RESUMEN
Autosomal dominant hypercholesterolemia (ADH), one of the most frequent hereditary disorders, is characterized by an isolated elevation of LDL particles that leads to premature mortality from cardiovascular complications. It is generally assumed that mutations in the LDLR and APOB genes account for ADH. We identified one large French pedigree (HC2) and 12 additional white families with ADH in which we excluded linkage to the LDLR and APOB, implicating a new locus we named "FH3." A LOD score of 3.13 at a recombination fraction of 0 was obtained at markers D1S2892 and D1S2722. We localized the FH3 locus to a 9-cM interval at 1p34.1-p32. We tested four regional markers in another set of 12 ADH families. Positive LOD scores were obtained in three pedigrees, whereas linkage was excluded in the others. Heterogeneity tests indicated linkage to FH3 in approximately 27% of these non-LDLR/non-APOB ADH families and implied a fourth locus. Radiation hybrid mapping located four candidate genes at 1p34.1-p32, outside the critical region, showing no identity with FH3. Our results show that ADH is genetically more heterogeneous than conventionally accepted.
Asunto(s)
Apolipoproteínas B/genética , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Adulto , LDL-Colesterol , Femenino , Marcadores Genéticos , Humanos , Escala de Lod , LinajeRESUMEN
In most cases, xeroderma pigmentosum group D (XP-D) and trichothiodystrophy (TTD) patients carry mutations in the carboxy-terminal domain of the evolutionarily conserved helicase XPD, which is one of the subunits of the transcription/repair factor TFIIH (refs 1,2). In this study, we demonstrate that XPD interacts specifically with p44, another subunit of TFIIH, and that this interaction results in the stimulation of 5'-->3' helicase activity. Mutations in the XPD C-terminal domain, as found in most patients, prevent the interaction with p44, thus explaining the decrease in XPD helicase activity and the nucleotide excision repair (NER) defect.
Asunto(s)
ADN Helicasas/genética , Reparación del ADN , Proteínas de Unión al ADN , Enfermedades del Cabello/genética , Proteínas/genética , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Xerodermia Pigmentosa/genética , ADN Helicasas/metabolismo , Humanos , Mutación , Conformación Proteica , Proteínas/metabolismo , Factor de Transcripción TFIIH , Proteína de la Xerodermia Pigmentosa del Grupo DAsunto(s)
ADN Helicasas/genética , Reparación del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Mutación , Factores de Transcripción TFII , Factores de Transcripción/farmacología , Xerodermia Pigmentosa/genética , Humanos , Factor de Transcripción TFIIH , Xerodermia Pigmentosa/enzimologíaRESUMEN
We describe here the methodology developed in our laboratory to study the role of TFIIH, a multisubunit protein complex, in the various mechanisms of cell life: transcription, DNA repair, and cell cycle regulation. Protocols are given to purify TFIIH and to study its various enzymatic activities as well as its transcription and nucleotide excision repair activities.
Asunto(s)
Reparación del ADN , Factores de Transcripción TFII , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Células HeLa , Humanos , Factor de Transcripción TFIIH , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
TFIIH is a multiprotein factor involved in transcription and DNA repair and is implicated in DNA repair/transcription deficiency disorders such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Eight out of the nine genes encoding the subunits forming TFIIH have already been cloned. We report here the identification, cDNA cloning and gene structure of the 52 kDa polypeptide and its homology with the yeast counterpart TFB2. This protein, along with p89/XPB, p62, p44 and p34, forms the core of TFIIH. Moreover, using in vitro reconstituted transcription and nucleotide excision repair (NER) assays and microinjection experiments, we demonstrate that p52 is directly involved in both transcription and DNA repair mechanisms in vitro and in vivo.
Asunto(s)
Reparación del ADN , Proteínas de Saccharomyces cerevisiae , Factores Asociados con la Proteína de Unión a TATA , Factor de Transcripción TFIID , Factores de Transcripción TFII , Factores de Transcripción/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Western Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 6 , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Humanos , Hibridación Fluorescente in Situ , Microinyecciones , Datos de Secuencia Molecular , Saccharomyces cerevisiae/química , Alineación de Secuencia , Análisis de Secuencia , Factor de Transcripción TFIIH , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
A deletion of 7q21.2-q22.1 has been found in a patient with split foot and developmental retardation. Molecular analysis using polymerase chain reaction (PCR) showed deletion of three microsatellite markers, D7S527, D7S479 and D7S554, in the patient's paternal chromosome. These results pinpoint the critical region for an ectrodactyly locus (SHFD1) on chromosome 7.
Asunto(s)
Anomalías Múltiples/genética , Deleción Cromosómica , Cromosomas Humanos Par 7 , Deformidades Congénitas del Pie/genética , Deformidades Congénitas de la Mano/genética , Discapacidad Intelectual/genética , Preescolar , Eliminación de Gen , Marcadores Genéticos , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Sindactilia/genéticaRESUMEN
Ectrodactyly (split hand/split foot, SHSF) is characterized by the absence of middle rays of the hand or the foot. Cytogenetic analyses of some of the cases have indicated an association between chromosomal rearrangements involving 7q21.3-q22 and ectrodactyly. Based on these observations, an autosomal dominant form of ectrodactyly is assumed to reside in this region and the locus has been designated SHFD1 (split hand/split foot disorder). Here we report a large family where split hand/split foot long bone deficiency (SHFLD) segregates in an autosomal dominant mode. Linkage analysis, using microsatellite markers located in 7q21-q22, excludes this region from containing the gene responsible for SHFLD in this family. These results would appear to indicate genetic heterogeneity exists in autosomal dominant SHSF.
Asunto(s)
Anomalías Múltiples/genética , Cromosomas Humanos Par 7 , Deformidades Congénitas del Pie/genética , Ligamiento Genético , Deformidades Congénitas de la Mano/genética , Femenino , Marcadores Genéticos , Humanos , Escala de Lod , Masculino , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
We have defined a radiation hybrid panel for Xq11-q22 characterized with 20 DNA markers and shown how the panel can be used in conjunction with Alu-PCR and a gridded X-specific cosmid library to isolate cosmids from a preselected subregion. We used Alu-PCR products derived from two radiation hybrids, IHB2-B30 and IHB1-A12, sharing a segment in proximal Xq13.1, containing DXS453 to CCG1, as the only human component in common. Used as probes, these Alu-PCR products identified 39 cosmids on the gridded X-cosmid libraries that were positive for both probes. The target specificity of the derived cosmids was very high, 11 of 13 cosmids tested mapped to the region of fragment overlap in the hybrids, as was determined by two translocation breakpoints bordering most of the target interval. Accounting for a redundancy of four in the libraries, the isolated 39 cosmids should correspond to about 10 independent loci derived from the region of fragment overlap defined by these radiation hybrids. In addition, a subset of five radiation hybrids having breakpoints in the target region could be used to further subdivide the 11 cosmids into an ordered set of five submapping intervals of Xq13.1.
