Bajwa W, Rudolph H, Hinnen A
Biotechnology Department, Ciba-Geigy AG., Basel, Switzerland.
Yeast. 1987 Mar;3(1):33-42. doi: 10.1002/yea.320030106.
To identify the sequences involved in the regulation of the yeast acid phosphatase gene (PHO5) we constructed a series of hybrid promoters. Increasing lengths of 5'-flanking sequences of the PHO5 gene were placed in front of the TATA-box of constitutively expressed acid phosphatase gene (PHO3). The PHO5/PHO3 promoter constructions were used to replace the entire PHO5, PHO3 gene cluster on chromosome II. Depending on the length of PHO5 5'-flanking sequences present the PHO3 gene driven by the hybrid promoter could now be derepressed in response to inorganic phosphate (low Pi) exactly as the PHO5 wild type gene. A critical regulatory element was located between position -402 to -351 (upstream from ATG) and sequences further downstream (from -351 to -300) could increase transcriptional activation. The transcription levels of PHO3 were determined by northern blot analysis, under repressed (high Pi) and derepressed (low Pi) conditions which was paralleled by an increase in extra-cellular acid phosphatase activity. Fully regulated promoter hybrids showed a 40-fold induction of mRNA levels, comparable to wild type PHO5 promoter. Sl-nuclease protection experiments revealed that the PHO5 5'-flanking sequences, placed in front of PHO3, did not change the PHO3 transcription initiation site/s.
为了鉴定参与酵母酸性磷酸酶基因（PHO5）调控的序列，我们构建了一系列杂种启动子。将PHO5基因5'侧翼序列长度不断增加的片段置于组成型表达的酸性磷酸酶基因（PHO3）的TATA框前。利用PHO5/PHO3启动子构建体替换II号染色体上的整个PHO5、PHO3基因簇。根据所存在的PHO5 5'侧翼序列的长度，由杂种启动子驱动的PHO3基因现在能够像PHO5野生型基因一样在无机磷酸盐（低磷）存在时去阻遏。一个关键调控元件位于-402至-351位（ATG上游）之间，而更下游的序列（从-351至-300）可增强转录激活。通过Northern印迹分析测定PHO3在阻遏（高磷）和去阻遏（低磷）条件下的转录水平，这与细胞外酸性磷酸酶活性的增加相平行。完全受调控的启动子杂种显示mRNA水平有40倍的诱导，与野生型PHO5启动子相当。S1核酸酶保护实验表明，置于PHO3前的PHO5 5'侧翼序列并未改变PHO3转录起始位点。
