Proc. Natl. Acad. Sci. USA Vol. 80, pp. 6495-6499, November 1983 Biochemistry

Mitochondrial DNA and nuclear DNA from normal rat liver have a common sequence (Southern blot/mammalian ) HERBERT I. HADLER, BOGOMIR DIMmRJEVIC, AND RAVI MAHALINGAM Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL 62901 Communicated by William S. Johnson, July 22, 1983 ABSTRACT Although Pst I does not cut the circular mito- investigation we observed that the mammalian mtDNA and chondrial genome of the rat, BamHI generates from this genome nDNA from normal rat liver have a common sequence. Irre- two unequal fragments of DNA. Each of these fragments was cloned spective of the validity of our hypothesis this experimental in pBR322. Nuclear DNA was digested from rat liver singly or finding with normal mammalian intracellular is sig- doubly with Pst I and BamHI, and it was demonstrated that nu- nificant in its own right and is the subject of this report. clear DNA shared a common sequence with the larger mitochon- drial DNA BamHI fragment. The cloned larger mitochondrial DNA MATERIALS AND METHODS fragment was further subdivided with HidMII into four pieces that were labeled and then used to probe the double-digested nuclear Isolation of mtDNA. Mitochondria were isolated from DNA. The hybridization data showed that the common sequence Sprague-Dawley rats (purchased from Holtzman, Madison, WI). is less than 3 kilobase pairs long and lies within the part of the The twice washed mitochondrial pellet was obtained as de- mitochondrial genome containing the D-loop and a portion of the scribed (22) except 250 mM sucrose/100 mM Tris-EDTA, pH rRNA genes. It therefore appears that, as in lower , 7.5 replaced 250 mM sucrose. Once-banded mtDNA was iso- there are shared sequences between the nuclear and mitochon- lated by a modification of the method of Bogenhagen and Clay- drial genomes in mammals. ton (23). The mitochondria were not subjected to hypotonic treatment. The lysis buffer was 100 mM Tris.EDTA/10 mM In current editorial reviews (1, 2) the first evidence of the NaCl, pH 7.5. An additional step to remove protein and colored movement of DNA between intracellular genomes of eukary- substances was included. After adding CsCl to the lysate and otes during evolution was collected. Thus an ATPase gene had centrifuging at 20,000 x g for 30 min at 40C we adjusted the migrated from the mitochondria to the nucleus during the evo- cleared lysate (corresponding to 13.5 g of liver) to a density of lution of Saccharomyces cerevisiae and Neurospora crassa (3). 1.3 g/ml of CsCl and a volume of 3.0 ml and layered it (in a Mitochondria and of maize possess a closely ho- 5-ml polyallomer tube) over 1 ml of CsCl density 1.6 g/ml and mologous 12-kilobase-pair (kbp) segment of DNA (4). Mito- 1 ml of CsCl density 1.7 g/ml. After spinning in a Beckman SW chondria and nuclei of S. cerevisiae have multiple common 50.1 rotor for 20 hr at 170,000 X g at 20°C, the nucleic acids fragments of DNA (5). Mitochondria and nuclei of the sea ur- were pelleted. The bottom of the tube was pierced with an 18- chin (Strongylocentrotous purpuratus) have common pieces of gauge needle directed from the top, the lower viscous 1.5 ml DNA (6). This is also true for the locust Locusta migratoria (7). was collected and subjected to CsCl/ethidium bromide (1.6 g/ Wright and Cummings (8) ascribe the senescence of a fungus, ml and 150 ,ug/ml, respectively) isopycnic centrifugation (160,000 Podespora anserina, to the active mobilization of genetic ele- X g, 48 hr, Beckman SW 50.1 rotor, 20°C). The lower band was ments from the mitochondrion to the nucleus. It is cogent to collected with an 18-gauge needle, the dye was completely re- note that the observation by Chilton et al. (9) dealing with trans- moved by four extractions with isoamyl alcohol and dialyzed formation is analogous. These workers reported that when plant overnight with 4 changes of 10 mM Tris-HCl/1 mM Na2EDTA, cells are transformed by infecting bacteria, DNA from the bac- pH 7.5 (TE buffer). The DNA was precipitated by the addition terial becomes integrated into the nuclear genome of of 1/10 vol of 3 M NaOAc (pH 7.0) and 2 vol of ethanol and the host. then washed by centrifugation with 70% ethanol and, after brief We also have been examining the putative movement of drying in vacuo at room temperature, was dissolved in TE buff- mtDNA into the nuclear genome of rat because of a hypothesis er and stored at 5°C. that we presented some twelve years ago (10). This hypothesis Cloning of mtDNA. The once-banded mtDNA was digested stated that as a result of pressure on the mitochondria by car- with BamHI to yield a mixture of linear fragments 10.8 and 5.2 cinogens and their metabolites mitochondrial genetic material kbp. Transformation of Escherichia coli strain HB1O1 was car- may be integrated into nuclear DNA (nDNA) just like the ge- ried out as described (24). The clones were selected by growing netic material of an oncogenic virus. Since 1971 many other the ampicillin-resistant colonies on tetracycline plates (25). After examples of such pressure have been observed (11-20). Indeed the plasmid DNA was digested with BamHI, electrophoresis such pressure now has added significance because of the recent showed that only colonies with the 5.2-kbp piece of mtDNA report that human oncogenes express a mitochondrial and bac- inserted into pBR322 had been isolated. To obtain a recom- terial ATPase (21). binant of pBR322 with the 10.8-kbp piece of mtDNA as the We chose to test our hypothesis by using probes of normal insert, the mixture of mtDNA cut with BamHI was sized by gel mtDNA to compare nDNA derived from normal tissue with electrophoresis and the 10.8-kbp fragment was recovered from nDNA derived from tumor tissue. During the course of this the gel (26). In addition the pBR322 that had been linearized with BamHI was dephosphorylated with alkaline phosphatase The publication costs of this article were defrayed in part bypage charge payment. This article must therefore be hereby marked "advertise- Abbreviations: nDNA, nuclear DNA; kbp, kilobase pairs; NaCl/Cit, ment" in accordance with 18 U. S.C. §1734 solely to indicate this fact. standard saline citrate. 6495 Downloaded by guest on September 30, 2021 6496 Biochemistry: Hadler et al. Proc. Natl. Acad. Sci. USA 80 (1983) (27). Two colonies selected as before had the 10.8-kbp piece of orange G. The voltage gradient was 0.7 V/cm. A glass plate was mtDNA inserted into pBR322. carefully placed on the gel after the DNA had been driven into Growth, Isolation, and Characterization of Plasmid DNA. the gel. The buffer was not circulated. Plasmid pBR322 or recombinant with the 10.8 kbp or The method of Southern (31) as described by Jeffreys and 5.2 kbp of mtDNA inserted into the BamHI site were grown Flavell (32) was used to transfer the cut DNA to nitrocellulose, in E. coli strain HB101 in L-broth fortified with ampicillin. for hybridization (650C for 2 days in a box), for washing the strips Chloramphenicol was added to increase the yield of plasmid (18 x 4 cm), and for autoradiography. During hybridization, 50 DNA. Lysis at pH 8.0 used Tris-HCl, sucrose, lysozyme, ml of the solution in the box contained 2.5 Ag of probe of spe- Na2EDTA, and 2% Triton X-100. The lysate, which was cleared cific activity 1 x 107 cpm/,ug of DNA. The following wash so- at 40,000 rpm for 1 hr at 100C in a Beckman SW 41Ti rotor, was lutions, modelled after Denhardt (33), were used at 650C to treat extracted with phenol and then with isoamyl alcohol. The nu- the nitrocellulose before and after hybridization. Before hy- cleic acid was precipitated with NaOAc and ethanol, washed bridization the strips of nitrocellulose were washed for 30 min with 70% ethanol, dried, and dissolved in TE buffer. After in 3X standard saline citrate (NaCl/Cit; lx NaCl/Cit is 150 treatment with RNase and extraction in succession with phenol mM NaCl/15 mM trisodium citrate) then treated for 3 hr with and isoamyl alcohol the nucleic acids were precipitated again as 1OX Denhardt's solution (0.2% Ficoll/0.2% polyvinylpyrolli- described above. The was now banded in TE buffer done/0.2% bovine serum albumin in 3x NaCl/Cit). This so- with CsCl (1.56 g/ml)/ethidium bromide (150 jig/ml) in a ver- lution was replaced by a similar solution supplemented with tical head (Sorvall TV-850) at 40,000 rpm at 150C for 24 hr. The denatured and sheared salmon testes DNA at 50 pkg/ml (Sigma), lower band was collected, processed, and stored as described poly A at 10 tkg/ml (Sigma), and 0.1% NaDodSO4 and incu- for the preparation of mtDNA. bated for 1 hr. After hybridization the strips were successively Plasmid pBR322 and the two recombinant plasmids were washed at 650C as follows: 3 washes for 5 min then 2 washes for characterized by digestion with BamHI followed by electro- 30 min with 3X NaCl/Cit/lX Denhardt's solution/0.1% phoresis with a submerged horizontal, 6-mm thick 1% agarose NaDodSO4 and 2 more washes for 45 min each with 0.1X NaClI gel (28) after 16 hr in 40 mM Tris base/20 mM NaOAc/1 mM Cit/0.1% NaDodSO4. Na2EDTA adjusted to pH 7.9 with HOAc (E buffer) at a voltage The molecular weight markers were various restriction frag- gradient of 1.3 V/cm. The gel was stained in E buffer with ments of pBR322 and recombinant DNA. ethidium bromide at 0.5 ,ug/ml for 10 min and photographed Other portions of nDNA were processed as described above with a transilluminator using a UV light, 254 nm, a UV filter except Pst I was replaced by BamHI or a combination of BamHI for the camera, Polaroid film and a Polaroid MP-7 camera. with Pst I. Isolation of nDNA. A homogenate of rat liver in 250 mM Autoradiography was for either 3 days or 7 days at -70°C. sucrose/i mM MgCl2 was spun at 900 x g for 15 min at 4°C. The x-ray film was Kodak XAR-5 without preflash. Dupont The pellet from 1.5 g of liver was suspended (homogenizer) in Cronex Lightning Plus intensifying screens were used. 10 ml of 2.2 M sucrose/30 mM MgCl2 and layered over 22 ml To obtain sharp reproducible marker bands of unfragmented of the same medium and spun at 112,500 x g for 1 hr at 4°C mtDNA it was necessary to digest once-banded mtDNA with in a Beckman SW 28 rotor. The clear gelatinous pellet of nuclei Pst I, presumably to digest away accompanying nDNA. (29) was processed as the mitochondrial pellet described above Preparationof p3.0, p4.0, p2.1, and pl.7. The large recom- to yield nDNA in TE buffer. There was only a single broad band binant was digested with BamHI and the desired fragments of after isopycnic centrifugation. DNA were separated in E buffer on 1% agarose by electro- Labeling of Probes. Probes were labeled by the method of phoresis and electroelution (34). The aqueous solution was suc- Rigby et al. (30) and were purified by extraction with phenol cessively extracted with phenol and then isoamyl alcohol, and and then by chromatography on Sephadex 100. the DNA was precipitated as described above with NaOAc and Hybridization of Labeled Probes to Southern Blots of Sized ethanol. The precipitate was dissolved in low-salt buffer (200 Restriction Fragments of nDNA Digested with Restriction mM NaCl/20 mM Tris.HCl/1 mM Na2EDTA, pH 7.4) and . The nDNA (500 ,ug) in 1 ml was incubated at 370C passed through an ELUTIP-d column (according to the direc- for 16 hr with 420 units (5-fold excess) of Pst I (Bethesda Re- tions of the manufacturer, see ref. 35). The DNA adhered to search Laboratories) in a buffer recommended by the manu- the column. The DNA was eluted with high-salt buffer (1.0 M facturer. Completeness of digestion was confirmed by incu- NaCl/20 mM Tris.HCl/1 mM Na2EDTA, pH 7.4) precipitated bating in parallel 1 jg of pBR322 added to a 50-,ul aliquot of with NaOAc and ethanol as described above and stored in TE the main digest and running a gel. After completeness of diges- buffer. Labeling with 32P was as described above. tion was confirmed, the reaction mixture, which had been stored at 40C, was extracted first with phenol and then with isoamyl alcohol, and the DNA was precipitated in the cold (-70°C for RESULTS 2 hr) by the addition of NaOAc and ethanol. The precipitate As recorded by others (36) and noted in Fig. 1 circular mtDNA was collected by centrifugation (16,300 X g for 10 min at 0°C), from rat contains 16 kbp. Digestion of mtDNA with BamHI washed with 70% ethanol, dried briefly in vacuo at room tem- yields two linear pieces (36-39)-namely, 10.8 and 5.2 kbp. Pst perature, and redissolved in TE buffer to give a solution of once- I does not cut rat liver mtDNA (36). HindIII cuts the 10.8-kbp digested nDNA. To obtain twice-digested nDNA, the above fragment into four fragments (36-39), which are 3.0, 4.0, 2.1, digestion with 2.5-fold excess of and monitoring for and 1.7 kbp. These enzymes do not discriminate between the completeness of digestion was carried out with 250 ,ug of once- two polymorphic forms of rat mtDNA (36-39). digested nDNA. The twice-digested nDNA was also extracted, Each fragment of mtDNA obtained by digestion with BamHI precipitated, washed, dried, and dissolved in TE buffer. was cloned into the BamHI site of pBR322. Data confirming Electrophoresis was carried out in a horizontal 6-mm thick this are shown in Fig. 2. Each recombinant of DNA was la- slab of 1% agarose submerged 1 cm in E buffer at room tem- beled with 32P by nick-translation (30) to yield one probe with perature for 112 hr. The slots were 7.5 x 2 X 5 mm. Each slot the 10.8-kbp insert (lp) and another probe with the 5.2-kbp in- was loaded with 40 /.l of a solution that contained the desig- sert (sp). These probes of cloned mtDNA were of course free nated amount of nDNA and 2% Ficoll, 2 mM Na2EDTA, and of any contaminating nDNA. In addition, five other probes of Downloaded by guest on September 30, 2021 Biochemistry: Hadler et al. Proc. Natl. Acad. Sci. USA 80 (1983) 6497

3.OKbp HI woo

15.1 @ 10.8 _

lam Hi P I 1.7Kbp 7.2_ . ^ Hindill FIG. 1. Features of rat liver mtDNA. The various fragments of 5.7 * 1 mtDNA used to prepare labeled probes are shown. The organization and restriction enzyme sites have been described (36-38). FIG. 3. Hybridization of lp to Southern blot of sized restriction DNA were used-pBR322 alone and the four fragments (3.0, fragments of nDNA digested with Pst I. These data are from a single 4.0, 2.1, and 1.7 kbp) derived from the cloned 10.8-kbp piece blot and the alignment of all lanes is identical to that on the original single piece ofnitrocellulose. Lanes a, b, andcwere hybridized with one by digestion with HindIII. The four probes are designated p3.0, preparation of lp and exposed to x-ray film for 3 days at -700C; lanes p4.0, p2.1, and p1.7, respectively. d, e, f, g, h, and i were hybridized with a second preparation of lp and The results of hybridizing blots of Pst I digests of nDNA with exposed to a second x-rayfilm for 7 days. Guide lines were used to align Ip are shown in Fig. 3. Similar hybridization was carried out the two exposed films before photography. Lanes a and e contain size with sp but the results are not shown. Bands 1 and 3 in lanes markers in kbp, indicated bythe numbers next to lane a. 0, origin. The f, g, h, and i, which hybridized to both lp and sp and are in the numbers 1, 3, and 5 identify certain bands discussed in the text. Lane b, 10 ug ofonce-digested nDNA. Lane c, 113 pg ofonce-banded mtDNA same position of marker mtDNA (lane b), were presumed to be that was digested withPst I. Lane d, 10 ug ofonce-digested nDNA and unfragmented nonintegrated mtDNA contaminating the digest 113 pgofonce-banded mtDNA that was digested withPst I. Lanes fand of nDNA. Because it migrated faster than band 1, band 3 was g, 10 pg each of nDNA that was digested once and twice, respectively. presumed to be form 1 of mtDNA and band 1 was presumed Lanes h and i, 30 gg each of nDNA that was digested once and twice, to be either form 2 or form 3 of mtDNA or a mixture of the two respectively. forms. Band 5, which was about 10.8 kbp and thus decidedly smaller than unfragmented mtDNA, was hybridized to lp and and g) was loaded with 10 ,g of nDNA digested once and twice not hybridized to sp. This band could contain aportion of mtDNA with excess of the same enzyme. The other pair of lanes (h and integrated into the nuclear genome. i) was loaded with 30 ,ug of nDNA digested once and twice with In this experiment and in those shown in Figs. 4 and 5, there excess of the same enzyme. Thus artifactual bands due to in- are two pairs of lanes with nDNA digests. One pair of lanes (f complete digestion should fade away after the successive diges- tions. a c e h The result of hybridizing blots of BamHI digests of nDNA b d f to lp are shown in Fig. 4. Similar hybridization was carried out with sp but the results are not shown. Preliminary experiments indicated that the bands present either at 10.8 kbp by hybrid- ization to lp or at 5.2 kbp by hybridization to sp would be so intense that they could obscure neighboring bands. Thus, to circumvent this difficulty the locating lanes (a, b, c) were first exposed to x-ray film and the section of the blot for lanes d, e, f, g, h, and i that correspond to the intense band was cut out and relocated at the top of the blot before exposing lanes d, e, f, g, h, and i to fresh x-ray film. Band 11 (about 14 kbp) was not due to incomplete digestion. It was larger than 10.8 kbp and very faintly hybridized to sp. DNA in this band could contain a portion of mtDNA covalently integrated into the nuclear ge- nome. Band 13, hybridized only to lp, was a piece of frag- mented nonintegrated mtQNA contaminating the digest of FIG. 2. Characterization ofrecombinant plasmid DNA by electro- nDNA. phoretic analysis ofrestriction fiagments. Lane a, pBR322. At 12.4 cm The results of hybridizing lp to blots of nDNA digested once is form 1 and at 7.8 cm is form 2 pBR322. Lane b, pBR322 digested with and twice by the combination of Pst I with BamHI are shown BamHI. At 8.5 cm is form 3 (linearized) pBR322. Lane c, recombinant in Fig. 5. Similar hybridization was carried out with sp but the DNA with the small insert. At 7.0 cm is form 1 ofthe recombinant and at 2.5 cm is form 2 of the smaller recombinant DNA. Lane d, smaller results are not shown. Band 17 likely is the same as band 11 recombinant DNA digested with BamHI. At 8.5 cm is form 3 pBR322 because it was much less intense with sp. Although this band andat 7.6 cm isthe 5.2-kbpinsertofmtDNA. Lane e, recombinant DNA could contain a portion of mtDNA integrated into the nuclear with the large insert. At 3.8 cm is form 1 and at 1.0 cm is form 2 ofthe genome, the result is equivocal because of the fading of the large recombinant DNA. Lane f, larger recombinant DNA digested with band after extra digestion (lanes g and i). Band 19, which hy- BamHI. At 8.5 cm is form 3 pBR322 and at 3.7 cm is the 10.8-kbp piece bridized to was mtDNA of ofmtDNA. Lane g, stored and undigested once-banded mtDNA. There only lp, nonintegrated fragmented is a faint band at 2.5 cm (form 1 mtDNA) and an intense band at 0.7 10.8 kbp. Band 21 (8.6 kbp) hybridized to lp and not to sp. This cm (form 2 mtDNA). Lane h, mtDNA digested with BamHI. At 7.7 cm band survived extra digestion and was absent when the en- isthe 5.2-kbp amentand at 3.7 cm isthe 10.8 kbpfagmentofmtDNA. zymes were singly Pst I or BamHI. Band 21 accordingly is a Downloaded by guest on September 30, 2021 6498 Biochemistry: Hadler et al. Proc. Natl. Acad. Sci. USA 80 (1983)

a b c d e f p1.7 p2.1 p3.0 P4.0

l * t3**13 U,0 a Mn 4 p -I 0 15.1 _ a _ 10.8 -0 W

15.1 - 3* 1 .t 10.8___d 5.7 7.2 _ FIG. 6. Hybridization ofp3.0, p4.0, p2.1, and pl.7 to Southern blots 5.7 _ 9* of sized restriction fragments of nDNA digested simultaneously with ..10 Pst I and BamHI. The procedure is described in the legend to Fig. 3. Fourdifferentblots are shown. The probes usedwitheachblot, from left to right, were pl.7, p2.1, p3.0, and p4.0. The left lane of each blot con- FIG. 4. Hybridization of lp to Southern blot of sized restriction tains size markers in kbp, indicated by numbers on left. 0, origin. The arrow fragments ofnDNA digested withBamHI. These data are from a single right lane for each blot had 30 pg of twice-digested nDNA. The blot and the alignment of all lanes is identical to that on the original indicates the 8.6-kbp band. single piece ofnitrocellulose. The procedure is similar to that described for Fig. 3, except the nDNA was digested with BamHI and the strip of placed by its HindIII fragments p3.0, p4.0, p2.1, and p1.7. As nitrocellulose at the 10.8-kbp mark was cut out and relocated on top shown in Fig. 6 the nDNA was again digested successively with before exposure to the second x-ray film. Lanes a and e contain size the combination of Pst I and BamHI. Each of the four blots has markers in kbp, indicated by numbers next to lane a. 0, origin. The an numbers 11 and 13 identify certain bands discussed in the text. Lane intense band at about 10.8 kbp because of nonintegrated b, 10 jig ofonce-digested nDNA. Lane c, 113 pg ofonce-banded mtDNA fragmented mtDNA. This is a positive control, which showed that was digested with BamHI. Lane d, 10 Aug of once-digested nDNA that each probe was effective. Only p3.0 hybridized to the 8.6- and 113 pg of once-banded mtDNA that was digested withPst I. Lanes kbp band. The intensity of the band at 10.8 kbp was so di- f and g, 10 jig each of nDNA that was digested once and twice, re- minished, that it was no longer necessary to cut out and relocate spectively. Lanes h and i, 30 A.g each of nDNA that was digested once any exposure to x-ray film. and twice, respectively. bands before the final of each blot In the 8.6-kbp piece of DNA there are at least 5.6 kbp of nDNA. It is important to note these results were obtained under portion of mtDNA covalently integrated into nDNA with one that conditions of high stringency compatible with perfectly matched end a Pst I site and the other end a BamHI site. (32). To further narrow down the region of homology lp was re- hybrids Additional blots with 30 pug of twice-digested nDNA cor-

a b c d e f g h i responding to the enzymes used in Figs. 3, 4, and 5 were hy- -19_ .4* bridized to a probe derived from pBR322. No bands were de- tected (data not shown). The results represented by Figs. 3, 4, O -i 5, and 6 were obtained with nDNA prepared from at least two different groups of rats. Only the bands seen in Fig. 6 were present even after exposing the four blots to x-ray film for 4 months. .._ 15.1 The results do not eliminate the possibility that there are other _ i17 a w portions of mtDNA covalently integrated into the nuclear ge- 10.8 __ nome.

21 - DISCUSSION rat liver after 7.2 _ The results show that nDNA derived from yield, double digestion with Pst I and BamHI, an 8.6-kbp piece of DNA that contains a mtDNA sequence found within the 3.0- 5.7 _ kbp segment of the mitochondrial genome (Fig. 1). This piece of nDNA has a Pst I site at one end and a BamHI site at the other end and is not homologous with mtDNA, which is either FIG. 5. Hybridization of lp to Southern blot of sized restriction clockwise or counterclockwise to the 3-kbp mitochondrial seg- fragments of nDNA digested simultaneously with Pst I and BamHI. ment. These data are from a single blot and the alignment ofall lanes is iden- Further investigation could describe with greater precision tical to that on the original piece ofnitrocellulose. The procedure is sim- ilar to that described for Fig. 4, except the nDNA was digested simul- the sequence of covalently integrated mtDNA and its possible taneously with Pst I and BamHI. Lanes a and e contain size markers function in evolution, development, transformation, and aging. in kbp, indicated by numbers next to lane a. 0, origin. The numbers 17, Our experimental approach, which is applicable to isolable nDNA 19, and 21 identify certain bands discussed in the text. Lane b, 10 ,ig contaminated with nonintegrated mtDNA, also can be used to ofonce-digestednDNA. Lanec, 113 pg ofonce-bandedmtDNAthatwas investigate pathological conditions because, unlike other strat- digested with Pst I and BamHL. Lane d, 10 ug of once-digested nDNA egies (5), it is not restricted to mutant organisms deficient in and 113 pg ofonce-banded mtDNA that was digested withPst I. Lanes con- f and g, 10 ,ug each of nDNA that was digested once and twice, re- mitochondria. In such mutants the isolable nDNA is not spectively. Lanes h and i, 30 pig each of nDNA that was digested once taminated with nonintegrated mtDNA. Is the observation true and twice, respectively. for humans, other mammals, and other higher eukaryotes? Is Downloaded by guest on September 30, 2021 Biochemistry: Hadler et al. Proc. Natl. Acad. Sci. USA 80 (1983) 6499

the homology due to a portion of the 16S rRNA gene, the 12S 12. Hadler, H. I. & Daniel, B. D. (1972) Res. 32, 1037-1041. rRNA gene, the D-loop region (see Fig. 1), or a tRNA gene? 13. Hadler, H. I. & Daniel, B. D. (1973) Cancer Res. 33, 117-122. The results thus far do not favor either tRNA or rRNA genes. 14. Hadler, H. I. & Demetriou, J. M. (1975)J. Antibiot. 28, 809-818. 15. Hadler, H. I. & Demetriou, J. M. (1975) Biochemistry 14, 5374- Because tRNA genes are scattered around the whole mito- 5378. chondrial genome (38) possibly more than one of the probes 16. Hadler, H. I. & Mueller, K. W. (1977)J. Environ. Pathol. Toxicol. would be expected to show homology with the nuclear genome. 1, 75-85. Such was not the case. The same argument holds for rRNA genes 17. Hadler, H. I. & Cao, T. M. (1978) Chem. Biol. Interact 20, 219- as p4.0, which contains much of the mitochondrial 16S rRNA 226. gene, did not show homology with the 8.6-kbp piece of nDNA. 18. Hadler, H. I. & Cook, G. L. (1978) J. Environ. Pathol. Toxicol. 1, In addition, Rothblum et al. (40) isolated and characterized clones 419-432. 19. Hadler, H. I. & Cook, G. L. (1979)1. Environ. Pathol. Toxicol. 2, of rat rRNA nuclear genes. The largest piece in the cloned DNA, 601-612. with one end a BamHI site and the other end a Pst I site, is 4.0 20. Hadler, H. I. & Cook, G. L. (1979)1. Environ. Pathol. Toxicol. 2, kbp (12.7-8.7). Thus the 8.6-kbp piece of nDNA cannot be 1343-1350. present in the DNA that was cloned. 21. Gay, N. J. & Walker, J. E. (1983) Nature (London) 301, 262-264. The remaining section of mtDNA, which could be homol- 22. Hadler, H. I., Alt, S. K. & Falcone, A. B. (1966)1. Biol. Chem. ogous with the 8.6-kbp piece of the nuclear genome, includes 241, 2886-2890. 23. Bogenhagen, D. & Clayton, D. A. (1974)J. Biol. Chem. 249, 7991- the D-loop region. The D-loop region in mammalian mtDNA 7995. has attracted considerable attention (41-45). It contains the or- 24. Cohen, S. D., Chang, A. C. Y. & Hsu, L. (1972) Proc. Nati. Acad. igin of replication for the H strand. The region has been com- Sci. USA 69, 2110-2114. pletely sequenced for man (41, 42), cow (44), and mouse (45) 25. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., and partially sequenced for rat (43). This region in rat (43) has Heynecker, H. L., Boyer, H. W, Crossa, J. H. & Falkors, S. (1977) 2-fold rotational symmetry, palindrome structures, a G Gene 2, 95-113. cluster, 26. Finkelstein, M. & Rownd, R. H. (1978) Plasmid 1, 557-562. A+T-rich regions, possible single-stranded hairpin structures, 27. Ullrich, A., Shine, J., Chirgwin, J., Pictet, R., Tischer, E., Rut- and is largely noncoding. Because this region of mtDNA is so ter, W. J. & Goodman, H. M. (1977) Science 196, 1313-1319. species specific, Clayton (46) suggested that the D-loop region 28. Mickel, S., Arena, V., Jr., & Bauer, V. (1977) Nucleic Acids Res. is a candidate for an interactive role with the nuclear genome. 4, 1465-1482. Our findings support this view. 29. Sung, M. & Smithies, 0. (1969) 7, 39-58. 30. Rigby, P. W. J., Diekman, M., Rhodes, C. & Berg, P. (1977)J. Mol. This work would not have been possible without Professor Robert Biol. 113, 237-251. Williamson's interest and support. The discussions with Drs. Janet Ar- 31. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. rand and Julian Crampton were crucial. Other helpful discussions were 32. Jeffreys, A. J. & Flavell, R. A. (1977) Cell 12, 429-439. with Drs. James Bailey, Jerry L. Slightom, Michael T. Sung, Howard 33. Denhardt, D. T. (1966) Biochem. Biophys. Res. Commun. 23, 641- Temin, and Donald Sleeter. Mr. Jed Clampet helped to design and build 646. the equipment. Mr. Kenneth Boger prepared the final photographs. 34. Smith, H. 0. (1980) Methods Enzymol. 65, 371-380. The Fogarty Foundation, National Institutes of Health, provided H.I.H. 35. Schleicher & Schuell Inc. 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