Moore K R, Blakely R D
Emory University School of Medicine, Atlanta, GA.
Biotechniques. 1994 Jul;17(1):130-5, 137.
To evaluate structure/function relationships among neurotransmitter transporters, chimeric cDNAs were formed between antidepressant- and cocaine-sensitive norepinephrine and serotonin transporters. To eliminate experimenter bias in the positioning of chimeric junctions, we utilized a novel method that involves the in situ formation of chimeras in transformed Escherichia coli from linearized plasmids that bear a single copy of both parental transporter cDNAs. Colonies recovered after growth on selective media frequently contain plasmids bearing chimeric inserts. Coupled with the vaccinia-T7 expression system, this method allowed us to rapidly generate and evaluate multiple transporter chimeras without the need to introduce compatible restriction sites or the time and expense involved in formation of individual chimeric cDNAs. Transporter chimeras with switch points proximal but not distal to the middle of putative transmembrane domain 1 retain serotonin or norepinephrine transport and high-affinity antagonist recognition. Loss of substrate and antagonist recognition despite normal levels of transporter protein by distal chimeras suggests important and divergent interactions between multiple transmembrane domains in forming ligand binding sites. The method of chimera synthesis applied in these studies is likely to be of generic utility particularly when the formation of a library of chimeric cDNAs is desired.
为了评估神经递质转运体之间的结构/功能关系，在对抗抑郁药和可卡因敏感的去甲肾上腺素和5-羟色胺转运体之间构建了嵌合cDNA。为了消除实验者在嵌合连接定位上的偏差，我们采用了一种新方法，该方法涉及从携带两个亲本转运体cDNA单拷贝的线性化质粒在转化的大肠杆菌中进行嵌合体的原位形成。在选择性培养基上生长后回收的菌落通常含有携带嵌合插入片段的质粒。结合痘苗-T7表达系统，该方法使我们能够快速生成和评估多个转运体嵌合体，而无需引入兼容的限制性酶切位点，也无需耗费时间和成本来构建单个嵌合cDNA。在假定的跨膜结构域1中间近端而非远端具有切换点的转运体嵌合体保留了5-羟色胺或去甲肾上腺素转运以及高亲和力拮抗剂识别能力。远端嵌合体尽管转运体蛋白水平正常，但底物和拮抗剂识别能力丧失，这表明在形成配体结合位点时多个跨膜结构域之间存在重要且不同的相互作用。这些研究中应用的嵌合体合成方法可能具有普遍实用性，特别是当需要构建嵌合cDNA文库时。
