University of Groningen

The Restriction Fragment Map of Rat-Liver Mitochondrial DNAPepe, G.; Bakker, H.; Holtrop, M.; Bollen, J.E.; Bruggen, E.F.J. van; Cantatore, P.; Terpstra,P.; Saccone, C.Published in:Biochimica et Biophysica Acta %28BBA%29 - Nucleic Acids and Protein Synthesis

DOI:10.1016/0005-2787(77)90177-0

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Citation for published version (APA):Pepe, G., Bakker, H., Holtrop, M., Bollen, J. E., Bruggen, E. F. J. V., Cantatore, P., Terpstra, P., &Saccone, C. (1977). The Restriction Fragment Map of Rat-Liver Mitochondrial DNA: A Reconsideration.Biochimica et Biophysica Acta %28BBA%29 - Nucleic Acids and Protein Synthesis, 478(2).https://doi.org/10.1016/0005-2787(77)90177-0

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Biochimica et Biophysica Acta, 478 (1977) 128--145 © Elsevier /North-Hol land Biomedical Press

BBA 99000

THE RESTRICTION FRAGMENT MAP OF RAT-LIVER MITOCHONDRIAL DNA: A RECONSIDERATION

A.M. K R O O N a, G. PEPE c, H. B A K K E R a, M. HOLTRO P a, J.E. BOLLEN b E.F.J . VAN B R U G G E N b, p. C A N T A T O R E c, p. T E R P S T R A a and C. SACC()NE c

Laboratories o f a Physiological Chemistry and b Biochemistry, State University, Groningen (The Netherlands) and c Laboratory o f Biological Chemistry, University o f Bari (Italy)

(Received March 9th , 1977)

Summary

1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Barn HI and 11 for the restriction endonuclease Hap II.

2. The physical map of the restriction fragments of Eco RI, Hind III, Barn HI and Hap II is constructed on the basis of: (a) the analysis of partially restricted fragments; (b) analysis of the double digests of total mtDNA; (c) the digestion of isolated restriction fragments with other restriction endonucleases; (d) the identification of fragments of complete single and double digestions and of par- tially digested fragments containing the base sequences complementary to the 12-S and 16~S RNAs of rat-liver mitochondrial ribosomes.

3. The genes for the ribosomal RNAs are shown to be closely linked. This result differs from data previously reported (Saccone, C., Pepe, G., Cantatore, P., Terpstra, P. and Kroon, A.M. (1976) in The Genetic Funct ion of Mitochon- drial DNA, pp. 27--36, Elsevier/North-Holland Biomedical Press, Amsterdam).

4. The origin of replication (D-loop) is localized in the vicinity of the small ribosomal RNA gene and the direction of replication is distant from this gene.

5. The mitochondrial tRNA genes are scattered over the genome as in other animal mtDNAs. The approximate minimal number of tRNA genes is 16--20.

6. We concluded previously that the Eco RI restriction fragments A and D are no t adjacent and failed to show the overlap of the 16 S rRNA gene for the Eco RI fragment D and Hind III fragment A. This misinterpretation was due to the fact that the two smallest Eco RI fragments could no t be detected with the

Postal addresses: a Bloemsingel 10, Groningen; b Zernikelaan, Groningen, The Netherlands; c Via Amendola 165/A, Bari 70126, Italy. Abbreviat ions: m t D N A , mi tochondr ia l DNA; mtRNA, mitochondrial RNA; SDS, sodium dodecyl- sulphate; SSC, 0.15 M NaCI/0.015 M trisodium citrate, pH 7.0.

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methods applied and to the lower specific radioactivity of the ribosomal RNAs used in the first series of hybridization experimentS.

Introduct ion

Recently we presented a restriction endonuclease cleavage map of mitochon- drial DNA (mtDNA) from rat liver [2]. This map was based on the calculation of the molecular weights of the fragments in complete and partial digests of mtDNA with the restriction enzymes Eco RI and Hind III. The Eco RI frag- ments of DNA from qb29 and Neurospora crassa mitochondria were used as standards. It was further based on the analysis of double digests of total mtDNA and on the results of digestion of isolated Eco RI fragments with Hind III. There appeared to be a high degree of consistency between the vari- ous results. In a subsequent series of experiments we hybridized the mitochon- drial ribosomal RNAs to the isolated fragments and observed that the DNA fragments carrying the ribosomal RNA genes were not adjacent but about 4000 basepairs apart [1]. This result appeared exceptional in the sense that other animal mtDNAs tested bear their ribosomal RNA genes on closely linked parts of the genome [3,4]. Inclusion' of Barn HI digestion confirmed part of the order of fragments of the map. However, definitive proof of that part of the map containing the ribosomal RNA genes was not obtained in this way, because the two ribosomal RNAs mapped on the same Barn HI fragment. Also the enzymes Hae III, Hin f and Hap II did not settle the question easily, because the number of cleavage sites for these enzymes was quite high. One of the main arguments remained, therefore, the typical pattern of partial digestion products with the enzyme Eco RI. The composit ion of these partials was inferred from their molecular weights and not analyzed directly. Since the presence of one or more other small fragments could undermine our conclusion, we decided to reinvestigate the matter using the ribosomal RNAs as indicators for the pres- ence of the various fragments in the partial restriction products. The aim of this paper is to present the results of this reinvestigation and to extend the previous observations with data regarding the localization of the origin of mtDNA repli- cation and of mitochondrial tRNAs.

Methods and Materials

Preparation of mitochondrial DNA from rat liver mtDNA was prepared from the livers of male albino rats (Wistar strain)

exactly as described previously [2]. The method is essentially based on the iso- lation of the closed circular mtDNA by CsCl-ethidium bromide density gradient centrifugation of the lysate of mitochondria prepared by differential centri- fugation.

Preparation of mitochondrial RNA from rat liver 55-S mitochondrial r ibosomes were isolated as described elsewhere [5], the

rRNAs were extracted from the ribosomes with a SDS/phenol procedure. Poly(A)-containing mtRNA was obtained by oligo(dT) cellulose chromatog- raphy, as presented in detail recently [6]. Mitochondrial tRNA was prepared as

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the 4 S RNA fraction from the total RNA extracted from lysed mitochondria. The method used has been described for the isolation of tRNA from yeast mitochondria [7,8] and has proved useful as well for the isolation of mitochon- drial tRNAs from N. crassa [9].

Enzymatic fragmentation and gel electrophoresis o f mitochondrial DNA from rat liver

The mtDNA was digested at 37 ° C for 3 h or longer with appropriate amounts of the restriction endonucleases Eco RI, Hind III, Bam HI or Hap II. The latter enzyme was a gift of Dr. J.G.G. Schoenmakers (University of Nijmegen), the former 3 were obtained from New England Biolabs, Beverly, Ma., U.S.A. The media for digestion contained: 100 mM Tris • HC1, 50 mM NaC1, 5 mM MgC12 and 0.1 mg/ml gelatine for Eco RI; 7 mM Tris • HC1, 60 mM NaC1, 7 mM MgC12 for Hind III; 6 mM Tris. HCI, 50 mM NaC1, 6 mM MgC12, 6 mM mercapto- ethanol and 0.1 mg/ml gelatine for Bam HI and 6 mM Tris • HC1, 6 mM NaC1, 7 mM MgCl 2 and 6 mM mercaptoethanol for Hap II. The pH of these media was 7.6. The final volume of about 50/~1 contained 3--5/2g mtDNA. Electrophoresis was performed using composite slabgels of 20 × 30 cm. In most cases the gels consisted of a small sealing layer of 10% polyacrylamide, a layer of about 8--10 cm of 3% polyacrylamide and a 20 cm layer of 0.7% agarose. In some experi- ments we used longer 10% and 3% polyacrylamide layers and only a small agarose layer to improve the detectability of very short restriction fragments. All further details were the same as described in our previous paper [2].

Iodination o f the RNAs Iodination of the various RNAs was performed according to Getz et al. [10]

in a final volume of 50 pl containing 0.1 M sodium acetate (pH 5.0), 62.5 pM KI, 2.3 mM TIC13, 5--20 #g RNA and 0.5--1.0 mCi of 125I (carrier free 033 L, New England Nuclear). Incubation was for 20 min at 60°C. After chilling to 0°C 1/zl of 0.1 M Na2SO3 and 50 #1 ammonium acetate (pH 9.3) were added and the incubation was continued for 20 min at 70°C. The 12SI-labelled RNA was purified by the procedure of Prensky et al. [11], slightly modified as described elsewhere [9,12].

Hybridization procedure The restriction fragments were denatured and transferred onto nitrocellulose

filterstrips (Sartorius, 0.45 /zm pore size) exactly as described by Southern [13]. The filters were baked at 80°C for 4 h. Hybridization was performed either in a vessel similar to that described by Southern [13] or in a glasstube of 200 mm length and a diameter of 6 mm. In the latter case the filter was inserted gently, one end was closed with a plastic stopper, the hybridization mixture (2.5 ml 3 × SSC, 0.2% SDS} was introduced, the wet filter was pressed down against the wall, the other end of the tube closed and the whole tube clamped tightly in a glass tray in a horizontal position. The whole assembly was sub- merged in a waterbath at 65°C with the axis in the shaking direction. The hybridization time was 19 h; the input DNA per gel slot was approx. 3/~g. After the hybridization the filters were washed three times with 2 × SSC at 25°C, once with 2 × SSC at 60°C and then treated with a mixture of 10 pg/ml

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DNAase-free RNAase A and 25 units/ml RNAase T1 for I h at 20°C. After the RNAase-treatment the filters were washed again three times with 2 × SSC at 25°C, dried and finally subjected to autoradiography for various times. The autoradiograms were aligned with a true size photograph of the gel. In some experiments the radioactivity associated with the various fragments was quanti- fied by liquid scintillation counting after cutting the stripfilters into pieces.

Fragment nomenclature The terminal fragments are designated by capital letters in order of increas-

ing electrophoretic mobility. In combination with the fragment designation letter, the following abbreviations have been used for the restriction enzymes: E for Eco RI, H for Hind III, B for Barn HI and Hap for Hap II. The new frag- ments in double digests are designated by arabic numerals in order of increasing electrophoretic mobility preceded by the combined abbreviations of the endo- nucleases used. Partial digestion products are designated by the abbreviation of the enzyme used, followed by the letter p and an arabic numeral in order of increasing electrophoretic mobility. This nomenclature was also used in our previous papers on rat-liver mtDNA [ 1,2].

Electron microscopy (A) Restriction endonuclease fragments were made after glyoxal fixation of

the D-loop as described by Brown and Vinograd [14]. These fragments were prepared for electron microscopy by a formamide modification of the basic protein film technique [15]. The spreading solution contained 2~g/ml of DNA, 60% formamide, 0.4 M ammonium acetate, 8 mM EDTA (pH 8.3) and 0.01% cytochrome c. For the spreading distilled water, redistilled twice over quartz, was used as hypophase.

The protein nucleic acid film was picked up on 400 mesh copper grids cov- ered with parlodion. Grids were dehydrated and stained by dipping into 5 • 10-: mM uranyl acetate, 5 • 10 -2 mM hydrochloric acid in 90% ethyl alco- hol for 30 s, followed by a brief rinse in isopentane. Finally they were rotary shadowed with platinum at an angle of 5 ° .

(B) The DNA-fragments cleaved by the restriction endonuclease Hap II {Fig. 7). were spread by absorption to charged carbon films and rotary sha- dowed with Pt at an angle of 5 ° as decribed by Bracket al. [16].

The specimens were examined in a Philips EM200. Pictures were taken at magnifications 5000--17000 on 35 mm Kodak FRP film. Calibration of mag- nification was done with a grating replica (Ladd. 2160 lines/mm). Length mea- surements were made with a Hewlett-Packard 9864A Digitizer connected to a 9820A calculator system. Full-length mtDNA molecules were added after enzymatic restriction as internal size standards.

Results and Discuss ion

Stripfilter hybridization o f mtDNA fragments with 16 S RNA o f mitochon. drial ribosomes

The sedimentation values of the two large ribosomal RNAs of the ribosomes from animal mitochondria are 16 and 12 S [5]. Stripfilters were prepared con-

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taining the fragments of complete and partial digests of mtDNA treated with the various restriction endonucleases. It was hoped that the method of strip- filter hybridization [13] should be useful and sensitive enough to indicate also the partials containing the genes of either o f t h e two ribosomal RNAs. The results of a number of these experiments are shown in Fig. 1. In this and in some of the further figures the ultraviolet photographs of the original slabgels are combined with the autoradiograms of the stripfilters after hybridization with the ~2SI-labelled RNAs. Fig. 1A shows that the Hind III fragments A and B contain sequences complementary to the 16 S rRNA. The relative amount of radioactivity bound to the fragment HB was 3 times higher than that bound to the fragment HA. Of the Hap II fragments (Fig. 1B) radioactivity was bound at the position of HapB, HapE and HapH. It should be noted that the HapH band contains 2 fragments of the same electrophoretic mobility. The only Eco RI fragment, binding the 16 S rRNA, was ED (Fig. 1C). The conclusion of these

A B C D E

Fig. 1. H y b r i d i z a t i o n of m i t o c h o n d r i a l 16 S r R N A wi th res t r i c t ion f r a g m e n t s of m t D N A . Str ipf i l ters con- ta in ing the d e n a t u r e d res t r i c t ion f r a g m e n t s of rat- l iver m t D N A were i n c u b a t e d wi th 12Si . iod ina te d 16 S r R N A f r o m rat- l iver m i t o c h o n d r i a l r i bosomes . F o r detai ls , see the Me th o d s sec t ion . The specific ac t iv i ty of 16 S r R N A was 1.4 - 106 cpm/Atg. The inpu t was 0 .56 • 106 c p m p e r fi l ter. Div ided over the var ious f r a g m e n t s , b e t w e e n 1.5 a nd 2.5% of the i npu t c o u n t s were b o u n d to the filters. P h o t o g r a p h s of the au to- r a d i o g r a m s al igned w i t h t rue size p h o t o g r a p h s of the original gels a re given. Th e a u t o r a d i o g r a m s are indi- c a t ed w i th the capi ta l le t ters . The pos i t ions of the var ious f r a g m e n t s visible on the original gels and au to- r a d i o g r a m s are i nd i ca t ed b y the smal l b lack lines. Solid lines: b an d s p re sen t on b o t h gel an d au to rad io - g r am; b r o k e n lines: b a n d s p r e s e n t only in gel. The length of the var ious f r a g m e n t s is g iven in the Tables I and I I . Q u e s t i o n m a r k s indica te the pos i t ions of bands of par t ia l d iges t ion p ro d u c t s , wh ich are n o t f u r t h e r cha rac t e r i zed . A, h y b r i d i z a t i o n wi th a Hind I I I digest ; B0 h y b r i d i z a t i o n wi th a Hap II digest; C, hybr id iza - t i on w i t h an E co RI digest; D, h y b r i d i z a t i o n wi th an i n c o m p l e t e Eco R I digest ; E, h y b r i d i z a t i o n wi th a d o u b l e digest of Eco RI a nd Hind I I I .

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experiments is that the Hind fragments A and B are adjacent and that the cleav- age site between these 2 fragments is located on Eco RI fragment D. In the same area the Hap II fragments B, E and H (one or both) are situated. In Fig. 1D the hybridization pattern of 16 S rRNA and the fragments of a partial digest with Eco RI are shown. The partials previously indicated as E p l l and Epl2 (2) bind 16 S rRNA and do, therefore, contain ED. Epl0 is not labelled, but radioactivity is found at a position between EC and Epl0. This partial digestion product was not previously detected and interpreted as such. Another interesting feature is the presence of an ED containing partial at the position of EA: it is clearly not EA itself that is labelled as can be concluded from Fig. 1C.

To substantiate the conclusion that Eco RI fragment D contains a Hind III cleavage site hybridization was performed using a stripfilter containing the frag- ments of mtDNA digested with both Eco RI and Hind III (Fig. 1E). The frag- ments EH3 and EH6 (not previously detected) are labelled. These fragments derive from HB and ED and from HA and ED respectively. This was shown in separate experiments (cf. Table II). The bands just below HC are considered to be the partial digestion products comparable to E p l l and Epl2. The data ob- tained with double digests of Eco RI or Hind III with Barn H1 are notshown. The larger part of the HA fragment (HB1, cf. Table II) and the smaller fragment of EA (EB2, cf. Table II) contained the sequence complementary to the 16 S rRNA.

The findings presented above are different from those presented earlier [ 1,2]. In the previously published [1] map the Hind III fragments A and B were sepa- rated by the fragment HD. If we reconsider our earlier hybridization data, it can be seen that the fragment HA has indeed bound some RNA but in the sys- tem used at that time the binding was not significantly different from the back- ground. The procedure of iodination used in the present study, however, led to much higher specific radioactivity of the RNAs. A strong argument for the pre- vious ordering of fragments was the pattern of partial digestion products with Eco RI. We have now detected still another partial digestion product with a lower molecular weight than EB (designated Epl0a in Fig. 1D). The only reasonable manner to explain the high number of partials smaller than EB is to assume that more than two small Eco RI fragments have to be present. Once one accepts this assumption it is clear that the reasoning for the previous order- ing of fragments becomes invalid.

The presence o f more than two small Eco R I fragments As outlined in the preceeding paragraph, the only possible explanation for

the results of the stripfilter hybridization experiments with 16 S rRNA impli- cates the presence of more than 2 small Eco RI fragments. By overloading the gelslots and by using a different type of composite slabgel we were, indeed, able to detect two smaller fragments with a length of less than 150 basepairs each. With the same technique the fragment HF and the fragment HapI and HapJ could be visualized. The existence of HF was previously deduced from the presence of a partial digestion product consisting of HE plus HF [2]. Photo- graphs of the gels containing these smaller fragments are shown in Fig. 2. When the restriction endonucleolytic digestion was incomplete, we were able to detect faint bands of partial digestion products as well. These are shown in Fig.

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A B C D

Fig. 2. Slabgel e lec txophoreses of r e s t r i c t ion f r a g m e n t s of m t D N A . The c o m p o s i t i o n of the gels was as fol- lows: a l ayer of 1 0 - - 1 5 c m of 10% p o l y a c r y l a m i d e , a l aye r of 15 - -10 c m of 3% p o l y a c r y l a m i d e an d a layer of a b o u t 5 c m of 0 .7% agarose . To d e t e c t smal l f r a g m e n t s the gels we re l oaded wi th 2 0 - - 2 5 Dg m t D N A per slot . A and B are par t s f r o m the s a m e gel, C a nd D are t a k e n f r o m t w o d i f f e r en t gels. T h e pos i t ion of the va r ious f r a g m e n t s is i nd i ca t ed wi th b lack lines; the i n t e r r u p t e d lines ind ica te the pos i t ion of par t ia l d iges t ion p r o d u c t s . F o r f u r t h e r detai ls see the M e t h o d s sec t ion an d ref . 2. A, a H ind I I I digest; B and C, Eco R I digests; D, a H ap I I digest .

2B and C. The fragment lengths of the smaller fragments were roughly esti- mated by extrapolation, using the fragments EE, EF, HE and HF as the mark- ers. In this way we arrived at lengths of 120 and 100 basepairs for the frag- ments EG and EH, respectively. The pattern of partials was consistent with these molecular weights and with a clustering of these fragments in one area of the genome, in between the Eco RI fragments D and C. The most likely order in this cluster is EHFG. The inferred composition of the partials indicated (Fig. 2B and C) is as follows: Epl3 : EE + EF + EG + EH; Epl4 : EE + EH; Ep15: EF + EG + EH; Ep l6 : EF + EG; Epl7 : EF + EH. The order of all Eco RI frag- ments becomes, than: ADEHFGCBA.

The lengths of all fragments obtained by single and double digestion of rat- liver mtDNA with the four restriction enzymes are summarized in Tables I and II. The data of double digestion are obtained in two ways. In the first place total mtDNA was incubated with two different enzymes in two steps of incuba-

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T A B L E I

L E N G T H OF T H E R E S T R I C T I O N E N Z Y M E F R A G M E N T S OF R A T - L I V E R M I T O C H O N D R I A L D N A

For exper imenta l details and nomenc la ture see the Methods sect ion.

Barn HI fragments

M X 10 -6 Basepairs

Eco RI fragments

M × 10 -6 Basepairs

BA 6 .65 10 000 BB 2.96 4 450

9.61 14 450

Hap II fragments

M X 10 -6 . Basepairs

HapA 2.32 3 500 HapB 1.89 2 850 HapC 1.16 1 750 H a p D 1.08 1 630 H a p E 1.03 1 550 H a p F 0 .74 1 120 H a p G 0 .64 970 HapHa 0 .45 680 HapHb 0 .45 680 HapI 0 .12 180 H a p J 0 .07 110

9 .95 15 020

EA * 3 .64 5 500 EB * 2.36 3 550 EC * 1.76 2 650 ED * 1.20 1 800 EE 0 .43 6 5 0 EF 0 .27 400 EG 0 .08 125 EH 0.07 100

9.81 14 775

Hind III fragments

M X 10 -6 Basepairs

H A * 3.96 5 950 HB * 2 .50 3 750 HC * 1.53 2 3 0 0 HD * 1.26 1 9 0 0 HE 0 .53 8 0 0 H F 0 .10 150

9.68 14 850

* Fragments containing t R N A genes (see text) .

tion without isolation of the various fragments. In the second place the frag- ments of an incubation with one enzyme were separated on gels and reisolated for digestion with the second enzyme. For this purpose we used slabgels with large 3% polyacrylamide layers, because in our hands the method of fragment extraction from polyacrylarnide with buffer as used by Van den Hondel and Schoenmakers [17] gave a better yield of fragments than the other methods tried [ 18,19].

The localization o f the 12 S ribosomal RNA gene We have shown previously [1], that the gene for the 12 S rRNA of mito-

chondrial ribosomes is localized on a part of the genome that is shared by the fragments A of Eco RI, Hind III and Bam HI. This observation could be con- firmed with the stripfilter hybridization approach. With the 12 S rRNA we met the problem of contamination of the RNA preparation with fragments of 16 S rRNA. In most of the experiments 16 S rRNA was added as competitor RNA. Fig. 3 shows the gels and autoradiograms of 12 S rRNA hybridized to filters containing the mtDNA fragments after digestion with Eco RI, Hind III and Hap II. In the experiments with Hind III and Hap II no competitor 16 S rRNA was present in these cases. The main hybridization is with the fragments HA (Fig. 3B) and HapE (Fig. 3C). In separate experiments (not shown) it was ob- served that the 12 S rRNA hybridized with fragment Barn A. From experiments

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T A B L E II

L E N G T H O F T H E N E W R E S T R I C T I O N F R A G M E N T S O F R A T - L I V E R M I T O C H O N D R I A L D N A

O B T A I N E D IN D O U B L E D I G E S T S W I T H D I F F E R E N T E N D O N U C L E A S E S

R a t - l i v e r m t D N A was d i g e s t e d in t w o succes s ive i n c u b a t i o n s w i t h a c o m b i n a t i o n o f t w o of t h e r e s t r i c t i o n

e n z y m e s E c o R I , H i n d I I I , Barn HI a n d H a p II . T h e r e s t r i c t i o n f r a g m e n t s o f e i t h e r e n z y m e , n o t c l e a v e d b y

t h e o t h e r are n o t g i v e n in t h i s t a b l e . In a s e p a r a t e se r ies o f e x p e r i m e n t s t he r e s t r i c t i o n f r a g m e n t s we re iso-

l a t e d a f t e r d i g e s t i o n w i t h t h e f i r s t e n z y m e a n d s u b s e q u e n t l y f u r t h e r d i g e s t e d w i t h a s e c o n d e n z y m e . F o r

f u r t h e r d e t a i l s a n d n o m e n c l a t u r e see t h e M e t h o d s s e c t i o n .

E c o R I + Barn HI H i n d I I I + Barn HI Barn HI

f r a g m e n t b a s e p a i r s f r a g m e n t h a s e p a i r s f r a g m e n t

EB 1 * 1 9 5 0

E B 2 * 2 6 0 0

E B 3 * 1 9 5 0

E B 4 1 6 5 0

EB1 + E B 2 = E A + HB1 + H B 2 = H A +

E B 3 + E B 4 = EB ÷ H B 3 + H B 4 = HC ÷

E B I + E B 4 = BB +

+ Hap II

b a s e p a i r s

HB1 * 3 0 5 0 B H a p l 3 1 0 0

H B 2 * 2 9 0 0 B H a p 2 1 2 2 0

H B 3 1 5 5 0 B H a p 3 5 4 0

H B 4 * 9 2 0 B H a p 4 4 3 0

B H a p l + B H a p 4 = H a p A

B H a p 2 + B H a p 3 = H a p C

Eco R I + H i n d I I I Ec o R I + Ha p II

f r a g m e n t ba sepa ix s f r a g m e n t b a s e p a i r s

E H 1 * 5 4 5 0 E H a p l 2 0 5 0

E H 2 * 1 4 5 0 E H a p 2 1 5 0 0

E H 3 * 1 4 0 0 E H a p 3 1 3 6 0

E H 4 * 1 2 5 0 E H a p 4 1 3 6 0

E H 5 3 7 0 E H a p 5 9 0 0 E H 6 3 5 0 E H a P 6 4 1 0

E H a p 7 2 6 0 E H 1 + E H 6 = H A +

E H 2 + E H 5 = H D + E H a p l + E H a p 2 = H a p A *

E H 2 + E H 4 = EC ÷ E H a p 4 + E H a p 7 = H a p D +

E H 3 + E H 6 = E D + H a p B c o n t a i n s EE , E F ,

E H a p 3 a n d E H a p 6 +

H a p E c o n t a i n s E H a p 5 a n d

a f r a g m e n t o f 6 8 0 bP*

H i n d I I I + Hap II

f r a g m e n t h a s e p a i r s

H H a p l 1 3 6 0

H H a p 2 1 3 2 0

H H a p 3 1 2 7 0

H H a p 4 9 5 0

H H a p 5 2 7 5

H H a p 6 1 6 0

H H a p 3 + H H a p 5 = H a p E +

H H a p 4 + H H a p 6 = H a p F ÷

H a p A c o n t a i n s H H a p l , H H a p 2

a n d H E +

+ I n d e p e n d e n t l y c o n f i r m e d b y d i g e s t i o n o f i s o l a t e d t e r m i n a l r e s t r i c t i o n f r a g m e n t s .

* F r a g m e n t s c o n t a i n i n g m t R N A genes .

with double digests of Bam H1 with either Hind III or Eco RI in the presence of competi tor , it could be concluded that the 12 S rRNA hybridized exclu- sively with the larger part of HA (HB1, cf. Table II) and the smaller part of EA (EB2, cf. Table II). This observation is compatible with the conclusion reached above, that the cleavage site between EA and ED lies on HA. In the cases of Hind III and Hap II (Fig. 3B and C) we need some correction for the contribu- tions of (fragments of) contaminating 16 S rRNA. Comparison with Fig. 1A and B shows that the fragments labelled are the same (HA and HB; HapB, HapE and HapH). The amount of radioactivity bound to the various fragments was quanti tated. The radioactivity found on 12 S rRNA-stripfilters was corrected for 16 S rRNA contamination assuming that all counts bound to HB and HapB were to be ascribed to the 16 S rRNA contaminants and further assuming the same relative contribution of the contaminants to all labelled fragments. In both cases this led to the conclusion that about 50% of the counts were due to 16 S tRNA contamination. It further revealed that the fragments HapE and HapH con- tain sequences complementary to the 12 S rRNA, about 5/6 of the gene lying on

137

Fig. 3. H y b r i d i z a t i o n o f m i t o c h o n d r i a l 1 2 S r R N A w i t h r e s t r i c t i o n f r a g m e n t s o f m t D N A . F o r de ta i l s see t h e M e t h o d s s e c t i o n a n d the l e g e n d to Fig . 1. The spec i f ic a c t i v i t y o f t he 12 S r R N A w a s 1 2 8 5 0 0 c p m / D g (a) a n d 1 5 6 0 0 0 cpm//~g (B a n d C). The i n p u t w a s 4 4 0 0 0 c p m (A) a n d 1 4 0 0 0 0 c p m (B a n d C) p e r f i l ter . 1 . 1 , 11 a n d 2 .5% o f t he i n p u t c o u n t s we re h o u n d t o t h e f i l te rs in A , B a n d C, r e spec t i ve ly . 1 0 - f o l d excess c o m p e t i t o r 1 6 S r R N A w a s p r e s e n t in the e x p e r i m e n t i l l u s t r a t e d in A , b u t n o t in t h o s e o f B a n d C. P h o t o - g r a p h s o f t h e a u t o r a d i o g r a m s ( i n d i c a t e d b y the cap i t a l l e t t e r s ) a l i gned w i t h t r u e Size p h o t o g r a p h s o f t h e o r ig ina l gels a re g iven . A , h y b r i d i z a t i o n w i t h a n E c o RI d iges t : B, h y b r i d i z a t i o n w i t h a H i n d III d iges t ; C, h y b r i d i z a t i o n w i t h a H a p II d iges t .

HapE. Since HapE also contains about half of the 16 S rRNA gene it follows that the two ribosomal RNA genes are very closely linked on the mtDNA, leav- ing a gap in between the two rRNA cistrons of only about 200 basepairs. The data for this calculation are given in Table III. Taking the number of basepairs of HapH as 44% of the length of the 16 S rRNA we arrive at a molecular weight equivalent to 1500 ribonucleotides which is close to the value of 0.52 • 106 ob- tained by Kleinow [20] for Locusta mt~ribosomes and of 0.54 • 106 by Hamil- ton and O'Brien [21] for bovine mt-ribosomes, but lower than the value of 0.65 • 106 reported by Groot et al. [22]. The calculated space occupied by the 16 S rRNA on the mtDNA by this method is in excellent agreement with elec- tron microscopical observations on mtDNA • mtRNA hybrids [3,4].

1 3 8

T A B L E II I

Q U A N T I T A T I O N OF T H E S T R I P F I L T E R H Y B R I D I Z A T I O N W I T H T H E 16-S A N D 12-S R N A s F R O M M I T O C H O N D R I A L R IBOS OM ES

S o m e of the s t r ipf i l ters s h o w n in Figs. 1 and 3 were cu t in pieces and assayed in to luene con ta in ing 0.4% PPO and 0:01% POPOP in a l iquid scint i l la t ion c o u n t e r . R e g i o n s o f the f i lters w i t h o u t DNA were used to o b t a i n b a c k g r o u n d values. The rad ioac t iv i ty in areas wi th label led b an d s was c o r r e c t e d for this back- g round . Fo r f u r t h e r detai ls , see the legend to Figs. 1 and 3, the M e t h o d s sec t ion and the t e x t .

Res t r i c t ion f r a g m e n t s hybr id iz ing with m t r R N A

H A HB HapB H a p E H a p H a H a p H b

F r a g m e n t l ength [1 ] in basepai rs 5 9 5 0 3 7 5 0 2850 1550 680 680

16 S r R N A (Fig. 1A and 1B) % h y b r i d i z a t i o n (p) 24 76 5 J 51 44 0

p / l X 103 n.r. n.r. 2 33 65 n.r.

12 S r R N A (Fig. 3B and C) % h y b r i d i z a t i o n

b e f o r e c o r r e c t i o n for c o n t a m i n a t i n g 16 S r R N A 61 39 2.5 68 *--29.5-* after c o r r e c t i o n for c o n t a m i n a t i n g 16 S r R N A 4 9 ( 1 0 0 ) 0 0 4 2 ( 8 4 ) 0 8 (16)

n.r. = n o t re levant .

The origin and direction of replication: a confirmation o f the fragment map After having localized the genes for the 2 risobomal RNAs on the mtDNA,

we were interested to search for other markers. Using standard techniques we were able to show that the origin of replication, visible as the D-loop, was local- ized on the fragment EA (Fig. 4A) and HA (Fig. 4B). It further appeared that the D-loop overbridged a region of the mtDNA containing a cleavage site for Hap II. Typical H-form molecules as described by Brown and Vinograd [14] were found in the Hap II digest (Fig. 4D). The D-loop was also found in partial digestion products of Hind III (Fig. 4C), Eco RI (not shown) and Hap II (Fig. 4E and 4F). From length measurements of a large number of different mole- cules it was concluded that the two Hap II fragments present in H-form mole- cules are HapC and one of the HapH fragments. By digestion of isolated Bam-, Eco- and Hind-fragments with Hap II we knew already that one of the Hap H fragments was localized on BA/EA/HA and the other on BA/ED/HB. Since the fragments HapC and HapA are cleaved by Bam H1 we coulc decide that the D-loop is localized quite close to the rRNA genes. In fact either the origin or the growing replication fork reside on the same fragment (HapHb) as part of the 12 S rRNA gene. The left hand parts of the EA and HA fragments illus- trated in Fig. 4A and 4B represent the parts of the mtDNA that contain the sequence compelentary to the 12 S rRNA. We further looked for molecules containing expanding D-loops. In Fig. 5 two HA fragments containing D-loops of different length are shown. It can be seen that the left hand parts are con- stant and the right hand parts are variable in length. The direction of replication is, therefore, from left to right, off from the region containing the ribosomal RNA genes. The fragment HapHb, therefore, contains the origin of replication,

139

Fig. 4. E lec t ron mic rographs of m t D N A f ragments bear ing a D-loop. Details o f the t r e a t m e n t of the m t D N A for e lec t ron mic roscopy are given in the Methods sect ion. The magni f ica t ion is the same for all molecules depic ted . The bar is 0.5 ~m. A, f r agmen t EA; B, f r agmen t HA; C, a par t ia l digest ion p r o d u c t of Hind III; D, the f ragments HapC a n d HapH, i n t e r connec t ed by the single DNA s t rand o f the D-loop (cf. ref. 14); E, a par t ia l d iges t ion p r o d u c t of Hap II wi th the length o f HapC plus HapH; F, 2 par t ia l digest ion p r o d u c t s of Hap II, i n t e r connec t ed b y the single DNA-s t rand of the D-loop.

1 4 0

Fig. 5. The e x p a n d i n g D- loop o f ra t - l iver m t D N A . T w o H A f r a g r a e n t s are s h o w n b e a r i n g D- loops of d i f fe r -

en t l eng th . T h e m a g n i f i c a t i o n is t he s a m e fo r A a n d B. The ba r is 0 . 5 / ~ m .

the fragment HapC the part of the D-loop that will act as the growing replica- tion fork, if replication proceeds.

Using the D-loop as a marker we have constructed the physical map of Hind III fragments by measuring the length of a large number a complete and partial digestion products, all containing the D-loop. The results are shown in Fig. 6. The only order of Hind III fragments compatible with the results of these mea- surements is ABDC(EF)A. This order is the same as the order arrived at by means of the fragment analyses and hybridization experiments reported above.

i I

38 'n 3 I

3

2 b o h 1 I o o h 2

b

b 19 b ~ ~ 3 I

86

11o1

I I~I~HclHol HB I ,HA I ~ H C l H O l HB I It , , , , = A i i , i i i i i i i i i

4 3 2 1 0 1 2 3 4 5 ( ~ m )

E Fig. 6. T h e o r d e r o f H i n d I I I f r a g m e n t s of ra t - l iver m t D N A as ba sed on e l ec t ron m i c r o s c o p i c a l l eng th m e a - s u r e m e n t s , m t D N A w a s t r e a t ed w i t h g lyoxa l a n d par t i a l ly d ige s t ed w i t h H i n d I I I . Molecu les c o n t a i n i n g D- loops w e r e m e a s u r e d . T h e f igure s h o w s the bes t f i t t i ng m u t u a l r e l a t ion b e t w e e n the m o l e c u l e s o f vari- ous l eng th . T h e r e s u l t i n g f r a g m e n t o rde r is i n d i c a t e d at t he b o t t o m p a r t o f t he f igure . T h e c o l u m n at the r i g h t i nd i ca t e s the n u m b e r o f m o l e c u l e s scored f o r each l eng th c a t e g o r y . Of t w o t he o re t i c a l l y poss ib le l e n g t h classes, no e x a m p l e was f o u n d . T h e n u m b e r 10 b e t w e e n b r a c k e t s i n d i c a t e s t he n u m b e r of mole - cules n o t f i t t i n g the p ic tu res . Mos t o f these were m o l e c u l e s b r o k e n ve ry close to the D- loop .

141

The method is not sensitive enough to decide on the order of HE and HF rela- tive to HA and HC. This sequence was already known from previous fragment analyses [2]. A similar approach for the construction of the restriction frag- ment map has been used by Upholt and Dawid for sheep and goat [23]. Recently also Koike et al. [24] have published the results of such an approach for the Eco RI fragments of rat-liver mtDNA. The fragment order is identical to the one presented in this paper. Unfortunately the details of their work are neither described in their paper nor presented at the conference to the proceed- ings of which the paper belongs. The fragments EG and EH were not detected by these authors.

The construction of the restriction fragment map of Hap H fragments Complete digestion of rat-liver mtDNA with the endonuclease Hap II gave

rise to 8 bands on standard agarose-polyacrylamide gels. In this pattern the fragment HapH was clearly present twice on a molar basis. By using gels with a 10% polyacrylamide layer and by overloading these gels another two bands rep- resenting smaller fragments were detected. Furthermore, the two HapH frag- ments can be separated under these conditions (see Fig. 2D). Also in the prepa- rations used for electron microscopical length measurements, a large number of fragments smaller than HapH could be detected as can be seen in Fig. 7. We estimate the length of these fragments in the order of 100 and 200 basepairs. With the exception of these two smaller fragments we have been able to con- struct the map of the Hap II fragments as well. The map is based on the hybrid- ization and electron microscopical experiments described above and further

60

_w

E

i E

2O

o; a2

h~:lp H

HopF ÷

I~pG

Hop C

Hop D ~E

HopB

0.6 08 10 1.2 frogment length (Hrn)

<- . . . . . Hap partiais . . . . . . >

Fig. 7. L e n g t h d i s t r ibu t ion of the fragments of rat- l iver m t D N A after endonuc l eo ly t i c digest ion w i t h Hap II . M e t h o d B (see Me t ho ds sec t ion) was used for spreading the f r agmen t s . A to ta l of about 4 0 0 molecu le s was scored. The fragment length was ca lcu la ted re la t ive to the length o f open ci rcular molecu les . 1 Mm was equ iva len t to 2 9 8 0 bascpairs u n d e r these e x p e r i m e n t a l condi t ions .

142

on the digestion of isolated Bam HI , Eco RI and Hind III fragments with Hap II. The data are included in Tables I and II. The order of fragments is: ADFBHaEHbCGA.

Stripfilter hybridization of mtDNA fragments with mitochondrial tRNAs and poly(A )-containing RNA

The 4 S RNA fraction from rat-liver mtRNA was isolated and iodinated with ~2sI. Stripfilter hybridization was carried out in the presence of excess mito- chondrial rRNAs as competitor. The results are shown in Fig. 8. On the filters containing the fragments of single digests as well as on those with the fragments of double digests most of the fragments coincide with spots on the autoradio- grams. It is clear that fragment ED (Fig. 8A) is very weakly labelled. The small- est amount of radioactivity bound to any fragment of the single and double digests with Eco RI, Barn HI and Hind III corresponded to about 5--6% of the total radioactivity bound. Assuming an even distribution of the iodine-label over the various tRNAs, and the presence of the various tRNAs in about equi- molar amounts, the lower estimate for the number of tl~NA genes on rat-liver mtDNA is in the order of 16--20. This is in good agreement with the number of tRNA genes so far recognized on the mtDNA from other animal sources [3,4].

Fig. 8. H y b r i d i z a t i o n of t o t a l m i t o c h o n d r i a l t R N A w i t h r e s t r i c t i o n f r a g m e n t s of m t D N A . F o r de ta i l s see the M e t h o d s s ec t i o n and the l egend to Fig . 1. The spec i f ic ac t i v i t y of t he t R N A p r e p a r a t i o n was 275 0 0 0 c p m / ~ g . The i n p u t was 180 0 0 0 c p m pe r f i l ter . A b o u t 0 .4% o f the i n p u t c o u n t s w e r e b o u n d to the f i l ters . C o r r e c t i o n s were m a d e for the b a c k g r o u n d o f a b o u t 10 c p m Per m m f i l t e r l eng th . 15- fo ld excess c o m - p e t i t o r 16 S + 12 S r R N A was p r e s e n t in the h y b r i d i z a t i o n m e d i u m . P h o t o g r a p h s of the a u t o r a d i o g r a m s ( i n d i c a t e d by the cap i t a l l e t te rs ) a l igned w i t h t rue size p h o t o g r a p h s of t he or ig ina l gels are given. A° hy- b r i d i z a t i o n w i t h an Eco R I d iges t ; B, h y b r i d i z a t i o n w i t h a H i n d I I I d iges t ; C, h y b r i d i z a t i o n w i t h a doub le d iges t o f H i n d I I I a n d B a m H1; D, h y b r i d i z a t i o n w i t h a d o u b l e d iges t o f Eco RI and Barn HI ; E, h y b r i d i z a - t i o n w i t h a d o u b l e d iges t o f Eco R I a n d H i n d I I I .

143

The fragments that were found to be able to bind tRNAs in the presence of compet i tor rRNA are marked with an asterisk in Tables I and II. As a general conclusion it appears that, also on mtDNA of the rat, the tRNA genes are scat. tered on the mitochondrial genome.

A similar random distribution was found for the poly(A)-containing mito- chondrial RNA. In this respect the stripfilter hybridization confirmed our pre- vious data [1]. However, if mitochondrial rRNAs and tRNAs were added as competitors, it was observed that the binding of the poly(A)-containing RNA to the fragments containing the rRNA-complementary sequences was consider- ably reduced. This was specially clear for fragment ED. Although contamina- tion with these RNAs may offer a simple and seemingly straightforward expla- nation, it may be recalled that mitochondrial transcription is thought to be a symmetrical process initially giving rise to large transcription products [25,26] , which may well contain the ribosomal RNA cistron and, moreover, a stretch of poly(A) at their 3' end [4,27]. These alternatives as well as the precise localiza- tion of specific mt- tRNAs are presently under investigation.

Concluding remarks

The data presented in this paper are summarized in Fig. 9. Our data show that it is very difficult to rely on the inferred composit ions of partial digestion products. This is especially true if the conditions of electrophoresis are such

Fig. 9. T h e re s t r i c t i on f r a g m e n t m a p o f rat-liver m t D N A a n d the p o s i t i o n o f a n u m b e r o f g e n e t i c m a r k e r s o n this m a p .

144

that small fragments and some partials can be missed or overlooked. This occurred to us in our original study, although at that t ime we already found 6 fragments with the enzymes Eco RI and Hind III, a number about twice as high as described at that t ime for mtDNA of other animal species [14,28]. However, Upholt and Dawid have described the presence of 5 cleavage sites for Eco RI in mtDNA of a goat [23]. Also Koike et al. have recently raised their number of Eco RI fragments of rat-liver mtDNA, in their case from 5 to 6 [24]. Unfortunately the gels with their partial digests, on which their experiments are partly based, have not ye t been published. Similar movements of fragments in the fragment maps of mtDNA have also been made for yeast by Sanders et al. [29] and N. crassa by Kiintzel et al. [30,31] .

In conclusion, the combined approach of electrophoretic restriction frag- ment analysis, stripfilter hybridization with marker RNAs, and electron micro- scopical length measurements has led to a consistent physical characterization. It has further revealed possibilities for the isolation of interesting parts of the mitochondrial genome. It is especially intriguing to further characterize the origin of replication. This is quite well feasible because the Hap Hb fragment can be easily obtained by Hap digestion of Hind III fragment A or by separa- tion from HapHa on 10% polyacrylamide gels. This fragment as it contains a replication point may well be interesting also from the viewpoint of genetic engineering.

Acknowledgements

These studies were supported in part by a grant to AMK from the Nether- lands Foundat ion for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO) and by a grant to CS from the Consiglio Nazionale delle Ricerche (CNR), Italy. The authors are indebted to Dr. Annika Arnberg for interest and advice, to Theo Deddens for drawing the figures and to Bert Tebbes and Klaas Gilissen for the photography.

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