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Cleavage maps of rat mitochondrial DNA genome
Gene, 2 (1977) 299--316 299 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
CLEAVAGE MAPS OF RAT MITOCHONDRIAL DNA GENOME (Rat mtDNA; replication origin; restriction endonucleases; EcoRI; HhaI; HindHI; physical maps)
KATSURO KOIKE and MIDORI KOBAYASHI
Cancer Institute, Japanese Foundation for Cancer Research, Kami-lkebukuro, Toshima-ku, Tokyo 170 (Japan) (Received July 12th, 1977) (Accepted September 12th, 1977)
SUMMARY
Physical maps of the rat mtDNA genome were constructed on the basis of specific cleavage by bacterial restriction endonucleases. The six fragments produced by restriction enzyme EcoRI from K coli were ordered by gel electrophoretic analysis of partially digested products and by analysis of an overlapping set of fragments produced by other restriction enzymes, HhaI or HindIII from Haemophilus haemolyticus and Haemophilus influenzae, respectively. Based on the alignment of partially digested forms with respect to the replication origin, the EcoRI cleavage sites were also mapped with electron micrographic analysis. With E¢oRI sites as reference points, the HhaI and Hind III cleavage sites were located on the map.
INTRODUCTION
Restriction endonucleases are site-specific DNAases that cleave doublestranded DNA at short, defined nucleotide sequences, and are extremely useful in dissecting genomes of a number of eukaryotic DNA~. The genome of rat mitochondria is a double-stranded closed-circular molecule of about 10 million daltons that replicates discontinuously by means of a Cairns-type intermediate (Koike and Wolstenholme, 1974). We have been studying the process of mtDNA replication in vitro (Koike et al., 1976a) and also analysing t h e i n vivo replicating structure with products obt~iined by restriction endonu~leases using electron micrographic and gel electrophoretic techniques. As was noted previously (Koike et al., 1975), the restriction endonuclease EcoRI Abbreviations: DTT, dithiothreitol; EtdBr, ethidium bromide; SDS, sodium dodecyl sulphate.
300
creates six double-stranded breaks at specific sites in the molecule. On the other hand, two and three restriction sites were obtained in mouse and human mtDNA, respectively (Robberson et al., 1974, Brown and Vinograd, 1974). In this report, we describe recent results concerning a physical mapping of the restt~iction fragments in the rat mitochondrial genome and its replicating. form. Rat mtDNA was cleaved into unique fragments by restriction enzymes EcoRI, HhaI and HindHI from E. coU, H. haemolyticus and H. influenzae, respectively. Sizes of the fragments were determined with data obtained by electron microscopy and agarose gel electrophoresis. On the basis of these data an(~ estimates of the sizes of the partially digested products, the physical maps were constructed. Being consistent with the previous observations (Koike et al., 1975), by analysing restriction fragments of in vivo Cairns-type intermediates, it was demonstrated that there is a fixed origin and the replication is unidiractional. According to our results of the physical mapping, we discuss a mode of replication of the rat mtDNA molecule. MATERIALSAND METHODS
Preparation and purification o f rat mtDNA Mitochondria were prepared from adult or newborn rats, Donryu strain, as described previously (Koike et al., 1974, 1976a; Fujisawa et al., 1977). The yield of mitochondria was about 25 mg mitochondrial protein per g of liver. Extraction of mtDNA was performed by the SDS-phenol method (Koike et al., 1974). The mitochondrial fraction was mixed with ow~tenth volume of 10% SDS and an equal volume of phenol saturated with 50 mM sodium phosphate buffer, pH 8.5 containing 0.15 M NaCI, 100 mM EDTA. The mixture was slowly rotated for 20 rain at room temperature and centrifuged to separate an aqueous phase. This extraction was repeated. The aqueous phase was concentrated and dialysed against 50 mM sodium phosphate buffer, pH 6.7 containing 0.1 M NaCI, 2 mM EDTA, at 4 ° C. Closed-circular mtDNA was purified by equilibrium centrifugation in CsCIEtdBr according to the procedure of Radloff et al. (1967) with minor modification. Solid CsCl and EtdBr were added to the DNA solution to a final concentration of 1.58 g/ml and 150 ~g/ml, respectively, and the mixture was centrifuged at 36 000 rev./min for 48 h in a Beckman SW50.1 rotor. After centrifugation, the closed circle-containing band was collected, and EtdBr was removed by a small column of purified Dowex-50 resin. The DNA, thus obtained, Was dialysed against 0.1 M Tris--HCl, pH 7.8, 0.5 mM EDTA. Under the electl'on microscope, about 22% of the closed circles from the newborn rat mitochondria were observed as D-loop molecules (Robberson et al., 1982). This waS about 200 times the frequency of that found in the adult (Koike and Kobayashi, 1973).
301
Cleavage of mtDNA with restriction endonucleases EcoRI endonuclease from E. coli was a gift from K. Matsubara, Laboratory of Molecular Genetics, Osaka University or obtained from Miles Biochemicals. HhaI endonuclease from H. haemolyticus and HapII from H. aphirophilus were gifts from M. Takanami, Chemical Institute, Kyoto University. HindIII endonuclease from H. influenzae was from Miles Biochemicals. One unit of EcoRI or HindIII is defined as an amount of the enzyme required to completely digest 1 ~g of kDNA in 1 h at 37 ° C. One unit of HhaI or HapII is an amount of the enzyme required to completely cleave 1 ~g of fdRFI DNA in l h at 37 ° C (M. Takanami, personal communication). EcoRI endonuclease reactions were usually carried out at 37 ° C in 0.1 M Tris--HCl pH 7.8, 7 mM MgCI2, 50 mM NaCI, 0.02 M potassium phosphate, 0.4 mM EDTA 0.28 mM ~-mercaptoethanol and 0.008% NP-40 for 2 h (Greene et al., 1974). For complete digestion, 2 units of EcoRI were added to 1 ~g of mtDNA. A partial cleavage of mtDNA was achieved by addition of the same amount of EcoRI at 10 ° C for 10 min. Reactions were stopped with 25 mM EDTA. The HhaI or HapII restriction buffer was 0.1 M Tris-HC1, pH 7.8, 7 mM MgCl2, 50 mM NaC1, 0.4 mM EDTA and 5% glycerol. The buffer for HindIII was 0.1 M Tris--HCl, pH 7.8, 7 mM MgCl2, 50 mM NaCl, 0.4 mM EDTA, 0.02 mM DTT, 0.2 mg/ml BSA an~ 5% glycerol. A partial cleavage with HhaI or HindIII was performed at 10 ° C for 10 min under a similar reaction condition, as described above. These reactions were stopped with 25 mM EDTA. . In some cases, digestion of the replicating form with EcoRI endonuclease was carried out using glutaraldehyde-fixed mtDNA from the newborn under following reaction conditions. DNA samples were dialysed against 50% formamide, 0.2 M KH~PO4, pH 7.5 and glutaraldehyde was then added to a final concentration of 0.1%. The mixture was incubated at 23 ° C for 20 rnin, and then dialysed extensively against 0.1 M Tris -HCI, pH 7.8, 0.5 mM EDTA at 4 ° C for subsequent treatment with EcoRI (Robberson et al., 1974). Agarose gel eleetrophoresis Agarose gels were prepared according to the procedure of Sharp et al. (1973) with a minor modifications. Solutions of 1.4% agarose (w/v) were prepared by dissolving agarose (Sea Kern) in electrophoresJs buffer (0.04 M Tris--HCl, pH 7.7, 0.02 M sodium acetate, 2 mM EDTA, and 0.5/~g/ml EtdBr/ ml). Gels were poured at 60 ° C into glass tubes (0.6 cm diameter) or glass slab (10 cm X 2 mm). After hardening, the top of the disk gel was sliced evenly to form a 9 cm gel, and a piece of cotton mesh was stretched over the bottom of the glass tube. Samples of 50--80/~l containing 5% glycerol or 1.2% sucrose and 0.005% bromophenol blue were layered under the electrophoresis buffer. The gels were run in the electrophoresis buffer at 5 mA per tube or at 8 V/cm per slab for 2.5--3.5 h. The gels were laid directly on the UV lamp (short or
302
long wavelength, Manasulu Light). The D N A bands were visualized by the fluorescence from bound EtdBr and photographed with a Polaroid land camera equipped with a Kodak No.23A red filterusing a Polaroid film type 107 or type 55P/N. The negatives were scanned using a Joyce Loebl microdensitometer. For the extraction of D N A fragments from the gels,the band regions were sliced,homogenized and incubated with 0.1 M Tris--HCl buffer. p H 7.8, containing 0.5 m M E D T A for 48 h at 0-4 ° C. Electron microscopy
Specimens were prepared according to the technique of Davis et al. (1971) with minor modifications (Koike and Kobayashi, 1973). The D N A was spread with the formamide spreading condition. Grids were shadowed with Pt-Pd. Electron micrographs were taken with a Hitachi HU-12 electron microscope at 75 kV. Either open-circularrntDNA or ColE1 D N A was used as an internal size standard. The contour length of m t D N A or ColE1 D N A was calibrated with grating replica(2000 lines/ram, OKEN).
9.1 x 10 6
1.3
2E 40
0.45 1.0
2.0
MICROMETERS
,
Rat
Mouse
Fig.l.(a) Length histograms of EcoRI generated fragments from rat mtDNA. Closedcircular mtDNA was digested with restriction endonuclease EcoRI, and restricted products were mounted for observation with electron microscopy by the formamide technique. Length histograms are given in micrometers. There are six peaks (A to F) represented in black rods. Intact circular genome of rat mtDNA forms a peak of 5.26 -+ 0.09 ~,m, but not shown in this histogram. (b) Electrophoretic pattern of EcoRI generated fragments from rat mtDNA. Electrophoretic conditions were 5 mA per tube for 2.5 h.
303 RESULTS
EcoRI endonuclease fragments of rat mtDNA A histogram of the EcoRI endonuclease cleavage products from a complete digestion of rat m t D N A is shown in F i g . l ~ Sizes of the fragments were estimated by electron micrographic measurements and are expressed in micrometer. There are at least six fragments, designated alphabetically (Eco A, B, C, D, E and F, A being the largest). Electrophoresis in 1.4% agarose can separate six fragments, as shown in Fig.lb. Measurements of intensi~y indicated that six bands are equimolar. Addition of more endonuclease and further incubation did n o t alter the profiles. Each fragment was recovered from the gel and examined by electron microscope. Table I summarises the results of length measurements. The sum of these six fragments is very close to the length of circular genome (5.26 :+- 0.09 gm, 10.4-106 daltons) using ColE1 DNA (2.04 -+ 0.07 ~m) as an internal size standard. Sizes of the six fragments were confirmed by a gel electrophoretic analysis using two EcoRI fragments of mouse m t D N A as standards of known molecular weight (1.3 and 9.1-106 daltons).
DNA fragments generated by HhaI, Hmdlll and HaplI digestions Gel electrophoresis separation of endonucleases HhaI, HindIII or HapH digests of rat m t D N A are shown in Fig.2a. Sizes of the DNA fragments were determined by their mobility relative to EcoRI fragments of rat mtDNA, as summarized in Table II. Endonuclease HhaI cleaves m t D N A into four fragments (//ha A, B, C and D) and HindIII into six (Hin A, B, C, D, E and F), but very small fragment, Hin F, was o u t of the gel under the conditions used. The gel of the HapII cleavage products showed eight resolved DNA bands (Hap A to H). On comparing the fluorescent intensity, the short fragment, Hap H had approximately twice the intensity of Hap G. There must be two TABLE I LENGTH ANALYSIS OF EcoRi FRAGMENTS Fragment
Number of molecules classified
Length in micrometers
A
85 84 120 84 99 95
2.01 ± 0.07 1.28 ± 0.05 0.95 _*0.06 0.60 ± 0.04 0.23 ± 0.07 0.17 ± 0.05 5.24± 0.06 5.26 ± 0.09
B C D
E F A+B+C+D+E+F Circular genome
38
304
(a)
(1)
(2)
• Eco R!
~o I0 'o •- 6 x 4
• Hhai
QS O
0.2 I 5
Eco Hha Hap
Eco Hind
I 10
,
I 15
Relative Mobility
Fig. 2.(a) Electrophoretic patterns of generated fragments from rat m t D N A by endonucleases HhaI, HindIII and HapII digestions. Electrophoretic conditions were 5 m A per tube for 3 h (1) and 3. 5 h (2). (b) Relationship between molecular weight and electrophoretic mobility of rat m t D N A fragments. Fragments were produced by cleavage with EcoRI, HhaI, HapII and HindIII. Molecular weights were calculated as described in Table II. Mobility o f fragment is given relative to the HhaI fragments.
TABLE II I~OLECULAR WEIGHT OF THE RESTRICTION ENZYME FRAGMENTS OF RAT mtDNA EcoRI fragment
M.W. •10 -*
//hal fragment
M.W. •10 - '
HindllI
M.W.
HapIl
M.W.
fragment
•10-S
fragment
.10-6
A B C D E F
3.98 2.53 1.88 1.18 0.45 0.33
A B C D
5.65 2.80 1.25 0.65
A B C D E F
4.08 2.70 1.65 1.32 0.50 0.14
A B C D E F G H (doublet)
2.58 2.05 1.23 1.11 1.03 0.77 0.63 0.48
305
fragments of about equal length. Therefore, numbers of the HapII cleavage sites on the rat mtDNA became at least 9. A plot of log molecular weight versus electrophoretic mobility of the restriction fragments is shown in Fig.2b. From the length of EcoRI fragments of rat mtDNA, it was also possible to construct a calibration curve of log fragment length versus mobility, since data from the gel electrophoresis were in good agreement with the electron microscopic measurements.
Position of the replication origin and unidirectional replication Restriction endonuclease cleavage provided a basis ~or electron micrographic examination of the initiation site of mtDNA replication. When the in vivo closed-circular DNA molecules containing replicating forms were extensively digested by EcoRI, a small replication eye was found exclusively on the Eco A (2.01/~m). The small eyes randomly selected were slightly heterogeneous in size and strandedness (Fig.3a, b). Analysis of these eye forms indicated that about 25% of them had two double-stranded branches (Fig.3b), and the rest had one each of single- and double-stranded branches (Fig.3a). Before EcoRI digestion, the closed-circular molecules contained 2 L 5 _+3.2% of D-loop molecules among the population. After cleavage, the frequency of resulting
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Fig. 3. Electron micrographs of Eco A with a replicating eye. Replicated region can be seen as an eye. The scale represents 0.5 ~m.
306 Origin I •
•
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.,
ms
Ill
m,
Origin !
i
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i
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I
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M ICROMETERS
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Fig.4. Location of small replicating eye in E¢o A fragment. The linear replicating Eco A are alignmed with short unreplicated portion to the left. The replicated region is represented by a black rect.ngie. Origin indicates that initiation of replication occurs on E¢o A at a position 0. 47/Am away from the proximal restriction site. Fig. 5. Location of expanded eye in Eco A. Linear replicating £¢o A are aligned with short unreplicated portions to the left. The replicated region is represented by a black rectangle. Replication proceeds unidirectionally away from origin throughout the length of Eco A.
linear molecule with the small eye became substantially lower than the initial frequency. This suggests that the replication eye in the linear form is probably lost through the process of rewinding or branch migration during the EcoRI digestion. Alignmen~ of these forms with a short unreplicated portion to the left indicated that initiation of the DNA replication occurs on Eco A at a fixed position of 0.47/~m away from the proximal restriction site (Fig.4). In addition, a location of the expanded eye in Eco A was also examined by electron microscopy (Fig.3c). Length measurements of such replicating forms also provided an array of molecules with increasing degree of replication, and it is indicated that replication proceeds unidirectionally away from the proximal restriction site throughout Eco A (Fig.5). A part of the data was previously described (Koike et al., 1975).
Physical map of the restriction endonuclease fr~/~ments (a) Determination of the order of EcoRl fragments A, B and C. In order to determine the order of EcoEI fragments, DNA molecules containing the small eye were cleaved by EcoRI under limited conditions, as described in
307 MATERIALS AND METHODS. The resulting partially digested products were examined by electron microscopy using the small eye as a marker. An example is shown in Fig.6. The replicated region located at a position of 1.1 ~m away from one end of this molecule. The partial digests were m e ~ u r e d similarly and aligned as shown in Fig.7. Taking the initiation site on the Eco A as the zero point, four out of the six EcoRI c~eavage sites could be mapped at the points, -8.9, 29.3, 53.6 and 71.7. Distance between each contiguous cleavage site corresponds to the percentage of Eeo A, B or C, calculated from the total length of mtDNA as 100%. Three EcoRI fragments could thus be placed in the following order: A-B-C, as shown in Fig.8. This combination was confirmed by digestion of the EeoRI fragments with other restriction endonuclease HhaI or HindIII, as vdll be shown later. As other EeoRI cleavage sites were less accurately located by electron micrographic analysis, mapping of small fragments D, E and F were carried out by gel electrophoretic analysis. (b) Order of the EeoRI fragments D, E and F. To deduce physical order of fragments generated by one endonuclease, the final products derived by the same enzyme from each partially digested segment were arranged in overlapping position. Partial digestion of mtDNA with EeoRI endonuclease revealed several intermediate bands in the gel, as shown in Fig.9. DNA lengths of those intermediates were determined from their mobility, as summarized
Fig.6. Electron mierograph of an intermediate with an eye generated by partial digestion of m t D N A with EcoRI. Partial digestion was carried out as described in the text. The replicated region can be seen as an eye, which is on the intermediate at a position 1.1 ~m away from the proximal restriction site.
308
-~0
,m
-2"0
w
6
.
~0
.
,.,
40
&
Percentage
Fig.7. Alignment of partially digested rat mtDNA molecules having small eyes. Rat mtDNA molecules containing ~he small eyes were cleaved by £coRI under limited conditions as described in the text, and the resulting partially digested forms were examined by electron micrographs using the initiation site as a marker. Taking the initiation site on the intermediate as the zero point, four out of the six cleavage sites could be located at -8.9, 29.3, 53.6 and 71.7% in order (from left to right)
in Table III. Further cleavage of the 0.42/~m intermediate I by EcoRI resulted into two fragments, Eco E and F. The 0.85/Jm intermediate II generated Eco D and E. Thus, the Eco E lies between Eco D and F. A third intermediate III with a size of 1.01 ~m consists of Eco D, E and F. Intermediate IV has a size of 1.14/~m and possibly cont.~ins two fragments C and F. Intermediate V of 1.43/~m suggested a combination between the fragments C, F and E. Taking account of other experiments by digestion of Eco C, D, E and F with restriction enzymes HhaI and HindIII, a combination of Eco A and D w ~ ot~tained, as also suggested by previous data (Fig.6). Details will be sho~n li~er. In this way, a combination of EcoRI fragments D-E-F-C.B-A, with A ~ d D being adjacent in the circular molecule, was oriented as shown in Fig.~o
F~g.8. A physical map of the rat mitochondrial genome constructed by the cleavages with endonucleases EcoRI, HhaI a n d HindIII. Replication proceeds clockwise from the origin.
309 TABLE HI LENGTH ANALYSIS OF INTERMEDIATE SPECIES GENERATED BY PARTIAL DIGESTION OF RAT mtDNA WITH EcoRI Intermediate species
M W. -10-6
Length in micrometers
Possible combination
I II HI IV V
0.83 1.68 2.00 2.25 2.85
0.42 0.85 1.01 1.14 1.43
E-F D-E I)-E-F C-F C-F-E
(1)
. . . . . . . . .
(2) (3)
|
Fig.9. Electrophoretic separation of the intermediate species generated by partial digestion of mtDNA with EcoRI. Partial digestions were carried out at 10 ° C for 5 min (1) and 10 rain (2), as described in the text. Electrophoretic condition was 5 mA per tube for 3 h. In (1) the same gel is shown in different exposure.
(c) Determination of the order of the Hhal fragments. Data for mapping of four HhaI fragments were obtained by gel electrophoretic analysis. After partial digestion of mtDNA with HhaI endonuclease, two intermediate species, smaller than Hha A (2.80/am), revealed as discrete bands, lengths of which were determined from their mobility. Each intermediate recovered from the gel was redigested with the same enzyme and was separated again. One intermediate (1.03/am) contained Hha C (0.66/am) and D (0.35/am), and the other (2.07/am) consisted of Hha B (1.41/am) and C (0.66/am). Hha C therefore,
310 TABLE IV CLEAVAGE OF EcoRI FRAGMENTS BY HhaI Number of HhaI cleavage sites
Length of generated pieces in micrometer
A
1
1.05,
0.97
B
0
C D E F
I (2) 1 0 0
0.56, 0.40
0.33 0.21
EcoRI fragment
lies between Hha B and D. Because of circular structure of mtDNA, Hha A becomes adjacent to Hha B and D. (d) Cleavage of EeoRI fragments by HhaI. To analyse cleavage site(s) of endonuclease HhaI on the EeoRI fragment, each EcoRI fragment was recovered from the gel and extensively digested with HhaI. The digest was again separated by gel e]ectrophoresis together witch a migture of EcoRI fragments as a marker. DNA lengths of resultant short pieces were determined from their mobflities. Location of HhaI cleavage site(s) on an EcoRI fragment is summarized in Table IV. Eco A yielded two close bands, whose DNA lengths were 1.05 #m and 0.97 ~m. These two together add up to a size equal to £eo A (2.01 #m). We concluded that there is one HhaI cleavage site in Eeo A. Digestion of Eco B yielded only one band, which was exactly corresponding to £co B. There is no cleavage site. Eco C revealed two distinct bands with sizes of 0.56 #m and 0.33 #m. Sum of these two was slightly shorter than the size of EcoC (0.95 ~m). A very short piece was therefore expected, but too short to be detected in the gel, under the conditions used, There is a t least one, possibly two, cleavage site. Eco D was separated into two bands with lengths of 0.40 #m and 0.21 #m, the sum of which is equal to the original length (0.60/lm). Cleavage of Eco E or F with HhaI did not make any difference from the original fragment, indicating that there is no cleavage site. This result corrects the previous data (Koike et al., 1976b). A conclusion derived from the present analyses is that each of Eco A, C and D contains one HhaI cleavage site. As there are four HhaI cleavage sites in the rat mtDNA (Table II), one cleavage site is ambiguously located on Eco C. (e) Cleavage of HhaI fragments by EcoRI. Each HhaI fragm~mt was digested by EcoP,I, and the resulting pieces were analysed in the gel, as summarized in Table V./tha A yielded three bands, the sum of these is exactly the Size of Hha A (2.85 ~m). The largest piece corresponded to Eco B, The second band (1.04 #m) was equal to one of the pieces obtained by HhaI cleavage of EcoA. The third band (0.54 ~m) corresponded ~o a piece derived from EeoC by HhaI.
311
TABLE V CLEAVAGE OF HhaI FRAGMENTS BY E c o R I HhaI fragment
Number of EcoI cleavage sites
Length of generated pieces in micrometer
A B C D
2 1 2(3) 0
1.28, 0.97, 0.23,
1.04, 0.42 0.19
0.54
This implies that Hha A localized in a region of Eco A, B and C, as shown in Fig.8. Hha B generated two distinct bands (0.97 and 0.42/~m) by EcoRI which corresponded to HhaI generated pieces from Eco A and Eco D, respectively (Table V). As described above, Eco A and D contain one each of HhaI cleavage site, and Hha B is contiguous to Hha A. Consequently, we located Hha B as shown in Fig.8. Digestion of Hha C (0.63/Jm) by EcoRI resulted in an appearance of two bands corresponding to sizes of 0.23/~m (Eco E) and 0.19 I~m, which migrates slightly slower than Eco F (0.17/~m). As described in the previous section that one HhaI cleavage site was ambiguously located on Eco C, such location would yield Eco F and a small piece, but both are not d e tected under the conditions used. On the basis of these observations, one can say that ~here are at least two, possibly three, EcoRI cleavage sites in Hha C. (f) Determination o f the order of Hindlll fragments. A combination of five HindIII fragments was examined by the partial digestion technique as before. Digestion of mtDNA by HindIII under limited condition yielded five intermediate species, smaller than Hin A (2.06 #m), in the gel. Based on their DNA lengths and further cleavage of intermediate species by HindIII, we found out their composition. As shown in Table VI, the first intermediate (2.01 ~m) contains Hin B (1.36/~m) and D (0.67 ~m). The second intermediate (1.81 /~m) consists ofHin C (0.83/~m), D (0.67/~m) E (0.25/~m) and F. The third TABLE VI LENGTH ANALYSIS OF INTERMEDIATE SPECIES GENERATED BY PARTIAL DIGESTION WITH HindIII Composition of
Intermediate species
Length in micrometer
HindIII fragments
I H HI IV V
2.01 1.81 1.54 1.14 1.07
B, C, C, C, C,
I) D, E, F D, F E, F E
312 intermediate (1.54 pm) contains HinC, D and F. The fourth intermediate (1.14/zm) contains Hin C, E and F. The fifth intermediate (1.07 pro) is of Hin C and E. The fragment F, therefore, has a size about 0.07 pm and lies between Hin C and D. Tnus, a possible combination of five fragments is the following: E-C-F-D-B. (g) Cleavage o f EcoRl fragments by HindlII. Each Eco RI fragment was digested by HindIII and resulting pieces were analysed by electrophoresis. As summarized in Table VII, Eco B, C and D were digested into smaller pieces. However, other fragments A, E and F were not digested. Digestion of Eco B (1.28 pm) by HindIII produced four bands corresponding to DNA lengths of 1.18 um, 0.97 pm, 0.29 p m and 0.15/~m, under the condition used. These were not final producm. The first band migrated slower than Hin F. The second band migrated to a position almost identical with that of Hin E (0.25 pm). The third band (0.97 pro) was slightly larger than that of Hin C plus F, indicating that Hin C and F are contained in Eco B. This is consistent with the observation that avev.! short fragment corresponding to Hin F lies between Hin C and D. It is indicated that at least three, possibly four, HindIII cleavage sites are located on Eco B. Eco C (0.95 pro) generated two dzstinct bands (0.52 pm and 0.45 pm) which sum up to the size of Eco C. There is one HindIII cleavage site in Eco C. According to above analyses of HindIII fragments (Table VI), Hin B is put bet;,veen Hin D and A. Eco D yielded a single band (0.56 pm) which migrat~ed only s'~ightly faster than Eco D (0.60 pm), indicating that one HindIII cleavage site locates closely "~othe junction of Eco A and D. This result was confirmed Oy a re-digestion experiment of Hin B by EcoEI. (h) Cleavage of HindlIl fragments by HhaI. Each HindIII fragment was redigested by HhaI endonuclease. Hin A (2.06/~m) produced two close bands corresponding to DNA lengths of 1.07 ~m and 0.99 pro. Sum of two pieces is exactly the size of Hin A. This is cotzsistent with the above observation that a
TABLE VII CLEAVAGE OF EcoRI FRAGMENTS BY
HindIII
!~oRI ~ragment
Number of HindIII cleavagesites
Length of generated pieces in micrometer
A B C D E F
0 3(4) 1 1 0 0
1.18, 0.52, 0.56
0.97, 0.45
0.29,
0.15
digestion of Eco A by HindIII yielded no small piece. Hin B (1.36/~m) was cleaved into a piece of DNA (0.30/~m), Hha C (0.63/~m) and D (0.33/~m), T~ese three together add up to a size of 1.26/~m, which is slightly shorte~ than Hin B. A tiny piece in the digest was out of the gel in this case. This
313
TABLE VIII CLEAVAGE OF HindIII FRAGMENTS BY HhaI HindIH fragment
Number of HhaI cleavage sites
Length of generated pieces in micrometer
A S
1 2(3)
1.07, 0.63,
c
0
D E F
0 0 0
0.99 0.33,
0.30
implies that three HhaI cleavage sites are located on Hin B. On the other hand, no HhaI cleavage site was found in Hin C, D, E and F. Therefore, four HindIII fragments were arranged inside Hha A in a following order: E-C-F-D. A HindIII cleavage map thus constructed is also shown in Fig.8. DISCUSSION
Cleavage maps of the rat mtDNA genome were constructed based on specific sites of the DNA molecule susceptible to bacterial restriction endonucleases. Methods we have used to order DNA fragments are analogous to those used for ordering peptides in a protein molecule. We were able to identify overlapping DNA fragments by the distinctive electrophoretic mobility to their final digestion products. DN.A fragments were also ordered by electron microscopic examinations of marker(s) of structural significance. The DNA molecules containing the small replication eye were cleaved by EeoRI under limited conditions, and the resulting intermediate species were examined by electron microscopy using the initiation site as a marker. However, ability to measure the duplex DNA sets a lower limit to the length of a fragment which can be measured in this way. Six fragments, obtained when the rat mtDNA was cleaved with EcoRI endonuclease, provided a convenient starting point to construct a physical map of this genome. This was achieved by a combination of methods that involved analysis and cleavage of intermediates obtained by a partial digestion of mtDNA with EcoRI, and the action of other enzyme, HhaI or HindIII, on both the fragments obtained when mtDNA was treated with EcoRI and the fraffments produced by digestion with HindlII or H ~ I . These methods helped to unambiguously locate restriction endonuclease fragments on the map as shown in Fig.8. DNA fragments can also be ordered by an electron microscopic examination of heteroduplexes formed between Eco A, B, C and Hha A. Hybridization of the denatured Eco A, B or C and Hhu A yielded a partially singlestranded molecule, thus confirming the location of EcoRI cleavage sites within //ha A (data not shown).
314
Earlier suggestions that the rat mtDNA replication is unidirectional were based on the electron microscopic observation of a single-stranded region at one fork of the replicating molecule (Wolstenholme et al., 1973, Koike and Wolstenholme, 1974), Koike etal. (1975), then, determined that the origin of DNA replication in rat mtDNA is located at 0.47/~m from the E c o R I cleavage ~ite on Eco A. This result, however, did not distinguish the origin being located on the right side or on the left side of the fragment (see Fig.3a, b). The results presented here definitely place the origin at 0.47 ~m from the Eco A-D junction on Eco A. One conclusion drawn in this paper is that rat mtDNA replication begins at a specific site and proceeds in one direction around the circular genome. Since the initiation site has been localized and the DNA fragment containing this site is available, it will be interesting to determine what special nucleotide sequences might represent a signal for the initiation. A region containing the origin of DNA replication might be conserved in various species for functional reasons (Robberson et al., 1974; Koike et al., 1975). When the mtDNA replication is compared with that of the SV40 viral DNA, the former replicates unidirectionally whereas the latter does bidirectionally (Danna and Nathans, 1972). What general significance uni- or bidirectional replication may have is not known. As the rat mtDNA replication is unidirectional, the termination region of DNA replication would be close to the initiation site in Eco A. As described previously (Koike et al., 1974, 1976b), a short pulse of [3H] thymidine to rat mitochondria, which are actively synthesizing DNA, resulted in an incorporation of label into the growing point of replicating DNA. During a pulse time, most of the label is incorporated into the nascent DNA associates with the closed-circular DNA, but open-circular molecule containing appropriate small gap is also labeled in such gap region before 4~-]osure [i.e. gap-filling synthesis after segregation of two daughter molecules before closure (Robberson et al., 1972; Berk and Clayton, 1976)]. Then there will be a concentration of labeling in such region of the DNA. For this purpose, the [3H] open-circular DNA among the pulse~labeled daughter molecules was selected and completely digested with EcoRI. Each fragment was analYsed for its radioactivity after !separation of the products by agarose gel electrophoresis. As previously shown (Koike et aL, 1976), there was a concentration of labeling. Further digestion of the [ 3H] labeled Eco A by other restriction enzymes is underway. Preliminary data indicate that the specific activity was highest in a small region of the Eco A, which is roughly located at a left side o~"the initiation origin (Fig.8). D~_ta are consistent with an idea that replication of rat mtDNA proceeds unidirectionally away from the origin, and terminates at a region adjacent to the initiation site on both parental strands. Such comparison of the order of synthesis wish the physical order of the restriction fragments may allow one to distinguish rat from mouse mode of replication. Rat mtDNA is similar to mouse mtDNA in having the unidirectional replication, but a degree of asymmetry is different from that-of mouse mtDNA, which is extremely asymmetric (Berk aild Clayton, 1974). Details will be published elsewhere.
315
A creation of the physical map of mtDNA will he!p an assignment of genetic markers c f biological significance, as has been achieved with HeLa ceil, Xe~opus or Drosophila mtDNA genome (Ojala and Attardi, 1974; David, 1976). From the preliminary results, the genes coding for two rat mitoribosomal RNA species can be located on the physical map, and are found to lie adjacent to each other (unpublished data). In the experiments described here, the mtDNA used was all derived from normal rat. The cleavage pattern of the restriction enzyr~es on mtDNA derived from rat tumor cells is significantly different ( m ~ u s c r i p t in preparation). The present maps are also currently in use for analyses of in vitro ~phage-mtDNA recombinant grown in E. coll. ACKNOWLEDGEMENT
We thank Dr. H. Sugano, Director of the Cancer Institute, for his interest and encouragement. This work was partly supported by a Grant-in-Aid for Scientific Research and a Grant-in-Aid for Cancer Research from the Ministry
of Education, Science and Culture, Japan. REFERENCES Berk, A.J. and Clayton, D.A., Mechanism of mitochondrial DNA replication in mouse L-cells: Asynchronous replication of strands, segregation of circular daughter molecules, aspects of topology and turnover of an initiation sequence, J. Mol. Biol., 86 (1974) 801--824. Brown, W.M. and Vinograd, J., Restriction endonuclease cleavage maps of animal mitochondrial DNAs, Proc. Natl. Acad. Sci. USA, 71 (1974) 4617--4621. Danna, K.J. and Nathans, D., Bidirectional replication of simian virus 40 DNA, Proc. Natl. Acad. Sci. USA, 69 (1972) 3097--3100. Davis, R.W., Simon, M. and Davidson, N., Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in Grossman, L. and Moldave, I~ (Eds.), Methods in Enzymology, Vol. 21D, Academic Press, New York, 1971, pp.413--428. Dawid, I.B., K]ucas, C.K., Ohi, S., Ramires, J.L. and Upholt, WoB., Structure and evolution of animal mitochondrial DNA, in Saccone, C. and Kroon, A.M. (Eds.), The Genetic Function of Mitochondrial DNA, Elsevier/North-Holland, Amsterdam, 1976, pp. 3--13. Fujisawa, T., Tanaka, S., Kobayashi, M. and Koike, K., Mitochondrial DNA polymerase from rat liver, Biochir~ Biophys. Acta, 475 (1977) 611--622. Greene, P.J., Betlach, M., Goodman, H.M. and Boyer, H., The Eco R I restriction endonuclease, in Wickner, R.B. (Ed.), Molecular Biology Series: DNA Replication and Biosynthesis, Vol.7, Dekker, New York, 1974, pp.87--111. Koike, K. and Kobayashi, M., Molecule contour length of partially single-stranded circular mitochondrial DNA from newborn rat liver, Biochim. Biophys. Acta, 335 (1973) 14--18. Koike, K. and Wolstenholme, D.R., Evidence for discontinuous replication of circular mitochondrial DNA molecules from Novikoff rat ascites hepatoma cells, J. Cell Biol., 61 (1974) 14--25. Koike, K., Kobayashi, M. and Fujisawa, T., Novel properties of two classes of nascent mitochondrial DNA formed in vitro, Biochim. Biophys. Acta, 361 (1974) 144--154. Koike, K., Kobayashi, M. and Fujisawa, T., Mode of extension of the daughter strands in the replication of closed circular mitochondrial DNA in vitro, Biochim. Biophys. Acta, 425 (1976a) 18--29.
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Koike, K , Kobayashi, M: Tanaka, S, and Mizusawa, H., Physical map and replication of rat mitoehond~1~l DNA, in Biicher, Th., Neupert, W., Sebald, W. and Warner, S. (Eds.), Genetics and Biogenesis of Chloroplastsand Mitoehondria, ElsevierINorth-Holland, Amsterdam, 1976b, pp.593--596. O]ala, D. and Attardi, G., Identification and partial characterization of multiple discrete polyadenylic acid-containing RNA components coded for by HeLa cell mitoehondrial DNA, J. Mol. Biol., 88 (1974) 205--219. Radloff, R., Bauer, W. and Vinograd, J., A dye-buoyant-density method for the detection and isolation of closed circular duplex DN:'~: The closed circular DNA in HeLa cells, Proc. Natl. head. SeL USA, 57 (1967) 1514--1521. Robberson, D.L., Kasan~tsu, H. and Vinogra~,'J., Replication of mitoehondrial DNA. Circular replicative intermediates in mouse L cells, Proe. Natl. Acad. Sei. USA, 69 (1972) 737--741. Robberson, D.L., Clayton, D..~ and Morrow, J.F., Cleavage of replicating forms of mitochondrial DNA by ~ o R I endonuclease, Froe. Natl. Acad. Sei. USA, 71 (1974) 4447-4451. Sharp, P.A., Sugdcn, ]3. and Sambrook, J., Detection of two restriction endonuclease activities in l-Iaemophilusparainfluenzae using analytical agarose-ethidium bromide electrophoresis, Biochemistry, 12 (1973) 3055--3063. Woistenholme, D.R., Koike, and Cochran-Fouts, P., Single strand-containing replicating molecules of circular mitochondrial DNA, J. Cell Biol. 56, (1973) 230--245. i
Communicated by If. Matsubara.
CLEAVAGE MAPS OF RAT MITOCHONDRIAL DNA GENOME (Rat mtDNA; replication origin; restriction endonucleases; EcoRI; HhaI; HindHI; physical maps)
KATSURO KOIKE and MIDORI KOBAYASHI
Cancer Institute, Japanese Foundation for Cancer Research, Kami-lkebukuro, Toshima-ku, Tokyo 170 (Japan) (Received July 12th, 1977) (Accepted September 12th, 1977)
SUMMARY
Physical maps of the rat mtDNA genome were constructed on the basis of specific cleavage by bacterial restriction endonucleases. The six fragments produced by restriction enzyme EcoRI from K coli were ordered by gel electrophoretic analysis of partially digested products and by analysis of an overlapping set of fragments produced by other restriction enzymes, HhaI or HindIII from Haemophilus haemolyticus and Haemophilus influenzae, respectively. Based on the alignment of partially digested forms with respect to the replication origin, the EcoRI cleavage sites were also mapped with electron micrographic analysis. With E¢oRI sites as reference points, the HhaI and Hind III cleavage sites were located on the map.
INTRODUCTION
Restriction endonucleases are site-specific DNAases that cleave doublestranded DNA at short, defined nucleotide sequences, and are extremely useful in dissecting genomes of a number of eukaryotic DNA~. The genome of rat mitochondria is a double-stranded closed-circular molecule of about 10 million daltons that replicates discontinuously by means of a Cairns-type intermediate (Koike and Wolstenholme, 1974). We have been studying the process of mtDNA replication in vitro (Koike et al., 1976a) and also analysing t h e i n vivo replicating structure with products obt~iined by restriction endonu~leases using electron micrographic and gel electrophoretic techniques. As was noted previously (Koike et al., 1975), the restriction endonuclease EcoRI Abbreviations: DTT, dithiothreitol; EtdBr, ethidium bromide; SDS, sodium dodecyl sulphate.
300
creates six double-stranded breaks at specific sites in the molecule. On the other hand, two and three restriction sites were obtained in mouse and human mtDNA, respectively (Robberson et al., 1974, Brown and Vinograd, 1974). In this report, we describe recent results concerning a physical mapping of the restt~iction fragments in the rat mitochondrial genome and its replicating. form. Rat mtDNA was cleaved into unique fragments by restriction enzymes EcoRI, HhaI and HindHI from E. coU, H. haemolyticus and H. influenzae, respectively. Sizes of the fragments were determined with data obtained by electron microscopy and agarose gel electrophoresis. On the basis of these data an(~ estimates of the sizes of the partially digested products, the physical maps were constructed. Being consistent with the previous observations (Koike et al., 1975), by analysing restriction fragments of in vivo Cairns-type intermediates, it was demonstrated that there is a fixed origin and the replication is unidiractional. According to our results of the physical mapping, we discuss a mode of replication of the rat mtDNA molecule. MATERIALSAND METHODS
Preparation and purification o f rat mtDNA Mitochondria were prepared from adult or newborn rats, Donryu strain, as described previously (Koike et al., 1974, 1976a; Fujisawa et al., 1977). The yield of mitochondria was about 25 mg mitochondrial protein per g of liver. Extraction of mtDNA was performed by the SDS-phenol method (Koike et al., 1974). The mitochondrial fraction was mixed with ow~tenth volume of 10% SDS and an equal volume of phenol saturated with 50 mM sodium phosphate buffer, pH 8.5 containing 0.15 M NaCI, 100 mM EDTA. The mixture was slowly rotated for 20 rain at room temperature and centrifuged to separate an aqueous phase. This extraction was repeated. The aqueous phase was concentrated and dialysed against 50 mM sodium phosphate buffer, pH 6.7 containing 0.1 M NaCI, 2 mM EDTA, at 4 ° C. Closed-circular mtDNA was purified by equilibrium centrifugation in CsCIEtdBr according to the procedure of Radloff et al. (1967) with minor modification. Solid CsCl and EtdBr were added to the DNA solution to a final concentration of 1.58 g/ml and 150 ~g/ml, respectively, and the mixture was centrifuged at 36 000 rev./min for 48 h in a Beckman SW50.1 rotor. After centrifugation, the closed circle-containing band was collected, and EtdBr was removed by a small column of purified Dowex-50 resin. The DNA, thus obtained, Was dialysed against 0.1 M Tris--HCl, pH 7.8, 0.5 mM EDTA. Under the electl'on microscope, about 22% of the closed circles from the newborn rat mitochondria were observed as D-loop molecules (Robberson et al., 1982). This waS about 200 times the frequency of that found in the adult (Koike and Kobayashi, 1973).
301
Cleavage of mtDNA with restriction endonucleases EcoRI endonuclease from E. coli was a gift from K. Matsubara, Laboratory of Molecular Genetics, Osaka University or obtained from Miles Biochemicals. HhaI endonuclease from H. haemolyticus and HapII from H. aphirophilus were gifts from M. Takanami, Chemical Institute, Kyoto University. HindIII endonuclease from H. influenzae was from Miles Biochemicals. One unit of EcoRI or HindIII is defined as an amount of the enzyme required to completely digest 1 ~g of kDNA in 1 h at 37 ° C. One unit of HhaI or HapII is an amount of the enzyme required to completely cleave 1 ~g of fdRFI DNA in l h at 37 ° C (M. Takanami, personal communication). EcoRI endonuclease reactions were usually carried out at 37 ° C in 0.1 M Tris--HCl pH 7.8, 7 mM MgCI2, 50 mM NaCI, 0.02 M potassium phosphate, 0.4 mM EDTA 0.28 mM ~-mercaptoethanol and 0.008% NP-40 for 2 h (Greene et al., 1974). For complete digestion, 2 units of EcoRI were added to 1 ~g of mtDNA. A partial cleavage of mtDNA was achieved by addition of the same amount of EcoRI at 10 ° C for 10 min. Reactions were stopped with 25 mM EDTA. The HhaI or HapII restriction buffer was 0.1 M Tris-HC1, pH 7.8, 7 mM MgCl2, 50 mM NaC1, 0.4 mM EDTA and 5% glycerol. The buffer for HindIII was 0.1 M Tris--HCl, pH 7.8, 7 mM MgCl2, 50 mM NaCl, 0.4 mM EDTA, 0.02 mM DTT, 0.2 mg/ml BSA an~ 5% glycerol. A partial cleavage with HhaI or HindIII was performed at 10 ° C for 10 min under a similar reaction condition, as described above. These reactions were stopped with 25 mM EDTA. . In some cases, digestion of the replicating form with EcoRI endonuclease was carried out using glutaraldehyde-fixed mtDNA from the newborn under following reaction conditions. DNA samples were dialysed against 50% formamide, 0.2 M KH~PO4, pH 7.5 and glutaraldehyde was then added to a final concentration of 0.1%. The mixture was incubated at 23 ° C for 20 rnin, and then dialysed extensively against 0.1 M Tris -HCI, pH 7.8, 0.5 mM EDTA at 4 ° C for subsequent treatment with EcoRI (Robberson et al., 1974). Agarose gel eleetrophoresis Agarose gels were prepared according to the procedure of Sharp et al. (1973) with a minor modifications. Solutions of 1.4% agarose (w/v) were prepared by dissolving agarose (Sea Kern) in electrophoresJs buffer (0.04 M Tris--HCl, pH 7.7, 0.02 M sodium acetate, 2 mM EDTA, and 0.5/~g/ml EtdBr/ ml). Gels were poured at 60 ° C into glass tubes (0.6 cm diameter) or glass slab (10 cm X 2 mm). After hardening, the top of the disk gel was sliced evenly to form a 9 cm gel, and a piece of cotton mesh was stretched over the bottom of the glass tube. Samples of 50--80/~l containing 5% glycerol or 1.2% sucrose and 0.005% bromophenol blue were layered under the electrophoresis buffer. The gels were run in the electrophoresis buffer at 5 mA per tube or at 8 V/cm per slab for 2.5--3.5 h. The gels were laid directly on the UV lamp (short or
302
long wavelength, Manasulu Light). The D N A bands were visualized by the fluorescence from bound EtdBr and photographed with a Polaroid land camera equipped with a Kodak No.23A red filterusing a Polaroid film type 107 or type 55P/N. The negatives were scanned using a Joyce Loebl microdensitometer. For the extraction of D N A fragments from the gels,the band regions were sliced,homogenized and incubated with 0.1 M Tris--HCl buffer. p H 7.8, containing 0.5 m M E D T A for 48 h at 0-4 ° C. Electron microscopy
Specimens were prepared according to the technique of Davis et al. (1971) with minor modifications (Koike and Kobayashi, 1973). The D N A was spread with the formamide spreading condition. Grids were shadowed with Pt-Pd. Electron micrographs were taken with a Hitachi HU-12 electron microscope at 75 kV. Either open-circularrntDNA or ColE1 D N A was used as an internal size standard. The contour length of m t D N A or ColE1 D N A was calibrated with grating replica(2000 lines/ram, OKEN).
9.1 x 10 6
1.3
2E 40
0.45 1.0
2.0
MICROMETERS
,
Rat
Mouse
Fig.l.(a) Length histograms of EcoRI generated fragments from rat mtDNA. Closedcircular mtDNA was digested with restriction endonuclease EcoRI, and restricted products were mounted for observation with electron microscopy by the formamide technique. Length histograms are given in micrometers. There are six peaks (A to F) represented in black rods. Intact circular genome of rat mtDNA forms a peak of 5.26 -+ 0.09 ~,m, but not shown in this histogram. (b) Electrophoretic pattern of EcoRI generated fragments from rat mtDNA. Electrophoretic conditions were 5 mA per tube for 2.5 h.
303 RESULTS
EcoRI endonuclease fragments of rat mtDNA A histogram of the EcoRI endonuclease cleavage products from a complete digestion of rat m t D N A is shown in F i g . l ~ Sizes of the fragments were estimated by electron micrographic measurements and are expressed in micrometer. There are at least six fragments, designated alphabetically (Eco A, B, C, D, E and F, A being the largest). Electrophoresis in 1.4% agarose can separate six fragments, as shown in Fig.lb. Measurements of intensi~y indicated that six bands are equimolar. Addition of more endonuclease and further incubation did n o t alter the profiles. Each fragment was recovered from the gel and examined by electron microscope. Table I summarises the results of length measurements. The sum of these six fragments is very close to the length of circular genome (5.26 :+- 0.09 gm, 10.4-106 daltons) using ColE1 DNA (2.04 -+ 0.07 ~m) as an internal size standard. Sizes of the six fragments were confirmed by a gel electrophoretic analysis using two EcoRI fragments of mouse m t D N A as standards of known molecular weight (1.3 and 9.1-106 daltons).
DNA fragments generated by HhaI, Hmdlll and HaplI digestions Gel electrophoresis separation of endonucleases HhaI, HindIII or HapH digests of rat m t D N A are shown in Fig.2a. Sizes of the DNA fragments were determined by their mobility relative to EcoRI fragments of rat mtDNA, as summarized in Table II. Endonuclease HhaI cleaves m t D N A into four fragments (//ha A, B, C and D) and HindIII into six (Hin A, B, C, D, E and F), but very small fragment, Hin F, was o u t of the gel under the conditions used. The gel of the HapII cleavage products showed eight resolved DNA bands (Hap A to H). On comparing the fluorescent intensity, the short fragment, Hap H had approximately twice the intensity of Hap G. There must be two TABLE I LENGTH ANALYSIS OF EcoRi FRAGMENTS Fragment
Number of molecules classified
Length in micrometers
A
85 84 120 84 99 95
2.01 ± 0.07 1.28 ± 0.05 0.95 _*0.06 0.60 ± 0.04 0.23 ± 0.07 0.17 ± 0.05 5.24± 0.06 5.26 ± 0.09
B C D
E F A+B+C+D+E+F Circular genome
38
304
(a)
(1)
(2)
• Eco R!
~o I0 'o •- 6 x 4
• Hhai
QS O
0.2 I 5
Eco Hha Hap
Eco Hind
I 10
,
I 15
Relative Mobility
Fig. 2.(a) Electrophoretic patterns of generated fragments from rat m t D N A by endonucleases HhaI, HindIII and HapII digestions. Electrophoretic conditions were 5 m A per tube for 3 h (1) and 3. 5 h (2). (b) Relationship between molecular weight and electrophoretic mobility of rat m t D N A fragments. Fragments were produced by cleavage with EcoRI, HhaI, HapII and HindIII. Molecular weights were calculated as described in Table II. Mobility o f fragment is given relative to the HhaI fragments.
TABLE II I~OLECULAR WEIGHT OF THE RESTRICTION ENZYME FRAGMENTS OF RAT mtDNA EcoRI fragment
M.W. •10 -*
//hal fragment
M.W. •10 - '
HindllI
M.W.
HapIl
M.W.
fragment
•10-S
fragment
.10-6
A B C D E F
3.98 2.53 1.88 1.18 0.45 0.33
A B C D
5.65 2.80 1.25 0.65
A B C D E F
4.08 2.70 1.65 1.32 0.50 0.14
A B C D E F G H (doublet)
2.58 2.05 1.23 1.11 1.03 0.77 0.63 0.48
305
fragments of about equal length. Therefore, numbers of the HapII cleavage sites on the rat mtDNA became at least 9. A plot of log molecular weight versus electrophoretic mobility of the restriction fragments is shown in Fig.2b. From the length of EcoRI fragments of rat mtDNA, it was also possible to construct a calibration curve of log fragment length versus mobility, since data from the gel electrophoresis were in good agreement with the electron microscopic measurements.
Position of the replication origin and unidirectional replication Restriction endonuclease cleavage provided a basis ~or electron micrographic examination of the initiation site of mtDNA replication. When the in vivo closed-circular DNA molecules containing replicating forms were extensively digested by EcoRI, a small replication eye was found exclusively on the Eco A (2.01/~m). The small eyes randomly selected were slightly heterogeneous in size and strandedness (Fig.3a, b). Analysis of these eye forms indicated that about 25% of them had two double-stranded branches (Fig.3b), and the rest had one each of single- and double-stranded branches (Fig.3a). Before EcoRI digestion, the closed-circular molecules contained 2 L 5 _+3.2% of D-loop molecules among the population. After cleavage, the frequency of resulting
b
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.
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Fig. 3. Electron micrographs of Eco A with a replicating eye. Replicated region can be seen as an eye. The scale represents 0.5 ~m.
306 Origin I •
•
,, •
•
.,
ms
Ill
m,
Origin !
i
•
i
[[
,.
|[
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I
II
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I
,,
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IJ
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l
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Fig.4. Location of small replicating eye in E¢o A fragment. The linear replicating Eco A are alignmed with short unreplicated portion to the left. The replicated region is represented by a black rect.ngie. Origin indicates that initiation of replication occurs on E¢o A at a position 0. 47/Am away from the proximal restriction site. Fig. 5. Location of expanded eye in Eco A. Linear replicating £¢o A are aligned with short unreplicated portions to the left. The replicated region is represented by a black rectangle. Replication proceeds unidirectionally away from origin throughout the length of Eco A.
linear molecule with the small eye became substantially lower than the initial frequency. This suggests that the replication eye in the linear form is probably lost through the process of rewinding or branch migration during the EcoRI digestion. Alignmen~ of these forms with a short unreplicated portion to the left indicated that initiation of the DNA replication occurs on Eco A at a fixed position of 0.47/~m away from the proximal restriction site (Fig.4). In addition, a location of the expanded eye in Eco A was also examined by electron microscopy (Fig.3c). Length measurements of such replicating forms also provided an array of molecules with increasing degree of replication, and it is indicated that replication proceeds unidirectionally away from the proximal restriction site throughout Eco A (Fig.5). A part of the data was previously described (Koike et al., 1975).
Physical map of the restriction endonuclease fr~/~ments (a) Determination of the order of EcoRl fragments A, B and C. In order to determine the order of EcoEI fragments, DNA molecules containing the small eye were cleaved by EcoRI under limited conditions, as described in
307 MATERIALS AND METHODS. The resulting partially digested products were examined by electron microscopy using the small eye as a marker. An example is shown in Fig.6. The replicated region located at a position of 1.1 ~m away from one end of this molecule. The partial digests were m e ~ u r e d similarly and aligned as shown in Fig.7. Taking the initiation site on the Eco A as the zero point, four out of the six EcoRI c~eavage sites could be mapped at the points, -8.9, 29.3, 53.6 and 71.7. Distance between each contiguous cleavage site corresponds to the percentage of Eeo A, B or C, calculated from the total length of mtDNA as 100%. Three EcoRI fragments could thus be placed in the following order: A-B-C, as shown in Fig.8. This combination was confirmed by digestion of the EeoRI fragments with other restriction endonuclease HhaI or HindIII, as vdll be shown later. As other EeoRI cleavage sites were less accurately located by electron micrographic analysis, mapping of small fragments D, E and F were carried out by gel electrophoretic analysis. (b) Order of the EeoRI fragments D, E and F. To deduce physical order of fragments generated by one endonuclease, the final products derived by the same enzyme from each partially digested segment were arranged in overlapping position. Partial digestion of mtDNA with EeoRI endonuclease revealed several intermediate bands in the gel, as shown in Fig.9. DNA lengths of those intermediates were determined from their mobility, as summarized
Fig.6. Electron mierograph of an intermediate with an eye generated by partial digestion of m t D N A with EcoRI. Partial digestion was carried out as described in the text. The replicated region can be seen as an eye, which is on the intermediate at a position 1.1 ~m away from the proximal restriction site.
308
-~0
,m
-2"0
w
6
.
~0
.
,.,
40
&
Percentage
Fig.7. Alignment of partially digested rat mtDNA molecules having small eyes. Rat mtDNA molecules containing ~he small eyes were cleaved by £coRI under limited conditions as described in the text, and the resulting partially digested forms were examined by electron micrographs using the initiation site as a marker. Taking the initiation site on the intermediate as the zero point, four out of the six cleavage sites could be located at -8.9, 29.3, 53.6 and 71.7% in order (from left to right)
in Table III. Further cleavage of the 0.42/~m intermediate I by EcoRI resulted into two fragments, Eco E and F. The 0.85/Jm intermediate II generated Eco D and E. Thus, the Eco E lies between Eco D and F. A third intermediate III with a size of 1.01 ~m consists of Eco D, E and F. Intermediate IV has a size of 1.14/~m and possibly cont.~ins two fragments C and F. Intermediate V of 1.43/~m suggested a combination between the fragments C, F and E. Taking account of other experiments by digestion of Eco C, D, E and F with restriction enzymes HhaI and HindIII, a combination of Eco A and D w ~ ot~tained, as also suggested by previous data (Fig.6). Details will be sho~n li~er. In this way, a combination of EcoRI fragments D-E-F-C.B-A, with A ~ d D being adjacent in the circular molecule, was oriented as shown in Fig.~o
F~g.8. A physical map of the rat mitochondrial genome constructed by the cleavages with endonucleases EcoRI, HhaI a n d HindIII. Replication proceeds clockwise from the origin.
309 TABLE HI LENGTH ANALYSIS OF INTERMEDIATE SPECIES GENERATED BY PARTIAL DIGESTION OF RAT mtDNA WITH EcoRI Intermediate species
M W. -10-6
Length in micrometers
Possible combination
I II HI IV V
0.83 1.68 2.00 2.25 2.85
0.42 0.85 1.01 1.14 1.43
E-F D-E I)-E-F C-F C-F-E
(1)
. . . . . . . . .
(2) (3)
|
Fig.9. Electrophoretic separation of the intermediate species generated by partial digestion of mtDNA with EcoRI. Partial digestions were carried out at 10 ° C for 5 min (1) and 10 rain (2), as described in the text. Electrophoretic condition was 5 mA per tube for 3 h. In (1) the same gel is shown in different exposure.
(c) Determination of the order of the Hhal fragments. Data for mapping of four HhaI fragments were obtained by gel electrophoretic analysis. After partial digestion of mtDNA with HhaI endonuclease, two intermediate species, smaller than Hha A (2.80/am), revealed as discrete bands, lengths of which were determined from their mobility. Each intermediate recovered from the gel was redigested with the same enzyme and was separated again. One intermediate (1.03/am) contained Hha C (0.66/am) and D (0.35/am), and the other (2.07/am) consisted of Hha B (1.41/am) and C (0.66/am). Hha C therefore,
310 TABLE IV CLEAVAGE OF EcoRI FRAGMENTS BY HhaI Number of HhaI cleavage sites
Length of generated pieces in micrometer
A
1
1.05,
0.97
B
0
C D E F
I (2) 1 0 0
0.56, 0.40
0.33 0.21
EcoRI fragment
lies between Hha B and D. Because of circular structure of mtDNA, Hha A becomes adjacent to Hha B and D. (d) Cleavage of EeoRI fragments by HhaI. To analyse cleavage site(s) of endonuclease HhaI on the EeoRI fragment, each EcoRI fragment was recovered from the gel and extensively digested with HhaI. The digest was again separated by gel e]ectrophoresis together witch a migture of EcoRI fragments as a marker. DNA lengths of resultant short pieces were determined from their mobflities. Location of HhaI cleavage site(s) on an EcoRI fragment is summarized in Table IV. Eco A yielded two close bands, whose DNA lengths were 1.05 #m and 0.97 ~m. These two together add up to a size equal to £eo A (2.01 #m). We concluded that there is one HhaI cleavage site in Eeo A. Digestion of Eco B yielded only one band, which was exactly corresponding to £co B. There is no cleavage site. Eco C revealed two distinct bands with sizes of 0.56 #m and 0.33 #m. Sum of these two was slightly shorter than the size of EcoC (0.95 ~m). A very short piece was therefore expected, but too short to be detected in the gel, under the conditions used, There is a t least one, possibly two, cleavage site. Eco D was separated into two bands with lengths of 0.40 #m and 0.21 #m, the sum of which is equal to the original length (0.60/lm). Cleavage of Eco E or F with HhaI did not make any difference from the original fragment, indicating that there is no cleavage site. This result corrects the previous data (Koike et al., 1976b). A conclusion derived from the present analyses is that each of Eco A, C and D contains one HhaI cleavage site. As there are four HhaI cleavage sites in the rat mtDNA (Table II), one cleavage site is ambiguously located on Eco C. (e) Cleavage of HhaI fragments by EcoRI. Each HhaI fragm~mt was digested by EcoP,I, and the resulting pieces were analysed in the gel, as summarized in Table V./tha A yielded three bands, the sum of these is exactly the Size of Hha A (2.85 ~m). The largest piece corresponded to Eco B, The second band (1.04 #m) was equal to one of the pieces obtained by HhaI cleavage of EcoA. The third band (0.54 ~m) corresponded ~o a piece derived from EeoC by HhaI.
311
TABLE V CLEAVAGE OF HhaI FRAGMENTS BY E c o R I HhaI fragment
Number of EcoI cleavage sites
Length of generated pieces in micrometer
A B C D
2 1 2(3) 0
1.28, 0.97, 0.23,
1.04, 0.42 0.19
0.54
This implies that Hha A localized in a region of Eco A, B and C, as shown in Fig.8. Hha B generated two distinct bands (0.97 and 0.42/~m) by EcoRI which corresponded to HhaI generated pieces from Eco A and Eco D, respectively (Table V). As described above, Eco A and D contain one each of HhaI cleavage site, and Hha B is contiguous to Hha A. Consequently, we located Hha B as shown in Fig.8. Digestion of Hha C (0.63/Jm) by EcoRI resulted in an appearance of two bands corresponding to sizes of 0.23/~m (Eco E) and 0.19 I~m, which migrates slightly slower than Eco F (0.17/~m). As described in the previous section that one HhaI cleavage site was ambiguously located on Eco C, such location would yield Eco F and a small piece, but both are not d e tected under the conditions used. On the basis of these observations, one can say that ~here are at least two, possibly three, EcoRI cleavage sites in Hha C. (f) Determination o f the order of Hindlll fragments. A combination of five HindIII fragments was examined by the partial digestion technique as before. Digestion of mtDNA by HindIII under limited condition yielded five intermediate species, smaller than Hin A (2.06 #m), in the gel. Based on their DNA lengths and further cleavage of intermediate species by HindIII, we found out their composition. As shown in Table VI, the first intermediate (2.01 ~m) contains Hin B (1.36/~m) and D (0.67 ~m). The second intermediate (1.81 /~m) consists ofHin C (0.83/~m), D (0.67/~m) E (0.25/~m) and F. The third TABLE VI LENGTH ANALYSIS OF INTERMEDIATE SPECIES GENERATED BY PARTIAL DIGESTION WITH HindIII Composition of
Intermediate species
Length in micrometer
HindIII fragments
I H HI IV V
2.01 1.81 1.54 1.14 1.07
B, C, C, C, C,
I) D, E, F D, F E, F E
312 intermediate (1.54 pm) contains HinC, D and F. The fourth intermediate (1.14/zm) contains Hin C, E and F. The fifth intermediate (1.07 pro) is of Hin C and E. The fragment F, therefore, has a size about 0.07 pm and lies between Hin C and D. Tnus, a possible combination of five fragments is the following: E-C-F-D-B. (g) Cleavage o f EcoRl fragments by HindlII. Each Eco RI fragment was digested by HindIII and resulting pieces were analysed by electrophoresis. As summarized in Table VII, Eco B, C and D were digested into smaller pieces. However, other fragments A, E and F were not digested. Digestion of Eco B (1.28 pm) by HindIII produced four bands corresponding to DNA lengths of 1.18 um, 0.97 pm, 0.29 p m and 0.15/~m, under the condition used. These were not final producm. The first band migrated slower than Hin F. The second band migrated to a position almost identical with that of Hin E (0.25 pm). The third band (0.97 pro) was slightly larger than that of Hin C plus F, indicating that Hin C and F are contained in Eco B. This is consistent with the observation that avev.! short fragment corresponding to Hin F lies between Hin C and D. It is indicated that at least three, possibly four, HindIII cleavage sites are located on Eco B. Eco C (0.95 pro) generated two dzstinct bands (0.52 pm and 0.45 pm) which sum up to the size of Eco C. There is one HindIII cleavage site in Eco C. According to above analyses of HindIII fragments (Table VI), Hin B is put bet;,veen Hin D and A. Eco D yielded a single band (0.56 pm) which migrat~ed only s'~ightly faster than Eco D (0.60 pm), indicating that one HindIII cleavage site locates closely "~othe junction of Eco A and D. This result was confirmed Oy a re-digestion experiment of Hin B by EcoEI. (h) Cleavage of HindlIl fragments by HhaI. Each HindIII fragment was redigested by HhaI endonuclease. Hin A (2.06/~m) produced two close bands corresponding to DNA lengths of 1.07 ~m and 0.99 pro. Sum of two pieces is exactly the size of Hin A. This is cotzsistent with the above observation that a
TABLE VII CLEAVAGE OF EcoRI FRAGMENTS BY
HindIII
!~oRI ~ragment
Number of HindIII cleavagesites
Length of generated pieces in micrometer
A B C D E F
0 3(4) 1 1 0 0
1.18, 0.52, 0.56
0.97, 0.45
0.29,
0.15
digestion of Eco A by HindIII yielded no small piece. Hin B (1.36/~m) was cleaved into a piece of DNA (0.30/~m), Hha C (0.63/~m) and D (0.33/~m), T~ese three together add up to a size of 1.26/~m, which is slightly shorte~ than Hin B. A tiny piece in the digest was out of the gel in this case. This
313
TABLE VIII CLEAVAGE OF HindIII FRAGMENTS BY HhaI HindIH fragment
Number of HhaI cleavage sites
Length of generated pieces in micrometer
A S
1 2(3)
1.07, 0.63,
c
0
D E F
0 0 0
0.99 0.33,
0.30
implies that three HhaI cleavage sites are located on Hin B. On the other hand, no HhaI cleavage site was found in Hin C, D, E and F. Therefore, four HindIII fragments were arranged inside Hha A in a following order: E-C-F-D. A HindIII cleavage map thus constructed is also shown in Fig.8. DISCUSSION
Cleavage maps of the rat mtDNA genome were constructed based on specific sites of the DNA molecule susceptible to bacterial restriction endonucleases. Methods we have used to order DNA fragments are analogous to those used for ordering peptides in a protein molecule. We were able to identify overlapping DNA fragments by the distinctive electrophoretic mobility to their final digestion products. DN.A fragments were also ordered by electron microscopic examinations of marker(s) of structural significance. The DNA molecules containing the small replication eye were cleaved by EeoRI under limited conditions, and the resulting intermediate species were examined by electron microscopy using the initiation site as a marker. However, ability to measure the duplex DNA sets a lower limit to the length of a fragment which can be measured in this way. Six fragments, obtained when the rat mtDNA was cleaved with EcoRI endonuclease, provided a convenient starting point to construct a physical map of this genome. This was achieved by a combination of methods that involved analysis and cleavage of intermediates obtained by a partial digestion of mtDNA with EcoRI, and the action of other enzyme, HhaI or HindIII, on both the fragments obtained when mtDNA was treated with EcoRI and the fraffments produced by digestion with HindlII or H ~ I . These methods helped to unambiguously locate restriction endonuclease fragments on the map as shown in Fig.8. DNA fragments can also be ordered by an electron microscopic examination of heteroduplexes formed between Eco A, B, C and Hha A. Hybridization of the denatured Eco A, B or C and Hhu A yielded a partially singlestranded molecule, thus confirming the location of EcoRI cleavage sites within //ha A (data not shown).
314
Earlier suggestions that the rat mtDNA replication is unidirectional were based on the electron microscopic observation of a single-stranded region at one fork of the replicating molecule (Wolstenholme et al., 1973, Koike and Wolstenholme, 1974), Koike etal. (1975), then, determined that the origin of DNA replication in rat mtDNA is located at 0.47/~m from the E c o R I cleavage ~ite on Eco A. This result, however, did not distinguish the origin being located on the right side or on the left side of the fragment (see Fig.3a, b). The results presented here definitely place the origin at 0.47 ~m from the Eco A-D junction on Eco A. One conclusion drawn in this paper is that rat mtDNA replication begins at a specific site and proceeds in one direction around the circular genome. Since the initiation site has been localized and the DNA fragment containing this site is available, it will be interesting to determine what special nucleotide sequences might represent a signal for the initiation. A region containing the origin of DNA replication might be conserved in various species for functional reasons (Robberson et al., 1974; Koike et al., 1975). When the mtDNA replication is compared with that of the SV40 viral DNA, the former replicates unidirectionally whereas the latter does bidirectionally (Danna and Nathans, 1972). What general significance uni- or bidirectional replication may have is not known. As the rat mtDNA replication is unidirectional, the termination region of DNA replication would be close to the initiation site in Eco A. As described previously (Koike et al., 1974, 1976b), a short pulse of [3H] thymidine to rat mitochondria, which are actively synthesizing DNA, resulted in an incorporation of label into the growing point of replicating DNA. During a pulse time, most of the label is incorporated into the nascent DNA associates with the closed-circular DNA, but open-circular molecule containing appropriate small gap is also labeled in such gap region before 4~-]osure [i.e. gap-filling synthesis after segregation of two daughter molecules before closure (Robberson et al., 1972; Berk and Clayton, 1976)]. Then there will be a concentration of labeling in such region of the DNA. For this purpose, the [3H] open-circular DNA among the pulse~labeled daughter molecules was selected and completely digested with EcoRI. Each fragment was analYsed for its radioactivity after !separation of the products by agarose gel electrophoresis. As previously shown (Koike et aL, 1976), there was a concentration of labeling. Further digestion of the [ 3H] labeled Eco A by other restriction enzymes is underway. Preliminary data indicate that the specific activity was highest in a small region of the Eco A, which is roughly located at a left side o~"the initiation origin (Fig.8). D~_ta are consistent with an idea that replication of rat mtDNA proceeds unidirectionally away from the origin, and terminates at a region adjacent to the initiation site on both parental strands. Such comparison of the order of synthesis wish the physical order of the restriction fragments may allow one to distinguish rat from mouse mode of replication. Rat mtDNA is similar to mouse mtDNA in having the unidirectional replication, but a degree of asymmetry is different from that-of mouse mtDNA, which is extremely asymmetric (Berk aild Clayton, 1974). Details will be published elsewhere.
315
A creation of the physical map of mtDNA will he!p an assignment of genetic markers c f biological significance, as has been achieved with HeLa ceil, Xe~opus or Drosophila mtDNA genome (Ojala and Attardi, 1974; David, 1976). From the preliminary results, the genes coding for two rat mitoribosomal RNA species can be located on the physical map, and are found to lie adjacent to each other (unpublished data). In the experiments described here, the mtDNA used was all derived from normal rat. The cleavage pattern of the restriction enzyr~es on mtDNA derived from rat tumor cells is significantly different ( m ~ u s c r i p t in preparation). The present maps are also currently in use for analyses of in vitro ~phage-mtDNA recombinant grown in E. coll. ACKNOWLEDGEMENT
We thank Dr. H. Sugano, Director of the Cancer Institute, for his interest and encouragement. This work was partly supported by a Grant-in-Aid for Scientific Research and a Grant-in-Aid for Cancer Research from the Ministry
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Koike, K , Kobayashi, M: Tanaka, S, and Mizusawa, H., Physical map and replication of rat mitoehond~1~l DNA, in Biicher, Th., Neupert, W., Sebald, W. and Warner, S. (Eds.), Genetics and Biogenesis of Chloroplastsand Mitoehondria, ElsevierINorth-Holland, Amsterdam, 1976b, pp.593--596. O]ala, D. and Attardi, G., Identification and partial characterization of multiple discrete polyadenylic acid-containing RNA components coded for by HeLa cell mitoehondrial DNA, J. Mol. Biol., 88 (1974) 205--219. Radloff, R., Bauer, W. and Vinograd, J., A dye-buoyant-density method for the detection and isolation of closed circular duplex DN:'~: The closed circular DNA in HeLa cells, Proc. Natl. head. SeL USA, 57 (1967) 1514--1521. Robberson, D.L., Kasan~tsu, H. and Vinogra~,'J., Replication of mitoehondrial DNA. Circular replicative intermediates in mouse L cells, Proe. Natl. Acad. Sei. USA, 69 (1972) 737--741. Robberson, D.L., Clayton, D..~ and Morrow, J.F., Cleavage of replicating forms of mitochondrial DNA by ~ o R I endonuclease, Froe. Natl. Acad. Sei. USA, 71 (1974) 4447-4451. Sharp, P.A., Sugdcn, ]3. and Sambrook, J., Detection of two restriction endonuclease activities in l-Iaemophilusparainfluenzae using analytical agarose-ethidium bromide electrophoresis, Biochemistry, 12 (1973) 3055--3063. Woistenholme, D.R., Koike, and Cochran-Fouts, P., Single strand-containing replicating molecules of circular mitochondrial DNA, J. Cell Biol. 56, (1973) 230--245. i
Communicated by If. Matsubara.