RESUMO
Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.
Assuntos
Evolução Molecular Direcionada , Plasmídeos/genética , Origem de Replicação , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano/genética , Proteínas de Escherichia coli/genética , Genes Bacterianos , Óperon Lac , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fenótipo , RNA Bacteriano/química , RNA Bacteriano/genética , Deleção de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND: The activating BRAF(V600E) mutation is the most common genetic alteration reported in papillary thyroid carcinoma (PTC). While some reports suggest the BRAF(V600E) mutation is associated with factors predicting a poor prognosis and recurrence, this remains a controversial issue. AIM: To determine whether the presence of the BRAF(V600E) mutation is a prognostic indicator for clinical recurrence in low-risk patients with conventional PTC. PATIENTS AND METHODS: The study involved 203 conventional PTC patients who underwent total or near-total thyroidectomy followed by immediate 131I ablation of the remnants. Patients with antithyroglobulin antibodies and those with extracervical metastases at presentation were excluded. DNA was extracted from paraffin-embedded tumour specimens, and the presence of the BRAF(V600E) mutation was evaluated using PCR amplification and direct sequencing. RESULTS: The BRAF(V600E) mutation was found to be present in 149 (73.4%) of 203 patients. The BRAF(V600E) mutation was correlated with male gender (P = 0.006) and with tumour size (P = 0.005). While there appeared to be an association between the BRAF(V600E) mutation and extrathyroid extension, this did not reach statistical significance (P = 0.062). During follow-up of the 203 patients (median 7.3 years; range 0.7-10.0 years), 36 (18%) patients experienced recurrence. While univariate analysis showed the BRAF(V600E) mutation was associated with tumour recurrence (21% with mutation vs 7% without mutation; P = 0.037), this association was not shown following multivariate analyses adjusting for the clinicopathological prognostic factors of age, gender, tumour size, extrathyroid extension, multifocality and lymph node metastasis. CONCLUSIONS: Although the BRAF(V600E) mutation was found to be associated with a higher clinical recurrence of disease in low-risk conventional PTC patients, it was not an independent predictor.
Assuntos
Carcinoma Papilar/genética , Recidiva Local de Neoplasia/genética , Mutação Puntual , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Carcinoma Papilar/mortalidade , Carcinoma Papilar/cirurgia , Feminino , Fluordesoxiglucose F18 , Seguimentos , Humanos , Radioisótopos do Iodo/uso terapêutico , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/mortalidade , Tomografia por Emissão de Pósitrons , Prognóstico , Modelos de Riscos Proporcionais , Compostos Radiofarmacêuticos/uso terapêutico , Análise de Sobrevida , Neoplasias da Glândula Tireoide/mortalidade , Neoplasias da Glândula Tireoide/cirurgia , TireoidectomiaRESUMO
BACKGROUND: The BRAF(V600E) mutation, the most common genetic alteration reported in papillary thyroid carcinoma, has been associated with poor prognostic factors. AIM: To determine whether the presence of the BRAF(V600E) mutation is associated with poor prognosis in Korean patients with conventional papillary thyroid microcarcinoma (micro-PTC). PATIENTS AND METHODS: DNA was extracted from paraffin-embedded thyroid tumour specimens taken from 60 patients with conventional micro-PTC, as well as from nine patients with follicular variant papillary carcinoma, six with nodular hyperplasia, four with follicular carcinoma (including one with Hürthle cell carcinoma), four with follicular adenoma (including two with Hürthle cell adenoma) and one each with medullary carcinoma, poorly differentiated carcinoma and anaplastic carcinoma. The presence of the BRAF(V600E) mutation was determined by polymerase chain reaction (PCR) amplification of exon 15 followed by direct sequencing. RESULTS: The BRAF(V600E) mutation was detected in tumour samples from 31 of 60 conventional micro-PTC patients (52%), but was not detected in patients with other types of thyroid tumours. The age distribution, tumour size, extrathyroid extension, multifocality and staging did not differ significantly between patients with and without the BRAF(V600E) mutation. CONCLUSION: In Korean patients with conventional micro-PTC, the presence of the BRAF(V600E) mutation was not significantly associated with prognostic factors.
Assuntos
Adenoma/genética , Mutação Puntual , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Adenoma/patologia , Adulto , Distribuição de Qui-Quadrado , Análise Mutacional de DNA , Feminino , Marcadores Genéticos , Humanos , Coreia (Geográfico) , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Neoplasias da Glândula Tireoide/patologiaRESUMO
A recent and intriguing discovery in plant biology has been that some transcription factors can move between cells. In Arabidopsis thaliana, the floral identity protein LEAFY has strong non-autonomous effects when expressed in the epidermis, mediated by its movement into underlying tissue layers. By contrast, a structurally unrelated floral identity protein, APETALA1, has only limited non-autonomous effects. Using GFP fusions to monitor protein movement in the shoot apical meristem and in floral primordia of Arabidopsis, we found a strong correlation between cytoplasmic localization of proteins and their ability to move to adjacent cells. The graded distribution of several GFP fusions with their highest levels in the cells where they are produced is compatible with the notion that this movement is driven by diffusion. We also present evidence that protein movement is more restricted laterally within layers than it is from L1 into underlying layers of the Arabidopsis apex. Based on these observations, we propose that intercellular movement of transcription factors can occur in a non-targeted fashion as a result of simple diffusion. This hypothesis raises the possibility that diffusion is the default state for many macromolecules in the Arabidopsis apex, unless they are specifically retained.