Nucleic Acids Research
Volume 4 Number S 1977
Microheterogeneity detected in circular dimer mitochondrial
DNAt
D. L. Robberson,* C. E. Wilkins,* D. A. Clayton+ and J. N. Doda+
*The University of Texas System
Cancer Center, M.D. Anderson Hospital and Tumor Institute Houston, TX 77030 and +Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA Received 7 January 1977
ABSTRACT Exhaustive EcoRI digests of circular dimer mitochondrial DNA (mtDNA) from mouse cell lines LD and LDTK- yield two major fragments whose average lengths are slightly smaller than the corresponding fragments of circular monomer mtDNA from mouse LA9 and LMTK cells. A third fragment approximately 400 nucleotide pairs in length is frequently produced in less than molar yield. Exhaustive EcoRI digests of circular dimer mtDNA from human acute myelogenous leukemiic leucocytes yield three major fragments. The presence of mtDNA resistant to cleavage as well as fragments of intermediate sizes indicatesmicroheterogeneity in the genomic positions of EcoRI recognition sequences in both mouse and human circular dimer mtDNA. Analysis of the distribution averages of circular contour lengths indicates microheterogeneity in the sizes of mouse LD and human mtDNAs. The denatured-renatured EcoRI fragments frequently contain a small loop(s) of single-strand DNA as would occur for deletion(s) or addition(s) of nucleotide sequences in some of the circular dimer molecules.
INTRODUCTION Covalent closed circular duplex DNA of mammalian mitochondria can be found to occur in oligomeric forms (1,2). In one type of oligomer, the monomer genome has been duplicated (1) and joined in head-to-tail fashion to form a circulardimer (CD) molecule (3). Circular dimers are found at only very low (0.1-0.3%) (4) or nondetectable frequencies (5) in healthy mammalian tissues but constitute significant proportions of the mtDNA population in leucocytes of human myelogenous leukemias (6) as well as in a variety of solid tumor tissues (7). Association of CD mtDNA with human malignancies has focused attention on the fine structure differences that may distinguish it from circulr monomer (CM) mtDNA. A previous study using electron microscopic and density gradient techniques applied to heteroduplexes between human CD and CM mtDNAs demonstrated that more than 90% sequence homology was shared by monomer and dimer forms (3). C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
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Nucleic Acids Research The application of restriction endonucleases to studies of mammalian mtDNA structure has provided an additional means of assessing sequence heterogeneity among a population of molecules. For example, the mtDNAs from two strains of human HeLa cells differ in their content of sequences sensitive to cleavage by the Hae III (8,9) restriction endonuclease(l0). Earlier studies of the EcoRI (9,11) cleavage pattern of human HeLa cell mtDNA (12,13) had revealed the presence of subpopulations of fragments resulting from cleavages in addition to those which define the majority population of linear duplexes (12). Also, in the case of EcoRI digests of mouse L cell mtDNA (12,13), a minor population of molecules was found which were cleaved at only one site or a few closely spaced sites on the genome (Fig. IA and 1B, reference 12). It was suggested that these molecules constitute a minor population of CM mtDNA in which at least one of the EcoRI cleavage sequences had been deleted or altered, thus giving rise to the apparent resistance to further cleavage under the exhaustive digestion conditions employed (12). The observation that some of these molecules appeared slightly shorter than one genome length also suggested the possibility of reiteration of the EcoRI cleavage sequence within the regions that defined the two major cleavage fragments of CM mtDNA from mouse cells (12). In plasmid recombinant DNA experiments, Brown and Vinograd (14) subsequently identified a third EcoRI fragment of mouse L cell mtDNA with a length of 160+10 nucleotide pairs (np). These combined studies have indicated a limited degree of heterogeneity in CM mtDNA with respect to EcoRI (12) and Hae III (10) cleavage sequences. In the study presented here, we find a similar level of microheterogeneity of EcoRI cleavage sequences among circular dimer mtDNAs and in addition detect microheterogeneity in the sizes of circular dimer molecules.
MATERIALS AND METHODS Preparation of mitochondria and mtDNA. Mitochondria and closed circular mtDNAs were isolated from mouse cell lines LD and LDTK growing exponentially in suspension culture by procedures previously described (15). The mitochondria and closed circular mtDNAs of human leucocytes were isolated from the peripheral blood of two patients with AML prior to the administration of chemotherapy or other therapeutic regimens essentially by procedures described earlier (6) with elimination of the DNAase I and RNAase A treatments of purified mitochondrial fractions (15). Isolated closed circular mtDNAswere rebanded in buoyant CsCl gradients containing
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Nucleic Acids Research saturating levels of ethidium bromide (16). After collection of lowerbands ethidium bromide was removed by dialysis against Dowex cation exchange resin as described (17). EcoRI cleavage of mtDNAs. Purified closed circular mtDNAs were dialyzed against either 0.1 M Tris, .01 M MgCl2, pH 7.6 (low salt digestion) or 0.1 M NaCl, 0.1M Tris, .01 MgCl2, pH 7.6 (high salt digestion). Each digestion mixture (75 -pl) contained 0.05-0.1 pg of DNA to which was added 1 pl of EcoRI and the sample incubated at 370C for 15 min. A second 1 pl aliquot of EcoRI was then added and the incubation continued for an additional 15 min. The digestion was then terminated by addition of 20 ul of 0.25 M disodium EDTA previously adjusted to pH 8.0 with sodium hydroxide and the sample was then dialyzed against STE (0.1 M NaCl, 0.05 M Tris, 0.01 M disodium EDTA, pH 8.5) (15) essentially as described (12). One microliter of EcoRI, prepared by the method of Yoshimori (11), would cleave 1 1ig of simian virus 40 DNA using similar reaction conditions. EcoRI was the kind gift of Dr. William R. Folk. Electron Microscopy. Samples were prepared for electron microscopy as described (12) before or after EcoRI treatment using the basic protein Kleinschmidt technique in the presence or absence of formamide (18) with the modifications previously noted (19,20). Homoduplexes of EcoRI treated mtDNA were constructed with renaturation conditions chosen to permit reassociation of 40-70% of the largest single-strands in a particular sample (21). The samples were then prepared for electron microscopy as described (21). Throughout these studies we have added PM2 DNA as an internal length standard just prior to spreading of the sample. Grids were rotary shadowed with Pt-Pd and examined in a Philips 300 electron microscope. Length measurements were made from tracings of enlarged images photographed on 35 mm film utilizing either a map measure or a Numonics digitizer. RESULTS Lengths of CD mtDNAs prior to EcoRI treatment. Examination of purified mtDNAs by the basic protein Kleinschmidt technique permits measurements of their contour lengths relative to an internal standard of PM2 DNA. The results of length measurements determined relative to this internal standard of PM2 DNA are presented in Table 1 for spreadings in the absence (aqueous technique) or presence of formamide (18) as described (19,20). The average lengths of CD mtDNAs from mouse or human sources are similar to each other and are approximately twice the size of average 1317
Nucleic Acids Research TABLE 1. Sizes of Circular Dimer Mitochondrial DNAs Determined by Electron M icroscopy.
Average Contour Length Source of mtDNA Mouse Line LD Mouse Line LD Mouse Line LDTKHuman AML Leucocytes, Sample 1 Human AML Leucocytes, Sample 2
Spreading Procedure
± Standard Deviation
In Nucleotide Pairs*
Number of Molecules Measured
Aqueous Formamide Aqueous
31,820 + 580 32,580 1,460 32,270 550
39 37 43
Aqueous
32,610
700
41
Formamide
31,450 ± 1,300
31
* Contour lengths were determined relative to an internal standard of PM2 DNA with an average length taken as 9,900 nucleotide pairs.
lengths measured for the corresponding CM mtDNAs from mouse LA9 (12,13) and LMTKI cells (12) or human HeLa cells (12,13) determined relative to the same internal standard of PM2 DNA (12). Examination of contour lengths after spreading in formamide permits assessment of closed circular displacement replicative forms (20) whose presence, unaccounted for in the sample, could have lead to incorrect estimates of average contour length determined by the aqueous technique. This apparently does not occur and we are thus able to use average lengths determined by the aqueous technique with reduced error from the smaller standard deviations observed. Histogram intervals will represent 0.01 or 0.02 fractions, respectively, of one-half these average dimer lengths. EcoRI cleavage of CD mtDNA of LD cells. The mtDNA purified from the LD mouse cell line contains more than 99% circular dimer forms (17) and, like the human circular dimer form of mtDNA, is a duplication of monomer genomes in head-to-tail arrangement (3). Treatment with EcoRI under high salt conditions produces linear duplex fragments (micrograph inset in Fig. 1A) whose frequency distribution of lengths (Fig. 1A) is similar to that observed for CM mtDNA (12,13). The major EcoRI fragment lengths of Cm mtDNA purified from mouse LA9 cells were previously determined to be 13,880+400 and 1,950+120 nucleotide pairs (np), respectively (12). These fragments constitute 86.5+2.5 and 12.2+0.8% of the monomer mitochondrial genome and were found to be in good agreement with those determined in a parallel study by Brown and Vinograd (13). We note that the average lengths of the CD mtDNA fragments (Table 2) are slightly smaller than lengths of corresponding fragments from CM mtDNAs. 1318
Nucleic Acids Research
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Fractional Length Frequency distributions of fragment lengths resulting from EcoRI digestion of circular dimer mitochondrial DNA from mouse LD cells before (A) (B) denaturation-renaturation. Micrograph inset in (A) illustrates the appearance of fragments before denaturation. Micrograph inset in (B) illustrates appearance of a minority population of renatured EcoRl fragments with one clean end and one short single-strand end (indicated by dashed line in
Figure
1.
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accompanying drawing). The bars indicate the intervals over which average lengths have been calculated. Magnification in (A) is indicated by the contour length of the longest molecule of 4.5 microns. In (B) the magnification 2.6 times.
has been increased
approximately
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Nucleic Acids Research TABLE 2. Sizes of Linear Duplex Fragments Resulting From Digestion of Circular Dimer Mitochondrial DNAs with EcoR I. Length of Fragments ± Standard Deviation in Nucleotide Pairs Source of mtDNA
Digest Condition
LD
High Salt
RI-1
Rl-2
13,270 ± 320 (83.4) ± (2.0)*
1,880 ± 130
[410 ± 1801
(11.8) ± (0.8)
[(2.6) ± (1.)jt
Rl-3
LDTK-
High Salt
13,450 ± 390 (83.4) ± (2.4)
1,860 ± 250 (11.5) ± (1.6)
r430
LDTK-
Low Salt
13,740 ± 450 (85.1) ± (2.8)
1,940 ± 210 (12.0) ± (1.3)
[410 ± 180 [(2.5) ± (1.1)
Human AML
High Salt
7,790 ± 160 (47.8) ± (1.0)
7,060 ± 170 (43.3) ± (1.0)
1,070 ± 140 (6.6) ± (0.9)
± 180 L(2.7) ± (1.1)]
* Numbers in parentheses are the calculated percentages ± standard deviations of one half the average circular dimer genome length. t Lengths within brackets were obtained for fragments released in less than molar equivalent yield and infrequently cyclize.
Furthermore, a third fragment (RI-3,Table 2) is present at a substantial frequency (Fig. lA) but is produced in less than molar quantity. If produced in molar quantity, fragment RI-3 should have been detected electron microscopically at approximately the same frequencies observed for fragments RI-2 and RI-l, respectively (22). The uniqueness of these major cleavage sites on LD mtDNA can be demonstrated by denaturation and renaturation of the EcoRI treated sample (21). The frequency distribution of lengths for these fragments (Fig. lB) is similar to that obtained prior to denaturation-renaturation with the same average lengths for the major EcoRI fragments having been obtained (Table 3). Fragment RI-3 is not renatured to the same extent, as expected from consideration of its reduced frequency and size (23). Some RI-l and RI-2 fragments possess a short single-strand terminus (micrograph inset in Fig. lB) whose size is close to that expected for the RI-3 fragment. Although the pattern of EcoRI cleavage of this CD mtDNA is basically similar to that of CM mtDNA from mouse LA9 cells, it is found that additional cleavages have occurred to produce not only the subpopulation of RI-3 fragments but also fragments (2% of total mtDNA mass) whose lengths lie between fragments RI-2 and RI-l (Fig. 1) and were not previously detected in cleavage of CM mtDNA from mouse cells (12,13). Furthermore, 1320
Nucleic Acids Research TABLE 3.
Sizes of Linear Duplex Fragments Resulting From Digestion of Circular Dimer Mitochondrial DNAs with EcoR Followed by Denaturation and Renaturation. Length of Fragment ± Standard Deviation in Nucleotide Pairs
Source of mtDNA LD Human AML Leucocytes
RI-1
Rl-2
Rl-3
13,360 ± 540 (84.0) ± (3.4)
1,700 ± 290 (10.7) ± (1.8)
[460 ± 100 1 [(2.9) ± (0.6)*|
7,760 ± 160 (47.6) ± (1.0)
6,970 ± 200 (42.7) ± (1.2)
980 ± 180 (6.0) ± (1.11)
* The length of renatured LD
RI-3 fragments reported here were determined in a separate
sampling.
there is a minor population of linear duplex molecules whose lengths are close to one monomer genome (Fig. 1A) similar in size and proportion (8% of total mtDNA mass) to the subpopulation found previously in cleavage of monomer mtDNA (12). We also find some molecules (6% of total mtDNA mass) with lengths betwen one and two monomer genomes (Fig. 1A) that have resisted further cleavage to produce the major monomer equivalent size fragments even under the excessive digestion conditions employed. EcoRI cleavage of CD mtDNA of LDTK-cells. The thymidine kinase minus derivative (LDTK ) of mouse LD cells (15) contains more than 95% of its mtDNA components as circulr dimer forms and constitutes a subpopulation of the parental LD cell line. Therefore, we examined the EcoRI fragmentation pattern of this mtDNA to probe for possible altered genome positions of the EcoRI cleavage sequence that may have been selected from within the original population of LD mtDNA. The pattern of fragmentation obtained under high salt digestion conditions (Fig. 2A) is similar to that obtained for LD mtDNA. We find two major fragments with average lengths (Table 2) that are slightly shorter than major fragment lengths determined previously for EcoRI digestion of CM mtDNA of LMTK- cells (12). The major EcoRI fragments of CM mtDNA purified from mouse LMTK cells have lengths of 13,810+320 and 2,270+160 np, respectively (12) and comprise 86.3+2.0 and 14.2+1.0% of this monomer mitochondrial genome (12). We also note that fragments with lengths between fragments RI-l and RI-2 are present at low frequencies, as well as fragments whose lengths are greater than fragment RI-l (Fig. 2A). As occurred with cleavage of LD mtDNA (Fig. 1), we detect a third EcoRI fragment that is frequently present but is produced in less than molar quantity (Fig. 2A). The size of this 1321
Nucleic Acids Research
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Fractional Length Figure 2. Frequency distribution of EcoRI fragment lengths after digestion of LDTK mtDNA using high salt (A) or (B) low salt conditions (See Materials and Methods). Mlcrographs inset in (A) and (B) illustrate appearance of EcoRI fragments after spreading at 6bC. Near the center of the micrograph inset at top in (A), fragment RI-2 appears cyclized. In the lower part of this micrograph fragment Rc-l appears cyclized (at left)and in linear form (at right). In the upper part ofthis micrograph an EcoRI fragment of intermediate site appears cyclized. Micrograph inset at bottomof (A) illustrates appearance (at positions indicated by arrows) of cyclized fragments which are smaller than fragment RI-2. In (B), the inset micrographs illustrate appearance of fragments RI-I (Top) and RI-2 (Bottom) which have dissociated to release a smaller fragment at the positions indicated by the arrows. Magnification is the same in all micrographs and indicated by the largest circle in (A) with a contour lengthof approximately 4.0 microns.
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Nucleic Acids Research fragment is similar to that observed for fragment RI-3 derived from EcoRI digestion of LD mtDNA (Table 2). It is possible that fragment RI-3 may have resulted from cleavage by additional nuclease activities in the EcoRI preparations (20,23). One such activity, designated EcoRI (25), has been reported to recognize the central tetranucleotide of the EcoRI sequence (25). This activity should be suppressed in the presence of 0.1 M sodium chloride for conditions (25) similar to those we have utilized in our high salt digests (see Materials and Methods). When digests are performed in the abscence of added sodium chloride (low salt conditions) the EcoRI activity will be expressed at both EcoRI and EcoRI recognition sequences (25). We therefore digested LDTK- mtDNA with EcoRI under low salt conditions as we have done previously for LMTK (12) and obtained a fragmentation pattern (Fig. 2B) which is very similar to that obtained for high salt digests (Fig. 2A). Two major fragments are produced with average lengths (Table 2) similar to those obtained for digests in high salt. The relative quantity of fragment RI-3 is increased by the low salt digestion and is detected at approximately the frequencies observed for fragments RI-2 or RI-1. This increased frequency of fragment RI-3 could reflect two or more additional cleavages near the termini of some of the larger EcoRI fragments. Fragments generated by EcoRI (26) or EcoRI (25) cleavages are provided with cohesive termini which can form stable intramolecular associations upon incubation at reduced temperatures. Such associations can be visualized by electron microscopy as circular duplexes when the spreading procedure is performed at reduced temperature (26). When LDTKmtDNA is exhaustively digested with EcoRI under low salt conditions and prepared for electron microscopy at 60C (26), fragments RI-1 and RI-2 were frequently observed to cyclize (micrograph inset in Fig. 2A), whereas fragment RI-3 was rarely observed in circular form (Fig. 2A,arrows). This is instriking contrast with the high frequencies of cyclization expected for an EcoRI fragment of this small size (24). Fragments RI-1 and RI-2 can cyclize and the cohesive termini then be pulled apart by the spreading forces in the mounting procedure for electron microscopy. Such molecules are sometimes seen to contain fragments the size of RI-3 within the region of opposed cohesive ends (micrographs inset in Fig. 2B). These combined observations suggest that fragment RI-3 is derived from sequences initially contained within and near the termini of fragments RI-l and RI-2, although the recognition sequence for cleavage(s) that produces RI-3 may not be 1323
Nucleic Acids Research identified with that for EcoRI or EcoRI enzyme activities. EcoRI cleavage of replicating LDTK mtDNA. If microheterogeneity in * genome position of EcoRI and EcoRI recognition sequences occurs among the population of CD mtDNA, we would expect to find variation in the apparent position for initiation of displacement synthesis (20) determined after EcoRI cleavage. The origin of DNA replication has been mapped at a unique site on the LMTK mitochondrial genome (12). This site is located on the largest EcoRI fragment at a position that is 1,890+250 np (11.8+1.2% of the monomer circular genome length) from the proximal restriction site (12). EcoRI fragmentation of LMTK mtDNA had been performed under conditions identical to the low salt digests we have utilized in this study and should have permitted the expression of both EcoRI and EcoRI activities. We therefore examined EcoRI digests of CD mtDNA from LDTK cells for the presence of D-loops (17) and expanded D-loops (20) which were iniated at sites on fragment RI-1 incommensurate with the previously determined unique initiation site on monomer mtDNA (12, and arrow at top of Fig. 3). In 21 of the 39 molecules sampled at random to construct the array presented in Fig. 3, the initiation site for DNA replication lies outside two standard deviations (2.3% of the monomer genome length indicated by a bar at top of Fig. 3) of the mean position for initiation on monomer mtDNA (12). We note that individual replicating molecules have significantly different lengths in contrast to the homogeneous population of replicating monomer mtDNA resulting from cleavage by EcoRI (12). We have arbitrarily grouped these molecules according to different extents of EcoRI cleavage which most closely approximates the loss or retention of fragment RI-3. The lenqth of fragment RI-3 comprises 2.5% of the equivalent circular monomer genome (Table 2) and approximates the standard deviation of length measurements for the total population of RI-l fragment lengths obtained in low salt digests (Table 2). Since the two initiations sites for DNA synthesis on mtDNA of LD cells have been shown to occur at diametrically opposed sites on this circular dimer genome (17), we conclude that apparent disparity in the initiation site for DNA synthesis on these molecules must have arisen from microheterogeneity of circular dimer mtDNA sequences cleaved by our preparation of EcoRI. EcoRI cleavage of human CD mtDNA. The microheterogeneity in circular dimer mtDNA we have detected in mouse cell lines could reflect mutational 1324
Nucleic Acids Research
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Nucleic Acids Research events that have accumulated with repeated passage of cells in culture. Thus, it became of interest to examine a population of CD mtDNA that had recently been formed from circular monomer genomes. Such a state occurs for mtDNAs isolated from the circulating white cells of patients with leukemia (1). We therefore prepared the total closed circular mtDNA components from the peripheral leucocytes of one patient with AML prior to administration of therapeutic regimens. This sample contained approximately 60% circular dimer forms and upon digestion with EcoRI under high salt conditions produces a fragmentation pattern (Fig. 4A) which is similar to that obtained for EcoRI digestion of CM mtDNA from HeLa cells (12,13). Three major fragments are produced with average lengths (Table 2) similar to those obtained by EcoRI digestion of HeLa mtDNA (12,13). EcoRI fragment lengths of CM mtDNA purified from human HeLa cells are 8,170+170, 7,370+150 and 1,070+70 np, respectively (12) and agree well with values determined in a parallel study by Brown and Vinograd (13). These HeLa cell mtDNA fragments comprise 49.2+1.0, 44.4+0.9 and 6.4+0.4% of the circular genome length (12). In the case of a mixed population of CD and CM mtDNAs as occurs for the human AML sample, we note that the length of fragment RI-l is slightly shorter (Table 2) than the corresponding fragment of HeLa cell mtDNA. Fragments RI-2 and RI-3 are essentially the same size for both AML and HeLa cell mtDNAs (Table 2). Although the standard deviations of measured lengths for fragments RI-l and RI-2 are very close to those reported for HeLa cell mtDNA, these two fragments are not well resolved in the frequency distribution of fragment lengths (Fig. 4A). There are fragments with lengths intermediate in size (4% of total mtDNA mass) as well as fragments with lengths greater than RI-1. In particular, we note two subpopulations of molecules (16% of total mtDNA mass) with lengths just slightly greater than the length of fragment RI-1 (Fig. 4A) which have resisted further EcoRI cleavage. As occurred in the cleavage of CD mtDNA from mouse cells, there are fragments with lengths near one monomer genome (6% of total mtDNA mass) as well as fragments with lengths between one and two monomer genomes (6% of total mtDNA mass). These observations suggest that the mixed population of CM and CD mtDNAs in human AML is also microheterogeneous with respect to the genome positions of the EcoRI cleavage sequence. Uniqueness of these cleavage sites on CM and CD mitochondrial genomes is demonstrated by denaturation and renaturation with recovery of the majority of renatured molecules as linear duplexes with clean ends (21).
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Nucleic Acids Research
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Figure 4. Frequency distribution of fragment lengths resulting from BcoRI digestion of human AML leucocyte mtDNA under high salt conditions before (A) and after denaturtsion-renaturation (B) compared to frequency distribuoton obtained with low salt digestion conditions (C). oicrograph inset in (A) illustrates appearance of 8coRI fragments before denaturation compared to circular PM2 DNA (lower part of field). Micrograph inset in (It) is at higher magnification and illustrates the appearance of aminor population of some retatured RI-I or Rl-2 fragments which possess one clean and one single-strand end (dashed line in drawing).
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Nucleic Acids Research When this is done, the frequency distribution of contour lengths obtained (Fig. 4B) is identified with that observed prior to denaturation (Fig. 4A). The average lengths of the denatured-renatured fragments (Table 3) are essentially the same as those observed for the major EcoRI fragments prior to denaturation (Table 2). We conclude that the majority population of CM and CD mtDNAs in this sample from human AML are cleaved by EcoRI at unique sites on the respective genomes. We infrequently find renatured molecules containing one clean end and one single-strand end (micrograph inset in Fig. 4B). The apparent length of this terminal single-strand segment suggests that a strand from one of the major EcoRI fragments, either RI-l or RI-2, has renatured with a complementary strand from one of the larger fragments in the subpopulations noted above which had resisted further cleavage by EcoRI. Cleavage of this mtDNA under low salt digestion conditions yields a frequency distribution of lengths with even poorer resolution of the two largest major EcoRI fragments (Fig. 4C). Although fragments of intermediate sizes persist, the subpopulations whose fragment lengths were greater than fragment RI-1 are largely removed. This suggests that these particular minor populations may represent fragments which contain sites sensitive to cleavage by EcoRI Microheterogeneity detected in the sizes of CD mtDNAs. The poor resolution of lengths determined in frequency distributions of human mtDNA fragments RI-1 and RI-2 could be the result of microheterogeneity in the sizes of some of the circular dimer molecules present in the sample prior to application of restriction endonuclease treatment. In order to examine this possibility, we have applied an observation from the statistics of polymers. It is invariably found that for a heterogeneous population of any given polymer, the number average molecular weight is less than the weight average molecular weight which in turn is less than the Z average molecular weight which in turn is less than the (Z + 1) average molecular weight etc., (27). In general, for a uniform mass per unit length as obtained for molecules measured electron microscopically we expect a heterogeneous population of molecular lengths to be distributed about average values such that L
Volume 4 Number S 1977
Microheterogeneity detected in circular dimer mitochondrial
DNAt
D. L. Robberson,* C. E. Wilkins,* D. A. Clayton+ and J. N. Doda+
*The University of Texas System
Cancer Center, M.D. Anderson Hospital and Tumor Institute Houston, TX 77030 and +Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA Received 7 January 1977
ABSTRACT Exhaustive EcoRI digests of circular dimer mitochondrial DNA (mtDNA) from mouse cell lines LD and LDTK- yield two major fragments whose average lengths are slightly smaller than the corresponding fragments of circular monomer mtDNA from mouse LA9 and LMTK cells. A third fragment approximately 400 nucleotide pairs in length is frequently produced in less than molar yield. Exhaustive EcoRI digests of circular dimer mtDNA from human acute myelogenous leukemiic leucocytes yield three major fragments. The presence of mtDNA resistant to cleavage as well as fragments of intermediate sizes indicatesmicroheterogeneity in the genomic positions of EcoRI recognition sequences in both mouse and human circular dimer mtDNA. Analysis of the distribution averages of circular contour lengths indicates microheterogeneity in the sizes of mouse LD and human mtDNAs. The denatured-renatured EcoRI fragments frequently contain a small loop(s) of single-strand DNA as would occur for deletion(s) or addition(s) of nucleotide sequences in some of the circular dimer molecules.
INTRODUCTION Covalent closed circular duplex DNA of mammalian mitochondria can be found to occur in oligomeric forms (1,2). In one type of oligomer, the monomer genome has been duplicated (1) and joined in head-to-tail fashion to form a circulardimer (CD) molecule (3). Circular dimers are found at only very low (0.1-0.3%) (4) or nondetectable frequencies (5) in healthy mammalian tissues but constitute significant proportions of the mtDNA population in leucocytes of human myelogenous leukemias (6) as well as in a variety of solid tumor tissues (7). Association of CD mtDNA with human malignancies has focused attention on the fine structure differences that may distinguish it from circulr monomer (CM) mtDNA. A previous study using electron microscopic and density gradient techniques applied to heteroduplexes between human CD and CM mtDNAs demonstrated that more than 90% sequence homology was shared by monomer and dimer forms (3). C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1315
Nucleic Acids Research The application of restriction endonucleases to studies of mammalian mtDNA structure has provided an additional means of assessing sequence heterogeneity among a population of molecules. For example, the mtDNAs from two strains of human HeLa cells differ in their content of sequences sensitive to cleavage by the Hae III (8,9) restriction endonuclease(l0). Earlier studies of the EcoRI (9,11) cleavage pattern of human HeLa cell mtDNA (12,13) had revealed the presence of subpopulations of fragments resulting from cleavages in addition to those which define the majority population of linear duplexes (12). Also, in the case of EcoRI digests of mouse L cell mtDNA (12,13), a minor population of molecules was found which were cleaved at only one site or a few closely spaced sites on the genome (Fig. IA and 1B, reference 12). It was suggested that these molecules constitute a minor population of CM mtDNA in which at least one of the EcoRI cleavage sequences had been deleted or altered, thus giving rise to the apparent resistance to further cleavage under the exhaustive digestion conditions employed (12). The observation that some of these molecules appeared slightly shorter than one genome length also suggested the possibility of reiteration of the EcoRI cleavage sequence within the regions that defined the two major cleavage fragments of CM mtDNA from mouse cells (12). In plasmid recombinant DNA experiments, Brown and Vinograd (14) subsequently identified a third EcoRI fragment of mouse L cell mtDNA with a length of 160+10 nucleotide pairs (np). These combined studies have indicated a limited degree of heterogeneity in CM mtDNA with respect to EcoRI (12) and Hae III (10) cleavage sequences. In the study presented here, we find a similar level of microheterogeneity of EcoRI cleavage sequences among circular dimer mtDNAs and in addition detect microheterogeneity in the sizes of circular dimer molecules.
MATERIALS AND METHODS Preparation of mitochondria and mtDNA. Mitochondria and closed circular mtDNAs were isolated from mouse cell lines LD and LDTK growing exponentially in suspension culture by procedures previously described (15). The mitochondria and closed circular mtDNAs of human leucocytes were isolated from the peripheral blood of two patients with AML prior to the administration of chemotherapy or other therapeutic regimens essentially by procedures described earlier (6) with elimination of the DNAase I and RNAase A treatments of purified mitochondrial fractions (15). Isolated closed circular mtDNAswere rebanded in buoyant CsCl gradients containing
1316
Nucleic Acids Research saturating levels of ethidium bromide (16). After collection of lowerbands ethidium bromide was removed by dialysis against Dowex cation exchange resin as described (17). EcoRI cleavage of mtDNAs. Purified closed circular mtDNAs were dialyzed against either 0.1 M Tris, .01 M MgCl2, pH 7.6 (low salt digestion) or 0.1 M NaCl, 0.1M Tris, .01 MgCl2, pH 7.6 (high salt digestion). Each digestion mixture (75 -pl) contained 0.05-0.1 pg of DNA to which was added 1 pl of EcoRI and the sample incubated at 370C for 15 min. A second 1 pl aliquot of EcoRI was then added and the incubation continued for an additional 15 min. The digestion was then terminated by addition of 20 ul of 0.25 M disodium EDTA previously adjusted to pH 8.0 with sodium hydroxide and the sample was then dialyzed against STE (0.1 M NaCl, 0.05 M Tris, 0.01 M disodium EDTA, pH 8.5) (15) essentially as described (12). One microliter of EcoRI, prepared by the method of Yoshimori (11), would cleave 1 1ig of simian virus 40 DNA using similar reaction conditions. EcoRI was the kind gift of Dr. William R. Folk. Electron Microscopy. Samples were prepared for electron microscopy as described (12) before or after EcoRI treatment using the basic protein Kleinschmidt technique in the presence or absence of formamide (18) with the modifications previously noted (19,20). Homoduplexes of EcoRI treated mtDNA were constructed with renaturation conditions chosen to permit reassociation of 40-70% of the largest single-strands in a particular sample (21). The samples were then prepared for electron microscopy as described (21). Throughout these studies we have added PM2 DNA as an internal length standard just prior to spreading of the sample. Grids were rotary shadowed with Pt-Pd and examined in a Philips 300 electron microscope. Length measurements were made from tracings of enlarged images photographed on 35 mm film utilizing either a map measure or a Numonics digitizer. RESULTS Lengths of CD mtDNAs prior to EcoRI treatment. Examination of purified mtDNAs by the basic protein Kleinschmidt technique permits measurements of their contour lengths relative to an internal standard of PM2 DNA. The results of length measurements determined relative to this internal standard of PM2 DNA are presented in Table 1 for spreadings in the absence (aqueous technique) or presence of formamide (18) as described (19,20). The average lengths of CD mtDNAs from mouse or human sources are similar to each other and are approximately twice the size of average 1317
Nucleic Acids Research TABLE 1. Sizes of Circular Dimer Mitochondrial DNAs Determined by Electron M icroscopy.
Average Contour Length Source of mtDNA Mouse Line LD Mouse Line LD Mouse Line LDTKHuman AML Leucocytes, Sample 1 Human AML Leucocytes, Sample 2
Spreading Procedure
± Standard Deviation
In Nucleotide Pairs*
Number of Molecules Measured
Aqueous Formamide Aqueous
31,820 + 580 32,580 1,460 32,270 550
39 37 43
Aqueous
32,610
700
41
Formamide
31,450 ± 1,300
31
* Contour lengths were determined relative to an internal standard of PM2 DNA with an average length taken as 9,900 nucleotide pairs.
lengths measured for the corresponding CM mtDNAs from mouse LA9 (12,13) and LMTKI cells (12) or human HeLa cells (12,13) determined relative to the same internal standard of PM2 DNA (12). Examination of contour lengths after spreading in formamide permits assessment of closed circular displacement replicative forms (20) whose presence, unaccounted for in the sample, could have lead to incorrect estimates of average contour length determined by the aqueous technique. This apparently does not occur and we are thus able to use average lengths determined by the aqueous technique with reduced error from the smaller standard deviations observed. Histogram intervals will represent 0.01 or 0.02 fractions, respectively, of one-half these average dimer lengths. EcoRI cleavage of CD mtDNA of LD cells. The mtDNA purified from the LD mouse cell line contains more than 99% circular dimer forms (17) and, like the human circular dimer form of mtDNA, is a duplication of monomer genomes in head-to-tail arrangement (3). Treatment with EcoRI under high salt conditions produces linear duplex fragments (micrograph inset in Fig. 1A) whose frequency distribution of lengths (Fig. 1A) is similar to that observed for CM mtDNA (12,13). The major EcoRI fragment lengths of Cm mtDNA purified from mouse LA9 cells were previously determined to be 13,880+400 and 1,950+120 nucleotide pairs (np), respectively (12). These fragments constitute 86.5+2.5 and 12.2+0.8% of the monomer mitochondrial genome and were found to be in good agreement with those determined in a parallel study by Brown and Vinograd (13). We note that the average lengths of the CD mtDNA fragments (Table 2) are slightly smaller than lengths of corresponding fragments from CM mtDNAs. 1318
Nucleic Acids Research
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Fractional Length Frequency distributions of fragment lengths resulting from EcoRI digestion of circular dimer mitochondrial DNA from mouse LD cells before (A) (B) denaturation-renaturation. Micrograph inset in (A) illustrates the appearance of fragments before denaturation. Micrograph inset in (B) illustrates appearance of a minority population of renatured EcoRl fragments with one clean end and one short single-strand end (indicated by dashed line in
Figure
1.
and after
accompanying drawing). The bars indicate the intervals over which average lengths have been calculated. Magnification in (A) is indicated by the contour length of the longest molecule of 4.5 microns. In (B) the magnification 2.6 times.
has been increased
approximately
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Nucleic Acids Research TABLE 2. Sizes of Linear Duplex Fragments Resulting From Digestion of Circular Dimer Mitochondrial DNAs with EcoR I. Length of Fragments ± Standard Deviation in Nucleotide Pairs Source of mtDNA
Digest Condition
LD
High Salt
RI-1
Rl-2
13,270 ± 320 (83.4) ± (2.0)*
1,880 ± 130
[410 ± 1801
(11.8) ± (0.8)
[(2.6) ± (1.)jt
Rl-3
LDTK-
High Salt
13,450 ± 390 (83.4) ± (2.4)
1,860 ± 250 (11.5) ± (1.6)
r430
LDTK-
Low Salt
13,740 ± 450 (85.1) ± (2.8)
1,940 ± 210 (12.0) ± (1.3)
[410 ± 180 [(2.5) ± (1.1)
Human AML
High Salt
7,790 ± 160 (47.8) ± (1.0)
7,060 ± 170 (43.3) ± (1.0)
1,070 ± 140 (6.6) ± (0.9)
± 180 L(2.7) ± (1.1)]
* Numbers in parentheses are the calculated percentages ± standard deviations of one half the average circular dimer genome length. t Lengths within brackets were obtained for fragments released in less than molar equivalent yield and infrequently cyclize.
Furthermore, a third fragment (RI-3,Table 2) is present at a substantial frequency (Fig. lA) but is produced in less than molar quantity. If produced in molar quantity, fragment RI-3 should have been detected electron microscopically at approximately the same frequencies observed for fragments RI-2 and RI-l, respectively (22). The uniqueness of these major cleavage sites on LD mtDNA can be demonstrated by denaturation and renaturation of the EcoRI treated sample (21). The frequency distribution of lengths for these fragments (Fig. lB) is similar to that obtained prior to denaturation-renaturation with the same average lengths for the major EcoRI fragments having been obtained (Table 3). Fragment RI-3 is not renatured to the same extent, as expected from consideration of its reduced frequency and size (23). Some RI-l and RI-2 fragments possess a short single-strand terminus (micrograph inset in Fig. lB) whose size is close to that expected for the RI-3 fragment. Although the pattern of EcoRI cleavage of this CD mtDNA is basically similar to that of CM mtDNA from mouse LA9 cells, it is found that additional cleavages have occurred to produce not only the subpopulation of RI-3 fragments but also fragments (2% of total mtDNA mass) whose lengths lie between fragments RI-2 and RI-l (Fig. 1) and were not previously detected in cleavage of CM mtDNA from mouse cells (12,13). Furthermore, 1320
Nucleic Acids Research TABLE 3.
Sizes of Linear Duplex Fragments Resulting From Digestion of Circular Dimer Mitochondrial DNAs with EcoR Followed by Denaturation and Renaturation. Length of Fragment ± Standard Deviation in Nucleotide Pairs
Source of mtDNA LD Human AML Leucocytes
RI-1
Rl-2
Rl-3
13,360 ± 540 (84.0) ± (3.4)
1,700 ± 290 (10.7) ± (1.8)
[460 ± 100 1 [(2.9) ± (0.6)*|
7,760 ± 160 (47.6) ± (1.0)
6,970 ± 200 (42.7) ± (1.2)
980 ± 180 (6.0) ± (1.11)
* The length of renatured LD
RI-3 fragments reported here were determined in a separate
sampling.
there is a minor population of linear duplex molecules whose lengths are close to one monomer genome (Fig. 1A) similar in size and proportion (8% of total mtDNA mass) to the subpopulation found previously in cleavage of monomer mtDNA (12). We also find some molecules (6% of total mtDNA mass) with lengths betwen one and two monomer genomes (Fig. 1A) that have resisted further cleavage to produce the major monomer equivalent size fragments even under the excessive digestion conditions employed. EcoRI cleavage of CD mtDNA of LDTK-cells. The thymidine kinase minus derivative (LDTK ) of mouse LD cells (15) contains more than 95% of its mtDNA components as circulr dimer forms and constitutes a subpopulation of the parental LD cell line. Therefore, we examined the EcoRI fragmentation pattern of this mtDNA to probe for possible altered genome positions of the EcoRI cleavage sequence that may have been selected from within the original population of LD mtDNA. The pattern of fragmentation obtained under high salt digestion conditions (Fig. 2A) is similar to that obtained for LD mtDNA. We find two major fragments with average lengths (Table 2) that are slightly shorter than major fragment lengths determined previously for EcoRI digestion of CM mtDNA of LMTK- cells (12). The major EcoRI fragments of CM mtDNA purified from mouse LMTK cells have lengths of 13,810+320 and 2,270+160 np, respectively (12) and comprise 86.3+2.0 and 14.2+1.0% of this monomer mitochondrial genome (12). We also note that fragments with lengths between fragments RI-l and RI-2 are present at low frequencies, as well as fragments whose lengths are greater than fragment RI-l (Fig. 2A). As occurred with cleavage of LD mtDNA (Fig. 1), we detect a third EcoRI fragment that is frequently present but is produced in less than molar quantity (Fig. 2A). The size of this 1321
Nucleic Acids Research
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25
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Fractional Length Figure 2. Frequency distribution of EcoRI fragment lengths after digestion of LDTK mtDNA using high salt (A) or (B) low salt conditions (See Materials and Methods). Mlcrographs inset in (A) and (B) illustrate appearance of EcoRI fragments after spreading at 6bC. Near the center of the micrograph inset at top in (A), fragment RI-2 appears cyclized. In the lower part of this micrograph fragment Rc-l appears cyclized (at left)and in linear form (at right). In the upper part ofthis micrograph an EcoRI fragment of intermediate site appears cyclized. Micrograph inset at bottomof (A) illustrates appearance (at positions indicated by arrows) of cyclized fragments which are smaller than fragment RI-2. In (B), the inset micrographs illustrate appearance of fragments RI-I (Top) and RI-2 (Bottom) which have dissociated to release a smaller fragment at the positions indicated by the arrows. Magnification is the same in all micrographs and indicated by the largest circle in (A) with a contour lengthof approximately 4.0 microns.
1322
Nucleic Acids Research fragment is similar to that observed for fragment RI-3 derived from EcoRI digestion of LD mtDNA (Table 2). It is possible that fragment RI-3 may have resulted from cleavage by additional nuclease activities in the EcoRI preparations (20,23). One such activity, designated EcoRI (25), has been reported to recognize the central tetranucleotide of the EcoRI sequence (25). This activity should be suppressed in the presence of 0.1 M sodium chloride for conditions (25) similar to those we have utilized in our high salt digests (see Materials and Methods). When digests are performed in the abscence of added sodium chloride (low salt conditions) the EcoRI activity will be expressed at both EcoRI and EcoRI recognition sequences (25). We therefore digested LDTK- mtDNA with EcoRI under low salt conditions as we have done previously for LMTK (12) and obtained a fragmentation pattern (Fig. 2B) which is very similar to that obtained for high salt digests (Fig. 2A). Two major fragments are produced with average lengths (Table 2) similar to those obtained for digests in high salt. The relative quantity of fragment RI-3 is increased by the low salt digestion and is detected at approximately the frequencies observed for fragments RI-2 or RI-1. This increased frequency of fragment RI-3 could reflect two or more additional cleavages near the termini of some of the larger EcoRI fragments. Fragments generated by EcoRI (26) or EcoRI (25) cleavages are provided with cohesive termini which can form stable intramolecular associations upon incubation at reduced temperatures. Such associations can be visualized by electron microscopy as circular duplexes when the spreading procedure is performed at reduced temperature (26). When LDTKmtDNA is exhaustively digested with EcoRI under low salt conditions and prepared for electron microscopy at 60C (26), fragments RI-1 and RI-2 were frequently observed to cyclize (micrograph inset in Fig. 2A), whereas fragment RI-3 was rarely observed in circular form (Fig. 2A,arrows). This is instriking contrast with the high frequencies of cyclization expected for an EcoRI fragment of this small size (24). Fragments RI-1 and RI-2 can cyclize and the cohesive termini then be pulled apart by the spreading forces in the mounting procedure for electron microscopy. Such molecules are sometimes seen to contain fragments the size of RI-3 within the region of opposed cohesive ends (micrographs inset in Fig. 2B). These combined observations suggest that fragment RI-3 is derived from sequences initially contained within and near the termini of fragments RI-l and RI-2, although the recognition sequence for cleavage(s) that produces RI-3 may not be 1323
Nucleic Acids Research identified with that for EcoRI or EcoRI enzyme activities. EcoRI cleavage of replicating LDTK mtDNA. If microheterogeneity in * genome position of EcoRI and EcoRI recognition sequences occurs among the population of CD mtDNA, we would expect to find variation in the apparent position for initiation of displacement synthesis (20) determined after EcoRI cleavage. The origin of DNA replication has been mapped at a unique site on the LMTK mitochondrial genome (12). This site is located on the largest EcoRI fragment at a position that is 1,890+250 np (11.8+1.2% of the monomer circular genome length) from the proximal restriction site (12). EcoRI fragmentation of LMTK mtDNA had been performed under conditions identical to the low salt digests we have utilized in this study and should have permitted the expression of both EcoRI and EcoRI activities. We therefore examined EcoRI digests of CD mtDNA from LDTK cells for the presence of D-loops (17) and expanded D-loops (20) which were iniated at sites on fragment RI-1 incommensurate with the previously determined unique initiation site on monomer mtDNA (12, and arrow at top of Fig. 3). In 21 of the 39 molecules sampled at random to construct the array presented in Fig. 3, the initiation site for DNA replication lies outside two standard deviations (2.3% of the monomer genome length indicated by a bar at top of Fig. 3) of the mean position for initiation on monomer mtDNA (12). We note that individual replicating molecules have significantly different lengths in contrast to the homogeneous population of replicating monomer mtDNA resulting from cleavage by EcoRI (12). We have arbitrarily grouped these molecules according to different extents of EcoRI cleavage which most closely approximates the loss or retention of fragment RI-3. The lenqth of fragment RI-3 comprises 2.5% of the equivalent circular monomer genome (Table 2) and approximates the standard deviation of length measurements for the total population of RI-l fragment lengths obtained in low salt digests (Table 2). Since the two initiations sites for DNA synthesis on mtDNA of LD cells have been shown to occur at diametrically opposed sites on this circular dimer genome (17), we conclude that apparent disparity in the initiation site for DNA synthesis on these molecules must have arisen from microheterogeneity of circular dimer mtDNA sequences cleaved by our preparation of EcoRI. EcoRI cleavage of human CD mtDNA. The microheterogeneity in circular dimer mtDNA we have detected in mouse cell lines could reflect mutational 1324
Nucleic Acids Research
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Nucleic Acids Research events that have accumulated with repeated passage of cells in culture. Thus, it became of interest to examine a population of CD mtDNA that had recently been formed from circular monomer genomes. Such a state occurs for mtDNAs isolated from the circulating white cells of patients with leukemia (1). We therefore prepared the total closed circular mtDNA components from the peripheral leucocytes of one patient with AML prior to administration of therapeutic regimens. This sample contained approximately 60% circular dimer forms and upon digestion with EcoRI under high salt conditions produces a fragmentation pattern (Fig. 4A) which is similar to that obtained for EcoRI digestion of CM mtDNA from HeLa cells (12,13). Three major fragments are produced with average lengths (Table 2) similar to those obtained by EcoRI digestion of HeLa mtDNA (12,13). EcoRI fragment lengths of CM mtDNA purified from human HeLa cells are 8,170+170, 7,370+150 and 1,070+70 np, respectively (12) and agree well with values determined in a parallel study by Brown and Vinograd (13). These HeLa cell mtDNA fragments comprise 49.2+1.0, 44.4+0.9 and 6.4+0.4% of the circular genome length (12). In the case of a mixed population of CD and CM mtDNAs as occurs for the human AML sample, we note that the length of fragment RI-l is slightly shorter (Table 2) than the corresponding fragment of HeLa cell mtDNA. Fragments RI-2 and RI-3 are essentially the same size for both AML and HeLa cell mtDNAs (Table 2). Although the standard deviations of measured lengths for fragments RI-l and RI-2 are very close to those reported for HeLa cell mtDNA, these two fragments are not well resolved in the frequency distribution of fragment lengths (Fig. 4A). There are fragments with lengths intermediate in size (4% of total mtDNA mass) as well as fragments with lengths greater than RI-1. In particular, we note two subpopulations of molecules (16% of total mtDNA mass) with lengths just slightly greater than the length of fragment RI-1 (Fig. 4A) which have resisted further EcoRI cleavage. As occurred in the cleavage of CD mtDNA from mouse cells, there are fragments with lengths near one monomer genome (6% of total mtDNA mass) as well as fragments with lengths between one and two monomer genomes (6% of total mtDNA mass). These observations suggest that the mixed population of CM and CD mtDNAs in human AML is also microheterogeneous with respect to the genome positions of the EcoRI cleavage sequence. Uniqueness of these cleavage sites on CM and CD mitochondrial genomes is demonstrated by denaturation and renaturation with recovery of the majority of renatured molecules as linear duplexes with clean ends (21).
1326
Nucleic Acids Research
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1.6
1.8 2.0
Figure 4. Frequency distribution of fragment lengths resulting from BcoRI digestion of human AML leucocyte mtDNA under high salt conditions before (A) and after denaturtsion-renaturation (B) compared to frequency distribuoton obtained with low salt digestion conditions (C). oicrograph inset in (A) illustrates appearance of 8coRI fragments before denaturation compared to circular PM2 DNA (lower part of field). Micrograph inset in (It) is at higher magnification and illustrates the appearance of aminor population of some retatured RI-I or Rl-2 fragments which possess one clean and one single-strand end (dashed line in drawing).
1327
Nucleic Acids Research When this is done, the frequency distribution of contour lengths obtained (Fig. 4B) is identified with that observed prior to denaturation (Fig. 4A). The average lengths of the denatured-renatured fragments (Table 3) are essentially the same as those observed for the major EcoRI fragments prior to denaturation (Table 2). We conclude that the majority population of CM and CD mtDNAs in this sample from human AML are cleaved by EcoRI at unique sites on the respective genomes. We infrequently find renatured molecules containing one clean end and one single-strand end (micrograph inset in Fig. 4B). The apparent length of this terminal single-strand segment suggests that a strand from one of the major EcoRI fragments, either RI-l or RI-2, has renatured with a complementary strand from one of the larger fragments in the subpopulations noted above which had resisted further cleavage by EcoRI. Cleavage of this mtDNA under low salt digestion conditions yields a frequency distribution of lengths with even poorer resolution of the two largest major EcoRI fragments (Fig. 4C). Although fragments of intermediate sizes persist, the subpopulations whose fragment lengths were greater than fragment RI-1 are largely removed. This suggests that these particular minor populations may represent fragments which contain sites sensitive to cleavage by EcoRI Microheterogeneity detected in the sizes of CD mtDNAs. The poor resolution of lengths determined in frequency distributions of human mtDNA fragments RI-1 and RI-2 could be the result of microheterogeneity in the sizes of some of the circular dimer molecules present in the sample prior to application of restriction endonuclease treatment. In order to examine this possibility, we have applied an observation from the statistics of polymers. It is invariably found that for a heterogeneous population of any given polymer, the number average molecular weight is less than the weight average molecular weight which in turn is less than the Z average molecular weight which in turn is less than the (Z + 1) average molecular weight etc., (27). In general, for a uniform mass per unit length as obtained for molecules measured electron microscopically we expect a heterogeneous population of molecular lengths to be distributed about average values such that L
