title: Transient expression of the cloned mouse c-Ha-ras 5' upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element.
abstract: The mouse Ha-ras oncogene is activated by point mutation and overexpressed in developing papillomas during two-stage skin carcinogenesis in SENCAR mice. One of our research aims is to characterize the factors regulating Ha-ras gene expression at the transcriptional level in SENCAR mouse epidermis. Towards this goal, we sequenced 1400 bp of the 5' upstream region of the mouse Ha-ras gene so as to characterize various cis-regulatory elements present in the gene. We identified seven sites with the proper consensus sequence for binding the SP1 transcription factor and three potential binding sites for the CTF-1 factor. In addition, we located a 13-base sequence with 92% homology to the consensus sequence for an estrogen response element and two hexamers with consensus sequences identical to the core sequence of the glucocorticoid response element. A series of transient gene expression vectors was constructed in which various regions of the mouse Ha-ras 5' upstream region were fused to the chloramphenicol acetyltransferase (CAT) gene. These expression plasmids were transfected into newborn and adult primary SENCAR epidermal cells, the epidermal cell population that presumably contains the stem cells involved in two-stage skin tumorigenesis. Transient gene expression assays carried out after 48-72 h indicated that a 2.3-kb Ha-ras 5' fragment produced CAT activity comparable to that produced by pSV2CAT and pdolCMVCAT, both of which are plasmids with strong viral promoters and enhancers driving CAT gene expression. Maintenance of transfected keratinocytes under both nondifferentiating (0.05 mM calcium) and differentiating (1.2 mM calcium) culture conditions demonstrated that the mouse Ha-ras upstream region was relatively unresponsive to changes in calcium concentration in transient expression assays carried out in either newborn or adult keratinocytes. Our results demonstrated the power of the cloned mouse Ha-ras promoter and upstream region in driving transient gene expression after transfection into primary keratinocytes.
authors.affiliation: Eppley Cancer Research Institute, University of Nebraska Medical Center, Omaha 68198-6805.
authors.forename: R
authors.initials: R
authors.lastname: Neades
authors.forename: N A
authors.initials: NA
authors.lastname: Betz
authors.forename: X Y
authors.initials: XY
authors.lastname: Sheng
authors.forename: J C
authors.initials: JC
authors.lastname: Pelling
mesh_terms: D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D011905:Genes, ras; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000502:physiology*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium