RESUMO
Expansions of trinucleotide repeats within gene transcripts are responsible for fragile X syndrome, myotonic dystrophy and spinal and bulbar muscular atrophy. To identify other human genes with similar features as candidates for triplet repeat expansion mutations, we screened human cDNA libraries with repeat probes and searched databases for transcribed genes with repeats. From both strategies, 40 genes were identified and 14 characterized. Five were found to contain repeats which are highly polymorphic including the N-cadherin, BCR, glutathione-S-transferase and Na+/K+ ATPase (beta-subunit) genes. These data demonstrate the occurrence of other human loci which may undergo this novel mechanism of mutagenesis giving rise to genetic disease.
Assuntos
Biblioteca Gênica , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico/genética , Sequência de Bases , Caderinas/genética , Clonagem Molecular , DNA Complementar/genética , Feminino , Glutationa Transferase/genética , Humanos , Masculino , Dados de Sequência Molecular , Família Multigênica/genética , Oligodesoxirribonucleotídeos , Linhagem , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Fragile X syndrome is the result of the unstable expansion of a trinucleotide repeat in the 5'-untranslated region of the FMR1 gene. Fibroblast subclones from a mildly affected patient, each containing stable FMR1 alleles with 57 to 285 CGG repeats, were shown to exhibit normal steady-state levels of FMR1 messenger RNA. However, FMR protein was markedly diminished from transcript with more than 200 repeats. Such transcripts were associated with stalled 40S ribosomal subunits. These results suggest that a structural RNA transition beyond 200 repeats impedes the linear 40S migration along the 5'-untranslated region. This results in translational inhibition by trinucleotide repeat expansion.
Assuntos
Síndrome do Cromossomo X Frágil/genética , Proteínas do Tecido Nervoso/genética , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA , Sequências Repetitivas de Ácido Nucleico/genética , Centrifugação com Gradiente de Concentração , Células Clonais , Regulação para Baixo/genética , Feminino , Fibroblastos/química , Proteína do X Frágil da Deficiência Intelectual , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
Transferrin (Tf) crystals diffract to only medium resolution. The mediocre quality of the crystals may be due to two factors: (1) the genetic variations naturally present in the primary sequence of Tf, and (2) the glycosylation of the protein. To control genetic variations and glycosylation of samples of Tf, it would be desirable to express the Tf gene from a recombinant clone. Additionally, expression of Tf from a clone would allow for manipulation of the structure of Tf. The cDNA encoding Tf has been cloned into the pL-based expression vector, pRE1, and the T7-based expression vectors, pRSETA and pET11A. The Tf expression plasmids, pTF-SSn and pTF-ESn, based on the T7 expression vectors, efficiently produce a 76-kDa protein that is approximately the same size as deglycosylated Tf, cross reacts with anti-Tf antibodies, and matches the deduced N-terminal amino acid sequence. Expression of Tf in Escherichia coli will allow the production of genetically pure, unglycosylated protein.