Expression plasmids were constructed in which the human β-globin gene or a variant of it precisely iacklng its two intmns was tmnscriM from its own promoter, the herpes simplex virus type 1 thymidine kinase (HSV-tk) promoter, or the immediate early promoter of human cytomegalovirus (CMV-IE). Forty two hours after transfection of these plasrnids into monkey kidney cells, nuclear and cytoplasmic RNA were isolated. Quentitative S1 nuclease mapping and primer extension analysis were us& to deternine the relative abundances, cellular locations, and leader sizes of the accumulated β-globin RNAs. Whereas transcripts of all of the intmn-containing genes accumulated in the cytoplasm to high levels, transcripts of their cDNA vadents were neither stably maintained in the nucleus nor accumulated in the cytoplasm, irrespective of the promoter from which transcdption was driven. We conclude that the intron requirement for cytoplasmic accumuitition of β-globin RNA can not be circumvented by synthesis from either the promoter of the intronless WSV-tk gene or the CMV-IE promoter.
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Dedicated to Paul Berg on the occasion of his 65th birthday. We thank Patrick Charnay, Dan Greenspan, Steve McKnight, and Michael Neuberger for gifts of plasmids and Paul Larnbert and members of our laboratory for discussions and helpful comments on the manuscript. This research was supported by U.S. Public Health Service grants CA47175 and CA22443 from the National Cancer Institute.
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