Boyce F M, Anderson G M, Rusk C D, Freytag S O
Mol Cell Biol. 1986 Apr;6(4):1244-52. doi: 10.1128/mcb.6.4.1244-1252.1986.
The human argininosuccinate synthetase locus is subject to metabolite-mediated repression by arginine in some cultured cell lines. To gain insight into the mechanism underlying this regulation, chloramphenicol acetyltransferase (CAT) minigenes under the transcriptional control of the human argininosuccinate synthetase promoter were constructed and tested for regulation. When the minigenes were introduced into RPMI 2650 cells, a human cell line that shows sixfold regulation of the argininosuccinate synthetase gene, CAT expression was repressed three- to fivefold when arginine was present in the culture medium. A minigene containing only 149 base pairs of 5'-flanking sequence was expressed at similar levels and regulated to the same degree as one having approximately 3 kilobases of 5'-flanking sequence. Therefore, the cis-acting sequences required for the arginine-mediated repression are likely to be located within the region of the transcription initiation site. The arginine-mediated repression of the CAT minigenes was not observed in canavanine-resistant variants of RPMI 2650 cells, and therefore they showed the appropriate cell-type specificity. Cultured cells having 200-fold-increased levels of argininosuccinate synthetase can be selected by growth in medium containing the arginine analog canavanine. It was previously demonstrated that the increased expression of argininosuccinate synthetase in canavanine-resistant human lymphoblasts was due to a trans-acting mechanism. To gain further support for a trans-acting mechanism, we tested our CAT minigenes for the trans induction in canavanine-resistant variants of RPMI 2650 cells. Transfection of the CAT minigenes into RPMI 2650 cells and canavanine-resistant variants of this cell line yielded no difference in transient CAT expression. Furthermore, cloned canavanine-resistant variant cells having integrated copies of the CAT minigenes expressed CAT at similar levels as compared to the parental cell lines. Since these cell lines do exhibit arginine-mediated repression of CAT but not trans induction, these data indicate that the argine-mediated repression is a regulatory event that occurs independently of the trans induction.
在一些培养的细胞系中，人类精氨琥珀酸合成酶基因座受到精氨酸代谢物介导的抑制作用。为深入了解这种调控的潜在机制，构建了在人类精氨琥珀酸合成酶启动子转录控制下的氯霉素乙酰转移酶（CAT）微型基因，并对其调控进行了测试。当将这些微型基因导入RPMI 2650细胞（一种精氨琥珀酸合成酶基因显示出六倍调控的人类细胞系）时，若培养基中存在精氨酸，CAT表达会被抑制三至五倍。一个仅包含149个碱基对5'侧翼序列的微型基因，其表达水平与一个具有约3千碱基5'侧翼序列的微型基因相似，且调控程度相同。因此，精氨酸介导的抑制所需的顺式作用序列可能位于转录起始位点区域内。在RPMI 2650细胞的刀豆氨酸抗性变体中未观察到CAT微型基因的精氨酸介导的抑制作用，因此它们显示出适当的细胞类型特异性。通过在含有精氨酸类似物刀豆氨酸的培养基中生长，可以选择精氨琥珀酸合成酶水平增加200倍的培养细胞。先前已证明，刀豆氨酸抗性人类淋巴母细胞中精氨琥珀酸合成酶表达的增加是由于一种反式作用机制。为进一步支持反式作用机制，我们在RPMI 2650细胞的刀豆氨酸抗性变体中测试了我们的CAT微型基因的反式诱导作用。将CAT微型基因转染到RPMI 2650细胞及其刀豆氨酸抗性变体中，瞬时CAT表达没有差异。此外，整合有CAT微型基因拷贝的克隆刀豆氨酸抗性变体细胞与亲本细胞系相比，CAT表达水平相似。由于这些细胞系确实表现出精氨酸介导的CAT抑制作用，但没有反式诱导作用，这些数据表明精氨酸介导的抑制是一个独立于反式诱导发生的调控事件。
