Abstract
Cloning of the 5 -flanking region of the rat pp120 gene has indicated that it is a housekeeping gene: it lacks a functional TATA box and contains several Sp1 binding sites and multiple transcription initiation sites at nucleotides -101, -71, -41, and -27 spread over a GC-rich area. A fragment between nucleotides -21 and -1609 exhibited promoter activity when ligated in a sense orientation into a promoterless luciferase reporter plasmid and transiently transfected into rat H4-II-E hepatoma cells. 5' progressive deletion and block substitution analyses revealed that the three proximal Sp1 boxes (boxes 3, 5, and 6) are required for basal transcription of the pp120 gene. Promoter activity was stimulated 2-3-fold in response to insulin, dexamethasone, insulin plus dexamethasone, and cAMP. Although unaltered by phorbol esters alone, promoter activity was stimulated 4-5-fold in response to phorbol esters plus cAMP. Several motifs resembling response elements for insulin (in the rat phosphoenolpyruvate carboxykinase gene), glucocorticoids, cAMP, and phorbol esters as well as a number of putative binding sites for activating proteins-1 (Jun/Fos) and -2, and liver-specific factors were detected. The role of these sites in tissue-specific expression of pp120 remains to be investigated.
Highlights
Pp120/ecto-ATPase,1 a substrate of the insulin receptor tyrosine kinase, is an integral liver plasma membrane glycoprotein [1,2,3,4]
Molecular cloning revealed that the rat pp120 gene consists of nine exons, the 7th of which (53 bp) is alternatively spliced during mRNA processing, generating two alternatively spliced variants that differ in the intracellular cytoplasmic domain [11]
Sequence Analysis of the 5Ј-Flanking Region and Intron 1 of the Rat pp120 Gene—XhoI treatment of -2 genomic DNA, a bacteriophage that contains exons 1– 4 including ϳ24 kb of the 5Ј-flanking region of the pp120 gene (Fig. 1 and Ref. 11), yielded several (0.4 –10 kb) DNA fragments that were ligated into pBluescript II KSϩ plasmid
Summary
Vol 271, No 15, Issue of April 12, pp. 8809 –8817, 1996 Printed in U.S.A. Cloning and Characterization of a Functional Promoter of the Rat pp120 Gene, Encoding a Substrate of the Insulin Receptor Tyrosine Kinase*. Several motifs resembling response elements for insulin (in the rat phosphoenolpyruvate carboxykinase gene), glucocorticoids, cAMP, and phorbol esters as well as a number of putative binding sites for activating proteins-1 (Jun/Fos) and -2, and liver-specific factors were detected. To understand the molecular basis for the regulation by glucocorticoids, we have cloned the 5Ј-flanking region and demonstrated a functional promoter activity of the pp120 gene This region lacks a TATA box and contains multiple transcription initiation sites as well as potential binding sites for basal and regulatory transcriptional elements. The mutant promoter fragments were cloned into the XhoI/HindIII sites of pGL3-BASIC plasmid Using this procedure, three mutant constructs were synthesized and designated as Ϫ249Mut3pLuc, Ϫ249Mut5pLuc, and Ϫ249Mut6pLuc containing block substitution mutation from nt Ϫ209 to Ϫ189, Ϫ169 to Ϫ149, and Ϫ148 to Ϫ128, respectively. After initial DNA denaturation at 94 °C for 5 min, 20 (first reaction) or 25–30 (second reaction) cycles of PCR were carried out as described previously [15]
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