RESUMO
The pattern of expression of a carrot dhfr-ts gene was evaluated in different plant organs, in somatic embryos, and in hypocotyl explants induced to dedifferentiate in vitro by the addition of the synthetic auxin 2,4 dichorophenoxyacetic acid. The promoter of this gene was also placed upstream of a uidA (GUS) reporter gene and, using biolistic and protoplasts transient expression assays, was shown to drive a particularly high level of expression in actively growing suspension cells. The results from these expression analyses combined with the presence of putative cell cycle-related cis-acting elements in the dhfr-ts promoter, strongly point to a cell division-dependent expression of this gene.
Assuntos
Daucus carota/enzimologia , Regulação da Expressão Gênica de Plantas/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Linhagem Celular , Proliferação de Células , Daucus carota/genética , Daucus carota/crescimento & desenvolvimento , Regulação Enzimológica da Expressão Gênica/genética , Hibridização In Situ , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismoRESUMO
The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.
Assuntos
Genes Arqueais , Glicosídeo Hidrolases/genética , Thermococcus/enzimologia , Sequência de Aminoácidos , Domínio Catalítico/genética , Passeio de Cromossomo , Clonagem Molecular , Escherichia coli/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Glicosilação , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Óperon/genética , Sinais Direcionadores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência , Homologia de Sequência de Aminoácidos , Thermococcus/genéticaRESUMO
PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases. The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis. The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity. The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal alpha-carboxy group plays a key role in the strict aminopeptidase activity.