Abstract
The organization, sequence, and transcriptional regulation of expression of the murine S100 beta gene are reported. The gene is approximately 9 kilobase pairs in length and is composed of three exons and two introns. The deduced murine S100 beta protein sequence differs from the human S100 beta protein by only 1 amino acid. The murine S100 beta gene contains a TATA box (AATAA) and a reverse CCAAT box (ATTGG) located at 30 nucleotides and 92 nucleotides upstream of the cap site, respectively. A 149-base pair DNA fragment (-157/-9) spanning the TATA box and the reverse CCAAT box functions as a promoter. The murine S100 beta promoter drives a 4-fold higher level of transcription in glial (C6) than in non-glial (3T3) cells, suggesting the existence of a potential cell type-specific regulatory element within the promoter region. The 5'-flanking region suppresses transcription from the homologous S100 beta as well as the heterologous SV40 promoters in an orientation-independent fashion. However, the 5'-flanking region exhibits cell type specificity when suppressing the S100 beta promoter-dependent transcription, indicating its involvement in the cell type-specific expression of S100 beta gene. In order to map cell type-specific regulatory elements, transcription analyses of various deletions of the 5'-region were carried out in C6 and 3T3 cells. Two cell type-specific negative regulatory elements, one active in non-glial cells and another active in glial cells, were mapped to the regions -1552/-1234 and -1234/-551, respectively. A strong negative regulatory element and a relatively weak negative element were located in the regions -551/-157 and -1669/-1552, respectively. The murine S100 beta gene is under complex transcriptional regulation involving tonic negative control exerted by combination of multiple cis-acting regulatory elements including cell type-specific elements.
Highlights
The organization,sequence, and transcriptional reg- SlOOB is a small acidic Ca2+-binding proteinwhich in the ulation of expression of the murine SlOOj3 gene are nervous system is expressed exclusively by glial cells (1-3)
In order to map cell type-specific regulatory elements, recently have biological functions been convincingly ascribed to this protein
Sloop has been shown to inhibit the phosa relatively weak negative lement were located in the phorylation of a number of protein kinase C substratesfound regions -5511-157 and -16691-1552, respectively. in the nervous system, including MARCKS
Summary
Reagents-Restriction enzymes and other DNA modifyingenzymes were purchased from Life Technologies, Inc., Boehringer Mannheim, Promega, Stratagene, United States Biochemical Corp., and International Biotechnologies Inc. A 2.2-kb SmaI restriction fragment (-1669/+470) from the murine Sloop genomic clone containing the entire 5'-flankingregion, exon I, and a B'-portion of intron Iwas cloned in the sense orientation upstream of the CAT gene in pCAT-Basic vector and the resulting plasmid was named pCB22A. To generate the pCES-167 construct, a 2.5-kb BamHI restriction fragment (-167/+2307) containing the potential promoter, entire exon I, and a 5'-portion of intron I from the murine Sloop genomic clone was first blunt-ended with T4 DNA polymerase (Life Technologies, Inc.) and cloned in the sense orientation upstream of the CAT gene in pCAT-Enhancer vector at theBglII site. To generate the PCP-1669A and PCP-1669B constructs, a 1.5-kb BamHI restriction fragment (-1669/-167) which contained most of the 5"flanking region of the murine Sloop gene was inserted upstream of the SV40 promoter in pCAT-Promotervector, in eithersense (PCP-1669A) or antisense (PCP-1669B) orientation. Murine SI OOP Gene Expression and pCBS-1669B constructs, a 1.5-kb EcoRI-ApaI restriction fragment (-1669/-142) of the 5’-flanking region of murine SlOO@ge-
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