ES2205989B1 - ADAMTS-15 HUMAN PROTEIN IDENTIFICATION PROCEDURE. - Google Patents
ADAMTS-15 HUMAN PROTEIN IDENTIFICATION PROCEDURE.Info
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- ES2205989B1 ES2205989B1 ES200102167A ES200102167A ES2205989B1 ES 2205989 B1 ES2205989 B1 ES 2205989B1 ES 200102167 A ES200102167 A ES 200102167A ES 200102167 A ES200102167 A ES 200102167A ES 2205989 B1 ES2205989 B1 ES 2205989B1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
Procedimiento de identificación de la proteína humana ADANTS-15. La invención consiste en identificar fragmentos de genes humanos similares a secuencias de genes de proteínas ADAM, amplificarlos mediante PCR de ARN de tejidos humanos, extender la secuencia de los fragmentos obtenidos hacia los extremos 5'' y 3'' y determinar la secuencia de los clones de ADNc generados. La secuencia identificada es SEQ ID NO :1 y se ha denominado ADAMTS-15. La aplicación de dicha secuencia está relacionada fundamentalmente con la diagnosis y el tratamiento de anomalías en los procesos de angiogénesis, hemostasis, adhesión celular y remodelación tisular.ADANTS-15 human protein identification procedure. The invention consists in identifying fragments of human genes similar to ADAM protein gene sequences, amplifying them by means of human tissue RNA PCR, extending the sequence of the fragments obtained towards the 5 '' and 3 '' ends and determining the sequence of the cDNA clones generated. The sequence identified is SEQ ID NO: 1 and has been called ADAMTS-15. The application of this sequence is fundamentally related to the diagnosis and treatment of anomalies in the processes of angiogenesis, hemostasis, cell adhesion and tissue remodeling.
Description
Procedimiento de identificación de la proteína humana ADAMTS-15.Protein Identification Procedure human ADAMTS-15.
La invención se adscribe al campo de los procesos biológicos de adhesión celular y remodelación tisular, incluyendo los asociados a condiciones fisiológicas como la respuesta inmune, la angiogénesis, la coagulación, la cicatrización de heridas, los procesos reproductivos, la implantación embrionaria, o el desarrollo fetal, así como procesos patológicos incluyendo los tumorales, artríticos, cardiovasculares, hematológicos y neurodegenerativos. En concreto, la presente invención versa sobre una proteína humana que contiene dominios de adhesión celular y metaloproteasa, sobre el gen que la codifica, y sobre sus posibles inhibidores. Más particularmente, la presente invención aborda la identificación de la proteína humana llamada ADAMTS-15, y el análisis de su estructura y de sus posibles funciones normales y patológicas.The invention is attached to the field of processes Biological cell adhesion and tissue remodeling, including those associated with physiological conditions such as the immune response, angiogenesis, coagulation, wound healing, reproductive processes, embryonic implantation, or fetal development, as well as pathological processes including Tumor, arthritic, cardiovascular, hematological and neurodegenerative Specifically, the present invention is about a human protein that contains cell adhesion domains and metalloprotease, about the gene that encodes it, and about its possible inhibitors More particularly, the present invention addresses the identification of the human protein called ADAMTS-15, and the analysis of its structure and its possible normal and pathological functions.
Las proteínas denominadas ADAMs (a disintegrin and metalloproteinase domain) o desintegrinas celulares, son una familia de enzimas que han adquirido una notable importancia dada su capacidad de participar en procesos biológicos que implican fenómenos de adhesión celular y proteolisis extracelular (Cell 90, 589, (1997)). Estas proteínas poseen una peculiar organización estructural con dominios de proenzima, metaloproteasa, desintegrina, rico en cisteína, factor de crecimiento epidérmico, transmembrana, y citoplasmático. Algunos de estos dominios son semejantes a los encontrados en una familia de proteínas aisladas de venenos de serpientes (Methods Enzymol. 248, 345, (1995)). Estas proteínas de serpientes junto con las ADAMs, constituyen la superfamilia de las reprolisinas, caracterizadas por la presencia de una secuencia HEXXHXXGXXHD en su dominio catalítico.The proteins called ADAMs ( a d isintegrin a nd m etalloproteinase domain) or cellular disintegrators, are a family of enzymes that have acquired a remarkable importance given their ability to participate in biological processes that involve phenomena of cell adhesion and extracellular proteolysis (Cell 90 , 589, (1997)). These proteins have a peculiar structural organization with domains of proenzyme, metalloprotease, disintegrin, rich in cysteine, epidermal growth factor, transmembrane, and cytoplasmic. Some of these domains are similar to those found in a family of proteins isolated from snake venoms (Methods Enzymol. 248 , 345, (1995)). These snake proteins, together with ADAMs, constitute the superfamily of reprolysins, characterized by the presence of a HEXXHXXGXXHD sequence in their catalytic domain.
Las ADAMs han sido identificadas en una variedad de tejidos de mamíferos, así como en otros organismos eucariotas como Xenopus laevis, Drosophila melanogaster y Caenorhabditis elegans, pero no en plantas, levaduras o bacterias. Inicialmente, las ADAMs se asociaron a procesos reproductivos, pero posteriormente su espectro de funciones se ha extendido considerablemente (Curr. Opin. Cell Biol. 10, 654, (1998)). Así, la meltrina- \alpha (ADAM-12) se ha implicado en fusión de mioblastos. Las meltrinas \alpha y \beta también participan en procesos de diferenciación y actividad osteoblástica. Otras ADAMs como las denominadas MS2 y decisina, participan en distintos procesos de la respuesta inmune. Además, estudios recientes han permitido caracterizar las propiedades enzimáticas y especificidad de sustrato de varias ADAMs como ADAM-9, ADAM-10 o ADAM-17 que actúan como proteasas implicadas en el procesamiento proteolítico de sustratos celulares relevantes, incluyendo precursores de citoquinas y factores de crecimiento.ADAMs have been identified in a variety of mammalian tissues, as well as in other eukaryotic organisms such as Xenopus laevis , Drosophila melanogaster and Caenorhabditis elegans , but not in plants, yeasts or bacteria. Initially, ADAMs were associated with reproductive processes, but subsequently their spectrum of functions has extended considerably (Curr. Opin. Cell Biol. 10 , 654, (1998)). Thus, meltrin-α (ADAM-12) has been implicated in myoblast fusion. The α and β meltrins also participate in differentiation processes and osteoblastic activity. Other ADAMs, such as those called MS2 and decisin, participate in different immune response processes. In addition, recent studies have allowed characterizing the enzyme properties and substrate specificity of several ADAMs such as ADAM-9, ADAM-10 or ADAM-17 that act as proteases involved in the proteolytic processing of relevant cell substrates, including cytokine precursors and increase.
La complejidad estructural y funcional de esta familia de proteínas se ha extendido considerablemnte tras el reciente hallazgo de una serie de nuevas proteasas relacionadas con las ADAMs y caracterizadas por la presencia en su secuencia de aminoácidos de varias copias de dominios trombospondina (J. Biol. Chem. 274, 25555, (1999)). El primer miembro de esta familia, denominado ADAMTS-1, se identificó como consecuencia de su asociación con el desarrollo de caquexia tumoral y de varios procesos inflamatorios. Posteriormente, se identificó la ADAMTS-2, con actividad de procolágeno I amino-proteasa y cuya deficiencia origina el síndrome de Ehlers-Danlos tipo VIIC (Am. J. Hum. Gen. 65, 308, (1999)). Otros miembros de la familia son las proteínas denominadas ADAMTS-4 y ADAMTS-11, las cuales poseen la actividad agrecanasa responsable de la degradación del cartílago articular en enfermedades artríticas. Por otra parte la ADAMTS-8 y la ADAMTS-1 han sido identificadas como proteínas capaces de inhibir los procesos de angiogénesis. Finalmente, otras proteínas como las ADAMTS-3, ADAMTS-5, ADAMTS-6, ADAMTS-7 y ADAM-TS12 sólo se han caracterizado al nivel estructural y sus funciones todavía no han sido aclaradas. Todas estas proteínas tienen una organización similar en dominios, pero difieren sustancialmente de la estructura prototipo de las ADAMs. Así, las ADAMTS-s carecen del dominio de factor de crecimiento epidérmico, la región transmembrana, y la cola citoplasmática características de las ADAMs, pero contienen una serie de copias de trombospondina, que representan la característica distintiva de los miembros de esta familia de proteínas. El hallazgo de que las ADAMTS pueden estar implicadas en una amplia variedad de procesos biológicos y patológicos ha estimulado la búsqueda de nuevos componentes de la familia.The structural and functional complexity of this family of proteins has extended considerably after the recent discovery of a series of new proteases related to ADAMs and characterized by the presence in their amino acid sequence of several copies of thrombospondin domains (J. Biol. Chem 274 , 25555, (1999)). The first member of this family, called ADAMTS-1, was identified as a consequence of its association with the development of tumor cachexia and various inflammatory processes. Subsequently, ADAMTS-2 was identified, with procollagen I amino-protease activity and whose deficiency causes Ehlers-Danlos syndrome type VIIC (Am. J. Hum. Gen. 65 , 308, (1999)). Other members of the family are proteins called ADAMTS-4 and ADAMTS-11, which have the aggrecanase activity responsible for the degradation of articular cartilage in arthritic diseases. On the other hand, ADAMTS-8 and ADAMTS-1 have been identified as proteins capable of inhibiting angiogenesis processes. Finally, other proteins such as ADAMTS-3, ADAMTS-5, ADAMTS-6, ADAMTS-7 and ADAM-TS12 have only been characterized at the structural level and their functions have not yet been clarified. All these proteins have a similar organization in domains, but differ substantially from the prototype structure of ADAMs. Thus, ADAMTS-s lack the epidermal growth factor domain, the transmembrane region, and the cytoplasmic tail characteristic of ADAMs, but contain a series of thrombospondin copies, which represent the distinctive characteristic of members of this family of proteins. . The finding that ADAMTS may be involved in a wide variety of biological and pathological processes has stimulated the search for new family components.
Una de las estrategias para la identificación de nuevas ADAMTS humanas consistiría en la aplicación de métodos de clonación por homología. Una de las múltiples formas de abordar este método, persigue en un primer paso la búsqueda en bancos de datos accesibles públicamente, de fragmentos de secuencias de nucleótidos de genes humanos generados de manera aleatoria y que tengan similitud con las secuencias de los genes de las desintegrinas ya conocidas. Tras su identificación, los hipotéticos fragmentos homólogos se pueden amplificar mediante PCR de ARN total de tejidos humanos en los que se sospeche la expresión de dichos genes, y utilizarlos como sondas para hibridar genotecas de ADNc preparadas a partir de ARN de los mismos tejidos. Alternativamente, se puede extender la secuencia hacia los extremos 5' o 3' mediante técnicas de amplificación rápida de los extremos de los ADNcs (RACE). Finalmente, la secuenciación y posterior caracterización de los clones humanos aislados mediante técnicas estándar de Biología Molecular, permitiría confirmar la identificación de nuevas ADAMTS y definir el posible papel de las proteínas codificadas por dichos clones en procesos normales y patológicos de adhesión celular o proteolisis. Basándose en esta idea, los autores de la invención, tras los pertinentes estudios experimentales, han llegado a los objetivos antes enumerados que constituyen los diversos aspectos de la presente invención.One of the strategies for identifying new human ADAMTS would consist of the application of methods of cloning by homology. One of the many ways to approach this method, pursues in a first step the search in banks of publicly accessible data, from sequence fragments of nucleotides of randomly generated human genes and that have similarity to the sequences of the genes of the Disintegrators already known. After identification, the hypothetical homologous fragments can be amplified by total RNA PCR of human tissues in which the expression of these is suspected genes, and use them as probes to hybridize cDNA libraries prepared from RNA of the same tissues. Alternatively, the sequence can be extended towards the 5 'or 3' ends by Rapid amplification techniques of cDNA ends (RACE) Finally, the sequencing and subsequent characterization of isolated human clones using standard biology techniques Molecular, it would confirm the identification of new ADAMTS and define the possible role of the proteins encoded by said clones in normal and pathological processes of cell adhesion or proteolysis Based on this idea, the authors of the invention, After the relevant experimental studies, they have reached objectives listed above that constitute the various aspects of the present invention.
Un objeto de la presente invención es identificar el gen humano que codifica una nueva proteína humana denominada ADAMTS-15.An object of the present invention is to identify the human gene that encodes a new human protein called ADAMTS-15.
Un segundo objeto de la invención es analizar la expresión en tejidos humanos del gen de la ADAMTS-15.A second object of the invention is to analyze the expression in human tissues of the gene of the ADAMTS-15.
Un tercer objeto de la invención es analizar la expresión del gen de la ADAMTS-15 en tumores humanos.A third object of the invention is to analyze the ADAMTS-15 gene expression in tumors humans.
El primer objeto de la invención consistió en la identificación de un gen humano que pudiera codificar una nueva ADAM humana. Para ello la secuencia de aminoácidos de regiones conservadas en las ADAMs descritas se comparó con la división de Expressed Sequence Tags (ESTs) de la base de datos GenBank utilizando el programa TBLASTN (J. Mol. Biol. 215, 403, (1990)). Se identificó en ADN genómico humano secuencias que podrían corresponder a regiones metaloproteasa y disintegrina de una nueva ADAMTS human. Para llevar a cabo su amplificación se sintetizaron dos oligonucleótidos, AD-1 (5'-GTCGAATCTCTTCTTGGAGC-3') y AD-2 (5'-AGCTGCTCTGTCATTGAGGACG-3'). Estos oligonucleótidos fueron utilizados para amplificar el fragmento de ADNc correspondiente, empleando como molde DNA total aislado a partir de una genoteca de ADNc humano de hígado fetal. Para ello se utilizaron 20 pmoles de cada oligonucleótido, aproximadamente 1 microgramo de ADNc, 0,2 mM dNTPs y 1,25 U de Taq DNA polimerasa en un volumen total de 50 microlitros de tampón ExpandLong 3 (Boehringer Mannheim). La amplificación se llevó a cabo en un aparato GeneAmp2400 de Perkin-Elmer, y consistió en 40 ciclos de desnaturalización (15 s, 94°C), hibridación (20 s, 64°C) y extensión (20 s, 72°C). El fragmento de DNA resultante, de 460 pares de bases (pb) se purificó mediante electroforesis en gel de agarosa y posterior extracción con GeneClean. La identidad del fragmento amplificado se verificó mediante su clonación en el vector pUC18 y posterior secuenciación de nucleótidos mediante técnicas estándar de Biología Molecular. La traducción conceptual del fragmento clonado indicó que se trataba de una nuevo miembro de la familia ADAMTS.The first object of the invention was the identification of a human gene that could encode a new human ADAM. For this, the amino acid sequence of regions conserved in the described ADAMs was compared with the division of Expressed Sequence Tags (ESTs) of the GenBank database using the TBLASTN program (J. Mol. Biol. 215 , 403, (1990)) . Sequences that could correspond to metalloprotease and disintegrin regions of a new human ADAMTS were identified in human genomic DNA. To carry out its amplification, two oligonucleotides were synthesized, AD-1 (5'-GTCGAATCTCTTCTTGGAGC-3 ') and AD-2 (5'-AGCTGCTCTGTCATTGAGGACG-3'). These oligonucleotides were used to amplify the corresponding cDNA fragment, using as a template total DNA isolated from a human fetal liver cDNA library. For this, 20 pmoles of each oligonucleotide were used, approximately 1 microgram of cDNA, 0.2 mM dNTPs and 1.25 U of Taq DNA polymerase in a total volume of 50 microliters of ExpandLong 3 buffer (Boehringer Mannheim). The amplification was carried out in a Perkin-Elmer GeneAmp2400 apparatus, and consisted of 40 cycles of denaturation (15 s, 94 ° C), hybridization (20 s, 64 ° C) and extension (20 s, 72 ° C) . The resulting 460 base pair (bp) DNA fragment was purified by agarose gel electrophoresis and subsequent extraction with GeneClean. The identity of the amplified fragment was verified by cloning into the pUC18 vector and subsequent nucleotide sequencing by standard Molecular Biology techniques. The conceptual translation of the cloned fragment indicated that it was a new member of the ADAMTS family.
Con el fin de obtener una secuencia de ADNc que contuviera la información codificante de la proteína completa, a partir del producto de PCR obtenido con los oligonucleótidos AD-1 y AD-2 se llevó a acabo la extensión de sus extremos 5' y 3 mediante técnicas de amplificación rápida de extremos de ADNcs utilizando ARN de hígado fetal humano y el método Marathon de Clontech. Tras una serie de amplificaciones sucesivas se obtuvo un fragmento que contenía un codón de terminación en la misma fase de lectura que el resto del ADNc identificado. Finalmente, el ADNc codificante completo se obtuvo por amplificación con los oligonucleótidos ADTS15F (5'-ATGCTTCTGCTGGGCATCCTA-3') y ADTS15R (5'-TCAGCACGGCCTCAGGACGCA-3'). El análisis informático de la secuencia obtenida reveló la existencia de una fase abierta de lectura, que codifica una proteína de 950 aminoácidos a la que denominamos ADAMTS-15. Su secuencia de aminoácidos, así como la secuencia nucleotídica que la codifica se muestra como SEQ ID NO:1. La comparación de esta secuencia de aminoácidos con todas las secuencias presentes en los bancos de datos accesibles públicamente demostró la existencia de un grado significativo de similitud con otras ADAMs y más específicamente con miembros de la familia de las ADAMTS (J. Biol. Chem. 274, 25555, (1999). Así, la proteína presenta todos los motivos característicos de estos enzimas incluyendo la secuencia señal, el propéptido, los dominios metaloproteasa, desintegrina y rico en cisteína, así como diversas repeticiones tipo trombospondina (TS).In order to obtain a cDNA sequence containing the complete protein coding information, from the PCR product obtained with the oligonucleotides AD-1 and AD-2 the extension of its 5 'and 3 ends was carried out by Rapid amplification techniques of cDNA ends using human fetal liver RNA and Clontech's Marathon method. After a series of successive amplifications, a fragment containing a termination codon in the same reading phase as the rest of the identified cDNA was obtained. Finally, the complete coding cDNA was obtained by amplification with the oligonucleotides ADTS15F (5'-ATGCTTCTGCTGGGCATCCTA-3 ') and ADTS15R (5'-TCAGCACGGCCTCAGGACGCA-3'). Computer analysis of the sequence obtained revealed the existence of an open reading phase, which encodes a protein of 950 amino acids, which we call ADAMTS-15. Its amino acid sequence, as well as the nucleotide sequence that encodes it, is shown as SEQ ID NO: 1. The comparison of this amino acid sequence with all the sequences present in the publicly accessible data banks demonstrated the existence of a significant degree of similarity with other ADAMs and more specifically with members of the ADAMTS family (J. Biol. Chem. 274 , 25555, (1999) Thus, the protein has all the characteristic motifs of these enzymes including the signal sequence, the propeptide, the metalloprotease, disintegrate and cysteine-rich domains, as well as various thrombospondine (TS) -type repetitions.
Un análisis más detallado de la secuencia de
aminoácidos deducida para la ADAMTS-15 determinó la
existencia de un prodominio en el que se localiza un residuo de
cisteína (posición 174) que podrían estar implicado en el
mantenimiento de la latencia enzimática. Este prodominio termina en
un motivo dibásico que podía corresponder al sitio de activación
por furina, que poseen estos enzimas. El dominio catalítico incluye
la secuencia HEXXHXXGXXHD (posiciones 361-372)
implicado en la coordinación del átomo de zinc en el centro activo
de las metaloproteasas, y con el residuo de ácido aspártico que
permite distinguir las reprolisinas de las MMPs. Este dominio
también posee el residuo de metionina (posición 390) que contribuye
a formar la estructura Met-giro presente en
reprolisinas y MMPs. Tras el dominio catalítico puede reconocerse
el dominio desintegrina, similar en tamaño al de otras ADAMTS y
con las ocho cisteínas altamente conservadas en dicha región.
Finalmente, el dominio rico en cisteínas muestra un alto porcentaje
de identidades (alrededor del 50%) con el dominio equivalente
presente en otras ADAMTS incluyendo los diez residuos de cisteína
conservados en todas ellas. Por todo ello, podemos concluir que la
proteína identificada pertenece a la familia de las ADAMTS y ha
sido denominada ADAMTS-15 La secuencia fue
depositada en el banco de datos EMBL con el número de acceso
AJ315733. Tanto el ADN aislado como el polipéptido codificado,
representados en SEQ ID NO:1, como secuencias parciales obtenidas
de ambos, pueden sintetizarse químicamente
también.A more detailed analysis of the deduced amino acid sequence for ADAMTS-15 determined the existence of a dominance in which a cysteine residue (position 174) is located that could be involved in the maintenance of enzymatic latency. This dominance ends in a dibasic motive that could correspond to the furin activation site, which these enzymes possess. The catalytic domain includes the sequence HEXXHXXGXXHD (positions 361-372) involved in the coordination of the zinc atom in the active center of the metalloproteases, and with the aspartic acid residue that makes it possible to distinguish reprolysins from MMPs. This domain also possesses the methionine residue (position 390) that contributes to the Met-spin structure present in reprolysins and MMPs. After the catalytic domain, the disintegrating domain can be recognized, similar in size to that of other ADAMTS and with the eight highly conserved cysteines in that region. Finally, the cysteine-rich domain shows a high percentage of identities (around 50%) with the equivalent domain present in other ADAMTS including the ten cysteine residues conserved in all of them. Therefore, we can conclude that the identified protein belongs to the ADAMTS family and has been called ADAMTS-15. The sequence was deposited in the EMBL database with the accession number AJ315733. Both the isolated DNA and the encoded polypeptide, represented in SEQ ID NO: 1, as partial sequences obtained from both, can be chemically synthesized.
too.
El segundo objeto de la invención es analizar la expresión en tejidos humanos del gen de la ADAMTS-15. Con este fin, se realizaron reacciones de amplificación mediante técnicas de PCR de ADNcs de diversas genotecas de tejidos humanos adultos (próstata, cerebros, mama, glándula submaxilar, endotelio, placenta, hígado, aorta, ovario) y fetales (corazón, pulmón, hígado y riñón). Para ellos se utilizaron 20 pmoles los oligonucleótidos específicos AD-1 y AD2 Para ello se utilizaron 20 pmoles de cada oligonucleótido, aproximadamente 1 microgramo de ADNc, 0,2 mM dNTPs y 1,25 U de Taq DNA polimerasa en un volumen total de 50 microlitros de tampón ExpandLong 3 (Boehringer Mannheim). La amplificación se llevó a cabo en un aparato GeneAmp2400 de Perkin-Elmer, y consistió en 40 ciclos de desnaturalización (15 s, 94°C), hibridación (20 s, 64°C) y extensión (20 s, 72°C). Como puede observarse en la figura 1, tras hibridación con la sonda de ADAMTS-15, se detectó un producto de amplificación en la genotecas de ADNc de hígado y riñón fetales. La confirmación de que se trataba de ADAMTS-15 se hizo mediante la secuenciación directa del producto de amplificación y posterior traducción conceptual de la secuencia obtenida.The second object of the invention is to analyze the expression in human tissues of the gene of the ADAMTS-15. To this end, reactions were performed of amplification by PCR techniques of cDNAs of various Adult human tissue libraries (prostate, brains, breast, submaxillary gland, endothelium, placenta, liver, aorta, ovary) and fetal (heart, lung, liver and kidney). For them they were used 20 pmoles specific oligonucleotides AD-1 and AD2 For this, 20 pmoles of each oligonucleotide were used, approximately 1 microgram of cDNA, 0.2 mM dNTPs and 1.25 U of Taq DNA polymerase in a total volume of 50 microliters of buffer ExpandLong 3 (Boehringer Mannheim). The amplification led to out on a Perkin-Elmer GeneAmp2400 device, and consisted of 40 cycles of denaturation (15 s, 94 ° C), hybridization (20 s, 64 ° C) and extension (20 s, 72 ° C). How can observed in figure 1, after hybridization with the probe ADAMTS-15, an amplification product was detected in the fetal liver and kidney cDNA libraries. The confirmation that it was about ADAMTS-15 was done through the direct sequencing of the amplification product and subsequent conceptual translation of the sequence obtained.
El tercer objeto de la invención consistió en el estudio de la expresión del gen de la ADAMTS-15 en muestras obtenidas de tumores humanos. Se realizó de forma similar a la anterior, utilizando ADNc de genotecas de carcinoma mamario y de osteosarcoma.The third object of the invention consisted of the study of the expression of the ADAMTS-15 gene in samples obtained from human tumors. It was performed similarly to the previous one, using cDNA from mammary carcinoma libraries and of osteosarcoma.
Figura 1. Análisis de la expresión de ADAMTS15 en las diversas genotecas de ADNc analizadas.Figure 1. Analysis of ADAMTS15 expression in the various cDNA libraries analyzed.
INFORMACIÓN GENERAL:GENERAL INFORMATION:
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- NOMBRE: Universidad de OviedoNAME: Oviedo University
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- CALLE: San Francisco, 3STREET: San Francisco, 3
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- PAÍS: EspañaCOUNTRY: Spain
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- CÓDIGO POSTAL: 33003POSTAL CODE: 33003
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- TÍTULO DE LA INVENCIÓN: Procedimiento de identificación de la proteína humana ADAMTS-15TITLE OF THE INVENTION: Human protein identification procedure ADAMTS-15
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- LONGITUD: 2853LENGTH: 2853
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- TIPO: ácido nucleicoTYPE: acid nucleic
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- CARACTERÍSTICA:CHARACTERISTIC:
\vskip0.500000\baselineskip\ vskip0.500000 \ baselineskip
- NOMBRE/CLAVE: secuencia codificanteNAME / KEY: coding sequence
\vskip0.500000\baselineskip\ vskip0.500000 \ baselineskip
- LOCALIZACIÓN: 1..2850LOCATION: 1..2850
\vskip0.800000\baselineskip\ vskip0.800000 \ baselineskip
- CARACTERÍSTICA:CHARACTERISTIC:
\vskip0.500000\baselineskip\ vskip0.500000 \ baselineskip
- NOMBRE/CLAVE: codón de paradaNAME / KEY: stop codon
\vskip0.500000\baselineskip\ vskip0.500000 \ baselineskip
- LOCALIZACIÓN: 2851..2853LOCATION: 2851..2853
\vskip0.800000\baselineskip\ vskip0.800000 \ baselineskip
- DESCRIPCIÓN DE LA SECUENCIA: SEQ ID NO:1 (Depositada en el GeneBank Database el 23 de Junio de 2001, con número de acceso AJ315733)DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 (Deposited in the GeneBank Database on 23 June 2001, with accession number AJ315733)
Claims (10)
- a)to)
- Comparar la secuencia de nucleótidos de regiones conservadas en proteínas ADAMTS con las secuencias parciales de nucleótidos presentes en las bases de datos de genes expresados.Compare the nucleotide sequence of regions conserved in ADAMTS proteins with sequences Partial nucleotides present in gene databases expressed.
- b)b)
- Identificar fragmentos homólogos y amplificarlos mediante PCR de RNA total de tejidos humanos en los que se puedan expresar dichas secuencias génicas.Identify homologous fragments and amplify them by PCR of total RNA from human tissues in the that these gene sequences can be expressed.
- c)C)
- Utilizar los fragmentos amplificados como sondas para hibridar genotecas de ADNc humano o como moldes informativos para extender la secuencia hacia los extremos 5' o 3'.Use the amplified fragments as probes to hybridize human cDNA libraries or as molds informative to extend the sequence towards the 5 'ends or 3'.
- d)d)
- Aislar los clones de ADNc obtenidos y determinar su secuencia completa de nucleótidos.Isolate the cDNA clones obtained and Determine your complete nucleotide sequence.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200102167A ES2205989B1 (en) | 2001-09-24 | 2001-09-24 | ADAMTS-15 HUMAN PROTEIN IDENTIFICATION PROCEDURE. |
| PCT/JP2002/009771 WO2003027282A1 (en) | 2001-09-24 | 2002-09-24 | Adamts-15, -16, -17, -18 and -19 |
| JP2002277172A JP2003180384A (en) | 2001-09-24 | 2002-09-24 | Adamts-15, -16, -17, -18 and -19 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200102167A ES2205989B1 (en) | 2001-09-24 | 2001-09-24 | ADAMTS-15 HUMAN PROTEIN IDENTIFICATION PROCEDURE. |
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| Publication Number | Publication Date |
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| ES2205989A1 ES2205989A1 (en) | 2004-05-01 |
| ES2205989B1 true ES2205989B1 (en) | 2005-08-16 |
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| ES200102167A Expired - Fee Related ES2205989B1 (en) | 2001-09-24 | 2001-09-24 | ADAMTS-15 HUMAN PROTEIN IDENTIFICATION PROCEDURE. |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000053774A2 (en) * | 1999-03-08 | 2000-09-14 | Neurocrine Biosciences, Inc. | Metalloproteinases and methods of use therefor |
| WO2001034785A1 (en) * | 1999-11-11 | 2001-05-17 | Yamanouchi Pharmaceutical Co., Ltd. | Novel metalloprotease having aggrecanase activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6040353A (en) * | 1998-11-25 | 2000-03-21 | Dow Corning Corporation | Radiation curable silicone compositions |
| JP2003533977A (en) * | 2000-02-14 | 2003-11-18 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフトング | Laryngeal cancer-related protein LARCAP-1 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000053774A2 (en) * | 1999-03-08 | 2000-09-14 | Neurocrine Biosciences, Inc. | Metalloproteinases and methods of use therefor |
| WO2001034785A1 (en) * | 1999-11-11 | 2001-05-17 | Yamanouchi Pharmaceutical Co., Ltd. | Novel metalloprotease having aggrecanase activity |
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| Publication number | Publication date |
|---|---|
| ES2205989A1 (en) | 2004-05-01 |
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