RESUMO
The activin type II receptor (ACVR2) contains two identical microsatellites in exons 3 and 10, but only the exon 10 microsatellite is frameshifted in mismatch repair (MMR)-defective colonic tumors. The reason for this selectivity is not known. We hypothesized that ACVR2 frameshifts were influenced by DNA sequences surrounding the microsatellite. We constructed plasmids in which exons 3 or 10 of ACVR2 were cloned +1 bp out of frame of enhanced green fluorescent protein (EGFP), allowing -1 bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells, and subsequent non-fluorescent cells were sorted, cultured and harvested for mutation analysis. We swapped DNA sequences flanking the exon 3 and 10 microsatellites to test our hypothesis. Native ACVR2 exon 3 and 10 microsatellites underwent heteroduplex formation (A(7)/T(8)) in hMLH1(-/-) cells, but only exon 10 microsatellites fully mutated (A(7)/T(7)) in both hMLH1(-/-) and hMSH6(-/-) backgrounds, showing selectivity for exon 10 frameshifts and inability of exon 3 heteroduplexes to fully mutate. Substituting nucleotides flanking the exon 3 microsatellite for nucleotides flanking the exon 10 microsatellite significantly reduced heteroduplex and full mutation in hMLH1(-/-) cells. When the exon 3 microsatellite was flanked by nucleotides normally surrounding the exon 10 microsatellite, fully mutant exon 3 frameshifts appeared. Mutation selectivity for ACVR2 lies partly with flanking nucleotides surrounding each microsatellite.
Assuntos
Pareamento Incorreto de Bases/genética , Reparo do DNA/genética , DNA Intergênico/genética , Éxons/genética , Repetições de Microssatélites/genética , Mutagênese/genética , Receptores de Activinas Tipo II/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Proteína 1 Homóloga a MutL , Proteínas Nucleares/deficiênciaRESUMO
Complex II of mitochondria contains succinate dehydrogenase and subunits to link this enzyme directly to the inner mitochondrial membrane. The four peptides of this complex are the flavoprotein (Fp) and iron-sulfur protein (Ip) of the dehydrogenase, and two integral membrane proteins referred to as C(II-3) and C(II-4). Their respective genes in mammals are SDHA, SDHB, SDHC and SDHD) in order of decreasing molecular weights of the peptides. In this paper we describe the identification of two pseudogenes and the complete characterization and mapping of the active SDHC gene in humans. The active gene, encoding a small peptide of 15.5 kDa, has six exons and five introns extending over 35 kb. It has been mapped at position 1q21, adjacent to the pericentric heterochromatin on the long arm of chromosome 1. Approximately I kb of the promoter region has also been sequenced and examined for sequence motifs suggesting the binding of known transcription factors. Several potential sites for the nuclear respiratory factors NRF-1 and NRF-2 were identified.
Assuntos
Cromossomos Humanos Par 1/genética , Genes , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Mapeamento Cromossômico , Proteínas de Ligação a DNA/metabolismo , Éxons , Fator de Transcrição de Proteínas de Ligação GA , Biblioteca Gênica , Humanos , Hibridização in Situ Fluorescente , Íntrons , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Fator 1 Relacionado a NF-E2 , Fator 1 Nuclear Respiratório , Fatores Nucleares Respiratórios , Regiões Promotoras Genéticas , Pseudogenes , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH and complex II) of the respiratory chain is highly conserved in evolution [Gould et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1934-1938]. We have cloned the entire human Ip cDNA, as well as the Ip-encoding gene (SDH-B) from two genomic human libraries. The cDNA contains a coding sequence of 840 nt, flanked by a 5'-UTR of 133 nt and a 3'-UTR of 123 nt. The entire transcript is encoded by eight exons within approx. 40 kb. The seven introns range in size from 0.75 kb to > 11 kb, and they appear to be of the 'late' intron class. Approx. 5 kb of upstream sequence was also cloned, and approx. 2.4 kb of the promoter region were sequenced and analyzed for consensus elements binding potential transcription factors and transcriptional activators.
Assuntos
Proteínas Ferro-Enxofre/genética , Mitocôndrias/enzimologia , Succinato Desidrogenase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Éxons/genética , Biblioteca Genômica , Humanos , Íntrons/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Conformação Proteica , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A partial human cDNA clone for the iron-protein (IP) subunit of succinate dehydrogenase (EC 1.3.99.1) was used in Southern analyses of restriction enzyme digests of genomic human and hamster DNA as well as hamster-human hybrids containing a limited number of human chromosomes. The gene for this protein was mapped to human chromosome 1. Digestion of genomic DNA with several restriction enzymes yielded two fragments detectable on a Southern blot, in contrast to the expectations based on the sequence of the cDNA clone. A preliminary analysis of a genomic clone with most of the IP gene has indicated the presence of several introns containing restriction sites detected by the Southern analysis. This genomic clone was also used for subregional mapping by fluorescence in situ hybridization (FISH) to human metaphase chromosomes. A single locus in the region 1p35-36.1 was identified.
