RESUMO
A DNA fragment containing the SRM12/ADA1 gene sequence inserted into a recombinant circular plasmid improves its maintenance in budding yeast (Saccharomyces cerevisiae) cells. Plasmid stabilization caused by the integrated SRM12 sequence does not require the SRM12 function complementing the srm12 mutation and depends on the orientation of the inserted fragment in the vector. This stabilization is mainly due to a decrease in spontaneous plasmid underreplication/copy loss rather than an increase in the fidelity of mitotic plasmid segregation.
Assuntos
Mitose , Plasmídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Proteínas Adaptadoras de Transdução de Sinal , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismoRESUMO
The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.
