Hadfield C, Cashmore A M, Meacock P A
Gene. 1987;52(1):59-70. doi: 10.1016/0378-1119(87)90395-7.
An efficiently transforming chloramphenicol-resistance (CmR) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics. The complete nucleotide (nt) sequence of the CmR marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence. SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E. coli. Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx. 0.1% of the total soluble protein as CAT in haploid yeast cells. When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast. When the marker was on a multicopy plasmid in yeast, up to 2.1% of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells. Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure. In E. coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity. The level of expression provided by these promoters was equivalent to that of an average E. coli gene.
一种用于酿酒酵母和大肠杆菌的高效转化氯霉素抗性（CmR）穿梭标记已根据其一级结构和表达特性进行了表征。给出了CmR标记的完整核苷酸（nt）序列，包括限制酶切位点、两种生物的明显表达信号以及Cm乙酰转移酶（CAT）编码序列的翻译细节。SDS-聚丙烯酰胺凝胶电泳和蛋白质免疫印迹证实该标记在酵母和大肠杆菌中产生了相同的CAT蛋白。该标记的每个拷贝，无论是多拷贝还是单拷贝，在单倍体酵母细胞中产生的CAT约占总可溶性蛋白的0.1%。与同一ADC1启动子驱动的醇脱氢酶（ADH-I）同源表达相比，这代表CAT表达降低了27倍，这是酵母中异源基因表达的典型情况。当该标记位于酵母中的多拷贝质粒上时，产生的CAT最多占总可溶性细胞蛋白的2.1%，但这对宿主细胞的生长没有不利影响。培养基中Cm浓度的增加并未导致质粒数量或产生的CAT蛋白量增加，表明质粒拷贝数和标记表达与选择压力无关。在大肠杆菌中，发现ADC1酵母启动子DNA同时具有正向和反向启动子活性。这些启动子提供的表达水平与平均大肠杆菌基因的表达水平相当。
