RESTRICTION SITE-INDEPENDENT FORMATION OF CHIMERAS FROM HOMOLOGOUS NEUROTRANSMITTER-TRANSPORTER CDNAS

To evaluate structure/function relationships among neurotransmitter tr ansporters, chimeric cDNAs were formed between antidepressant- and coc aine-sensitive norepinephrine and serotonin transporters. To eliminate experimenter bias in the positioning of chimeric junctions, we utiliz ed a novel method that involves the in situ formation of chimeras in t ransformed Escherichia coli from linearized plasmids that bear a singl e copy of both parental transporter cDNAs. Colonies recovered after gr owth on selective media frequently contain plasmids bearing chimeric i nserts. Coupled with the vaccinia-T7 expression system, this method al lowed us to rapidly generate and evaluate multiple transporter chimera s without the need to introduce compatible restriction sites or the ti me and expense involved in formation of individual chimeric cDNAs. Tra nsporter chimeras with switch points proximal but not distal to the mi ddle of putative transmembrane domain 1 retain serotonin or norepineph rine transport and high-affinity antagonist recognition. Loss of subst rate and antagonist recognition despite normal levels of transporter p rotein by distal chimeras suggests important and divergent interaction s between multiple transmembrane domains in forming ligand binding sit es. 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.