Evidence For A Second Promoter

In our work, we used a genomic walking protocol to determine if the promoter described by Levinson et al. was connected to the 196 bp of Chelly et al. DNA from a human genomic library was amplified using a primer that anchored nucleotides in the 196-bp region. The experiments succeeded

Fig. 4. Organization and mapping of the MNK 5' flanking region. (A) Two separate loci with contiguous promoters linked to a common untranslated exon which through splicing links to the coding region; (B) two separate loci each with a different promoter. Only P-2 connects with exon 0. Exon 1 is common to both loci. In (A) a suspected PvuII sites flanking P-1 gives rise to a 2.0-kb DNA fragment extending from exon 1. This fragment when cloned was found to contain P-1, the promoter described by Levinson et al. (17). The 0.8-kb fragment is believed to originate in an unknown PvuII site between upstream exon 1 and exon 0.

Fig. 4. Organization and mapping of the MNK 5' flanking region. (A) Two separate loci with contiguous promoters linked to a common untranslated exon which through splicing links to the coding region; (B) two separate loci each with a different promoter. Only P-2 connects with exon 0. Exon 1 is common to both loci. In (A) a suspected PvuII sites flanking P-1 gives rise to a 2.0-kb DNA fragment extending from exon 1. This fragment when cloned was found to contain P-1, the promoter described by Levinson et al. (17). The 0.8-kb fragment is believed to originate in an unknown PvuII site between upstream exon 1 and exon 0.

in amplifying a genomic fragment that had Chelly et al.'s sequences and 1.3 kb of nucleotides upstream, in all, about 1.5 kb upstream from exon 1. Partial sequence data for this segment is shown in Fig. 3A. None of the bases beyond the first seven matched the sequences of Levinson et al. (Fig. 3B). Instead, the first 600 bases contained 14 sites that matched cis-acting sites in promoters including 2 TATA boxes, a CAAT site, and AP-1 and AP-3 sites, all significant regulatory sites for controlling eukary-otic gene expression.

In further studies, we took advantage of the fact that the promoter of Levinson et al. lacked a PvuII restriction site and repeated the upstream analysis on a PvuII digest of the genomic DNA. A second genomic walking analysis on the fragments again used the exon 1 primer as a downstream anchor. Two equally strong well-defined bands of 0.8 and 2.0-kb were generated. The 2.0-kb band upon cloning and sequencing matched the sequences reported by Levinson et al. (Fig. 4B), confirming a connection between Levinson et al.'s promoter and exon 1. No evidence for a second promoter sequences were found in the 2.0-kb fragment. The data, therefore, suggest two distinctly dissimilar promoters in the region flanking exon 1. That neither the sequences reported by Chelly et al. nor Levinson et al. had a PvuII site leads us to conclude that the 0.8-kb band may a fragment that joins exon 1 to an intron that is proximal to exon 1. This hypothesis is currently being investigated.

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