Structural and replicative forms of mitochondrial DNA from human leukocytes in relation to age

Mechanisms of Ageing and Development, 3 (1978) 351-365

351

©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

STRUCTURAL AND REPLICATIVE FORMS OF MITOCHONDRIAL DNA F R O M H U M A N L E U K O C Y T E S IN R E L A T I O N T O A G E

LAJOS PIK0 and ROXANNE MEYER Developmental Biology Laboratory, Veterans Administration Hospital, Sepulveda, California 91343 (U.S.A.)

JOSEPH EIPE and NICOLAS COSTEA Department of Medicine, U.C.L.A. San Fernando Medical Program, Veterans Administration Hospital, Sepulveda, California 91343 (U.S.A.)

(Received August 15, 1977)

SUMMARY The structure and replication of human leukocyte mitochondrial DNA (mtDNA) was investigated in healthy young adult males (23-37 years old), middle-aged males (42-52 years old) with secondary polycythemia, and elderly males (80-89 years old) who exhibited different degrees of age-related disease syndromes. The distribution of the various cell types within the white cell population was within normal limits in all samples. Total mtDNA was isolated in ethidium bromide--CsC1 gradients and examined by electron microscopy after spreading by the aqueous and formamide techniques. The individual frequencies of catenated forms ranged from 2 to 6% but showed relatively little change (declining slightly) with age. The individual frequencies of circular dimers varied from 0 to 0.1% in the young adult and polycythemic groups and in 10 out of the 12 elderly individuals. One elderly individual had a circular dimer frequency of 0.3% (including a circular molecule of tetramer size) and another had 4.5%. This finding suggests that agerelated cellular pathology may exist in the blood-forming system in some cases. The mode of replication of leukocyte mtDNA agrees well with that described for mouse L cells. There was no evidence of aberrant mtDNA replication as a result of aging.

INTRODUCTION In recent years the properties and function of the mitochondrial genetic system have been under active investigation. The basic genetic unit in the mitochondria of higher animals and man is a circular duplex DNA of about 5 tam perimeter. The mitochondrial DNA (mtDNA) codes for unique mitochondrial ribosomal RNAs, transfer RNAs and a distinct set of messenger-like RNA species. The products of mitochondrial protein

352 synthesis are similar in as widely different cells as yeast and human cells and are required for the normal assembly and function of the mitochondrial electron-transport system (see reviews by Borst [ 1], Schatz and Mason [2] and Attardi et aL [3]). The major portion of the mtDNA in normal cells occurs in the form of simple 5/~m circular molecules whereas a minor portion occurs in the form of complex molecules. Two or more circular units may be topologically interlocked like the links in a chain to form catenated dimers and higher oligomers, or two monomer units may be attached in tandem to form double-size circular dimers. Catenated mtDNA has been found in all normal tissues at frequencies ranging from 2 to 10%, but the frequency of circular dimers generally does not exceed 0.1 to 0.2%. The frequency of complex forms ofmtDNA can increase considerably in malignant or otherwise pathological tissues, virus-transformed cells or in cells exposed to abnormal physiological conditions [4-7]. One of the beststudied examples is that of human leukemic leukocytes in which the frequency of circular dimers may reach 50% [6, 8, 9]. The mechanisms of mtDNA replication in cultured mouse L cells has been elucidated recently in considerable detail [10-13]. Synthesis of one of the mtDNA strands (the H-strand) is initiated at a unique origin and proceeds unidirectionaUy, displacing the unreplicated parental H-strand. The synthesis of the second daughter strand (the L-strand) follows with considerable delay and can be initiated at any point. Characteristic replicarive intermediates, revealed by electron microscopy, are circular duplex molecules containing completely or partially single-stranded displacement loops of varying sizes and circular daughter-molecules composed of double-stranded and single-stranded segments. An early replicating intermediate containing a displacement loop of 3-5% genome size (termed D-loop DNA) usually occurs at a high frequency. This mode of replication is generally applicable to mtDNA, but the degree of asynchrony in the replication of the two strands may vary [14]. The frequencies and types of the various replicative forms appear to be characteristic for a given tissue [ 15]. Although an age-related deterioration in the f'me structure and respiratory function of the mitochondria has been described in several tissues [ 16-19], there is little information available at present on possible disturbances in the structure and replication of mtDNA during aging. In a recent study involving four mouse tissues (brain, heart, kidney and liver), the frequency of catenated mtDNA changed little as a result of aging. On the other hand, the frequency of circular dimers showed a significant age-related increase from about 0.05% in adult tissues to 0.3% in kidney, 0.5% in liver, 0.6% in heart and 1.9% in brain of senescent mice. In the same study, the frequencies and types of the various replicative intermediates of mtDNA were unaffected by donor age, suggesting that the rates of synthesis and turnover of mtDNA remained relatively unchanged in the senescent tissues [ 15]. In the present work, we have studied the structural and replicative forms ofmtDNA in circulating white cells from three donor groups: young adult males, middle-aged males with secondary polycythemia, and elderly males (over 80 years old). There was little if any age-related effect revealed by this study in the occurrence ofcatenated forms or the frequencies and types of the replicative intermediates of mtDNA. The incidence of

353 circular dimers was very low (less than 0.1%) in the individuals of the three groups; however, one elderly individual had a somewhat elevated frequency (0.3%) and another a relatively high frequency (4.5%) of circular dimers. These rare cases may well be attributable to some age-related pathological conditions affecting leukocyte formation. However, an increased frequency of circular dimers is not a usual feature of aging of the hematopoietic system in man.

MATERIALS AND METItODS

Blood donors Three groups contributed blood for these studies: 5 healthy young males (designated C-1 to C-5), 23 to 37 years old; 5 males with polycythemia (P-1 to P-5), 42 to 52 years old; and 12 elderly males (A-1 to A-12), 80 to 89 years old. The individuals with polycythemia were outpatients at the VA Hospital, Sepulveda; the polycythemia was secondary to chronic obstructive lung disease. The elderly individuals were all patients in the medical ward of the same hospital. The immediate cause for their admission varied, but all exhibited various degrees of one or several of the following conditions: atherosclerosis, arthritis, diabetes mellitus, cerebrovascular accidents and organic heart disease. All of the elderly patients had complete blood tests done at the time of the blood donation: seven had mild to moderate anemia, and one (A-4) had chronic leukopenia. The distribution of white cell types was within normal limits in all cases. The one patient (A-2) with a high frequency of circular dimers, 4.5%, had a typical medical record. He was admitted to the hospital for chest pain and shortness of breath. Medical history included diabetes mellitus (controlled by diet), cataracts, ulcers and organic heart disease. His hematological values and arterial 02 tension at the time of the blood donation were normal. Isolation of mitochondria White blood cells were isolated from freshly drawn blood (50 ml) by a modification of the procedure of B6yum [20]. This procedure generally resulted in the recovery of 80% or more of the white cells present in the blood sample. The citrated blood sample was mixed with 1/6 volume of prewarmed (37 °C) Plasmagel (Roger Bellon Laboratories, Neuilly, France) and layered over an equal volume of warm (37 °C) dextran-Renografm hypophase with a density of 1.071 ; the latter was obtained by adding 2 parts of 6% (w/v) dextran (Pharmacia, Uppsala, Sweden) in distilled water to 1 part of 34% (v/v) Renografm-76 (E. R. Squibb & Sons Inc., Princeton, N.J.) also diluted with distilled water. The blood was allowed to settle for about 45 min at room temperature. The hyperphase containing the white blood cells was collected and centrifuged at 750 X g in a Sorvall HB-4 rotor (DuPont Instruments, Newton, CT) for 5 min. The leukocyte pellet was resuspended in ice-cold 0.25 M sucrose, 0.01 M Tris--HCl, 0.005 M EDTA, pH 8.0 (STE buffer) and recentrifuged as above. The cellswere swollen in 10 X volume of 0.01 M Tris--HC1, 0.001 M EDTA, pH 7.6 for about 10 min at room temperature. A solution of 2 M sucrose in distilled water was then added to a final concentration of 0.25 M sucrose, and the cells

354 were homogenized in a Potter-Elvehjem homogenizer with a motor-driven loose fitting Teflon pestle. The homogenization was monitored by light microscopy of samples stained by methyl green and pyronin Y until more than 95% of the cells were raptured. The homogenate was diluted to 50 ml with STE buffer and centrifuged at 750 × g two times to remove nuclei. The supernatant was then spun at 12,000 × g for 10 min to pellet the mitochondria. The supernatant was decanted and the mitochondrial pellet was suspended in 2.0 ml of 0.25 M sucrose, 0.01 M Tris-HC1, 0.005 M MgC12, pH 7.6 containing 100 pg pancreatic DNAase (2500 U/mg, electrophoretically pure, Worthington Biochemicals, Freehold, N.J.) and incubated at 37 °C for 20 min to digest extra-mitochondrial DNA. The DNAase was quenched with STE buffer and the mitochondria were repelleted at 12,000 × g for 10 min. Isolation of mitoehondrial DNA The procedures used for the isolation ofmtDNA were described [7] and are briefly as follows. The mitochondrial pellet was suspended in 1.0 ml of 0.5 M NaCI, 0.1 M TrisHC1, 0.01 M EDTA, pH 9.0 (NTE buffer) and lysed with 0.5 ml of warm (37 °C) 4% sodium dodecyl sulfate in distilled water. The lysate was centrifuged in a CsC1--ethidium bromide gradient (final volume 3.5 ml) for about 24 h at 40 krpm, 20 °C, in the Beckman SW50.1 rotor. The total DNA spanning the upper and lower bands was collected; the bulk of the DNA (about 70%) was located in the lower band in these experiments. The mtDNA solution was dialyzed in NTE buffer to remove the CsCI, concentrated to about 0.5 ml in the dialysis tube with dry Sephadex G-200 (Pharmacia, Uppsala, Sweden), and purified by band-velocity sedimentation through a CsC1 gradient containing 100 pg/ml ethidium bromide [7]. The contents of the tube except for the upper portion of the gradient located above the nicked mtDNA band was collected and dialyzed as above. The DNA, in NTE buffer, was pelleted in the SW50.1 rotor at 45 krpm for 4 h, 20 °C. The supernatant was removed to about 50 pl and the mtDNA pellet (which still contained some ethidium bromide) was resuspended for electron microscopic examination. Electron microscopy The complex forms of mtDNA were analyzed on grids prepared with the aqueous basic protein film technique [21]. Before spreading, the mtDNA was photochemicaUy nicked by illuminating the DNA solution in a small glass tube at a distance of 5 cm from a high intensity lamp for about 1 h at 10 °C. The mtDNA-protein film was transferred to a padodion-coated 200-mesh copper "finder" grid (Polaron Instruments, Inc., Warrington, Pa.), stained in alcoholic uranyl acetate, rotary shadowed with Pt-Pd, and examined in a Philips EM200 operated at 40 kV. About 2000 mtDNA molecules per sample were selected at random and evaluated for the frequencies of complex forms as described [22]. Usually more than 80% of the mtDNA molecules were present in the nicked (relaxed) form. To reduce the chance of accidental overlapping, only those grids with less than 400 molecules per grid square were evaluated. All circular dimers and catenated oligomers were photographed; the negatives of all circular dimers were traced on a Nikon 6C projection comparator and measured with a map measure for final verification.

355 TABLE I FREQUENCY OF COMPLEX mtDNA IN LEUKOCYTES FROM YOUNG ADULT MALES* Donor

C-I C-2 C-3 C-4 C-5 Average~"

Monomer

94.4 ± 0.9 94.5 ± 1.0 94.8 ± 0.9 96.1 ± 0.8 96.4 -+0.8 95.2 ± 0.9

Orcular dimer

0.04 ± 0.08 0 0.08 ± 0.11 0 0.05 ± 0.10 0.04 ± 0.03

Catenated dimer

oligomer

5.0 5.1 4.5 3.5 3.1 4.2

0.6 0.4 0.7 0.4 0.4 0.5

± 0.9 ± 1.0 ± 0.8 ± 0.8 ± 0.8 ± 0.9

± 0.2 ± 0.3 ± 0.2 ± 0.2 ± 0.2 ± 0.1

Total molecules scored

2273 1988 2389 1994 2046 10690

*Frequencies are given as percent of total number of molecules scored ± the statistical sampling error at the 95% confidence limit. "l'Average ± standard deviation of individual frequencies. The replicative forms o f mtDNA were visualized by formamide spreading [23]. Randomly taken electron micrographs were printed at a final magnification o f 30,000 × and scored for the frequencies of replicative forms. The lengths o f single- and doublestranded regions were measured from negative tracings enlarged to about 80,000 ×. The within-group variability o f the individual frequencies o f molecular classes was analyzed in X2 tests using 2 × C contingency tables. The mean frequencies o f molecular classes among the three groups were compared using Student's t test [24].

RESULTS Frequency o f complex mtDNA Table I summarizes the distribution o f circular dimers and catenated forms o f mtDNA in the leukocyte mtDNA preparations o f the five young adult males examined. The frequency o f circular dimers was exceedingly low, ranging from zero to about 0.1%. Overall, a total o f four circular dimers were observed in a population of 10,690 mtDNA molecules. The frequency o f catenated dimers varied from 3.1 to 5.0% and that of total catenated forms from 3.5 to 5.6%. The within-group heterogeneity o f the frequencies o f total catenated forms, as calculated by the ×2 method, is statistically significant (e < 0.005).

Table II shows the frequencies o f complex mtDNA in the leukocytes of the five (middle-aged) male patients with secondary polycythemia. The circular dimer frequency was again extremely low, only two circular dimers having been seen in the mtDNA preparation o f one individual. The frequency o f total catenated forms ranged from 2.4 to 3.6%; the within-group heterogeneity o f these frequencies is not statistically significant. When compared in Student's t test, the average frequency o f total catenated forms o f mtDNA in the polycythemic group is significantly lower than that in the young adult group (P < 0.02).

356 T A B L E II FREQUENCY OF COMPLEX mtDNA IN LEUKOCYTES FROM POLYCYTHEMIC MALES* Donor

Monomer

P-1 P-2 P-3 PA P-5 Average t

96.3±0.8 96.3±0.8 96.5±0.8 97.6±0.7 96.7±0.8 96.7±0.6

Circular dimer

0.1

±0.1 0 0 0 0 0.02±0.04

Catenated dimer

oligomer

3.2±0.8 3.1±0.8 3.4±0.8 2.2±0.6 3.1±0.7 3.0±0.5

0.4±0.2 0.6±0.2 0.2±0.2 0.2±0.2 0.3±0.2 0.3±0.2

Total molecules scored

2023 2018 2029 2105 2036 10211

*'t'See footnotes in Table I. T A B L E III FREQUENCY OF COMPLEX mtDNA IN LEUKOCYTES FROM ELDERLY MALES* Donor

Monomer

A-1 A-2 A-3 A-4 A-5 A-6 A-7 A-8 A-9 A-10 A-11 A-12 Average t

97.2 92.2 95.9 97.5 93.5 96.0 96.6 97.8 94.7 97.5 97.0 97.1 96.1

± 0.7 +_ 1.7 ± 0.9 ± 0.7 ± 1.1 +_0.8 +- 0.8 ± 0.7 ± 1.0 ± 0.7 ± 0.7 ± 0.7 ± 1.8

O'rcu lar dimer

0.3 4.5 0.1 0.1 0.05 0.05 0 0 0 0 0.05 0.05 0.4

± 0.2** ± 1.3 ± 0.1 ± 0.1 ± 0.1 ± 0.1

± 0.1 ± 0.1 ± 1.3

Catena ted dimer

oligomer

2.5 3.1 3.6 2.2 5.5 3.6 3.1 2.0 5.0 2.3 2.7 2.6 3.2

0.2 ± 0.2 0.2 ± 0.2 0.4 ± 0.2 0.2-+ 0.2 0.9 ± 0.1 0.5 ± 0.2 0.3 ± 0.2 0.2 ± 0.2 0.3 ± 0.2 0.3 ± 0.2 0.2 ± 0.2 0.3 ± 0.2 0.3 ± 0.2

± 0.7 +- 1.1 ± 0.8 ± 0.7 ± 1.0 ± 0.8 ± 0.7 ± 0.6 ± 0.9 ± 0.7 ± 0.7 ± 0.7 ± 1.1

Total molecules scored

1975 1003 1953 1943 1969 2046 2200 1939 2131 2016 2054 2056 23285

*tSee footnotes in Table I. **This frequency includes 2 single circular dimers, 2 circular dimers catenated to each other and a circular molecule of tetramer size (see Fig. 1)

T a b l e III p r e s e n t s t h e d i s t r i b u t i o n o f c o m p l e x f o r m s o f m t D N A in t h e l e u k o c y t e m t D N A p r e p a r a t i o n s from 12 elderly males. T h e f r e q u e n c y o f circular d i m e r s is very low in 10 i n d i v i d u a l s (A-3 t o A-12), ranging f r o m zero to 0.1%. T h e circular d i m e r f r e q u e n c y is s o m e w h a t elevated, 0.3%, in o n e d o n o r ( A - I ) ; in a d d i t i o n , a u n i q u e m o l e c u l e o f t e t r a m e r size, p r e s u m e d t o be a circular t e t r a m e r o f m t D N A , was also o b s e r v e d in this sample (Fig. 1). T h e size o f this m o l e c u l e was 3.93 X larger t h a n t h e average size o f 10 a d j a c e n t circular m o n o m e r s . A m u c h h i g h e r f r e q u e n c y o f circular dimers, 4.5%, was f o u n d in one elderly d o n o r ( A - 2 ) ; t h e f r e q u e n c y o f c a t e n a t e d f o r m s in t h e sample was 3.3% w h i c h is close to t h e average f r e q u e n c y o f 3.5% for t h e elderly g r o u p as a w h o l e . T h e v a r i a t i o n in

357

Fig. 1. Electron micrograph of complex mtDNA molecules from human leukocytes. A circular dimer (2×) and a circular molecule of tetramer size (4×) is shown together with a circular monomer (IX). The mtDNA was prepared for electron microscopy by the aqueous protein film technique. × 24, 000.

the frequency of total catenated forms within the elderly group, from 2.2 to 6.4%, is statistically significant (P < 0.01). The average frequency of total catenated forms is significantly lower (P < 0.05) than that in the young adult group but does not differ significantly from that in the polycythemic group. The average frequency of circular dimers in the elderly group, 0.4%, is significantly higher (P < 0.02) than that in the other two groups. However, if the unusually high frequency observed in the sample from A-2 is omitted, there is no significant difference in the frequency of circular dimers among the three groups.

358

Fig. 2. Electron micrographs of replicative intermediates of mtDNA from human leukocytes. (a) Molecule with a small single-stranded expansion of 0.15 genome size; (b) molecule with a single-stranded expansion of 0.7 genome size; (c) a totally double-stranded Cairns' form with a 0.4 genome size expansion; (d) gapped circle with a single-stranded segment of 0.4 genome size (between arrowheads); (e) two monomer-size circular duplexes connected by a small single-stranded junction loop possibly resulting from cross-strand exchange, Magnification × 30,000. Insert: enlarged picture of the junction region of the molecules shown in (e); X55,000.

Frequency of replicative forms of mtDNA The frequencies o f the various types o f replicative intermediates o f leukocyte mtDNA were evaluated in electron micrographs o f mtDNA spread by the forrnamide technique. Duplex circles o f mtDNA containing displacement loops of 5% genome size or smaller were classified as D-loop molecules. The larger replicative intermediates included expanded D molecules (mtDNA duplexes containing displacement loops larger than 5% genome size) and gapped circles (mtDNA circles containing duplex and single-stranded segments). Examples o f larger replicative forms o f mtDNA are illustrated in Fig. 2. About 20% o f expanded D molecules in these preparations exhibited different degrees o f

359 TABLE IV FREQUENCY OF REPLICATIVE FORMS OF mtDNA IN LEUKOCYTES FROM YOUNG ADULT MALES* Donor

Clean duplex circle

D loop

Expanded D loop

Gapped circle

Total molecules scored

C-1 C-2 C-3 C-4 C-5 Averaget

56.8±3.9 61.9±4.1 66.7±4.6 72.2±5.7 80.3±5.3 67.6±9.1

38.8±3.8 33.1±4.0 29.1±4.4 20.7±5.1 18.3±5.2 28.0±8.5

2.7±1.3 3.8±1.6 3.2±1.7 4.6±2.6 1.4±1.6 3.1±1.2

1.6±1.0 1.1±0.9 1.2±1.1 2.5±2.0 0.5±0.9 1.4±0.7

621 531 403 241 214 2010

*The monomeric units of catenated forms are scored as separate molecules. Frequencies are given as percent of total number of monomers scored ± the statistical sampling error at the 95% confidence limit. The terminology of replicative forms follows that of Robberson et al. [ 13]. tAverage ± standard deviation of individual frequencies. TABLE V FREQUENCY OF REPLICATIVE FORMS OF mtDNA IN LEUKOCYTES FROM POLYCYTHEMIC MALES* Donor

Clean duplex circle

D loop

Expanded D loop

Gapped circle

Total molecules scored

P-1 P-2 P-4 P~ Averaget

67.0±6.4 64.1±4.6 77.7±4.4 72.4±4.9 70.3±6.0

31.0±6.3 32.0±4.5 19.4±4.2 21.9±4.6 26.1±6.4

2.0±1.9 2.4±1.5 1.7±1.4 4.1±2.2 2.6±1.1

2.5±2.1 1.5±1.2 1.2±1.1 1.6±1.4 1.7±0.6

205 412 345 315 1277

*tSee footnotes in Table IV. branch migration (single-stranded or duplex linear segments emerging from the replicating forks; see Matsumoto et al. [7]). Table IV shows the frequencies o f replicative forms o f leukocyte mtDNA in the young adult group. The average frequency o f D-loop DNA was 28% (ranging from 18 to 39%), that o f expanded D molecules 3.1% (1.4 to 4.6%) and that o f gapped circles 1.4% (0.5 to 2.5%). The within-group variability o f the individual frequencies is statistically significant (P < 0.001) for D-loop DNA but not for the other two forms. Table V gives the frequencies for the replicative forms o f leukocyte mtDNA in the polycythemic group. The average frequencies are very similar to the one obtained in the young adult group. Again the variation in the individual frequencies is statistically significant for D-loop DNA (P < 0.001) but not for the two larger replicative intermediates. Table VI shows the distribution of replicative forms o f leukocyte mtDNA in the elderly group. The average frequencies o f the various forms agree well with those observed in the other groups. When the variation in the individual frequencies is tested, a

360 TABLE VI FREQUENCY OF REPLICATIVE FORMS OF mtDNA IN LEUKOCYTES FROM ELDERLY MALES*

Donor

Clean duplex circle

D loop

Expanded D loop

Gapped circle

Total molecules scored

A-1 A-2 A-3 A4 A-5 A~ A-7 A-8 A~ A-10 A-II A-12 Average t

54.1±4.5 65.3±6.1 71.3±3.5 68.3±4.5 73.2±5.7 78.7±5.6 61.1±5.8 51.6±7.2 71.6±5.4 75.3±4.5 59.5±5.8 73.0±5.7 66.9±8.6

43.5±4.4 31.4±5.9 22.1±3.2 27.1±4.3 24.2±5.5 18.3±5.3 35.6±5.7 39.7±7.1 23.5±5.1 21.3±4.3 32.6±5.5 20.4±5.2 28.4±8.2

1.2±1.0 1.7±1.6 3.5±1.4 3.4±1.7 1.3±1.5 1.0±1.4 1.9±1.6 4.3±2.9 2.6±1.9 0.6±0.8 5.7±2.7 3.9±2.5 2.6±1.6

1.2±1.0 1.7±1.6 3.2±1.4 1.2±1.0 1.3±1.5 2.0±1.9 1.5±1.4 4.3±2.9 2.2±1.8 2.8±1.7 2.2±1.7 2.6±2.1 2.2±0.9

481 236 634 417 231 202 270 184 268 352 279 230 3784

*tSee footnotes in Table IV.

statistically significant heterogeneity is obtained for D-loop DNA as well as for expanded D molecules (P < 0.001 in both cases) but not for gapped circles.

Analysis of duplex synthesis The pattern of duplex synthesis on the displaced strand was analyzed in 98 expanded D molecules selected at random in leukocyte mtDNA preparations from 8 elderly males and in 78 such molecules from 4 young adult males. Figures 3(a) and 3(b) show the extent of duplex synthesis in these molecules in relation to the size of the expansion. It is clear that replication of the displaced strand is highly asynchronous with respect to displacement synthesis in the leukocyte mtDNA of both groups: only about 32% of the expansions had double-stranded regions in the elderly group and about 28% did so in the young adult group. The initiation of duplex synthesis appears to be random and may occur at any time during expansion. Multiple duplex segments, indicating multiple initiations, were observed in about 6% of the expanded D molecules in each group. Among a total of 118 gapped circles examined in the two groups (data not shown) 16 molecules or about 14% had two or more double-stranded segments. Totally double-stranded expansions or Cairns' forms (Fig. 2c) were rare, 1/98 expanded D molecules in the elderly group and 3/78 in the young adult group; the difference between these frequencies is not statistically significant. In the search for larger replicating forms in mtDNA samples spread by the formamide technique, an unusual molecule illustrated in Fig. 2(e) was encountered. This molecule consists of two monomer-size circular duplexes joined together through a small singlestranded junction loop (Fig. 2e, insert). The junction loop measures about 800 A and is apparently the result of the partial melting of the four duplexes forming the junction.

361

z"o ~ '8 . ~

~

0

0.6

X -J

,,, >

©

©

~ 0.2

OO

®

© ® 0.2 (a)

0.4 RELATIVE

0.6 0.8 EXPANSION

1.0

0.8 co w "r

~

0

.

6

x

©

w _.J

©

g o.4 O w

_>

C,o oo, 0.2 (b)

0.4 RELATIVE

0.6 EXPANSION

0.8

1.0

Fig. 3. Relative duplex synthesis in expanded D molecules of human leukocyte mtDNA from (a) young adult and (b) elderly individuals. The length of duplex segment(s) on the displaced strand is plotted as a function of the total length of the expansion. The diagonal line indicates the position of fully double-stranded expansions (Cairns' forms). The numbers within the c~cles give the number of overlapping data points. The units refer to the size of the monomer genome.

Junctions having a similar free structure were observed between circular subunits o f lambda phage DNA and were interpreted as intermediates o f genetic recombination involving cross-strand exchange between homologous DNA strands [25]. There is evidence indicating frequent recombination between m t D N A molecules in animal cells, at least under certain experimental conditions, such as in rodent-human somatic cell hybrids [26, 2 7 ] . The apparent scarcity o f m t D N A forms suggesting recombinational events could be

362 due to the very short existence of such forms or to the lack of their detection under the usual conditions of electron microscopic examination. For example, in the absence of denaturation of the junction region, the nature of the junction would remain unrecognized. The molecule described above was the only one of its kind detected during the scanning of about 6000 mtDNA molecules.

DISCUSSION Cytological evaluation of the blood samples used in the present study showed a normal distribution of the various cell types within the white cell population. We estimate that the bulk of the mtDNA (roughly 800) in these samples was derived from granulocytes and a minor portion originated from lymphoid cells and monocytes. An interesting feature of this system is that the granulocyte population circulating in the blood stream has a half-life of only a few hours and is being constantly replenished by the hematopoietic system of the bone marrow [28, 29]. Therefore, the circulating granulocytes are very young cells. Transplantation studies in mice indicate that the proliferative capacity of bone marrow stem cells is not significantly reduced during the lifetime of the individual [30, 31]. The frequency of catenated forms of mtDNA varied within relatively narrow limits, from about 2 to 6%, throughout this study. Despite this narrow range, the within-group variability of the individual frequencies is statistically significant in the young adult and the elderly groups. Since special care was taken to avoid accidental overlapping of the molecules in the preparations for electron microscopy, we consider the individual variation in the frequency of catenanes real and not due to an artifact of preparation. Tke average frequency of catenated forms is significantly higher in the young adult group as compared with either of the other two groups. Nevertheless, the difference is quite small and, in view of the individual variability that exists, it can only be regarded as suggestive evidence. Similar results were obtained in a previous study for various mouse tissues, in which the occurrence ofcatenated mtDNA was affected only slightly or not at all by donor age [15]. In contrast, the proportion of catenated forms was found to be generally elevated in the leukocytes of leukemic patients [6, 9] and also, to a lesser extent, in the leukocytes of patients with some non-malignant proliferative disorders of the blood-forming system such as myeloid metaplasia [9], mononucleosis and Burkitt's lymphoma [32]. The data on the frequency of circular dimers in the three groups examined in the present study are of particular interest, because of the very high incidence of these forms, up to 50%, in leukocyte mtDNA from patients with granulocytic leukemia [6, 8, 9]. The very low frequency of circular dimers in the young adult group (0.04%) and in patients with polycythemia (0.02%) corroborate the previous findings of Clayton and his associates and others (see ref. [6]) on the apparent absence of these forms in mtDNA from normal leukocytes. Our data for the elderly group are less clearcut. Although a very low incidence of circular dimers of mtDNA also appears to be the rule for this group, one

363 sample with a relatively high content of circular dimers (4.5%) was also found. Another sample had a slightly increased frequency of circular dimers (0.3%) and also contained a circular tetramer which appears to be the first example of the occurrence of a higher circular oligomer of mtDNA. These Findings suggest that, although an increased incidence of circular dimers is not a usual feature of leukocyte intDNA from aging individuals, age-related pathological conditions may exist which in some cases promote the accumulation of these forms. There is now substantial evidence to support the view that an increase in circular dimer frequency is generally indicative of cellular pathology such as may occur in malignant and some other physiologically abnormal cells [5, 6]. A previous report of a high incidence of circular dimers in normal thyroid tissues was not corroborated by a comparative study of thyroid mtDNA [7]. An age-related increase in the occurrence of circular dimers of mtDNA was also found in several mouse tissues, mouse brain being the most affected (about 2% circular dimers [15] ). At this time there are insufficient data to determine whether the type of cell (for example, non-dividing versus proliferating cells) has a significant influence on the structural alterations of mtDNA during the aging process. The nature of the cellular aberration leading to circular dimer formation is also unknown. However, the existence of chronic mild hypoxia (an arterial O2 tension of 70-80 Torr versus the normal range of 80-95 Tort), as was the case for the patients with secondary polycytheinia in the present study, apparently has no adverse effect on the structure of intDNA of circulating leukocytes. The replicative intermediates of leukocyte mtDNA observed in the present study are in good agreement with the model ofintDNA replication described for mouse L cells [ 10-13]. The replication of the two strands of leukocyte intDNA is highly asynchronous, resembling the type of intDNA replication observed in L cells. The average frequency of D-loop DNA is similar in the three groups (about 30%) as is the frequency of larger replicative forms (about 5%). However, there is a significant heterogeneity in the individual frequencies of D-loop DNA in each group. The frequencies of larger replicative forms also show a significant variability in the elderly group, but not in the other two groups, suggesting a greater individual variation in the rate of mtDNA replication as a result of aging. Nevertheless, the pattern of duplex synthesis is similar in the young adult and the elderly groups. All in all, the results of this study suggest that the frequencies and types of the various replicative forms and the pattern of mtDNA replication in human leukocytes are little affected by the aging process. A similar Finding was made earlier with respect to mtDNA replication in tissues from adult and senescent mice [ 15].

ACKNOWLEDGEMENTS It is a pleasure to acknowledge the excellent technical assistance of Phyllis Hotchkin. This work was supported in part by research funds from the Clinical and Research Center on Aging, Veterans Administration Hospital, Sepulveda, California.

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