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Journal Articles: Biochemistry & Molecular Biology Biochemistry & Molecular Biology
10-2000
Genomic structure of murine mitochondrial DNA polymerase- gamma.
Justin L. Mott University of Nebraska Medical Center, [email protected]
Grace Denniger Saint Louis University
Steve J. Zullo National Institute of Mental Health
H. Peter Zassenhaus Saint Louis University
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Recommended Citation Mott, Justin L.; Denniger, Grace; Zullo, Steve J.; and Zassenhaus, H. Peter, "Genomic structure of murine mitochondrial DNA polymerase-gamma." (2000). Journal Articles: Biochemistry & Molecular Biology. 3. https://digitalcommons.unmc.edu/com_bio_articles/3
This Article is brought to you for free and open access by the Biochemistry & Molecular Biology at DigitalCommons@UNMC. It has been accepted for inclusion in Journal Articles: Biochemistry & Molecular Biology by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. DNA AND CELL BIOLOGY Volume 19, Number 10, 2000 Mary Ann Liebert, Inc. Pp. 601–605
Genomic Structure of Murine Mitochondrial DNA Polymerase-g
JUSTIN L. MOTT,1 GRACE DENNIGER,1 STEVE J. ZULLO,2 and H. PETER ZASSENHAUS1
ABSTRACT
We have sequenced a genomic clone of the gene encoding the mouse mitochondrial DNA polymerase. The gene consists of 23 exons, which span approximately 13.2 kb, with exons ranging in size from 53 to 768 bp. All intron–exon boundaries conform to the GT-AG rule. By comparison with the human genomic sequence, we found remarkable conservation of the gene structure; the intron–exon borders are in almost identical lo- cations for the 22 introns. The 59 upstream region contains approximately 300 bp of homology between the mouse and human sequences that presumably contain the promoter element. This region lacks any obvious TATA domain and is relatively GC rich, consistent with the housekeeping function of the mitochondrial DNA polymerase. Finally, within the 59 flanking region, both mouse and human genes have a region of 73 bp with high homology to the tRNA-Arg gene.
INTRODUCTION the genomic sequence (GenBank Accession No. AC005316) of the human Pol g gene (POLG) are known, the genomic se- HE MITOCHONDRIAL GENOME is a circular DNA molecule quence of the mouse Polg locus provides information about the Tthat is replicated and maintained independently of the nu- conservation of the genomic structure. Furthermore, the pro- clear genome. Multiple genomes can exist within each mito- moter can be tentatively assigned by studying the conserved chondrion, and there are hundreds to thousands of mitochon- flanking DNA. dria per cell. The essential role that mitochondrially encoded proteins play in energy metabolism underscores the importance of maintaining this extranuclear genome. To replicate the mi- tochondrial DNA (mtDNA), mitochondria have a dedicated MATERIALS AND METHODS DNA polymerase, termed polymerase gamma (Pol g; Lecrenier et al ., 1997). Like most mitochondrial proteins, Pol g is en- Identification, isolation, and sequencing coded by the nucleus and imported into mitochondria (Voos et al ., 1999). The murine gene is located on chromosome 7, and Colony arrays obtained from Research Genetics were probed the human homolog localizes to a syntenic region on the long for Polg using a probe generated from the cDNA. Positive arm of chromosome 15 (Zullo et al ., 1997). To improve our un- colonies were obtained (Research Genetics) and subcloned ac- derstanding of the Pol g gene, we have identified and sequenced cording to the manufacturer’s instructions. Southern blotting of a genomic clone containing the mouse homolog (Polg). The DNA minipreps revealed that BAC 408-F6 contained both 59 cDNA sequence for mouse Pol g was previously reported by and 39 termini of the Polg cDNA. The BAC DNA was mini- us (GenBank Accession No. U53584)* and used to identify the prepped according to the manufacturer’s instructions and di- intron–exon junctions of this gene. rectly sequenced using dye terminator technology (ABI) run on As both the cDNA (GenBank Accession No. 002693) and an ABI Prism 310 Genetic Analyzer by capillary flow.
1Department of Molecular Microbiology and Immunology, St. Louis University Health Sciences Center, St. Louis, Missouri. 2Laboratory of Biochemical Genetics, National Institute of Mental Health, Bethesda, Maryland. *Sequences previously determined and referenced in this paper are mouse Polg cDNA (U53584), human POLG cDNA (002693; also U60325 and D84103), human POLG genomic (AC005316), Drosophila tRNA-Arg (L09199), and human tRNA-Arg (Z69590). The nucleotide sequence data reported in this article have been submitted to GenBank and have been assigned the accession numbers AF268970, AF268971, AF268972, AF268973, AF268974, and AF268975.
601 602 MOTT ET AL.
Sequence comparisons the exons ranges from 71% to 95%, while for introns, the range is from 55% to 82%. Alignments and percent homology were determined using the GCG program (Genetics Computer Group, Madison, WI), Putative promoter region while related sequences were searched using BLAST (Altschul et al ., 1997). All alignments were further subjected to review On the basis of homology between the mouse and human 59 to determine precise junction sites. To generate simultaneous upstream regions, a putative promoter region has been identi- alignments, the ClustalW program was used (Thompson et al ., fied. There are 297 bp of highly homologous sequence imme- 1994). diately upstream of the transcription start site, with 74% iden- tity from mouse to human. There is no TATA box, but a putative 9 RESULTS transcription initiator element (5 GCAGACG; Lo and Smale, 1996) overlaps the start site (based on the first nucleotide of the cDNA sequence). There is an Sp1-binding site at 292 to Genomic structure of Polg 287, and the promoter’s overall GC content is relatively high Approximately 14.8 kb of mouse genomic DNA were se- (63%). The TATA-less promoters with high GC content often quenced to determine the structure of Polg . The genomic clone drive the expression of housekeeping genes, consistent with a was in the form of a bacterial artificial chromosome (BAC 408- constitutive need for mitochondrial DNA replication. Promoter F6; Research Genetics) identified by colony blotting to contain elements known to influence expression of nuclear genes en- Polg (not shown). The intron–exon junctions were deduced by coding mitochondrial proteins such as OXBOX-REBOX comparison with the cDNA sequence using the GAP function (Chung et al ., 1992), Mt3 and Mt4 (Suzuki et al ., 1991), and of the GCG program. As illustrated in Figure 1, the gene is NRF-1 (Evans and Scarpulla, 1990) were all absent from the composed of 23 exons and 22 introns. All donor–acceptor junc- promoter region. The corresponding region of the human ge- tions conformed to the GT-AG rule (Table 1). nomic sequence is also TATA-less and GC rich (64%) and has Table 1 illustrates the striking conservation of intron–exon a single Sp1-binding site. There is not a consensus concerning structure between the mouse and human genes. Of the 20 in- the transcription start site for the human mRNA (compare ternal coding exons, 14 are of identical length with the same cDNA sequences 002693, U60325, and D84103). It is worth intron placement. Length differences within the remaining 6 in- mentioning that the sequence of the putative transcription ini- ternal exons are minor, the longest being a 9-bp insertion in tiator element in the mouse gene is 100% conserved in the hu- exon 12 of the human gene. Interestingly, besides insertions of man and is located 29 bp upstream of the earliest cDNA start single-codon multiples, one or two nucleotides are inserted (ex- site reported for the human (Lecrenier et al ., 1997). ons 4 and 15) into one exon, only to be deleted from the sub- Interestingly, 73 bp of absolutely conserved sequence define sequent exon. Exon 2 is significantly shorter in the mouse gene the upstream boundary of homology between mouse and hu- secondary in part to the absence of a CAG-repeat region (Lecre- man genes (Fig. 2A). This high-homology area (the longest nier et al ., 1997; Rovio et al ., 1999). Mouse exons range from stretch of 100% identity within the ,14.8 kb of genomic se- 53 to 768 bp in length. Homology from mouse to human within quence) is also highly homologous to the tRNA-Arg genes of
FIG. 1. Genomic structure of murine Polg. Exons are numbered and indicated by boxes, while introns and flanking sequences are represented by a line. The transcription start site (5 9) and the poly(A) site (AAAA) are indicated, as are the translation start (ATG) and stop codons (TAA). Brackets mark the borders of homology between murine Polg and human POLG. The location of a tRNA-Arg homolog is denoted by the cloverleaf symbol near the 59 end (see text for details). GENOMIC STRUCTURE OF MURINE POL c 603
TABLE 1. INTRON –EXON JUNCTIONS FOR MURINE POLG
Exon Exon Intron Intron Exon identity Position size Splice Intron identity Splice size No. (%) in cDNA (bp) donor no. (%) acceptor (bp) Mouse 1 79 1-124 124 GAGgtttn 1 70 tttagGAC 707 Human 1-123 123 ...... c ...... T 759 Mouse 2 86 125-892 768 CTGgtaag 2 61 ttcagGTA .1128 Human 124-941 818 ...... c...... 2817 Mouse 3 89 893-1088 196 CAGgtaag 3 62 tccagGAC .666 Human 942-1137 196 ...... ct....GT 970 Mouse 4 86 1089-1254 166 GCGgtgag 4 59 ctcagAAT 156 Human 1138-1305 168 ...... c....TC 111 Mouse 5 83 1255-1403 149 CAGgtatg 5 56 gctagGAT 127 Human 1306-1452 147 ...... c....C 149 Mouse 6 89 1404-1483 80 GAGgtgag 6 82 cttagGTG 186 Human 1453-1532 80 ...... ca..... 1106 Mouse 7 95 1484-1666 183 GAGgtagt 7 76 tgcagGTA 103 Human 1533-1715 183 ...... c ..t..... 103 Mouse 8 89 1667-1815 149 AAGgtggg 8 67 cacagATC 136 Human 1716-1867 152 ...... C. 173 Mouse 9 76 1816-1942 127 TGGgtaag 9 59 aatagGTG 762 Human 1868-1994 127 .....g.. cg...A.. 926 Mouse 10 81 1943-2179 237 CAGgtaag 10 58 cacagAGC .513 Human 1995-2231 237 ...... c...... 1034 Mouse 11 88 2180-2300 121 ACGgtgag 11 57 tctagGTA 195 Human 2232-2352 121 ...... c..... 205 Mouse 12 83 2301-2378 78 CTGgtgag 12 65 cacagCCT 286 Human 2353-2439 87 ...... A.. 303 Mouse 13 82 2379-2486 108 AAGgtatg 13 64 cacagGAC 444 Human 2440-2547 108 .....g.. tc.....T 501 Mouse 14 90 2487-2647 161 CAGgtagg 14 62 tacagTTC 583 Human 2548-2708 161 .....g.. c....C.. 726 Mouse 15 89 2648-2700 53 CCAgttat 15 58 tccagGGC .208 Human 2709-2762 54 .AG..atg a.....CA 108 Mouse 16 87 2701-2819 119 CGGgtata 16 72 tgcagCCT 419 Human 2763-2880 118 ...... g ca...... 475 Mouse 17 90 2820-2955 136 ATGgtaag 17 62 tccagGCT 87 Human 2881-3016 136 .....g...... 116 Mouse 18 90 2956-3202 247 CCGgtgag 18 56 cccagGTA .442 Human 3017-3263 247 .T...... 1415 Mouse 19 79 3203-3325 123 GAAgtgag 19 55 cgcagGTC 78 Human 3264-3386 123 .....a.. .tt..... 128 Mouse 20 88 3326-3494 169 GAGgtgag 20 60 tctagTTT 109 Human 3387-3555 169 .....atc c.c..... 181 Mouse 21 92 3495-3703 209 CAGgtatg 21 55 ttcagGTG 899 Human 3556-3764 209 ...... 1005 Mouse 22 93 3704-3864 161 AGGgtgag 22 62 tacagGTG 449 Human 3765-3925 161 ...... t...... 548 Mouse 23 71 3865-4521 657 ———— Human 3926-4435 510
Identical bases are indicated with (.). Exons whose sizes differ in mouse and human are in bold. both Drosophila melanogaster (GenBank Accession No. moters, termed box A and box B (Sharp et al ., 1981), both of L09199) and humans (GenBank Accession No. Z69590). Up- which are present in the tRNA-Arg homolog. Furthermore, the stream of the tRNA-like sequence, homology from mouse to PolIII termination signal (TTTTT) is also present, 13 bases human is about 42%. Because the Polg promoter is defined here downstream of the alignment shown. as the homologous region between the mouse and human 59 flanking regions, the tRNA-like stretch is contained within the Additional features promoter. The tRNA-like sequence is coded on the minus strand of the chromosome (with respect to Polg) from 2225 to 2297, At the 39 end of the gene, homology ends abruptly at the hu- so its polarity is opposite that of Polg . RNA polymerase III man polyadenylation signal sequence within exon 23 (Fig. 2B). (PolIII) transcripts such as tRNAs contain two internal pro- This homology border helps explain the relatively low overall 604 MOTT ET AL.
FIG. 2. Sequence alignments at the borders of homology. (A) The 59 flanking region of the mouse (M.m.) and human (H.s.) Pol g genes contains a sequence related to both Drosophila (D.m.) and human tRNA-Arg genes. This defines the upstream bor- der of homology between the Pol g genes. Boxed nucleotides are shared by three or more of the sequences. The sequences are presented and numbered as if coding for the tRNA; for mouse Polg, the sequence starts at 2225 and ends at 2297 relative to the transcription start site. (B) The downstream border of homology between mouse and human lies within exon 23, at the hu- man poly(A) signal sequence (shaded). The noncanonical poly(A) signal sequence for the mouse (also shaded) lies 185 bp down- stream (gap indicated by . . . ). Polyadenylation sites are denoted by the large A at the end of each sequence.
homology of exon 23, as the first 459 bases of this exon share leaves a footprint, so we examined the genomic sequence for ,85% homology and the last 198 bases have ,40% homol- such clues. The three main features of retroposed genes are; (1) ogy. Thus, both the 59 and 39 borders of homology are sharp, direct repeats (7–21 bp) flanking both ends of the gene; (2) the stepping from .85% to ,40% homology at a defined point. remains of a poly(A) tail; and (3) absence of introns (Weiner Of note, the human poly(A) signal sequence (5 9-AAUAAA) et al ., 1986). Because of the evolutionary distance between the is conserved in the mouse, yet the mouse cDNA clone previ- retroposition event and the emergence of mammals, the reten- ously sequenced is not polyadenylated until 202 bases down- tion of direct repeats is unlikely. There is the remnant of a stream. Twenty-five bases upstream of the mouse poly(A) start poly(A)-like tail at the 39 end of the gene (Fig. 2B; 59- site is the sequence 59-UAUAAA, which is most likely the AATAAAAACAAACACCAAA), which, interestingly, de- poly(A) signal sequence for the transcript. The possibility that fines precisely the 39 border of homology between the mouse both poly(A) signal sequences are functional in the mouse, re- and human genes. Finally, Polg does have introns, but pre- sulting in transcripts that differ in size by about 200 bp, is sup- sumably these were added after the retroposition event. For ported by the existence of two similarly sized mRNA species some retroposons, tRNAs serve as primers for reverse tran- by Northern blotting of mouse heart RNA (Zhang et al ., in scription (Marquet et al ., 1995). In that light, it is interesting press). that the highly conserved tRNA-Arg sequence constitutes the 59 border of homology between the mouse and human genes. However, the polarity of the tRNA-like element is opposite DISCUSSION what is expected for it to have functioned as a primer for retroposition. Furthermore, such primers are usually degraded, Here, we present the genomic structure of the mouse Polg whereas this sequence remains remarkably intact. Overall, the gene, which encodes the mitochondrial DNA Pol g. This en- structure of Polg is consistent with a distant retroposition event. zyme is related to prokaryotic DNA polymerases, the prototype The tRNA-like sequence present at the 59 end of Polg pos- being PolI of E. coli (Ye et al ., 1996). All of these proteins sesses features necessary for transcription by PolIII, namely have polymerase activity, as well as 39–59 exonucleolytic proof- boxes A and B in the D stem/loop and G-T-C-C loop, respec- reading activity. Each domain is encoded by three regions of tively, and an oligo(T) termination signal. The anticodon (UCG) conserved gene sequence (Lecrenier et al ., 1997). is intact, as is the invariant U at position 8. However, it does Because Polg was likely originally encoded by the early mi- not code the known tRNA-Arg for either mice or humans. Fur- tochondrial endosymbiont (Margulis, 1970), its nuclear loca- ther work is necessary to test the possibility that this is a cod- tion may have arisen by retroposition. Such an event often ing gene for tRNA-Arg. GENOMIC STRUCTURE OF MURINE POL c 605
The putative promoter of Polg lacks the promoter elements SCHULTZ, R.A., SWOAP, S.J., McDANIEL, L.D., ZHANG, B., commonly seen in nuclear genes that encode mitochondrial pro- KOON, E.C., GARRY, D.J., LI, K., WILLIAMS, R.S. (1998). Dif- teins. The absence of these elements is consistent with the data ferential expression of mitochondrial DNA replication factors in mammalian tissues. J Biol Chem 273, 3447–3451. of Schultz and associates, who showed that Pol g mRNA is not SHARP, S., DEFRANCO, D., DINGERMANN, T., FARRELL, P., upregulated by stimuli that increase mitochondrial biogenesis SOLL, D. (1981). Internal control regions for transcription of eu- but rather is constitutively expressed in all tissues (Schultz et karyotic tRNA genes. Proc Natl Acad Sci USA 78, 6657–6661. al ., 1998). Thus, the genomic sequence of Polg reveals a gene SUZUKI, H., HOSOKAWA, Y., NISHIKIMI, M., OZAWA, T. (1991). structure conserved from mouse to human, with features con- Existence of common homologous elements in the transcriptional sistent with a housekeeping function for Pol g. regulatory regions of human nuclear genes and mitochondrial gene for the oxidative phosphorylation system. J Biol Chem 266, 2333– 2338. THOMPSON, J.D., HIGGINS, D.G., GIBSON, T.J. (1994). CLUSTAL ACKNOWLEDGMENTS W: Improving the sensitivity of progressive multiple sequence align- ment through sequence weighting, position-specific gap penalties and This work was supported by grants to HPZ from the weight matrix choice. Nucleic Acids Res 22, 4673–4680. Alzheimer’s Association, the American Heart Association, and VOOS, W., MARTIN, H., KRIMMER, T., PFANNER, N. (1999). the Aging and the Heart, Lung, and Blood Institutes of the NIH Mechanisms of protein translocation into mitochondria. Biochim Bio- and a grant to JLM from the American Diabetes Association. phys Acta 1422, 235–254. WEINER, A.M., DEININGER, P.L., EFSTRATIADIS, A. (1986). Nonviral retroposons: Genes, pseudogenes, and transposable ele- ments generated by the reverse flow of genetic information. Annu REFERENCES Rev Biochem 55, 631–661. YE, F., CARRODEGUAS, J.A., BOGENHAGEN, D.F. (1996). The ALTSCHUL, S.F., MADDEN, T.L., SCHAFFER, A.A., ZHANG, J., gamma subfamily of DNA polymerases: Cloning of a developmen- ZHANG, Z., MILLER, W., and LIPMAN, D.J. (1997). Gapped tally regulated cDNA encoding Xenopus laevis mitochondrial DNA BLAST and PSI-BLAST: A new generation of protein database polymerase gamma. Nucleic Acids Res 24, 1481–1488. search programs. Nucleic Acids Res 25, 3389–3402. ZHANG, D., MOTT, J.L., CHANG, S., DENNINGER, G., FENG, Z., CHUNG, A.B., STEPIEN, G., HARAGUCHI, Y., LI, K., and WAL- ZASSENHAUS, H.P. (2000). Construction of transgenic mice with LACE, D.C. (1992). Transcriptional control of nuclear genes for the tissue-specific acceleration of mitochondrial DNA mutagenesis. Ge- mitochondrial muscle ADP/ATP translocator and the ATP synthase nomics 69, in press. beta subunit: Multiple factors interact with the OXBOX/REBOX pro- ZULLO, S.J., BUTLER, L., ZAHORCHAK, R.J., MACVILLE, M., moter sequences. J Biol Chem 267, 21154–21161. WILKES, C., MERRIL, C.R. (1997). Localization by fluorescence EVANS, M.J., and SCARPULLA, R.C. (1990). NRF-1: A trans-acti- in situ hybridization (FISH) of human mitochondrial polymerase vator of nuclear-encoded respiratory genes in animal cells. Genes gamma (POLG) to human chromosome band 15q24 R q26, and of Dev 4, 1023–1034. mouse mitochondrial polymerase gamma (Polg) to mouse chromo- LECRENIER, N., VAN DER BRUGGEN, P., FOURY, F. (1997). Mi- some band 7E, with confirmation by direct sequence analysis of bac- tochondrial DNA polymerases from yeast to man: A new family of terial artificial chromosomes (BACs). Cytogenet Cell Genet 78, 281– polymerases. Gene 185, 147–152. 284. LO, K., and SMALE, S.T. (1996). Generality of a functional initiator consensus sequence. Gene 182, 13–22. Address reprint requests to: MARGULIS, L. (1970). Origin of Eukaryotic Cells. (Yale University Dr. H. Peter Zassenhaus Press, New Haven, CT). Department of Molecular Microbiology and Immunology MARQUET, R., ISEL, C., EHRESMANN, C., EHRESMANN, B. St. Louis University Health Sciences Center (1995). tRNAs as primer of reverse transcriptases. Biochimie 77, 1402 S. Grand Boulevard 113–124. St. Louis, MO 63104 ROVIO, A., TIRANTI, V., BEDNARZ, A.L., SUOMALAINEN, A., SPELBRINK, J.N., LECRENIER, N., MELBERG, A., ZEVIANI, M., POULTON, J., FOURY, F., JACOBS, H.T. (1999). Analysis of E-mail: [email protected] the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals. Eur J Hum Received June 6, 2000; received in revised form and accepted Genet 7, 140–146. July 19, 2000.