RESUMEN
BACKGROUND/OBJECTIVES: The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes. SUBJECTS/METHODS: We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox (Hox) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed. RESULTS: Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance (P<0.001) and insulin sensitivity (P<0.05). MeDIP-seq data analysis revealed a uniform distribution of differentially methylated regions (DMR) through the entire adipocyte genome, with a higher number of hypermethylated regions in HFD mice (P<0.005). This different methylation profile was accompanied by increased expression of the Dnmt3a DNA methyltransferase (Dnmt; P<0.05) and the methyl-CpG-binding domain protein Mbd3 (P<0.05) genes in HFD mice. Gene ontology analysis revealed that, in the HFD-treated mice, the Hox family of development genes was highly enriched in differentially methylated genes (P=0.008). To validate this finding, Hoxa5, which is implicated in fat tissue differentiation and remodeling, has been selected and analyzed by bisulphite sequencing, confirming hypermethylation in the adipose tissue from the HFD mice. Hoxa5 hypermethylation was associated with downregulation of Hoxa5 mRNA and protein expression. Feeding animals previously exposed to the HFD with a standard chow diet for two further months improved the metabolic phenotype of the animals, accompanied by return of Hoxa5 methylation and expression levels (P<0.05) to values similar to those of the control mice maintained under standard chow. CONCLUSIONS: HFD induces adipose tissue abnormalities accompanied by epigenetic changes at the Hoxa5 adipose tissue remodeling gene.
Asunto(s)
Tejido Adiposo/metabolismo , Metilación de ADN , Dieta Alta en Grasa , Regulación hacia Abajo , Proteínas de Homeodominio/genética , Fosfoproteínas/genética , Transcripción Genética , Animales , Modelos Animales de Enfermedad , Epigénesis Genética , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , Factores de TranscripciónRESUMEN
The gene RPGR was previously identified in the RP3 region of Xp21.1 and shown to be mutated in 10-20% of patients with the progressive retinal degeneration X-linked retinitis pigmentosa (XLRP). The mutations predominantly affected a domain homologous to RCC1, a guanine nucleotide exchange factor for the small GTPase Ran, although they were present in fewer than the 70-75% of XLRP patients predicted from linkage studies. Mutations in the RP2 locus at Xp11.3 were found in a further 10-20% of XLRP patients, as predicted from linkage studies. Because the mutations in the remainder of the XLRP patients may reside in undiscovered exons of RPGR, we sequenced a 172-kb region containing the entire gene. Analysis of the sequence disclosed a new 3' terminal exon that was mutated in 60% of XLRP patients examined. This exon encodes 567 amino acids, with a repetitive domain rich in glutamic acid residues. The sequence is conserved in the mouse, bovine and Fugu rubripes genes. It is preferentially expressed in mouse and bovine retina, further supporting its importance for retinal function. Our results suggest that mutations in RPGR are the only cause of RP3 type XLRP and account for the disease in over 70% of XLRP patients and an estimated 11% of all retinitis pigmentosa patients.
Asunto(s)
Proteínas Portadoras/genética , Exones/genética , Proteínas del Ojo , Retinitis Pigmentosa/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN/química , ADN/genética , Análisis Mutacional de ADN , Salud de la Familia , Peces , Ligamiento Genético , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Sistemas de Lectura Abierta , Polimorfismo Conformacional Retorcido-Simple , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Células Tumorales Cultivadas , Cromosoma X/genéticaRESUMEN
The X and Y chromosomes that maintain human dimorphism are thought to have descended from a single progenitor, with the Y chromosome becoming largely depleted of genes. A number of genes, however, retain copies on both X and Y chromosomes and escape the inactivation that affects most X-linked genes in somatic cells. Many of those genes are present in two pseudoautosomal regions (PARs) at the termini of the short (p) and long (q) arms of the sex chromosomes. For both PARs, pairing facilitates the exchange of information, ensuring the homogenisation of X and Y chromosomal material in these regions. We report here a strikingly different regulation of expression of a gene in Xq PAR. Unlike all Xp PAR genes studied so far, a synaptobrevin-like gene, tentatively named SYBL1, undergoes X inactivation. In addition, it is also inactive on the Y chromosome, thereby maintaining dosage compensation in an unprecedented way.
Asunto(s)
Proteínas de Arabidopsis , Compensación de Dosificación (Genética) , Proteínas de la Membrana/genética , Cromosoma X , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Artificiales de Levadura , Regulación de la Expresión Génica , Humanos , Células Híbridas , Masculino , Proteínas de la Membrana/biosíntesis , Datos de Secuencia Molecular , Proteínas R-SNARE , Ribonucleasas , Homología de Secuencia de Aminoácido , Transcripción Genética , Cromosoma YRESUMEN
X-linked retinitis pigmentosa (xlRP) is a severe progressive retinal degeneration which affects about 1 in 25,000 of the population. The most common form of xlRP, RP3, has been localised to the interval between CYBB and OTC in Xp21.1 by linkage analysis and deletion mapping. Identification of microdeletions within this region has now led to the positional cloning of a gene, RPGR, that spans 60 kg of genomic DNA and is ubiquitously expressed. The predicted 90 kD protein contains in its N-terminal half a tandem repeat structure highly similar to RCC1 (regulator of chromosome condensation), suggesting an interaction with a small GTPase. The C-terminal half contains a domain, rich in acidic residues, and ends in a potential isoprenylation anchorage site. The two intragenic deletions, two nonsense and three missense mutations within conserved domains provide evidence that RPGR (retinitis pigmentosa GTPase regulator) is the RP3 gene.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Proteínas del Ojo , Proteínas de Unión al GTP/genética , Factores de Intercambio de Guanina Nucleótido , Retinitis Pigmentosa/genética , Cromosoma X , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Femenino , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Humanos , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Linaje , Reacción en Cadena de la Polimerasa , Prenilación de Proteína , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Xenopus , Proteínas de XenopusRESUMEN
We report a novel PPARG germline mutation in a patient affected by colorectal cancer that replaces serine 289 with cysteine in the mature protein (S289C). The mutant has impaired transactivation potential and acts as dominant negative to the wild type receptor. In addition, it no longer restrains cell proliferation both in vitro and in vivo. Interestingly, the S289C mutant poorly activates target genes and interferes with the inflammatory pathway in tumor tissues and proximal normal mucosa. Consistently, only mutation carriers exhibit colonic lesions that can evolve to dysplastic polyps. The proband presented also dyslipidemia, hypertension and overweight, not associated to type 2 diabetes; of note, family members tested positive for the mutation and display only a dyslipidemic profile at variable penetrance with other biochemical parameters in the normal range. Finally, superimposing the mutation to the crystal structure of the ligand binding domain, the new Cys289 becomes so closely positioned to Cys285 to form an S-S bridge. This would reduce the depth of the ligand binding pocket and impede agonist positioning, explaining the biological effects and subcellular distribution of the mutant protein. This is the first PPARG germline mutation associated with dyslipidemia and colonic polyp formation that can progress to full-blown adenocarcinoma.
Asunto(s)
Dislipidemias/genética , Mutación de Línea Germinal , Pólipos Intestinales/genética , PPAR gamma/genética , Adenocarcinoma/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Células COS , Chlorocebus aethiops , Neoplasias del Colon/genética , Cartilla de ADN/genética , Dislipidemias/metabolismo , Femenino , Humanos , Técnicas In Vitro , Pólipos Intestinales/metabolismo , Pérdida de Heterocigocidad , Masculino , Ratones , Persona de Mediana Edad , Modelos Moleculares , Células 3T3 NIH , PPAR gamma/química , PPAR gamma/metabolismo , Linaje , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Adulto JovenRESUMEN
The past few years have seen rapid advances in our understanding of the genetics and molecular biology of cerebral cavernous malformations (CCM) with the identification of the CCM1, CCM2, and CCM3 genes. Recently, we have recruited a patient with an X/3 balanced translocation that exhibits CCM. By fluorescent in situ hybridization analysis, sequence analysis tools and database mining procedures, we refined the critical region to an interval of 200-kb and identified the interrupted ZPLD1 gene. We detected that the mRNA expression level of ZPLD1 gene is consistently decreased 2.5-fold versus control (P=0.0006) with allelic loss of gene expression suggesting that this protein may be part of the complex signaling pathway implicated in CCM formation.
Asunto(s)
Cromosomas Humanos Par 3 , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal/fisiología , Translocación Genética , Adulto , Línea Celular , Rotura Cromosómica , Bases de Datos de Proteínas , Femenino , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/fisiología , Imagen por Resonancia Magnética , Fenotipo , Insuficiencia Ovárica Primaria/complicaciones , Insuficiencia Ovárica Primaria/genética , ARN Mensajero/metabolismo , Inactivación del Cromosoma X/genéticaRESUMEN
PRDM2/RIZ is a member of a superfamily of histone/protein methyltransferases (PRDMs), which are characterized by the conserved N-terminal PR domain, with methyltransferase activity and zinc finger arrays at the C-terminus. Similar to other family members, two main protein types, known as RIZ1 and RIZ2, are produced from the PRDM2 locus differing by the presence or absence of the PR domain. The imbalance in their respective amounts may be an important cause of malignancy, with the PR-positive isoform commonly lost or downregulated and the PR-negative isoform always being present at higher levels in cancer cells. Interestingly, the RIZ1 isoform also represents an important target of estradiol action downstream of the interaction with hormone receptor. Furthermore, the imbalance between the two products could also be a molecular basis for other human diseases. Thus, understanding the molecular mechanisms underlying PRDM2 function could be useful in the pathophysiological context, with a potential to exploit this information in clinical practice.
RESUMEN
Retinitis pigmentosa is the most common form of retinal degeneration and is heterogeneous both clinically and genetically. The autosomal dominant forms (ADRP) can be caused by mutations in 12 different genes. This report describes the first simultaneous mutation analysis of all the known ADRP genes in the same population, represented by 43 Italian families. This analysis allowed the identification of causative mutations in 12 of the families (28% of the total). Seven different mutations were identified, two of which are novel (458delC and 6901C-->T (P2301S), in the CRX and PRPF8 genes, respectively). Several novel polymorphisms leading to amino acid changes in the FSCN2, NRL, IMPDH1, and RP1 genes were also identified. Analysis of gene prevalences indicates that the relative involvement of the RHO and the RDS genes in the pathogenesis of ADRP is less in Italy than in US and UK populations. As causative mutations were not found in over 70% of the families analysed, this study suggests the presence of further novel genes or sequence elements involved in the pathogenesis of ADRP.
Asunto(s)
Genes Dominantes , Retinitis Pigmentosa/genética , Adolescente , Adulto , Edad de Inicio , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas Portadoras/genética , Niño , Preescolar , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas del Ojo/genética , Familia , Frecuencia de los Genes , Proteínas de Homeodominio/genética , Humanos , Italia/epidemiología , Proteínas Asociadas a Microtúbulos , Persona de Mediana Edad , Mutación , Prevalencia , Proteínas de Unión al ARN , Retinitis Pigmentosa/clasificación , Retinitis Pigmentosa/epidemiología , Rodopsina/genética , Transactivadores/genéticaRESUMEN
PURPOSE: To report the clinical and functional characteristics of an autosomal dominant retinitis pigmentosa (ADRP) family with a novel point mutation (P2301S) in the PRPF8 gene. METHODS: PRPF8 gene analysis and complete ophthalmologic examination in an ADRP family. RESULTS: Clinical examination revealed the typical RP phenotype in all family members. Electroretinography showed preserved ERG photopic responses. Genetic analysis showed that the P2301S missense mutation segregated with the disease in all subjects. CONCLUSIONS: Unlike previously reported families, the PRPF8 gene mutation in our family is associated with a mild phenotype in which cone function is partially preserved.
Asunto(s)
Proteínas Portadoras/genética , ADN/genética , Mutación , Retinitis Pigmentosa/genética , Electrorretinografía , Femenino , Humanos , Italia , Masculino , Oftalmoscopía , Linaje , Fenotipo , Proteínas de Unión al ARN , Retinitis Pigmentosa/diagnósticoRESUMEN
X-linked Retinitis Pigmentosa (XLRP) shows a huge genetic heterogeneity with almost five distinct loci on the X chromosome. So far, only two XLRP genes have been identified, RPGR (or RP3) and RP2, being mutated in approximately 70% and 10% of the XLRP patients. Clinically there is no clearly significative difference between RP3 and RP2 phenotypes. In the attempt to assess the degree of involvement of the RP2 gene, we performed a complete mutation analysis in a cohort of patients and we identified five novel mutations in five different XLRP families. These mutations include three missense mutations, a splice site mutation, and a single base insertion, which, because of frameshift, anticipates a stop codon. Four mutations fall in RP2 exon 2 and one in exon 3. Evidence that such mutations are different from the 21 RP2 mutations described thus far suggests that a high mutation rate occurs at the RP2 locus, and that most mutations arise independently, without a founder effect. Our mutation analysis confirms the percentage of RP2 mutations detected so far in populations of different ethnic origin. In addition to novel mutations, we report here that a deeper sequence analysis of the RP2 product predicts, in addition to cofactor C homology domain, further putative functional domains, and that some novel mutations identify RP2 amino acid residues which are evolutionary conserved, hence possibly crucial to the RP2 function.
Asunto(s)
Ligamiento Genético/genética , Mutación/genética , Retinitis Pigmentosa/genética , Cromosoma X/genética , Secuencia de Aminoácidos , Secuencia de Bases , Estudios de Cohortes , Secuencia Conservada/genética , Análisis Mutacional de ADN , Etnicidad/genética , Exones/genética , Femenino , Heterogeneidad Genética , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de SecuenciaRESUMEN
X-linked congenital stationary night blindness (CSNBX) is a hereditary non-progressive retinal disorder, which can appear in two different clinical forms, complete and incomplete, associated with CSNB1 and CSNB2 loci on Xp. We describe a Sardinian family with complete CSNBX and define better the limits of the CSNB1 genetic locus on Xp11.4 through linkage analysis. Haplotype analysis showed two key recombinants, which restrict the CSNB1 locus to a region of about 3 cM limited by markers DSX1068 and DSX6810 respectively. The locus that we describe is included in the CSNB1 locus defined by previous reports referring to the same clinical form of the disease. These results, in addition to other recent mapping reports about families from different geographical areas, confirm the genetic homogeneity of X-linked complete CSNB.
Asunto(s)
Ceguera Nocturna/congénito , Ceguera Nocturna/genética , Cromosoma X , Mapeo Cromosómico , Haplotipos , Humanos , Italia , Escala de LodRESUMEN
Rod-specific cGMP phosphodiesterase (PDE) is a key enzyme of the phototransduction cascade, and mutations in its catalytic subunits have been associated with retinal degenerative diseases. The bovine delta-subunit solubilises the normally membrane-bound PDE and is the only subunit expressed in extraocular tissues. We isolated the human and mouse orthologs, and found 78% identity at the DNA level and 98% identity at the protein level. The Caenorhabditis elegans homolog shows 69% identity at the protein level. The human PDED gene consisted of 5 exons spanning at least 30 kb of genomic DNA. Northern blot analysis showed a 1.3 kb transcript in human retina, heart, brain, placenta, liver, and skeletal muscle. Fluorescence in situ hybridisation (FISH) and radiation hybrid mapping localised the human PDED gene to chromosome 2q37. A preliminary screen of all 5 exons in 20 unrelated patients with autosomal recessive retinitis pigmentosa revealed no PDED mutations.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , ADN Complementario , Exones , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Mutación , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis characterised by facial dysmorphisms, mental retardation and multiple congenital anomalies. SLOS is caused by mutations of the human Delta7-sterol reductase (DHCR7) gene and, so far, 19 different mutations have been described. Among these, mutations impairing the activity of the C-terminus appear to be the most severe. Here we report the mutational analysis of the DHCR7 gene in nine Italian SLOS patients. The T93M mutation, previously reported in one patient, results the most frequent one (7/18 alleles) in our survey. Furthermore, we identified three novel mutations, two missense mutations (N407Y and E448K), and a 33 bp deletion spanning part of exon 5 and the donor splice site of intron 5.
Asunto(s)
Mutación Missense , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Oxidorreductasas/genética , Síndrome de Smith-Lemli-Opitz/genética , Adolescente , Alelos , Preescolar , Colesterol/biosíntesis , Análisis Mutacional de ADN , Cara/anomalías , Femenino , Eliminación de Gen , Humanos , Lactante , Discapacidad Intelectual/genética , Italia , Masculino , LinajeRESUMEN
The RPGR (retinitis pigmentosa GTPase regulator) gene has been shown to be mutated in 10-20% of patients with X-linked retinitis pigmentosa (XLRP), a severe form of inherited progressive retinal degeneration. A total of 29 different RPGR mutations have been identified in northern European and United States patients. We have performed mutation analysis of the RPGR gene in a cohort of 49 southern European males affected with XLRP. By multiplex SSCA and automatic direct sequencing of all 19 RPGR exons, seven different and novel mutations were identified in eight of the 49 families; these include three splice site mutations, two microdeletions, and two missense mutations. RNA analysis showed that the three splice site defects resulted in the generation of aberrant RPGR transcripts. Six of these mutations were detected in the conserved amino-terminal region of RPGR protein, containing tandem repeats homologous to the RCC1 protein, a guanine nucleotide-exchange factor for Ran-GTPase. Several exonic and intronic sequence variations were also detected. None of the RPGR mutations reported in other populations were identified in our series. Our results are consistent with the notions of heterogeneity and minority causation of XLRP by mutations in RPGR in Caucasian populations.
Asunto(s)
Proteínas Portadoras/genética , Proteínas del Ojo , Ligamiento Genético , Mutación , Retinitis Pigmentosa/genética , Cromosoma X , Secuencia de Bases , Análisis Mutacional de ADN , Europa (Continente)/epidemiología , Exones , Femenino , Eliminación de Gen , Variación Genética , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Mutación Missense , Linaje , Polimorfismo Genético , Empalme del ARN , Retinitis Pigmentosa/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estados Unidos/epidemiologíaRESUMEN
The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.
Asunto(s)
Clonación Molecular , ADN/genética , Escherichia coli/genética , Glucosafosfato Deshidrogenasa/biosíntesis , Secuencia de Bases , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/genética , Glucosafosfato Deshidrogenasa/inmunología , Humanos , Isopropil Tiogalactósido/farmacología , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Represoras/genética , Transformación GenéticaRESUMEN
The dbl oncogene is generated by substitution of the 5' portion of its normal counterpart with an unrelated human sequence. To analyze the genomic structure and transcriptional regulation of the dbl proto-oncogene, we have isolated human genomic clones containing the entire human proto-dbl gene, localized in Xq26. Restriction mapping of a 600kb YAC clone (yWXD311) placed proto-dbl about 50kb telomeric to the coagulation Factor IX gene. The genomic DNA fragment containing the 5' end of proto-dbl was subcloned into plasmid vectors and the nucleotide sequences of exon 1, the flanking intronic region and genomic DNA 5' of the first codon were determined. Sequence analysis of 85119bp from the region revealed the genomic structure of proto-dbl. It contains 25 exons coding for a 4.7kb transcript including large 5'- and 3'- (1218bp and 701bp, respectively) untranslated regions (UTRs). RNase protection and primer extension assays on RNA from medullary thyroid carcinoma (TT) cells, which normally express dbl, revealed a transcription start site 1218bp upstream of the ATG of the first exon. A 1.6kb genomic 5' of the translation start sites drives the expression of a CAT-reporter in transient transfections in the TT cell line, though lacking TATA or CAAT boxes.
Asunto(s)
Proteínas Proto-Oncogénicas/genética , Transcripción Genética , Cromosoma X/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN/química , ADN/genética , Exones , Genes/genética , Factores de Intercambio de Guanina Nucleótido , Humanos , Intrones , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
STSs, which have been used to build and format clone contigs, have been used here to assemble a transcriptional map across a cytogenetic band. Of fifty one STSs in Xq28, 20 were positive by RT-PCR. Thus, an additional 20 possible ESTs were detected among the STSs, and seven of these also identified cDNAs in at least one library. The transcripts confirm the high expression level of this region, correlated with its GC compositional map and CpG island content.
Asunto(s)
Lugares Marcados de Secuencia , Transcripción Genética , Cromosoma X , Secuencia de Bases , Northern Blotting , ADN Complementario , Expresión Génica , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
SYBL1 is a gene in the 320kb human pseudo-autosomal region at the terminus of Xq and Yq. In contrast to other pseudoautosomal genes, SYBL1 is inactivated on one X in every female cell, and is also inactive on the Y of male cells. Hypermethylation of the CpG island associated with the human gene is involved in this phenomenon. In an attempt to further examine its regulation, the genomic organization of the X-linked mouse Sybl1 homolog was analyzed and compared with the human gene. Human and mouse show the same exon number, exon-intron junctions and a highly conserved basal promoter. The structural and functional conservation of basal regulatory regions suggests that inactivation is imposed by similar auxiliary epistatic regulatory mechanism.
Asunto(s)
Genes/genética , Proteínas de la Membrana/genética , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN/química , ADN/genética , Exones , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Humanos , Intrones , Masculino , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas R-SNARE , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Transcripción GenéticaRESUMEN
We describe the structure, genomic organization, and some transcription features of a human brain-specific gene previously localized to the genomic region involved in temporal lobe epilepsy and spastic paraplegia on chromosome 10q24. The gene, which consists of six exons disseminated over 16 kb of genomic DNA, is highly homologous to the porcine tmp83.5 gene and encodes a putative transmembrane protein of 141 amino acids. Unlike its porcine homolog, from which two mRNAs with different 5'-sequences are transcribed, the human gene apparently encodes three mRNA species with 3'-untranslated regions of different sizes. Mutation analysis of its coding sequence in families affected with temporal lobe epilepsy or spastic paraplegia linked to 10q24 do not support the involvement of this gene in either diseases.
Asunto(s)
Encéfalo/metabolismo , Cromosomas Humanos Par 10/genética , Epilepsia del Lóbulo Temporal/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Paraplejía/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario/química , ADN Complementario/genética , Exones , Expresión Génica , Genes/genética , Humanos , Intrones , Datos de Secuencia Molecular , Mutación , Proteínas de la Mielina , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
AIMS: To describe the clinical phenotype of X linked juvenile retinoschisis in eight Italian families with six different mutations in the XLRS1 gene. METHODS: Complete ophthalmic examinations, electroretinography and A and B-scan standardised echography were performed in 18 affected males. The coding sequences of the XLRS1 gene were amplified by polymerase chain reaction and directly sequenced on an automated sequencer. RESULTS: Six different XLRS1 mutations were identified; two of these mutations Ile81Asn and the Trp122Cys, have not been previously described. The affected males showed an electronegative response to the standard white scotopic stimulus and a prolonged implicit time of the 30 Hz flicker. In the families with Trp112Cys and Trp122Cys mutations we observed a more severe retinoschisis (RS) clinical picture compared with the other genotypes. CONCLUSION: The severe RS phenotypes associated with Trp112Cys and to Trp122Cys mutations suggest that these mutations determine a notable alteration in the function of the retinoschisin protein.