History of the Tfam gene in primates

Gene 362 (2005) 125 – 132 www.elsevier.com/locate/gene

History of the Tfam gene in primates Ilenia D’Errico a, Maria Maddalena Dinardo b, Oronzo Capozzi c, Caterina De Virgilio a, Gemma Gadaleta a,* a

Dipartimento di Biochimica e Biologia Molecolare, Universita` degli studi di Bari, via Orabona 4/A, 70125, Bari, Italy b Dipartimento di Fisiologia generale ed ambientale, Universita` di Bari, Italy c Dipartimento di Genetica e Microbiologia, Universita` di Bari, Italy Received 7 April 2005; received in revised form 5 July 2005; accepted 7 July 2005 Available online 30 September 2005 Received by G. Bernardi

Abstract Tfam is a single copy nuclear gene mapping on chromosome 10 in human and mouse, 20 in rat and 12 in Presbytis cristata. It encodes for an HMG (high-mobility-group) protein showing a high affinity with the two transcriptional promoters and other mitochondrial DNA regions. It is an activator of mitochondrial transcription acting in the presence of mitochondrial RNA polymerase and of transcription factor B. Other interesting features of Tfam gene in human and rat are reported such as the existence of a smaller isoform, originated by an alternative splicing mechanism of the exon 5 (D5 isoform) and the presence of different processed pseudogenes in addition to the active copy of the gene. In order to widen knowledge about Tfam gene and the appearance of some of its properties in the evolutionary history of primates, we have studied some aspects of this gene in different species. In particular we have determined its chromosomal localization, suggesting that its locus is highly conserved; we have searched for the presence of the D5 isoform, demonstrating that it is present only in hominids; we have provided evidence of Tfam processed pseudogenes in the majority of the analysed genomes. Sequence data from this article have been deposited in the EMBL nucleotide database. D 2005 Elsevier B.V. All rights reserved. Keywords: Alternative splicing; Processed pseudogene; Fluorescence in situ hybridization

1. Introduction Tfam is a single copy nuclear gene mapping on chromosome 10 in human (Tiranti et al., 1995) and mouse (Larsson et al., 1997), 20 in rat (Rantanen et al., 2001) and 12 in Presbytis cristata (D’Errico et al., 2005). It spans about 10 kb and is structured in seven exons and six introns (Tominaga et al., 1993; Larsson et al., 1997; Mezzina et al., 2002; Reyes et al., 2002; D’Errico et al., 2005). This gene encodes for a mitochondrial transcription factor, member of Abbreviations: mtDNA, mitochondrial DNA; Tfam, mitochondrial transcription factor A; LSP, light strand promoter; HSP, heavy strand promoter; HMG, high mobility group. * Corresponding author. Tel.: +39 080 5443471; fax: +39 080 5443317. E-mail address: [email protected] (G. Gadaleta). 0378-1119/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.07.007

the HMG (high-mobility-group) protein family, having two HMG boxes. It shows a high affinity with the light and heavy strand promoters, LSP and HSP, bending and unwinding mitochondrial transcriptional promoters in the D-loop region (Fisher et al., 1987, 1989; Fisher and Clayton, 1988) and other mtDNA regions (Gadaleta et al., 1996; Pierro et al., 1999), a characteristic typical of the HMG family of proteins. Tfam enhances mtDNA transcription by mitochondrial RNA polymerase in the presence of mitochondrial transcription factor B (Falkenberg et al., 2002; McCulloch et al., 2002) and is essential for mtDNA maintenance (Shadel and Clayton, 1997; Maniura-Weber et al., 2004). Recently, it has been reported that human Tfam is abundant enough to wrap entire mtDNA (Takamatsu et al., 2002) and that most Tfam molecules associate with mtDNA (Alam et al., 2003).

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An interesting feature of the Tfam gene was reported in 1993 by Tominaga and co-workers about the existence of a smaller isoform in human tissues. This originates from an alternative splicing mechanism of the exon 5, which removes part of the HMG box2 and produces an isoform (D5 isoform) uniformly distributed and constituting about 30% of Tfam transcripts in all human tissues. We have since also shown the presence of this isoform in different rat tissues in which it represents about 10% of the Tfam transcripts (Mezzina et al., 2002). Other studies of the Tfam gene have shown, in addition to the active copy, the existence of different homologous sequences identified as processed pseudogenes in the human and rat genomes (Mezzina et al., 2002; Reyes et al., 2002). The term ‘‘pseudogene’’ comprises a wide group of non-functional loci with a marked diversity of characteristics (D’Errico et al., 2004). They have been described as dead genes, because they are homologous to their functional gene but contain nucleotide changes that prevent the production of a functional protein. Pseudogenes are created by two mechanisms: tandem duplication or retrotransposition from a functional gene. Tandem duplication originates non-processed pseudogenes, which are usually linked to their functional gene and conserve the exon – intron structure. The second mechanism creates processed pseudogenes or retropseudogenes through reverse transcription from partial or completely processed messenger RNAs. Processed pseudogenes are typically characterized by an absence of both the 5V-promoter sequence and introns, the presence of a 3V-polyadenylation tract and flanking direct repeats (Rogers, 1985; Vanin, 1985). Through Southern blotting hybridization and in silico mapping, using a probe of Tfam sequence, it was determined that about 20 Tfam processed pseudogenes exist in human genomic DNA (Reyes et al., 2002) and at least 12 copies in the rat genome (Mezzina et al., 2002) were detected by in vitro hybridization technique. In this work we have tried to expand the range of information about the Tfam gene and outline its evolutionary history studying some of its features and their arising during the evolution of the primates. Molecular and morphological studies (Shoshani et al., 1996; Goodman et al., 1998) divide the primates into strepsirrhines (lemuriforms and loriforms) and haplorhines (tarsiers and anthropoids), the anthropoids into platyrrhines (New World monkeys) and catarrhines, and the catarrhines into cercopithecids (Old World monkeys) and hominids (apes and humans). We have chosen species belonging to different families of the primate order: cercopithecids (Papio anubis and Presbytis cristata), hylobatids (Hylobates lar), hominids (Pongo pygmaeus, Pan troglodytes, Pan paniscus, and Homo sapiens) and lemurids (Lemur catta). In these ones we have determined: i) the chromosomal localization of Tfam gene, by fluorescence in situ hybridisation (FISH); ii) the presence of alternative splicing products and in particular of the well documented alternative isoform (D5

isoform) previously found in human and rat tissues (Tominaga et al., 1993; Mezzina et al., 2002); and iii) the presence of Tfam processed pseudogenes in the genome of all the primates studied.

2. Materials and methods 2.1. Fluorescence in situ hybridization Metaphase preparations of primates’ cultured cells were hybridized with BAC probes belonging to the RP11 de Jong libraries. The fluorescence in situ hybridization experiment was performed essentially as described by Lichter et al. (1990). Digital images were obtained using a Leica DMRXA epifluorescence microscope equipped with a cooled CCD (charge coupled device) camera (Princeton Instruments, N. J.). The Cy 3 and 4V, 6-diamidino-2phenylindole (DAPI) fluorescence signals detected with specific filters were recorded separately as grey-scale images. Pseudocoloring and merging of images were performed using Adobe Photoshop software. 2.2. Nucleic acids extraction from cultured cells Lymphoblastoid cell lines were used for all the species except for Lemur catta whose genomic DNA and RNA was extracted from fibroblastoid cell cultures. Genomic DNAs were extracted using the Wizard Genomic DNA Purification Kit (Promega) and treated with RNase free DNase (Roche). Total RNAs were prepared using the RNeasy Mini Kit (Qiagen). The cells were cultured at 37 -C in RPM1-1640 medium supplemented with 10% foetal bovine serum, penicillin (100 U/ml) and streptomycin (100 U/ml) and 5  106 cells were used for RNA and DNA extraction. The extracted RNAs were treated with Rnase-free Dnase (Roche). Genomic DNAs were treated with Dnase-free Rnase (Roche). 2.3. Reverse transcription-polymerase chain reaction In order to analyse Tfam coding sequences in different species, as described in a previous work (Mezzina et al., 2002), we have carried out a search of the known Tfam sequences available in the EMBL database. Thus, Tfam cDNAs have been multialigned and then primers have been designed on highly conserved regions on exons 2 and 7 (Ex2for 5V-GAA CA(AG) CTA CCC A(AT)A TTT AAA GC-3V and Ex7rev 5V-CAT (CT)TG (CT)TC TTC CCA (AG)GA (CT)TT CAT TTC ATT-3V). Total RNAs (1.5 Ag) were reverse transcribed with the Super Scripti III One-Step RT-PCR containing Platinum Taq (Invitrogen) using a standard protocol. For radioactive RT-PCR experiments, the same kit and conditions were used as above but including 10 ACi of a32P dCTP in the reaction mix.

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2.4. Polymerase chain reaction amplification Total genomic DNAs (100 ng) were amplified with a recombinant Taq polymerase (Promega) and primers cited before following a standard protocol.

with LINEUP program, both from the GCG package (GCG, 1994). Based on this multialignment, the percentages of similarity between sequences were obtained using the DISTANCES program (GCG, 1994).

2.5. Cloning and sequencing

3. Results

PCR and RT-PCR products were electrophoresed on 1% agarose gels, purified using the High Pure PCR Product Purification Kit (Roche) and cloned in pGEM-T easy vector (Promega). At least five clones for each PCR and RT-PCR product were completely sequenced in both directions. Sequencing of cloned fragments was performed with an automated sequencer (Seq 4  4, Amersham) using Thermo Sequenase Cy 5.5 Dye Terminator Cycle Sequencing Kit (Amersham). The specific cycle sequencing parameters depend on the primer sequence and the amount and purity of the template added to the reactions.

3.1. In situ hybridization of the Tfam gene

2.6. Data analysis The partial CDS sequences of the Tfam gene of the different species analysed were multialigned using the PILEUP program and adjusted manually, when necessary,

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We carried out fluorescence in situ hybridization experiments on metaphase preparations of cultured cells. Pongo pygmaeus, Pan troglodytes, Papio anubis, Hylobates lar, Pan paniscus, Presbytis cristata and Homo sapiens metaphase preparations were hybridized with BAC probe RP11-373P23. It contains a tract of chromosome 10 stretching from position 59 432 030 to position 59 627 477 and including the Tfam gene (from position 59 489 779 to position 59 500 500); Lemur catta metaphase preparation was hybridized using three contiguous BAC clones (RP11-373P23 and the two adjacent ones RP11-644O12 and RP11-1056J5). Fig. 1 shows in detail the chromosome mapping of the Tfam gene (red signals) and the evolutionary distances between the species analysed.

Fig. 1. Tfam gene mapping. The chromosomes containing the Tfam locus in the primates analysed and their divergence times in Million years ago (Mya); NWN=new world monkeys; OWM=old world monkeys.

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3.2. Tfam splicing isoform research

Table 1 Comparison of partial Tfam CDS sequences

We carried out RT-PCR amplification of total RNA, with and without the incorporation of radioactive nucleotides. We performed the experiments using the pair of primers cited in Materials and methods. RT-PCR in the presence of radioactive nucleotides allowed the detection of small amounts of the fragments in the exponential phase of amplification. As shown in Fig. 2a, after a 2-h exposure we have obtained a fragment of about 480 bp, in all the species analysed (lanes 1–8), and an additional band of about 380 bp only in the two chimps Pan paniscus and Pan troglodytes (lanes 6, 7) and in Homo sapiens (lane 8). After a longer exposure of 60 h (Fig. 2b), a clear signal became visible in the lane 5 corresponding to Pongo pygmaeus. The size of the shorter band corresponds exactly to the dimension of D5 isoform detected in human (Fig. 2c). We have cloned and sequenced all the 480 bp fragments, obtained in non-radioactive RT-PCR experiments, and the sequences corresponding to the partial CDS of Tfam submitted in EMBL nucleotide database under the accession numbers AJ841766-AJ841771, AJ830015. Table 1 shows the similarity percentages calculated among these sequences.

Species

PTR

PAN

HSA

PPY

PCR

HLA

LCA

PPA PTR PAN HSA PPY PCR HLA

99.79

98.95 98.95

98.95 98.95 98.74

97.46 97.67 96.81 96.59

94.81 95.04 94.59 95.26 93.46

93.25 93.25 92.79 93.47 90.93 93.69

69.28 69.59 69.59 69.90 69.90 70.45 69.59

experiments using the pair of primers cited above. As shown in Fig. 3a we obtained a fragment of about 480 bp in all the species and in Presbytis cristata and Homo sapiens shorter fragments of about 380 bp. We have cloned all the products and completely sequenced at least five clones. These sequences were submitted in EMBL under accession numbers AJ841771 – AJ841784. The analysis of the corresponding sequences revealed that all the amplified bands lack introns and show a high similarity (81.43% –96.23%) with human Tfam cDNA. In the Fig. 3b we have reported the exact PCR products sizes deduced by sequencing. Some fragments, obtained in the same species (Pan troglodytes and Hylobates lar), present different sequences in spite of the identical size. The sequence of the shorter bands obtained in Homo sapiens and Presbytis cristata has revealed that these fragments show a high similarity with human Tfam

3.3. Tfam processed pseudogenes isolation and analysis To verify the presence of Tfam processed pseudogenes in the genome of the species analysed, we carried out PCR

(a)

mu Le

r

tt ca

a

e Pr

1

sb

sc yti

s eu s es us lar ma dit bis ien g s u isc y e ap n glo t n p s a o a a o p tr mo lob n ng n pio Pa Po Pa Hy Pa Ho

a tat ris

2

3

4

5

6

7

8

480 bp

Tfam full length CDS

380 bp

Tfam ∆5 splicing isoform

(b)

Ominidae

(c) Exon1

Exon2

Exon3

Exon4

Exon5

Exon6

Exon7 Tfam gene

IntronII IntronIII

IntronI

2

1

3

IntronIV 4

5

IntronV IntronVI 7

6

Tfam full length CDS 1

2

3

4

6

7 Tfam ∆5 splicing isoform

Fig. 2. RT-PCR of Tfam transcripts. Radioactive RT-PCR was performed using a pair of primers designed in exons 2 and 7 of human Tfam cDNA. The amplified fragments were separated in 5% polyacrylamide gel. The gel was autoradiographed with intensifing screen at 80 -C. a) Autoradiography after 2 h exposure; two molecular weight marker sizes are reported on the left of the figure, the names of the amplified fragments on the right; b) Autoradiography after 60 h exposure; c) Schematisation of RT-PCR products. From the top: the structure of: Tfam gene, Tfam full length CDS and Tfam D5 splicing isoform; the two sizes are reported on the left of the figure. Boxes represent exons and solid line introns. The dotted lines indicate the alternatively spliced exon 5.

I. D’Errico et al. / Gene 362 (2005) 125 – 132 ta s eu es sta s r de bis cus gma lar odit s cri ien d s l i u s p a y sa an ani o p ate rog byt pl 0 b pio p ng lob n t es mo 10 Pa Pan Po Hy Pa Pr Ho

(a)

(b)

bp

500 bp 400 bp

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480 bp 380 bp

Species

Ps

Length (bp)

Homo sapiens

2

383;479

Pan troglodytes

2

478;478

Pan paniscus

3

480;481

Pongo pygmaeus

2

476;480

Hylobates lar

1

476;480;480

Papio anubis

3

480

Presbytis cristata

3

377; 478;479

Lemur catta

--

--

Fig. 3. PCR detection of Tfam processed pseudogene. PCR experiments were performed using a pair of primers designed on exons 2 and 7 of the Tfam gene: a) Agarose gel showing PCR products obtained in the primates analysed. The names of the species are reported on the top, the two molecular weight marker sizes on the left, the dimensions of PCR fragments obtained on the right. b) Species names, number and length of pseudogenes deduced by sequencing.

cDNA despite lacking exon 5 and part of exon 4, respectively.

4. Discussion 4.1. Identification of Tfam gene chromosomal position In order to analyse the chromosomal position of the Tfam gene, we first used an in silico approach comparing its position in Rattus norvegicus, Mus musculus, Homo sapiens, and Pan troglodytes whose genomes are available in the Ensembl database (http://www.ensembl.org/). This revealed that the gene is located in the same syntenic blocks in all the species analysed (Fig. 4). The position of these blocks confirms reports in the literature (Tiranti et al., 1995; Larsson et al., 1997; Rantanen et al., 2001) in fact, the blocks containing Tfam, map on chromosomes 10 in human and mouse and on 20 in rat. In Pan troglodytes the oneblock chromosome 8 corresponds entirely to human chromosome 10. These results suggest that the Tfam locus is highly conserved among mammals. Therefore with the aim of investigating the history of the Tfam locus in the primate order, we carried out fluorescence in situ hybridization (FISH) experiments on metaphase preparations of the different primates. Studies on the evolutionary history of chromosomes homologous to human chromosome 10 (PHYL-10) in primates (Carbone et al., 2002) have revealed that, in the primate ancestor, PHYL-10 was organized in two separate chromosomes: one corresponding to the short arm of human Chr.10 and one corresponding to the long arm. During the evolution of the primate order a fusion event gave rise to a unique chromosome with a marker order corresponding to the orang form (Pongo pygmaeus) (Carbone et al., 2002). The chromosomal localization obtained through FISH experiments (Fig. 1) allowed us to verify the correspondence between the position of the human Tfam gene on chromosome 10 and the other primate

species analysed. As we verified after CDS sequencing, due to the evolutionary distance between Lemur catta and Homo sapiens, the sequence of the Tfam gene in these two species is divergent enough (see Table 1). This led us to use three contiguous BAC clones for the localization of the Tfam locus in Lemur catta. As shown in Fig. 1, the Tfam gene maps on chromosome 8 in the two chimps Pan troglodytes (PTR) and Pan paniscus (PPA), 7 in Pongo pygmaeus (PPY), 2 in Hylobates lar (HLA), 12 in Presbytis cristata (PCR), 10 in Papio anubis (PAN) and 12 in Lemur catta (LCA). All these data are in perfect agreement with those previously reported about the evolutionary history of PHYL-10 (Carbone et al., 2002) confirming that the Tfam locus is highly conserved among those species. 4.2. Spread of Tfam D5 isoform in primate species In 1993, Tominaga and co-workers reported that a smaller isoform of human Tfam produced by alternative splicing of exon 5 (D5 isoform) is widely distributed in human tissues representing about 30% of Tfam transcripts. The same isoform was detected in different rat tissues where it constitutes about 10% of Tfam transcripts (Mezzina et al., 2002). As other isoforms of transcriptional factors do, even in the case of Tfam, a hypothetical dimer formation could occur (Tominaga et al., 1993). Often, the expression of these isoforms depends on the tissue and developmental stage. In some cases, shorter isoforms, generated by alternative splicing, function as negative regulators (Foulkes et al., 1991). The function and the spread of the Tfam D5 isoform are still unknown in mammals. For this reason, we were interested in its distribution in different primates, with the aim of understanding whether it is a common characteristic shared by various species, or if it is a peculiar feature that appeared at a particular moment in the evolutionary history of mammalian species.Through RT-PCR experiments, carried out using radioactive nucleotides, we have detected the Tfam gene transcriptional products. After a 2-h exposure

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Fig. 4. Tfam locus synteny conservation. Conserved syntenic blocks between Homo sapiens chromosome 10 and: Rattus norvegicus chromosomes (a), Mus musculus chromosomes (b), Pan troglodites chromosomes (c). In (d), the position of the Tfam locus on Homo sapiens chromosome 10. Black arrows indicate the block containing Tfam on Homo sapiens chromosome 10.

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(Fig. 2a) of the radioactive gel we have seen in all the lanes (1– 8) a band corresponding to the Tfam full length CDS spanning between the primers cited before and an additional band corresponding to the D5 splicing isoform only in three hominids (lanes 6 – 8). All the other species (lanes 1 –5) did not show this band. Nevertheless the lack of this additional fragment could be due both to the absence of the D5 splicing isoform or to lower efficiency of RT-PCR reaction or of primers annealing. Since a long exposure allows the revealing of weak radioactive signals, due for example to a sub-optimal RT-PCR reaction, we have extended exposure time until 60 h. As shown in Fig. 2b, under this very long condition of exposure, the band corresponding to the D5 splicing isoform, has appeared only in Pongo pygmaeus (lane 5) but not in the other species (lanes 1 –4). Moreover, we exclude an unsuccessful annealing of the primers used in RT-PCR, because the annealing sites belong to high conserved regions of the Tfam gene (see Section 2.3). The general high level of conservation of Tfam CDS sequences, revealed by similarity percentages always very high (see Table 1), confirm the spare possibility of the primers annealing failure. The sequencing of the shorter fragments obtained by non-radioactive RT-PCR in Pan troglodytes and Pan paniscus (AJ971485, AJ971484) has confirmed that these additional products correspond to Tfam CDS lacking exon 5, as expected, and show a high similarity percentage (about 99%) with human Tfam. Although Tfam D5 splicing isoform has been found in rat (Mezzina et al., 2002), the lack of an alternative splicing isoform in some species should not surprise because the alternative splicing of a particular exon is not always conserved (Pan et al., 2005). Another possible explanation for the lack of Tfam D5 splicing isoform in some primates could be ascribed to the sequence elements (belonging to introns and/or exons of the gene), which may act positively or negatively on splice site recognition and pairing (Martinez-Contreras et al., 2003). Till now, we do not know what kind of signals determine the formation of the Tfam short isoform, but it is possible that these signals are not shared by all the species, particularly if they belong to intronic regions. Anyway we did not have the possibility to analyse Tfam transcripts in other tissues, so we cannot currently exclude that alternative splicing on the above-mentioned exon could exist in other tissues. 4.3. Origin and structure of Tfam processed pseudogenes The Tfam gene presents a series of properties which correspond to genes that produce processed pseudogenes: its mRNA is widely distributed in different tissues, highly conserved, short (741 bp) and GC poor (Tominaga et al., 1993; Mezzina et al., 2002). Moreover, previous studies have reported that HMG genes could have a large number of retroposed copies (Strichman-Almashanu et al., 2003). According to these data, we previously demonstrated the presence of Tfam processed pseudogenes in rat (Mezzina et

131

al., 2002) and human (Reyes et al., 2002) and we determined that about 20 copies exist in human genomic DNA and at least 12 in the rat genome. PCR analysis produced a fragment of about 480 bp in length in all the primates analysed, except in Lemur catta 8 (Fig. 3b). Fragments of this size obviously do not correspond to the Tfam gene amplification since such a product should be of approximately 9 kb long. Moreover, we have obtained shorter fragments, of 377 and 383 bp, respectively, in Presbytis cristata and Homo sapiens (Fig. 3a). Since all PCR products lack intronic sequences and show a high similarity with human Tfam cDNA, we suggest that they correspond to Tfam processed pseudogenes. Lemur catta belongs to the suborder strepsirrhins that, from an evolutionary point of view, is far from the primates’ suborder catarrhins, which includes all the others species analysed. This evolutionary distance could correspond to a sequence divergence. So, the absence of a PCR product in Lemur catta could be due to the non-availability of primer annealing sites, or to a deletion at the level of the annealing site of one primer, or to the longer size of the region spanning between exons 2 and 7. This does not imply the absence of Tfam processed pseudogenes in this primate. We suggest that fragments of about 480 bp originate from a retro-transposition of the not alternatively spliced Tfam mRNA and that the shorter one amplified in human results from the retro-transcription and integration of the D5 splicing isoform. In fact, the analysis of this product has revealed that the deletion involves exon 5, as it happens in the D5 splicing isoform. This could mean that Tfam processed pseudogenes have derived also from the alternative spliced mRNA. We have also proposed a similar hypothesis for the shorter fragment obtained in Presbytis cristata, lacking part of exon 4, but we did not reveal the presence of a similar splicing isoform in this species (D’Errico et al., 2005). It may be that this shorter pseudogene originated from a spliced mRNA that appeared only at a certain moment in primate or Presbytis Cristata evolution and that has now been lost, but we cannot shed light upon this hypothesis. The interesting aspect of such a splicing isoform is its symmetry compared to the welldocumented exon 5 splicing. While the latter involves the removal of a part of the second HMG box of the Tfam transcript, the putative splicing in Presbytis Cristata would have concerned part of the first HMG box. All these observations about the Tfam gene, have underlined that some of its features, such as the chromosomal position and the presence of processed pseudogenes, are highly conserved, while others, such as splicing isoforms, seem to be species-specific characteristics. Thus, we can deduce from our observations that in the history of Tfam gene in the primates order the gene locus remained conserved in all the species. However, the processed pseudogenes, derived from Tfam transcripts, appeared during evolution of the primates and become a common characteristic shared by all the species but we do

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not know if they represent the result of a single or of different retroposition events. Later, at a particular moment in the evolutionary history of the primate order, a splicing isoform (Tfam D5 isoform) appeared in the hominid family fixing itself as a permanent characteristic. Perhaps, other splicing isoforms, now lost, also appeared during primate history, leaving traces of their appearance only through processed pseudogenes that we can detect today. Acknowledgements This work was supported by Fondi Ateneo FDinamica evolutiva di geni e famiglie geniche nei vertebrati_. References Alam, T.I., et al., 2003. Human mitochondrial DNA is packaged with TFAM. Nucleic Acids Res. 31, 1640 – 1645. Carbone, L., Ventura, M., Tempesta, S., Rocchi, M., Archidiacono, N., 2002. Evolutionary history of chromosome 10 in primates. Chromosoma 111, 267 – 272. D’Errico, I., Gadaleta, G., Saccone, C., 2004. Pseudogenes in metazoa: origin and features. Brief. Funct. Genomics Proteomics 3, 157 – 167. D’Errico, I., Reyes, A., Dinardo, M.M., Gadaleta, G., 2005. Study of the mitochondrial transcription factor A (Tfam) gene in the primate Presbytis cristata. Gene 354, 117 – 124. Falkenberg, M., Gaspari, M., Rantanen, A., Trifunovic, A., Larsson, N.G., Gustafsson, C.M., 2002. Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA. Nat. Genet. 31, 289 – 294. Fisher, R.P, Clayton, D.A., 1988. Purification and characterization of human mitochondrial transcription factor 1. Mol. Cell. Biol. 8, 3496 – 3509. Fisher, R.P., Topper, J.N., Clayton, D.A., 1987. Promoter selection in human mitochondria involves binding of a transcription factor to orientationindependent upstream regulatory elements. Cell 50, 247 – 258. Fisher, R.P., Parisi, M.A., Clayton, D.A., 1989. Flexible recognition of rapidly evolving promoter sequences by mitochondrial transcription factor 1. Genes Dev. 3, 2202 – 2217. Foulkes, N.S., Borrelli, E., Sassone-Corsi, P., 1991. CREM gene: use of alternative DNA-binding domains generates multiple antagonists of cAMP-induced transcription. Cell 64, 739 – 749. Gadaleta, G., D’elia, D., Capaccio, L., Saccone, C., Pepe, G., 1996. Isolation of a 25-kDa protein binding to a curved DNA upstream the origin of the L strand replication in the rat mitochondrial genome. J. Biol. Chem. 271, 13537 – 13541. Goodman, M., et al., 1998. Toward a phylogenetic classification of Primates based on DNA evidence complemented by fossil evidence. Mol. Phylogenet. Evol. 9, 585 – 598. Larsson, N.G., Barsh, G.S., Clayton, D.A., 1997. Structure and Chromosomal localization of the mouse mitochondrial transcription factor A gene (Tfam). Mamm. Genome 8, 139 – 140.

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