Abstract
Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.
Highlights
Fragments of 6”flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with an improved low background chloramphenicolacetyltransferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M.,and Ebina, Y. (1988) Nucleic Acids Res. 16,1627).Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 is sufficient for maximal promoter activity
Insulin binding to the a subunit of the receptor results in stimulation of intrinsic tyrosine kinase activity of the p subunit [6, 7], which is essential for key physiological responses of the peptide hormone [8,9,10]
The efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Spl to four G-C boxes located -593 to -618 base pairs upstream ofthe ATG translation initiation codon
Summary
Deletion and Insertion Analysis of the HumanIR Promoter [4] and theHindIII-NcoI fragment (575 bp) of the promoter region Using the Low Background pSVOOCATSystem-To investiof the human IR gene [11]were ligated with a HindIII-EcoRI frag- gate weak promoter activities of eukaryotic genes, we conment (2.9 kb) of pcDVl [25] to construct pEA5. The p0.63kb-00CAT was digested with HindIII, and a 0.63-kb HindIII-Hind111 fragment was inserted into a HindIII siteof pMT21 This plasmid was digested relative promoter activities in the transientexpression assay (comparedwithpSphI-OOCAT)are showninFig, 1. 2112-bp HincII-Hind111cDNA fragment of Spl inserted into pUC118 bp DNA fragment of four putative S p l bindingsitesjust upstream of the HindIII site When this 0.63-kb region was used as a promoter, CAT was expressed at theoriginal level of pSphI-OOCAT activity. The digestion of DNA was initiated by the addition of DNase I (3 ng of DNase I for control (no crude lysate) and 10 ng for 20 and 100 pg of crude lysates), and the samples were incubated for sequence from -420 to -436 When this region was removed from p0.63kb-OOCAT, and the resultant plasmid, p0.63kb~vuIJ-NruI-OOCATw, as transfected, the CAT activity did not differ from the activity seen at the p0.63kb-. The gelwas dried and autoradi- DNase I Footprinting with Spl-The results of the CAT ographed at -70 "C
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