Functional domains of chicken mitochondrial transcription factor A for the maintenance of mitochondrial DNA copy number in lymphoma cell line DT40.
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.
Matsushima
Yuichi
Y
Graduate Courses for Regulation of Biological Signals, Nagoya University, Nagoya-shi, Japan.
Matsumura
Kiyoshi
K
Ishii
Shoji
S
Inagaki
Hidetoshi
H
Suzuki
Tomohiro
T
Matsuda
Yoichi
Y
Beck
Konrad
K
Kitagawa
Yasuo
Y
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004272:DNA, Mitochondrial / Q000235:genetics*; D004268:DNA-Binding Proteins; D018628:Gene Dosage; D015870:Gene Expression; D006609:High Mobility Group Proteins; D006801:Humans; D008223:Lymphoma; D051379:Mice; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics*; D014407:Tumor Cells, Cultured; D029867:Xenopus Proteins; D014982:Xenopus laevis
D018076:DNA, Complementary; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D024101:Mitochondrial Proteins; D009687:Nuclear Proteins; C044544:TFAM protein, Xenopus; C499631:TFAM protein, human; C081930:Tfam protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; C097078:XL-MTTFA protein, Xenopus; D029867:Xenopus Proteins; C422562:mitochondrial transcription factor A