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Proximal Rat Prolactin Promoter Sequences Direct Optimal, Pituitary Cell-Specific Transcription

Thomas Lufkin^*,, Arthur E. Jackson, Wayne T. Pan and Carter Bancroft

Department of Physiology and Biophysics, Mount Sinai School of Medicine of City University of New York New York, New York 10029

Address requests for reprints to: Dr. Carter Bancroft, Department of Physiology and Biophysics, Mount Sinai School of Medicine of The City University of New York, New York, New York 10029.

Abstract

Previous studies have shown that transferred hybrid constructs containing the PRL promoter are expressed specifically in rat pituitary (GH) cell lines. However, it is not yet clear which DNA region(s) is primarily responsible for expression directed by this promoter in pituitary cells. In the present studies we have examined the DNA sequences required for cell type-specific transcription of the rat PRL (rPRL) promoter either during transient expression in intact cells or in nuclear chromatin extracts. RNase protection and nuclear run-on transcription assays showed directly that a PRL-chloramphenicol acetyltransferase (CAT) construct containing about two kilobasepairs of the rPRL promoter region (pPRL-CAT) is transcribed specifically in pituitary (GH₃) cells. Analysis by transient expression in GH₃ cells of pPRL-CAT and its 5' deletions showed that 1) deletion of sequences between positions –1957 and –958 did not significantly affect CAT activity; 2) the first 187 basepairs (bp) of the rPRL promoter directs full CAT activity; and 3) 98% of this activity is accounted for by rPRL DNA sequences between positions –187 and –113, containing two GH₃ chromatin footprinting sites. Analysis in GH₃ cell nuclear extracts showed that transcription of PRL-CAT constructs is unaffected by successive 5' deletions from position –1957 to –187, and that further deletion to –75 yielded only a moderate (2-fold) decrease in transcription. Furthermore, the latter highly deleted construct, containing only a single GH₃ cell footprint site plus a TATA box, is transcribed in vitro in a highly cell type-specific fashion, exhibiting activity in GH₃ extracts more than 1000-fold higher than that in human HeLa extracts and more than 10-fold higher than that in extracts of either of two nonpituitary rat cell lines examined. Thus, results obtained with whole cells or nuclear extracts imply that proximal promoter sequences direct pituitary cell-specific transcription of the PRL gene.

FOOTNOTES

This work was supported by NIH Grant GM-36847, an Irma T. Hirschl Career Scientist Award (to C.B.), and National Research Service Award Fellowship DK-08071 (to A.E.J.).

^* Present address: Laboratoire de Genetique Moleculaire des Eucaryotes, Institut de Chimie Biologique, Faculte de Medecine, 11 rue Humann, Strasbourg, France.

The first two authors contributed equally to the studies described here.

Received for publication October 7, 1988. Revision received December 1, 1988. Accepted for publication December 1, 1988.