

TRENDANGEOUS MODELS PflRfl Lñ DIsBETES HELLITUS TYPE II Fs- * a invention refers to a process for altering gene-t i carnen + e lines of cells of animals and animals so that they express the roteo.na encoded by the flrmloid Polypeptide gene of human Tslote (TflPP). IAPP, formerly known as arniline, is the main protein component of the loine arnid of the pancreatic islet that is formed in the pancreas of patients with Diabetes Rail or Non-Dependent Insulin (MTDDM). The latest studies of the characteristics are structural and functional of TflPP suggest that TflPP, along with insulin and other hormones, plays an important role in the metabolism of carbohydrates. TflPP is a product, stored and secreted by pancreatic β cells in the islets of Langerhans. This can mimic the phenomenon of insulin resistance in MIDDM inhibiting glucose uptake and glycogen synthesis in muscle and liver tissue. The generation of arniloi deposits is in humans believed to be due to the capacity of the peptide's central portion (amino acids 20 to 29) to form a β-folded sheet structure. The IflPP of rodents differs from human IflPP in that the sequences of this protein on the other hand very well conserved between amino acids 20 and 29 is not conserved and the aryloid deposits are not formed in the rodent pancreas. A working hypothesis is that the overexpression of human TflPP leads to insulin resistance in peripheral tissues and to the formation of deposits in Loides. Tumor-like animals, especiaLment or mice, have proved to be very useful in the detailed analysis of complex systems to generate new information relevant to human disease. The selective expression of human genes in such mice has generated new model systems for the study of the disease, especially when the overpressure of a gene produces a disease state * rats can be applied * to problems relating to (1) the speci fi city of the expression tissue; (2) test the hypothesis that overexpression of a particular gene leads to disease; (3) the number and identity of tissues / organs that are affected by * this overexpression; and (4) effects of various treatment, including drugs on the progression or alleviation of the disease phenotype. The generation of transgenic mice expressing human TflPP has been described in the literature, thinking that none of these animals developed a diabetes phenotype.

Niles Fox et al. (FEBS Letters, 323., 40-44, [1993J] constructed a transformed gene that fused the rat insulin promoter sequence with a genomic flDN fragment containing the complete human TflPP gene (exons 1-3 d mtrons 1 and 2 ) . The expression of the flRN of the transformed gene is I detect in the pancreas, anterior pituitary and brain. Although levels of IflPP in plasma were 5 times higher in reLacLon with non-transgenic parent litters, no metallic consequence of this elevation was observed. C.B. Ver-chore et al. (Diabetologia 37., 725-729 ri994J) used a 600 base pair fragment encoding the complete human proTflPP sequence. Their transgenic animals showed a higher pancreatic content of both IflPP and insulin in relation to the non-transgenic progenitor control baits. The greater secretion of both hormones was also detected in studies of peritoneal pancreas. No clinical manifestations of this increased secretion and storage were observed. Hoppener et al. (Diabetologia, 3_5, 1258-1265 C19933) describe the generation of multiple transgenic lines that expressed either human or rat IFlPP in the mouse endocrine pancreas. The Hoppener group used a 703 bp insulin II promoter fragment to drive the expression of human or rat IFlPP from genomic flDN fragments. IflPP levels in plasma multiplied by 15 but no hyperglucerma or hyperpulsulinernia was observed. In a later study, no accumulation of live arniloid plaque was observed, but intra- and extracellular arniloid fibrils were formed when these transgenic islets were cultured in vitro under conditions that mimicked hyperglycemia (De Konmg et al., Proc. Nati. Acad., 91, 8467-8471 ["1994].) In one embodiment, the present invention is directed to recombinant flDN comprising a non-promoter of IflPP, a sequence (jue encodes human IflPP or one of its proteins operably linked to a sequence that encodes the human albumin mtron T, a sequence encoding the termination of human GflPDH and a sequence encoding the poly demiation of human GAPDH, said flDN producing the expression of a diabetic phenotype when incorporated into a suitable host. a recombinant flDN in which the non-promoter of TflPP is selected from the group consisting of * the promoters of the rat insulin I genes, rat insulin II, human insulin a, mouse IAPP, glucocmase specific for rat beta cells, glucose transporter 2, human tyrosine arnide transferase, human albumin, mouse albumin, rat liver specific glucokinase and mouse mouse lotion. In addition, recombinant DNA is preferred in which said promoter is the rat IT insulin promoter. Especially preferred is also the recornobinante AUN in which said sequence encoding human IAPP or one of its active fragments has the characteristics of a cDNA. Additionally, a recombinant DNA is preferred in which said sequence encoding human IAPP or one of its fragments has the characteristics of a genomic DNA. It is also further preferred to have a recornbinating DNA in which said sequence is that of SEO ID No. 4. It is also additionally preferred in particular a combin DNA or e in which said cDNA sequence is that of SEO ID No .. r -. In another embodiment, the DNA sequence encoding the human EAPP is replaced by a DNA sequence encoding mouse LAPP or one of its active fragments, said mouse DNA preferably having the characteristics of cDNA. The present invention is also directed to vectors comprising recombinant DNA of the present invention (SEO, ID No. 1). The present invention is also directed to a line of eukaryotic cells comprising recornbinating DNA of the same invention, the cell lines being selected from the group consisting of rat insolorna cells (RIN), Hamster Insulmotna (HIT) cells. and mouse msulinorna cells [3-TC3. The present invention is also directed to transgenic non-human mammals comprising recombinant DNA of the present invention, mice and rats being especially preferred as transgenic mammals, said transgenic mammals having a diabetes phenotype. In another embodiment, the present invention is directed to a method of treating an animal having a disease characterized by an overexpression of an IAPP gene product comprising administering a therapeutically effective amount of an over expression of said product. 1 PP gene to said ma i fero. In yet another embodiment, the present invention is directed to a method for analyzing the effect of a treatment comprising administering said treatment and analyzing the effect of said treatment on the overexpression product of a gene encoding IAPP. The method in which said treatment is administered to an animal is preferred, the animal being especially preferred a human. The present invention is also directed to a method for determining whether a patient has a risk of diabetes or obesity which comprises examining said patient regarding the overexpression of an IAPP gene product, said overexpression being a risk indicator. In yet another embodiment, the present invention is directed to a method for analyzing an animal model for a disorder or disease state comprising determining whether an IAPP gene in said animal model is expressed at a predetermined level., a preferred method being one in which said level is greater than the level in a wild type or normal animal. Figure la is a linear apa of the transformed gene RIPHAT. The human IAPP cDNA sequence is represented in black; the rat insulin II promoter is represented as a white box, the human albumin intron is a shaded box of dark, the poly ladeni 1 aci region of GAPDH (poly fl ated) is represented as a shaded box of color. Clear. Figure Ib is an extension of the ends of the coding region that presents the restriction sites that can be used * to replace alternative cDNAs for human IAPP. Figure 1 is an extension of the ends of the promoter region that presents the restriction sites that can be used to substitute * the alternative promoters for RIP II in the RIPHAT-formed ran gene. Figure 2 is a circular map * of the plasmid pSV2Dogl. PSV2Dogl was constructed by inserting the modified CAT gene (BspM I / BarnH I fragment) downstream of the SV4 promoter (partial fragment BarnH T / Nco I from? LuxF3). The resulting plasmid contains the sequence encoding the CAT gene fused at its 5 'end with optimal mammalian translation sequences and fused at its 3' end with the 3 'untranslated region of firefly lucy fea and the addition site of poly A. Expression of the modified CAT gene is driven by the SV40 promoter. Figure 3 is a circular map of the plasmid pSV2Dogll which contains the polyhydration dehydrogenase region of gl iceraldehyde 3 phosphate used to construct the pDog 15 plasmid. The pSV2Dogll was constructed by inserting the 3'-untranslated region of gl-ceraldehyde-3 dehydrogenase - human phosphate amp Lifi each by PCR in pSV2Dogl digested with Spe I / BarnH 1. These sequences do not encode the 3 'sites of GAPDH downstream of the region encoding CflT. Figure 3 is a schematic drawing of the cloning strategy used to build pRTPHATl, starting with the piasmid pDogld. Figure 4 is an autoradiogram of a Southern blot of 6-tail DNA genomic flDNs digested with the restriction endonuclease EcoRT and hybridized to the 32 P-labeled human GAPDH fragment within the t ransgemeo DNA of RTPHAT. The 6 bands represent a bait of anima Les produced from a cross of RG male with transgemea line of RIPHflT and female wild type FVB / N. Figure 5 is an autoradiogram of a No LTR transference of total pancreatic RNA isolated from RHA, RHF and RHG transgenic lines together with pancreatic RNA from Human Pancreas and a non-transgenic mouse. The trans¬ brerence was hybridized against a fragment of human IAPP cDNA labeled with 20. FIG. 6 is an electron micrograph of a ß pancreatic cell. The arrows mark a deposit of amyloid plaque. Figure 7 is an electron microscopy showing a immunological staining with purple at 37,000 magnifications of the micellular arniloid plate by means of a rabbit anti-human lAPP antibody.

FIG. 8 is a graphic representation of the appearance of hyperglycemia in 3 male RHF furnace mice compared to 3 non-transgermal male mice (FVB / N race). Figure 9 represents the result of an oral glucose tolerance test carried out on 5-week RHF bacteregous transgenic males (Bait NQ RHF11, n = 5) and females (bait N RHF11, n = 3) compared with FVB mice / N not t ransgenicos matched by age.

The plasmids: The plasmid pRIPHAT (gene transformed from human IAPP rat insulin promoter) (SEO ED NQ i) contains DNA fragments from 5 different sources, three from human genes, the fourth from rat and the fifth a vector from a commercially available plasmid. These are the rat IT insulin promoter (876 p.b.); (SEO, ID NQ 2), sequence encoding human 1APP (270 bp) (SEO ID NQ 3), mtron I of human albumin (720 bp) (SEO ID NQ 4), and the site of polyadenylation of the dehydrogenase gene of gl? ceraldeh? do ~ 3- human phosphate (GAPDH) and the RNA termination region (546 bp) (SEO ID NQ 5). The commercial plot available is Bluescppt SK (-) (Stratagene, La 3olla, CA) (SEO ID NQ 6). The enzymatic manipulations of the recornbinant DNA, including the junctions, restriction endonuclease digestions, DNA synthesis reactions and transformations of E. coli were carried out according to well-demonstrated procedures as described in Saint-1 * ook, 3., Fritsch, EF, and Maniatis, T. Molecular Cloning: Laboratory Manu L. 2Q Ed. Cold Spring Harbor Laboratory Press, New York, 1989. The intron I of human albumin (SEO ID NQ 4) and the fragments of the GAPDH gene (SEO ID HQ.5) were obtained from the plasmid pSV2Dog! 5 by digestion of this plasmid with Barn HI and ApaLI and isolating the 1262 bp fragment that it contained these two regions. PSV2Dogl5 was built by David Lloyd and John Thompson of Pfizer, Tnc. They generated the human albumin intron I sequence by a polyunerase chain reaction (PCR) amplification (Innis, MA et al., Eds PRC Protocols, Acadernic Press, New York 1990) of this portion of the albumin gene using Human genomic DNA (obtained from Clontech, Palo Alto, CA) and DNA oligomers 18505.022 (sequence 5 'CCCTCTAGAAGCTTGTCTGGGCAAGGGAAGAAAA 3') (SEO ID NQ 8) and 18505.024 (5 'sequence GGGAAGCTTCTfl-GflCTTTCGTCGflGGTGCflCGTAflGflfl 3') (SEO TD NQ 9). Since these oligorneros included exogenous Xba I sites at their ends, the resulting PRC product was digested with Xba I and inserted into the compatible Spe I site in? SV2Dogll. This plasmid, constructed by David Lloyd, in turn contained the polyadenylation region of human GAPDH. This was also generated by PCR cloning using human genomic DNA as a template and the oligorneros 18970,246 (sequence 5'CAAACCG-GATCCGCCCTGACTTCCTCCACCTGTCAGC 3 ') (SEO ID No. 10) and 18970.244 1 L (sequence 5 'CACAACACTAGTGACCCCTGGACOflCCAGCCCCAGC 3') (SEO TD NQ Ll) as PCR primers. The PCR product generated in this way was subjected to digestion with Spe 1 and Bam Hl and inserted in? SV2Dogl digested with Spel / Barn Hl (see Figure 2). The hybrid fragment of poly A region of GAPDH mtron I of albumin LI LI / Barn Hl of 1262 p.b. ligated to the fragment of flDN amplified with PCR of 278 p.b. containing the region that codes for the protein product of prepro IAPP. This fragment was amplified using a TAPP cDNA (hIAPP-cl from phage DNA Larnbda, obtained from Sietse MosseLman, Rij ^ suniversiteit te Utrecht, The Netherlands and described in Mosselrnan S. et al., Febs Lett. 247, 154-158 [1989]) as mold and oligorneros 19383,288 (sequence 5 '~ GTCATGTGCACCTAAAGGGGCAAGTAATTCA 3') (SEO ID NQ 12) and 19987, -116 (sequence 5 '-GAAGCCATGGGCATCCTGAAGCTGCAAGTA 3') (SEO ID NQ 13) as the primers of PCR The fragment of 1523 p.b. The resultant was joined to pSuperLuc (pSL) to generate the plasmid pSLfllO. PSL is a DNA plasmid containing the reporter gene of luciferase (Mosselrnan, S. et al, FEBS Lett 271, 33-36 [1990]). In this case, the plasmid was used only to determine the presence of the appropriate Bam Hl and Neo I restriction sites. The rat IT insulin promoter (SEO ID NQ 2) and the 5 'untranslated head region were generated by rat genomic DNA PCR amplification (obtained from Clontech, cat. NQ 6750-1) using oligorneros 19303,284 ( sequence 5'-GTCAGGAATTCGGATCCCCCAACCACTCCAAGT 3 ') (SEO ID NQ LO and 19383.292 (sequence 5' -ACAGGGCCATGGTGGAACAATGACCTGGAAGATA 3 ') (SEO ID NQ 15), The olí gomeros 19383.292 contains a point mutation; mode for introducing a Neo E site at the 3 'end of the fragment by altering two 5' residues of a nucleotide (fl a C) of the start codon.The 883 bp PCR product is cleaved with Neo I to generate a fragment of 175 pb with blunt end and a 708 bp fragment with two Neo r ends The pSLA 10 plasmid was digested with Xba T. The resulting 5 'ends were supplemented with Klenow polymerase and NTPd to generate blunt ends. digestion with Neo I and joined the fragment of insu lina II of rat with blunt end and Neo I of 175 p.b. to generate pSLAll (see Figure 3). PSLfIII was digested with Neo I and ligated to a rat TI Neo I insulin fragment of 708 p.b. to generate the plasmid pSLA12. The appropriate orientation of the Neo T fragment of 708 p.b. by digestion of the plasmid with Eco Rl and Barn Hl. The chimeric transformed gene (rat insulin II promoter and the region encoding IAPP of untranslated head of the 5 'end, mtron I of albumin, polyadenylation region of GAPDH) (SEO ID No. 7) was transferred from the structure of pSL to Bluescppt SK (-) aligned with Barn Hl by partial digestion of Barn Hl from pSLA12 to generate pRIPHAT I (SEO ID NQ i). The rat insulin II promoter and the as-de-labeled region of the 5 'terminal and the region encoding the 1APP were sequenced by the blood dideoxy chain termination procedure to ensure that no mutations were introduced. .. To make sure that the t ansformed gene was expressed in mouse cells, it transfected transiently pRIPHAT I (SEO ID Q i) into f3TC cells by means of electroporation as described by Osselman et al. (FEBS Lett 271, 33-36, 1990). Total RNA was isolated by * known procedures (Chornczyns i and Sachi, Anal. Biochern., 162, 156-159) 24 hours later. He Specific RNA of the transgene was detected by PCR amplification of the cDNA derived from this total RNA by reverse transcription (Innis, 1 * 1 A. et al., Eds PRC Protocols, flcadernic Press, New York 1990). The size and quantity of PRC product showed that the transformed gene had been expressed and that the mtron portion of the human albumin of the transformed gene was efficiently treated in these cells.

The lines of Stably Transfected Cells. The above described plasmids were stably introduced into RIN and βTC3 cells by electroporation together with a plasmid conferring geneticin resistance (G418) to the host cell. ßTC3 cells were obtained from Shirnon Efrat and Cold Sppng Harbor * Labora tory, Cold Spring Harbor, N.Y. and are described in Efrat, s. et al, Proc. Nati Acad. Sci. 85, 9037-9041 (1988). Cells were prepared by electroporation by tppsi nization of serníconfluent inonolapas, to Lomerando twice, with L washing in RPMI 1640 medium free of serum and resuspension in this medium at a concentration of? x 107 eelulas / inl. In general, 50 μg of the appropriate plamid was added together with 3.3 μg of the selected plasmid pHA2.3neo (which confers resistance to G418, Dr. Peter Hobart, Pfizer, Inc.) to 0.5 μl of cells in a cuvette. Electroporation (Bethesda Research Labs [BRL]), Gaithersburg, MD) and subjected to a field of 250 v / cm at 800 [mu] F and adjusted to low resistance in an electroporation unit BRL Cell-Porator. The cells were allowed to stand for 2 minutes. Then, these were diluted with 2 volumes of 10% fetal bovine serum in RPMI 1640 and transferred to T25 flasks. After 36 hours, the viable cells were transferred to clustered 6-well plates and reproduced at a concentration of 2 x 10 5 cells in the selection medium (the same as above with 500 ug / rnL of active Geneticin). The colonies appeared after 3 weeks and were isolated by known procedures using trypsin and fat-coated porcelain cloning rings. The clones that survived this procedure were reproduced for mass culture, frozen and stored in liquid nitrogen. Confirmation of the expression of the transformed gene was obtained by PCR amplification of the cDNA derived from the total RNA of the clones. In addition, a radioinmunoassay (Case NQ RIK-7321 from Peninsula Labs, Belmont, CA) was carried out both in the total cellular protein and in the surrounding medium to confirm the increase in EAPP content and secretion.

Transgenic mice. Mouse embryos of the FVB / N strain (Taketo, M. et al., Proc. Nati, Acad Sci 88, 2065-2069 [1991]) were injected with linear flDN fragments that were isolated from the plasmid described above. The fragment of flDN RTPHAT of 2395 p.b. it was separated from the plasmid by * cleavage with the restriction endonucleases Xba F and Xho T. The transformed gene fragment of 2395 p.b. it was isolated by electroelution (65 v, 3 hours) after two rations of electrophoresis in agarose gel (0.9% GTG agarose, FNC Bioproducts, Rocl-land, Me) of the product of the digestion reaction. The fragment was further purified on a Schleicher and Schuell Elutip-d column following the manufacturer's protocol Elutip-d Basic for the purification of flDN before it was injected into the embryos. The injection of the embryos was carried out according to published procedures, as described in Hogan, B. et al., Manipulating the Mouse Ernbryo, Cold Spring Harbor Laboratories, New York, 1986.

BEST EXPRESSION AND PREFERRED EMBODIMENTS The plasmids that have been described above can be altered to optimize the expression of the transformed odo gene such as with several insertions, deletions and / or substitutions of one or several base pairs. This includes alterations of base pairs in the forward region of the lucifer-asa start codon to optimize translation efficiency. The promoter region * within pRIPHAT 1 can be exchanged for other promoters, such as human TAPP, rat insulin T, mouse insulin, mouse IAPP, rat glucokinase (specific for liver and / or β cells), gastrin human, human or mouse albumin, mouse neonate, and human tyrosine arnitransferase (Figure Ib). The rat insulin II promoter is represented as a shaded box of dark * color, the coding region as a black box, the human albumin intron as a white box, and the polyadenylation region of human GAPDH as a striped box. TAPP cDNAs from other species such as mice, or mutated functional forms of human IAPP that retain their amyloidogenic portions or portions that induce insulin resistance, can be substituted for the IAPP cDNA region within pRIPHAT. 1 (Figure le). The DNAs of transformed genes can be injected into embryos of other races of mice and mutants thereof, which include db / +, ob / +, A "» 1 or A ", either on a C57BL / 63 or C57BL / Ks background . Alternatively, these transgenic mice can be mated with races of these genetic traits. Preferred cell lines include ßTC3 (Cold Spring Harbor Laboratories), RINrndf (Gazdar, A.F. and others, PNAS L7 77, 3519-3523 [1980] obtained from W. Chick, U. Mass., Uorcester Massachusetts) and HIT (Santerro, R.F. and others, PNAS 78, I339-4343 [19811; obtained from ATCC Rockville MD). Utility Stably transfected cell lines can be used to screen drugs for their ability to alter transcription, rRNA levels, translation, accumulation or secretion of human IAPP. In particular, steady-state levels of RNA from transformed genes can be traced in a very efficient manner using PCR detection methods. The cells can also be used to determine the mechanism of action of possible drugs that are found to alter the aforementioned processes. Transgenic animals can be used to screen drugs for their ability to alter levels of human IAPP in cells, tissues, organs and / or plasma. These can also be used * to study * the pathological consequences of overexpression of human IAPP in whole animals.

Experimental Materials and Procedures Restriction enzymes including ApaLI, Barn Hl, Hmd III, Neo, Not I, Xba I, Xho I and Eco Rl were obtained from New England Biolabs. DNA-modifying enzymes that include Li-ASA DNA T4 and DNA Kinase T5 were obtained from the same supplier. Bacterial alkaline phosphatase was obtained from Boehpnger Mannheirn. All the enzymes obtained commercially were used under the conditions described as optimal by the supplier.

Means The means of growth of E. coli consisted of LO g of Bacto-tpptona, 5 g of Àacto-e yeast tract and 5 g of NaCl. The pH was adjusted to 7.5 with NaOH LO N after adding the rest of the ingredients.

Ethanol Precipitation from ñDN Sodium acetate (NaOAc) from a 3M standard solution at pH 5.2 was added to a DNA sample to bring the final NaOAc concentration to 200 mM. Two and a half volumes of cold absolute ethanol (~ 20 ° C) were added to a volume of aqueous DNA sample and the sample was placed at -70 ° C for 15 minutes or at -20 ° C overnight.

RDN electrophoresis was mixed with 0.20 volumes of DNA loading tarnpon in 10 rnM Tris-HCl, pH 7.6 (or Hepes, pH 7.6), 1 mM EDTA. the loading tarnpón consisted of 30% glycerol, 10 mM Tris-HCl, pH 7.6, ethylene diamine tetraacetic acid (EDTA) 20 rnM, 0.25% brornophenol blue (w / v) and 0.25% cyanol xylene (p. / v). DNA was electrophoresed through GTG agarose (FMC Bioproducts, Rockland, ME) at 0.8-1.2% (w / v) at 5 to 10 volts per c distance between the electrodes in Tris-Borate buffer 1 x and AEDT ( TBE) (89 inM of Tris, pH 8.3, 89 rnM of borate, 2 rnM of AEDT).

FlDN electroelduction The DNA bands were extracted from the gels by cutting a strip of gel containing the band "the edges with a razor blade" with the clean edge and placing the gel strip in a dialysis tube of 6.35 nm. in diameter (BRL Life Technologies, Inc .. Gaithersburg, MD) (length varied with the size of the gel strip) which was filled with 0.5 x TBE buffer and then sealed at both ends with dialysis tube staples. The filled tube was placed in an electrophoresis gel box filled with tarnpon TBE 0.5 x and the DNA eluted out of the gel strip on the inner side of the dialysis tube by applying a voltage of 10 volts / crn distance between the electrodes during the 3 hours. At the end of this time, the tarnpon solution containing the DNA was transferred to an Eppendorf tube, concentrated in a Speed-Vac apparatus (Savant Instrunents Inc. Farrningdale, N.Y.) to reduce the volume to a minimum of 100 μl. This volume was loaded onto a pre-filled sephadex G-50 spin column (Pharmacia, Piscataway, N.J.) and centrifuged for 3 minutes at 600 G force in an IEC bench-top clinical centrifuge (International Equipment Company, Needharn HTS, MA). This allowed the extraction of the borate salt from the DNA sample. The sample was then precipitated in ethanol and resuspended in Tris 10 inM, pH 7.6 and EDTA 1 rnM.

Visualization of the FlDN Bands in the Gels For the AON to be subjected to elect roforesis in agarose gels, 0.1 volumes of a solution of ethidium bromide (EtBr) of 1 mg / inl were added to the sample. The DNA bands were visualized after the laser electrodes by placing the gel in a UV transilluminator that emitted UV light with a wavelength of 320 nm. By this procedure, a bleaching treatment is not needed. The DNA bands on the gel strips isolated for preparative purposes were electroeluted as described above. Et Bru bound to the DNA was extracted by the ethanol precipitation procedure.

Precipitation of the flDN of the Bacterial Plasmid Maxiprep procedure (for yields of 100 to 2000 μg of plasmid DNA) This DNA was prepared by the alkaline lysis procedure described in Mamatis, Molecular Cloning: A Laboratory Manual. 0.5 liters of media Luna, described as "media" with a volume of 0.1 rnl of a stationary phase culture of the appropriate E. coli strain, was inoculated. After adding? 1 L25 μg of dry ampicillin powder to the incipient media, the bacteria were coated at 7 ° C with shaking overnight. The next morning, the bacteria were agglomerated by centrifugation on a Sorvall GS3 rotor (Dupont Instrument Products, Eiornedical Division, Newton, CT) at 5,000 rprn for 10 minutes at 4 ° C. The agglomerated bacteria were suspended in 20 rnl of a solution composed of 50 mM glucose, 25 rnM Tris-HCl, pH 8.0, 10 mM EDTA and 5 rnin / ln lirin. Bacteria were left at room temperature for 10 minutes, after which time 40 ml of 0.2 M NaOH / 1% SDS was added. The lysed bacteria were allowed to stand at room temperature for another 10 minutes, after which time 20 ml of ice cold 3 M sodium acetate, pH 5.2 was added and the mixture was incubated on ice for 10 minutes. The white precipitate was agglomerated by centrifugation in the original tubes for 10 minutes at 5000 rprn in the GS3 rotor. The supernatant was collected and the volume was determined. The flDN was precipitated by the addition of an equal volume of isopropanol and was agglomerated by centrifugation in a Sorvall HSA rotor at 7500 rprn for 15 minutes at 4 ° C. The agglomerate was dissolved in 2 rnM Tns-HCl 10 rnM and added to a 5 ml polystyrene tube containing 3.10 g of CsCl. The sample was transferred to a Beckman heat-sealed tube of 3.9 rnl containing 50 μl of a 10 ng / ml solution of EtBr, filled with water, placed in a Beckman MRT 100 rotor and centrifuged at 100,000 rpm for 4 hours in a Beckman Optuna TLX Ultracentrí desktop leak (Beckrnan Instruments, PaLo Alto CA). the DNA bands of the plasinid were visible to the human and were ex- tracted with a 20G needle and a tuberculin syringe of 1 crn3 The EtBr was removed by extracting the plasmid solution with saturated 3M NaCl-isopropanol. The DNA was then precipitated in ethanol and stored at -20 ° C. Mimprep procedure (for yields of 1 to 20 μg of plasmid DNA). This ttDN was prepared by the boiling water procedure described in Mamatis Molecular Cloning: fl Laboratory Manual. 1.5 ml of stationary E. coli culture was poured into an Eppendorf 1.5-ml tube. The rest of the culture was stored at 4 ° C. The tube was centrifuged at 12,000 x G for 15 seconds in an Eppendorf microcentrifuge at room temperature. The supematant was removed by aspiration and the agglomerate was resuspended in 0.4 rn 1 of tarnpon STET: 8% sucrose, 0.5% Triton X-100 detergent, 50 mM EDTA, 50 rnM Tris-HCl, pH 8.0. 30 μl of lysozrin at 10 rng / rnl was added to the resuspended cells. The tube was immediately placed in water at 100 ° C for 1 minute. The tube was removed from the boiling water and centrifuged at 12,000 x G for 15 minutes. 200 ul of supernatant was transferred to a new tube and mixed with 200 μl of isopropanol. The sample was stored at -70 ° C for 15 minutes, time after which the precipitated DNA was recovered by centrifugation at 12,000 x G at 4 ° C for 10 minutes. The DNA agglomerate was swollen with 70% ethanol and allowed to dry * in the air. The pellet was pelleted in 50 μl of 10 ml Tps-HCl, pH 7.6, 1 mM EDTA and stored at 4 ° C.

Oral Glucose Tolerance Test The mice to be tested * were fasted for 12 hours or more; blood samples were taken from the retroorbital eye; glucose determinations were carried out using a single-use Glucornetro (Lifescan Tnc. Milpitas CA). The blood samples were taken before the administration of a glucose sample (t = 0) and 30, 75 and 120 minutes after the glucose sample. The glucose sample was composed of a dextrose solution of 200 rng / ml administered orally at a dose of 1 mg / g body weight by means of a syringe of 1 cm3 and oral dosage needle of rnupna. The bacterial plasmid DNA was prepared by the alkaline lysis procedure described in Maniatis, Molecular Cloning: A Laboratory Manual.

Transformation in E. coli Bacterial strains were composed of cells SURE obtained from Stratagene, Inc. or DH5 cells from Bethesda Research Labs, Gaithersburg, MD. Suitable cells were prepared according to the CaCl2 procedure (Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratories, 2i? 4 Ed. 1989), they were quickly frozen in liquid nitrogen and stored at -70 ° C. The transferability of these strains with isolate plasmids was usually carried out by incubating LO ul of the binding mixture with HO ul of competent cells followed by a heat stroke at 37 ° C for 2 minutes and subsequent incubation at 37 ° C. ° C for one hour after adding 0.8 rnl of half a moon. Typically, 100 nrl of this mixture was incubated in I. B plates containing 50 ug / ml of arnpicillin co or selection agent. The colonies were collected after incubation overnight at 37 ° C.

EXAMPLE 1 Construction of pRIPHflT Construction of pSV2Dog! 5 A fragment of DNA containing the polyadenylation region of glyceral dehydrogenase deh? Do-3-phosphate / transcription fi nalization (SEO LD NQ 5) was generated by * polymerase chain reaction amplification (PCR) ). The oligonucleotides 18970.244 (SEO ID NQ) and 18970.246 (SEO ID No. 10) were incubated with 3 ug of human genomic DNA (Clontech, San Carlos CA) under standard PCR conditions: primers 1 uM, DNA target 3 ug , NTPd 200 rnM, 2.5 units of Amplitaq DNA polymerase, 10 rnM TPS-HCl, pH 8.3, 50 rnM KC1, 1.5 rnM MgCl2. The amplification conditions were adjusted to 25 cycles, 1 minute at 96 ° C, 2 minutes at 58 ° C, 3 minutes at 72 ° C. The fragment of 545 p.h. The resulting sample was digested with 10 units of Spe I and 10 units of Barn Hl (37 ° C / 30 minutes) and bound to 1 μg of pSV2DogL of vector DNA digested with Spe I / Bam phosp roast to generate * the plasmid? SV2Dogll "The pSV2Dogll itself was then digested with Spe T and treated with alkaline phosphatase (0.25 units of Boehpnger * Mannheirn in 50 mM T-HCl, pH 8.5 at 55 ° C for 2 hours). A DNA fragment containing human albumin mtron I (SEO TD NQ 3) was generated by PCR amplification of 3 ug of human genomic DNA (Clontech) using the oligonucleotides 18505.022 and L85f) 5.024 under the standard conditions described previously. The fragment of 740 p.b. The resultant was digested with Spe I and ligated to? SV2 Dogll linearized with Spe I under the standard ligation conditions to generate the plasmid pSV2Dogl5. Optronization of the mtron fragment was confirmed by digestion of? SV2Dogl5 with restriction endonuclease Aat I. Digeperon was 20 ug of? SV2Dogl5 with 60 units of Bam HT and 22.5 units of Neo I to isolate a fragment of Neo I / Barn Hl by elect roelucion. Three microorganisms of this fragment were successively digested with 20 units of Apa Ll for 4 hours at 37 ° C. The resulting digestion products were separated on a 0.8% GTG agarose gel (FMC Bioproducts, Rockland, ME). A fragment of 1265 p.b. of Barn HI / Apa Ll by electroelution (stay conditions: 60 V / 60 minutes in 0.5 X TBE (Tris borate- EDT pH 8.3) with the gel strip in a dialysis bag). The eluted flDN was purified by rotary column chromatography using ream 650, followed by ethanol precipitation. This fragment contained the intr-on of human albumin fused at its terminal end '!' with the human GAPDH polyadelation region.

Construction of pSLfllO A portion of human IAPP cDNA (SEO ID NQ 2) containing only the protein encoding IAPP message region was generated by PCR. The oligonucleotides 198383,288 (SEO ID No. 12) and 1.9383.292 (SEO ID No. 15) at a concentration of 1 uM were incubated with 0.1 ng of Hsiappl of cloned IAPP cDNA (Mosselman et al.) As standard. The buffer and the conditions of the standard cycles were used, as described above. The reaction products were extracted with chloroform to remove residual mineral oil and precipitated in ethanol. The precipitated DNA was resuspended in 20 ul of restriction endonuclease tarnpón IX NEB 4 and 10 units of restriction endonuclease Apa Ll. The digestion products were subjected to electrophoresis on a 1% GTG agarose gel, visualized by staining with ethidium bromide and recovered by electroelution with a yield of 1 μl. This fragment was ligated to the 1265 p.b. fragment. Ban HI / Apa Ll from? SV2Dogl5 in a total volume of 20 ul in binding tarnpon IX BRL plus ligase units of T4 DNA. The reaction was incubated at 16 ° C for 3 days. The residual ligase activity present? 7 in this reaction after incubation it is deactivated by heat (65 ° C / 10 minutes). The binding reaction was then diluted to 100 ul in the presence of salt restriction buffer (100 mM NaCl Tris lOrnM, pH 7.6, MgCl 2 LO rnM) and digested with 5 units of Neo I for 2 hours at 37 ° C. . The resulting fragment (1533 p.b.) was isolated by electrophoresis followed by electroelution. The shuttle vector for cloning this fragment was prepared by digestion of 20 pg of plasmid generator pSuper-Luo (pSL) with 9 units of Neo I and 20 units of Bam III at 37 ° C for 3 hours in a vo Lumen of reaction of LOO ul. To this reaction, 5 ul of 1 M Tris, pH 8.0 and 22 units of alkaline phosphatase were added. The reaction was now allowed to proceed at 50 ° C for 2 hours to remove the phosphate groups at the 5 'ends and thus prevent * the recircularization of the vector only in the subsequent binding steps. The phosphatase reaction was terminated by phenol / chloroform extraction followed by ethanol precipitation. The DNA was resuspended in 10 mM Hepes, pH 7.6, AEDT (HE) 1 rnM at a concentration of 0.2 ug / ul. The IAPP / mtron fragment of alburnin na / poly A of GAPDH of 1533 p.b. was cloned into pSL cut by * Neo I / Barn Hl by binding of 1.1 ug of insert against 0.2 ug of the vector pSL in a volume of 20 ul in ligation tarnpon IX BRL and 400 units of flDN T4 ligase. The reaction was incubated at 16 ° C overnight. The next morning, the SURE cells (Stratagene, San diego, CA) of competent E. coli were incubated with Lt) ul of binding unit to üc > C for 20 minutes followed by a 2 minute heat stroke at 37 ° C. 0 .8 ml of half Luna was added to the mixture and incubated at 37 ° C for 60 minutes. 100 ul of this mixture was distributed on a Half Moon Agar plate containing 200 ug / ml of arnpicillin. Sixteen colonies resistant to the armpi-ill were collected and cultured in liquid medium; half of these cultures developed plasmids with the appropriate insert as determined by Miniprep DNA digestion (as described in Materials and Procedures). A culture (Mimprep NQ 3) was reproduced up to 0.5 liters in half Moon for a DNA Maxiprep and its plasmid was denoted pSLAlO.

Construction of pSLfIII The next step involved the insertion of rat insulin II promoter (RIP) (SEO ID NQ 2) into pSLAlO. Because the RIP contains an internal Ncol site, this procedure was carried out in two stages. The RIP DNA fragment itself was synthesized (876 bp: 700 bp of the 5 'terminal plus 176 bp of the 5' untranslated head region including the first intron) by PCR under conventional (previous) conditions using 1 uM of each one of the oligonucleotides 19383,284 (SEO ID Q 14) and 19383,292 (SEO ID NQ 15); 3 ug of rat genomic DNA (Clontech) was used as a template. Oligonucleotide 19383,292 contains a single basic alteration (A to C) of two nucleotides 5 'of the codon of inido to allow the binding of the RIP to the region encoding the IAPP by the Neo T site. The PCR product is extracted a chloroform and precipitated in ethanol L. Fl DNA was resuspended in 20 ul of restriction buffer IX NEB (New England Biolabs) together with 5 units of Neo I and incubated at 37 ° C for 2 hours. Two DNA fragments of this digestion recovered by electrophoresis in 1.0% GTG agarose, visualized by staining with ethidium bromide and electroelution of the DNA of the gel strips: a DNA of 708 p.b. with 2 Neo T ends containing the transcription start site and the 5 'front region and a 168 p.b. fragment. with end / Neo T containing the most 5 'side sequence of the rat IT insulin promoter. The plasmid pSLAlO was first cleaved with Xba I. Ten mi crograms of pSLAlO digested with 20 units of Xba I in a volume of 100 ul of restriction tarnpon highly psalm IX (100 mM NaCl, 10 mM Tris, pH 7.6, 10 mM MgCl2) for 2 hours at 37 ° C. The reaction was stopped by phenol / chloroform extraction followed by ethanol precipitation. The 5 'single stranded DNA leaving fragments completed by a polymerization step: the DNA was resuspended in 10 ul of 10 M Hepes, pH 7.6, 1 nMn AEDT and added to a final reaction volume of 100 ul containing Tris 10 rnM, pH 7.6, MgCl2 10 rnrn, NaCl 50 rnM, 5 units of Klenow enzyme and NTPd 25 uM. This reaction was allowed to proceed at room temperature overnight. The next morning, the filler reaction was heated at 65 ° C for 10 minutes to deactivate the Klenow enzyme; after this 2 ul of 5 M NaCl and 5 units of Neo I restuption endonuclease added and the reaction was allowed to proceed at 37 ° C for 2 hours. The cleavage reaction was stopped by the addition of 20 ul of gel loading tarnpon; composed of 30% glycerol, Tps-HCl 10 rnM, pH 7.6, ethylene diarrhea tetraacetic acid (EDTA) 20 rnM, brornophenol blue 0.25% (w / v) and xi cyanol 0.25% (w / v) , the resulting mixture was electrophoresed through a 0.8% GTG agarose gel. The linear form of the digested pSLA10 plasmid was recovered by electroelution followed by centrifugation through a rotating G-50 column and ethanol precipitation. The precipitated DNA was resuspended in 10 ul of Hepes 10 mil, pH 7.6, AEDT 1 rnM. Two rnicrolitres (1 ug) of this solution incubated with 0.25 ug of the fragment with end / Neo I of 174 p.b. of the rat insulin promoter, 5 units of DNA T4 NEB and tarnpon BNA IX binding (Bethesda Research Laboratories) in a final volume of 20 ul and incubated at 16 ° C overnight. The next morning 10 ul of the binding reaction was used to transform the competent E. coli SURE cells. Mimprep DNA was prepared (as described in Materials and Procedures) from the cultures of 16 colonies; 2 showed Bam Hl fragments of the correct size. One of these clones was reproduced as a Maxiprep DNA as described in Materials and Procedures for preparing more DNA from the plasmid and designated as pSLAll. The pSLAll was incubated with 7.5 units of Neo I in a volume of 200 μl in buffer IX NEB4 at 37 ° C dur-ante 2 hours followed by the addition of LO ul of Tris 1 rnM pH 8.0 and 22 units of alkaline phosphatase Boehpnger Mannheim and subsequent incubation at 50 ° C for 2 hours. It was continued with 3 sequential extractions in phenol / chloroform and precipitation with ethanol. 3.32 ug of this linearized form of pSLAll was bound at 0.5 ug of the rat insulin promoter II fragment of 760 p.b. described above in a volume of 20 μl in IX BRL binding buffer containing 20 units of T4 NEB DNA ligase; The reaction was incubated at 16 ° C overnight. Ten microliters of this ligation reaction was used to transform SURE cells into competent E. coli (as described in Mamatis, Molecular Cloning: A Labora * ory Manual). Of the 8 colonies that were produced from this transformation, a miniprep flDN preparation showed bands of the appropriate size as determined by comparison to flDN molecular weight markers supplied by Bethesda Research Laboratories, Bethesda, MD, when they were digested with restriction endonuclease. n Eco Rl. This plasmid was partially digested with Bam Hl and the insert of the transformed gene, 2.4 kb in length, was bound to Bluescript SK (-) digested with Barn Hl: generating pRIPHAT (SEO. ID No. 1) to facilitate the determination of the sequence of DNA. One of the 5 independently derived clones did not present mutations)? inadequate and was used to prepare the insert of the transformed gene for the microinjection. Preparation of RIPHAT DNA by Microjection. Three hundred grams of pRIPHAT (SEO ID No. 1) were digested with 300 units of each of the restriction endonuclease Xba I and Xho 1 in a total reaction volume of 600 ul at 37 ° C for 2 hours. The reaction was stopped by the addition of EDTA at a concentration of 21 nM, followed by the addition of a loading buffer and 2 treatments of electrophoresis on a 0.5% GTG agarose gel. The gel wrath containing the 2.4 kb DNA fragment was removed and the DNA isolated by electroelution (65V, 3 hours). The fragment of the transformed RIPHAT gene (2.4 kb (SEO, ID No. 7)) was further purified using a Schleicher and Schuell Elutip-d column and following the manufacturer's protocol. The yield was 5.6 ug of purified RIPHAT fragment. The pRIPHATl was deposited at the American Type Culture Collection on April 27, 1995 and received ATCC assignment No. 69794. This DNA was supplied to Pfiezer's transgenic equipment for microinjection. Microinjection of Mouse Embryos and Generation of Transgenic Mice. The embryo mouse embryos and the generation of transgenic mice were carried out by published procedures. Detailed procedures describing the preparation of the mice, the procedures of microinjection, the reimplantation of the injected embryos, the maintenance of the milk mothers and the recovery and maintenance of the transgenic lines can be found in Gordon, 3. and Ruddle , F., Methods m Enzyrnology, 101, 411-433 (1983). The embryos were isolated from the progeny of females Fl of the inbred crossbreed RVB / N race. The actual injection procedure was carried out as described in Uagner, T. et al., PNAS 78 6376-6380 (1981), except that the injected eggs were transferred directly to the donor females instead of the 5 day incubations. in culture tubes. The mice resulting from the reimplantation were tested for the presence of the transformed gene in their genomic DNA by coupling / Southern blotting of the DNA isolated from the tailed biopsies. Positive trials were crossed with non-transgerucos RVB / N mice of opposite sex. The descendants of these crosses were tested for the transmission of the transformed gene, obtaining the tails biopsies, isolating the genomic DNA thereof and amplifying by PCR the sequences of the transformed gene using the bases 22018- 134-L (5 '-CGAGTGGGCTATGGGTTTGT- 3 ') (SEO ID No. 16) and 22018-134-2 (5'-GTCATGTGCACCTAAAGGGGCAAGTAATTCA-3') (SEO ID No. 17) to generate a DNA product by PCR of 883 bp of diagnosis. Determination of the Transgenic Lines. The devices to assay the descendants for the presence of the transformed gene were reticulated with FVB / N mice to determine the transgene lines. The injection of 280 FVB / N embryos provided the generation of LO cases in which RTPHAT was found. Six of these cases could transmit the transformed gene to their descendants, as determined by PCR amplification of the sequences of the transformed genomic DNA gene isolated from the biopsies of the descendants' tails. Identification of Lines Expressing the Transformed Gene Total RNA was prepared from various tissues of descendants of the 6 lines (including the pancreas, liver and kidney). The RNA was isolated by oven-heating in polytron (Brinkrnann Instruments, lestbury, N.Y.) of each of the tissues in 2 rnl of TRISOLVTM denaturant (Biotecx Laboratories, Houston TX) for 60 seconds. The addition of 4 rnl of chloroform allowed the separation of the furnace in upper water phase and a lower phase of phenol (chlorine form.) The RNA was precipitated by the addition of an equal volume of isopropanol to the aqueous phase of each furnace. Isopropanol was centrifuged at 12,000 x G for 10 minutes at 4 ° C, washed once with 75% ethanol and allowed to air dry. The RNA samples were resuspended to 200 μl of 1 mM EDTA and their concentration determined by UV spectrophotoinetry. The Northern analysis was carried out as described in Mamatis, using agarose gels in 1.0% GTG forrnaldehyde, transferring to Nylon membranes and hybridizing the transfer to a DNA fragment labeled with 32 p that corresponds to the polyadeny region. lation of human GAPDH in the transformed RTPHAT gene. The RIPHAT specific RNA was detected in RHA, RHF and RHC lines, showing with the RHA and RHF lines 10 times greater pancreatic expression than with the RHC Line. The RHF line was selected for the expansion of colonies. Generation of the Hornocigotos of the RHF Line The descendants of transgenic pharyngeal horns RHF underwent matings between brother and sister to generate transgenic descendants to heinicigotes to furnaces in a 1: 2: 1 ratio. Transgemnous descendants were identified by pro-TCP analysis of tailed biopsies; the kiln-like of this group were identified by a cross-breeding trial of transgemeos with wild type FVB / N and the animals that generated non-transgemnous descendants (> 20 descendants by putative homozygote) were identified. The kiln-killers that were identified in this way crossed each other to generate a colony of RHF kilncigotes. Determination of IAPP in Plasma and Insulin Levels The animals of the same representative non-transgenic bait, herní ci góticos and homozygotes were sacrificed by asphyxia with CO2. Whole blood was drawn by puncture in the Cava vein of the asphyxiated animals with a 20G needle and a 1 ml tuberculin syringe. The whole blood was transferred to 1.0 rnl Microtamer Plasma separator tubes (Becton Dickmson, Rutherford, NJ) to prevent coagulation and centrifuged at 2000G for 2 minutes to allow the plasma to be isolated. The plasmas were rapidly frozen in dry ice and stored at -70 ° C until assay. Use of Ozone Killers with RHF Line for Drug Evaluation Normally, animals are divided into groups of 10 for each dose of a given test compound. Their plasma glucose levels are determined by punctures in the retroorbital eye one day before dosing. The dosage is carried out daily, e.g. to 0.1, 1.0 and 10 rng / kg on days 1 to 4. On the fifth day, the animals are bled to To determine its level of fasting plasma glucose in order to detect a diminishing effect of glucose.

Alternatively, the animals are subjected to an oral glucose tolerance test (OGTT) to demonstrate improved glucose tolerance. The animals undergo the - L5 blood draw to measure plasma insulin levels and demonstrate a decrease in insulin concentration.

LIST OF THE SEQUENCES (1), .- General Information: Applicant: Soeller, Ualter C. Car and, Maynard D. Kreu, David K. (n) Title of the Invention: Transgenic Animal Models for Diabetes Mellitu lipo II (m) Number of Sequences: 17 dv) Address for Correspondence: (A) Recipient: Pfizer- Inc. (B) Street: 235 East 42nd Street, 20th Floor (C) City: New York (D) State: New York (E) Country United States (F) Zip Code: 10017-5755 (v) Computer Reading Form: (A) Media Type: Flexible Disk (B) Computer: IBM PC Compatible (C) Operating System: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reléase 1..0 Version .25 (vi) Data of the Present Application: (A) Application Number: US N / A (B) Date of Submission: (C) ) Classification: (vm) Information about the Agent: (A) Name: Shey a, Robert F. (B) Registration Number: 31.304 (Or Reference Number / File: PC8153 (ix) Information on Telecommunications: ( A) Telefon o: (212) 573-1189 (B) Telefax: (212) 573-1939 (C) felex: N / D (2) Information for SEO TD \ Q i (i) Sequence Characteristics: (A) Longitude: 5356 base pairs (B) Type: Nucleic Acid (C) Chain: Double (D) Topology: Circular (n) Molecule Type: DNA (Genomic) (x) Sequence description: SEO ID NQ 1: CTG? CGCGCC CTCTAGCGGC GC? TTA? CCG CGGCGGGTGT GGTGGTT? CG CGCAGCGTGA 60 CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG 120 CC? OGTTCGC CGGCTTTCCC CßTC ?? ßCTC T ?? TCGGGG GCTCCCTTT? GGGTTCCG? T 180 TT? GTGCTTT ACGGC? CCTC GACCCC ???? AACTTß? TT? GOOTG? TßßT TCACGTAGTG 240 OGCCATCGCC CTGAT? ß? CO OTTTTTCOCC CTTTO? CGTT GOAGTCCACG TTCTTT ?? T? 300 GTOß? CTCTT GTTCC ??? CT GG ?? C ?? C ?? C TC ?? CCCT? T CTCGGTCT? T TCTTTTO? TT 360 T? T ?? CGG? T TTTGCCGATT TCGGCCTATT GGTT ?????? TG? ßCTß? TT T ?? C ????? T 420 TT ?? CGCß ?? TTTT ?? C ??? ATATTAACOC TTAC ?? TTTC CATTCOCCAT TC? ßßCTGCG 480 C? CTOTTGO G ?? ßGGCß? T CGCTGCOGOC CTCTTCGCT? TTACGCC? ßC TGGCCAAAOG S40 GGG? TGTGCT GC ?? OßCG? T T ?? OTTGGGT AACGCCAGGG TTTTCCC? ßT CACGACGTTG 600 T ???? CC? CG GCCAGTß? ßC GCGCGT ?? T? Cß? CTC? CT? TAGßßCG ?? T TGGGTACCGG 660 GCCCCCCCTC GAGGTCß? CG GTATCG? T ?? OCTTO? T? TC GAATTCCTGC AGCCCGGßßß 720 ATCCCCC? C CACTCCAACT GC? ßGCTGAß AAAOOTTTTO TAOCTGGOTA G? GT? TGT? C 780 T? G? ß? TOß AG? C? ßCTGG CTCTGAGCTC T? ?? OCAAGC? CCTCTTATC G? CAGTTGCT 840 G? CCTTC? GG TßC ??? TCT? Aß? T? CT? C? GG? ß ?? T? C? CC? TGGßßCT TC? ßCCC? ßT 900 TGACTCCCO? GTGGGCTATG OOTTTOTOß? ? OO? ßAG? T? G ?? G? G ?? ßG GACCTTTCTT 960 CTTGAATTCT GCTTTCCTTC TACCTCTß? ß GGTß? ßCTßß GGTCTC? ßCT G? ßGT? Β? 1020 C? C? GCT? TC? OTGGG ?? CT GT? ?? C ?? C AGTTCAAGOG? C ?? OTT? C T? ßCTCCCCC 1080 ?? C ?? CTGC? GCCTCCTGGG G ?? TGATOTG ß ????? TGCT C? ßCC ?? Gß? C ??? G ?? ßGC 1140 CTCACCCTCT CTß? G? C ?? T GTCCCCTGCT ßTO ?? CTGCT TCATCAGGCC ACCCAGCAGC 1200 CCCTATT ?? ß ACTCTAATTA CCCT ?? GGCT AA? TGAGCT GTTOTTGTCC AATC? GCACT 1260 TTCTßC? G? C CT? GC? CCAG? C? GTGTTT?? CTGC? GCTTCAGCCC CTCTßßCCAT 1320 CTGCTGATCC ACCCTT ?? TG GG? C ??? C? ß C ??? ßTCC? ß GGGTC? ßßßG GGGGTGCTTT 1380 GG? CT? T ??? GCT? GTGGGG ATTCAGTAAC CCCCAGCCCT ?? OTß? CC? O CT? C? ßTCGG 1440 A ?? CC? TC? G CAAOCAGGTA TOTACTCTCC? ßßOTOGGCC TGGCTTCCCC? GTC ?? ß? CT 1500 CC? GGC? TTT G? CGG? CGCT GTGGGCTCTT CTCTTACATG TACCITTTOC T?? CCTC ?? C 1560 CCTG? CT? TC TTCC? GGTC? TTGTTCC? CC ATGGGCATCC Tß ?? ßCTGC? AOTATTTCTC 1620CTOTTOCATT G ?? CC? TCT ??? šCT? C? CCATTCAAAG TCATCAGGTO 1680 C ???? GCCC? ?? TGC ?? C? TGCCAC? TGT GC ?? CGC? GC GCCTGGC ??? TTTTTTAGTT 1740 C? TTCC? GC? ? C ?? CTTTGG TGCCATTCTC TC? TCT? CC? ACGTGGGATC CAATACATAT 1800 GGCAAGAGOA ATGC? ßTAß? GOTTTT ??? ß ACAGAGCCAC TGAATT? CTT GCCCCTTTAG_1860_GTGC? COT ?? GAAATCCATT TTTCTATTCT TC ?? CTTTT? TTCT? TTTTC CCAGTAAAAT 1920 A ?? GTTTT? G T ??? CTCTGC ATCTTTAAAG AATTATTTTÃ GCATTTATTT CT ???? TGGC 1980 AT? GC? TTTT GTATTTGTGA ACTCTT? C ?? GGTTATCTT? TTAATAAAAT TCAAACATCC 2040 TAOGTAAAAA? AAAAGGTC? GAATTGTTT? GTGACTOTAA TtTTCTTTTO COC? CT ?? G 2100 AAAGTGCAAA GTAACTTACA GTß? CTß ??? CTTC? C? ß ?? TAGGGTTCAA GATTGAATTC 2160 ATAACTATCC C ??? ß? CCT? TCCATTOCAC T? TGCTTT? T TT ????? CC? C ???? CCTGT 2220 GCTGTTG? TCATAAATAG? CTTGT? TT T? T? TTT? TT TACATJCTTAG TCTGTCTTCT 2280 TGßTTGCTGT TGAT? G? C? C T ???? ß? GT? TTAGATATTA TCTAAOTTTO AATATAAGCC 2340 TATAAATATT TAATAATTTT TA ?? ATAGT? TTCTTGGTAA TTß ?? TT? TT CTTCTGTTT? 2400 A? ßßC? ß ?? G AAATAATTGA ACATCATCCT G? OTTTTTCT GT? GOAATCA C? OCCCAATA 2460 TTTTG ??? C? AATOCATAAT CTAAGTCAAA TGß ??? ß ??? T? T ???? AGT AACATTATTA 2520 CTTCTTGTTT TCTTCAGTAT TTAAC ?? TCC TTTTTTTTTCT TCCCTTGCCC AGACAAGCTT 2580 CTAGTGACCC CTGG? CC? CC AGCCCC? ßC? ? Or? GCAC ?? ß AGG ?? GAO? O? ß? CCCTC? C 2640 TGCTGGßßAß TCCCTGCCAC ACTCAGTCCC CCACCACACT G ?? TCTCCCC TCCTCACAGT 2700 TGCCATCTAG? CCCCCTC ?? GAGGGGAGGG GCCT? GGGAG CCGCACCTTO TCATOTACCA 2760 TCAATAAAGT ACCCTßTOCT CAACC? ßTTA CTTGTCCTCT CTTATTCTAO GGTCTGGGGC 2820 AGAGGGGAGG G ?? GCTGGGC TTGTGTC ?? O GTGAGACATT CTTGCTOCGO? GGCACCTGG 2880 TATGTTCTCC TCAGACTGAG GOT? OGGCCT CC ??? C? GCC TTßCTTCCTT Cß? ß ?? CC? T 2940 TTOCTTCCCC CTCAOACGTC TTß? ßTGCT? C? ßß ?? ßCTO ßC? CC? CT? C TTC? G? ß ?? C 3000 A? ßGCCTTTT CCTCTCCTCß CTCCAGTCCT? ßßCT? TCTß CTGTTßßCC? AACATGCAAG 3060 ?? GCT? TTCT OTOOGC? ßCT CCAGGGAOGC T?? C? ß? Tß?? ß? ?? OTC? ß OGCGGATCCA 3120 CT? ßTTCT? ß? ßCGßCCßCC ACCOCOOTßß? ßCTCC? ßCT TTTßTTCCCT TT? ßTßAßßß 3180 TTA? TTßCGC GCTTßßCßT? ATCATOGTCA T? ßCTßTTTC CTGTGTGAA? TTOTT? TCCß 3240 CTCACAATTC CACACAACAT? Cß? ßCCßß? AGCATAAACT GTAAAGCCTG GßCTGCCTAA 3300 TG? ßTG? GCT AACTCACATT A? TTßOTTT? COCTC? CT? CCGCTTTCCA GTCGCCAAAC 3360 CTCTCCTGCC AGCTßCATTA ATß ?? TCßßC C ?? CßCßCßß Gß? CAGGCOG TTTGCCTATT 3420 GßßCßCTCTT CCGCTTCCTC GCTC? CTß? C TCCCTGCGCT CGGTCGTTCG GCTCCGGCG? 3480 GCGGT? TC? G CTC? CTC ??? GGCGOTAATA COOTTATCC? C? ß ?? TC? ßG Gß? T ?? CßCA 3540 GG ??? ß ?? C? TGTGAßC ??? AßßCC? ßC ?? TO? ßCC? GG? ? CCCT ????? ßßCCßCCTTß 3600 CTßßCGTTTT TCCAT? ßßCT CCGCCCCCCT G? Cß? ßC? TC AC ?? A ?? TCG? CGCTCAAGT 3660 C? CA0GT6GC G ??? CCCß? C Aßß? CT? T ?? AOATACCAOO COTTTCCCCC TGO ?? GCTCC 3720 CTCGTGCßCT CTCCTßTTCC G? CCCTGCCG CTT? CCGG? T ACCTßTCCßC CTTTCTCCCT 3780 TCGßß ?? GCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT? TCTCAGTTC GGTGTAGGTC 3840 GTTCOCTCCA AGCTßGßCTC TGTOCACß ?? AGGCTGACCß CTßCGCCTT? 3900 TCCGGT ?? CT ATCCTCTTO? GTCC? CCCO GTA? ß? CACG ACTT? TCGCC? CTOßCAGC? 3960 GCCACTGGT? ACAGGATTAG CAGAGCGAOG T? TCTAGGCG GTOCTAC? ß? ßTTCTTG ?? G 4020 TGGTGGCCT? ? CT? CßßCT? C? CT? ß ?? ßß ACAGTATTTC GT? TCTGCOC TCTGCTG ?? G 4080 CC? ßTT? CCT TCGG ????? ß AßTTOOT? ßC TCTTGATCCC GC ??? C ??? C C? CCGCTCCT 4140 AGCGGTGGTT TTTTTTGTTTG C? ßC? ßC? ß ATTACGCGCA ß ??????? ßß ATCTCAACAA 4200 G? TCCTTTGA TCTTTTCTAC GßßßTCTß? C GCTC? ßTßß? ? Cß ???? CTC ACGTT ?? ßßß 4260 ATTTTßOTC? TOAGATTATC A ???? GG? TC TTCACCTAGA TCCTTTT ??? TT ????? TG? 4320 AGTTTT ??? T C ?? TCT ??? ß TATATATGAß T ??? CTTGGT CTGAC? ßTT? CCAATCCTTA 4380 ATCAGTOAGG CACCTATCTC AGCßATCTOT CTATTTCGTT CATCCATAGT TGCCTß? CTC 4440 CCCGTCGTCT AGATAACTAC GAT? Cßßß? ß GßCTT? CC? T CTGGCCCC? ß TGCTGCAATG 4S00 ATACCGCß? ß ACCC? CßCTC ACCCGCTCCA CATTTATCAC CAATAAACCA GCCAGCCGGA 4560 AGOGCCGAGC GCAG ?? GTßG TCCTGC ?? CT TTATOCGCCT CCATCCAGTC TATTAATTCT 4620 TßCCGßß ?? G CTAG? ßT ?? ß T? OTTCGCC? GTT ?? T? OTT TOCGCAACOT TOTTOCCATT 4680 GCT? CAßßC? TCßTGGTßTC ACGCTCGTCß TTTCOTATGC CTTCATTCAG CTCCGGTTCC 4740 CAACGATCAA GGCß? ßTT? C ATGATCCCCC ATGTTOTCCA ????? GCGGT T? ßCTCCTTC 4800 GßTCCTCCGA TCGTTGTC? ß A ßT ?? ßTTO GCCGCAGTOT TATCACTCAT OOTTATOGCA 4860 GC? CTßCAT? ATTCTCTTAC TOTCATGCCA TCCGT ?? G? T GCTTTTCTGT C? CTGGTG? ß 4920 TACTC ?? CC? AGTCATTCTO Aß ?? T? GTGT ATOCCGCCAC CC? CTTGCTC TTGCCCGGCC 4980 TC ?? T? COGG AT ?? T? CC? C GCC? C? TAG?? ß ?? CTTT ?? A? ßTGCTC? T C? TTCG ???? 5040 CßTTCTTCGG GßCß ???? CT CTC ?? ßß? TC TT? CCGCTOT Tß? O? TCC? ß TTCGATGTAA 5100 CCCACTCOTG C? CCC ?? CTG? TCTTC? ßC? TCTTTTTACTT TC? CC? GCGT TTCTOGGT? 5160 GCAAA ?? C? G G? A WC ???? TGCCSC ???? A? Oßß ?? T ?? GGGCß? C? OO OAAATOTTOA 5220 ATACTC? T? C TCTTCCTTTT TCAATATTAT Tß ?? ßC? TTT ATCAOßßTTA TTGTCTCATQ S280 AGCOß? TAC? TATTTG ?? TG TATTT? ß ??? AATA ?? CA ?? T? ßGßßTTCC GCGCACATTT 5340 CCCCGAAAAG TGCCAC (2) Information for the SEO ID NQ 2: (i) Sequence Characteristics: (A) Length: 076 stops of bases (B) Type: Nucleic Acid (C) Chain: Double (D) Topology: Linear (n) ) Molecule Type: DNA (Genornic) (ix) Sequence Description: SEO TD NQ2: Oß? TCCCCC? ACCACTCC ?? GTOß? ßßCTG? ß ??? ßßTßTTT TOT? ßCTßßß T? ß? ßT? TGT 60? CT? ß? ß? T Oß? ß? CAßCT GOCTCTO? ßC TCTß ?? ßC ?? GCACCTCTTA TßßAGAGTTß 120 CTOACCTTCA GGTGC ??? TC T ?? G? T? CT? CAGCAG? ATA C? CC? TOGß? CTTCA? CCCA 180? -TT? CTCCC OAGTGGGCTA TG? STTTGT??? O????? T? ß ?? ß? ß ?? Oßß? CCTTTC 240 TTCTTßß TT CTßCTTTOCT TCT? CCTCTG? ßßßTGAßCT CßßßTCTC? ß CTß? ßßTGAO 300 ß? C? C? ßCT? TC? ßTßßß ?? CTOTOA ?? C? AC? OTTCAAG GCAC ??? GTT? CT? OCTCCC 360 CC ?? C ?? CTG CAGCCTCCTG GGGAATGATG TOG ????? TO CTC? ßCC ?? ß ß? C ??? ß ?? ß 420 ßCCTC? CCCT CTCTGAGACA ATGTCCCCTO CTOTß ?? CTG OTTCATCAßß CC? CCC? ßß? 480 OCCCCTATTA AOACTCTAAT T? CCCT ?? GG CT ?? ßT? ß? G GTGTTGTTGT CCAATGAGCA 540 CTTTCTßC? G ACCT? ßC? CC AGGCAAGTCT TTßG ??? CTG C? GCTTC? ßC CCCTCTßßCC 600 ATCTGCTGAT CCACCCTTAA Tßßß? C ??? C? ßC ??? ßTCC AßßßßTCTC? ßßßßßßßßßTßCT 660 TTCGACTATA AAGCTAGTOG GOATTC? GT? ? CCCCC? ßCC CTAAGTGACC AGCTACAGTC 720 Gß ??? CC? TC AGCAACC? ßß T? TGT? CTCT CC? ßßßTCGß CCTßßCTTCC CCAGTC ?? G? 780 CTCCAOOGAT TTCAOOCACß CTßTßOßCTC TTCTCTTAC? TGTACCTTTT GCT? ßCCTC? 840 ACCCTGACTA TCTTCC? GCT CATTCTTCCA CCATßß 876 (2) Information for SEO ID NQ3 (i) Sequence Characteristics: (A) Length: 278 base pairs (B) Type: Nucleic Acid (C) Chain: Double (D) Topology : Linear (11) Molecule Type: cDNA (? X) Sequence description: SEO TD HQ 3: CC? TCGCC? T CCTGAAGCTG CAAGTATTTC TCATTOTGCT CTCTGTTGCA TTGAACCATC 60 Tß ??? šCT? C ACCCATTGAA AGTCATCAGG TGß ???? GCß G ??? TGC ?? C ACTGCCACAT 120 GTßC ?? CGC? GCGCCTGGC? AATTTTTT? C TTC? TTCC? G C ?? C ?? CTTT GGTGCC? TTC 180 TCTCATCTAC C ?? CGTGGG? TCCAATACAT ATGGCAAGAG G ?? TßCACTA G? GGTTTTA? 240 Aß? ß? ß? ßCC ACTG? TT? C TTGCCCCTTT AGßTßCAC 278 (2) Information for SEO ID NQ 4 (i) Sequence Characteristics: (A) Length: 720 base pair (s) Type: Nucleic Acid (C) Chain: Double (D) Topology: Linear (n) ) Molecule Type: DNA (Genomic) dx) Sequence description: SEO TD NQ 4: GTGC? COTAA GAAATCCATT TTTCTATTOT TCAACTTTTA TTCTATTTTC CC? ßT ???? T 60 ??? ßTTTT? ß T ??? CTCTßc ATCTGTAAAO AATTATTTTO GCATTTAGTT CTAAAATGOC 120 ATAßC? TTTT GTATTTGTGA AßTCTTACAA GOTT? TCTT? TT ?? T ???? T TC ??? C? TCC 180 TAOGT ???? A ????? ßßTC? Or ?? TTOTTT? OTO? CTCTAA TTITCTTTl'U CßCACTAACG 240 A ?? ßTGCAAA GTAACTTAGA GTOACTOAAA CTTCACACAA TAOGCTTO ?? G? TTCA? TTC 300 ATAACTATCC CAAAGACCTA TCCATTOCAC T? TOCTTTAT TT ????? CCA CA ??? CCTCT 360 GCTßTTOATC TCATA ?? T? ß AACTTGTATT TATATTTATT TACATTTT? O TCTGTCTTCT 420 TßßTTßCTßT Tß? T? ß? C? C T ???? ß? ßT? TTAGAT? TT? TCT ?? GTTTO ?? T? T ?? ßGC 480 T? TAAATATT TAATAATTTT T ???? T? OT? TTCTTOGTAA TTO? ATTATT CTTCTOTTTA 540 A? ßßC? ß ?? ß A ?? T ?? TTß? ACATCATCCT G? ßTTTTTCT GT? G? TC? G? ßCCC? T? 600 TTTTO? AACA AATGCATAAT CTAAGTC? AA TOO ??? ß ??? T? T ????? OT ?? C? TT? TT? 660 CTTCTTOTTT TCTTCAGTAT TT ?? C ?? TCC TTTTTTTTTCT TCCCTTGCCC A? C ?? GCTT 720 (2) Information for SEO ID NQ 5 (1) Sequence Characteristics: (A) Length: 545 base pairs (B) Type: Nucleic Acid (C) Chain: Double (D) Topology: Linear (ii) Type of Molecule: DNA (Genomic) (ix) Description of the Sequence: SEO ID NQ 5: ?? ßCTTCTAß TGACCCCTGG ACC? CC? GCC CCAGC ?? CAC C? C ?? ß? GC? ? ß? ß? ß? G? C 60 CCTC? CTGCT OßGß? ßTCCC TßCC? CACTC AßTCCCCC? C CACACT? T CTCCCCTCCT 120 C? C? ßTTOCC ATCTACACCC CCTß? ß? ßß ßß? ßßßßCCT? OGC? GCCGC? CCTTCTCAT 180 OT? CC? TC ?? T ??? CT? CCC TGTOCTC ?? C C? OTT? CTTO TCCTCTCTT? TTCT? GGGTC 240 TGGGGC? G? ß GGC? GG? ?? G CTGOGCTTGT OTC? AßßTGA GACATTCTTG CTßßßß? ßßß 300 ACCTßßT? Tß TTCTCCTC? O ACTOAOGGTA CGGCCTCCA? ? CAGCCTTGC TTGCTTCCAG 360 A? CC? TTTGC TTCCCGCTC? G? CGTCTTO? OTOCT? CAGG AAGCTGGCAC C? CT? CTTC? 420 G? G ?? C ?? ßß CCTTTTCCTC TCCTCOCTCC AGTCCTAOGC T? TCTGCTOT TGGCC ??? C? 480 TOOAAGAAGC TATTCTCTOC GCAGCTCC? ß Gß? ßßCTG? C? ßßTßß? ßß? ? GTC? GGGCC 540 GATCC 545 (2) Information for SEO ID NQ 6 (i) Sequence Characteristics: (A) Length: 2961 base pairs (B) Type: Nucleic Acid (C) Chain: Double (D) Topology: Circular (n) Type of Molecule: DNA (Genomic) dx) Sequence description: SEO ID NQ 6: CTGT? GCGOC GCATT? ACCG CGGCGGCTGT GGTCGTTACC CGCAGCCTCA 60 CCßCT? C? CT T? CC?? C? CC? CT?? C? CCC? CTCCTTTC? C TTTCTT? CTC TCCTTTCTCO 120 CC? CGTTCGC CßßCTTTCCC CGTC ?? OCTC T ??? TCßßß? GCTCCCTTT? GGCTTCCGAT 180 TT? ßTGCTTT ACGßCACCTC GACCCC ???? ?? CTTG? TT? Oß? Tß? TOOT TC? CßT? ßTß 240 GGCC? TCßCC CTGAT? ß? Cß GTTTTTCGCC CTTTOACOTT ßß? ßTCC? Cß TTCTTT ?? T? 300 GTßß? CTCTT GTTCC ??? CT OOAACAACAC TC ?? CCCTAT CTCO? TCTAT TCTTTTOATT 360 TAT ?? ßßß? T TTTßCCßATT TCßßCCT? TT? ßTT ?????? Tß? ßCTGATT T ?? C ????? T 420 TT ?? OGCCAA TTTT ?? C ??? ATATT ?? COC TT? C ?? TTTC CATTC? CC? T TCAGGCTGCG 480 CAACTOTTOC G? ßßßCß? T CßOTßCTOßC CTCTTCOCT? TT? CßCC? ßC TßßCß ??? ßß 540 Gßß? TGTGCT GC ?? ßßCß? T TAAOTTOGGT ?? CTCC? ßßß TTTTCCC? ßT C? CGACGTTG 600 T ???? CG? CG GCC? ßTG? GC GCCCOTAATA C?? CTC? CT? T? OGGCG? AT TGGGT? CCGC 660 GCCCCCCCTC CAGGTCGACG OTATCCATAA GCTTGATATC GAATTCCTGC? ßCCCßßßßßß 720 ATCC? CTACT TCT? ß? ßCßß COOCC? CCßC GßTßß? ßCTC CAGCTTTTOT TCCCTTTAGT 780 GAGOGTTAAT TGCßCßCTTß GCGTAATCAT GGTCAT? GCT CTTTCCTGTG Tß? AATTGTT 840 ATCCßCTC? C A? TTCC? C? C A? CAT? C?? ß COO? ßC? T AA? ßTGT ?? A ßCCTßßßOTß 900 CCT? TCAGT G? GCT? CTC ACATTA? TTO CCTTCCGCTC? CTGCCCOCT TTCCAGTCGG 960 ß ??? CCTGTC GTGCCAGCTG CATT? ATCAA TCß? CC? C? CGC? B???? GGCGGTTTOC 1020 GTATTOGGCC CTCTTCCßCT TCCTCßCTC? CTOACTOGCT GCßCTCßßTC GTTCßßCTßC 1080 GGCß? ßCßßT? TC? ßCTC? C TC ??? OOCßß T ?? T? CßßTT ATCC? C? ß ?? TC? ßßßß? T? 1140? CGC? ßß ??? GAACATOTOA GCA? AAOßCC? ßC ???? ßOC CAGGAACCGT AAAAAGGCCG 1200 CGTTßCTGGC GTTTTTCC? T AGGCTCCGCC CCCCTO? Cß? GCATCACAAA AATCCACOCT 1260 CAAGTCAGAC GTGGCO ??? C CCC? CAGGAC T? TAA? ßATA CCAGGCOTTT CCCCCTßß ?? 1320 GCTCCCTCGT GCGCTCTCCT CTTCCG? CCC T? CCGCTT? C CGGATACCTG TCCCCCTTTC 1380 TCCCTTCGGG ?? GCGTßßCC CTTTCTC? T? GCTCACGCTG TAGGTATCTC AGTTCGGTCT 1440 AßßTCßTTCß CTCC ?? ßCTG GGCTGTGTGC ACßAACCCCC CGTTCAGCCC ß? CCGCTGCG 1500 CCTTATCCGG TAACTATCOT CTTG? ßTCC? ACCCGOTAAG? C? Cß? CTT? TCGCCACTGG 1560 C? ßC? ßCC? C TGGT ?? C? ßß ATTAGCAGAG CCAGCTATOT AGGCO? TOCT? C? ß? ßTTCT 1620 TOAAOTOOTC GCCT ?? CT? C G? CT? C? CT? O? ßC? C? GT? TTTGGT? TC TGCGCTCTGC 1680 TO ?? ßCC? ßT T? CCTTCßß? AA ?? ßAGTTß ßT? ßCTCTTG? TCCßßC ??? C ??? CC? CCG 1740 CTGGT? GCCG TOGTTTTTTT GTTTGC ?? ßC AOCAGATT? C GCGC? ß ???? AAAGGATCTC 1800 A? ß ?? ß? TCC TTT? TCTTT TCT? CßßßßT CTOACGCTCA GTßß ?? Oß ?? ?? CTC? CGTT 1860 A? ßGß? TTTT GGTCATC? ß? TTATCAA ??? GGATCTTCAC CT? GATCCTT TT? AATTAAA 1920 A? Tß ?? ßTTT TAAATCAATC TAAAGTATAT ATOAGT ?? AC TTOCTCTC? C AGTTACCAAT 1980 GCTTA? TC? ß TGAGGC? CCT ATCTC? ßCß? TCTGTCT? TT TCGTTC? TCC AT? ßTT? CCT 2040 G? CTCCCCCT CßTGT? ß? T? ACTACß? T? C ßßß? ßßßCTT? CCATCTCGC CCC? GTGCTG 2100 CAATCATACC CCG? ß? CCC? CGCTC? CCGG CTCC? C? TTT? TCAGCAATA AACCAGCCAO 2160 CCCCAAGGCC CGAGCGC? ß? AGTGGTCCTO CAACTTTATC CGCCTCCATC C? GTCTATTA 2220 ATTOTTßCCO ßß? AOCT? ß? GT ?? ßT? ßTT CßCC? ßTT ?? T? ßTTTGCCC ?? CGTTOTTG 2280 CC? TTGCT? C AGCCATCGT? GTGTC? C? CT CGTCOTTTG? T? TOGCTTC? TTC? GCTCCß 2340 GTTCCC ?? Cß ATC ?? ßOCO? GTTACATGAT CCCCCATGTT OTßCA ????? GCOOTT? OCT 2400 CCTTCGGTCC TCCß? TCßTT GTC? ß ?? ßT? AGTTGGCCCC? OTGTT? TC? CTCATGGTTA 2460 TGGC? GC? CT GCATAATTCT CTT? CTGTCA TCCCATCCGT ?? O? TOCTTT TCTCTCACTG 2520 GTGAGTACTC A? CC ?? ßTC? TTCTß? ß ?? T AOTOTATGCO ßCOACCGAGT TOCTCTTCCC 2580 COßOOTCAAT ACGGßAT ?? T ACCßCTCC? C ATAGCAGAAC TTTAA? AOTG CTCATCATTO 2640 G? AACGTTC TTCGßßßCO? AAACTCTCAA GO? TCTT? CC OCTßTTG? ß? TCC? GTTCC? 2700 TGTAACCCAC TCOTßC? CCC AACTGATCTT CAOCATCTTT T? CTTTCACC? CCGTTTCTC 2760 4b GßTß? ßC ??? AACAGOAAOG CA ??? TOCCß C ?????? ß? ß ?? T ?? ßßßCß? C? Oßß ??? T 2820 OTTOAATACT C? T? CTCTTC CTTTTTC ?? T? TT? TT? ß C? TTT? TC? ß ßßTT? TTßTC 2880 TC? TO? ßCO? T? C? T? TTT? TGT? TTT A? ????? T ?? ? C ??? T? ßßß GTTCCOCOCA 2940 CATTTCCCOO A ??? ßTßCC? C 2961 (2) Information par-a l SEO ID NQ 7 (i) Sequence Characteristics: (A) Length: 2395 base pairs (B) Type: Nucleic Acid (C) Chain: Double (D) Topology: Linear di) Molecule Type: DNA (Genornic) (ix) Sequence Description: SEO TD NQ 7: OATCCCCC ?? CC? CTCC? O TOOA? CTOA GA? ßOTTTT GTAOCTOOOT AßAOTATßT? 60 CT? JMM? T? G? ß? CACCT? ßCTCT?? CTCT? AßßC? ß CACCTCTTAT OßAβ? OTTGC 120 T? CCTTC? ß CTOCAAATCT A? ß? T? CTAC AGOA? T? C? CCATOOT? C TTCAOCCCAG 180 T OACTCCOG AGTOGOCTAT OOOTTTTO AAQßAQAGAT Aß ?? ß? ß ?? ß GßACCTTTCT 240 TCTTOAATTC TGCTTTCCTT CTACCTCTß? Oß? Tß? ßCTO OOOTCTCAOC TOAßßTGAßß 300 ACACACCTAT C? ßTOGßAAC TOTO ?? AC ?? C? ßTTC ?? ßß G? C ??? ßTTA CTAOGTCCCC 360 C ?? C ?? CTOC? ßCCTCCTOO Oß? ATß? TßT Oß ????? TOC TC? OCC ?? ßß AC ??? ßA? Oß 420 CCTCACCCTC TCTG? ß? C ?? TßTCCCCTOC TOTGAACTOO TTCATCAOßC CACCCAOGAO 480 CCCCTATTAA GACTCTAATT ACCCTAAOGC T? OTAO? ßß TQTTßTTGTC CAATG? ßC? C 540 TTTCTOCAGA CCTAOCACCA QOC? AOTOTT TOO ??? CTOC AGCTTC? OCC CCTCTGOCCA 600 TCTOCTOATC CACCCTTAAT Gß? CAAAC? OCAAAOTCCA OGOGTCAOCG GßßßßTOCTT 660 TGGACTAT ?? AGCTAßTOOa OATTCAOTAA OCOCC? OCCC T ?? ßTß? CCA OCTACACTCG 720 C ??? CCATC? GCAAGCAGCT ATGT CTCTC CAOßOTOOOC CTOOCTTCCC CAOTCAAGAC 780 TCCAOOGATT TOAOOßAOOC TOTOOCCTCT TCTCTTACAT ßTACCTTTTG CTAßCCTC ?? 840 CCCTOACTAT CTTCCACGTC ATTßTTCCAC CATOOßCATC CTCAAOCTGC AAGTATTTCT 900 CATTGTOCTC TCTOTTOCAT TOAACCATCT G ?? AOCSAC? CCC? TTOAAA OTCATCACCT 960 GOA ??? OCOO A? ATGC ?? C? CTOCCACATO TOCAAOGCAO OOCCTGGC ?? ATTTTTTAOT 1020 TCATTCCACC? AC ?? CTTTG QTCCCATTCT CTCATCTACC AAOOTOOGAT CCAAT? CATA 1080 TOßCA? OAßß AATOCAOTAG AGGTTTTAAA O? OAßAGCC? CTGAATTACT TOCCCCTTTA 1140 GßTGCAOGTA Aß ??? TCC? T TTTTCTATTO TTCAACTTTT ATTCTATTTT CCCACTAAAA 1200 T ??? ßTTTT? CTAAACTCTC CATCTTTA ?? G ?? TTATTTT GGC? TTTATT TCTA ??? TGG 1260 CATAGCATTT TOTATTTOTO AAGTCTTACA AOGTTATCTT ATT? ATAAAA TTCAAACATC 1320 CTAßOTAAAA AAAAA? ßßTC AOAATTOTTT AOTß? CTßTA ATTTTCTTTT GOßCACTAAG 1380 G ??? ßTGCA? ATATAACTTAO AOTOACTCAA ACTTC? CAß? ATAOGOTTGA AGATTCAATT 1440 CATAACTATC CCAA? ßACCT ATCCATTGC? CTATOCTTTA TTTAA ??? CC? C ???? CCTG 1500 TGCTGTIGAT CTCATAAATA GAACTTGTAT TTAT? TTTAT TTACATTTTA OTCTOTCTTC 1560 TTOOTTOCTO TTOATAß? CA CTA? AO? ßT ATTAßATATT ATCTAAOTTT G? TATAAGß 1620 CTATA? TAT GTAATAATTT TTA? ATAOT ATTCTTOOTA ATT? TTAT TCTTCTCTTT 1680 AAAOOCAO? A OAAATAATTO AACATCATCC TßAOTTTTTC TGTAOGAATC Aß? ßCCCAAT 1740 ATTTTGA? AC AAATOCATAA TCT ?? ßTC ?? ? TOO ??? ßA? AT? TAA ?? Aß TAACATTATT 1800 ACTTCTTGTT TTCTTCAGTA TTTA? C ?? TC CWTWTGTC TTCCCTTGCC C? ß? C ?? šCT 1860 TCTXOTGACC CCTßß? CC? C CAGCCCC? ßC ?? ß? ßC? C ?? ß? ßß ?? ß? ß? ß? ß? CCCTC? 1920 CTGCTGOOOA GTCCCTGCC? C? CTC? OTCC CCC? CC? C? C T? TCTCCC CTCCTCACAO 1980 TTGCCATGTA ß? CCCCCTß? ? ß? ßßßß? Oß OOCCTAGCC? GCCßC? CCTT OTCATCT? CC 2040 ATC ?? T ??? ß TACCCTOTOC TC ?? CC? ßTT ACTTOTCCTO TCTT? TTCT? OOGTCTßOßß 2100 C? B? B? OOTß? G? C? T TCTTGCTGGG ßAGGOACCTß 2160 GT? TßTTCTC CTC? ß? CTß? GßßT? ßGGCC TCC ??? CAGC CTTGCTTGCT TCß? ß ?? CC? 2220 TTTßCTTCCC OCTCAß? COT CTTGAOTOCT ACAOOAAGCT OGCACCACTA CTTC? ß? ß ?? 2280 C? AO? CCTTT TCCTCTCCTC ßCTCC? ßTCC T? ßßCTATCT GCTGTTGGCC ??? C? Tßß ?? 2340 G? GCT? TTC TCTOOGC? ßC TCCAOGCAGG CTC? C? CGTG G? GG ?? GTCA GGGCG 2395 (2) Information for the SEO ID NQ 8 (i) Sequence Characteristics: (A) Length: 34 base pairs (B) Type: Nucleic Acid (C) Chain: Simple (D) Topology: Linear (11) Molecule Type: DNA (Genornic) (x) Sequence description: SEO ID N 8: CCCTCTAGAA GCTTGTCTGG GCAAGGGAAG AAAA (2) Information for SEO TD NQ 9 (i) Sequence Characteristics: (A) Length: 38 base pairs (B) Type: Nucleic Acid (C) Chain: Simple (D) Topology: Linear in) Type of Molecule: DNA (Genomic) (ix) Description of the Sequence: SEO ID NQ qr.

GGGAAGCTTC TAGACTTTCG TCGAGGTGCA CGTAAGAA (2) Information for the SEO ID NQ 10: (i) Characteristics of the Sequence: (A) Length: 37 base pairs (B) Type: Nucleic Acid (C) Chain: Simple (D) Topology: Linear di) Molecule type: DNA (Genornic) (ix) Sequence description: SEO ID NQ 10; CAAACCGGAT CCGCCCTGAC TTCCTCCACC TGTCAGC (2) Information for SEQ ID NQ 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) Type: Nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) Molecule Type: DNA (Genomic) dx) Sequence description: SEO TD NQ 11: CACAACACTA GTGACCCCTG GACCACCAGC CCCAGC (2) Information for SEO ID NQ 12: (i) Sequence Characteristics: (A) Longi: 31 base pairs (B) Type: Aci or Nucleic (C) Chain: Simple (D) Topology: Linear ( n) Ti or Molecule: DNA (Genoinic) dx) Sequence description: SEO ID NQ 12: GTCATGTGCA CCTAAAGGGG CAAGTAATTC A (2) Information for SEO TD N9 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) Type: Nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) Molecule Type: DNA (Genornic) (x) Sequence Description: SEO ED NQ 13 GAAGCCATGG GCATCCTGAA GCTGCAAGTA (2) Information for SEO TD NQ 14: (i) Caractepsti cas of the Sequence: (A) Length: 33 base pairs (B) Type: Nucleic Acid (O String: Simple (D) Topology: Linear (n) Molecule Type: DNA (Genornic) dx) Sequence description: SEO ID NQ 14: GTCAGGAATT CGGATCCCCC AACCACTCCA AGT (2) Information for SEO ID NQ 15: (i) Sequence Characteristics: (A) Length: 34 base pairs (B) Type: Nucleic Acid (C) Chain: Single (D) ) Topology: Linear di) Type of MolecuLa: DNA (Genornico) dx) Sequence description: SEO TD NQ 15 ACAGGGCCAT GGTGGAACAA TGACCTGGAA GATA (2) Information for SEO TD NQ 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) Type: Nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear di) Type of Molecule: DNA (Genornico) dx) Sequence Description: SEO ID NQ 16 CGAGTGGGCT ATGGGTTTGT (2) Information for the SEO ID NQ 17: (i) Characteristics of the Sequence; (A) Length: 31 base pairs (B) Type: Nucleic Acid (C) Chain: Single (D) Topology: Line L (11) Molecule Type: DNA (Genornic) (ix) Sequence Description: SEO LD NQ 7 GTCATGTGCA CCTAAAGGGG CAAGTAATTC A

Claims (18)

hl NOVELTY OF THE INVENTION CLAIMS

1. - DNA recornbinant e comprising a non-promoter of EAPP, a sequence encoding human IAPP or one of the active fragments thereof linked functionally to a sequence encoding human albumin intron 1, a sequence encoding the termination of human GAPDH and a sequence encoding human GAPDH polyadelation , said DNA providing the expression of a diabetic phenotype when incorporated into a suitable host.

2. Recombining DNA according to claim 1, wherein the non-IAPP promoter is selected from the group consisting of promoters for the rat insulin I genes, r-ata insulin II, human insulin, mouse IAPP, glucocmase specific for rat B cells, transporter _? of glucose, human transferase transferase from human tyrosma, human albumin, mouse albumin, rat liver specific glucocmase and mouse rnetalothionein.

3. Recombinant DNA according to claim 2, wherein said promoter is the rat insulin II promoter.

4. Recombinant DNA according to claim 1, wherein said sequence encoding the human IAPP or one of its active fragments has the characteristics of a genoinic DNA ..

5.- Recombinant DNA according to claim 5, in the that said sequence encoding the human IAPP or one of its active fragments has the cDNA characteristics.

6. - Recombinant DNA according to claim 1, wherein said sequence is that of SEO ID No. 1.

7. - Recombinant DNA according to claim 5, wherein said cDNA sequence is that of SEO ED NQ 3.

8. Recombinant DNA according to La rei vi ntion 1, in which the sequence encoding the human IAPP is replaced with a sequence encoding the mouse IAPP or one of its active fragments.

9. Recombinant DNA according to claim 8, wherein said DNA is cDNA.

10. A vector comprising recombinant DNA according to claim 1.

11. A vector comprising recombinant DNA according to claim 4.

12. A vector comprising recornbinante DNA according to claim 5.

13. - A line of eucanotic cells comprising recornbinant DNA according to claim 1.

14. A line of eukaryotic cells comprising recornbinante DNA according to claim 4.

15. A line of eucapotic cells comprising Recombinant DNA according to claim 5.

16. - A cell line according to claim 13, wherein the cells are selected from the group consisting of Rat Insulin Cells (RIT), Rat Irisuloma Cells (HIT) and Msulinorin Cells. ra ton ß-T03.

17. A cell line according to claim 14, wherein the cells are selected from the group consisting of insulin rat iran rat (RIT) cells, rat msulinorna cells (HIT) and mouse insulussin cells. TC3.

18. A cell line according to claim 13, wherein the cells are selected from the group consisting of rat insulinoma cells (RIT), rat insulin cells (HIT) and mouse n ß msulinorna cells. -TC3. L9. ~ A transgenic non-human mammal comprising reclosing DNA according to claim 1. 20.- A transgenic non-human mammal comprising recornbinating DNA according to claim 4. 21.- A transgeruco non-human mammal comprising RECOMBINANT DNA according to claim 5. 22. A transgenic non-human mammal according to claim 19, wherein said animal is a mouse. 23. A transgenic non-human mammal according to claim 20, wherein said animal is a mouse. 24.- A non-human transgenic mammal according to Ü- + Claim 2L, wherein said animal is a mouse. 25. The use of an inhibitor * of the overexpression of a gemco product of IAPP in the preparation of compositions for treating an animal having a disease characterized by * an overexpression of a lAPP gene product. 26.- A method to 'analyze * the effect of a treatment that includes analyzing, in a sample returned from the animal, the effect of said treatment on the product of overexpressing a gene encoding TAPP. 27. The use of claim 25, wherein the treated animal is a mammal. 28. The use of claim 25, wherein said animal is a human being. 29. A method to determine if a patient is at risk of suffering from diabetes or obesity, which comprises examining a sample of said patient with respect to the overexpression of a TAPP gene product, said overexpression being a risk indicator. 30. A method for analyzing an animal model with respect to a disorder or disease state comprising determining in a sample thereof whether an IAPP gene in said animal model is expressed at a predetermined level. 31. The method of claim 30, wherein said level is greater than the level in a wild type or normal animal.