Current Genetics 1, 13-19 (1979)
Current Genetics © by Springer-Verlag 1979
A Detailed Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA* Horst Feldmann and Riidiger Grogkopf Institut ffir Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universitiit, Goethestrasse 33, D-8000 Mtinchen 2, Federal Republic of Germany
Summary. A restriction endonuclease cleavage map of rat liver mitochondrial DNA is presented for the following enzymes: Xba I, Bgl II, Hae II, Barn HI, Hpa I, Hha I, Bcl I, Hind II, Hind III, Eeo RI, Hpa II, Hae III, and Sau 314. It was derived from complete and partial digestions with these enzymes, double digestions, and redigestions of defined fragments obtained with one enzyme with other restriction enzymes. By the use of these and further enzymes (Taq YI, Alu I) the mitochondrial DNA (ca. 15.6 Kb) can be dissected into a large number of defined fragments.
Key words: Mitochondrial DNA - Restriction mapping.
Introduction Mitochondria represent the smallest replication units which contain genetic information for rRNAs, tRNAs and mRNAs. The evolution and maintenance of this extrachromosomal system seems due to the fact that the mitochondrial gene products possess unique features. During biogenesis of mitochondria, the limited number of mitochondrial products has to be complemented by a large number of components encoded in nuclear DNA. This affords a strict interplay between the two genetic systems (summary, Bandlow et al., 1977). For these reasons, studies on the organization of mitochondrial genomes on a molecular level are of particular interest.
Offprint requests to: H. Feldmann * Abbreviations: mtDNA, mitochondrial DNA; bp or Kbp, base pairs or kilo base pairs, respectively. Other abbreviations follow IUB-IUPAC conventions.
Animal mtDNAs are the smallest mitochondrial genomes (ca. 10 - 106 daltons in molecular weigth). Studies in several systems (e.g. Attardi et al., 1976; Dawid et al., 1976; Klukas and Dawid, 1976; Saccone et al., 1977; Battey and Clayton, 1978) have shown that they code for two ribosomal RNAs, an apparently full complement of tRNA, and only a small number of mRNAs. Since genetic experiments in animal mitochondria are limited, the main approachto tackle the problem of their genomic organization consists in constructing genetic maps by the use of electronmicroscopic techniques and/or restriction endonucleases together with hybridization techniques. Physical mapping is a prerequisite if one wants to locate specific genes by hybridization of specific RNAs with restriction endonuclease cleavage fragments. Rat liver mitochondrial DNA has been investigated by several authors and data have been obtained With respect to: (i) restriction endonuclease cleavage maps (Saccone et al., 1976, 1977; Kroon et al., 1977a and b;Parker and Watson, 1977; Koike et al., 1976; Buzzo et al., 1978), (ii) location of the D-loop (Koike et al., 1976 ; Buzzo et al., 1978), and (iii) location of the rRNA genes and the tRNA genes (Saccone et al., 1976, 1977; Kroon et al., 1977a and b). However, several discrepancies exist among the reported restriction maps, which in part may be due to the fact that two different molecular types of rat liver mtDNA are existent. This interesting observation was first reported by Francisco and Simpson (1977) and has been confirmed by several authors (Buzzo et al., 1978; Hayashi et al., 1978; Kroon et al., 1978). The divergence in sequences was detected through the differences in the number of specific restriction sites. It seems that there is only one type of DNA in one animal and that this is maternally inherited (Buzzo et al., 1978; Kroon et al., 1978). O172-8083/79/0001/0013/$01.40
14
H. Feldmann and R. GroiSkopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA For a detailed study of the mitochondrial genome
f r o m rat liver it s e e m e d desirable t o o b t a i n a r e s t r i c t i o n m a p for a large n u m b e r o f e n z y m e s w h i c h t h e n w o u l d allow t o dissect t h e m t D N A i n t o d e f i n e d f r a g m e n t s o f small sizes. In this p a p e r we wish t o r e p o r t t h e result o f this analysis.
hours. The fluid from the fluorescent band containing the circular DNA (as visualized under a UV lamp) was collected by punching the centrifuge tubes with a syringe needle. Ethidium bromide was removed by at least 5 extractions with propanol-2/butanol-1 (1 : 1, v/v). The aqueous phase was dialyzed against TE buffer (10 mM Tris-HC1, pH 7.4, 1 mM EDTA) and the DNA precipitated with ethanol and centrifuged. The pellet was dissolved in TE buffer to a concentration of ca. 250 #g/ml.
Digestions o f mtDNA with Restriction Endonucleases. For anaMaterials a n d M e t h o d s Female rats, Sprague-Dawley strain, were purchased from WIGA, Vet suclistieranstalt, Sulzfeld. Seakam agarose and Seaplaque agarose were products of Marine Colloids Inc., Rockland Maine. Acrylamide and N,N'-bismethylen-acrylamide were from Serva, Heidelberg. Sucrose (RNase-free), CsC1, and proteinase K were obtained from Merck, Darmstadt. All chemicals were of analytical grade. [32p] phosphate, carrier-free, without HC1, was bought from New England Nucl. Corp. Polynucleotide kinase was obtained from Boehringer Mannheim GmbH, alkaline phosphatase from Worthington, Biochemical Corp. Restriction endonucleases from several sources were used: Eco RI, Bam H1, Hind 111, and Hpa 11 were from Boehringer Mannheim GmbH. Hind Ili, Bcl 1, and Taq Y1 were from Microbiological Research Establ., Porton, Eco R1 and Hind H were gifts of Dr. Igo-Kemenes; Bsu I and Bsp were gifts of Dr. H6rz; Hha 1, A lu 1, Bgl 11, Sau 3/11, Pst 1, Sal 1, Xba 1, and Mbo H were gifts of Dr. Streeck; Bal 1, Hae 11, Hae Ili, Barn HI, Xma I were gifts of Dr. Hanggi (all in this laboratory) ; Hpa I and Hpa H were prepared according to the method reported by Bickte et al. (1977) including a rechromatography on DEAE-ceUulose at pH 8.4. Xdvl, Xdv21, and PM2 DNA were gifts of Dr. Streeck, T5 DNA was from Dr. Hiinggi. The Bsp fragments of kdvl (Streeck and Zachau, 1978) served as standards. Dr. Zolg supplied cells of Haemophilus parainfluenzae.
Preparation of Rat Liver Mitochondria. If not mentioned otherwise, the livers from 20 rats ( 1 8 0 - 2 0 0 g) were batchwise disrupted in a Potter homogenizer in ca. 600 ml of the following buffer: 0.25 M sucrose, 10 mM Tris-HC1, 50 mM EDTA, pH 7.3 (Kleinow and Neupert, 1970). The homogenate was centrifuged at 3,000 rpm in a Sorvall SS 34 rotor for 5 min to remove cell debris and nuclei. The supernatant was then centrifuged at 12,000 rpm for 15 min. The crude mitochondrial pellet was resuspended in the above buffer, and the procedure of low and high speed centrifugations was repeated three times. All steps were performed at 4 ° C. The final mitochondrial pellet was used immediately for the preparation of DNA.
Preparation ofMitochondriaIDNA. Mitochondria were suspended om 150 ml of the following buffer: 25 mM Tris-HCl, 50 mM EDTA, 75 mM NaCI, pH 7.4. Lysis was achieved similar to GrossBellard at al. (1973) by adding 2% of sodium dodecylsulphate and 20 ~tg/ml proteinase K, and incubating the mixture for 2 hours at 37 °C. The clear lysate was extracted twice with an equivolume of chloroform/i-amylalcohol (24:1, v/v) and the DNA precipitated with 3 volumes of cold ethanol (plus 3% potassium acetate). The DNA pellet was dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and dialyzed against the same buffer overnight. 15.6 ml aliquots of this solution were mixed with 15 g CsCI, then 9.6 mg of ethidium bromide (0.1% solution) was added. Centrifugation was carried out in a Beckman ultracentrifuge at 15 °C (33,000 rpm, rotor Ti 60) for 65
lytical purposes, 2 - 3 hour incubations were routinely done in 25 to 100 #1 of theappropriate buffer, using 1 - 5 tag DNA or DNA fragments and 1 - 5 units of enzyme. For partial digestions, the samples were incubated for only short periods of time ( 1 10 min.). After incubation, the samples were extracted with phenol, the fragmented DNA was precipitated with cold ethanol (made 3% in potassium acetate and 10 mM in Mg-acetate), pelleted, and redissolved in 10 to 20 #1 TE buffer. For the preparation of defined restriction fragments 1 0 20 #g of DNA were digested in 2 0 0 - 5 0 0 #1 incubation mixture and treated similarly as described above. Double digests with two restriction endonucleases were performed in two ways: (i) the DNA was preincubated for 1 hour with the first enzyme, then the buffer conditions were adapted for the second enzyme and incubation continued for an additional 2 hours (ii) the two enzymes were added together, if the buffer conditions for digestion were similar.
Gel Electrophoreses. Electrophoreses on 0.4 to 2.2% agarose slab gels (15 x 24 x 0.3 cm, 15 slots each 1 cm wide) and tube gels (7 x 180 mm or 13 x 18 mm) were performed in different buffer systems: (A) 0.08 M Tfis-borate, 3 mM EDTA, pH 8.3 (Peacock and Dingman, 1967); (B) 0.0036 M Tris-phosphate, 1 mM EDTA, pH 7.7 (de Wachter and Fiefs, 1971); (C) 0.02 M Tris-acetate, 0.04 M Na-acetate, 1 mM EDTA, pH 7.4 (Loening, 1969). Gels were either run without the addition of ethidium bromide and stained after electrophoresis, or 0.1 #g/ml ethidium bromide was added to all buffers. Gels in buffer systems (A) and (B) were operated at 4 to 5 volts/cm, gels in buffer system (C) at 2 volts/cm. On a preparative scale (e.g. for redigestions), DNA fragments were obtained by electrophoreses of the digests in buffer system (C) on Seaplaque agarose and extraction of the fragments from gel slices. Up to 100 #g DNA could be loaded onto the larger tube gels. Minced gel slices containing one fragment were extracted twice with an appropriate volume of 0.5 M NaCI solution. The extract was filtered through Whatman 3 MM paper and the DNA was precipitated with 2.5 volumes of ethanol. Gel electrophoreses on polyacrylamide gels were performed in buffer system (C). Appropriate gel concentrations were 3 or 4% acrylamide mixed with N,N'-bismethylenacrylamide in a ratio of 20 : 1 or 30 : 1. Quantitative Evaluations of Gels. Gels after staining with ethidium bromide were photographed by the use of an ultraviolet light source, a Durst laborator camera and AGFA film 230p (Pfeiffer et al., 1975). The films were evaluated photometrically in a Gilford spectrophotometer equipped with an automatic scanning device. Standards for calibration of fragment lengths were always run in parallel and sometimes together with fragmented mtDNA. Analysis for Fragments 5'-labeled with 32p. Restriction fragments extracted from Seaplaque gels (see above) were treated with bacterial alkaline phosphatase and rephosphorylated by reaction with [7"32p]ATP (Glynn and Chappell, 1964) and polynucleotide kinase (Lillehaug and Kleppe, 1975). The sub-
H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA fragments produced by cleavage with further restriction endonucleases were separated by electrophoresis on polyacrylamide gels. Gels were stained with ethidium bromide and radioautographed by the use of Kodak X-Omat R film.
15
This information allowed to properly arrange the subfragments and was also helpful to establish the relative location of restriction sites which were produced by different enzymes, but are located very close to each other.
Results and Discussion In this study we have used female rats from an inbread strain (Sprague-Dawley type), which had largely type A mtDNA (Francisco and Simpson, 1977) according to our analyses with restriction endonucleases Eco RI, Hha I, and Hind II. The strategy for the restriction mapping of this mtDNA was the following: (i) Digestions of mtDNA with single restriction enzymes and determination of fragment sizes by electrophoreses on agarose slab gels of varied concentrations (0.4 to 2.2%) together with appropriate standards. The following standards (see Methods) were used: (a) Bsp (or Hae III) fragments of Xdvl which cover a range from 1686 to 34 bp; the fragment sizes have been accurately determined (Streeck and Zachau, 1978), (b) Hind III fragments of T5 DNA (van Gabain et al., 1976) (17.3 to 0.91 Kb) which were used for a size estimation of the larger fragments. (ii) Double digestions with various enzymes and determination of the fragment sizes. For most of the enzymes used (except Bgl II, Taq YI, Alu I, and Sau 3AI) combinations with two to five of the other enzymes were evaluated. (iii) Redigestions of defined fragments obtained from digestions with one enzyme by several other enzymes and analysis of the products. In some cases also a second redigestion was performed on the subfragments. Defined fragments were obtained on a preparative scale by the use of Seaplaque agarose gels, from which slices were cut out and the restriction fragments eluted (see Methods). Redigestions could be carried out on this material directly, since it was free from contaminants which otherwise have been observed to inhibit further enzymatic steps on DNA extracted from agarose gels. Redigestions were mainly done with those enzymes that produce relatively short restriction fragments. In some cases, we have used conditions (see Methods) for the digestion or in the redigestion of defined fragments, which lead to partial cleavages thus facilitating the arrangement of the subfragments. Futhermore, we took advantage of the techniques to 5'-label some restriction fragments with 32p prior to redigestion(s). Such fragments were then digested with other restriction enzymes either completely or partially, following the method devised by Smith and Birnstiel (1976). The subfragments were separated by electrophoresis on polyacrylamide gels, the gels were stained and radioautographed.
Table 1. Fragments of mtDNA derived from restriction endonuclease cleavages The fragments are designated by capitals and listed in decreasing length, their sizes (in bp) as determined through gel electrophoreses are given in parentheses. Some additional very short fragments for Hae III or Sau 3AI have been observed but not yet positioned. BgIH
A 23b-17a(14,000), B 17b-23a(1,700)
Hae H
A 15-47(9,600), B 48-14(6,100)
XbaI
A 24-4(9,800), B 5-23(6,000)
BamHI
A 27-13(10,800), B 14-26(4,950)
HpaI
A 46-35(13,400), B 36-45(2,450)
HhaI
A 15-44(8,400), B 58-14(4,250), C 48-57(1,950), D 45-47(1,050)
BclI
A 35-53(4,200), B 19-34(4,000), C 9-18(3,600), D59-8(2,850),E54 58(730)
HindlI
A 12-32(7,100), B 46-7(4,950), C 36-45(2,450), D 8-11(820), E 33-35(280)
HindlII
A 2-20(6,250), B 44-1(4,100), C 25-33 (2,450), D 34-43(1,950), E 23-24(780), 21-22 a
Hind III
(partial digest, in addition to fragments A to E)
23-43(6,200), 34-1(5,900), 25-43(4,400), 23-33(3,200) Eco R I
A 3-21(5,900), B 22-38(3,700), C 39-46(2,700), D 55-2(1,850), E 52-54(680), F 49-50(460), G 47-48(130), H 51 (110)
Eco R I
(partial digest, in addition to fragments A to H) 47-38(12,800), 3-46(12,350), 39-21(11,900), 3-38(9,550), 22-51(6,900), 22-46(6,400), 39-54(4,050), 39-51(3,400), 47-2(3,250),. 52-2(2,550), 47-54(1,380)
HpaH
A 17-29(3,700), B 43-56(3,100), C 11-15(1,820), D 38-42(1,700), E 1-5(1,650), F 31-37(1,080), G 16 (960), H 57-60(720), H' 6-10(720), J 30(80)
HaeIII
A 13-17(2,550), B 42-49(2,100), C 20-25(1,850), (D), E 4-6(980), F 60-3(960), G 37-39(820), H 32-36(800), J 56-59(780), (K), L 18-19(680), M 7-9(600), M' 10-12(600), N 53-55(500), O 26-27(530), P 50-52(420), Q 29-31(410), R 40(360), S 28(240), T 41(200)
Sau 3AI
A lb-8(2,100), B 42b-49a(1,780), C 14-17a(1,690),
D 27 34(1,500), E 9-13a(1,160), F 25b-26(960), F' 35-40a(960), F" 49b--53(960), G 19-20a(720), H 59-1a(640), J 54-57a(620), K 17b-18(500), L 20b-23a(475), M 40b-41a(420), N 4 lb-42a(360), O 23b-24a(295), O' 13b(280), P 24b-25a(160), P' 57b-58(150) a
cf. text
16
H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA
Table 2. Restriction patterns from combined cleavages of mtDNA with two restriction endonucleases The nomenclature is a in Fig. 1. The observed fragmentation pattern is documented in a simplified manner: the fragments run up to and include the indicated subfragments. Detailed data can be requested from the authors.
XbaI,
nhaI: 4/14/23/44/47/57/, Hpa11: 4/5/10/15/16/23/29/30/37/42/56/60/
BclL
nhaI: 8/14/18/34/44/47/53/57/58/,Hind11I: 1/8/18/21/24/33/34/43/53/58/,EcoRI: 2/8/18/21/34/38/46/47/50/51/53/ 54/58/, Hpa11: 5/8/10/15/16/18/29/34/37/42/53/56/58/60/
Bam H1, Hae11: 13/14/26/47/, HhaI: 13/14/26/44/47/57/, Hind11: 7/11/13/26/32/35/45/, Hind 11I: 1/13/20/22/24/26/33/43/, Eco Rh 2/13/21/26/38/46/48/50/51/54/, Hpa I1: 5/10/13/15/16/26/29/30/37/42/56/ 60/, Hae11I: allHaeI11fragments except A and O, instead fragments 13, 14-17, 26 Hae11,
HhaL" 14/44/47/57/,Hpa1: 14~35~45~47~,Hind111: 1/14/20/22/24/33/43/47/,EcoRl: 2/14/21/38/46/48/50/51/54/
Hpa L
Hha L" 14/35/44/45/47/57/, Hind IlI: 1/20/22/24/33/35/43/45/, Hae 11L"all Hae 111 fragments except B and H, instead fragments 32-35, 42-45, 4 6 - 4 9
Hhal,
Hindll: 7/11/14/32/35/44/45/47/57/,HindllI: 1/14/20/22/24/33/43/44/47/57/,EcoR1: 2/14/21/38/44/46/47/48/50/51/ 54/57/, Hpa 1I." 5/10/14/15/16/29/30/37/42/44/47/56/57/60/, Hae 111: all Hae 1II fragments except A, B and J, instead fragments 13-14, 17, 42-44, 47, 49, 56-57, 59
Hind III, Eco RI: 1/2/21/22/24/33/38/43/46/48/50/51/54/, Hpa 11: 1/5/10/15/16/20/24/29/30/33/37/42/43/56/60/, Hae I11: 1/3/6/9/12/17/19/22/24/25/27/28/31/33/37/39/40/41/43/49/52/55/59/ EcoRI,
Hpa11: 2/5/lO/15/16/21/29/30/37/38/42/46/48/50/51/54/56/60/,Hae11I: 2/3/6/9/12/17/19/21/25/27/28/31/36/38/39/ 40/41/46/48/50/51/54/55/59/
B
t
B
A
I
A
I
B
A
D
I D /
I t
H
F
I
C
g
I
ii E I I A
A H'
E
I M I M'I
C
G
I A
I
I
A
I
Hpa I (GTT'AAC) Hoe II (PuGCGC'Py)
iO1'
I IEI I
C B
F IF iO ISIQI H
C
D
i A C D
C
I
B B FII E t
p C
I D I I t G tRITI B
I I E I
Hha l (GCG'C) Bcl I (T'GATCA) Hind II(GTPy'PuAC] t Hind I[I(A'AGCTT) !EcoRI[G'AATTC)
D
B
! I.PI N,,
H
i
J I
Hpa II (C'CGG] Hae t]I (GG'CC)
1~ 11711allg120,11123,h L 25 I~lleaI[lat32I~l1381 19 l~oI.F.~Id~Sl !6 ~1!~1[I521531!sq159N 6 8 10 12 14
I
i E
t I L I
i0,11~14 I s liE 82 11111~2E n3. II ~54 I I
I A
A "1 E
I
B
A
B
I I
Xba (T'CTAGA) Barn H I ( G'GATCC )
A
I
I
C
I
I B I I K I G I L iO1~ ~ F
I
i
D
A I F'
i
IMtNI
I
B I I i
IKbp
t F" I J it/'H ~ I I
i
Bgl ]i [A'GATCT) Sau 3A [(GATC] "'Taq YI (TCGA)
Fig. 1. Restriction endonuclease cleavage map of rat liver mitochondrial DNA (linearized presentation). The series of fragments which were obtained by digestion of mtDNA with a particular restriction endonuclease are designated by capitals. The subfragments produced by the enzymes listed in the upper part of the map are designated by arabic numerals taking the restriction site between the Hpa I1 fragments H and E as a reference point. In the lower part of the map those fragments and sites are listed which were derived from redigestions of isolated fragments with further enzymes (see also Table 3). This additional fragmentation is referred to in Table 3 by the small letters a and b (left and rigth portion of a subfragment, respectively)
F o r simplicity, the results o f t h e above m e n t i o n e d analyses are summarized in tabular f o r m (Tables 1, 2, and 3). F r o m these data we have deduced t h e restriction m a p shown in Fig. 1. Altogether, we were able to locate t h e sites for the following enzymes: Xba 1, Barn 111, Hae
11, Bgl II, Hpa L Bcl L Hind 11, Hha L Hind Ill, Eco R L Hpa 1I, Hae Ill, and Sau 3.41. Sau 3A1 is o f particular
interest as its recognition sequence is contained within the recognition sequences o f Barn HI, Bcl 1, and Bgl II. In addition we have p e r f o r m e d digestions o n m t D N A w i t h o t h e r restriction enzymes such as Taq YI, Alu I, and Mbo 11. However, the f r a g m e n t a t i o n patterns were found to be rather c o m p l e x , as a large n u m b e r o f fragm e n t s is produced. On t h e o t h e r hand, we have used Taq
H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA
17
Table 3. Redigestions of isolated restriction endonuclease cleavage fragments from mtDNA with further restriction enzymes Presentation of the data is as in Table 2. The isolated fragments on which digestions were performed with a second enzyme are listed by indicating the first and the last subfragment in brackets. In some cases (for Taq YI orAlu 1) the order of the subfragments h.as not yet been established, only fragment sizes are listed. An asterisk refers to such fragments which have also been Y-labeled with [ 32pj posphate for analysis. Hha I
A (15-44) Xba h 23/, Bell: 18/34/, Hae IIL" 17/19/25/27/28/31/36/39/40/41/, Hpa I: 36/, Bgl Ih 17a/23a/, Sau 3A: 17a/ 18/20a/23a/24a/26/34/40a/41a/4 2a/ (22-38).Hpa IL" 30/37/, Hae III: 25/27/28/31/36/ EeoRI: (15-21)HpaIL" 16/21/,HaeIII: 17/19/ (39-44) Hpa H: 42/, Hae III: 39/40/41
HhaI
B (58-14)BglII:noeleavage, XbaI: 4/,BelI: 8/,HaeIII: 3/6/9/12/14/59/,Sau3A: la/8/13a/13b/ EcoRL f (3-14)HpaII: 5/10/ k (58-2)Hpalh 60/ (1-5) Hae llI: 3/ (11-14) Hae HL" 12/ HpaII: (6-10)HaeIIh 7/9/, Taq YI: 430/170/110/ (58-60) Hae IlL" 59/
Hha I
C (48-57) Xba I, Bgl IL" no cleavage, Bcl I: 53/, Hae III: 49/52/55/, Taq YI: 52a/56a/,
Sau 3A: 49a/53/57a/, Eeo RI (partial): 48,48-50,48-54,49-50,51,51-54,51-57,52-54,55-57 f (52-54) Hae IlL" 52/ Eeo RI: (55-57) BraeIII: 55/ (49-50) Hae III: 49/ HhaI
D (45-47) XbaLBelLHaeIII, Sau3AL'nocleavage, Hpah 45/,EcoRI." 46/,Taq YI: 45a/46a/
Hpa II A (17 -29)* BglII: 17a/23a/, Xba I: 23/, BelL" 18/, Hae III: 17/19/25/28/, Hae III (partial, in addition): 18-25, 26-.29, HindIIL" 20/24/,Aluh 890, 750, 570, 510, 320, 280, 230, 130, HpaH A (17-29) Eeo RI: ~ (17-21) Sau 3A: 18/20a/ l (22-29) Sau 3A: 23b/24b/25b/27/ f
Hpa II B (43-56)* Xba l, Bgl IL" no cleavage, Bel h 53/, Hpa I: 45/, Eeo RI." 46/48/50/51/54/, Hae HI." 49/52/55/, Taq YI: 44a/45a/46a/52a/56a/, Sau 3AI: 49a/53/ Hpa II C (11-15)* Xba L Bell, Bgl II." no cleavage, Hha I: 14/, Hae III: 12/, Sau 3AI: 13a/13b/ Hpa [I D (38-42)* Xba L Bell, Bgl IL" no cleavage, Eeo RI: 38/, Hae IH: 39/40/41/, Taq YL" 860, 640, 210 Sau 3Ah 40/41a/42a/ Hpa I I E
(1-5)* Xba I: 4/, B e l l Bgl IL" no cleavage, Eeo RI: 2/, Hae III: 3/, Sau 3AI: la/
Hpa II F (31-37)* Xba I, Bgl II: no cleavage, Bel I: 35/, Hae III: 31/36/, Taq Yh 670, 335, 135, Sau 3AI: 34/ Hpa II G (16) Xba i, Bell, Bgl lI, Sau 3AI, Hae III: no cleavage, Alu h 490, 455 Hpa II H (57-60) Xba I' Bgl II: no cleavage, Hae IIL' 59/, Hha I: 58/, Sau 3AI: 57a/58/, Taq YI: 610, 105 Hpa H H' (6-10)* Xba i, Hha i, Bgl lI: no eleavage, Hae IIh 6/9/, Sau 3AL" 8/, Taq Yh 430, 170, 110 BelI
B (19-34)HindtII: 22/24/
Bel I
C (9-18) Hind III: no eleavage
BelI
D (59-8)HindlIl: 1/
Y I and Alu I for the redigestion of several Hha I fragments and Hpa I I fragments in order to derive their sizes by additional restriction analyses (see Table 3). In this way the positions of additional restriction sites were determined (see Table 3 and Fig. 1). The average length of the mtDNA by summing up the fragment lengths from the various digestions was calculated to be 15,600 -+ 150 bp. The average size o f the fragments located i n Fig. 1 is ca. 200 bp and the restriction sites represent ca. 2.4% o f sequence information.
The restriction endonucleases Hae III, Bsp L and Bsu I recognize the same DNA sequence. We found that also on mtDNA they may be used alternatively, since they produce identical restriction patterns. Bal I (CGGCCG) which contains the recognition sequence for these three enzymes cuts mtDNA only once in position 31/32 (Fig. 1). Xma I which is another enzyme having a recognition site composed o f only C and G (CCCGGG) does not cut mtDNA. There is also no cleavage site for Pst I (CTGCAG) in mtDNA.
18
H. Feldmann and R. GrofSkopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA
There is rather good agreement between our determination of fragment lengths and the data reported by Buzzo et al. (1977) and Koike et al. (1976), who measured some fragments in the electron microscope. In contrast, the values given for total length and for the sizes of the larger fragments by Kroon et al. (1977) and Saccone et al. (1977) may be too low. Our arrangement of the larger Eco RI fragments and of the fragments produced with Barn HI, Hha I, and Hind III (Fig. 1) is the same as reported by Saccone et al. (1977) (type A mtDNA). With respect to the fragments obtained with Barn HI, Hpa I, Hae II, Hind III, and Hind H (= Hinc I1) our map is also compatible with that given for type B mtDNA by Parker and Watson (1977). On the other hand, we arrive at some alterations of the restriction maps presented so far. This we attribute to the fact that we have used a large number of restriction enzymes, many double digestions, and the redigestion of defined fragments, which allowed a more precise positioning of sites. Nevertheless, a few points remain to be discussed. For example, in complete Hind III digests from some mtDNA preparations we found a 370 bp fragment. However, in the redigestion of defined fragments, which should contain this particular fragment, it was absent. Moreover, in summing up the lengths of the sub fragments, there was no room for a 370 bp fragment. Instead there may be a very short Hind III fragment between Hind IIi fragments A and E. As an explanation, we assume that the 370 bp fragment originates from a contamination of mtDNA with satellite DNA (Philippsen et al., 1974). The fragment pattern obtained from mtDNA with
Hae III (or Bsp 1) even on prolonged incubations always contained the fragments D (1,200 bp) and K (680 bp) (Table 1), but in much less than a molar ratio. We found the same result in the analysis of mtDNA from a single individual. There are two possible explanations: (i) existence of microheterogeneities in the mtDNA of one animal, or (ii) less accessibility of some sites in whole length mtDNA. Although we cannot exclude the first possibility, we favour the second one, because we have not observed fragments D and K in redigestions of other fragments, and because it is possible to consider them as partial products (D = G + R or O + S + Q, K = Q + S, Fig. 1). Further detailed analyses will clarify the points raised above. The results presented here have enabled us to dissect the mtDNA into a large number of defined fragments. Hybridization experiments according to the method of Southern (1975) with tRNAs fractionated by our 2Delectrophoresis system (Fradin et al., 1975) have led to the location of most of the mt tRNA genes. These results will be reported elsewhere.
Acknowledgements. This work has been supported by the Deutsche Forschungsgemeinschaft (Forschergruppe "Genomorganisation") and by Fonds der Chemischen Industrie. We are grateful to Mrs. C. Bleifug and Miss K. Flaig for skilled technical assistance. We thank Drs. R. E. Streeck, U. J. H/inggi, T. IgoKemenes, W. H6rz, and W. Zolg from this laboratory for supplying restriction enzymes and helpful discussions.
References Attardi, G., Albring, M., Amalric, F., Gelfand, R., Griffith, J., Lynch, D., Merkel, C., Murphy, W., Ojala, D.: In: Genetics and Biogenesis of Chloroplasts and Mitochondria. Biicher, Th., Neupert, W., Sebald, W., Wemer, S. (eds), pp. 573585. Amsterdam: North-Holland Publ. Company, 1976 Bandlow, W., Schweyen, R. J., Wolf, K., Kaudewitz, F.: Genetics and Biogenesis of Mitochondria. Berhn, New York: W. d. Gruyter 1977 Battey, J., Clayton, D. A.: Cell 14, 143-156 (1978) Bickle, T. A., Pirrotta, V., Imber, R.: Nucl. Acids Res. 4, 2561-2572 (1977) Bogenhagen, D., Clayton, D. A.: J. Biol. Chem. 249, 79917995 (1974) Buzzo, K., Fouts, D. L., Wolstenholme, D. R.: Proc. Nat. Acad. Sci. USA 75,909-913 (1978) Dawid, I. B., Klukas, C. K., Ohi, S., Ramirez, J. L., Upholt, W. B.: In: The Genetic Function of Mitochondrial DNA. Saccone, C., Kroon, A. M. (eds), pp. 3-13. Amsterdam: NorthHolland Publ. Company 1976 de Wachter, R., Fiers, W.: Methods Enzymol. 21, 167-178 (1971) Fradin, A., Gruhl, H., Feldmann, H.: FEBS Lett. 50, 185-189 (1975) Francisco, J. F., Simpson, M. V.: FEBS Lett. 79, 291-294 (1977) Glynn, I. M., ChappeU, J. B.: Biochem. J. 90, 147-149 (1964) Gross-BeUard, M., Oudet, P., Chambon, P.: Eur. J. Biochem. 36, 32-38 (1973) Hayashi, J., Yonekawa, H., Gotoh, O., Motohashi, J., Tagashira, Y.: Biochem. Biophys. Res. Commun. 81,871-877 (1978) Kleinow, W., Neupert, W.: Z. Physiol. Chem. 351, 1205-1214 (1970) Klukas, C. K., Dawid, I. B.: Cell 9,615-625 (1976) Koike, K., Kobayashi, M., Tanaka, S., Mizusawa, H.: In: Genetics and biogenesis of chloroplasts and mitochondria. Biicher, Th., Neupert, W., Sebald, W., Werner, S. (eds.), pp. 593-596. Amsterdam: North-Holland Publ. Company 1976. Kroon, A. M., Bakker, H., Holtrop, M., Terpstra, P.: Biochim. Biophys. Acta 474, 61-68 (1977) Kroon, A. M., Pepe, G., Bakker, H., Holtrop, M., Bollen, J. E., van Bruggen, E. F. J., Cantatore, P., Terpstra, P., Saccone, C.: Biochim. Biophys. Acta 478, 128-145 (1977) Kroon, A. M., de Vos, W. M., Bakker, H.: Biochim. Biophys. Acta 519, 269-273 (1978) Lillehaug, J. R., Kleppe, K.: Biochemistry 14, 1225-1229 (1975) Loening, U. E.: Biochem. J. 113,131-138 (1969) Parker, R. C., Watson, R. M.: Nucl. Acids Res. 4, 1291-1299 (1977) Peacock, A. C., Dingman, C. W.: Biochemistry 6, 1818-1827 (1967) Pfeiffer, W., H6rz, W., lgo-Kemenes, T., Zachau, H. G.: Nature 258,450-452 (1975)
H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA Philippsen, P., Streeck, R. E., Zachau, H. G.: Eur. J. Biochem. 45,479-488 (1974) Saccone, C., Pepe, G., Cantatore, P., Terpstra, P., Kroon, A. M.: In: The genetic function of mitochondrial DNA. Saccone, C., Kroon, A. M. (eds.), pp. 27-36. Amsterdam: NorthHolland Publ. Company 1976 Saccone, C., Pepe, G., Bakker, H., Kroon, A.: In: Genetics and Biogenesis of Mitochondfia. Bandlow, W., Schweyen, R. J., Wolf, K., Kandewitz, F. (eds.), pp. 303-315. Berlin, New York: W. d. Gruyter 197~7 Smith, H. O., Birnstiel, M. L.: Nucleic. Acids Res. 3, 23872398 (1976)
19
Southern, E. M.: J. Mol. Biol. 98,503-517 (1975) Streeck, R. E., Zachau, H. G.: Eur. J. Biochem. 89, 267-279 (1978) van Gabain, A., Hayward, G. S., Bujard, H.: Mol. Gem Genet. 143,279 290 (1976)
C o m m u n i c a t e d b y F. Kaudewitz Received April 26, 1979
Current Genetics © by Springer-Verlag 1979
A Detailed Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA* Horst Feldmann and Riidiger Grogkopf Institut ffir Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universitiit, Goethestrasse 33, D-8000 Mtinchen 2, Federal Republic of Germany
Summary. A restriction endonuclease cleavage map of rat liver mitochondrial DNA is presented for the following enzymes: Xba I, Bgl II, Hae II, Barn HI, Hpa I, Hha I, Bcl I, Hind II, Hind III, Eeo RI, Hpa II, Hae III, and Sau 314. It was derived from complete and partial digestions with these enzymes, double digestions, and redigestions of defined fragments obtained with one enzyme with other restriction enzymes. By the use of these and further enzymes (Taq YI, Alu I) the mitochondrial DNA (ca. 15.6 Kb) can be dissected into a large number of defined fragments.
Key words: Mitochondrial DNA - Restriction mapping.
Introduction Mitochondria represent the smallest replication units which contain genetic information for rRNAs, tRNAs and mRNAs. The evolution and maintenance of this extrachromosomal system seems due to the fact that the mitochondrial gene products possess unique features. During biogenesis of mitochondria, the limited number of mitochondrial products has to be complemented by a large number of components encoded in nuclear DNA. This affords a strict interplay between the two genetic systems (summary, Bandlow et al., 1977). For these reasons, studies on the organization of mitochondrial genomes on a molecular level are of particular interest.
Offprint requests to: H. Feldmann * Abbreviations: mtDNA, mitochondrial DNA; bp or Kbp, base pairs or kilo base pairs, respectively. Other abbreviations follow IUB-IUPAC conventions.
Animal mtDNAs are the smallest mitochondrial genomes (ca. 10 - 106 daltons in molecular weigth). Studies in several systems (e.g. Attardi et al., 1976; Dawid et al., 1976; Klukas and Dawid, 1976; Saccone et al., 1977; Battey and Clayton, 1978) have shown that they code for two ribosomal RNAs, an apparently full complement of tRNA, and only a small number of mRNAs. Since genetic experiments in animal mitochondria are limited, the main approachto tackle the problem of their genomic organization consists in constructing genetic maps by the use of electronmicroscopic techniques and/or restriction endonucleases together with hybridization techniques. Physical mapping is a prerequisite if one wants to locate specific genes by hybridization of specific RNAs with restriction endonuclease cleavage fragments. Rat liver mitochondrial DNA has been investigated by several authors and data have been obtained With respect to: (i) restriction endonuclease cleavage maps (Saccone et al., 1976, 1977; Kroon et al., 1977a and b;Parker and Watson, 1977; Koike et al., 1976; Buzzo et al., 1978), (ii) location of the D-loop (Koike et al., 1976 ; Buzzo et al., 1978), and (iii) location of the rRNA genes and the tRNA genes (Saccone et al., 1976, 1977; Kroon et al., 1977a and b). However, several discrepancies exist among the reported restriction maps, which in part may be due to the fact that two different molecular types of rat liver mtDNA are existent. This interesting observation was first reported by Francisco and Simpson (1977) and has been confirmed by several authors (Buzzo et al., 1978; Hayashi et al., 1978; Kroon et al., 1978). The divergence in sequences was detected through the differences in the number of specific restriction sites. It seems that there is only one type of DNA in one animal and that this is maternally inherited (Buzzo et al., 1978; Kroon et al., 1978). O172-8083/79/0001/0013/$01.40
14
H. Feldmann and R. GroiSkopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA For a detailed study of the mitochondrial genome
f r o m rat liver it s e e m e d desirable t o o b t a i n a r e s t r i c t i o n m a p for a large n u m b e r o f e n z y m e s w h i c h t h e n w o u l d allow t o dissect t h e m t D N A i n t o d e f i n e d f r a g m e n t s o f small sizes. In this p a p e r we wish t o r e p o r t t h e result o f this analysis.
hours. The fluid from the fluorescent band containing the circular DNA (as visualized under a UV lamp) was collected by punching the centrifuge tubes with a syringe needle. Ethidium bromide was removed by at least 5 extractions with propanol-2/butanol-1 (1 : 1, v/v). The aqueous phase was dialyzed against TE buffer (10 mM Tris-HC1, pH 7.4, 1 mM EDTA) and the DNA precipitated with ethanol and centrifuged. The pellet was dissolved in TE buffer to a concentration of ca. 250 #g/ml.
Digestions o f mtDNA with Restriction Endonucleases. For anaMaterials a n d M e t h o d s Female rats, Sprague-Dawley strain, were purchased from WIGA, Vet suclistieranstalt, Sulzfeld. Seakam agarose and Seaplaque agarose were products of Marine Colloids Inc., Rockland Maine. Acrylamide and N,N'-bismethylen-acrylamide were from Serva, Heidelberg. Sucrose (RNase-free), CsC1, and proteinase K were obtained from Merck, Darmstadt. All chemicals were of analytical grade. [32p] phosphate, carrier-free, without HC1, was bought from New England Nucl. Corp. Polynucleotide kinase was obtained from Boehringer Mannheim GmbH, alkaline phosphatase from Worthington, Biochemical Corp. Restriction endonucleases from several sources were used: Eco RI, Bam H1, Hind 111, and Hpa 11 were from Boehringer Mannheim GmbH. Hind Ili, Bcl 1, and Taq Y1 were from Microbiological Research Establ., Porton, Eco R1 and Hind H were gifts of Dr. Igo-Kemenes; Bsu I and Bsp were gifts of Dr. H6rz; Hha 1, A lu 1, Bgl 11, Sau 3/11, Pst 1, Sal 1, Xba 1, and Mbo H were gifts of Dr. Streeck; Bal 1, Hae 11, Hae Ili, Barn HI, Xma I were gifts of Dr. Hanggi (all in this laboratory) ; Hpa I and Hpa H were prepared according to the method reported by Bickte et al. (1977) including a rechromatography on DEAE-ceUulose at pH 8.4. Xdvl, Xdv21, and PM2 DNA were gifts of Dr. Streeck, T5 DNA was from Dr. Hiinggi. The Bsp fragments of kdvl (Streeck and Zachau, 1978) served as standards. Dr. Zolg supplied cells of Haemophilus parainfluenzae.
Preparation of Rat Liver Mitochondria. If not mentioned otherwise, the livers from 20 rats ( 1 8 0 - 2 0 0 g) were batchwise disrupted in a Potter homogenizer in ca. 600 ml of the following buffer: 0.25 M sucrose, 10 mM Tris-HC1, 50 mM EDTA, pH 7.3 (Kleinow and Neupert, 1970). The homogenate was centrifuged at 3,000 rpm in a Sorvall SS 34 rotor for 5 min to remove cell debris and nuclei. The supernatant was then centrifuged at 12,000 rpm for 15 min. The crude mitochondrial pellet was resuspended in the above buffer, and the procedure of low and high speed centrifugations was repeated three times. All steps were performed at 4 ° C. The final mitochondrial pellet was used immediately for the preparation of DNA.
Preparation ofMitochondriaIDNA. Mitochondria were suspended om 150 ml of the following buffer: 25 mM Tris-HCl, 50 mM EDTA, 75 mM NaCI, pH 7.4. Lysis was achieved similar to GrossBellard at al. (1973) by adding 2% of sodium dodecylsulphate and 20 ~tg/ml proteinase K, and incubating the mixture for 2 hours at 37 °C. The clear lysate was extracted twice with an equivolume of chloroform/i-amylalcohol (24:1, v/v) and the DNA precipitated with 3 volumes of cold ethanol (plus 3% potassium acetate). The DNA pellet was dissolved in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and dialyzed against the same buffer overnight. 15.6 ml aliquots of this solution were mixed with 15 g CsCI, then 9.6 mg of ethidium bromide (0.1% solution) was added. Centrifugation was carried out in a Beckman ultracentrifuge at 15 °C (33,000 rpm, rotor Ti 60) for 65
lytical purposes, 2 - 3 hour incubations were routinely done in 25 to 100 #1 of theappropriate buffer, using 1 - 5 tag DNA or DNA fragments and 1 - 5 units of enzyme. For partial digestions, the samples were incubated for only short periods of time ( 1 10 min.). After incubation, the samples were extracted with phenol, the fragmented DNA was precipitated with cold ethanol (made 3% in potassium acetate and 10 mM in Mg-acetate), pelleted, and redissolved in 10 to 20 #1 TE buffer. For the preparation of defined restriction fragments 1 0 20 #g of DNA were digested in 2 0 0 - 5 0 0 #1 incubation mixture and treated similarly as described above. Double digests with two restriction endonucleases were performed in two ways: (i) the DNA was preincubated for 1 hour with the first enzyme, then the buffer conditions were adapted for the second enzyme and incubation continued for an additional 2 hours (ii) the two enzymes were added together, if the buffer conditions for digestion were similar.
Gel Electrophoreses. Electrophoreses on 0.4 to 2.2% agarose slab gels (15 x 24 x 0.3 cm, 15 slots each 1 cm wide) and tube gels (7 x 180 mm or 13 x 18 mm) were performed in different buffer systems: (A) 0.08 M Tfis-borate, 3 mM EDTA, pH 8.3 (Peacock and Dingman, 1967); (B) 0.0036 M Tris-phosphate, 1 mM EDTA, pH 7.7 (de Wachter and Fiefs, 1971); (C) 0.02 M Tris-acetate, 0.04 M Na-acetate, 1 mM EDTA, pH 7.4 (Loening, 1969). Gels were either run without the addition of ethidium bromide and stained after electrophoresis, or 0.1 #g/ml ethidium bromide was added to all buffers. Gels in buffer systems (A) and (B) were operated at 4 to 5 volts/cm, gels in buffer system (C) at 2 volts/cm. On a preparative scale (e.g. for redigestions), DNA fragments were obtained by electrophoreses of the digests in buffer system (C) on Seaplaque agarose and extraction of the fragments from gel slices. Up to 100 #g DNA could be loaded onto the larger tube gels. Minced gel slices containing one fragment were extracted twice with an appropriate volume of 0.5 M NaCI solution. The extract was filtered through Whatman 3 MM paper and the DNA was precipitated with 2.5 volumes of ethanol. Gel electrophoreses on polyacrylamide gels were performed in buffer system (C). Appropriate gel concentrations were 3 or 4% acrylamide mixed with N,N'-bismethylenacrylamide in a ratio of 20 : 1 or 30 : 1. Quantitative Evaluations of Gels. Gels after staining with ethidium bromide were photographed by the use of an ultraviolet light source, a Durst laborator camera and AGFA film 230p (Pfeiffer et al., 1975). The films were evaluated photometrically in a Gilford spectrophotometer equipped with an automatic scanning device. Standards for calibration of fragment lengths were always run in parallel and sometimes together with fragmented mtDNA. Analysis for Fragments 5'-labeled with 32p. Restriction fragments extracted from Seaplaque gels (see above) were treated with bacterial alkaline phosphatase and rephosphorylated by reaction with [7"32p]ATP (Glynn and Chappell, 1964) and polynucleotide kinase (Lillehaug and Kleppe, 1975). The sub-
H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA fragments produced by cleavage with further restriction endonucleases were separated by electrophoresis on polyacrylamide gels. Gels were stained with ethidium bromide and radioautographed by the use of Kodak X-Omat R film.
15
This information allowed to properly arrange the subfragments and was also helpful to establish the relative location of restriction sites which were produced by different enzymes, but are located very close to each other.
Results and Discussion In this study we have used female rats from an inbread strain (Sprague-Dawley type), which had largely type A mtDNA (Francisco and Simpson, 1977) according to our analyses with restriction endonucleases Eco RI, Hha I, and Hind II. The strategy for the restriction mapping of this mtDNA was the following: (i) Digestions of mtDNA with single restriction enzymes and determination of fragment sizes by electrophoreses on agarose slab gels of varied concentrations (0.4 to 2.2%) together with appropriate standards. The following standards (see Methods) were used: (a) Bsp (or Hae III) fragments of Xdvl which cover a range from 1686 to 34 bp; the fragment sizes have been accurately determined (Streeck and Zachau, 1978), (b) Hind III fragments of T5 DNA (van Gabain et al., 1976) (17.3 to 0.91 Kb) which were used for a size estimation of the larger fragments. (ii) Double digestions with various enzymes and determination of the fragment sizes. For most of the enzymes used (except Bgl II, Taq YI, Alu I, and Sau 3AI) combinations with two to five of the other enzymes were evaluated. (iii) Redigestions of defined fragments obtained from digestions with one enzyme by several other enzymes and analysis of the products. In some cases also a second redigestion was performed on the subfragments. Defined fragments were obtained on a preparative scale by the use of Seaplaque agarose gels, from which slices were cut out and the restriction fragments eluted (see Methods). Redigestions could be carried out on this material directly, since it was free from contaminants which otherwise have been observed to inhibit further enzymatic steps on DNA extracted from agarose gels. Redigestions were mainly done with those enzymes that produce relatively short restriction fragments. In some cases, we have used conditions (see Methods) for the digestion or in the redigestion of defined fragments, which lead to partial cleavages thus facilitating the arrangement of the subfragments. Futhermore, we took advantage of the techniques to 5'-label some restriction fragments with 32p prior to redigestion(s). Such fragments were then digested with other restriction enzymes either completely or partially, following the method devised by Smith and Birnstiel (1976). The subfragments were separated by electrophoresis on polyacrylamide gels, the gels were stained and radioautographed.
Table 1. Fragments of mtDNA derived from restriction endonuclease cleavages The fragments are designated by capitals and listed in decreasing length, their sizes (in bp) as determined through gel electrophoreses are given in parentheses. Some additional very short fragments for Hae III or Sau 3AI have been observed but not yet positioned. BgIH
A 23b-17a(14,000), B 17b-23a(1,700)
Hae H
A 15-47(9,600), B 48-14(6,100)
XbaI
A 24-4(9,800), B 5-23(6,000)
BamHI
A 27-13(10,800), B 14-26(4,950)
HpaI
A 46-35(13,400), B 36-45(2,450)
HhaI
A 15-44(8,400), B 58-14(4,250), C 48-57(1,950), D 45-47(1,050)
BclI
A 35-53(4,200), B 19-34(4,000), C 9-18(3,600), D59-8(2,850),E54 58(730)
HindlI
A 12-32(7,100), B 46-7(4,950), C 36-45(2,450), D 8-11(820), E 33-35(280)
HindlII
A 2-20(6,250), B 44-1(4,100), C 25-33 (2,450), D 34-43(1,950), E 23-24(780), 21-22 a
Hind III
(partial digest, in addition to fragments A to E)
23-43(6,200), 34-1(5,900), 25-43(4,400), 23-33(3,200) Eco R I
A 3-21(5,900), B 22-38(3,700), C 39-46(2,700), D 55-2(1,850), E 52-54(680), F 49-50(460), G 47-48(130), H 51 (110)
Eco R I
(partial digest, in addition to fragments A to H) 47-38(12,800), 3-46(12,350), 39-21(11,900), 3-38(9,550), 22-51(6,900), 22-46(6,400), 39-54(4,050), 39-51(3,400), 47-2(3,250),. 52-2(2,550), 47-54(1,380)
HpaH
A 17-29(3,700), B 43-56(3,100), C 11-15(1,820), D 38-42(1,700), E 1-5(1,650), F 31-37(1,080), G 16 (960), H 57-60(720), H' 6-10(720), J 30(80)
HaeIII
A 13-17(2,550), B 42-49(2,100), C 20-25(1,850), (D), E 4-6(980), F 60-3(960), G 37-39(820), H 32-36(800), J 56-59(780), (K), L 18-19(680), M 7-9(600), M' 10-12(600), N 53-55(500), O 26-27(530), P 50-52(420), Q 29-31(410), R 40(360), S 28(240), T 41(200)
Sau 3AI
A lb-8(2,100), B 42b-49a(1,780), C 14-17a(1,690),
D 27 34(1,500), E 9-13a(1,160), F 25b-26(960), F' 35-40a(960), F" 49b--53(960), G 19-20a(720), H 59-1a(640), J 54-57a(620), K 17b-18(500), L 20b-23a(475), M 40b-41a(420), N 4 lb-42a(360), O 23b-24a(295), O' 13b(280), P 24b-25a(160), P' 57b-58(150) a
cf. text
16
H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA
Table 2. Restriction patterns from combined cleavages of mtDNA with two restriction endonucleases The nomenclature is a in Fig. 1. The observed fragmentation pattern is documented in a simplified manner: the fragments run up to and include the indicated subfragments. Detailed data can be requested from the authors.
XbaI,
nhaI: 4/14/23/44/47/57/, Hpa11: 4/5/10/15/16/23/29/30/37/42/56/60/
BclL
nhaI: 8/14/18/34/44/47/53/57/58/,Hind11I: 1/8/18/21/24/33/34/43/53/58/,EcoRI: 2/8/18/21/34/38/46/47/50/51/53/ 54/58/, Hpa11: 5/8/10/15/16/18/29/34/37/42/53/56/58/60/
Bam H1, Hae11: 13/14/26/47/, HhaI: 13/14/26/44/47/57/, Hind11: 7/11/13/26/32/35/45/, Hind 11I: 1/13/20/22/24/26/33/43/, Eco Rh 2/13/21/26/38/46/48/50/51/54/, Hpa I1: 5/10/13/15/16/26/29/30/37/42/56/ 60/, Hae11I: allHaeI11fragments except A and O, instead fragments 13, 14-17, 26 Hae11,
HhaL" 14/44/47/57/,Hpa1: 14~35~45~47~,Hind111: 1/14/20/22/24/33/43/47/,EcoRl: 2/14/21/38/46/48/50/51/54/
Hpa L
Hha L" 14/35/44/45/47/57/, Hind IlI: 1/20/22/24/33/35/43/45/, Hae 11L"all Hae 111 fragments except B and H, instead fragments 32-35, 42-45, 4 6 - 4 9
Hhal,
Hindll: 7/11/14/32/35/44/45/47/57/,HindllI: 1/14/20/22/24/33/43/44/47/57/,EcoR1: 2/14/21/38/44/46/47/48/50/51/ 54/57/, Hpa 1I." 5/10/14/15/16/29/30/37/42/44/47/56/57/60/, Hae 111: all Hae 1II fragments except A, B and J, instead fragments 13-14, 17, 42-44, 47, 49, 56-57, 59
Hind III, Eco RI: 1/2/21/22/24/33/38/43/46/48/50/51/54/, Hpa 11: 1/5/10/15/16/20/24/29/30/33/37/42/43/56/60/, Hae I11: 1/3/6/9/12/17/19/22/24/25/27/28/31/33/37/39/40/41/43/49/52/55/59/ EcoRI,
Hpa11: 2/5/lO/15/16/21/29/30/37/38/42/46/48/50/51/54/56/60/,Hae11I: 2/3/6/9/12/17/19/21/25/27/28/31/36/38/39/ 40/41/46/48/50/51/54/55/59/
B
t
B
A
I
A
I
B
A
D
I D /
I t
H
F
I
C
g
I
ii E I I A
A H'
E
I M I M'I
C
G
I A
I
I
A
I
Hpa I (GTT'AAC) Hoe II (PuGCGC'Py)
iO1'
I IEI I
C B
F IF iO ISIQI H
C
D
i A C D
C
I
B B FII E t
p C
I D I I t G tRITI B
I I E I
Hha l (GCG'C) Bcl I (T'GATCA) Hind II(GTPy'PuAC] t Hind I[I(A'AGCTT) !EcoRI[G'AATTC)
D
B
! I.PI N,,
H
i
J I
Hpa II (C'CGG] Hae t]I (GG'CC)
1~ 11711allg120,11123,h L 25 I~lleaI[lat32I~l1381 19 l~oI.F.~Id~Sl !6 ~1!~1[I521531!sq159N 6 8 10 12 14
I
i E
t I L I
i0,11~14 I s liE 82 11111~2E n3. II ~54 I I
I A
A "1 E
I
B
A
B
I I
Xba (T'CTAGA) Barn H I ( G'GATCC )
A
I
I
C
I
I B I I K I G I L iO1~ ~ F
I
i
D
A I F'
i
IMtNI
I
B I I i
IKbp
t F" I J it/'H ~ I I
i
Bgl ]i [A'GATCT) Sau 3A [(GATC] "'Taq YI (TCGA)
Fig. 1. Restriction endonuclease cleavage map of rat liver mitochondrial DNA (linearized presentation). The series of fragments which were obtained by digestion of mtDNA with a particular restriction endonuclease are designated by capitals. The subfragments produced by the enzymes listed in the upper part of the map are designated by arabic numerals taking the restriction site between the Hpa I1 fragments H and E as a reference point. In the lower part of the map those fragments and sites are listed which were derived from redigestions of isolated fragments with further enzymes (see also Table 3). This additional fragmentation is referred to in Table 3 by the small letters a and b (left and rigth portion of a subfragment, respectively)
F o r simplicity, the results o f t h e above m e n t i o n e d analyses are summarized in tabular f o r m (Tables 1, 2, and 3). F r o m these data we have deduced t h e restriction m a p shown in Fig. 1. Altogether, we were able to locate t h e sites for the following enzymes: Xba 1, Barn 111, Hae
11, Bgl II, Hpa L Bcl L Hind 11, Hha L Hind Ill, Eco R L Hpa 1I, Hae Ill, and Sau 3.41. Sau 3A1 is o f particular
interest as its recognition sequence is contained within the recognition sequences o f Barn HI, Bcl 1, and Bgl II. In addition we have p e r f o r m e d digestions o n m t D N A w i t h o t h e r restriction enzymes such as Taq YI, Alu I, and Mbo 11. However, the f r a g m e n t a t i o n patterns were found to be rather c o m p l e x , as a large n u m b e r o f fragm e n t s is produced. On t h e o t h e r hand, we have used Taq
H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA
17
Table 3. Redigestions of isolated restriction endonuclease cleavage fragments from mtDNA with further restriction enzymes Presentation of the data is as in Table 2. The isolated fragments on which digestions were performed with a second enzyme are listed by indicating the first and the last subfragment in brackets. In some cases (for Taq YI orAlu 1) the order of the subfragments h.as not yet been established, only fragment sizes are listed. An asterisk refers to such fragments which have also been Y-labeled with [ 32pj posphate for analysis. Hha I
A (15-44) Xba h 23/, Bell: 18/34/, Hae IIL" 17/19/25/27/28/31/36/39/40/41/, Hpa I: 36/, Bgl Ih 17a/23a/, Sau 3A: 17a/ 18/20a/23a/24a/26/34/40a/41a/4 2a/ (22-38).Hpa IL" 30/37/, Hae III: 25/27/28/31/36/ EeoRI: (15-21)HpaIL" 16/21/,HaeIII: 17/19/ (39-44) Hpa H: 42/, Hae III: 39/40/41
HhaI
B (58-14)BglII:noeleavage, XbaI: 4/,BelI: 8/,HaeIII: 3/6/9/12/14/59/,Sau3A: la/8/13a/13b/ EcoRL f (3-14)HpaII: 5/10/ k (58-2)Hpalh 60/ (1-5) Hae llI: 3/ (11-14) Hae HL" 12/ HpaII: (6-10)HaeIIh 7/9/, Taq YI: 430/170/110/ (58-60) Hae IlL" 59/
Hha I
C (48-57) Xba I, Bgl IL" no cleavage, Bcl I: 53/, Hae III: 49/52/55/, Taq YI: 52a/56a/,
Sau 3A: 49a/53/57a/, Eeo RI (partial): 48,48-50,48-54,49-50,51,51-54,51-57,52-54,55-57 f (52-54) Hae IlL" 52/ Eeo RI: (55-57) BraeIII: 55/ (49-50) Hae III: 49/ HhaI
D (45-47) XbaLBelLHaeIII, Sau3AL'nocleavage, Hpah 45/,EcoRI." 46/,Taq YI: 45a/46a/
Hpa II A (17 -29)* BglII: 17a/23a/, Xba I: 23/, BelL" 18/, Hae III: 17/19/25/28/, Hae III (partial, in addition): 18-25, 26-.29, HindIIL" 20/24/,Aluh 890, 750, 570, 510, 320, 280, 230, 130, HpaH A (17-29) Eeo RI: ~ (17-21) Sau 3A: 18/20a/ l (22-29) Sau 3A: 23b/24b/25b/27/ f
Hpa II B (43-56)* Xba l, Bgl IL" no cleavage, Bel h 53/, Hpa I: 45/, Eeo RI." 46/48/50/51/54/, Hae HI." 49/52/55/, Taq YI: 44a/45a/46a/52a/56a/, Sau 3AI: 49a/53/ Hpa II C (11-15)* Xba L Bell, Bgl II." no cleavage, Hha I: 14/, Hae III: 12/, Sau 3AI: 13a/13b/ Hpa [I D (38-42)* Xba L Bell, Bgl IL" no cleavage, Eeo RI: 38/, Hae IH: 39/40/41/, Taq YL" 860, 640, 210 Sau 3Ah 40/41a/42a/ Hpa I I E
(1-5)* Xba I: 4/, B e l l Bgl IL" no cleavage, Eeo RI: 2/, Hae III: 3/, Sau 3AI: la/
Hpa II F (31-37)* Xba I, Bgl II: no cleavage, Bel I: 35/, Hae III: 31/36/, Taq Yh 670, 335, 135, Sau 3AI: 34/ Hpa II G (16) Xba i, Bell, Bgl lI, Sau 3AI, Hae III: no cleavage, Alu h 490, 455 Hpa II H (57-60) Xba I' Bgl II: no cleavage, Hae IIL' 59/, Hha I: 58/, Sau 3AI: 57a/58/, Taq YI: 610, 105 Hpa H H' (6-10)* Xba i, Hha i, Bgl lI: no eleavage, Hae IIh 6/9/, Sau 3AL" 8/, Taq Yh 430, 170, 110 BelI
B (19-34)HindtII: 22/24/
Bel I
C (9-18) Hind III: no eleavage
BelI
D (59-8)HindlIl: 1/
Y I and Alu I for the redigestion of several Hha I fragments and Hpa I I fragments in order to derive their sizes by additional restriction analyses (see Table 3). In this way the positions of additional restriction sites were determined (see Table 3 and Fig. 1). The average length of the mtDNA by summing up the fragment lengths from the various digestions was calculated to be 15,600 -+ 150 bp. The average size o f the fragments located i n Fig. 1 is ca. 200 bp and the restriction sites represent ca. 2.4% o f sequence information.
The restriction endonucleases Hae III, Bsp L and Bsu I recognize the same DNA sequence. We found that also on mtDNA they may be used alternatively, since they produce identical restriction patterns. Bal I (CGGCCG) which contains the recognition sequence for these three enzymes cuts mtDNA only once in position 31/32 (Fig. 1). Xma I which is another enzyme having a recognition site composed o f only C and G (CCCGGG) does not cut mtDNA. There is also no cleavage site for Pst I (CTGCAG) in mtDNA.
18
H. Feldmann and R. GrofSkopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA
There is rather good agreement between our determination of fragment lengths and the data reported by Buzzo et al. (1977) and Koike et al. (1976), who measured some fragments in the electron microscope. In contrast, the values given for total length and for the sizes of the larger fragments by Kroon et al. (1977) and Saccone et al. (1977) may be too low. Our arrangement of the larger Eco RI fragments and of the fragments produced with Barn HI, Hha I, and Hind III (Fig. 1) is the same as reported by Saccone et al. (1977) (type A mtDNA). With respect to the fragments obtained with Barn HI, Hpa I, Hae II, Hind III, and Hind H (= Hinc I1) our map is also compatible with that given for type B mtDNA by Parker and Watson (1977). On the other hand, we arrive at some alterations of the restriction maps presented so far. This we attribute to the fact that we have used a large number of restriction enzymes, many double digestions, and the redigestion of defined fragments, which allowed a more precise positioning of sites. Nevertheless, a few points remain to be discussed. For example, in complete Hind III digests from some mtDNA preparations we found a 370 bp fragment. However, in the redigestion of defined fragments, which should contain this particular fragment, it was absent. Moreover, in summing up the lengths of the sub fragments, there was no room for a 370 bp fragment. Instead there may be a very short Hind III fragment between Hind IIi fragments A and E. As an explanation, we assume that the 370 bp fragment originates from a contamination of mtDNA with satellite DNA (Philippsen et al., 1974). The fragment pattern obtained from mtDNA with
Hae III (or Bsp 1) even on prolonged incubations always contained the fragments D (1,200 bp) and K (680 bp) (Table 1), but in much less than a molar ratio. We found the same result in the analysis of mtDNA from a single individual. There are two possible explanations: (i) existence of microheterogeneities in the mtDNA of one animal, or (ii) less accessibility of some sites in whole length mtDNA. Although we cannot exclude the first possibility, we favour the second one, because we have not observed fragments D and K in redigestions of other fragments, and because it is possible to consider them as partial products (D = G + R or O + S + Q, K = Q + S, Fig. 1). Further detailed analyses will clarify the points raised above. The results presented here have enabled us to dissect the mtDNA into a large number of defined fragments. Hybridization experiments according to the method of Southern (1975) with tRNAs fractionated by our 2Delectrophoresis system (Fradin et al., 1975) have led to the location of most of the mt tRNA genes. These results will be reported elsewhere.
Acknowledgements. This work has been supported by the Deutsche Forschungsgemeinschaft (Forschergruppe "Genomorganisation") and by Fonds der Chemischen Industrie. We are grateful to Mrs. C. Bleifug and Miss K. Flaig for skilled technical assistance. We thank Drs. R. E. Streeck, U. J. H/inggi, T. IgoKemenes, W. H6rz, and W. Zolg from this laboratory for supplying restriction enzymes and helpful discussions.
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H. Feldmann and R. Grot~kopf: Restriction Endonuclease Cleavage Map of Rat Liver Mitochondrial DNA Philippsen, P., Streeck, R. E., Zachau, H. G.: Eur. J. Biochem. 45,479-488 (1974) Saccone, C., Pepe, G., Cantatore, P., Terpstra, P., Kroon, A. M.: In: The genetic function of mitochondrial DNA. Saccone, C., Kroon, A. M. (eds.), pp. 27-36. Amsterdam: NorthHolland Publ. Company 1976 Saccone, C., Pepe, G., Bakker, H., Kroon, A.: In: Genetics and Biogenesis of Mitochondfia. Bandlow, W., Schweyen, R. J., Wolf, K., Kandewitz, F. (eds.), pp. 303-315. Berlin, New York: W. d. Gruyter 197~7 Smith, H. O., Birnstiel, M. L.: Nucleic. Acids Res. 3, 23872398 (1976)
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C o m m u n i c a t e d b y F. Kaudewitz Received April 26, 1979
