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1.
Cytogenet Genome Res ; 113(1-4): 230-7, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16575185

RESUMEN

Genomic imprinting at the H19/Igf2 locus is governed by a cis-acting Imprinting-Control Region (ICR), located 2 kb upstream of the H19 gene. This region possesses an insulator function which is activated on the unmethylated maternal allele through the binding of the CTCF factor. It has been previously reported that paternal transmission of the H19(SilK) deletion, which removes the 3' portion of H19 ICR, leads to the loss of H19 imprinting. Here we show that, in the liver, this reactivation of the paternal H19 gene is concomitant to a dramatic decrease in Igf2 mRNA levels. This deletion alters higher-order chromatin architecture, Igf2 promoter usage and tissue-specific expression. Therefore, when methylated, the 3' portion of the H19 ICR is a bi-functional regulatory element involved not only in H19 imprinting but also in 'formatting' the higher-order chromatin structure for proper tissue-specific expression of both H19 and Igf2 genes.


Asunto(s)
Regulación de la Expresión Génica , Factor II del Crecimiento Similar a la Insulina/genética , ARN no Traducido/genética , Animales , Cruzamientos Genéticos , Femenino , Impresión Genómica , Corazón/fisiología , Hígado/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Especificidad de Órganos , ARN Largo no Codificante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
2.
Dig Dis Sci ; 46(10): 2231-8, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11680602

RESUMEN

Bile acid malabsorption (BAM) has been described in patients with collagenous colitis. There are no similar studies in lymphocytic colitis. The possibility that BAM might not necessarily be part of the microscopic colitis process and that both entities could simply be concomitant has not been evaluated. Our aim was to assess the frequency and severity of BAM in patients with microscopic colitis as well as in patients with previously unexplained functional chronic diarrhea. Likewise, we wanted to investigate the effect of cholestyramine on the induction and maintenance of remission of these conditions. A [75Se]HCAT abdominal retention test was performed in 26 patients with collagenous colitis, 25 with lymphocytic colitis, and 32 with previously unexplained functional chronic diarrhea. Patients with microscopic colitis who had BAM as well as a subgroup of eight collagenous colitis patients without BAM received treatment with cholestyramine. All patients with previously unexplained chronic diarrhea who had BAM were treated with cholestyramine. Twenty-two (43.1%) patients with microscopic colitis and 24 (75%) patients with previously unexplained functional chronic diarrhea presented with BAM. The frequency of BAM was higher in lymphocytic colitis than in collagenous colitis (60% vs 27%; P = 0.025). Cholestyramine induced clinical remission in 19 of 22 patients with microscopic colitis and BAM, none of eight patients with collagenous colitis without BAM, and all patients with previously unexplained chronic diarrhea and BAM. In conclusion, BAM seems to be common in patients with microscopic colitis-mainly in lymphocytic colitis-and in those with previously unexplained functional chronic diarrhea, suggesting that idiopathic BAM and microscopic colitis are often concomitant conditions. In this setting, cholestyramine seems to be highly effective in stopping diarrhea.


Asunto(s)
Ácidos y Sales Biliares/metabolismo , Colitis/fisiopatología , Enfermedades Funcionales del Colon/fisiopatología , Diarrea/fisiopatología , Absorción Intestinal , Anciano , Enfermedad Crónica , Colitis/etiología , Diarrea/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos
3.
Mech Dev ; 101(1-2): 133-41, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11231066

RESUMEN

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.


Asunto(s)
Metilación de ADN , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , Factores de Edad , Alelos , Animales , Northern Blotting , Southern Blotting , Cruzamientos Genéticos , Padre , Femenino , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Modelos Genéticos , Madres , Músculo Esquelético/metabolismo , ARN Largo no Codificante , ARN no Traducido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Distribución Tisular
4.
Oncogene ; 19(50): 5810-6, 2000 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-11126368

RESUMEN

H19 is a paternally imprinted gene whose expression produces a 2.4 kb RNA in most tissues during development and in mammalian myoblastic cell lines upon differentiation. Deletion of the active maternal allele of H19 and its flanking regions in the mouse leads to biallelic methylation and loss of imprinting of the neighbouring Igf2 gene. The function of H19 RNA remains unknown and, although polysome-associated, the absence of a conserved open reading frame suggests that it does not encode a protein product. We describe a novel post-transcriptional regulation of H19 gene expression which, in spite of this lack of coding capacity, is dependent on translational activity. We show that stabilization of the RNA is solely responsible for its accumulation during in vitro muscle cell differentiation. This conclusion is based on the finding that inhibition of protein synthesis results in a dramatic destabilization of H19 RNA in proliferating mouse C2C12 myoblastic cells but not in differentiated cells, and on run-on experiments which showed that the rate of transcription of H19 RNA remains constant during muscle cell differentiation. This mechanism could also be involved in H19 gene expression during mouse development in addition to its transcriptional activation which we have shown to occur.


Asunto(s)
Músculo Esquelético/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN no Traducido/biosíntesis , ARN/metabolismo , Animales , Diferenciación Celular/genética , División Celular/genética , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/fisiología , Impresión Genómica , Ratones , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , ARN/genética , ARN Largo no Codificante , ARN no Traducido/genética , Regulación hacia Arriba
5.
Proc Natl Acad Sci U S A ; 94(19): 10243-8, 1997 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-9294195

RESUMEN

Recent investigations have shown that the maintenance of genomic imprinting of the murine insulin-like growth factor 2 (Igf2) gene involves at least two factors: the DNA (cytosine-5-)-methyltransferase activity, which is required to preserve the paternal specific expression of Igf2, and the H19 gene (lying 90 kb downstream of Igf2 gene), which upon inactivation leads to relaxation of the Igf2 imprint. It is not yet clear how these two factors are related to each other in the process of maintenance of Igf2 imprinting and, in particular, whether the latter is acting through cis elements or whether the H19 RNA itself is involved. By using Southern blots and the bisulfite genomic-sequencing technique, we have investigated the allelic methylation patterns (epigenotypes) of the Igf2 gene in two strains of mouse with distinct deletions of the H19 gene. The results show that maternal transmission of H19 gene deletions leads the maternal allele of Igf2 to adopt the epigenotype of the paternal allele and indicate that this phenomenon is influenced directly or indirectly by the H19 gene expression. More importantly, the bisulfite genomic-sequencing allowed us to show that the methylation pattern of the paternal allele of the Igf2 gene is affected in trans by deletions of the active maternal allele of the H19 gene. Selection during development for the appropriate expression of Igf2, dosage-dependent factors that bind to the Igf2 gene, or methylation transfer between the parental alleles could be involved in this trans effect.


Asunto(s)
Metilación de ADN , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , Madres , Proteínas Musculares/genética , ARN no Traducido , Alelos , Animales , Femenino , Amplificación de Genes , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , ARN Largo no Codificante , Eliminación de Secuencia
6.
J Biol Chem ; 271(39): 23985-91, 1996 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-8798632

RESUMEN

U1 small nuclear ribonucleoprotein (snRNP) is an important ribonucleoprotein involved early in the spliceosome formation to commit pre-mRNAs to the splicing pathway. We have determined the association and dissociation kinetics of the 5' splice site-U1 snRNP interaction using purified U1 snRNP and a short RNA oligonucleotide comprising the 5' splice site (5'-SS) consensus sequence of pre-mRNAs (5'-SS RNA oligo). The association is rapid, does not require ATP, and is almost irreversible. Surprisingly, oligonucleotide-directed cleavage of the U1 small nuclear RNA (snRNA) 5' end sequence with RNase H has no significant effect on the rate of association of the 5'-SS RNA oligo, but it does lead to rapid dissociation. This provides evidence that U1-specific snRNP proteins are critical for the 5' splice site recognition while base pairing ensures the stability of the interaction. The recognition of the 5' splice site by U1 snRNP does not result from the individual action of one or more proteins but rather from their organization around U1 snRNA. A consequence of this organization is that the U1-C protein makes direct contacts with the site, as it becomes cross-linked to the RNA oligo upon exposition of the reactions to shortwave UV light.


Asunto(s)
Empalme del ARN , ARN Nuclear Pequeño/fisiología , Ribonucleoproteína Nuclear Pequeña U1/fisiología , Sistema Libre de Células , Secuencia de Consenso , Células HeLa , Humanos , Enlace de Hidrógeno , Cinética , Sustancias Macromoleculares , Oligonucleótidos/metabolismo , Relación Estructura-Actividad
7.
Nature ; 381(6577): 80-2, 1996 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-8609994

RESUMEN

Several metazoan splicing factors are characterized by ribonucleoprotein (RNP) consensus sequences and arginine-serine repeats (RS domain) which are essential for their function in splicing. These include members of the SR-protein family (SC35, SF2/ASF), the U1 small nuclear (sn) RNP protein (U1-70K) and the U2 snRNP auxiliary factor (U2AF). SR proteins are phosphorylated in vivo and the phosphorylation state of U1-70K's RS domain influences its splicing activity. Here we report the purification of a protein kinase that is specific for SR proteins and show that it is DNA topoisomerase I. This enzyme lacks a canonical ATP-binding motif but binds ATP with a dissociation constant of 50 nM. Camptothecin and derivatives, known to be specific inhibitors of DNA topoisomerase I, strongly inhibit the kinase activity in the presence of DNA and affect the phosphorylation state of SR proteins. Thus, DNA topoisomerase I may well be one of the SR protein kinases operating in vivo.


Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Camptotecina/análogos & derivados , Camptotecina/farmacología , Línea Celular , ADN-Topoisomerasas de Tipo I/genética , Células HeLa , Humanos , Fosforilación/efectos de los fármacos , Proteínas de Unión al ARN , Proteínas Recombinantes/metabolismo , Factores de Empalme Serina-Arginina , Topotecan
8.
Biochimie ; 78(6): 436-42, 1996.
Artículo en Inglés | MEDLINE | ID: mdl-8915533

RESUMEN

In the spliceosome, the pre-mRNA, U2 and U6 snRNAs fold into a catalytic structure exhibiting striking similarities with domain V and VI of group II introns. Building of this tripartite structure implies that an evolutionary conserved base pairing between U4 and U6 snRNAs should be disrupted to allow potentially U6 catalytic residue to interact with U2 snRNAs and the pre-mRNA. The steps leading to U4/U6 disruption have been recently discovered and have been shown to involve a modification of the 3' end of U6 snRNA and the hnRNP C protein.


Asunto(s)
Precursores del ARN/metabolismo , Empalme del ARN/genética , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/metabolismo , Empalmosomas/metabolismo , Composición de Base , Ribonucleoproteína Heterogénea-Nuclear Grupo C , Ribonucleoproteínas Nucleares Heterogéneas , Conformación de Ácido Nucleico , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/metabolismo
10.
J Biol Chem ; 270(27): 16476-81, 1995 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-7608220

RESUMEN

Due to 3' end modifications, mammalian U6 small nuclear RNA (snRNA) is heterogeneous in size. The major form terminates with five U residues and a 2',3'-cyclic phosphate, but multiple RNAs containing up to 12 U residues have a 3'-OH end. They are labeled in the presence of [alpha-32P]UTP by the terminal uridylyl transferase activity present in HeLa cell nuclear extracts. That these forms all enter the U6 snRNA-containing particles, U4.U6, U4.U5.U6, and the spliceosome, has been demonstrated previously. Here, we report an interaction between the heterogeneous nuclear ribonucleoprotein (hnRNP) C protein, an abundant nuclear pre-mRNA binding protein, and the U6 snRNAs that have the longest uridylate stretches. This U6 snRNA subset is free of any one of the other snRNPs, since anti-Sm antibodies failed to immunoprecipitate hnRNP C protein. Furthermore, isolated U4.U6 snRNPs containing U6 snRNAs with long oligouridylate stretches are disrupted upon binding of hnRNP C protein either purified from HeLa cells or produced as recombinant protein from Escherichia coli. In view of these data and our previous proposal that the U6 snRNA active in splicing has 3'-OH end, we discuss a model where the hnRNP C protein has a decisive function in the catalytic activation of the spliceosome by allowing the release of U4 snRNP.


Asunto(s)
Conformación de Ácido Nucleico , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteínas/metabolismo , Composición de Base , Centrifugación por Gradiente de Densidad , Reactivos de Enlaces Cruzados , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo C , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Oligorribonucleótidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Empalme del ARN , ARN Nuclear Pequeño/efectos de la radiación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Rayos Ultravioleta , Nucleótidos de Uracilo/metabolismo
11.
Biochemistry ; 32(38): 10005-14, 1993 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-8399127

RESUMEN

The properties of polymerization and interaction of the G-actin-myosin S1 complexes (formed with either the S1(A1) or the S1(A2) isoform) have been studied by light-scattering and fluorescence measurements in the absence and in the presence of DNase I. In the absence of DNase I, the G-actin-S1(A1) and G-actin-S1(A2) complexes were found to be characterized by different limiting concentrations (l.c.), defined as the complex concentrations above which the polymerization occurs spontaneously within 20 h at 20 degrees C in a "no salt" buffer (l.c. = 0.42 and 8.8 microM for G-actin-S1(A1) and G-actin-S1(A2), respectively). The occurrence of a limiting concentration for either complex together with the kinetic properties of the polymerization led us to conclude that the G-actin-S1 polymerization occurs via a nucleation-elongation process. Fluorescence titrations and proteolysis experiments revealed that G-actin interacts with S1 with a 1:1 stoichiometry (independently of the presence of ATP) with dissociation constants, in the absence of nucleotide, of 20 and 50 nM for the G-actin-S1(A1) and G-actin-S1(A2) complexes, respectively. In the presence of at least a 1.5-fold excess of DNase I, the polymerization of the G-actin-S1 complexes was blocked even at high protein concentration or in the presence of salts. In addition, the affinity of either S1 isoform to actin was reduced 4-5-fold by DNase I, while the stoichiometry of the G-actin-S1 complexes was not changed. However, since the dissociation constants remain in the submicromolar range, we could demonstrate the existence of ternary DNase I-G-actin-S1 complexes stable under polymerizing conditions. Finally, the study of the effect of nucleotides and of various salts on the G-actin-S1 interaction further showed significant differences between the G-actin-S1 and F-actin-S1 interactions.


Asunto(s)
Actinas/metabolismo , Desoxirribonucleasa I/farmacología , Subfragmentos de Miosina/metabolismo , Actinas/química , Actinas/aislamiento & purificación , Animales , Desoxirribonucleasa I/metabolismo , Cinética , Luz , Sustancias Macromoleculares , Músculos/metabolismo , Subfragmentos de Miosina/química , Subfragmentos de Miosina/aislamiento & purificación , Unión Proteica , Conejos , Dispersión de Radiación , Espectrometría de Fluorescencia , Factores de Tiempo
12.
Mol Cell Biol ; 13(3): 1641-50, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8441402

RESUMEN

Mammalian U6 small nuclear RNA (snRNA) is heterogeneous with respect to the number of 3' terminal U residues. The major form terminates with five U residues and a 2',3' cyclic phosphate. Because of the presence in HeLa cell nuclear extracts of a terminal uridylyl transferase, a minor form of U6 snRNA is elongated, producing multiple species containing up to 12 U residues. In this study we have used glycerol gradients to demonstrate that these U6 snRNA forms are assembled into U6 ribonucleoprotein (RNP), U4/U6 snRNPs, and U4/U5/U6 tri-snRNP complexes. Furthermore, glycerol gradients combined with affinity selection of biotinylated pre-mRNAs led us to show that elongated forms of U6 snRNAs enter the spliceosome and that some of these become shortened with time to a single species having the same characteristics as the major form of U6 snRNA present in mammalian nuclear extracts. We propose that this elongation-shortening process is related to the function of U6 snRNA in mammalian pre-mRNA splicing.


Asunto(s)
Empalme del ARN , ARN Nuclear Pequeño/metabolismo , Empalmosomas/metabolismo , Uridina Monofosfato/metabolismo , Composición de Base , Núcleo Celular/metabolismo , Sistema Libre de Células , Variación Genética , Células HeLa , Humanos , Sustancias Macromoleculares , Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/aislamiento & purificación , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Transcripción Genética , UDP-Glucosa-Hexosa-1-Fosfato Uridiltransferasa/metabolismo
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