RESUMO
Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome caused by mutations in the adenomatous polyposis coli (APC) gene. Clinical genetic testing fails to identify disease causing mutations in up to 20% of clinically apparent FAP cases. Following the inclusion of multiplex ligation-dependent probe amplification (MLPA) probes specific for APC promoter 1B, seven probands were identified with a deletion of promoter 1B. Using haplotype analysis spanning the APC locus, the seven families appear to be identical by descent from a common founder. The clinical phenotype of 19 mutation carriers is classical FAP with colectomy at an average age of 24. The majority of cases had a large number of duodenal and gastric polyps. Measurements of allele-specific expression of APC mRNA using TaqMan assay confirmed that relative expression in the allele containing the promoter 1B deletion was reduced 42-98%, depending on tissue type. This study confirms the importance of APC promoter deletions as a cause of FAP and identifies a founder mutation in FAP patients from the United States.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Regiões Promotoras Genéticas , Deleção de Sequência , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/química , Adulto , América , Efeito Fundador , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
The two EF-Tu encoding genes, tufA and tufB, of Salmonella typhimurium have been sequenced. Nearly all the differences from their Escherichia coli counterparts are third position changes which do not alter the encoded amino acids. Unexpectedly, most of the changes in one Salmonella tuf gene are paralleled by changes in the other tuf gene perhaps due to gene repair despite the distance separating the genes. Three mutants which cause mis-framing, have their substitutions at codon 375. Explanations for mutants which cause mis-framing are considered and the mechanism of normal reading frame maintenance discussed.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Fator Tu de Elongação de Peptídeos/genética , Biossíntese de Proteínas , Salmonella typhimurium/genética , Sondas de Oligonucleotídeos , Fator Tu de Elongação de Peptídeos/metabolismo , Homologia de Sequência do Ácido NucleicoAssuntos
Polipose Adenomatosa do Colo/genética , Genes APC , Éxons , Feminino , Conversão Gênica , Rearranjo Gênico , Humanos , Íntrons , Masculino , MutaçãoRESUMO
An unusual, spontaneously arising mutant of Escherichia coli tRNA(2Arg) has been deduced to have two extra nucleotides in its anticodon loop and a duplication of ten nucleotide residues in the TFC loop. This conclusion is based on its gene sequence, Northern blot analysis of isolated tRNA and the size of the in vitro-processed tRNA product. In vitro analyses showed that the mutant precursor is processed normally, albeit inefficiently, to a mature tRNA species 12 nucleotides longer than the wild-type. In addition, the mature tRNA functions as a frameshift suppressor in vivo. Several related mutants with more conservative changes within the gene sequence were similarly shown to be accurately processed, albeit with varying degrees of efficiency less than that of the wild-type. These results indicate that in spite of the high degree of evolutionary conservation of the tertiary structure of tRNAs, and the fact that no such naturally-occurring variant has been found, a greatly enlarged tRNA is capable of functioning in protein synthesis. The data also indicate that recognition sites for correct processing of precursor tRNAs may be unexpectedly tolerant of unusual context, and may depend on some specific features of the tertiary structure rather than the overall structure for accurate processing.
Assuntos
Escherichia coli/genética , Mutação , Conformação de Ácido Nucleico , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , RNA de Transferência de Arginina/química , RNA de Transferência de Arginina/genética , Sequência de Bases , Northern Blotting , Clonagem Molecular , Modelos Estruturais , Dados de Sequência Molecular , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Aminoacil-RNA de Transferência/isolamento & purificação , RNA de Transferência de Arginina/isolamento & purificaçãoRESUMO
The mutant tRNA(2Arg) encoded by the genetically-selected frameshift suppressor, sufT621, inserts arginine and causes a +1 reading-frame shift at the proline codon, CCG(U). There is an extra base, G36.1, in argV beta, one of the four identical genes for tRNA(2Arg) in the position between bases 36 and 37, corresponding to the 3' side of the anticodon. The new four-base anticodon, predicted from DNA sequencing to be 3' GGCA 5', is complementary to the four-base codon CCGU. Quadruplet translocation promoted by mutant argV does not require perfect complementarity between the codon and the anticodon since synthetic genes encoding derivatives of tRNA(2Arg) and tRNA(1Pro), with four-base anticodons complementary to three out of the four bases of CCGU, were also shown to be capable of frameshifting. Two other mutants of argV, inferred to have normal-size, seven-base anticodon loops, were also found to be capable of four-base-decoding demonstrating that quadruplet translocation promoted by mutant argV does not require an enlarged anticodon loop. Other alleles of argV, predicted to have nine bases in the anticodon loop, were also found to cause frameshifting. The DNA sequence of two of these showed in addition, either a deletion of G24, or a ten-base duplication in the region corresponding to the TFC arm. A general finding is that mutations in the DHU arm of tRNA(2Arg) are compatible with, and in one case necessary for, frameshifting.
Assuntos
Anticódon/genética , Escherichia coli/genética , Mutação da Fase de Leitura/genética , RNA de Transferência de Arginina/genética , Salmonella typhimurium/genética , Sequência de Aminoácidos , Sequência de Bases , Genes Supressores/genética , Genes Sintéticos/genética , Óperon Lac/genética , Modelos Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Transferência de Prolina/genéticaRESUMO
The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons. A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity. In this study, the mechanism of this trmD3-induced frameshift involving mutant tRNA(Pro) and tRNA(Leu) species has been investigated. Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain. Hybrid beta-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing. The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNA(Pro) deficient in m1G is the frameshifting agent. Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3-dependent +1 frameshifting. Of the four potential sites for tRNA(Leu) (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant. Thus, species of tRNA(Leu) may also act as +1 frameshift suppressors. No -1 frameshifting activity was found with the trmD3 mutant.
Assuntos
Mutação da Fase de Leitura/genética , RNA Bacteriano/genética , RNA de Transferência/genética , Salmonella typhimurium/genética , Supressão Genética/genética , Sequência de Aminoácidos , Sequência de Bases , Guanosina/análogos & derivados , Guanosina/deficiência , Dados de Sequência Molecular , RNA de Transferência de Leucina/genética , RNA de Transferência de Prolina/genética , beta-Galactosidase/genética , beta-Lactamases/genéticaRESUMO
We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.
Assuntos
Fator Tu de Elongação de Peptídeos/genética , Salmonella typhimurium/genética , Sequência de Bases , Sítios de Ligação , Resistência Microbiana a Medicamentos , Genes Bacterianos , Genes Letais , Dados de Sequência Molecular , Mutação , Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Conformação ProteicaRESUMO
Mutations in the DMD gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Readily available clinical tests detect only deletions of one exon or greater, which are found in approximately 60% of cases. Mutational analysis of other types of DMD mutations, such as premature stop codons and small frameshifting insertions or deletions, has historically been hampered by the large size of the gene. We have recently reported a method that allows the rapid and economical sequencing of the entire coding region of the DMD gene, and that is more sensitive than methods based on single-strand conformational polymorphism (SSCP) screening or other preliminary screening steps. Here we use single condition amplification/internal primer (SCAIP) sequencing analysis, in combination with multiplex amplifiable probe hybridization (MAPH) analysis of duplications, to report the frequency of mutations in a large cohort of unselected dystrophinopathy patients from a single clinic. Our results indicate that 7% of dystrophinopathy patients do not have coding region mutations, suggesting that intronic mutations are not uncommon. The availability of rapid and thorough mutation analysis from peripheral blood samples, along with an improved estimate of the percentage of non-coding region mutations, will be of benefit for improved genetic counseling and in identification of cohorts for clinical trials.
Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Mutação , Códon sem Sentido , Estudos de Coortes , DNA/química , DNA/genética , Análise Mutacional de DNA/métodos , Éxons/genética , Mutação da Fase de Leitura , Deleção de Genes , Duplicação Gênica , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Mutagênese Insercional , Mutação de Sentido Incorreto , Fenótipo , Polimorfismo Conformacional de Fita SimplesRESUMO
A method for concatenating exons from genomic DNA, thereby skipping large stretches of intron sequence, has been developed using the polymerase chain reaction (PCR) with primers based on known intron-exon junction sequences. The use of genomic DNA circumvents the need for cDNA preparation for many purposes, including cDNA construction and mutational analysis. This PCR method also facilitates the concatenation of nonconsecutive exons, allowing different (known or hypothetical) splice-forms to be amplified. We have used this technique to obtain concatamers of exons 3-9A of APC, a tumor suppressor gene that is mutated in sporadic colorectal cancers and in the germline of individuals with adenomatous polyposis coli. This method also facilitates the generation of any polymorphic derivative of a known sequence, even where the derivative differs from the available sequence at several positions.
Assuntos
Éxons/genética , Genoma , Íntrons/genética , Fases de Leitura Aberta , Reação em Cadeia da Polimerase/métodos , Processamento Alternativo , Primers do DNA , Genes APC , Polimorfismo GenéticoRESUMO
Codon usage data has been compiled for 110 yeast genes. Cluster analysis on relative synonymous codon usage revealed two distinct groups of genes. One group corresponds to highly expressed genes, and has much more extreme synonymous codon preference. The pattern of codon usage observed is consistent with that expected if a need to match abundant tRNAs, and intermediacy of tRNA-mRNA interaction energies are important selective constraints. Thus codon usage in the highly expressed group shows a higher correlation with tRNA abundance, a greater degree of third base pyrimidine bias, and a lesser tendency to the A+T richness which is characteristic of the yeast genome. The cluster analysis can be used to predict the likely level of gene expression of any gene, and identifies the pattern of codon usage likely to yield optimal gene expression in yeast.
Assuntos
Códon , Regulação da Expressão Gênica , RNA Mensageiro , Saccharomyces cerevisiae/genética , Composição de Bases , Escherichia coli/genética , Histonas/genética , Proteínas Ribossômicas/genéticaRESUMO
Members of the sufS class of -1 frameshift suppressors have alterations of the GGA/G-decoding tRNA(2Gly). Suppressor-promoted frameshifting at GGA was shown in this study to be directly mediated by the mutant tRNA(2Gly). We disproved the possibility that, in the presence of the compromised mutant tRNA(2Gly), either wild-type tRNA(1Gly), wild-type tRNA(3Gly), a GGA-reading mutant form of tRNA(3Gly), or any other agent suppresses the frameshift mutation trpE91.
Assuntos
Escherichia coli/genética , Mutação da Fase de Leitura/genética , Genes Supressores/genética , RNA de Transferência de Glicina/genética , Mutação/genética , Reação em Cadeia da Polimerase , Translocação GenéticaRESUMO
Previous work of others reported an untranslated stretch of 12 nucleotides in the 5' coding sequence of carA from Pseudomonas aeruginosa. However, N-terminal protein sequencing of carA-lacZ translational fusions shows that these 12 nucleotides are normally translated in a continuous triplet manner, both in P. aeruginosa and in Escherichia coli.
Assuntos
Sequência de Bases , Ligases/biossíntese , Ligases/genética , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , Deleção de Sequência , Sequência de Aminoácidos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Suppressor mutants that cause ribosomes to shift reading frame at specific and new sequences are described. Suppressors for trpE91, the only known suppressible -1 frameshift mutant, have been isolated in Escherichia coli and in Salmonella typhimurium. E. coli hopR acts on trpE91 within the 9-base-pair sequence GGA GUG UGA, is dominant, and is located at min 52 on the chromosome. Its Salmonella homolog maps at an equivalent position and arises as a rarer class in that organism as compared with E. coli. The Salmonella suppressor, hopE, believed to be in a duplicate copy of the same gene, maps at min 17. The +1 suppressor, sufT, acts at the nonmonotonous sequence CCGU, is dominant, and maps at min 59 on the Salmonella chromosome.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Mutação , Salmonella typhimurium/genética , Supressão Genética , Sequência de Bases , Clonagem Molecular , Cruzamentos Genéticos , Genótipo , Dados de Sequência Molecular , Plasmídeos , Transdução GenéticaRESUMO
Guanylin is a pro-secretory hormone that is expressed in intestinal epithelia. Previously, we mapped the guanylin gene to mouse and human chromosomal regions containing multiple intestinal tumor-modifying loci. Here, we investigate whether guanylin expression is downregulated in precancerous human and mouse intestinal adenomas and whether diminished guanylin expression increases adenoma susceptibility in an animal model of intestinal cancer, the multiple intestinal neoplasia (Min) mouse. In situ hybridization analysis indicated diminished guanylin expression in both mouse and human adenomas. Northern analysis of mouse intestinal tissues showed strain-specific levels of guanylin expression but no correlation with the resistance or susceptibility of each strain to adenoma formation. Similarly, cDNA sequence analysis indicated no inactivating mutations or polymorphisms common to either the high or low adenoma-risk groups. Nonetheless, we have shown that significant loss of guanylin RNA in adenomas of mouse and human is a marker of intestinal epithelial cell transformation.