Abstract
A region of the rat growth hormone gene and 5' flanking DNA has been identified which promotes accurate, thyroid hormone-regulated transcriptional initiation. GC rat pituitary tumor cells were transfected with chimaeric plasmids containing various lengths of rat growth hormone gene and 5' flanking DNA fused to the coding region of the dominant selectable marker gene neo. Thyroid hormone induction of rGH-neo RNA was observed by Northern and dot blot analysis of cells transfected with rGH-neo chimaeric genes sharing the rat growth hormone gene and upstream regions from -235 to +11. Initiation of rGH-neo transcription was mapped by S1 nuclease protection to the in vivo initiation site of the natural growth hormone gene. Transcription of the most deleted thyroid hormone responsive construct involved an induction-attenuation cycle qualitatively similar to the response of the natural gene. However, the 3,5,3'-triiodo-L-thyronine responsiveness of this deleted construct was approximately 2- to 3-fold less than that of less deleted rGH-neo genes tested. These results suggest that, at a minimum, the sequences required for the cyclic 3,5,3'-triiodo-L-thyronine transcriptional response are located within the region of the gene from -235 to +11. Other sequences essential for full responsiveness appear to be located elsewhere in the 5'-flanking DNA. Rat growth hormone promoter utilization appears to be strongly cell-type dependent. We obtained stable transfectants with rGH-neo constructs only in GC cells.
Highlights
A region of the rat growth hormone gene and 5‘ siveness to neighboringpromoters
All was observedby Northern and dot bloatnalysis of cells transfected with rGH-neo chimaeric genes sharing the rat growth hormone gene and upstream regions from -235 to +11
The 3,5,3’-triiodo-~-thyroninreesponsiveness of this deleted construct was approximately 2- to %foldless than thatof less deleted rGH-neo genes tested. These results suggest that, at a minimum, the the neogenecoding sequence and SV40 intronand polyadenylation signals from pSV2neo [9] to the BamHI site of pUC18 [10]. prGH2004neo was constructed by replacing the XbaI to BamHI fragment of prGHeh
Summary
All was observedby Northern and dot bloatnalysis of cells transfected with rGH-neo chimaeric genes sharing the rat growth hormone gene and upstream regions from -235 to +11. The XbaI sequences required for the cyclic 3,5,3’-triiodo-~-thy- site is within the first intron of the rGH gene, 4 nucleotides from the ronine transcriptional response are located within the acceptor junction. Ligation of this XbaI site to another such site region of the gene from -235 to +11. After treatment for the indicated lengths of timein hypomedium supplemented with nM T3, cells were harvested, nuclei were prepared, and in vitro transcription and filter hybridization reactions were performed as previously described [5]. RNA was prepared from the cytoplasmic fractions [5]. subjected to dotblotting, probed with the radioactive neo or GH probe andvisualized as describedabove, and the intensities of the dots were scanned, digitized, and analyzed asdescribed [17]
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