MXPA98004115A - Purification of more elevated order transcription compositions from transgenic animals no huma - Google Patents

Abstract

The invention relates to transgenes comprising DNA encoding the TATA box binding protein (TBP) tagged with epitope, to the production of transgenic animals expressing epitope-tagged TBP and to the use thereof for affinity purification of Transcription factors and (novel) transcription complexes from various types of eukaryotic cells and tissues

Description

PURIFICATION OF HIGHER ORDER TRANSCRIPTION COMPLEXES FROM NON-HUMAN TRANSGENIC ANIMALS. The invention relates to a transgene comprising DNA encoding a TATA box binding protein (TBP), epitopically labeled, to the production of transgenic animals expressing an epitopically-labeled TBP, and to the use thereof for the purification, by affinity, of (new) transcription factors and transcription complexes from a variety of tissues and eukaryotic cell types. For the regulation of the eukaryotic transcription process, the gradual formation and activity of the pre-initiation complex are fundamental. This is a large complex of multiple subunits that is necessary for the correct placement and initiation of the RNA polymerase II enzyme at the site of the start of transcription. In recent years, many of the general transcription factors (GTFs) of this complex have been characterized from eukaryotic nuclei, including TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH [Zawel and Reinberg (1995), Ann. Rev, Biochem. 64: 533-561; Serizawa et al. (1994) in Transcription: Mechanis s and Regulation (Transcript: Mechanisms and Regulation), 45--66, Raven Press]. In promoters that contain TATA, the TFIID has a specific affinity for the sequence of the TATA box. It is through the recognition of this sequence, that TFIID is the first element to bind to the promoter in basal transcription by RNA polymerase II, thus forming the nucleus for complex formation [B. Lewin (1990), Cell 61: 1161-1164]. The other GTFs come together in a defined and gradual way, which results in a complete pre-initiation complex. Subsequently, this large complex recruits and correctly places RNA polymerase II (Pol II) at the site of transcription start to initiate basal transcription. It has become clear that TFIID, itself a complex of multiple subunits, plays a key role in the regulation of "activated transcription", vaguely defined as high levels of mRNA production in the presence of transcription activators. Such activators may be determinants present in nature that bind to enhancers, such as the USF binding box E [Sawadogo and Roeder (1985), Cell 43: 165-175; Kirschbaum et al. (1992), Mol. Cell. Biol. 12: 5.094-5.100], or viral factors such as VP16 [Stringer et al. (1990), Nature 345: 783-786]. The TFIID is composed of the TATA-binding protein (TBP) and various factors associated with TBP (TAFjj ?, from the English, TBP-Associated Factors) (in the application, the "TAFXIs" include any kind of transcription factor, activator of transcription and transcription inhibitor). Up to 20 different TAFI3-s have been characterized to date, and TFIID complexes containing different combinations of TAFI] s have been observed: [Zawel and Reinberg (1995), Ann. Rev. Biochem. 64: 533-561; R. Hori and M. Carey (1994), Curr. Opinion Gen. Dev. 4: 236-244]. In addition, different combinations of TAFj-jS confer distinct properties to the TFIID complex. In Drosophila, for example, the Hunchback (HB) and bicoid (BCD) proteins, which are involved in the formation of development patterns, absolutely depend on the presence of TAFu60, TAFjjllO and TAFjj-250 in the TFIID complex [F. Sauer et al. (1995), Science 270, 1783-1788], These TFIID components act as co-activators for the HB and BCD proteins linked to the upstream enhancer. It has been shown that the neurogenic factor NTF-1 requires a minimum complex of TBP, TAF1: r150 (to which it binds) and TAF ?, 250 for activated transcription, while SPI requires the additional factor TAFI1; its activation [J.-L. Chen et al. (1994), Cell 79: 93-105]. Another study has made it possible to identify TAFjj28, whose presence is necessary for the activation of transcription by the nuclear receptors of estrogen and vitamin D3 [M. May et al. (1996), EMBO 15: 3.093-3.104]. Is it likely that TAF?; [; s act as transcription adapters, transmitting regulatory information from activator / repressor factors to the cen- tral initiation complex through protein-protein interactions. As regulated transcription is now seen, the expression of individual genes and / or small groups of closely related loci is controlled by definable sets of subunits of the transcription complex. Although some of the factors are ubiquitous and present in most transcription processes, eg, GTFs, increasing numbers of regulated transcription elements specific for genes and cells are now being described. There are several proven methods that have been used to identify transcription factors. The first strategies, which discovered the RNA Pol II and the seven GTFs (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH and TFIIJ), involved mainly the fractionation of nuclear preparations from cell lines in a column [Zawel and Rein- berg (1995); Serizawa et al. (1994); R. G. Roeder (1996), TIBS 21: 327-335]. These fractions produced semi-purified proteins with varying levels of transcription capacity. It seemed that the major, part of these fractions were absolutely necessary for basal transcription. At that time it was learned that the TFIID fraction was responsible for the recognition of the TATA box. However, attempts to isolate a single protein with ability to bind to TATA were unsuccessful. A break occurred when it was shown that a single yeast component was able to replace the TFIID in reconstituted basal transcription assays, which led to the isolation and cloning of a 27 kDa TATA-binding protein (TBP) [S. Buratowski et al. (1998), Nature 334: 37-42; B. Cavallini et al. (1988), Proc. Nat. Acad. Sci. 86: 9803-9.809], The use of degenerate primers led to the subsequent identification of genes for the TBP subunit of human TFI1D [C, C. Kao et al. (1990), Science 248: 1646-1649; A. Hoffmann et al. (1990), Nature 346: 387-390; M. G. Peterson et al. (1990), t-Science 248: 1625-1630], from Drosophila [T. Hoey et al. (1990), Cell 61: 1179-1166; M. L. Munich et al. (1,990), Proc. Nat. Acad. Sci. 87: 9.148-9.152] and mouse [T. Tamura et al. (1991), Nuc. Acids Res. 19: 3861-3.865]. The availability of the cDNA for TBP of different species made possible a great variety of investigations, including the overexpression of these proteins in a cell culture. Hela cell lines were produced constitutively expressing a TBP protein with a FLAG marker or the epitope marker of the hemagglutinin (HA) of the influenza virus, added to its amino terminus [Q. Zhou et al. (1993), Genes & Development 7, 180-187; Chiang et al. (1993), EMBO 12: 2,749-2,762]. The FLAG marker is an epitope consisting of a synthetic sequence of eight amino acids. The HA marker is a natural epitope with the amino acid sequence SEQ ID No. 1: "MGYPYDVPDYAV" (one letter code). In addition, a shorter peptide from the natural HA (10 amino acids of the haemagglutinin (HA) of influenza virus) has been used for the expression of a fusion protein containing TBP in a Drosophila cell line [Colgan et al. Manley (1992), Genes Dev. 6: 304-331; Trivrdi et al. (1996), Mol. Cell, Biol. 16: 6,909--6,916]. In bacteria, TBP proteins with two epitopes aggregated as markers have been expressed at their amino terminus, the FLAG epitope and the HA epitope [Chiang et al. (1993), EMBO 12: 2,749-2,762]. FLP-tagged TBP proteins have also been expressed under the control of an inducible promoter [Wu et al. (1996), BioTechniques 21: 718-725]. However, monoclonal antibodies against the epitope / epitopes were used to purify complexes associated with TBP from nuclear extracts, thereby co-complexing factors of the TFIID complex associated with TBP (TAFSIs) [Zhou et al. (1993), Genes Dev. 7: 180-187].

Research continues along these lines in many laboratories, having identified and characterized a recent wave of new TAFs and new factors that interact with TAF from nuclear extracts of Hela cells and from yeasts [Hori and Carey (1994), Curr . Op. Gen. Dev. 4: 236-244; Zawel and Reinberg (1995), Ann. Rev. Biochem. 64: 533-561; R. G. Roeder (1996), Trends Biochem. Sci. 21: 327-335]. The TAFs are known from humans: TAF?; R68, TAFI; [55, TAFI2: 30, TAF- ^ 28, TAF? A-20 and TAFjjld [Mengus et al. (1,995), EMBO 14: 1,520-1,531; Bertolotti et al. (1996), EMBO 15: 5,022-5,031; Wu and Chiang (1996), BioTechniques 21: 718-725]. It can not be overemphasized that, although many factors have been found with these methods, the research is confined to transcription complexes, TAFs and factors that interact with TAF that are particular to the type of cell line or the yeast strain. which is being used. The invention relates to a "more universal system" in which a TBP is applied epitomically marked to the puri-fission, by affinity, of new transcription complexes and transcription factors, TAFs and factors that interact with TAF, in the context of a complete animal and, therefore, from a variety of different tissues and eukaryotic cell types. The invention relates to a non-human transgenic animal that has the ability to express an epitopically labeled TATA box binding protein (TBP). In another aspect, the invention relates to the use of the transgenic non-human animal, preferably for the identification and isolation of higher order transcription complexes and for the identification and isolation of associated proteins in the higher order transcription complex (TAFs). and factors that interact with TAF, for example, transcription factors). In another aspect, the invention relates to the preparation of the non-human transgenic animal introducing a transgene in the germ line and / or somatic cells of the non-human transgenic animal, preferably at a particular stage of development. The invention also relates to a transgene that can be used to prepare non-human transgenic animals. Therefore, one embodiment of the invention provides a transgene encoding an epitopically labeled TBP. Preferably, the transgene comprises a first DNA sequence encoding one or more epitope markers and comprising a second DNA sequence encoding a TBP. The transgene may comprise another DNA sequence (s) encoding an epitope tag (s). Preferably, the DNA encoding TBP is a cDNA. The DNA encoding TBP could be any DNA present in nature, a derivative thereof or a part thereof. The DNA, preferably cDNA, can be derived from, for example, eukaryotes, including birds, amphibians, reptiles, yeasts, C. elegans, mammals, etc. For example, DNA encoding TBP, from rodents, sheep, dogs, cows, pigs, primates - and humans, or a part thereof - can be used. The use of human TBP (hTBP) -cDNA is preferred. The invention further comprises the use of any DNA not present in nature, such as, for example, a derivative of TBP-cDNA. A DNA derivative could have, for example, an altered sequence, eg, a mutated or modified sequence, and / or could comprise modified nucleotides. A DNA derivative can also be a salt, preferably a tolerable physiological salt. The transgene comprises one or more DNA sequences encoding one, two, three, four, five or more epitope markers. Preferably, the transgene comprises the DNA encoding two epitope markers. The DNAs encoding the individual epitope markers may be located at the 5 'end and / or the 3' end of the DNA encoding TBP and / or at any suitable position within the DNA sequence encoding TBP. The DNAs encoding the individual epitope markers may be separated and / or arranged in tandem or may be directly adjacent to each other, respectively. Any natural or synthetic peptide can be used as an epitope marker. Each epitope tag is expressed as a fusion protein with TBP, where the epitope tag may be connected, for example, directly to the TBP or via a spacer peptide. Preferably, an epitope tag should offer the possibility of affinity purification of the TBP or the fusion protein, and of affinity purification of the proteins that are associated with the TBP or the fusion protein, such as TAFs and factors that interact with TAF. In addition, an epitope tag should not destroy the functional activity of TBP when expressed as a fusion protein with TBP. For this purpose, epitope markers preferably employ short peptides; they may comprise from about 1 to 50 or more amino acids, with peptides comprising from 5 to 15 amino acids being used in particular. Non-limiting examples of peptides that can be used as epitope markers are: the FLAG epitope, the HA epitope, multiple residues of histidine [His tag (from 6 to 10 histidine residues or more, preferably 6 histidine residues)], the marker Myc [Stone et al. (1996), Nature 384: 129-134], streptavidin markers and others. For this purpose, shorter peptides of natural epitopes can also be used; for example, the use of the HA epitope includes the use of the epitopes "MGYPYDVPDYA" (ID SEQ NS 2), "GYPYDVPDYA" (ID SEQ No. 3), "YPYDVPDYA (ID SEQ P 4) and other peptides from the epitope. In one embodiment of the invention, the transgene contains the CDNA of human TBP and two DNA sequences that encode an epitope tag.Preferably, the first DNA sequence encodes the HA epitope, which can serve as an epitope for immunoreaction with, for example, a commercially available monoclonal antibody [Kolodziej and Young (1991), Meth. Enzym 194: 508-519], Just 3 'to the sequence encoding the HA marker, a DNA sequence encoding a fragment of 6 histidine residues (His tag) The His tag can form a reversible and non-covalent complex with Ni2 + ions For example, a commercially available material of Ni2 + -agarose for affinity columns is routinely used to purify His-tagged proteins. [E. Hochuli et al. (1987), J. Chromato-graphy 411: 177-184; R. Janknecht et al. (1991), Proc. Nat. Acad. Sci. 74: 4.835]. In a special embodiment of the invention, the transgene comprises the DNA sequence SEQ ID No. 13. ID SEQ No. 13 provides a transgene encoding a fusion protein consisting of double-labeled hTBP. In a preferred embodiment, the invention provides a transgene encoding an epitopically labeled TBP and comprising a promoter for the expression of the fusion protein. The transgene may comprise one or more gene regulatory sequences in addition to a DNA encoding TBP (eg, TBP cDNA or a derivative thereof) and a DNA sequence (s) encoding a (os) epitopic marker (s). Said gene regulatory sequences are, for example, natural or synthetic promoters or parts thereof and / or cis acting elements (eg, potentiators and silencers). For this purpose, promoters of mammalian animals, such as, for example, the promoter of the mouse transferrin gene, the neuron-specific enolase gene (NSE) promoter, can be used. S. Forss-Petter et al. (1990), Neuron 5: 187-197] or the promoter of the thymidine kinase gene. Viral promoters can also be used, such as, for example, the promoters of the cytomegalovirus genes or the SV 40 early gene. Preferably inducible or constitutive promoters or derivatives thereof are used. A constitutive promoter such as, for example, the promoter of the human elongation factor-1 alpha (EF) gene [T. Uetsuki et al. (1989), J. Biol. Chem. 264: 5.791-5.798], An inducible promoter can be used, such as, for example, the metallothionein (MT) promoter, which has various cis elements that are sensitive to heavy metals [ R. D. Palmiter (1987), Experi-mentia Supplementum 52: 63-80, Birkhuser Verlag]. In addition, a promoter of a gene that is expressed in a form, for example, cell cycle-specific, cell-type-specific or developmental-specific may be used for this purpose. In a special embodiment of the invention, the transgene comprises the hTBP cDNA and DNA sequences encoding the HA epitope (eg, 9 amino acids of the natural HA epitope) and the His epitope (eg, the 6xhis tag) and a constitutive promoter. . For example, the expression of TBP is controlled by the promoter for human elongation factor-1 alpha (EF) [T. Uetsuki et al. (1989), J. Biol. Chem, 264: 5,791-5,798]. The EF promoter is a promoter without TATA that has been used to express transgenes in mice with moderate but constant levels [K. Hanaoka et al. (1.991), Differentiation: 183-189]. In a special embodiment of the invention, the transgene comprises the DNA sequence encoding the double-labeled hTBP (HA and His epitopes) and the promoter sequence of the EF; in particular, the transgene has the DNA sequence SEQ ID No. 14. In another special embodiment of the invention, the transgene comprises a DNA encoding TBP, preferably the hTBP cDNA, and DNA sequences encoding the HA epitope ( example, 9 amino acids of the natural HA epitope) and the His epitope (eg, 6xhis) and an inducible promoter. Preferably, the inducible promoter is the metallothionein (MT) promoter. For example, in the event that the introduction of an additional sequence encoding TBP into the genome of an animal and the subsequent expression of TBP or of a TBP fusion protein result, respectively, to be toxic, this embodiment of the invention will allow the animal adapts to the transgene that encodes the TBP fusion protein, leaving it silent until the promoter is introduced. The promoter can be induced, for example, when the animal is fully developed or at any stage of development of interest with, for example, in the case of the MT promoter, an interperitoneal injection containing divalent cations such as Zn +, Mg2 +, Mn2 + and Cd2 +. As shown in other models, the gene directed to MT will then be expressed at high levels [R. D. Palmiter (1982), Cell 29: 701-710], In a particular embodiment of the invention, the transgene comprises a DNA sequence encoding the doubly labeled hTBP (HA and His epitopes) and the MT promoter; in particular, the transgene has the DNA sequence: SEQ ID No. 15. The invention further relates to a method for pre-stopping a transgene by connecting the DNA sequence (s) encoding one or more epitope markers, to the DNA sequence that encodes the TBP protein. The invention also relates to the use of the transgene. The transgene can be used, for example, for the preparation of a recombinant vector. The invention also relates to a method for preparing a recombinant vector. This method comprises integrating the transgene into an appropriate vector, such as, for example, a vector containing regulatory sequences. Expression vectors and retroviruses or derivatives thereof are examples of vectors. The invention also relates to the use of recombinant vectors comprising the transgene. In particular, the invention relates to the use of a recombinant vector comprising a transgene (containing, for example, a cDNA of TBP or a derivative thereof, in particular hTBP, and DNA sequences encoding markers). epitopes, such as, for example, the DNA sequences encoding the HA and His epitopes). The invention relates to the use of the vector for introducing the transgene into a eukaryotic cell, in particular for introduction into a mammalian animal cell. The invention also relates to the use of a vector comprising said transgene for the multiplication of the transgenic DNA in bacteria or in eukaryotic cells. For the heterologous / transgenic expression of the transgene, a eukaryotic cell in which a vector containing the transgene has been introduced can also be used. The eukaryotic cell (host cell) could be part of a transgenic animal. In a main embodiment of the invention, the transgene is used for the preparation of a non-human transgenic animal. Therefore, a transgene or a recombinant vector comprising the transgene is introduced into a host cell and / or an animal. In another aspect, the invention relates to a transgenic non-human animal that has been produced by introducing the transgene into the animal or into a cell thereof. A transgenic animal according to the invention has the ability to express or overexpress a TBP or a fusion protein comprising, or consisting of, an epitopically labeled TBP. For the preparation of a transgenic animal, a non-human animal is used as a host animal. Said non-human animal includes vertebrate animals such as rodents, non-human primates, sheep, goats, dogs, cows, pigs, birds, amphibians, reptiles, etc. Preferred animals are selected from non-human species of mammalian animals, preferably from animals of the rodent family, including rats and mice, most preferably mice. A non-human transgenic animal according to the invention comprises any animal in whose genome one or more copies of a transgene (s) that direct (s) the expression of, or that encode, TBP or derivatives thereof, such as a fusion protein consisting of, or comprising, TBP and epitope markers. The transgenic animal should have the ability to express the epitopically labeled TBP protein. In a particular embodiment of the invention, the transgenic animal may have a disruption or transgenic alteration of the gene (s) of endogenous TBP (s) (deficient animal). The transgenic animal according to the invention is an animal in which, by non-natural means (i.e., by human manipulation), one or more TBP genes (transgenes according to the invention) that are not naturally found have been introduced. in the animal, such as, for example, a foreign TBP gene or a derivative thereof, such as an endogenous or foreign genetically constructed TBP gene. The TBP gene that is not found in nature is called a transgene. The transgene can come from a species that is the same or different from that of the animal, but in any case, the transgene is not found naturally in the animal in the configuration and / or the chromosomal locus conferred by the transgene. The transgene (transgenetic DNA) may comprise a foreign gene encoding TBP, ie, sequences not normally found in the genome of the host animal, such as a TBP gene or a cDNA obtained from a different animal species. Alternatively or additionally, a transgene may comprise an endogenous gene encoding TBP, such as, for example, DNA sequences that are abnormal because they have been transposed or mutated in vitro in order to alter the normal pattern of in vivo expression of the TBP gene. , or to alter or eliminate the biological activity of endogenous TBP. The invention also relates to expression vectors comprising the transgene and which can be used to prepare the transgenic animal. The invention further relates to a method for pre-stopping a transgenic animal according to the invention. A transgenic animal according to the invention can be produced by introducing a transgene and / or a vector, for example, an expression vector comprising the transgene, in the germline or in a germline cell, respectively, and / or in a somatic cell of the non-human animal. For example, to introduce the transgene of the invention, target embryonic cells can be used at different stages of development. Depending on the stage of development of the target embryonic cell (s), different methods may be applied. Are some examples: . . . . 1. Zygote micromjection is a preferred method for incorporating a transgene into the genome of an animal in the course of bringing the invention into practice. Microinjection involves the isolation of embryos at the stage of a single cell. Therefore, the zygote, a fertilized egg that has not undergone fusion of the pronuclei or subsequent cell division, is the preferred target cell for the microinjection of transgenic DNA. The pronucleus of the murine male reaches a size of approximately 20 micrometers in diameter, a characteristic that allows the reproducible injection of 1-2 picoliters of a solution containing transgenic DNA. The use of a zygote for the introduction of a transgene has the advantage that, in most cases, the injected transgenic DNA will be incorporated into the genome of the host animal before the first cell division [Brinster et al. (1985), Proc. Nati Acad. Sci. 82: 4,438-4,442]. As a consequence, all the cells of the resulting transgenic animals (founder animals) stably carry a transgene incorporated, called transgenic allele, in a particular genetic locus. The transgenic allele demonstrates Mendelian inheritance: half of the progeny resulting from the crossing of a transgenic animal with a non-transgenic animal will inherit the transgenic allele, according to Mendel's rules on random distribution. The procedures currently used for the manipulation of embryos and for the microinjection of transgenic DNA are described in detail in "Transgenic Animal Technology - A Laboratory Handbook" (Technology on Transgenic Animals - A Laboratory Manual), compiled by Cari A. Pinkert, Academic Press, Inc. (1994). 2. Viral integration can also be used to introduce a transgene according to the invention into an animal. Developing embryos are cultured in vitro until the stage of development known as blastocyst. At that time, blastomeres can be infected with vectors that contain the transgene (transgenic DNA / DNA constructs); for this purpose, for example, an appropriate viral or retroviral vector can be used [R. Jaenich (1976), Proc. Nati Acad. Sci. (USA) 73: 1260-1264]. Transformation or infection of blastomeres can be enhanced by enzymatic elimination of the zona pellucida [Hogan et al. (1986), Manipulating the Mouse E bryo (Manipulating the Mouse Embryo), Cold Spring Harbor Press, Cold Spring Harbor, New York]. If the transgene is introduced into blastomeres by means of viral vectors, said vectors are typically defective in replication but remain competent for the integration of transgenic DNA sequences, which are linked to vector sequences, in the genome of the host animal [ Jahner et al. (1985), Proc. Nati Acad. Sci. (USA) 82: 6,927-6,931; Van der Putten et al. (1985), Proc. Nati Acad .. Sci.- (USA) 82: 6.148-6.152]. Transfection is obtained simply and efficiently by culturing blastomeres in a monolayer of cells that produce the vector containing the transgene [Van der Putten et al. (1985), Proc. Nati Acad. Sci. (USA) 82: 6,148-6,152; Stewart et al. (1987), EMBO 6: 383-388], Alternatively, the infection can be carried out at a later stage, such as in a blastocell [D. Jahner et al. (1982), Nature 298: 623-628]. In any case, most of the transgenic founder animals are produced by retroviral or viral integration in only a subset of all the cells that make up the transgenic founder animal. In addition, multiple (retro) viral integration processes can be produced in a single founder animal, generating multiple transgenic alleles that will be segregated in future generations of the progeny. By this method, the introduction of a transgene into germline cells is possible but probably occurs with low frequency [D. Jahner et al. (1982), Nature 298: 623-628], However, once a transgene has been introduced into germline cells by this method, a progeny can be produced in which the transgenic allele is present in all cells of the germline. animal, that is, both in somatic cells and germline cells. 3. Embryonic stem cells (embryonic stem cells) can also serve as target cells for the introduction of a transgene according to the invention in animals. ES cells are obtained from preimplantation embryos that can be cultured in vitro [M. J. Evens et al. (1981), Nature 292: 154-156; M. O. Bradley et al. (1984), Nature 309: 255-258; Gossler et al. (1986), Proc. Nati Acad. Sci. (USA) 83: 9,065-9,069; Robertson et al. (1986), Nature 322: 455-448; E. J. Robertson in Teratocarcinas and Embryonic Stem Cells: A Practical Approach (Teratocarcinomas and Embryonic Pluripotenal Cells: A Practical Approach), compiled by E. J. Robertson, IRL Press, Oxford (1987), pages 71-112]. ES cells, which are commercially available (from, for example, Genome Systems, Inc., St. Louis, Missouri, USA), can be transformed with one or more transgenes by established methods [R. H. Lovell-Badge in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, compiled by EJ Robertson, IRL Press, Oxford (1987), pages 153-182], Transformed ES cells can be combined with a blastocyst of the animal, after which ES cells colonize the embryo and contribute to the germ line of the resulting animal, which is a chimera (composed of cells from two or more animals) [R. Jaenisch (1988), Science 240: 1,468-1,474; A. Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, compiled by EJ Ro-bertson, IRL Press, Oxford (1987), pages 113-151], Again, once a transgene has been introduced into cells of the By using this method, a progeny can be produced in which the transgenic allele is present in all the cells of the animal, that is, both in the somatic cells and in the cells of the germline. Whatever happens, the initial introduction of a transgene is a non-Mendelian process. However, a transgene of the invention can be stably integrated into germline cells and transmitted to the progeny of the transgenic animal such as Mendelian loci. Integration into the germ line is essential for the production of transgenic animals that can transmit genetic information to their progeny in a Mendelian manner, and to use these transgenic animals as perpetual animal models. Other transgenic techniques give rise to transgenic mosaic animals, in which some cells carry the transgene and other cells do not. In mosaic transgenic animals in which germline cells do not carry the transgene, transmission of the transgene to the progeny does not occur. However, with the transgenic mosaic animals, phenotypes associated with the transgene can be demonstrated, and said animals can be used for the purification, by affinity, of particular transcription complexes, TAFs and factors that interact with TAF. The invention relates to transgenic mosaic animals that contain the described transgene of the invention. The invention also relates to transgenically introduced mutations; this comprises null ("deficient") alleles in which the DNA sequence encoding the species-specific TBP is deleted and / or replaced by a genetically altered TBP sequence (eg, a transgene according to the invention) of the same species or a different species, under the control of the promoter (s) / enhancer (s) of choice. The invention relates to a transgenic animal in which a transgene comprising the DNA encoding a marked epitopicin-labeled TBP has been introduced, in, if necessary, the context of a constitutive promoter or an inducible promoter. In particular, the invention relates to a transgenic animal in which a transgene comprising the DNA encoding an hTBP labeled with the HA and His epitopes and the DNA sequence of the EF promoter has been introduced. In another special embodiment, the invention relates to a transgenic animal in which a transgene comprising the DNA encoding an hTBP labeled with the HA and His epitopes and the DNA sequence of the MT promoter has been introduced. In a special embodiment of the invention, the transgenic animal contains the transgene with the sequence SEQ ID No. 13. Another transgenic animal of the invention contains the transgene with the sequence SEQ ID N2 14. Another transgenic animal of the invention contains the transgene with SEQ ID Ne 15. In particular, the invention relates to a transgenic animal having the transgene, for example, the DNA sequence SEQ ID N2 13, ID SEQ Ns 14 and / or ID SEQ Ns 15, stably integrated in its genome Progeny that have inherited the transgene can be distinguished from litters that have not inherited the transgene by analyzing the genetic material of the progeny in terms of the presence of biomolecules comprising unique sequences corresponding to sequences of, or encoded by, the transgene. Thus, for example, the biological fluids containing the polypeptides (e.g., the epitopically labeled TBP) only encoded by a transgene according to the invention can be inanalyzed with respect to the presence of the polypeptide encoded by the transgene. , such as, for example, the TBP epitopically marred. A simpler and more reliable means to identify the transgenic progeny comprises obtaining a sample of tissue from a limb of an animal, for example, from the tail, and analyzing the sample for the presence of nucleic acid sequences corresponding to the DNA sequence of one or a few unique portions of the transgene of the invention. The presence of said nucleic acid sequence can be determined by, for example, a hybridization analysis ("Southern", "Northern") with DNA sequences corresponding to unique portions of the transgene, the analysis of the reaction products. in polymerase chain (PCR; English, polymerase chain reaction) using DNA sequences from a sample as substrates and oligonucleotides from the DNA sequence of the transgene, etc. Therefore, the invention also relates to tests in which possible first generation transgenic animals (G0) as well as all the transgenic animals of later generations (Gl t G2, G3, G4 ...), or animals of transgenic lines. Transgenic animals can be analyzed for the presence of the transgene with, for example, standard PCR reactions. For this purpose, genomic DNA can be extracted from tissue of the animal, for example, from tail tissue, after a treatment with proteinase K and RNase. For such PCR reactions, the sequence of the primers should correspond to parts of the transgene sequence: for example, within the promoter regions, the DNA region (s) encoding the marker (s) (is) epitopic (s) and / or the unique DNA regions for the construction of particular TBP. In mice, for example, with said PCR reactions the corresponding PCR product can be detected in about 25% of the injected eggs; that is, approximately 25% of the mice produce a positive progeny. Another method to verify whether or not the animals carry the transgene refers to an assay relative to the presence of transgenic mRNA in possible transgenic animals / lines of transgenic animals. The initial assay can be carried out, for example, with SI analysis, which is very sensitive to small levels of mRNA [A. J. Berk and P. A. Sharp (1977), Cell 12: 721]. For this purpose, labeled antisense oligonucleotides are hybridized with total RNA that has been isolated from an animal tissue. The subsequent treatment with the nuclease SI produces the digestion of all nucleic acids, DNA and RNA, single chain. The double-stranded DNA and the DNA-RNA hybrids remain intact. If any mRNA of the transgene is present, it hybridizes with the antisense oligonucleotide and, thus, "protects" it from digestion by the nuclease SI, The invention comprises that the presence of transgenic mRNA is detected in tissue preparations, for example, in preparations of total liver mRNA, using SI protection assays. Antisense oligonucleotides that are complementary to a part of the transgenic mRNA can be synthesized (for example, the mRNA corresponding to the epitope-labeled TBP DNA sequence). For example, oligonucleotides are labeled, for example, labeled at the 5 'end, radioactively with 32S, 33P, 35P, 3H or 14C or with fluorescent labels or other types of labels, such as biotin and digoxigenin. The labeled oligonucleotides are mixed with transgenic mouse mRNA under selective hybridization conditions according to standard protocols (Sambrook et al., 1989). The mixture is then treated with nuclease SI. A short unpaired sequence region, for example, at the 3 * end of the oligonucleotide, should always be digested, and provides an internal control to show that the nuclease SI actually digests all the available nucleic acid from a single strand. In the presence of transgenic mRNA, the labeled oligonucleotide should be protected from digestion, and its presence and size could easily be determined by, for example, denaturing gel sequencing-type electrophoresis [e.g. by polyacrylamide gel electrophoresis (PAGE from English, p_olyacrylamide cjel electrophoresis) with urea 8 M]. The presence of the labeled and undigested oligonucleotide can then be detected by, for example, exposing the gel to a film. The absence of transgenic mRNA would not give any band since the unprotected oligonucleotide would be digested by the nuclease SI. The invention implies that different tissues and cells related to different cell types are prepared from the animals and analyzed for the presence of transgenic mRNA. Another embodiment of the invention relates to examining transgenic animals by Northern analysis. Very preferably, the transgenic animals in which they were found to be positive for the transgenic mRNA by PCR or nuclease SI mapping were additionally examined by Northern analysis [G. K. McMaster and Carmichael (1977), Proc. Nati Acad. Sci. 74: 4.835]. For this purpose, the total RNA from different tissues and / or cell types can be isolated and subjected to separation by low sizes, for example, denaturing conditions in an agarose gel. The RNA can then be bound to, for example, a membrane or a filter using, for example, capillary transfer, and cross-linked by UV radiation. The bound RNA can be probed, using conventional Northern blotting conditions, with a labeled probe corresponding to the coding region of the transgene or parts thereof.; for example, with a DNA probe corresponding to the 5 'end of the transgene according to SEQ ID N-13. Said DNA probes preferably have a length of about 20 to 1,000 base pairs (bp; ). Most preferably, they have a length of about 100, 200, 300, 400 and 500 bp. If necessary, the excess of the labeled probe is washed away and the membrane exposed to a film. The probes can be labeled in the manner described above for the oligonucleotides. In addition, oligonucleotides can sometimes be used for Northern blot experiments. A transgenic animal, preferably said transgenic animal that has tested positive in any other molecular biological assay, is analyzed for the presence of the transgenic TBP protein by a specific immunoreaction, for example, by means of a Western blot or an assay of enzyme-linked immunosorbent assay (ELISA, enzyme linked jLmmunosorbent assay). Therefore, from tissue or cells of the animals, preferably from liver tissue or any other soft tissue, nuclei can be isolated by standard procedures, such as, for example, a sucrose gradient ultracentrifugation. From said cores the total nuclear protein can be collected by, for example, treatment of the cores under conditions of high salinity (eg, 400 mM KCl) and with a nonionic surfactant (eg, NP-40). The nuclear protein can then be subjected to size separation by, for example, an appropriate denaturing gel electrophoresis, preferably by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE). Said gels can be transferred, for example, electrotransferred, to a solid surface such as, for example, membranes or filters, preferably a nitrocellulose membrane. The membranes or filters can then be pre-blocked and probed with appropriate antibodies according to standard protocols [Sam-brook et al., "Molecular Cloning", second edition (1989), - Cold Spring Harbor Laboratory Press]. For this purpose, polyclonal and / or monoclonal antibodies generated in, for example, mouse, rabbit, rat, sheep, goat, horse, birds, etc. can be applied. Detection could be carried out directly with antibodies that recognized the TBP fusion protein and that were coupled to an enzyme or a label, such as, for example, alkaline phosphatase, a fluorescent label, biotin or digoxigenin, or a radioactive marker The detection can also be carried out indirectly using a second antibody that recognizes a conserved region of the primary antibody; for example, when the first antibody is generated in a mouse, the second antibody must be an anti-mouse antibody generated in, for example, a sheep. Said second antibody can also be coupled to an enzyme or other labels for detection. The invention further relates to the use of the transgene or a part thereof, or of the encoded fusion protein or a portion thereof, for the production of antibodies that bind to the TBP epitopically labeled and encoded by the transgene; for example, for the preparation of monoclonal or polyclonal antibodies. The antibodies with respect to the invention are antibodies that recognize one or more epitopes of the TBP fusion protein. Said antibodies could be directed against an individual epitope (s) belonging to the TBP and / or could be directed against the epitopic marker (s). An embodiment of the invention relates to an antibody that recognizes the amino terminal region of the TBP fusion protein. Another embodiment of the invention relates to an antibody that recognizes the carboxyl terminal region of the TBP fusion protein. Another embodiment of the invention relates to an antibody that recognizes the epitope that is contiguous with the part of the amino acid sequence in which the TBP and the epitope marker (s), and / or in which the two epitope markers are connected to each other. An embodiment of the invention relates to an antibody that recognizes an epitope of the fusion protein consisting of hTBP and two epitope markers. In particular, the antibody recognizes an epitope of the fusion protein consisting of the His and HA markers and hTBP. Most preferably, the antibody recognizes an epitope (s) at the amino terminus of TBP labeled with HA and His. A special embodiment of the invention relates to an antibody that recognizes the amino acid sequence ID SEQ N2 16 or an epitope of the correctly folded fusion protein having the amino acid sequence ID SEQ N2 16 or a part thereof. The invention relates to an antibody (anti-TBP antibody) which recognizes the amino acid sequence ID SEQ N2 17 or the correctly folded epitope thereof, preferably in the context of a TBP fusion protein. The antibodies of the invention can be polyclonal or monoclonal. An antibody can be generated in all species of non-human animals, preferably in mice, rats, rabbits, sheep, goats, horses and birds (for example, in their eggs). This antibody can be generated according to standard protocols [Hurlow and Lane, "Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Press.] If necessary, the antibodies can be purified by affinity using the original immunization peptide (epitope) A special embodiment of the invention relates to polyclonal antibodies produced in rabbit, which are generated by immunization of the rabbit with the peptide having the sequence SEQ ID Ns 17 (for example, coupled to a protein suitable carrier) The polyclonal antibody (anti-TBP antibody) recognizes the fusion protein of hTBP (HA and His markers) An antibody relative to the invention can be applied for a Western blot analysis as well as for purification, affinity, of the TBP fusion protein and of higher order scription complexes that are associated with the TBP fusion protein The higher order complexes comprise TAFs that are associated with the TBP fusion protein and factors that interact with TAF. The invention relates to the use of the transgenic animal. A transgenic animal according to the invention can be used for the copurification, by affinity, of higher order transcription complexes, TAFs and factors that interact with TAF, coming from tissue of the transgenic animal and / or cultured cells of the transgenic animal. Such higher order transcription complexes can be isolated and purified from a variety of different tissues and / or cell types. Preferably, the nuclear preparations are carried out from said tissue / said cell types, most preferably from homogenized cells, according to standard protocols [Dignam et al. (1983), Nuc. Acids Res. 11: 1.575; Lichtenstein et al. (1987), Cell 51: 963-973; Gorsky et al. (1986), Cell 47: 767-776]. If necessary, the nuclear proteins can then be accumulated by standard methods. Therefore, the invention also relates to methods for affinity copurification of higher order transcription complexes, TAFs and factors that interact with TAF from transgenic animals. For example, the invention relates to the purification, by affinity, of higher order transcription complexes, TAFs and factors that interact with TAF, using epitope-specific antibodies, or charged materials (positively or negatively charged). For example, the antibodies already described in detail can be used for this purpose. One of the most preferred copurification methods for higher order transcription complexes comprising TBPs, preferably hTBPs, labeled with the HA and His epitopes includes affinity purification using Ni2 + and / or anti-HA antibodies (eg, commercially available antibodies). available) and / or antibodies that recognize the epitope according to the sequence SEQ ID NO. 17. Said antibodies or said Ni2 + could be coupled to a suitable column material so that affinity purification could be carried out using, for example, Ni2 + columns or columns with specific antibodies, such as, for example, anti-HIV antibodies. -HA or anti-TBP antibodies, In another embodiment, the invention relates to a TBP fusion protein, the epitopically labeled TBP. Preferably, the TBP fusion protein is expressed in a transgenic animal. In particular, the invention relates to a fusion protein of hTBP. A TBP fusion protein can be isolated and purified from a transgenic animal. One embodiment of the invention is a TBP protein labeled with HA and His, in particular the hTBP protein labeled with HA and His. Another embodiment of the invention is the protein having the amino acid sequence ID SEQ Ns 16. In another embodiment of the invention, the TBP fusion protein comprises one or more cleavage sites for a proteinase / peptidase, for example, a site of cleavage for thrombin. Preferably, the cleavage sites of the amino acid sequence of the fusion protein are located between the epitope tag (s) and the TBP protein. The invention relates to a method for preparing a TBP fusion protein in which a transgene according to the invention is expressed in a suitable host cell, which is preferably part of a transgenic animal. transgenic mRNA and / or a TBP fusion protein encoded by the transgene and / or higher order transcription complexes associated with the TBP fusion protein can be isolated from different tissue types and / or cell types found in the animal transgenic, such as, for example, brain, heart, kidney, liver, lung, nervous system, muscle, glands, bone marrow, cells belonging to the immune system, skin, etc. The invention relates to the use of the TBP fusion protein, in particular for the isolation of higher order transcription complexes from the transgenic animal and for the characterization of higher order transcription complexes isolated, obtained from different species, different tissues and / or different cell types. Therefore, the TBP fusion protein and a transgenic animal according to the invention can be used for the isolation and characterization of individual proteins, such as TAFs and factors that interact with TAF, which are associated in the different complexes of order. higher. For example, the associated proteins in a particular higher order transcription complex can be dissociated and separated so that individual TAFs and individual factors associated with TAF can be identified. The composition of the TAFs and the factors that interact with TAF is different, at least to some degree, in the different complexes of higher order depending on the tissue type and / or the cell type and / or the stage of development and / or the transgene that is expressed. TAFs and factors that interact with TAF, in particular tissue-specific factors that have already been characterized and for which antibodies already exist, can be quickly identified and evaluated as to the degree of association with the transcription complex. An example of this would be the Bob-I / OCA-B factor, which is thought to be a specific tissue coactivator responsible for a B-cell-restricted activation [M. Gstaiger et al. (1996), EMBO 15: 2781-2790], although without intrinsic DNA binding capacity and a requirement for nearly ubiquitous Oct factors, the appearance of this tissue-restricted factor confers a specific activation of transcription to B cells by medium of a protein-protein mechanism. In addition, in a higher order transcription complex, new TAFs and new factors interacting with TAF can be identified, in particular TAFs and factors that interact with TAF that are tissue specific and / or cell type, and / or developmental specific ( stage of development), and / or specific to the cell cycle. As mentioned above, there is abundant data strongly suggesting that many tissue-specific enhancer binding factors are capable of exerting influence and, consequently, tissue specificity on the transcription activity of a gene. Therefore, tissue-specific, cell-specific, "cell-specific" factors specific to the developmental stage that regulate gene-specific expression could be identified. The "universal" transgenic system also offers a powerful tool for investigating the extent to which such TAFs and factors that interact with TAF (eg, coactivators) are associated with TBP and / or TAFs and the transcription complex in a variety of tissues and cell types.

The invention also relates to a method for the identification and characterization of different higher order transcription complexes, in which the epitopically labeled TBP with which higher order transcription complexes are associated is isolated from a transgenic animal. A method for characterizing the composition of the different higher order transcription complexes may be, for example, a) the introduction of a transgene according to the invention in a non-human animal, b) the isolation of the epitopically-labeled TBP from a non-human animal. different tissues of the animal and / or different cell types of the animal, optionally at different stages of development of the animal; and c) determination of the composition of higher order transcription complexes. A method for isolating a higher order transcription complex from a transgenic non-human animal comprises affinity purification using at least one of the epitopes that mark TBP. Preferably, the higher order transcription complex and the TAFs and factors that interact with TAF that are associated in this complex are copurified-when the epitopically labeled TBP is isolated. For example, a higher order transcription complex can be isolated when the epitopically labeled TBP is purified by binding one of its epitopes, preferably an epitope tag, to a material to which the epitope specifically binds. This material may be, for example, a Ni2 + column (to which a His epitope binds) or antibodies, such as, for example, anti-HA antibodies (which bind to an HA epitope) or antibodies that bind to an epitope, of epitopically labeled TBP, having the sequence SEQ ID No. 17 or a portion thereof (eg, an epitope of sequence SEQ ID N2 17). The invention also relates to a method for identifying a new and / or specific TAF and / or factor that inter-act with TAF. Preferably, a higher order transcription complex is isolated from a transgenic animal according to the invention. Said method may comprise, for example, a) the introduction of a transgene according to the invention into a non-human animal, b) the isolation of an epitopically-labeled TBP from a particular tissue of the animal and / or a particular cell type of the animal. animal, optionally at a particular stage of development of the animal, and c) dissociation and separation of a TAF and / or a factor that interacts with TAF that are associated in the higher order transcription complex with the epitopically-labeled TBP, and , if necessary, d) the determination of the amino acid sequence of the TAF and / or the factor that interacts with TAF. In addition, to corroborate already known mechanisms, the transgenic model has the advantage, with respect to current cell culture systems, of the finding and characterization of new TAFs or factors associated with TAF. As mentioned above, the TAF proteins that have been affinity copurified to date have come from studies in Hela cells and yeast. TAA cDNAs have also been found for other organisms, but with time-consuming library scanning methods by interaction. The "complete organism" aspect of the transgenic model makes this universal transgenic system especially sensitive to the recognition of new tissue-specific activation elements by remaining very "close" to the true transcription process in vivo. A transgenic animal expressing labeled TBP, eg, a mouse, could contribute to the area of drug development.A considerable pharmaceutical interest can be attributed to any specific "cell-specific", cell-specific "specific" factors of the tissue. and specific stages of development (TAFs and factors that interact with TAF) of a transcription complex of a given gene, especially if it is a gene related to a disease. The identification and characterization of "key" TAFs and "key" factors that interact with TAF that are involved in the control of the transcription of one or some genes related to a disease is a successful strategy to develop therapeutic compounds that alleviate said genetic diseases. Once said TAF or said factor that interacts with TAF has been characterized and cloned, various screening procedures can be undertaken to identify, molecular species / substances that interact specifically with TAF or the factor associated with TAF. A pharmaceutically useful species / substance would be one that potentiates or represses in vitro and / or in vivo the natural activity of TAF or the factor that interacts with TAF, thereby allowing therapeutic manipulation of a related gene. The TAFs identified and the factors identified that interact with TAF could then also be applied as tools for biochemistry and molecular biology; for example, said proteins from non-human transgenic animals could be used as probes for the isolation of the corresponding human TAFs and the corresponding human factors that interact with TAF, or their cDNAs. Human TAFs and human factors that interact with TAF could then also be applied to the exploration of highly specific new drugs. The present invention is described in more detail in the following non-restrictive examples.

Example 1: Construction of transgenic animals Possible sources of animals The suitable animals for the transgenic experiments were obtained from standard commercial sources: Charles River (Wilmington, Massachusetts, USA), Taconic (Germantown, New York, USA) and The Jackson Laboratory (Bar Harbor, Maine, USA). B6SJL / F1 mice were used for the recovery and transfer of embryos. B6SJL / F1 males can be used for mating and Swiss Webster stallions undergoing vasectomy can be used to stimulate pseudopregnancy.

Transgenic mice Six-week-old female mice were caused to superovulate by an injection of 5 IU (intraperitoneal 0.1 cm3) of pregnant mare serum gonadotropin [PMSG; of English, pregnant mare serum gonadotropin (for example, from Sigma, Saint Louis, Missouri, USA)], followed, 48 hours later, by an injection of 5 IU (0.1 cm3 intra-peritoneal) of gonadotropin human chorionic acid [hCG; from English, human chorionic gonadotropin (for example, from Sigma)]. The females are placed with the males immediately after the injection of hCG. Twenty-one hours after the injection of hCG, mated females are sacrificed by asphyxiation with C02 or by cervical dislocation, and the embryos are recovered from the excised oviducts and placed in M2 medium (for example, from Sigma). The surrounding cluster cells are removed with hyaluronidase (1 mg / ml). The pronuclear embryos are then washed and placed in M16 medium (for example, from Sigma) and then placed in an incubator at 37 ° C with a humidified atmosphere, with 5% C02, 02, and 90% N2, until the moment of injection.

Example 2; Preparation of constructions for transfections and microinjections DNA clones for microinjection were excised with the appropriate enzymes, the DNA clones comprising the MT-hTBP transgene (doubly labeled, according to the sequence of Table 3) with Co. I and BamHl , and those comprising the EF-hTBP transgene (doubly labeled, according to the sequence of Table 2) with Eco RI / Eco RI, and DNA fragments of the appropriate size were subjected to electrophoresis in agarose gels at pH 1. % in TBE buffer [Sambrook et al, (1989)]. The DNA bands are visualized by staining with ethidium bromide, extracted and placed in dialysis bags containing 0.3 M sodium acetate, pH 7.0. The DNA is electroeluted in the dialysis bags, extracted with phenol-chloroform (1: 1), and precipitated by means of two volumes of ethanol. The DNA is redissolved in TE buffer (10 mM Tris, pH 7.4, and 1 mM EDTA) and purified on a DEAE-Sephacel column (e.g., from Pharmacia, Uppsala, Sweden), the column is first prepared with 0.5 ml of high salinity buffer (1.5 M NaCl, 10 mM Tris, pH 7.4, and 1 mM EDTA), which is followed by a wash with 3 ml of low salinity buffer (NaCl 0, 15 M, 10 mM Tris, pH 8.0, and 1 M EDTA). The salt content of the DNA solutions is adjusted to 0.15 M NaCl and then the solutions are passed through the column so that the DNA binds to the column matrix. After three washes with 3 ml of low salinity buffer, the DNA is eluted in aliquots of 4 x 0.3 ml of high salinity buffer and is precipitated by two volumes of ethanol. The fractions were collected by dissolving in 200 μl of TE, subjected once to extraction with phenol: chloroform and subjected twice to extraction with chloroform, and the DNA was then allowed to precipitate overnight with ethanol. The DNA was resuspended in TE buffer for microinjection (10 mM Tris, pH 7.4, 0.1 mM EDTA), and the DNA concentration was adjusted to 2 ng / μl and visualized against known DNA standards, by Electrophoresis in an agarose gel. Microinjection The DNA of the transgene (MT-hTBP or EF-hTBP), purified by DEAE, was dissolved in buffer for microinjection up to 2 ng / μl. Microneedles and holding pipettes were placed in a Flaming Brown extractor of micropipettes, for example, Model P87 (Sutter Inst. Co.). The clamp pipettes were then broken and polished on a fi brbrune-type microform (for example, from Technical Product Inst. Inc.). Pipettes were mounted in micromanipulators (eg, from Leitz) that were fixed to a Zeiss Axiovert® 135 microscope. The injection pipette (eg, from Medical System Corp.) filled with air was loaded with the DNA solution through of the tip. The embryos were placed, in groups of 40-50, in 200 μl of M2 medium, under silicone oil for micromanipulation. The embryo was oriented and held with the holding pipette and then the injection pipette was inserted into the pronucleus closest to the injection pipette. The injection was verified by swelling of the pronucleus. After the injection, the group of embryos was placed in M16 medium until their transfer to recipient females.

Example 3: Embryo transfer by microinjection Adult female mice of random cycle are mated with males subjected to vasectomy. For this purpose, the Swiss Webster strain or another comparable strain can be used. The receiving females mate at the same time as the donor females. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The oviducts are exposed through a single incision in the middle dorsal area. Then an incision is made through the body wall, directly on the oviduct. The ovarian bag is then torn open by watchmaker's eyes. The embryos to be transferred are placed in M16 and then in the tip of a transfer pipette (approximately 10-12 embryos). The tip of the pipette is inserted into the infundibulum and the embryos are transferred. After the transfer, the incision is closed by two sutures and the skin is sewn with staples. The receiver left to recover for three hours in a heating tray was then placed in the colony until delivery. For MT-hTBP, a total of 469 pronuclear embryos were microinjected and 170 offspring were born, and, for EF-hTBP, a total of 407 pronuclear embryos produced 76 offspring.

Example 4; Detection of transgenic DNA in founder mice 4a) DNA extraction Genomic DNA from possible founder mice was extracted from a small piece of tail tissue (approximately 1 cm), cut from progeny at 2-4 weeks of age. Tail sections were incubated overnight in a shaker, in 500-750 μl of Tampon for Tails at 54 ° C (Tampon for Tails: 10 mM Tris, pH 7.5, 100 mM NaCl, 10 mM EDTA, SDS 0.5%, 30 μg / ml proteinase K). An equivalent volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) was subsequently added to each glue sample. The samples were shaken gently by hand, or automatically in a Vortex mixer adjusted to a low level. All samples were centrifuged in a tabletop centrifuge for 10 minutes. The aqueous phase was transferred to a polypropylene tube (e.g., a Falcon® tube) of 5 ml capacity. A volume equivalent of ethanol was added slowly (dropwise) to the tube to allow the DNA to precipitate gradually. A second volume of ethanol was added at a time to mix the contents of the tube. The tubes were then inverted several times to ensure mixing. The genomic DNA was then wrapped around a flat pipette tip (gel tip sequencing) and transferred to an individual well of a microtiter plate. The DNA was then allowed to air dry on the plate for 4 hours. Then 200 μl of TE lx was added to each well (TE lx: 10 mM Tris, pH 7.4, 1 mM EDTA). Then it was left for 15-30 minutes for the genomic DNA to dissolve at room temperature, and then gentle mixing was made by raising and lowering the solution through a pipette. Prior to the PCR assay, microtiter plates were often stored at -20 ° C. Before the assay, 10 μl was taken and digested with EcoRI (for example, 10 μl of genomic DNA, 2 μl of EcoRI lOx buffer, 7 μl of H20, 1 μl of EcoRI, the enzyme and Boehringer buffer yielding -Mann-heim, Mannheim, Germany). 4b) PCR reactions The genomic DNA subjected to digestion was diluted 1: 3 with water. Then the samples were heated to 100 ° C. in a heating block for 10 minutes. Then 2 μl was used for the PCR reactions. The reactions were carried out in a volume of 50 μl consisting of 2 μl of genomic DNA, lx buffer of reaction, 1.5 mM MgCl, oligonucleotide primer forward 0.8 μM, reverse oligonucleotide primer 0.8 μM, 8 μl of nucleotide mixture containing 0.2 mM of each nucleotide (dATP, dCTP, dTTP and dGTP), and 2.5 units of Taq polymerase. PCR was carried out using, for example, Enzyme and AmpliTaq® buffer from Perkin Elmer (Perkin Elmer Norwalk, Connecticut, USA). Detection of the MT-hTBP transgene was carried out using the forward oligonucleotide primer (sense primer) 51 GGA GCA ACC GCC TGC TGG GTG C 31 (SEQ ID N2 5) and reverse oligonucleotide primer (antisense primer) 5 'CCT GTG TTG CCT GCT GGG ACG 3' (SEQ ID No. 6). Detection of the EF-hTBP transgene was carried out with the forward oligonucleotide primer 51 GGA GAC TGA AGT TAG GCC AGC 3 '(SEQ ID NO: 7). The same reverse primer was used as in the detection of MT-hTBP (51 CCT GTG TTG CCT GCT GGG ACG 3 '). Temperature cycles were carried out using a robot that generates temperature cycles (for example, from Stratagene, La Jolla, California, USA). The temperature cycles were: cycle 1 94 ° C-5 min 60 ° C-3 min 72 ° C-2 min cycle 2-25 94 ° C-1 min 60 ° C-2 min 72 ° C-3 min cycle 26 94 ° C-1 min 60 ° C-2 min 72 ° C-5 min or cycle 1-40 95 ° C-2 min 55 ° C-1 min 72 ° C-1 min The presence of the multiplied product was detected by performing a standard electrophoresis in agarose gel (for example, agarose at 1.2-1.5%) and staining the gel with ethidium bromide to visualize the DNA with UV light. The positive samples in MT-hTBP were distinguished by the presence of a multiplied DNA product of approximately 580 bp. Positive samples in EF-hTBP produced a multiplied DNA fragment of approximately 500 bp. The analysis relative to MT-hTBP of the DNA of 170 offspring, by PCR reaction, indicated that 35 samples of genetic DNA were positive for the transgene. Analysis of the genomic DNA of the 76 EF-hTBP pups by PCR analysis indicated that 9 mice contained the EF-hTBP transgene. 4d) Expansion of the transgenic model G0 DNA-positive founders were bred with non-transgenic B6SJL pairs and the genotypes of the resulting litters were determined by PCR. The G1 progeny were used for a continuous reproduction and maintenance of the individual lines and for a further analysis of the mRNA and protein expression.

Example 5: Detection of transgenic mRNA by the nuclease protection assay SI 5a) Specific products Oligonucleotides (oligo) complementary to the 5 * end and the 3 'end of the transgenic transcript were produced: Sequence of oligo 51 (primer sense) (ID SEQ Ns 8): 'GCGGCACCAGGCCGCTGCTGTGATGATGATGATGATGGCTGCTGCCCATGA CTGCGTAATGCGGTCATGACGCTTT 3 ' 3 'oligo sequence (antisense primer) (SEQ ID N g): 'GAAGGGGGTGGGGGAGGCAAGGGTACATGAGAGCCATTACGTCGTCTTCCT GAATCCCTTTAGCCGCTTTGCTCG 3"Underlined regions are sequences that do not hybridize.They were labeled by 5'40 ng of oligo with (32P gamma) -ATP (5,000 cpm / mM) using T4 polynucleotide kinase and buffer of, for example, Boehringer-Mannheim (Mannheim, Germany) The reaction was carried out in a 50 μl reaction volume at 30 ° C for 1 hour.The labeled oligo was isolated from unincorporated (32 P gamma) ATP using exclusion chromatography. of sizes (for example, Push-Columns®, Stratagene, La Jolla, California, USA) 5b) Induction of the promoter Before the RNA analysis, it was necessary to stimulate the MT-hTBP mice since the promoter is activated in the presence of Zn2 + In each mouse MT-hTBP two interperitoneal injections of ZnSO4 were made in H20, 18 hours before and again 4 hours before the liver was removed (the dose was 0.1 mg of ZnSO4 / 10 g of weight of mouse) 5c) RNA extraction Se extracted the total RNA from 1-5 g of tissue using the commercially available Trizol reagent (for example, from Gibco-BRL, Paisly., Great Britain). The tissue was homogenized with an Ultra-Turrax apparatus in 5 ml of Trizol solution in a polypropylene tube (e.g., a Fal-con® tube) of 12 ml capacity. 1 ml of chloroform was added, the tubes were capped and the tubes were shaken vigorously by hand for 15 seconds. The solutions were incubated at room temperature for 3 minutes and then centrifuged at 12,000 x g for 15 minutes at 4 ° C in a Sorvall SS-34 rotor. The supernatant was transferred to a new tube, 2 were added., 5 ml of isopropanol and the whole was mixed, which was followed by an incubation at room temperature for 10 minutes. The samples were centrifuged at 12,000 x g for 10 minutes at 4 ° C. The supernatant was removed and the RNA pellet was washed with 5 ml of 75% ethanol. The samples were mixed and centrifuged at 7,500 x g for 10 minutes. The RNA pellets were resuspended in RNase-free water, and the RNA content was subsequently quantified by measuring the absorbance at 260 nm. 5d) Protection of Nuclease Si Uniform amounts of RNA (10-20 μg) were taken up to 100 μl with RNase-free water. An amount of labeled oligo equivalent to 50,000-150,000 dpm (0.1-1 ng, depending on the effectiveness of labeling) was added. The RNA / oligo mixture was precipitated with 0.3 M sodium acetate and ethanol. The RNA / oligo pellet was washed and air dried. 23 μl of hybridization solution was added. { form-measure at 80%, PIPES 10 M, pH 6.4 [Sambrook et al. (1989)], 1 mM EDTA, 0.05% SDS} . The reaction mixture was stirred and denatured at 65 ° C for 20 minutes, and then 2 μl of 5 M NaCl was added to each sample, at 65 ° C. The samples were incubated 1 hour more at 65 ° C in an H20 bath and then the bath temperature was readjusted at 37 ° C. The gradual decrease in temperature from 65 ° C to 37 ° C (overnight) facilitated the specific hybridization of oligo-mRNA. The next day, 300 μl of SI buffer was added to each sample [SI buffer: 167 U / ml SI nuclease (for example, from Gibco BRL, Paisly, Great Britain) ", 0.3 M NaCl, 30 mM NaOAc (pH 4,5), 3 mM ZnSO4] The samples were incubated at room temperature for 1 hour and then (cold) 1 ml of ethanol was added to precipitate all the nucleic acid.The pellet was centrifuged and washed and dried briefly in a Speed-Vac device, then the pellet was resuspended in 12 μl of loading buffer for SI (85% formamide, 0.01% bromophenol blue, 0.01% xylene-cyanol, TBE lx). Samples were heated at 70 ° C for 5 minutes before being loaded and subjected to size separation using an electrophoresis in a thin, denaturing gel (urea 6).

M) of polyacrylamide. The autoradiography of the dried gel facilitated the detection of the protected, undigested bands. From 35 founders for MT-hTBP, 7 founders showed detectable mRNA levels as analyzed by the "SI. Pafa EF-hTBP protection assay, 3 founders showed detectable mRNA by SI analysis.

Example 6: Detection of transgenic mRNA by Northern blotting 6a) Synthesis of the hybridization probe by multiplication of TBP by PCR and labeling of the TBP probe Oligonucleotides / primers spanning a 498 bp fragment of the construct were designed and synthesized. of double-labeled hTBP. The forward oligo started 10 bases downstream of the "AUG" start site (site ATG in Table 1). The sequencing of the sense primer was: SEQ ID NS 10: 51 CCCTATGACGTCCCGGATTACG 3 '. The reverse primer ended 507 bp downstream of the "AUG" start site (ATG site in Table 1).

The sequence of the antisense primer was: SEQ ID N2 11: 51 GTGGAGTGGTGCCCGGCAAGGG 31. PCR reactions were carried out with 0.2 μg of pAG-17, 0.5 mM MgCl2, 0.8 μM of each primer, buffer lx for PCR (Perkin-Elmer), dATP, dTTP, dCTP and 0.2 mM dGTP, and 2.5 U of Enzyme AmpliTaq® (Perkin Elmer Norwalk, Connecticut, USA). Thermocycle generator program: cycles 1-35: 94 ° C-1 min 55 ° C-1 min 72 ° C-1 min; then 10 minutes at 72 ° C and then storage at 4 ° C. The multiplied bands were purified on a 0.7% agarose gel. The TBP-DNA probe was labeled with random primers and Klenow fragment from the compact Megaprime® labeling system (Amersham, Great Britain). 25-50 ng of probe DNA was combined with 5 μl of random hexamer primer (eg from Amersham) and 20 μl of H 0. DNA was subjected to denaturation at 100 ° C for 5 minutes, labeling mixture was added. to a final volume of 50 μl with lx buffer for reaction, dATP lx, dTTP lx, dGTP lx, 4 μl of 3,000 Ci / millimole of alpha-32P-dCTP (for example, from Amersham) and 2 U of Klenow fragment. The reaction was allowed to proceed at room temperature for 1 hour. The labeled DNA was isolated from unincorporated nucleotides using size exclusion chromatography (e.g., Push Columns®). 6b) Gel electrophoresis and transfer 10-20 μg of total RNA were precipitated with ethanol and the pellet was subjected to denaturation for 10 minutes, at 65 ° C, in loading buffer for RNA. { 65% formamide, 20% formaldehyde (37% solution), MOPS lx buffer [Sambrook et al. (1989)], 5% glycerol, 0.01% bromophenol blue, 0.01% xylene-cyanol, 0.1 mg / ml ethidium bromide} . The RNA was subjected to size fractionation on a 1% agarose gel containing 18% formaldehyde (37% solution) and MOPS lx development buffer (40 mM MOPS, pH 7.0, 10 mM sodium acetate, EDTA 1 mM). The gels were grown slowly overnight at 1 V / cm in MOPS lx development buffer containing 18% formaldehyde (37% solution). The gel was then treated in 0.05 M NaOH / 1.5 M NaCl for 30 minutes and then in 0.5 M Tris (pH 7.4) / 1.5 M NaCl for 20 minutes. The RNA was transferred to a nylon membrane (e.g., Hybond-N +, Amersham, Great Britain) using capillary techniques. The RNA bound to the membrane was crosslinked, for example, crosslinked by UV light using a Stratalinker® apparatus (Stratagene, La Jolla, California, USA). 6c) Hybridization and washing The prehybridization was carried out, for example, in "Rapid-Hyb" buffer (Amersham, Great Britain) in an oven for rotational hybridization (for example, Hybaid), at 65 ° C for 1-2 hours. Hybridization was carried out, for example, in 6-10 ml of "Rapid-Hyb" buffer containing 106-10x106 cpm of denatured probe, at 65 ° C for 2-3 hours. The membrane was washed twice at room temperature (10 minutes / wash) in 2x SSC / 0.1% SDS (SSC lx: 150 mM NaCl, 15 mM trisodium citrate, pH 7.0). The membrane was washed 2 more times at 65 ° C (15 minutes / wash) in SSC lx / 0.1% SDS. The next 2 washings were carried out at 65 ° C for 15 minutes in 0.5x SSC / 0.1% SDS, which was followed by 1 or 2 final washings (as necessary) at 65 ° C, of 15 minutes / wash, in 0.2x SSC / 0.1% SDS. An autoradiography of the washed membrane was performed.

Example 7: Generation of a polyclonal antibody, specific for labeled hTBP General strategy There is a significant amount of sequence homology between mouse TBP and human TBP. Therefore, most commercially available antibodies will cross-react, detecting both endogenous TBP and transgenic TBP. To distinguish between the two, a polyclonal antibody against the labeled region of the transgenic TBP (which corresponded to the cleavage site for thrombin and the His marker of the labeled hTBP) was generated: SEQ ID NS 12: H2N-MGSSHHHHHHSSGLVPRGC-COOH. This peptide was coupled to a carrier protein and injected into rabbits using standard protocols. Serum was collected at regular intervals and analyzed for its anti-hTBP titre using ELISA assays.

Example 8: Detection of transcrnic protein by Western transfer 8a) Preparation of liver nuclear extracts MT-hTBP mice were subjected to 2 intra-peritoneal injections of ZnSO4 (see 5b). The mice were killed by cervical dislocation and the livers were removed. The livers were immediately homogenized in 10 ml of buffer for homogenization. { 1.8 M sucrose, 10 mM HEPES, pH 7.4 [Sambrook et al. (1989), 25 mM KCl, 1 mM EDTA, 5% glycerol, 0.15 M spermine, 0.5 mM spermidine, 0.4 mM PMSF} . After homogenization, the volume was increased to 25 ml with the same buffer. The homogenization product was carefully arranged in the form of a layer on 7 ml of buffer for homogenization in centrifuge tubes, for example, in Ultra-Clear SW-28® tubes (Beckman, Palo Alto, California, USA). The samples were centrifuged for 1 hour at 25,000 rpm (4 ° C) in a SW-28 rotor. The supernatant was carefully removed and the pellet nuclei were resuspended in 200 μl of NEXB buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, glycerol 10%, 2 μg / ml aprotinin, 2 μg / ml leupeptin, 2 μg / ml pepstatin-A). The resuspended cores were incubated on ice for 15-30 minutes, with frequent mixing and movement up and down the solution through a pipette. The samples were subjected to 5 freeze / thaw cycles with a dry ice / ethanol bath and a H20 bath at 37 ° C. The samples were centrifuged during seconds at high speed and the protein content of the supernatant was measured with, for example, a reagent for protein analysis (eg, Bio-Rad, Hercules, California, USA). 8b) Electrophoresis and transfer 20-75 μg of extract was subjected to size separation by electrophoresis in a denaturing gel; resolution gel: 10% acrylamide (acrylamide: bisacrylate-ida in 1:37 ratio), 0.1% SDS, 375 mM Tris, pH 8.8; accumulation gel: 5% acrylamide, 0.1% SDS, 125 mM Tris, pH 8.3; development buffer: 25 mM Tris, pH 8.3, 192 mM glycol-coconut, 0.1% SDS. The protein was transferred to a pure nitrocellulose membrane (eg, from Bio-Rad) with a semi-dry transfer device (eg, from Hoefer, San Francisco, California, USA), using a Bjerrum transfer buffer. modified (48 mM Tris, 39 mM gli-coconut, 10% methanol, 0.0375% SDS, pH 9.2). The transfer was allowed to develop at 0.8 mA / cm during 2-4 hours 8c) Immunodetection The membranes were blocked for 1-2 hours at room temperature on an oscillating surface, in TBS lx. (20 mM Tris, pH 7.5, 500 mM NaCl) with 3% gelatin (for example, from Bio-Rad). The membranes were washed at room temperature for 10 minutes in TTBS lx (TBS lx with 0.05% Tween-20). Hybridization was carried out with primary antibody in 1% gelatin / TTBS lx on an oscillating surface, at room temperature, for a period of 4 hours overnight. The membranes were washed 2-3 times (5 minutes / wash) with TTBS lx. Hybridization with secondary antibody coupled with alkaline phosphatase (AP) was carried out for 2-3 hours; of English, alkaline phosphatase), in 1% gelatin / TTBS lx. The membranes were washed 2-3 times again (5 minutes / wash) in TTBS lx at room temperature, which was followed by a 5 minute wash in TBS lx buffer. The membranes were then incubated in lx buffer for development (eg, from Bio-Rad) containing the NBT / BCIP reagents, at room temperature, until a sufficient appearance of bands occurred. The PA reaction was stopped by a H20 wash.

ENUMERATION OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Hoechst Aktiengesellschaft (B) STREET: - (C) CITY: Frankfurt (D) STATE: - (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): 65926 (G) TELEPHONE: 069-305-7072 (H) TELEFAX: 069-35-7175 (I) TÉLEX: - (ii) TITLE OF THE INVENTION: Purification of higher order transcription complexes from non-human transgenic animals. (iii) NUMBER OF SEQUENCES: 17 (iv) LEGIBLE FORM BY COMPUTER: (A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay n = 1.0, version n = 1.25 (EPO) (2) INFORMATION FOR ID SEQ N2 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) TRAIT: (A) NAME / KEY: Peptide (B) SITUATION: 1..12 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N2 1: Met Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Val 1 5 10 (2) INFORMATION FOR ID SEQ N2 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) TRAIT: (A) NAME / KEY: Peptide (B) SITUATION: 1..11 (xi) DESCRIPTION OF THE SEQUENCE: ID SEQ N = 2: Met Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 10 (2) INFORMATION FOR ID SEQ N2 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) TRAIT: (A) NAME / KEY: Peptide (B) SITUATION: 1..10 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N2 3: Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 10 (2) INFORMATION FOR ID SEQ N 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) TRAIT: (A) NAME / KEY: Peptide (B) SITUATION: 1..9 (xi) SEQUENCE DESCRIPTION: ID SEQ N = 4: Tyr Pro Tyr Asp Val Pro Asp Tyr Wing 1 5 (2) INFORMATION FOR ID SEQ 2 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..22 (xi) DESCRIPTION OF THE SEQUENCE: ID SEQ N = 5: GGAGCAACCG CCTGCTGGGT GC 22 (2) INFORMATION FOR ID SEQ N = 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: ID SEQ N = 6: CCTGTGTTGC CTGCTGGGAC G 21 (2) INFORMATION FOR ID SEQ N2 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..21 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N2 7: GGAGACTGAA GTTAGGCCAG C 21 (2) INFORMATION FOR ID SEQ 2 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 76 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..76 (xi) DESCRIPTION OF THE SEQUENCE: ID SEQ N2 8: GCGGCACCAG GCCGCTGCTG TGATGATGAT GATGATGGCT GCTGCCCATG ACTGCGTAAT 60 GCGGTCATGA CGCTTT 76 (2) INFORMATION FOR ID SEQ N2 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 75 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..75 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N = 9: GAAGGGGGTG GGGGAGGCAA GGGTACATGA GAGCCATTAC GTCGTCTTCC TGAATCCCTT 60 TAGCCGCTTT GCTCG 75TION FOR ID SEQ N2 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..22 (xi) SEQUENCE DESCRIPTION: ID SEQ N2 10: CCCTATGACG TCCCGGATTA CG 22 (2) INFORMATION FOR ID SEQ N2 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..22 (xi) DESCRIPTION OF THE SEQUENCE: ID SEQ N- 11: GTGGAGTGGT GCCCGGCAAG GG 22 (2) INFORMATION FOR ID SEQ 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) TRAIT: (A) NAME / KEY: Peptide (B) SITUATION: 1..19 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N2 12: Met Gly Ser Ser His His His His His His Ser His Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Cys (2) INFORMATION FOR ID SEQ N2 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1,310 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) TRAIT: (A) NAME / KEY: exon (B) SITUATION: 1..1310 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N2 13: CCATGGGCTA TCCCTATGAC GTCCCGGATT ACGCAGTCAT GGGCAGCAGC CATCATCATC 60 ATCATCACAG CAGCGGCCTG GTGCCGCGCG GCAGCCATAT GGATCAGAAC AACAGCCTGC 120 CACCTTACGC TCAGGGCTTG GCCTCCCCTC AGGGTGCCAT GACTCCCGGA ATCCCTATCT 180 TTAGTCCAAT GATGCCTTAT GGCACTGGAC TGACCCCACA GCCTATTCAG AACACCAATA 240 GTCTGTCTAT TTTGGAAGAG CAACAAAGGC AGCAGCAGCA ACAACAACAG CAGCAGCAGC 300 AGCAGCAGCA GCAGCAACAG CAACAGCAGC AGCAGCAGCA GCAGCAGCAG CAGCAGCAGC 360 AGCAGCAGCA GCAGCAGCAA CAGGCAGTGG CAGCTGCAGC CGTTCAGCAG TCAACGTCCC 420 AGCAGGCAAC ACAGGGAACC TCAGGCCAGG CACCACAGCT CTTCCACTCA CAGACTCTCA 480 CAACTGCACC CTTGCCGGGC ACCACTCCAC TGTATCCCTC CCCCATGACT CCCATGACCC 540 CCATCACTCC TGCCACGCCA GCTTCGGAGA GTTCTGGGAT TGTACCGCAG CTGCAAAATA 600 TTGTATCCAC AGTGAATCTT OGTTGTAAAC TTGACCTAAA GACCATTGCA CTTCGTGCCC 660 GAAACGCCGA ATATAATCCC AAGCGGTTTG CTGCGGTAAT CATGAGGATA AGAGAGCCAC 720 GAACCACGGC ACTGATTTTC AGTTCTGGGA AAATGGTGTG CACAGGAGCC AAGAGTGAAG 780 AACAGTCCAG ACTGGCAGCA AGAAAATATG CTAGAGTTGT ACAGAAGTTG GGTTTTCCAG 840 CTAAGTTCTT GGACTTCAAG ATTCAGAACA TGGTGGGGAG CTGTGATGTG AAGTTTCCTA 900 TAAGGTTAGA AGGCCTTGTG CTCACCCACC AACAATTTAG TAGTTATGAG CCAGAGTTAT 960 TTCCTGGTTT AATCTACAGA ATGATCAAAC CCAGAATTGT TCTCCTTATT TTTGTTTCTG 1020 GAAAAGTTGT ATTAACAGGT GCTAAAGTCA GAGCAGAAAT TTATGAAGCA TTTGAAAACA 1080 TCTACCCTAT TCTAAAGGGA TTCAGGAAGA CGACGTAATG GCTCTCATGT ACCCTTGCCT 1140 CCCCCACCCC CTTCTTTTTT TTTTTTTTAAA CAAATCAGTT TGTTTTGGTA CCTTTAAATG 1200 GTGGTGTTGT GAGAAGATGG ATGTTGAGTT GCAGGGTGTG GCACCAGGTG ATGCCCTTCT 1260 GTAAGTGCCC CTTCCGGCAT CCCGGAATTC CTGCAGCCCA ACGCGGCCGC 1310 (2) INFORMATION FOR ID SEQ N2 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4,286 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA 5 (ix) RASK: (A) NAME / KEY: exon (B) SITUATION: 1..4286 (xi) DESCRIPTION OF THE SEQUENCE: ID SEQ 2 14: GAATTCCCCT GCAGGTCACT TAGCGTTGGC CACATAGTAG GTTCTCAAAT ACTTGTTAAT 60 AAATAAGTTT GTTCGAGAAG CTGGGCAATG ATATTCTACA GCTGGAAGAA GAAACATAAT 120 _1.5c GATCTAGTAA TTAGCTCAAT TAAAAATAAA CGTTCTTCTT TCCTCAGAGG AGCATTTCCC 180 AAGGCCTGCC TTGATAGCCA TCCAAAAAGG CCAAGCTCAT CCAATCTTGC CCTAGATTTA 240 TGCTAAAATG CAGTTACAAT CGATAGGATG ACAGAAAACG ACAGCACTTA TTTAAATATA 300 ATAGGCACTT ATTTAAATAG GAGAAGCTGT GACTTCATAG CAAGTGTTGG GGTTAGGAAA 360 20 CTGGGTGGAT AAACTTGCTG ATGCTGTAGA TCTTAGCCTC TACATGAGAT CATGTGGAAA 420 ATCTGAAAGC ATTTTAGGTT CCTTATGTTT GCAATCAAAT AACTGTACAC CTTTTAATTT 480 AAAAAGTACC ATGAGGCACA CACACACACT CGCAGGAACT TTTTGGCGTA ACAAAACTAG_540_ AATTAGATCT AAAAGCTAAC TGTAGGACTG AGTCTATTCT AAACTGAAAG CCTGGACATC 600 TGGAGTACCA GGGGGAGATG ACGTGTTACG GGCTTCCATA AAAGCAGCTG GCTTTGAATG 660 GAAGGAGCCA AGAGGCCAGC ACAGGAGCGG ATTCGTCGCT TTCACGGCCA TCGAGCCGAA 720 CCTCTCGCAA GTCCGTGAGC CGTTAAGGAG GCCCCCAGTC CCGACCCTTC GCCCCAAGCC 780 CCTCGGGGTC CCCGGGCCTG GTACTCCTTG CCACACGGGA GGGGCGCGGA AGCCGGGGCG 840 GAGGAGGAGC CAACCCCGGG CTGGGCTGAG ACCCGCAGAG GAAGACGCTC TAGGGATTTG 900 TCCCGGACTA GCGAGATGGC AAGGCTGAGG ACGGGAGGCT GATTGAGAGG CGAAGGTACA 960 CCCTAATCTC AATACAACCT TTGGAGCTAA GCCAGCAATG GTAGAGGGAA GATTCTGCAC 1020 GTCCCTTCCA GGCGGCCTCC CCGTCACCAC CCCCCCCAAC CCGCCCCGAC CGGAGCTGAG 1080 , Q AGTAATTCAT ACAAAAGGAC TCGCCCCTGC CTTGGGGAAT CCCAGGGACC GTCGTTAAAC 1140 TCCCACTAAC GTAGAACCCA GAGATCGCTG CGTTCCCGCC CCCTCACCCG CCCGCTCTCG 1200 TCATCACTGA GGTGGAGAAG AGCATGCGTG AGGCTCCGGT GCCCGTCAGT GGGCAGAGCG 1260 CACATCGCCC ACAGTCCCCG AGAAGTTGGG GGGAGGGGTC GGCAATTGAA CCGGTGCCTA 1320 GAGAAGGTGG CGCGGGGGTAA ACTGGGAAAG TGATGTCGTG TACTGGCTCC GCCTTTTTCC 1380 CGAGGGTGGG GGAGAACCGT ATATAAGTGC AGTAGTCGCC GTGAACGTTC TTTTTCGCAA 1440 CGGGTTTGCC GCCAGAACAC AGGTAAGTGC CGTGTGTGGT TCCCGCGGGC CTGGCCTCTT 1500 TACGGGTTAT GGCCCTTGCG TGCCTTGAAT TACTTCCACG CCCCTGGCTG CAGTACGTGA 1560 TTCTTGATCC CGAGCTTCGG GTTGGAAGTG GGTGGGAGAG TTCGAGGCCT TGCGCTTAAG 1620 GAGCCCCTTC GCCTCGTGCT TGAGTTGAGG CCTGGCCTGG GCGCTGGGGC CGCCGCGTGC 1680 1 ° GAATCTGGTG GCACCTTCGC GCCTGTCTCG CTGCTTTCGA TAAGTCTCTA GCCATTTAAA 1740 ATTTTTTGATG ACCTGCTGCG ACGCTTTTTT TCTGGCAAGA TAGTCTTGTA AATGCGGGCC 1800 AAGATCTGCA CACTGGTATT TCGGTTTTTGGGGCCGCGGG CGGCGACGGG GCCCGTGCGT 1860 CCCAGCGCAC ATGTTCGGCG AGGCGGGGCC TGCGAGCGCG GCCACCGAGA ATCGGACGGG 1920 GGTAGTCTCA AGCTGGCCGG CCTGCTCTGG TGCCTGGCCT CGCGCCGCCG TGTATCGCCC 1980 CGCCCTGGGC GGCAAGGCTG GCCCGGTCGG CACCAGTTGC GTGAGCGGAA AGATGGCCGC 2040 __ TTCCCGGCCC TGCTGCAGGG AGCTCAAAAT GGAGGACGCG GCGCTCGGGA GAGCGGGCGG 2100 GTGAGTCACC CACACAAAGG AAAAGGGCCT TTCCGTCCTC AGCCGTCGCT TCATGTGACT 2160 CCACGGAGTA CCGGGCGCCG TCCAGGCACC TCGATTAGTT CTCGAGCTTT TGGAGTACGT 2220 CGTCTTTAGG TTGGGGGGAG GGGTTTTATG CGATGGAGTT TCCCCACACT GAGTGGGTGG 2280 AGACTGAAGT TAGGCCAGCT TGGCACTTGA TGTAATTCTC CTTGGAATTT GCCCTTTTTG 2340 AGTTTGGATC TTGGTTCATT CTCAAGCCTC AGACAGTGGT TCAAAGTTTT TTTCTTCCAT 2400 TTCAGGTGTC GTGAGGAATT GCCCGGGGGA TCCATGGGCT ATCCCTATGA CGTCCCGGAT 2460 TACGCAGTCA TGGGCAGCAG CCATCATCAT CATCATCACA GCAGCGGCCT GGTGCCGCGC 2520 GGCAGCCATA TGGATCAGAA CAACAGCCTG CCACCTTACG CTCAGGGCTT GGCCTCCCCT 2580 CAGGGTGCCA TGACTCCCGG AATCCCTATC TTTAGTCCAA TGATGCCTTA TGGCACTGGA 2640 CTGACCCCAC AGCCTATTCA GAACACCAAT AGTCTGTCTA TTTTGGAAGA GCAACAAAGG 2700 CAGCAGCAGC AACAACAACA GCAGCAGCAG CAGCAGCAGC AGCAGCAACA GCAACAGCAG 2760 CAGCAGCAGC AGCAGCAGCA GCAGCAGCAG CAGCAGCAGC AGCAGCAGCA ACAGGCAGTG 2820 40 GCAGCTGCAG CCGTTCAGCA GTCAACGTCC CAGCAGGCAA CACAGGGAAC CTCAGGCCAG 2880 GCACCACAGC TCTTCCACTC ACAGACTCTC ACAACTGCAC CCTTGCCGGG CACCACTCCA 2940 CTGTATCCCT CCCCCATGAC TCCCATGACC CCCATCACTC CTGCCACGCC AGCTTCGGAG 3000 AGTTCTGGGA TTGTACCGCA GCTGCAAAAT ATTGTATCCA CAGTGAATCT TGGTTGTAAA 3060 CTTGACCTAA AGACCATTGC ACTTCGTGCC CGAAACGCCG AATATAATCC CAAGCGGTTT 3120 GCTGCGGTAA TCATGAGGAT AAGAGAGCCA CGAACCACGG CACTGATTTT CAGTTCTGGG 3180 AAAATGGTGT GCACAGGAGC CAAGAGTGAA GAACAGTCCA GACTGGCAGC AAGAAAATAT 3240 GCTAGAGTTG TACAGAAGTT GGGTTTTCCA GCTAAGTTCT TGGACTTCAA GATTCAGAAC 3300 ATGGTGGGGA GCTGTGATGT GAAGTTTCCT ATAAGGTTAG AAGGCCTTGT GCTCACCCAC 3360 CAACAATTTA GTAGTTATGA GCCAGAGTTA TTTCCTGGTT TAATCTACAG AATGATCAAA 3420 CCCAGAATTG TTCTCCTTAT TTTTGTTTCT GGAAAAGTTG TATTAACAGG TGCTAAAGTC 3480 AGAGCAGAAA TTTATGAAGC ATTTGAAAAC ATCTACCCTA TTCTAAAGGG ATTCAGGAAG 3540 ACGACGTAAT GGCTCTCATG TACCCTTGCC TCCCCCACCC CCTTCTTTTT TTTTTTTTTAA 3600 ACAAATCAGT TTGTTTTGGT ACCTTTAAAT GGTGGTGTTG TGAGAAGATG GATGTTGAGT 3660 TGCAGGGTGT GGCACCAGGT GATGCCCTTC TGTAAGTGCC CCTTCCGGCA TCCCGGATAT 3720 CCTGCAGCCC AACACGGCCG CTCGAGCATG CATCTAGAGA ACGTCACGGC CGCGATCCCC 3780 CTGTGCCTTC TAGTTGCCAG CCATCTGGTT GTTTGCCCCT CCCCCGTGCC TTCCTTGACC 3840 CTGGAAGGTG CCACTCCCAC TGTCCTTTCC TAATAAAATG AGGAAATTGC ATCGCATTGT 3900 CTGAGTAGGT GTCATTCTAT TCTGGGGGGT GGGGTGGGGC AGGACAGCAA GGGGGAGGAT 3960 TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT CTATGGGTAC CCAGGTGCTG 4020 AAGAATTGAC CCGGTTCCTC CTGGGCCAGA AAGAAGCAGG CACATCCCCT TCTCTGTGAC 4080 ACACCCTGTC CACGCCCCTG GTTCTTAGTT CCAGCCCCAC TCATAGGACA CTCAACTTGG 4140 AGCGGTCTCT CCCTCCCTCA TCAGCCCACC AAACCAAACC TAGCCTCCAA GAGTGGGAAG 4200 AAATTAAAGC AAGAAGGCTA TTAAGTGCAG AGGGAGAGAA AATGCCTCCA ACATGTGAGG 4260 AAGTAATGAT AGAAATCATA GAATTC 4286 ) INFORMATION FOR ID SEQ N2 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3,263 base pairs (B) TYPE: nucleic acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) RASK: (A) NAME / KEY: exon (B) SITUATION: 1..3263 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N 15: ATCGATAAGC TGAGATCCGG CTAGAAACTG CTGAGGGCTG GACCGCATCT GGGGACCATC 60 TGTTCTTGGC CCTGAGCGGG GCAGGAACTG CTTACCGCAG ATATCCTGTT TGCCCCAATT 120 CAGCTGTTCC ATCTGTTCTT GGCCCTGAGC GGGGCAGGAA CTGCTTACCA CAGATATCCT 180 GTTTGGCCCA TATTCAGCTG TCTCTCTGTT CCTGACCTTG ATCTGAACTT CTCTATTCTC 240 AGTTATGTAT TTTTCCCATG CCTTGCAAAA TGGCGTTACT TAAGCTAGCT TGCCAAACCT 300 ACGGCTGGGG TCTTTCACGT TTATATCTAT GAGGGGAAGG ACCCAGAGTG GGGAAGCTGG 360 GATCTTGGGA ACACGCTTCT CTACATGGCA TTGTCTGCAC GGTGGAGTCC GGATCTGAGC 420 TTGGCTTGGT TTTTAAAACC AGCCTGGAGT AGAGCAGATG GGTTAAGGTG AGTGACCCCT 480 CAGCCCTGGA CATTCTTAGA TGAGCCCCCT CAGGAGTAGA GAATAATGTT GAGATGAGTT 540 CTGTTGGCTA AAATAATCAA GGCTAGTCTT TATAAAACTG TCTCCTCTTC TCCTAGCTTC 600 GATCCAGAGA GAGACCTGGG CGGAGCTGGT CGCTGCTCAG GAACTCCAGG AAAGGAGAAG 660 CTGAGGTTAC CACGCTGCGA ATGGGTTTAC GGAGATAGCT GGCTTTCCGG GGTGAGTTCT 720 CGTAAACTCC AGAGCAGCGA TAGGCCGTAA TATCGGGGAA AGCACTATAG GGACATGATG 780 TTCCACACGT CACATGGGTC GTCCTATCCG AGCCAGTCGT GCCAAAGGGG CGGTCCCGCT 840 GTGCACACTG GCGCTCCAGG GAGCTCTGCA CTCCGCCCGA AAAGTGCGCT CGGCTCTGCC 900 AGGACGCGGG GCGCGTGACT ATGCGTGGGC TGGAGCAACC GCCTGCTGGG TGCAAACCCT 960 TTGCGCCCGG 'ACTCGTCCAA CGACTATAAA GAGGGCAGGC TGTCCTCTAA GCGTCACCAC 1020 GACTTCAACG TCCTGAGTAC CTTCTCCTCA CTTACTCCGT AGCTCCAGCT TCACCAGATC 1080 CTCGAGAACG TCTCCCATGG GCTATCCCTA TGACGTCCCG GATTACGCAG TCATGGGCAG 1140 CAGCCATCAT CATCATCATC ACAGCAGCGG CCTGGTGCCG CGCGGCAGCC ATATGGATCA 1200 GAACAACAGC CTGCCACCTT ACGCTCAGGG CTTGGCCTCC CCTCAGGGTG CCATGACTCC 1260 CGGAATCCCT ATCTTTAGTC CAATGATGCC TTATGGCACT GGACTGACCC CACAGCCTAT 1320 TCAGAACACC AATAGTCTGT CTATTTTGGA AGAGCAACAA AGGCAGCAGC AGCAACAACA 1380 ACAGCAGCAG CAGCAGCAGC AGCAGCAGCA ACAGCAACAG CAGCAGCAGC AGCAGCAGCA 1440 GCAGCAGCAG CAGCAGCAGC AGCAGCAGCA GCAACAGGCA GTGGCAGCTG CAGCCGTTCA 1500 GCAGTCAACG TCCCAGCAGG CAACACAGGG AACCTCAGGC CAGGCACCAC AGCTCTTCCA 1560 CTCACAGACT CTCACAACTG CACCCTTGCC GGGCACCACT CCACTGTATC CCTCCCCCAT 1620 GACTCCCATG ACCCCCATCA CTCCTGCCAC GCCAGCTTCG GAGAGTTCTG GGATTGTACC 1680 GCAGCTGCAA AATATTGTAT CCACAGTGAA TCTTGGTTGT AAACTTGACC TAAAGACCAT 1740 TGCACTTCGT GCCCGAAACG CCGAATATAA TCCCAAGCGG TTTGCTGCGG TAATCATGAG 1800 GATAAGAGAG CCACGAACCA CGGCACTGAT TTTCAGTTCT GGGAAAATGG TGTGCACAGG 1860 AGCCAAGAGT GAAGAACAGT CCAGACTGGC AGCAAGAAAA TATGCTAGAG TTGTACAGAA 1920 GTTGGGTTTT CCAGCTAAGT TCTTGGACTT CAAGATTCAG AACATGGTGG GGAGCTGTGA 1980 TGTGAAGTTT CCTATAAGGT TAGAAGGCCT TGTGCTCACC CACCAACAAT TTAGTAGTTA 2040 TGAGCCAGAG TTATTTCCTG GTTTAATCTA CAGAATGATC AAACCCAGAA TTGTTCTCCT 2100 TATTTTTGTT TCTGGAAAAG TTGTATTAAC AGGTGCTAAA GTCAGAGCAG AAATTTATGA 2160 AGCATTTGAA AACATCTACC CTATTCTAAA GGGATTCAGG AAGACGACGT AATGGCTCTC 2220 ATGTACCCTT GCCTCCCCCA CCCCCTTCTT TTTTTTTTTT TAAACAAATC AGTTTGTTTT 2280 GGTACCTTTA AATGGTGGTG TTGTGAGAAG ATGGATGTTG AGTTGCAGGG TGTGGCACCA 2340 GGTGATGCCC TTCTGTAAGT GCCCCTTCCG GCATCCCGGA ATTCCTGCAG CCCAACGCGG 2400 CCGCTTCGAG GGATCTTTGT GAAGGAACCT TACTTCTGTG GTGTGACATA ATTGGACAAA 2460 CTACCTACAG AGATTTAAAG CTCTAAGGTA AATATAAAAT TTTTAAGTGT ATAATGTGTT 2520 AAACTACTGA TTCTAATTGT TTGTGTATTT TAGATTCCAA CCTATGGAAC TGATGAATGG 2580 GAGCAGTGGT GGAATGCCTT TAATGAGGAA AACCTGTTTT GCTCAGAAGA AATGCCATCT 2640 AGTGATGATG AGGCTACTGC TGACTCTCAA CATTCTACTC CTCCAAAAAA GAAGAGAAAG 2700 GTAGAAGACC CCAAGGACTT TCCTTCAGAA TTGCTAAGTT TTTTGAGTCA TGCTGTGTTT 2760 AGTAATAGAA CTCTTGCTTG CTTTGCTATT TACACCACAA AGGAAAAAGC TGCACTGCTA 2820 TACAAGAAAA TTATGGAAAA ATATTCTGTA ACCTTTATAA GTAGGCATAA CAGTTATAAT 2880 CATAACATAC TGTTTTTTCT TACTCCACAC AGGCATAGAG TGTCTGCTAT TAATAACTAT 2940 GCTCAAAAAT TGTGTACCTT TAGCTTTTTA ATTTGTAAAG GGGTTAATAA GGAATATTTG 3000 ATGTATAGTG CCTTGACTAG AGATCATAAT CAGCCATACC ACATTTGTAG AGGTTTTACT 3060 TGCTTTAAAA AACCTCCCAC ACCTCCCCCT GAACCTGAAA CATAAAATGA ATGCAATTGT 3120 TGTTGTTAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA 3180 TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA 3240 TGTATCTTAT CATGTCTGGA TCC 3263 (2) INFORMATION FOR ID SEQ N2 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 371 amino acids (B) TYPE: amino acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) TRAIT: (A) NAME / KEY: Protein (B) SITUATION: 1..371 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N2 16: Met Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Val Met Gly Ser Ser 1 5 10 15 His His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His 20 - 25 30 Met Asp Gln Asn Asn Ser Leu Pro Pro Tyr Wing Gln Gly Leu Wing Ser 35 40 45 Pro Gln Gly Wing Met Thr Pro Gly lie Pro lie Phe Ser Pro Met Met 50 55 60 Pro Tyr Gly Thr Gly Leu Thr Pro Gln Pro lie Gln Asn Thr Asn Ser 65 70 75 80 Leu Ser lie Leu Glu Gln Gln Gln Arg Gln Gln Gln Gln Gln Gln Gln 85 90 95 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 100 105 110 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Wing 115 120 125 Val Wing Wing Wing Val Gln Gln Ser Thr Ser Gln Gln Wing Thr Gln 130 135 140 Gly Thr Ser Gly Gln Wing Pro Gln Leu Phe His Ser Gln Thr Leu Thr 145 150 155 160 Thr Ala Pro Leu Pro Gly Thr Thr Pro Leu Tyr Pro Ser Pro Met Thr 165 170 175 Pro Met Thr Pro lie Thr Pro Wing Thr Pro Wing Ser Glu Ser Ser Gly 180 185 190 lie Val Pro Gln Leu Gl n Asn lie Val Ser Thr Val Asn Leu Gly Cys 195 200 205 Lys Leu Asp Leu Lys Thr lie Wing Leu Arg Wing Arg Asn Wing Glu Tyr 210 215 220 Asn Pro Lys Arg Phe Wing Wing Val He Met Arg He Arg Glu Pro Arg 225 230 235 240 Thr Thr Ala Leu He Phe Ser Gly Lys Met Val Cys Thr Gly Wing 245 250 255 Lys Ser Glu Glu Gln Ser Arg Leu Ala Wing Arg Lys Tyr Wing Arg Val 260 265 270 Val Gln Lys Leu Gly Phe Pro Wing Lys Phe Leu Asp Phe Lys He Gln 275 280 285 Asn Met Val Gly Ser Cys Asp Val Lys Phe Pro He Arg Leu Glu Gly 290 295 300 Leu Val Leu Thr His Gln Gln Phe Ser Ser Tyr Glu Pro Glu Leu Phe 305 310 315 320 Pro Gly Leu He Tyr Arg Met He Lys Pro Arg He Val Leu Leu He 325 330 335 Phe Val Ser Gly Lys Val Val Leu Thr Gly Wing Lys Val Arg Wing Glu 340 345 350 He Tyr Glu Wing Phe Glu Asn He Tyr Pro He Leu Lys Gly Phe Arg 355 360 365 Lys Thr Thr 370 (2) INFORMATION FOR ID SEQ N2 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) CHAIN CLASS: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) TRAIT: (A) NAME / KEY: Protein (B) SITUATION: 1..18 (xi) DESCRIPTION OF THE SEQUENCE: ID SEQ N-17: Met Gly Ser Ser His His His His His His Ser His Ser Gly Leu Val Pro 1 5 10 15 Arg Gly

Claims (47)

1. CLAIMS I. A non-human transgenic animal that has the ability to express an epitope-tagged TATA (TBP) binding protein.
2. 2. A non-human transgenic animal according to claim 1, wherein the TBP is expressed as a fusion protein with two epitope markers.
3. 3. A transgenic non-human animal according to one or more of claims 1 and 2, wherein the fusion protein comprises human TBP (hTBP). .
4. A non-human transgenic animal according to one or more of claims 1 to 3, wherein the fusion protein comprises an HA epitope and a His epitope.
5. 5. A transgenic non-human animal according to claim 4, wherein the fusion protein has the sequence SE SEQ Ne 16.
6. 6. A non-human transgenic animal according to one or more of claims 1 to 5, in that the transgenic animal is a mouse.
7. A method for preparing a non-human tranegenic animal according to one or more of claims 1 to 6, introducing a transgene into the germ line and / or into somatic cells of a non-human animal.
8. 8. A method for preparing a non-human transgenic animal according to claim 7, microinjecting Transgenic DNA in a zygote of a non-human animal.
9. 9. A method for preparing a non-human transgenic animal according to claim 7, by transfecting a blastocyst with a vector containing a transgene.
10. 10. A method for preparing a non-human transgenic animal according to claim 7, by introducing a transgene into an embryonic pluripotenclal cell.
11. II. A transgene that encodes an epitopically labeled TBP and that can be used to prepare a non-human transgenic animal according to one or more of claims 1 to 6.
12. 12. A transgene according to claim 11, comprising a first DNA sequence encoding TBP and a second DNA sequence encoding one or more epitope markers.
13. 13. A transgene according to one or more of claims 11 and 12, wherein the DNA sequence encoding TBP is a cDNA.
14. 14. A transgene according to claim 13, wherein the DNA sequence is the human TBP cDNA (hTBP).
15. 15. A transgene according to one or more of claims 11 to 14, wherein the second sequencing of DNA encodes two epitope markers.
16. 16. A transgene according to claim 15, wherein the epitope markers are an HA epitope and a His epitope.
17. 17. A transgene according to one or more of claims 15 and 16, wherein the HA epitope has one of the sequences SEQ ID N = 1, ID SEQ NS 2, ID SEQ N 3, and SEQ ID NS 4.
18. 18. A transgene according to one or more of claims 11 to 17, comprising the sequence SEQ ID N2 13.
19. 19. A transgene according to one or more of claims 11 to 18, comprising the DNA of a inducible promoter or a constitutive promoter.
20. 20. A transgene according to claim 19, wherein the promoter is the promoter of the EF gene.
21. 21. A transgene according to claim 19, wherein the promoter is the MT gene promoter.
22. 22. A transgene according to one or more of claims 11 to 22, wherein the transgene has the sequence ID SEQ Ns 14 or ID SEQ N-15.
23. 23. The use of a transgene according to one or more of claims 11 to 22, for preparing a non-human transgenic animal.
24. 24. A method for preparing a transgene according to one or more of claims 11 to 22, by connecting the DNA sequence (s) encoding (n) one or more epitope markers to the DNA sequence encoding the TBP protein .
25. 25. The use of a transgene according to one or more of claims 11 to 22, to prepare epitopically labeled TBP.
26. 26. The use of a transgenic animal according to one or more of claims 1 to 6, to express epitopically labeled TBP.
27. 27. An epitopically labeled TBP, expressed in a transgenic animal according to one or more of claims 1 to 6.
28. 28. An epitopically labeled TBP according to claim 27, comprising two epitopes.
29. 29. An EPT epitopically marked TBP are one or more of claims 27 and 28, wherein the TBP is hTBP.
30. 30. An epitopically labeled TBP according to one or more of claims 27 to 29, wherein the TBP is linked to an HA epitope and a His epitope.
31. 31. An epitopically tagged TBP according to one or more of claims 27 to 30, wherein the HA epitope has one of the sequencies SE SEQ Ne 1, SEQ ID N = 2, SEQ ID Ns 3, and ID SEQ NS 4.
32. 32. An epitopically labeled TBP according to one or more of claims 27 to 31, having the sequence SEQ ID Ns 16.
33. 33. A method for preparing an epitopically labeled TBP according to one or more of the claims 27 to 32, introducing a transgene according to one or more of claims 11 to 22 in the germline and / or in somatic cells of an animal.
34. 34. A method for preparing an epitopically labeled TBP according to claim 33, by introducing a transgene comprising a constitutive promoter in a non-human animal, after which the epitopically-labeled TBP is expressed in particular cell types and / or tissues of the animal , optionally in a particular stage of development of the animal.
35. 35. A method for preparing an epitopically labeled TBP according to claim 33, introducing a transgene comprising an inducible promoter in a non-human animal, and expressing the epitopically labeled TBP upon induction of the promoter.
36. 36. The use of an epitopically labeled TBP, for the isolation of higher order transcription complexes from a transgenic animal, and for the identification of TAFs and factors that interact with TAF.
37. 37. The use of a non-human transgenic animal according to one or more of claims 1 to 6, to express epitopically labeled TBP.
38. 38. The use of a non-human transgenic animal according to one or more of claims 1 to 6, for the isolation of higher order transcription complexes from different tissues and / or cell types, optionally at different stages of development of the animal.
39. 39. The use of a non-human transgenic animal to identify new and / or specific TAFs and / or factors that interact with TAF.
40. 40. A method for the identification and characterization of different higher order transcription complexes, in which the epitopically labeled TBP with which higher order transcription complexes are associated is isolated from a transgenic animal.
41. 41. A method for characterizing the composition of different higher order transcription complexes, wherein a) a transgene is introduced according to one or more of claims 11 to 22 in a non-human animal, b) a TBP is isolated epitopically labeled from different tissues of the animal and / or different cell types of the animal, optionally at different stages of development of the animal, and c) the composition of higher order transcription complexes is determined.
42. 42. A method for identifying a new and / or specimen TAF and / or factor that interacts with TAF, wherein a) a transgene is introduced according to one or more of claims 11 to 22 in a non-human animal , b) an epitopically labeled TBP is isolated from a particular tissue of the animal and / or a particular cell type of the animal, optionally at a particular stage of development, and c) the TAF and / or the factor interacting with TAF are dissociated and separated. which are associated in the higher order transcription complex with the epitopically labeled TBP, and, optionally, d) the amino acid sequence of a TAF and / or a factor that interacts with TAF is determined.
43. 43. A method for isolating different higher order transcription complexes from a non-human transgenic animal according to one or more of claims 1 to 6, wherein a higher order transcription complex that is associated with an epitopically labeled TBP is affinity co-purified when epitopically-labeled TBP is affinity purified using at least one of the epitopes that mark TBP.
44. 44. A method for isolating a higher order transcription complex, according to claim 43, wherein the epitopically labeled TBP is purified by binding its His epitope to a Ni + column.
45. 45. A method for isolating a higher order transcription complex, according to one or more of claims 43 and 44, wherein the epitopically-labeled TBP is purified by binding its HA epitope to anti-HA antibodies .
46. 46. A method for isolating a higher order transcription complex, according to one or more of claims 43 to 45, wherein the higher order transcription complex is isolated using a column of affinity with antibodies that recognize an epitope of epitopically labeled TBP, having the sequence SEQ ID N = 17.
47. 47. An antibody that recognizes an epitope of epitopically labeled TBP, having the sequence SEQ ID NS 17.