Johnson P, Friedmann T
Center for Molecular Genetics, UCSD, La Jolla 92093.
Gene. 1990 Apr 16;88(2):207-13. doi: 10.1016/0378-1119(90)90033-n.
Typical of other housekeeping genes, the promoter for the human hypoxanthine phosphoribosyl transferase-encoding gene (HPRT) is G + C-rich, lacks a TATA box and has multiple transcription start points. To test the hypothesis that these features may result in relaxed control over the direction of transcription, we examined the effect of orientation on the ability of the HPRT promoter to control expression of the following reporter genes in transfected cells: luc (firefly luciferase), cat (bacterial chloramphenicol acetyltransferase) and neo (neomycin resistance). A 376-bp fragment containing the HPRT promoter efficiently expressed the luc gene irrespective of orientation, and the 5' ends of luciferase RNA produced in cells transfected with inverted promoter constructs mapped to within the HPRT promoter, indicating that the HPRT promoter has bidirectional activity. However, in the presence of two divergently-flanking reporter genes expression from the inverted HPRT promoter was only 10-20% compared to the noninverted orientation. Furthermore, the inverted HPRT promoter expressed cat less well than luc, and was unable to express neo sufficiently well to produce any colonies under appropriate selection conditions. Attempts to detect endogenous divergent HPRT transcripts were unsuccessful. The promoter of another housekeeping gene, encoding 3-phosphoglycerate kinase (PGK), expressed moderate levels of cat (40%) but not luc (less than 5%) in the inverted orientation. By comparison, two TATA-box containing promoters functioned extremely poorly when inverted. This study indicates that two plasmid-borne housekeeping promoters have at least a limited potential for bidirectional activity, but the functional significance of this is unclear if the corresponding endogenous housekeeping promoters express divergent transcripts at similarly low levels. The poor activity of the HPRT and PGK promoters in the inverted orientation suggests that there is a mechanism which influences the direction of transcription from these promoters.
与其他管家基因一样，人类次黄嘌呤磷酸核糖转移酶编码基因（HPRT）的启动子富含G + C，缺乏TATA盒且具有多个转录起始点。为了验证这些特征可能导致转录方向控制松弛这一假设，我们研究了方向对HPRT启动子在转染细胞中控制以下报告基因表达能力的影响：luc（萤火虫荧光素酶）、cat（细菌氯霉素乙酰转移酶）和neo（新霉素抗性）。包含HPRT启动子的376 bp片段无论方向如何都能有效表达luc基因，并且用反向启动子构建体转染的细胞中产生的荧光素酶RNA的5'端定位在HPRT启动子内，表明HPRT启动子具有双向活性。然而，在存在两个侧翼方向相反的报告基因的情况下，与非反向方向相比，反向HPRT启动子的表达仅为10 - 20%。此外，反向HPRT启动子对cat的表达不如luc，并且在适当的选择条件下无法充分表达neo以产生任何菌落。检测内源性反向HPRT转录本的尝试未成功。另一个管家基因3 - 磷酸甘油酸激酶（PGK）的启动子在反向时表达中等水平的cat（40%），但不表达luc（低于5%）。相比之下，两个含有TATA盒的启动子反向时功能极差。这项研究表明，两个质粒携带的管家启动子至少具有有限的双向活性潜力，但如果相应的内源性管家启动子以同样低的水平表达反向转录本，其功能意义尚不清楚。HPRT和PGK启动子在反向时活性较差表明存在一种机制影响这些启动子的转录方向。
