Kasunic D A, Kushner S R
Gene. 1980 Dec;12(1-2):1-10. doi: 10.1016/0378-1119(80)90009-8.
The PstI and BamHI fragments, containing the HIS3 (imidazoleglycerol phosphate dehydratase) gene of yeast obtained from pYehis2, and the ColE1-derived plasmid pBR322 were ligated in vitro and used to transform hisB463 strains of Escherichia coli K-12. Expression of the cloned HIS3 gene from yeast was markedly enhanced (3--5-fold) in polynucleotide phosphorylase (pnp)-deficient strains of E. coli. The levels of both HIS3 and plasmid-encoded mRNAs were increased in pnp- strains carrying the chimeric plasmids, whereas there was little difference in the levels of pBR322-specific mRNAs in pnp+ and pnp- strains. This increase in HIS3 mRNA appeared to be related to specific stabilization of the eukaryotic message due to its unique structural features, since the half-life of the HIS3 mRNA increased from 1.5 to 18.7 min, whereas no increase in the half-lives of pBR322 vehicle mRNAs was observed. A physical map of the plasmid pYehis2 was constructed using restriction endonuclease and molecular cloning techniques.
从pYehis2获得的含有酵母HIS3（咪唑甘油磷酸脱水酶）基因的PstI和BamHI片段，与源自ColE1的质粒pBR322在体外连接，并用于转化大肠杆菌K - 12的hisB463菌株。在大肠杆菌的多核苷酸磷酸化酶（pnp）缺陷菌株中，克隆的酵母HIS3基因的表达显著增强（3至5倍）。携带嵌合质粒的pnp - 菌株中HIS3和质粒编码的mRNA水平均升高，而pnp + 和pnp - 菌株中pBR322特异性mRNA水平几乎没有差异。HIS3 mRNA的这种增加似乎与其独特的结构特征导致真核信息的特异性稳定有关，因为HIS3 mRNA的半衰期从1.5分钟增加到18.7分钟，而未观察到pBR322载体mRNA半衰期的增加。使用限制性内切酶和分子克隆技术构建了质粒pYehis2的物理图谱。
