We have synthesised the @-subunit of the bovine GABAA receptor in stable, continuous insect (Spodoptera fnrgiperda) cell lines. A cDNA was integrated randomly into the insect cell genome under control of a baculovirus immediate early (IE-1) gene promoter. Transformed cells were obtained by co-transfection of the insect cells with pIEK1 .GR/Il, encoding thejI1 subunit cDNA, and pIEKl .neo, encoding the neomycin resistance gene. G-418-resistant clones were selected and expanded into continuous cell lines synthesising functional, GABA-gated, homo-oligomeric chloride channels. These cell lines had significant advantages over the transient baculovirus expression system for the characterisation of receptors using electrophysiological recording techniques.

A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3'-untranslated region (3'-UTR) and inserted into 3'-UTR or 3' flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5' flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5' and 3' flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3'-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.

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