Determination of the genomic structures of two nonallelic preproinsulin genes in Xenopus laevis using the polymerase chain reaction

General and Comparative Endocrinology
A R ShuldinerL A Scott

Abstract

We have previously cloned cDNAs that correspond to two different nonallelic preproinsulin genes in the amphibian, Xenopus laevis (Shuldiner et al., 1989, J. Biol. Chem. 264, 9428-9432). The coding regions of the two genes are very similar (i.e., 93% amino acid identity). While both preproinsulin genes are expressed coordinately in the adult pancreas, they are differentially expressed in prepancreatic embryos during neurulation (Shuldiner et al., 1991, Proc. Natl. Acad. Sci. USA 88, 7679-7683). We now report the use of the polymerase chain reaction (PCR) to investigate the genomic structures of both Xenopus preproinsulin genes. First, the nucleotide sequence of a portion of the 5' untranslated region that was lacking in our cDNA clones was obtained using rapid amplification of cDNA ends (5' RACE). Then, oligonucleotide primers were designed that flanked putative exon-intron splice boundaries, and intronic sequences in each of the two nonallelic genes were amplified. We determined that both Xenopus preproinsulin genes contain two introns, one interrupting the 5' untranslated region, and the second interrupting the region encoding the C-peptide. The introns are similar in position, but of greater length than those reported in most...Continue Reading

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