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Functional pleiotropy of an intramolecular triplex-forming fragment from the 3'-UTR of the rat Pigr gene

¹ Department of Bioquimica y Biologia Molecular, Instituto de Bioquimica, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain
² Department of Pharmacology
³ Center For Molecular Genetics, School of Medicine, University of California, San Diego, La Jolla, California 92093
⁴ Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
⁵ Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
⁶ Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

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.

genome regulators; polypurine-polypyrimidine elements; d(GGA)_n and d(GAA)_n triplet repeats

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