The antisense RNA strategy can be used in prokaryotes in order to regulate the expression of specific genes. This phenomenon, however, is still under study. In particular, only few details are known about the determinants leading to effective artificial antisense molecules. In order to identify new structural elements essential for the silencing effectiveness in E. coli, different aRNAs against the lacZ mRNA have been tested in vivo. Fragments of lacZ gene were cloned into the pBAD vector under the control of the ara promoter. The inhibition of beta-galactosidase synthesis was analysed by comparing the protein concentration and the mRNA level in bacterial cells expressing these aRNAs. Moreover, the in vivo stability of the anti-lacZ molecules was measured and the decay rate was calculated by using rifampicin to stop the transcriptional process.
Antisense RNA structural features improving silencing efficiency in E. coli.
STEFAN, ALESSANDRA;HOCHKOEPPLER, ALEJANDRO
2005
Abstract
The antisense RNA strategy can be used in prokaryotes in order to regulate the expression of specific genes. This phenomenon, however, is still under study. In particular, only few details are known about the determinants leading to effective artificial antisense molecules. In order to identify new structural elements essential for the silencing effectiveness in E. coli, different aRNAs against the lacZ mRNA have been tested in vivo. Fragments of lacZ gene were cloned into the pBAD vector under the control of the ara promoter. The inhibition of beta-galactosidase synthesis was analysed by comparing the protein concentration and the mRNA level in bacterial cells expressing these aRNAs. Moreover, the in vivo stability of the anti-lacZ molecules was measured and the decay rate was calculated by using rifampicin to stop the transcriptional process.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
