Title: Functional Domains of Chicken Mitochondrial Transcription Factor A for the Maintenance of Mitochondrial DNA Copy Number in Lymphoma Cell Line DT40
Abstract: Nuclear and mitochondrial (mt) forms of chicken mt transcription factor A (c-TFAM) generated by alternative splicing of a gene (c-tfam) were cloned. c-tfam mapped at 6q1.1-q1.2 has similar exon/intron organization as mouse tfam except that the first exons encoding the nuclear and the mt form-specific sequences were positioned oppositely. When cDNA encoding the nuclear form was transiently expressed in chicken lymphoma DT40 cells after tagging at the C terminus with c-Myc, the product was localized into nucleus, whereas the only endogenous mt form of DT40 cells was immunostained exclusively within mitochondria. c-TFAM is most similar to Xenopus (xl-) TFAM in having extended C-terminal regions in addition to two high mobility group (HMG) boxes, a linker region between them, and a C-terminal tail, also found in human and mouse TFAM. Similarities between c- and xl-TFAM are higher in linker and C-terminal regions than in HMG boxes. Disruption of both tfam alleles in DT40 cells prevented proliferation. The tfam + /tfam – cells showed a 50 and 40–60% reduction of mtDNA and its transcripts, respectively. Expression of exogenous wild type c-tfam cDNA in the tfam + /tfam – cells increased mtDNA up to 4-fold in a dose-dependent manner, whereas its transcripts increased only marginally. A deletion mutant lacking the first HMG box lost this activity, whereas only marginal reduction of the activity was observed in a deletion mutant at the second HMG box. Despite the essential role of the C-terminal tail in mtDNA transcription demonstrated in vitro, deletion of c-TFAM at this region reduced the activity of maintenance of the mtDNA level only by 50%. A series of deletion mutant at the tail region suggested stimulatory and suppressive sequences in this region for the maintenance of mtDNA level. Nuclear and mitochondrial (mt) forms of chicken mt transcription factor A (c-TFAM) generated by alternative splicing of a gene (c-tfam) were cloned. c-tfam mapped at 6q1.1-q1.2 has similar exon/intron organization as mouse tfam except that the first exons encoding the nuclear and the mt form-specific sequences were positioned oppositely. When cDNA encoding the nuclear form was transiently expressed in chicken lymphoma DT40 cells after tagging at the C terminus with c-Myc, the product was localized into nucleus, whereas the only endogenous mt form of DT40 cells was immunostained exclusively within mitochondria. c-TFAM is most similar to Xenopus (xl-) TFAM in having extended C-terminal regions in addition to two high mobility group (HMG) boxes, a linker region between them, and a C-terminal tail, also found in human and mouse TFAM. Similarities between c- and xl-TFAM are higher in linker and C-terminal regions than in HMG boxes. Disruption of both tfam alleles in DT40 cells prevented proliferation. The tfam + /tfam – cells showed a 50 and 40–60% reduction of mtDNA and its transcripts, respectively. Expression of exogenous wild type c-tfam cDNA in the tfam + /tfam – cells increased mtDNA up to 4-fold in a dose-dependent manner, whereas its transcripts increased only marginally. A deletion mutant lacking the first HMG box lost this activity, whereas only marginal reduction of the activity was observed in a deletion mutant at the second HMG box. Despite the essential role of the C-terminal tail in mtDNA transcription demonstrated in vitro, deletion of c-TFAM at this region reduced the activity of maintenance of the mtDNA level only by 50%. A series of deletion mutant at the tail region suggested stimulatory and suppressive sequences in this region for the maintenance of mtDNA level. Transcription of mitochondrial (mt) 1The abbreviations used are: mt, mitochondrial; kb, kilobase pair(s); HMG, high mobility group; MPA, mycophenolic acid-resistant gene; neo r, neomycin-resistant gene; PBS, phosphate-buffered saline; SSC, standard saline citrate solution; TFAM, mt transcription factor A. DNA is best understood for Saccharomyces cerevisiae (1Jaehning J.A. Mol. Microbiol. 1993; 8: 1-4Crossref PubMed Scopus (56) Google Scholar, 2Shadel G.S. Clayton D.A. J. Biol. Chem. 1993; 268: 16083-16086Abstract Full Text PDF PubMed Google Scholar), in which a core RNA polymerase of M r ∼ 145 with sequence similarity to viral RNA polymerases of T3, T7, and SP6 (3Masters B.S. Stohl L.L. Clayton D.A. Cell. 1987; 51: 89-99Abstract Full Text PDF PubMed Scopus (313) Google Scholar) initiates transcription together with a dissociable 39-kDa mt transcription factor B (sc-mtTFB) (4Schinkel A.H. Groot Koerkamp M.J.A. Tabak H.F. EMBO J. 1988; 7: 3255-3262Crossref PubMed Scopus (62) Google Scholar, 5Jang S.H. Jaehning J.A. J. Biol. Chem. 1991; 266: 22671-22677Abstract Full Text PDF PubMed Google Scholar). This protein functions like a bacterial σ factor (6Cliften P.F. Park J.Y. Davis B.P. Jang S.H. Jaehning J.A. Genes Dev. 1997; 11: 2897-2909Crossref PubMed Scopus (44) Google Scholar, 7Cliften P.F. Jang S H. Jaehning J.A. Mol. Cell. Biol. 2000; 20: 7013-7023Crossref PubMed Scopus (19) Google Scholar, 8Mangus D.A. Jang S.H. Jaehning J.A. J. Biol. Chem. 1994; 269: 26568-26574Abstract Full Text PDF PubMed Google Scholar), but amino acid sequence comparisons (9Carrodeguas J.A. Yun S. Shadel G.S. Clayton D.A. Bogen-hagen D.F. Gene Expr. 1996; 6: 219-230PubMed Google Scholar) and mutational analyses (10Shadel G.S. Clayton D.A. Mol. Cell. Biol. 1995; 15: 2101-2108Crossref PubMed Scopus (57) Google Scholar) do not strongly support the hypothesis that sc-mtTFB is homologous to bacterial σ factor. The Xenopus equivalent (xl-mtTFB) having this activity has been characterized (11Bogenhagen D.F. J. Biol. Chem. 1996; 271: 12036-12041Abstract Full Text Full Text PDF PubMed Google Scholar, 12Bogenhagen D.F. Insdorf N.F. Mol. Cell. Biol. 1988; 8: 2910-2916Crossref PubMed Scopus (25) Google Scholar), but direct evidence confirming the structural similarity of xl-mtTFB to sc-mtTFB has not been reported. Human mt-TFB has been cloned and shown to be structurally related to RNA adenine methyltransferase (13McCulloch V. Seidel-Rogol L. Shadel G.S. Mol. Cell. Biol. 2002; 22: 1116-1125Crossref PubMed Scopus (166) Google Scholar). These two classes of proteins bind cooperatively to a nonanucleotide promoter sequence (4Schinkel A.H. Groot Koerkamp M.J.A. Tabak H.F. EMBO J. 1988; 7: 3255-3262Crossref PubMed Scopus (62) Google Scholar, 14Schinkel A.H. Groot Koerkamp M.J.A. Touw E.P.W. Tabak H.F. J. Biol. Chem. 1987; 262: 12785-12791Abstract Full Text PDF PubMed Google Scholar) present at multiple locations in the 75-kb circular yeast mt genome (15Christianson T. Rabinowitz M. J. Biol. Chem. 1983; 258: 14025-14033Abstract Full Text PDF PubMed Google Scholar). Thus, mtTFBs acts as a specific factor enabling the RNA polymerase to locate the appropriate start sites. Our knowledge about the transcription of human mtDNA, mainly based on the in vitro transcription system, is somewhat different and depends on a transcription factor (h-TFAM; formerly referred to as h-mtTFA) containing two HMG boxes (2Shadel G.S. Clayton D.A. J. Biol. Chem. 1993; 268: 16083-16086Abstract Full Text PDF PubMed Google Scholar, 16Fisher R.P. Clayton D.A. J. Biol. Chem. 1985; 260: 11330-11338Abstract Full Text PDF PubMed Google Scholar, 17Parisi M.A. Clayton D.A. Science. 1991; 252: 965-969Crossref PubMed Scopus (444) Google Scholar). The yeast homologue sc-mtTFA is an abundant protein composed almost entirely of two HMG boxes separated by a rather short linker region (18Fisher R.P. Lisowsky T. Parisi M.A. Clayton D.A. J. Biol. Chem. 1992; 267: 3358-3367Abstract Full Text PDF PubMed Google Scholar). sc-mtTFA was originally described as ABF2, which could bind to the replication origin of yeast nuclear DNA but was later found to be localized in mitochondria (19Diffley J.F. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7864-7868Crossref PubMed Scopus (261) Google Scholar). Disruption of the ABF2 gene led to a loss of respiratory competence and mtDNA when cells were grown in the presence of glucose. Because expression of h-TFAM in the yeast abf2 strain improved the phenotype, a potential functional homology of h-TFAM to sc-mtTFA was confirmed (20Parisi M.A. Xu B. Clayton D.A. Mol. Cell. Biol. 1993; 13: 1951-1961Crossref PubMed Scopus (162) Google Scholar). Nonetheless, the role of sc-mtTFA in mtDNA transcription remains unclear since in vitro it can hardly activate transcription. By replacing the various regions of h-TFAM with the corresponding parts of the yeast homologue, the linker region and the C-terminal tail abbreviated in sc-mtTFA were shown to be significant for the recognition and transcriptional activation of the human mtDNA (21Dairaghi D.J. Shadel G.S. Clayton D.A. J. Mol. Biol. 1995; 249: 11-28Crossref PubMed Scopus (157) Google Scholar). Mouse (22Larsson N.-G. Garman J.D. Oldfors A. Barsh G.S. Clayton D.A. Nat. Genet. 1996; 13: 296-302Crossref PubMed Scopus (125) Google Scholar) and Xenopus mtTFA (25Bogenhagen D.F. Romanelli M.F. Mol. Cell. Biol. 1988; 8: 2917-2924Crossref PubMed Scopus (31) Google Scholar) (will be referred to as m- and xl-TFAM hereafter, respectively) have been cloned and shown to have the long linker region and the C-terminal tail as h-TFAM does. xl-TFAM stimulates transcription in vitro up to 10-fold for the H-strand, but only ∼3-fold for the L-strand (23Antoshechkin I. Bogenhagen D.F. Mol. Cell. Biol. 1995; 15: 7032-7042Crossref PubMed Scopus (74) Google Scholar). For better understanding of the function of TFAM, the evolutionary variation of the control region of mtDNA may be worthwhile to be considered. This is the region between the genes for tRNAPro (tRNAGlu in birds) and tRNAPhe containing the H-strand replication origin, the promoters for transcription activation, and H- and L-strand start sites (24Clayton D.A. Annu. Rev. Cell Biol. 1991; 7: 453-478Crossref PubMed Scopus (533) Google Scholar). In Xenopus, the distance of the transcription start sites of the L- and H-strands is rather short, with 50 bp (25Bogenhagen D.F. Romanelli M.F. Mol. Cell. Biol. 1988; 8: 2917-2924Crossref PubMed Scopus (31) Google Scholar) versus 150 bp in the case of human (26Montoya J. Christianson T. Levens D. Rabinowitz M. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7195-7199Crossref PubMed Scopus (207) Google Scholar). Lower dependence of Xenopus mtDNA transcription on xl-TFAM compared with the human system can be due to this difference of the control region. From this aspect, chicken mtDNA provides an opportunity to expand our knowledge of mtDNA transcription in higher vertebrates. The chicken mitochondrial genome has a characteristic organization of the genes compared with other vertebrates (27Desjardins P. Morais R. J. Mol. Biol. 1990; 212: 599-634Crossref PubMed Scopus (862) Google Scholar), and its H- and L-strand sites have no separating base pair (28L'Abbé D. Duhaime J.-F. Lang B.F. Morais R. J. Biol. Chem. 1991; 266: 10844-10850Abstract Full Text PDF PubMed Google Scholar). We have cloned and determined the cDNA sequence encoding chicken (c-)TFAM, which is more similar to xl-TFAM than to the human and mouse proteins. Targeted disruption of both c-tfam alleles in a chicken cell line DT40 showed that TFAM is essential for proliferation of this cell line. In cells with heterozygous disrupted c-tfam, mtDNA and its transcripts were reduced. Expression of exogenous wild type c-tfam cDNA in those cells increased mtDNA up to 4-fold in a dosage-dependent manner, whereas its transcripts increased only marginally. We thus show that c-TFAM is important for maintenance of the mtDNA copy number. Cell Culture, Gene Transfection, and Screening—DT40 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin at 37 °C under a humidified atmosphere of 5% CO2. Cells were split to 1 × 105/ml every third day. For DNA transfection, 107 cells were suspended in 0.5 ml of PBS containing 30 μg of linearized plasmid and electroporated with a Gene Pulser (Bio-Rad) at 550 V and 25 microfarads. The cells were transferred to 20 ml of fresh medium and incubated for 24 h. After resuspending in 100 ml of medium containing either 30 μg/ml mycophenolic acid or 2 mg/ml G418, cells were divided into five 96-well plates and cultured for 7–10 days. Drug-resistant colonies were transferred to 50 ml of T-flasks for preparation of genomic DNA to confirm heterozygous disruption of tfam alleles by Southern blotting. For screening of DT40 clones having both tfam alleles disrupted, the colonies with heterozygous disruption were subjected to another round of transfection/screening cycle using a medium supplemented with 50 μg/ml uridine and 110 μg/ml sodium pyruvate. To obtain the colony expressing exogenous c-tfam cDNA and its deletion mutants under the background of heterozygous tfam allele disruption, plasmids having either of these sequences and neomycin-resistance gene (neo r) in tandem, both under the control of β-actin promoters, were transfected to the cells with either tfam allele targeted by MPA, and the G418-resistant colonies were selected. The colonies positive to exogenous c-TFAM were further selected by Western blotting of the cell lysate. Screening and Sequencing of cDNA and Genomic DNA Clones—A chicken cDNA library in which mRNAs isolated from the day 5 embryos were reverse-transcribed using oligo-dT primers, and double-stranded cDNA were inserted into EcoRI-XhoI sites of λZAPII vector was a gift from Dr. Atsushi Kuroiwa. ∼1 × 107 phages were screened using the SalI fragment of h-tfam cDNA (17Parisi M.A. Clayton D.A. Science. 1991; 252: 965-969Crossref PubMed Scopus (444) Google Scholar) as a probe in a buffer containing 50 mm Tris-HCl (pH 7.5), 5× SSC, 5× Denhardt's solution, 1% SDS, and 100 μg/ml salmon sperm DNA. After hybridization (16 h, 53 °C), filters were washed 3 times with 2× SSC containing 0.1% SDS at room temperature and once with 0.2× SSC containing 0.1% SDS at 53 °C for 30 min and exposed to x-ray film (Fuji). Because the cDNA clones isolated from this library were found to encode only nuclear c-TFAM (GenBank™ accession number AB21166), the 5′ sequence encoding the N terminus of mt c-TFAM (GenBank™ accession number AB059657) was obtained by reverse transcription of RNA extracted from DT40 cells with SMART rapid amplification of cDNA ends cDNA amplification kit (Clontech) using a primer of 5′-CACCAGCTCCAAGCTGTTCAG-3′. The amplified fragment was sequenced with a primer of 5′-GCTGGTTGTCCCTCAGGAAG-3′. For transient expression of the nuclear or mt c-tfam cDNA tagged with a c-Myc sequence at 3′ end, either cDNA clone was amplified with 3′ primer of 5′-GCGGGATCCTTACAAGTCTTCTTCAGAAATAAGCTTTTGTTCTTCTTCAGATTTTTTCAGCTTCAGTTTCGCCAG-3′ paired with 5′-primer of 5′-CCCggTCgACACCGCGGTGGCGGCCGCCATGGGGCGGAGGGGCGGGCTG-3′ or 5′-GGCGGTCGACTCCGTGGGGAGGGTGGGCTGCGGCATG-3′, respectively. The amplified fragments were inserted downstream of β-actin promoter after SalI/BamHI digestion. A DT40 genomic DNA library with Sau3A partial digests (15–25 kb) inserted into the XhoI site of a λFIXII vector was a gift from Dr. Tatsuo Nakayama. ∼1 × 106 phages were screened at a hybridization temperature of 65 °C using a c-tfam cDNA clone as a probe. Genomic DNA clones were analyzed by restriction endonuclease mapping and Southern blot analysis. Pertinent restriction fragments were subcloned into pBluescript. DNA was sequenced by the dideoxynucleotide method on an Applied Biosystems (model 377) DNA sequencer. Preparation of Anti-chicken TFAM Antiserum—To express c-TFAM in Escherichia coli, the SacI-HindIII fragment of c-tfam cDNA coding for amino acid residues Ala-42 to Leu-248 was cloned into the SacI-HindIII sites of pQE31 (Qiagen, Chatsworth, CA). A bacterial culture harboring the plasmid was grown in LB medium containing 100 μg/ml ampicillin, and expression was induced by 0.1 mm isopropil-1-thio-β-d-galactoside. The N-terminal His6 tag was utilized for purification of the product on a nickel nitrilotriacetic acid column according to the manufacture's instructions. New Zealand White rabbits were immunized by intramuscular injections of 0.3 mg of purified protein in complete Freund's adjuvant and boosted once with 0.3 mg of protein in incomplete Freund's adjuvant. Sera were collected 4 weeks after initial injection. Indirect Immunofluorescence Microscopy—DT40 cells were grown on coverslips. After labeling with MitoTracker Red CMXRos (Molecular Probes) according to the manufacturer's instructions, cells were fixed with methanol/water/acetic acid (95:4:1, v/v) for 15 min, permeabilized with cold methanol for 10 min, incubated with anti-c-TFAM antiserum or anti-c-Myc rabbit polyclonal IgG (Santa Cruz Biotechnology) diluted 1:500 or 1:100, respectively, in buffer A (PBS containing 0.1% Tween 20) for 1 h, washed 4 times with PBS, incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG (Cappel; 1:100 dilution in buffer A) for 1 h, washed 4 times with PBS, and mounted in 90% (v/v) glycerol containing 2 mm Tris-HCl (pH 8.0), 0.2 m 1,4-diazabicyclo-(2,2,2)octane. Western Blotting—Cell extracts corresponding to 10 μg of total protein were separated in 15% SDS-polyacrylamide gels and transferred to Hybond enhanced chemiluminescence (ECL) nitrocellulose (Amersham Biosciences). Filters were preincubated with 5% skim milk in PBS, incubated with anti-c-TFAM (1:500 in buffer A) for 1 h, washed 4 times with buffer A, incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences) for 1 h, and washed with buffer A. Protein bands were visualized using the ECL Western blotting reagents (Amersham Biosciences). Chromosome Preparation and in Situ Hybridization—Chromosomal assignment of c-tfam was performed using the direct R-banding fluorescence in situ hybridization method as described (29Matsuda Y. Harada Y.N. Natsuume-Sakai S. Lee K. Shiomi T. Chapman V.M. Cytogenet. Cell Genet. 1992; 61: 282-285Crossref PubMed Scopus (231) Google Scholar, 30Matsuda Y. Chapman V.M. Electrophoresis. 1995; 16: 261-272Crossref PubMed Scopus (249) Google Scholar) with minor modifications. Mitogen-stimulated splenocytes were cultured at 39 °C and synchronized by a thymidine block, and 5-bromodeoxyuridine was incorporated during the late replication stage for differential replication staining after release of excessive thymidine. R-bands were stained by exposure of Hoechst 33258-stained chromosome slides to UV light. Chromosome slides were heated at 65 °C for 2 h, denatured at 70 °C in 70% formamide in 2× SSC, and dehydrated in a 70, 85, 100% ethanol series at 4 °C. The 15-kb chicken genomic DNA fragment inserted into the SalI site of pBluescript was labeled by nick translation with biotin 16-dUTP (Roche Applied Science), ethanol-precipitated together with chicken whole genomic DNA, salmon sperm DNA, and E. coli tRNA, and denatured at 75 °C in 100% formamide for 10 min. The denatured probe was mixed with an equal volume of hybridization solution to a final concentration of 50% formamide, 2× SSC, 10% dextran sulfate, and 2 mg/ml bovine serum albumin. 20 μl of the solution containing 250 ng of labeled DNA were put on the denatured slide, covered with Parafilm, and incubated overnight at 37 °C. Slides were washed in 50% formamide in 2× SSC at 37 °C for 20 min, and 2× and 1× SSC at room temperature for 20 min. After rinsing in 4× SSC, slides were incubated with Cy2-labeled streptavidin (Amersham Biosciences) at 1:500 dilution in 1% bovine serum albumin, 4× SSC at 37 °C for 1 h. After washing with 4× SSC, 0.1% Nonidet P-40 in 4× SSC, and 4× SSC for 10 min each on a shaker, slides were rinsed with 2× SSC and stained with 0.75 μg/ml propidium iodide. Hybridization was visualized by excitation at 450–490 nm (Nikon filter set B-2A) and ∼365 nm (UV-2A), and recorded on Kodak Ektachrome 100 film. Southern Blotting—Genomic DNA was purified from DT40 cells with DNAZOL (Invitrogen). 10 μg of DNA were separated by 1% agarose gel electrophoresis after digestion with EcoRI, SalI, and/or PstI and transferred to Hybond N+ (Amersham Biosciences). Hybridization was performed in a buffer containing 50 mm Tris-HCl (pH 7.5), 5× Denhardt's solution, 1% SDS, and 0.1 mg/ml salmon sperm DNA at 65 °C for 16 h. Filters were washed 3 times with 2× SSC containing 0.1% SDS at room temperature for 10 min and once with 0.2× SSC containing 0.1% SDS at 65 °C for 30 min and analyzed with a Fuji BAS 2000 image analyzer. For the screening of DT40 clones, of which tfam was disrupted, a 0.5-kb probe (probe 1 in Fig. 3A) was prepared by amplifying the SalI-EcoRI fragment inserted in pBluescript KS(+) with a paired primer of 5′-TAGTTCCCTTTCTGTCAAAG-3′ and T7 primer and digesting it with EcoRI. For semi-quantification of mtDNA, the EcoRI-StuI fragment of chicken ATPase 6/8 cDNA was used. A 5-kb EcoRI fragment of tfam (probe 2 in Fig. 3A) was used for the normalization of the quantification of mtDNA. Because this probe sequence is localized outside of the targeted region, its signal cannot be affected by the disruption of tfam in DT40 cells. Northern Blotting—Total RNA was extracted by the acid guanidinium thiocyanate/phenol/chloroform method (31Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). 10 μg of RNA was separated by 1.5% formaldehyde agarose gel electrophoresis and transferred to Hybond N+. Filters were hybridized to 32P-labeled probes in 5× standard saline phosphate -EDTA, 0.5% SDS, 5× Denhardt's solution, 50% formaldehyde, 0.1 mg/ml of denatured salmon sperm DNA at 42 °C for 16 h. Filters were washed 3 times with 2× SSC containing 0.1% SDS at room temperature and once with 0.1× SSC containing 0.1% SDS at 65 °C for 30 min and analyzed with a Fuji Film BAS 2000 Image Analyzer. Probes for c-tfam, ATPase α, ATPase γ, and ATPase 6/8 mRNAs were individual cDNA clones isolated in our laboratory. Probes for β-actin RNA (5′-ATGGATGATGATATTGCTGC-3′; 5′-TTCATCGTACTCCTGCTTGC-3′), 12 S/16 S rRNA (5′-AAGCTAGGACCCAAACTGG-3′; 5′-GTGAAGAGTTGTGGTCTGTG-3′), and ND6 (NADH dehydrogenase subunit VI) mRNA (5′-CAACCCACGCACAAGCTC-3′; 5′-GTAGCGTCTGTGATAGG-3′) were prepared by reverse transcription-PCR of chicken RNA with primers designed based on the reported sequences. Targeting Vector Construction—As a source of targeting vectors, plasmids with MPA (32Mulligan R.C. Berg P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2072-2076Crossref PubMed Scopus (636) Google Scholar) and neo r (33Buerstedde J.M. Takeda S. Cell. 1991; 67: 179-188Abstract Full Text PDF PubMed Scopus (484) Google Scholar) under the control of the chicken β-actin promoter were inserted into pBluescript II. Plasmids were gifts of Dr. Shunichi Takeda. Selection marker genes were sandwiched by a 1.8-kb sequence upstream of exon IM and a 4.6-kb sequence spanning the 3′ terminus of exon II through intron VI as left and right arms, respectively (cf. Fig. 3A). For the left arm insertion, a PstI site of the 1.8-kb SalI-PstI fragment was modified to a SalI site. For the right arm insertion, a BamHI-BamHI fragment was prepared by PCR using paired primers of 5′-CCCGGGATCCCTTCCTGAGGGACAACCAGC-3′ and 5′-CCCGGGATCCCACCTGTGCAGTACTCATCC-3′. Expression Vectors for TFAM Deletion Mutants—Paired c-tfam cDNA fragments that sandwich the domain to be deleted were prepared by PCR so as to have restriction ends of 5′-SalI/blunt-3′ or 5′-blunt/BamHI and were inserted downstream of β-actin promoter by a three-piece ligation technique. The deleted sequences of c-TFAM were Lys-46–Try-112 for the first HMG box, Lys-z13–Val-146 for the linker, Leu-147–Trp-213 for the second HMG box, and Glu-214–Glu-262E for the tail. A series of deletion mutants at the tail was also prepared; Val-218 to Glu-262 for del M, Ser-228 to Glu-262 for del R, Ser-244 to Glu-262 for del G, and Lys-254 to Glu-262 for del A. The vectors for expression of the mutants had neo r under the control of β-actin promoter for the selection of positive colonies. Sequence Analysis—General sequence editing and analysis were performed using MacDNASIS, version 3.7 (Hitachi Software). Similarity searches against the non-redundant GenBank™ data base (June 1999) were done using BLAST 2.0 (34Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). HMG boxes were compared with those of the Pfam data base (version 4.0; Ref. 35Bateman A. Birney E. Durbin R. Eddy S.R. Finn R.D. Sonnhammer E.L. Nucleic Acids Res. 1999; 27: 260-262Crossref PubMed Scopus (478) Google Scholar). Multiple sequence alignments were performed with ClustalW (36Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). Pairwise comparisons were done using the BLOSUM62 matrix and default values for gap penalties. Quantification of the Blot Signals—A Southern blot was analyzed by a Fuji Film BAS 2000 Image Analyzer, and the radioactivity was quantified by determining the photostimulated luminescence count with a Fuji Film Image Gauge, version 3.4. The ECL signal Western blotting was semiquantified with Image ID program (Amersham Biosciences). cDNA Cloning of Nuclear and mt c-TFAMs—Low stringency screening of 1 × 107 independent cDNA clones of mRNAs expressed in the day 5 chicken embryos with h-tfam cDNA probes yielded five selected clones. Partial sequencing and restriction fragment analysis of the inserts showed that they share identical 3′-sequences with different extensions in the 5′ direction. Three clones had the same sequence (AB021166) of 1324 bp, whereas the other two clones contained sequences of 1174 and 1008 bp, respectively. The longest sequence encodes an open reading frame of 792 bp starting with ATG at the fourth base and terminating at TAA at the 769th base to code for a 264-residue amino acid sequence. Neither poly(A)-adding signal nor poly(A) tail was found. Attempts of rapid amplification of cDNA ends (37Frohman M.A. Methods Enzymol. 1993; 218: 340-356Crossref PubMed Scopus (465) Google Scholar) using mRNA extracted from DT40 cells as the template yielded 5′-extended sequence (AB059657) encoding different N-terminal sequence (Fig. 1A), suggesting that chicken cells express two forms of c-TFAM. When these cDNA sequences obtained from the day 5 chicken embryos were transiently expressed in DT40 cells after tagging with the c-Myc sequence at the C terminus, indirect immunofluorescence staining of the cells with anti-c-Myc polyclonal IgG showed localization of the product into the nucleus, whereas the endogenous form in DT40 cells was localized in mitochondria (Fig. 1B; see below). We thus cloned mt and nuclear forms of c-TFAM. For mouse spermatocytes, a testis-specific TFAM isoform resulting from alternative splicing of a different exon IT has been characterized in addition to mt mouse m-TFAM, and this isoform is targeted to the nucleus (22Larsson N.-G. Garman J.D. Oldfors A. Barsh G.S. Clayton D.A. Nat. Genet. 1996; 13: 296-302Crossref PubMed Scopus (125) Google Scholar). Screening of a DT40 genomic DNA library with the mt c-TFAM cDNA sequence as a probe yielded three overlapping fragments. Analysis of restriction sites and partial sequencing resulted in the exon/intron organization of c-tfam as shown in Fig. 1A. The N-terminal sequence of nuclear c-TFAM is encoded by exon IN, which is directly connected to exon II without insertion of an intron, whereas that of mt c-TFAM is encoded by exon IM and continued to the sequence encoded by exon II by splicing out of the exon IN. Thus, the mechanism of generating the nuclear and mt c-TFAMs is similar to m-tfam, but the exons encoding the mt and nuclear form-specific sequences (exon IN and exon IM) are positioned oppositely. The downstream cDNA sequence is encoded by additional six exons. All exon/intron boundaries had well conserved consensus sequences for splicing both at the 5′- and 3′-end of introns (not shown). Immunostaining of DT40 cells with antiserum prepared against c-TFAM showed staining of mitochondria well overlapping with that of living dye for mitochondria (MitoTracker Red), and no signal was indicated in the nuclear region (Fig. 1C). Sequence Comparison of c-TFAM with xl-, m-, and h-TFAMs—Alignment of the deduced c-TFAM amino acid sequence with those of xl- (23Antoshechkin I. Bogenhagen D.F. Mol. Cell. Biol. 1995; 15: 7032-7042Crossref PubMed Scopus (74) Google Scholar), h- (17Parisi M.A. Clayton D.A. Science. 1991; 252: 965-969Crossref PubMed Scopus (444) Google Scholar), and m- (22Larsson N.-G. Garman J.D. Oldfors A. Barsh G.S. Clayton D.A. Nat. Genet. 1996; 13: 296-302Crossref PubMed Scopus (125) Google Scholar) TFAMs in Fig. 2A shows an identity of 42, 37, and 40%, respectively. In contrast to h- and m-TFAMs, c-TFAM shares with xl-TFAM the C terminus-extended regions. Its calculated molecular mass (M r = 29,985) is larger than that of h- (M r = 29,097) and m-TFAM (M r = 27,988) but smaller than that of xl-TFAM (M r = 35,501). Its theoretical isoelectric point of 10.7 is comparable with that of Xenopus (pI ∼ 10.2), which is slightly more basic than that of h- and m-TFAMs (pI ∼ 9.7). As do other TFAMs, the chicken sequence contains two HMG boxes separated by a ∼35-amino acid residue linker. Whereas the HMG-box sequences show high correlations between the different species, the closest relationship between c- and xl-TFAMs in the whole sequences is mainly due to the similarities of the C-terminal extensions and the linker region. By replacing the various regions of h-TFAM with the corresponding parts of the yeast homologue, the significance of the linker region and the C-terminal tail for the recognition and transcriptional activation of the human mtDNA promoter has been established (21Dairaghi D.J. Shadel G.S. Clayton D.A. J. Mol. Biol. 1995; 249: 11-28Crossref PubMed Scopus (157)