Asunto(s)
ADN Mitocondrial/genética , Insulina/sangre , Adulto , Anciano , Glucemia/análisis , ADN Mitocondrial/sangre , Diabetes Mellitus Tipo 2/etnología , Diabetes Mellitus Tipo 2/genética , Europa (Continente)/etnología , Ayuno/sangre , Femenino , Intolerancia a la Glucosa/etnología , Intolerancia a la Glucosa/genética , Humanos , Masculino , Persona de Mediana Edad , Nueva Zelanda/epidemiologíaRESUMEN
The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.
Asunto(s)
ADN Mitocondrial , ADN Polimerasa Dirigida por ADN/genética , Mitocondrias/enzimología , Repeticiones de Trinucleótidos , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , ADN Polimerasa gamma , ADN Complementario , Humanos , Datos de Secuencia MolecularRESUMEN
We have previously described the characterisation of an abundant mitochondrial protein (p40) that binds specifically to 5'-untranslated leaders of mitochondrial mRNAs in yeast. p40 consists of two polypeptides with M(r) of 40 and 39 kDa. Limited sequence analysis of p40 identifies it as the Krebs cycle enzyme NAD(+)-dependent isocitrate dehydrogenase (Idh). Both enzyme and RNA-binding activities are specifically lost in cells containing disruptions in either IDH1 or IDH2, the nuclear genes encoding the two subunits of the enzyme, thus conclusively identifying p40 as Idh and showing that both activities are dependent on the simultaneous presence of both subunits. Although we still must ascertain whether and how either function of Idh is regulated and whether the two functions are compatible or mutually exclusive, this combination of dehydrogenase activity and RNA-binding in a single protein may be part of a general regulatory circuit linking the need for mitochondrial function to mitochondrial biogenesis.
Asunto(s)
Isocitrato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Secuencia de Aminoácidos , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/metabolismo , Isocitrato Deshidrogenasa/inmunología , Datos de Secuencia Molecular , Mutagénesis Insercional , Péptidos/química , ARN/metabolismo , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae/enzimologíaRESUMEN
In site-specific recombination reactions catalyzed by Tn3 resolvase, the right and left arms of the res site are always religated to the correct partner. This poses the problem of how resolvase aligns the two sites correctly for the cleavage/religation reaction. We show that the "accessory" binding subsites II and III of res are important for correct alignment of the adjoining crossover subsite (subsite I). Deletion of subsites II and III from one of the two res sites removes a barrier to recombination between incorrectly aligned crossover subsites. Correct alignment does not require any DNA sequence asymmetry in the crossover subsite, DNA supercoiling, or covalent linkage of the two res sites. Our results suggest that correct subsite I alignment is determined by local, resolvase-mediated interactions of subsites II and III of both partners, consistent with a current model of the synapse. Surprisingly, the topological selectivity for intramolecular resolution in a supercoiled substrate does not require subsites II and III in both recombination partners.