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-HindIII fragment of rat genomic cosmid clone 9 which was 35 kb in length. This fragment was sequenced and found to contain approximately 940 base pairs (bp) of 5'-flanking sequence, exon 0, which was approximately 245 bp in length, and approximately 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 S1 protection assays of postnatal day 7 rat brain RNA identified 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 Sp1 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.
Highlights
Through analysis of rat genomic cosmid clones, the 5'-most exon of the rat neural cell adhesion molecule (NCAM) gene was identified
By using information derived from this fragment and the pR18 rat NCAM cDNA, the transcription initiation sites were determined with two assays
The probe was derived from p5B4-2 plasmid, which contains the partial sequence of the rat NCAM-180 cDNA [51]
Summary
Oligonucleotide primers 89.5 and 89.4 were synthesized by using phosphoramidite chemistry based on sequence from the 5' terminus of NCAM-140 cDNA clone pR18. The same oligonucleotides used for the primer extension assays (oligonucleotides 89.5 and 89.4) were used to synthesize single-stranded probes for the S1 assays. 32P_ 5'-end-labeled oligonucleotide 89.5 or 89.4 was annealed to a single-stranded M13 mpl template containing a 1.2-kb EcoRI-PstI subfragment of pR.9E.3.7 containing exon 0 and 940 bp of the upstream sequences. The probe was derived from p5B4-2 plasmid, which contains the partial sequence of the rat NCAM-180 cDNA [51]. Oligonucleotide 89.8 was synthesized based on the rat NCAM-120specific sequence from plasmid pR13.2 (unpublished data). Filters were probed with random primer 32P-labeled pR.9E.3.7 restriction fragments. Hybridization and washing conditions were identical to those described for the RNA dot blot analysis with the 0.78-kb AvaI-PstI fragment.
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