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In studies on the regulation of gene expression of mtTF1, we examined the steady state level of the mRNA in cultured HeLa cells. We found that in addition to the major mRNA, 30% of the mRNA of mtTF1 in the cells was a smaller isoform with a 96 base deletion. This smaller mRNA was also found in most human tissues. The region of the deletion corresponds to the second HMG-box, which may interact directly with DNA. We examined the structure of the genomic gene encoding the human mtTF1 to determine the mechanism of the deletion. We found that the gene is composed of 7 exons spanning over 10 kilobase-pairs and that its 5th exon is identical to the 96 bases skipped in the shorter mRNA. Therefore, the shorter mtTF1 is concluded to be generated by alternative splicing. 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