Abstract
In rat liver there are two types of serine:pyruvate aminotransferase (SPT) whose natures are indistinguishable but whose subcellular localization are different. One is a mitochondrial enzyme (SPTm) and the other a peroxisomal enzyme (SPTp). We compared, in this study, the structure of mRNAs encoding SPTm and SPTp by comparison of the sizes after removal of poly(A) tail by ribonuclease H and by means of RNA blot analysis and S1 nuclease protection assay. No differences were detected between these two mRNAs other than that about 100 nucleotides of the 5'-terminal sequence of SPTm mRNA are lacking in SPTp mRNA, and the length of the poly(A) tail is different. Southern blot analysis of rat genomic DNA showed that the SPT gene is single. Primer extension and S1 nuclease mapping analyses, using a DNA fragment of a genomic clone, revealed that the SPTm and SPTp mRNAs are transcribed from different initiation sites, about 70 nucleotides apart, in the same exon, exon 1. Ribonuclease protection assay performed with RNA hybridization probe corresponding to 5'-terminal portion of SPTm mRNA also showed that the 5'-terminal sequence of SPTp mRNA is about 70 nucleotides shorter than that of hormone-responsive SPTm mRNA. These results indicate that the different organelle distribution of SPTm and SPTp, the products of the same SPT gene, arises from transcription from different initiation sites, conferring N-terminal extension peptide, the mitochondrial targeting signal, only on the translation product of SPTm mRNA.
Highlights
IntroductionSouthern blot analysis of rat genomic DNA showed that the serine:pyruvate aminotransferase (SPT) gene is single
Comparison of SPTm mRNA with SPTp mRNA--In our previous papers [5, 6] it was reported that SPTm mRNA was approximately 200 nucleotides larger than SPTp mRNA, as judged by RNA blot analysis
We showed in this study that two kinds of serine:pyruvate aminotransferase (SPT) mRNA are generated from a single gene through differential initiation of transcription
Summary
Southern blot analysis of rat genomic DNA showed that the SPT gene is single. Primer extension and Sl nuclease mapping analyses, using a DNA fragment of a genomic clone, revealed that the SPTm and SPTp mRNAs are transcribed from different initiation sites, about 70 nucleotides apart, in the same exon, exon 1. These results indicate that the different organelle distribution of SPTm and SPTp, the products of the same SPT gene, arises from transcription from different initiation sites, conferring N-terminal extension peptide, the mitochondrial targeting signal, only on the translation product of SPTm mRNA. The mitochondrial enzyme (SPTm)’ and peroxisomal enzyme (SPTp) have very similar immunological [2,3], catalytic and physical [4] properties, but their responses to hormones or other stimuli are quite different.
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