RESUMEN
Fragile X syndrome (FRAXA) is characterized at the molecular level by an expansion of a naturally occurring 5'-(CGG)(n)-3' repeat in the promoter and 5'-untranslated region (5'-UTR) of the fragile X mental retardation (FMR1) gene on human chromosome Xq27.3. When expanded, this region is usually hypermethylated. Inactivation of the FMR1 promoter and absence of the FMR1 protein are the likely cause of the syndrome. By using the bisulfite protocol of the genomic sequencing method, we have determined the methylation patterns in this region on single chromosomes of healthy individuals and of selected premutation carriers and FRAXA patients. In control experiments with unmethylated or M- Sss I-premethylated DNAs, this protocol has been ascertained to reliably detect all cytidines or 5-methylcytidines as unmethylated or methylated nucleotides, respectively. Analyses of the DNA from FRAXA patients reveal considerable variability in the lengths of the 5'-(CGG)(n)-3' repeats and in the levels of methylation in the repeat and the 5'-UTR. In one patient (OEl) with high repeat length hetero-geneity ( n = 15 to >200), shorter repeats (n = 20-80) were methylated or unmethylated, longer repeats ( n = 100-150) were often completely methylated, but one repeat with n = 160 proved to be completely unmethylated. This type of methylation mosaicism was observed in several FRAXA patients. In healthy females, methylated 5'-CG-3' sequences were found in some repeats and 5'-UTRs, as expected for the sequences from one of the X chromosomes. The natural FMR1 promoter is methylation sensitive, as demonstrated by the loss of activity in transfection experiments using the unmethylated or M- Sss I-premethylated FMR1 promoter fused to the luciferase gene as an activity indicator.
Asunto(s)
Metilación de ADN , Síndrome del Cromosoma X Frágil/genética , Tamización de Portadores Genéticos , Discapacidad Intelectual/genética , Mosaicismo , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Proteínas de Unión al ARN , Repeticiones de Trinucleótidos , Cromosoma X , Regiones no Traducidas 5'/genética , Secuencia de Bases , Mapeo Cromosómico , ADN/sangre , Escherichia coli , Femenino , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Humanos , Luciferasas/genética , Masculino , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/deficiencia , Linaje , Proteínas Recombinantes de Fusión/biosíntesis , Valores de Referencia , Mapeo RestrictivoRESUMEN
Previous reports have described the human DNA CGG repeat-binding protein (CGGBP1 or p20), which binds specifically to nonmethylated, but not to methylated, 5'-(CGG)(n)-3' repeats in the promoter of the fragile X mental retardation 1 (FMR1) gene. The results of transfection experiments into human HeLa cells using a p20-green fluorescent protein fusion construct indicate that the p20 protein is targeted to the nucleus. By deletion analyses, a nuclear localization signal has been found between amino acids 80 and 84. Deletions between amino acids 69 and 71 and between 95 and 167 interfere with 5'-(CGG)(n)-3' binding. The results of electrophoretic mobility shift assays using DNA with 5'-(CGG)(n)-3' repeats of different lengths render it likely that oligomers of the p20 protein bind to the repeat. In cotransfection experiments, the activity of the FMR1 promoter is reduced by the presence of p20. Upon transfection of the p20 cDNA construct into HeLa cells, transcription of the endogenous FMR1 gene is decreased. The green fluorescent protein-p20 fusion protein associates preferentially with the telomeres of the short arms of human chromosomes 13, 14, 15, 21, and 22. Their telomeres carry the genes for the 28 S rRNA, which contain 5'-(CGG)(n)-3' repeats. The translated region of the p20 gene from three healthy, five fragile X syndrome, and five premutation-carrying individuals has been sequenced, but mutations have not been detected.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Cromosomas/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil/genética , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes , Microscopía Fluorescente , Datos de Secuencia Molecular , Señales de Localización Nuclear/genética , Unión Proteica/genética , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Eliminación de Secuencia , Telómero/metabolismo , TransfecciónRESUMEN
Binding of the Lac repressor to its operator DNA controls the expression of the genes of the lac operon of Escherichia coli. Lac repressor's affinity for the lac operator is diminished by an inducer that affects the structure of the repressor tetramer. Here we report the cloning and sequencing of the mutant Lac repressor i-t gene, whose product, the LacR-t repressor, shows a higher affinity for the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and a lower affinity for the lac operator than the wild-type repressor. We show that the altered phenotype is due to a single amino acid residue replacement; the alanine residue at position 110 in the wild-type is replaced by threonine in i-t. Other amino acid residues in position 110 have been shown to result in an i-s phenotype. For the i-s-substitution of alanine 110 with lysine we demonstrate an increase in the affinity for operator DNA and a decrease in the affinity for IPTG. Thus, A110--> K shows the opposite effect to A110-->T on the repressor protein. We explain the phenotype of the LacR mutants by displacements of the conformational equilibrium for the dimeric repressor unit between RR (high operator affinity, low inducer affinity) and R*R* (low operator affinity, high inducer affinity) towards R*R* in the i-t and towards RR in the i-s mutant in position 110 with respect to the wild-type. The putative structures of the wild-type and mutant Lac repressors confirm this conclusion.