RESUMO
The cDNA of a 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase) was isolated from rat liver RNA by reverse transcription and the polymerase chain reaction (PCR). The rat AICARFT/IMPCHase cDNA included 1928 bp containing a coding region of 1779 bp for a 592-amino acid polypeptide (Mr = 64 200). Rat and human AICARFT/IMPCHase cDNAs show 84 and 91% homology at the nucleotide and amino acid sequence level, respectively. The protein produced by the rat cDNA using pET-expression system catalysed the penultimate and final steps of de novo purine biosynthesis. Northern analysis identified a 2.8-kb AICARFT/IMPCHase mRNA and the level of the AICARFT/IMPCHase transcripts increased markedly at 24 h after partial (70%) hepatectomy.
Assuntos
DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/genética , Hidroximetil e Formil Transferases/genética , Nucleotídeo Desaminases/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Hepatectomia , Fígado/fisiologia , Regeneração Hepática/genética , Dados de Sequência Molecular , Fosforribosilaminoimidazolcarboxamida Formiltransferase , RNA Mensageiro/análise , Ratos , Proteínas Recombinantes , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The effects of ethanol on liver regeneration after partial hepatectomy were investigated. The injection of ethanol inhibited the increases in the activities of thymidylate synthase and thymidine kinase in regenerating rat liver at 24 h after partial hepatectomy in a dose-dependent manner, with a concomitant reduction in DNA content. Northern blot analysis showed that the inhibition of thymidylate synthase and thymidine kinase activities was caused by comparable decreases in their mRNA levels. The immunoblotting assay confirmed the protein levels of thymidylate synthase and thymidine kinase as proportional to the activity and mRNA levels. These findings suggest that ethanol inhibits DNA synthesis by the repression of mRNA levels of dTMP-synthesizing enzymes during liver regeneration.
