RESUMO
Genomic DNA from 97 cases of adult de novo acute myeloid leukaemia (AML) was screened using polymerase chain reaction (PCR) and conformation-sensitive gel electrophoresis (CSGE) for FLT3 exon 20 mutations. Initial sequencing of four cases, representing the spectrum of CSGE abnormalities, revealed changes affecting codon Asp835 in three cases and also an intron 20 A to G change. In order to identify all possible Asp835 alterations, as well as the frequency of the intronic change nucleotide 2541 + 57 A-->G, the patient PCR products were digested with EcoRV and NlaIII respectively. Seven cases (7.2%) possessed a mutation affecting Asp835; these were identified, following DNA sequencing, as Asp835Tyr (n = 5), Asp835His (n = 1) and Asp835del (n = 1). Alterations affecting Asp835 were not found in 80 normal control DNA samples. In contrast, the nucleotide 2541 + 57 A-->G change was shown to be a polymorphism, with an allelic frequency of 0.24 for the G and 0.76 for the A allele. This study reports, for the first time, point mutations in the human FLT3 gene that, because of their homology with other class III receptor tyrosine kinase mutations, probably result in constitutive activation of the receptor.
Assuntos
Leucemia Mieloide/genética , Mutação Puntual , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Doença Aguda , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ácido Aspártico/genética , Estudos de Casos e Controles , Eletroforese em Gel de Poliacrilamida/métodos , Feminino , Frequência do Gene , Humanos , Leucemia Mieloide/mortalidade , Masculino , Camundongos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Proto-Oncogênicas c-kit/genética , Ratos , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Homologia de Sequência de Aminoácidos , Estatísticas não Paramétricas , Análise de Sobrevida , Tirosina Quinase 3 Semelhante a fmsRESUMO
A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted. To date, such investigations can only be undertaken if the gene is not essential. We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated. The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene. The promoter showed an approximately 85-fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM. In the presence of either amino acid, expression was reduced to levels that were close to background. We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter. We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter. During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long-standing problem in this field.
