Distinct promoters in the rat insulin-like growth factor-I (IGF-I) gene are active in CHO cells.
In mammals, IGF-I mRNAs contain one of two different leader exon sequences that encode different 5'-untranslated regions (UTRs) and signal peptides. The pattern and regulation of expression of these exon 1 and exon 2-derived mRNAs suggests that the expression of each is controlled by a distinct regulatory region. In order to assess this possibility, DNA fragments consisting of sequences flanking and including the exon 1 and exon 2 transcription initiation sites were cloned into a luciferase expression vector and plasmid DNAs were transiently transfected into Chinese hamster ovary (CHO) cells. A fragment containing approximately 1.1 kb of sequence flanking the most upstream exon 1 transcription initiation site and 362 bp of exon 1 sequence did not stimulate luciferase activity. However, fragments containing 133 bp of 5'-flanking sequence and either 362 or 192 bp of exon 1 sequence stimulated luciferase activity significantly above that seen with a promoterless control plasmid. When the -133/+362 fragment was cloned in the opposite orientation with respect to the luciferase cDNA, the same level of promoter activity was observed. Removal of approximately 860 bp from the inactive fragment (i.e., approximately 782 bp of flanking sequence and approximately 74 bp of exon 1 sequence) resulted in promoter activity which was significantly greater than that seen with the promoterless luciferase expression vector, but which was less than that observed with fragments containing the proximal 133 bp of 5'-flanking sequence. Plasmids containing approximately 1.5 kb or 0.5 kb of flanking sequence and 44 bp of exon 2 sequence also significantly stimulated luciferase activity. These results constitute the first demonstration that both exon 1 and exon 2 transcription start sites are associated with distinct and potentially independently regulatable promoters and provide a molecular basis for the differential expression of these leader exons by developmental, tissue-specific and hormonal factors.
Adamo
M L
ML
Section on Molecular and Cellular Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Lanau
F
F
Neuenschwander
S
S
Werner
H
H
LeRoith
D
D
Roberts
C T
CT
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005091:Exons; D015870:Gene Expression; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D017384:Sequence Deletion; D016385:TATA Box; D014158:Transcription, Genetic; D014162:Transfection
D007334:Insulin-Like Growth Factor I