Transcription initiation sites and structural organization of the extreme 5' region of the rat neural cell adhesion molecule gene.

Abstract

Through analysis of rat genomic cosmid clones, the 5'-most exon of the rat neural cell adhesion molecule (NCAM) gene was identified. This exon, here named exon 0, contained the entire 5' untranslated region and the N-terminal signal sequence of the polypeptide. Exon 0 was isolated from a 1.6-kilobase (kb) EcoRI-HindIll fragment of rat genomic cosmid clone 9 which was 35 kb in length. This fragment was sequenced and found to contain -940 base pairs (bp) of 5'-flanking sequence, exon 0, which was -245 bp in length, and -400 bp of the following intron 0. By using information derived from this fragment and the pR18 rat NCAM cDNA, the transcription initiation sites were determined with two assays. Both primer extensions and nuclease Sl protection assays of postnatal day 7 rat brain RNA identffied seven initiation sites within a single 10-bp region at positions -195 to -186 relative to the translation start site. An additional minor site was found at position -329. In the immediate 5' region, no consensus TATA or CCAAT sequences were found. Potential regulatory elements within this region include Spl consensus binding sites and also a 178-bp homopurine-homopyrimidine sequence containing several mirror repeats. NCAM has multiple transcripts which are regulated in a developmental and tissue-specific fashion. To determine whether these transcripts are initiated at the same sites, transcription initiation sites were analyzed in postnatal day 7 and adult rat brain and also in cultured cell lines of neuronal, glial, and muscle phenotypes. These tissues and cells exhibited distinct NCAM transcript populations in Northern (RNA) dot blot analysis. In all cases similar transcription start sites were found, suggesting that all major NCAM transcripts have similar or identical initiation sites. These results provide essential information to begin analysis of NCAM regulation in different tissues and during development.

Sp1 Transcription Factor as a Molecular Target for Nitric Oxide– and Cyclic Nucleotide–Mediated Suppression of cGMP-Dependent Protein Kinase-Iα Expression in Vascular Smooth Muscle Cells

“…The importance of Sp1 for PKG gene expression may be related to studies showing that genes lacking TATA or CCAAT elements require Sp1 binding for transcription. 21,22 PKG-I␣ is one such gene that does not contain the putative TATA or CCAAT boxes. The mechanism by which NO directly modulates Sp1 transcriptional activity may be related to its ability to interact with Zn.…”

“…In many genes lacking a TATA box, a proximally positioned Sp1 site serves as the critical determinant of promoter activity and dictates the start site of transcription. 21,22 In this study, we have examined the effects of NO and cyclic nucleotide analogues on the modulation of PKG-I␣ expression at the transcriptional level. For this purpose, embryonic vascular smooth muscle cells (A7r5) were transfected with a human PKG-I␣ promoter (500 bp) and then treated with NO donors or cyclic nucleotide analogues.…”

Neural cell adhesion molecules in rat endocrine tissues and tumor cells: distribution and molecular analysis.

“…At least 20 major exons code for the different NCAM isoforms. The leader sequence is encoded by exon 0 (6)(7)(8). Exons l-14 are used to generate the extracellular domain of all three polypeptides.…”

Promoter Region of the Transcriptional Unit for Human α1‐Chimaerin, a Neuron‐Specific GTPase‐Activating Protein for p21rac

“…Primer extension was performed according to the method modified by Chen et al [13]. Briefly, total RNA (25 pg) and Y-~*Plabelled primer (1 X lo5 cpm) were mixed and heated at 80 "C for 5 min in 10 pl annealing buffer (0.25 M KC1 and 10 mM Tris/ HCl, pH 8.3), then allowed to anneal for 90 min at 55°C.…”