Two DNA elements which we have termed SAA and GAG have been shown to control expression of the rat amyloid precursor protein (APP) gene, and the region containing the SAA element has been shown to interact with nuclear proteins [Hoffman and Chernak (1994) Biochem. Biophys. Res. Commun. 201, 610-617]. In this report we study DNA sequences and proteins which influence the activity of the SAA element. An oligonucleotide containing the SAA element is specifically bound by nuclear proteins derived from rat PC12 cells, consistently forming four complexes designated C25, C30, C35 and C40 in electrophoretic mobility shift assays (EMSAs). We demonstrate that the C25, C30 and C40 complexes involve the binding of nuclear proteins to an SP1 consensus sequence located within the SAA element and that the C25 complex contains a protein antigenically related to the human SP1 protein. We establish further that the C35 complex requires a USF recognition site located within the SAA element and contains a protein antigenically related to the human upstream stimulatory factor (USF) protein. Using APP promoter/luciferase reporter gene constructs, we demonstrate that both the SP1 and the USF sites can play a role in the transcriptional activity of the SAA element. Finally, we show that complexes similar to the C25, C30 and C35 complexes are formed by rat cortex nuclear extracts and the SAA element in EMSA experiments, suggesting the relevance of our in vitro observations to the in vivo functioning of the rat APP promoter.

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We investigated the expression and functionality of a previously developed adenoviral vector carrying the rat cDNA for the dopamine D2 receptor (D2R), AdCMV.DopD2R. Comparative analysis of the autoradiographic images from the striatum injected with AdCMV.DopD2R and the contralateral striatum injected with a control vector, AdCMV.Null, in male rats indicated that D2R binding was increased by 40-60% on days 3 and 5 after injection, but then declined to baseline levels by day 21. When injected with apomorphine on days 3 and 7 after vector injection, experimental groups that had received unilateral striatal injections of AdCMV.DopD2R exhibited a distinct and significant laterality in rotational behavior. These results provide the first demonstration of an adenovirally mediated, intracerebral delivery of a functional neurotransmitter receptor.

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We have demonstrated both the synthesis and overproduction of the 5A protein encoded by the longest open reading frame of the bacterial insertion sequence ISS. Expression was obtained in vitro and in Escherichia coli maxicells from plasmids containing IS5 in either orientation, as well as in vitro from a restriction fragment containing exclusively IS5 DNA. When IS5 was cloned in the appropriate orientation downstream of a strong tac promoter, production of the 5A protein was increased to 10 to 20% of the total protein synthesized in vitro.

Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Althoclh there was no detectable expression from the 5B open reading fra.ne when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. 1-galactosidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80-85% of the total protein made in vitro and 20-25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vivo. Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.