Tfam is a single copy nuclear gene structured in seven exons and six introns coding for the mitochondrial transcription factor A. Tfam is a member of the HMG (high-mobility-group) protein family, having two HMG boxes. It enhances mtDNA transcription by mitochondrial RNA polymerase in the presence of mitochondrial transcription factor B and is essential for mtDNA maintenance. ft is known the presence of a smaller isoform of the Tfam gene originated by an alternative splicing mechanism of the exon 5 has been identified in human and rat constituting respectively about 30% and 10% of Tfam transcripts in ali tissues. Moreover the presence of a processed pseudogenes lacking exon 4 of the gene has been recently reported in Presbytis cristata. This one could be the trace of a now extinct alternative transcript of Tfam gene. Here we present the search of alternative splicing isoforms of Tfam gene in different Primate's species through several approaches. Materials and Methods: Lymphoblastoid (Pongo pyg-maeus, Pan troglodytes, Papio anubis, Hylobates lar, Pan paniscus, Presbytis cristata) and fibroblastoid (Lemur catta) celi lines were used. Total RNAs were reverse tran-scribed wíth the Super Script TM III One-Step RT PCR using a standard protocol. For radioactive experiments a 3Z P dCTP has been used. RESULTS: RT PCR experiments were carried out using radioactive nucleotides and primers designed on exons 2 and 7 of the gene revealing the presence of the Tfam 05 isoform only in hominids. In Presbytis cristata a standard RT PCR experiment with the same pair of primers was performed. The products were differentially hybridizated with two different probes separately, one corresponding to exon 4 and the other to exon 5. The result of these hybridizations shows only the presence of the band relating to the Tfam complete transcript. RT PCR experi-ments, using a pair of primers designed on exons 1 and 5 of Tfam gene has revealed the presence of a splicing isoform containing a tract of intron I of the gene. We have analyzed the three open reading frames of the new ísoformobserving several stop codons and so excluding that this isoform is translated

Spread of Tfam isoforms in primate species

DE VIRGILIO, Caterina;GADALETA, Gemma
2005

Abstract

Tfam is a single copy nuclear gene structured in seven exons and six introns coding for the mitochondrial transcription factor A. Tfam is a member of the HMG (high-mobility-group) protein family, having two HMG boxes. It enhances mtDNA transcription by mitochondrial RNA polymerase in the presence of mitochondrial transcription factor B and is essential for mtDNA maintenance. ft is known the presence of a smaller isoform of the Tfam gene originated by an alternative splicing mechanism of the exon 5 has been identified in human and rat constituting respectively about 30% and 10% of Tfam transcripts in ali tissues. Moreover the presence of a processed pseudogenes lacking exon 4 of the gene has been recently reported in Presbytis cristata. This one could be the trace of a now extinct alternative transcript of Tfam gene. Here we present the search of alternative splicing isoforms of Tfam gene in different Primate's species through several approaches. Materials and Methods: Lymphoblastoid (Pongo pyg-maeus, Pan troglodytes, Papio anubis, Hylobates lar, Pan paniscus, Presbytis cristata) and fibroblastoid (Lemur catta) celi lines were used. Total RNAs were reverse tran-scribed wíth the Super Script TM III One-Step RT PCR using a standard protocol. For radioactive experiments a 3Z P dCTP has been used. RESULTS: RT PCR experiments were carried out using radioactive nucleotides and primers designed on exons 2 and 7 of the gene revealing the presence of the Tfam 05 isoform only in hominids. In Presbytis cristata a standard RT PCR experiment with the same pair of primers was performed. The products were differentially hybridizated with two different probes separately, one corresponding to exon 4 and the other to exon 5. The result of these hybridizations shows only the presence of the band relating to the Tfam complete transcript. RT PCR experi-ments, using a pair of primers designed on exons 1 and 5 of Tfam gene has revealed the presence of a splicing isoform containing a tract of intron I of the gene. We have analyzed the three open reading frames of the new ísoformobserving several stop codons and so excluding that this isoform is translated
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/132194
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