RESUMO
A full-length cDNA clone representing the waxy protein (GBSSI) isolated from a hexaploid wheat developing grain cDNA library has been used to characterise the organisation and expression of the waxy genes in wheat. The genes are organised as a triplicate set of single copy homeoloci on chromosome arms 4AL, 7AS and 7DS. The genes are active throughout grain filling where the main 2.3 kb transcript accumulates to high levels. The 2.3 kb transcript is not expressed in leaves where the presence of a related, but less homologous, transcript of 1.6 kb suggests that a different set of genes operates. Gel analysis and purification of the waxy protein isolated from starch granules, followed by N-terminal amino acid sequencing in conjunction with data from hybrid select translation experiments and sequence analysis of the cDNA, shows that the mature protein has a molecular weight of 60kDa (615 amino acids) and that the preprotein includes a chloroplast/amyloplast transit peptide of 7kDa (75 amino acids). Analysis of the derived amino acid sequence and alignment with five other plant waxy proteins shows that they exhibit substantial homology. The wheat protein differs from all others in that it contains an 11 amino acid insertion towards the N-terminus. The protein contains the conserved motif KTGGL found in other waxy proteins and which has been implicated as the active site in glycogen synthase.
Assuntos
Genes de Plantas/genética , Proteínas de Plantas/genética , Sintase do Amido/genética , Triticum/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Clonagem Molecular , Expressão Gênica , Genoma , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Ploidias , Sinais Direcionadores de Proteínas/genética , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Análise de Sequência , Homologia de Sequência de Aminoácidos , Sintase do Amido/química , Sintase do Amido/isolamento & purificação , Distribuição Tecidual , Transcrição Gênica , Triticum/embriologiaRESUMO
A 23S rRNA gene of Listeria monocytogenes was cloned into pUC19 on a 6.2-kb Pst I fragment. Hybridisation studies demonstrated the presence of the 5S and partial 16S rRNA genes within the clone. The nucleotide sequence of the region encoding the 23S rRNA was found to be highly homologous with those of other low G + C Gram-positive bacteria. The 16S-23S intergenic spacer region was amplified using PCR technology and revealed two product sizes, the larger of which contained tRNA(Ala) and tRNA(Ileu) genes. Further tRNA genes were found downstream of the 5S rRNA gene.
