D'Errico I, Reyes A, Dinardo M M, Gadaleta G
Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Via Orabona, 4, 70126 Bari-Italy.
Gene. 2005 Jul 18;354:117-24. doi: 10.1016/j.gene.2005.03.044.
The mitochondrial transcription factor A (Tfam) is a member of the HMG-box protein family, necessary for both transcription and maintenance of mitochondrial DNA. The gene is structured in seven exons and six introns and it is estimated to span about 10 kb in mouse, human and rat. In addition to the full length mRNA of Tfam, a shorter mRNA isoform lacking exon 5 has been found to be widely distributed in human and rat tissues. Here we present the isolation and characterization of Tfam gene in the primate Presbytis cristata which belongs to the Cercopithecidae family. We have determined the complete CDS sequence, the size of all the six introns, the complete sequences of the three shorter ones (I, III, VI) and the partial sequences of the long introns (II, IV, V). The comparison with other available Tfam sequences from mammals has revealed a high degree of conservation (above 90%) both in CDS and introns. By in situ hybridization (FISH) experiments we have mapped Tfam gene on chromosome 12 which, according to other cytogenetics studies, is the homologous region of chromosome 10, where human Tfam has been mapped. Moreover we have searched for the presence of alternatively spliced isoforms through several approaches, such as RT-PCR and differential hybridization. In Presbytis cristata we have not detected the presence of any spliced isoforms lacking exons; however we have identified one isoform in which part of the intron I is retained in the mRNA. The inclusion of this portion of intron I would originate an early stop codon if translated.
线粒体转录因子A（Tfam）是HMG-box蛋白家族的成员，对于线粒体DNA的转录和维持均必不可少。该基因由7个外显子和6个内含子构成，在小鼠、人类和大鼠中估计跨度约为10 kb。除了Tfam的全长mRNA外，还发现一种缺少外显子5的较短mRNA异构体在人类和大鼠组织中广泛分布。在此，我们展示了在属于猕猴科的灵长类冠叶猴中Tfam基因的分离和特征。我们确定了完整的CDS序列、所有6个内含子的大小、3个较短内含子（I、III、VI）的完整序列以及长内含子（II、IV、V）的部分序列。与来自哺乳动物的其他可用Tfam序列进行比较后发现，CDS和内含子均具有高度保守性（超过90%）。通过原位杂交（FISH）实验，我们将Tfam基因定位到了12号染色体上，根据其他细胞遗传学研究，该染色体是10号染色体的同源区域，人类Tfam基因已定位在10号染色体上。此外，我们通过多种方法，如RT-PCR和差异杂交，搜索了选择性剪接异构体的存在情况。在冠叶猴中我们未检测到任何缺少外显子的剪接异构体的存在；然而，我们鉴定出一种异构体，其中内含子I的一部分保留在了mRNA中。如果进行翻译，内含子I的这部分包含会产生一个提前终止密码子。
