RESUMO
Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical and basic research purposes. In most cases, the 5' untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in this yeast. Because the effect of the AOX1 5'UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5'UTR clearly decreased expression of a ß-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5'UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5'UTR optimized for a higher level of expression may be challenging.
Assuntos
Regiões 5' não Traduzidas , Oxirredutases do Álcool/genética , Regulação Fúngica da Expressão Gênica , Pichia/metabolismo , Sequência de Bases , Sistema Livre de Células , Deleção de Genes , Perfilação da Expressão Gênica/métodos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Proteínas Recombinantes/metabolismo , beta-Galactosidase/metabolismoRESUMO
Historians have examined the significant contributions John and William Bartram made to 18th- and 19th-century knowledge of indigenous North American flora. However, the Bartrams' contribution to medicinal botanical knowledge, particularly William Bartram's compilation of Indians' knowledge on the preparation and use of medicinal botanicals, is not well-known. In addition, while William Bartram's contemporaries relied on his accounts of medicinal botanicals, they rarely acknowledged Bartram or Indians in their own works. Contemporaries plagiarized Bartram's writings and used his exquisite illustrations to ornament their own publications. This paper reconstructs William Bartram's careful collection and recording of medicinal botanical knowledge that became part of late 18th- and early 19th-century American pharmacology, as well as provides evidence for 54 Bartram-identified indigenous species and the pirating of William Bartram's work by contemporaries.
