161

Biochimica et Biophysica Acta, 349 ( 1 9 7 4 ) 1 6 1 - - 1 7 2 © Elsevier Scient i f ic Publ i sh ing C o m p a n y , A m s t e r d a m - - P r in t ed in The Ne the r l ands

BBA 9 7 9 9 5

REPLICATION OF THE KINETOPLAST DNA OF LEISHMANIA TARENTOLAE AND CRITHIDIA FASCICULA TA

L A R R Y SIMPSON, A G D A M. SIMPSON and R O N A L D D. WESLEY*

Department of Biology and the Molecular Biology Institute, University of California, Los Angeles, Calif. 90024 (U.S.A.)

(Received D e c e m b e r 10 th , 1973)

Summary

1. Replicating kinetoplast DNA networks from both Crithidia fasciculata and Leishmania tarentolae have an equilibrium density in ethidium bromide-- CsC1 which is less than that of covalently closed non-replicating networks. After a few hours of chase, these networks assume the covalently closed posi- tion in the gradient.

2. Pulse-labeled minicircles isolated from sonicated networks band in the upper position in ethidium bromide--CsC1 equilibrium gradients. Both "free" and network minicircles incorporate [3 H] thymidine at the same rate in a pulse. Labeled single stranded fragments of less than unit minicircle length are re- leased from pulse-labeled minicircles in alkali.

3. Intact networks of Leishmania and Crithidia can be isolated in the process of replication: replication involves a doubling of the surface area of the networks as visualized by spreading the kinetoplast DNA on glass slides, stain- ing and examining in the light microscope.

4. DNA replication within the kinetoplast DNA networks of both Leish- mania and Crithidia in all parts of the S phase is restricted to the periphery of the structures. In C. fasciculata the pulse-labeled DNA remains in position as the network enlarges by peripheral growth, and then becomes redistributed throughout the network sheet by an unknown mechanism after one cell genera- tion.

5. In the case of L. tarentolae it was demonstrated by density transfer experiments that all network minicircles replicate by an apparent semi-conser- vative pattern in one cell generation. This implies that there is some type of

Abbreviation: SSC, 0.15 M NaCI--O.015 M sodium citrate, pH 7.0. * Present address: Department of Molecular, Cellular & Developmental Biology, University of

Colorado, Boulder, Colo. 80302 (U.S.A.)

162

lateral mobility of molecules within the network, by which minicircles move past the peripheral locus of replication.

Introduct ion

Very little is known about the replication of the kinetoplast DNA of the hemoflagellates. The very complexity of the kinetoplast DNA structure -- a large "ne twork" of approx. 104 intercatenated minicircles and other molecules .... makes the problem formidable [1,2]. It has been suggested from qualitative electron microscope autoradiography studies of [3 HI thymidine incorporation in Trypanosoma lewisi [5] and Crithidia fasciculata [6] that DNA synthesis is limited to the end portions of the kinetoplast DNA in situ structure in thin sections of intact cells.

This report presents light microscope autoradiographic evidence for a peripheral replication pattern in kinetoplast DNA networks from two species of hemoflagellates (Leishmania tarentolae and Crithidia fasciculata). The replica- tion of kinetoplast DNA networks was also investigated in terms of the com- ponent molecules by means of density labeling and pulse-chase studies.

Methods

Cell culture L. tarentolae (clonal strain Lt-C1) and C. fasciculata (clonal strain Cf-C1)

cells were grown as described previously [4] in Difco brain heart infusion medium (BHI) or in defined media. The defined medium for L. tarentolae (Medium CA) was modified Medium C of Trager [10] containing 20 pg/ml adenine in place of the purine-pyrimidine mixture. The defined medium for C. fasciculata was that of Kidder and Dutta [11] .

Ethidium bromide--CsCl equilibrium gradients These were carried out, fractionated and counted as described elsewhere

[12].

Band velocity sedimentation in sucrose gradients This was performed as described previously both for neutral and alkaline

separations [8] .

Light microscopy o f kinetoplast DNA networks Purified networks in 0.15 M NaC1--0.015 M sodium citrate (SSC) were

mixed with 1% bovine serum albumin in SSC and smeared onto gelatin-subbed glass slides, air-dried, fixed in methanol for 5 min and stained in Giemsa for l h .

For autoradiography the unstained smears were coated with 1:1 diluted Ilford L-4 emulsion. Slides were developed for 4 min in D-19 developer, fixed and stained with Giemsa for 1 h. Micrographs were taken using Pan X film and a Zeiss Universal microscope with 100 × Neofluor phase contrast objective.

163

Analytical CsCl centrifugation This was carried out as described previously [2].

Isolation of kinetoplast DNA networks This was carried out by the hot sarkosyl-pronase lysis and differential

centrifugation technique described elsewhere [12].

Isolation of labeled reference DNAs ,4 C-labeled nuclear DNA was isolated from total cell lysate after removal

of the kinetoplast DNA networks by centrifugation. The isolation procedure has been described previously [2]. The cells had been grown in Medium CA with 0.5/aCi/ml [14 C] thymidine (36 Ci/M) for 4 days.

Partial synchronization of C. fasciculata cells Cells were treated with 200 pg/ml hydroxyurea in BHI medium for 6 h at

27°C and were then washed and resuspended in BHI without hydroxyurea. Samples were taken at different times and pulsed with 50 #Ci/ml [3 H]- thymidine in Medium CA to measure the rate of DNA synthesis per 106 cells. Smears were also prepared and stained with Giemsa for light microscopy, and the percentage of dividing cells was scored.

Results

Ethidium bromide--CsCl analysis of replicating networks It has been demonstrated that essentially all of the kinetoplast DNA net-

works from stationary phase L. tarentolae and C. fasciculata cells can be re- covered in a covalently closed configuration (ref. 12, and Simpson, A.M. and Simpson, L., unpublished results). However, a significant fraction of kine- toplast DNA networks from log phase cells possess an equilibrium density in ethidium bromide--CsC1 which is less than that characteristic of covalently closed molecules. This phenomenon was visualized by centrifuging purified L. tarentolae network DNA to equilibrium in ethidium bromide--CsC1, as shown in Fig. 1. The network preparation from early log phase cells in Fig. la showed an intermediate and upper region in the gradient in addition to the major lower band, whereas the network preparation from stationary phase cells in Fig. lb showed only a lower band.

The same phenomenon occurs in the replication of C. fasciculata kine- toplast DNA networks. Log phase cells were prelabeled with [14 C] thymidine for 18 h and were then pulsed with [3 H] thymidine for 10 rain and chased with unlabeled thymidine. The ethidium bromide-CsC1 equilibrium banding patterns of purified Crithidia kinetoplast DNA networks at several times during the chase are shown in Fig. 2. The pulse-labeled networks assumed a broad inter- mediate position in the gradient and then moved entirely to the covalently closed position by about 2 h into the chase. Another wave of nicking and subsequent covalent closure, due to the movement of the pulse-labeled cells through one entire cell cycle, can be seen in Fig. 2f and 2g. The labeled net- works from stationary phase cells in Fig. 2h are completely covalently closed. The absence of contaminating nuclear DNA was demonstrated both by the z 4 C

164

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Fig. 1. E th id ium bromide--CsCl equi l ibr ium g~adients of purif ied 3H-labeled k inetoplas t DNA from L. tarentolae cells at t w o t imes in the g~owth cuxve. 14C-labeled L. tarentolae nuclear DNA was added to e a c h tube as an in ternal u p p e r b a n d marker. Cent~fuga t ion condit ions: nD2 $o = 1.3876, 330 Dg e th id inm bromide /ml , 40 h at 4 0 0 0 0 rev,/min, 20°C, No. 50 rotor. Four drop f rac t ions w e r e c o l l e c t e d f r o m the b o t t o m o n t o Whatman No. 3MM filter discs, w h i c h w e r e t r i ch lo ro a ce t i c ac id -processed and c o u n t e d . (a) 8.5 h cells (28 • 106 cells/ml), (b) 2 day cells (82 • 106 celis/ml).

profile in each case, and also by analytical CsC1 equilibrium centrifugation of each sample (data not shown}.

Similar results were obtained when this experiment was repeated with L. tarentolae (data no t shown).

Pulse-labeling minicircles We examined the specific activities of the various molecular components

of kinetoplast DNA directly after pulsing L. tarentolae cells with [3H]- thymidine. In order to increase the amount of label taken up, the cells were partially synchronized with hydroxyurea prior to pulsing with 10 #Ci/ml [3 HI - thymidine for 18 min. As shown in Table I, essentially no label was incor- porated into closed minicircles, either " f ree" [7] or isolated from sonicated networks. Open " f ree" monomeric minicircles had a specific activity of 30 900 cpm/pg. The somewhat lower specific activities of the open minicircles, open catenanes and fragments derived f rom sonicated networks could be explained on the basis of the isolation technique, which yields peaks that are derived from broken or nicked closed minicircles as well as from originally open mini- circles. The data are consistent with the conclusions that only apparently open minicircles take up label in a pulse and that there is no difference between the labeling of ne twork open minicircles and "f ree" open minicircles. Since we do not known whether these pulse-labeled minicircles are identical in all respects to true open (Form II) minicircles, we shall designate pulse-labeled minicircles as Form II* molecules.

As shown in Fig. 3, sedimentation of pulse-labeled "f ree" II* minicircles in alkaline sucrose gradients demonstrated that the radioactivity was only in- corporated into linear strands, and that the labeled fragments were mostl~¢ smaller than unit length minicircle DNA.

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Fig. 2. E t h i d i u m b romide - - -CsCl e q u i l i b r i u m g r a d i e n t s o f p u r i f i e d k i n e t o p l a s t D N A f r o m p u l s e d a n d c.hased log p h a s e C. fasciculata cells. Cells w e r e p r e l a b e l e d in K i d d e r a n d D u t t a ' s m e d i u m f o r 1 8 h w i t h [ 1 4 C ] t h y m i d i n e (1 # C i / m l , 3 6 . 6 Ci /M) a n d w e r e t h e n w a s h e d a n d pu l sed w i t h [ 3 H I t h y m i d i n e f o r 1 0 r a i n ( 5 0 # C i / m l , 1 8 C i l m M ) . T h e cel ls w e r e t h e n w a s h e d in SBG w i t h 1 0 0 # g f m l o f u n l a b e l e d t h y m i d i n e a n d w e r e r e s u s p e n d e d in BHI m e d i u m w i t h 5 0 # g / m l o f u n l a b e l e d t h y m i d i n e . The k i n e t o p l a s t D N A w a s p u r i f i e d as d e s c r i b e d e l s e w h e r e [ 1 3 ] . C e n t r i f u g a t i o n c o n d i t i o n s : n D 2 s ° = 1 . 3 8 8 0 , 4 0 0 0 0 r e v . / m i n , 2 0 ° C 4 8 h , No. 50 r o t o r . F o u r - d r o p f r a c t i o n s w e r e c o l l e c t e d f r o m t h e b o t t o m o n t o W h a t m a n 3MM discs w h i c h were t r i c h l o r o a c e t i c a c i d - p r o c e s s e d a n d c o u n t e d . T h e d a t a wexe c o r r e c t e d f o r c r o s s o v e r a n d c o m p u t e r p l o t t e d . - - - - - - , 1 4 C . p r e l a b e l e d D N A ; , 3 H pu l s e - l abe l ed D N A . The c h a s e t i m e s w e r e as fo l l ows : (a) 0, (b) 0 . 5 h , (c) 1 .0 h , (d) 1 ,5 h , (e) 2 .0 h , (f) 5 . 0 h , (g) 6 . 0 h , (h) 3 . 0 d a y s .

T A B L E I

P U L S E - L A B E L I N G O F L. T A R E N T O L A E K I N E T O P L A S T D N A M I N I C I R C L E S W I T H [ 3H] T H Y M I D I N E

L. tarentolae cells in M e d i u m C A w e r e p a r t i a l l y s y n c h r o n i z e d w i t h h y d r o x y u r e a a n d i m m e d i a t e l y a f t e r re lease f r o m h y d r o x y u r e a i n h i b i t i o n we re p u l s e d w i t h [ 3 H ] t h y m i d i n e ( 1 0 # C i / m l , 1 8 C i / m M ) f o r 1 8 ra in . K i n e t o p l a s t D/CA n e t w o r k s we re i so l a t ed , s o n i c a t e d a n d s e p a r a t e d i n t o c lo sed a n d o p e n f r a c t i o n s b y e t h i d i u m b r o m i d e - - C s C 1 e q u i l i b i r u m c e n t r i f u g a t i o n . The v a r i o u s m o l e c u l a r spec ies we re r e s o l v e d b y b a n d ve loc i ty s e d i m e n t a t i o n in 5 - - 2 0 % s u c r o s e g r a d i e n t s in SSC. The a b s e n c e o f n u c l e a r D N A in the u p p e r e t h i d i u m b r o m i d e - - C s C 1 b a n d w a s d e m o n s t r a t e d b y a n a l y t i c a l CsCI e q u i l i b r i u m c e n t r i f u g a t i o n . " F r e e " min i c i r c l e s we re i s o l a t e d b y a lka l ine lys i s M e t h o d II o f Wesley a n d S i m p s o n [ 7 ] , a n d were s e p a r a t e d i n t o c lo sed a n d o p e n m o l e c u l e s b y b a n d v e l o c i t y s e d i m e n t a t i o n in 5 - - 2 0 % a lka l ine s u c r o s e g r a d i e n t s .

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( c p m / # g )

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Fig. 3. Band ve loc i ty s ed imenta t ion of [ 3 H ] t h y m i d i n e pulseqabeled "free" m o n o m e r i c II* mini- circles f rom L. t a r e n t o l a e in 5--20% alkaline sucrose gradient. The exper iment and the preparat ion techniques axe described in Table I. Centri fugat ion condit ions: 60 0 0 0 rev . /min, SW 50 rotor , 10 h, 4°C.

, absorbance at 254 n m and the f i l led circles the radioact iv i ty profi le .

Isolation of networks from partially synchronized cells The actual division process of kinetoplast DNA networks in thin sections

has been described for several species (See ref. 1 for review). This process implies a doubling of the surface area of the kinetoplast DNA network, as- suming a uni-minicircular thickness at all stages of replication.

As shown in Figs 4 and 5, networks isolated from hydroxyurea-syn- chronized [4] L. tarentolae and C. fascieulata cells at different points in the cell cycles showed the expected increase in surface area when spread on glass slides, stained with Giemsa and examined in the light microscope. The dif- ferences between the size distribution of early S phase networks, intermediate S phase networks, and late S phase networks are readily apparent. The net- works in Figs 4d and Fig. 5e were taken from cells at the end of S phase and

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Fig. 4. Size d is tr ibut ion h is tograms of L. t a r e n t o | a e kinetoplast D N A n e t w o r k s i so lated from h y d ~ o x y u r e a s y n c h r o n i z e d cells [4 ] at several t imes in the cell cyc le . The n e t w o r k s were processed for l ight m i c r o s c o p y as described in Methods . (a) Early S phase , 1 h after release f rom inhibi t ion. (b) Intea~nediate S phase 2 h after release f rom inhibi t ion. (c) Late S phase , 3 h after release f rom inhibit ion. (d) End o f S phase , beginning of cell d iv is ion, 4 h after release from inhibi t ion .

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Fig. 5. Size d i s t r i b u t i o n h i s t o g r a m s o f C. fasciculata kLne top ias t D N A n e t w o r k s i s o l a t e d f r o m h y d r o x y - u r e a s y n c h r o n i z e d cel ls a t severa l t i m e s in t h e cel l cyc le . The n e t w o r k s we re p r o c e s s e d f o r l i gh t m i c r o - s c o p y as described in Methods . (a) E a r l y S phase, 30 m i n a f t e r re lease f r o m i n h i b i t i o n . (b) I n t e r m e d i a t e S phase, 1 h after release f r o m i n h i b i t i o n . (c) I n t e r m e d i a t e S p h a s e , 1 . 5 h a f t e r r e l ease f r o m i n h i b i t i o n . (d) Late S p h a s e , 2 . 0 h after release f r o m i n h i b i t i o n . (e) Cell d iv i s ion , 3 h a f t e r re lease f r o m i n h i b i t i o n .

the beginning of cell division, and the decrease in the average ne twork size is consistent with kinetoplast DNA division. The larger size of Crithidia networks as compared to Leishmania networks is a striking difference between the spe- cies.

Localization of the sites o f DNA replication by autoradiography of [3H] thymidine labeled networks

Due to the large surface area of isolated networks when spread on glass slides, it was possible to localize the sites of DNA replication within the en- larging ne twork by means of light microscope autoradiography. This was per- formed with both L. tarentolae and C. fasciculata, although the kinetoplast DNA networks of Crithidia are larger and less fragile and therefore more suitable for this technique. Log phase C. fasciculata cells were pulsed for 10 min with [3H]thymidine and then chased with unlabeled thymidine. Kine- toplast DNA networks were isolated and processed for light microscope auto- radiography. Labeling patterns were classified into several basic types and the relative numbers of labeled networks showing these patterns were scored. The data from one representative experiment are shown in Table II and micrographs of representative labeled patterns observed are presented in Fig. 6. It should be emphasized, however, that the schematic classes of patterns given in Table II represent an artificial classification superimposed on a cont inuum of observed patterns. For example, the "gapped peripheral" networks had great variations in the size of the gap, and the "gapped central" networks merged into the "central" networks.

168

TABLE II

VARIATION IN PATTERNS OF PULSE-LABELED KINETOPLAST DNA NETWORKS OF C. FASCI- CULATA DURING A CHASE

Log phase cells were pulse-labeled with 50 pCi/ml of [3H] thymidine in Medium CA for 10 rain and then washed and chased in Medium CA containing 100 pg/ml of unlabeled thymidine. Kinetoplast DNA net- works were isolated from the cells at the indicated times and spread on glass slides for autoradiography. Labeling patterns: (a) peripheral, (b) gapped peripheral, (e) centripetal, (d) gapped centripetal, (e) central, (f) random, (g) miscellaneous.

Time (a) (b) (c) (d) (e) (f) (g) Total (h) coun-

Q O ~ ~ ~ O ~ ted

0 93% 3% 0% 0% 0% 0% 4% 370 3 11 46 14 14 13 1 1 304 4.5 1 26 14 24 24 10 1 201 6 1 10 2 17 8 60 3 301

Fig. 6. Light microscope autoradiogaphs of kinetoplagt D N A networks , isolated from C. fasciculata cells pulsed wi th [ 3 H ] t h y m i d t n e and chased wi th u,~imbeled thymidine . The experimental condit ions axe described in Table II. (a) and (b) pul~-Iabeled networks . (c) thzough (h) pulsed and chased networks . The ne tworks were selected to demonstrate the various types of labeling patterns observed in the chase situation.

169

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Fig . 7. Size d is tr ibut ion his tograms o f k inetoplast D N A n e t w o r k s i so lated from C. fasciculata cells pulse- labeled wi th [3HI thymid ine . The exper imenta l details are given in Table II . e , number of labeled net- w o r k s present in each size class. This represents on ly the zero t ime of Table I I .

Directly after the pulse, more than 90% of the labeled networks showed a definite peripheral labeling pattern, a fact which implies that networks in all portions of the S phase exhibited a peripheral replication pattern. This conclu- sion was strengthened by scoring the frequency distribution of peripherally labeled networks at zero time after the pulse as a function of network size as shown in Fig. 7.

With a progression of the chase the labeling patterns showed definite changes. As shown in Table II, the silver grains moved centripetally as the chase progressed to 4.5 h. Often gaps in the circular ring of grains were apparent. After 6 h of chase a significant portion of labeled networks exhibited a scatter- ed or random grain distribution pattern, implying a redistribution of labeled DNA throughout the network structure.

The control experiment for the random distribution of label over totally labeled networks was impossible to perform with C. fasciculata since it has been shown that any attempt at cont inuous labeling with [3 H] thymidine leads to an apparent pulse-chase situation due to a cell-mediated conversion of thymidine to thymine in the medium [13 ] . However, electron microscopic evidence has been presented by Renger and Wolstenholme [3] for a homo- geneous distribution of DNA molecules throughout the network structure of a related species, Crithidia acanthocephali.

Qualitatively identical results were obtained with L. tarentolae, but the localization of silver grains over kinetoplast DNA networks during the chase was difficult to quantitate due to the small size and fragility of the networks from this species.

Replication of L. tarentolae minicircles The apparent limitation of DNA replication to the periphery of the net-

work structure leads to an interesting prediction concerning molecules within the interior of the network, which apparently would not be replicated. In a

170

density transfer experiment using a heavy isotope to label the newly replicated DNA, one would expect to see a large amount of light/light (LL) parental molecules remaining after one generation and the simultaneous appearance of an equal amount of heavy/heavy (HH) daughter molecules.

The minicircle is the major molecular species of the network, Therefore, a density transfer experiment was performed using minicircles isolated from net- works of L. tarentolae, this species was employed due to the availability of techniques to isolate high yields of minicircles from the kinetoplast DNA [7 ].

Bromodeoxyuridine was employed as a density label. Cells were grown for three days in Medium CA with [3 H] thymidine and then transferred to Medium CA with 32 Pi and bromodeoxyur idine . After 1.0 (19.7 h) and 1.9 (29.5 h) cell divisions, cells were harvested and kinetoplast DNA networks were isolated. Closed minicircles were isolated from sonicated networks by band velocity sedimentation in alkaline sucrose and then centrifuged to equilibrium in neutral CsC1 as shown in Fig. 8a and 8b. Nuclear DNA was isolated from the cell lysates as an internal control and also centrifuged to equilibrium in neutral CsC1 (Fig. 8c and 8d).

The small LL minicircle shoulders in both gradients of Fig. 8a and 8b are probably derived from non-viable cells rather than from any non-replicated

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15

FRACTION

Fig. 8. P r e p a r a t i v e CsCI e q u i l i b r i u m g r a d i e n t s o f c losed monome]c i c m i n i c i r c l e s a n d n u c l e a r D N A f r o m L. tarentolae cells in a d e n s i t y t r a n s f e r e x p e r i m e n t . Cells w e r e g r o w n f o r 3 d a y s in M e d i u m C A w i t h [ 3 H ] t h y m i d i n e ( 1 0 /~Ci/ml, 2 ~tg/ml) a n d w e r e t h e n t r a n s f e r r e d t o M e d i u m C A w i t h 3 2 p i ( 1 0 / J C i / m l ) a n d b r o m o d e o x y u r i d i n e ( 2 0 0 /~g/ml). A f t e r 1 . 0 cel l d iv i s ions ( 1 9 . 7 h) a n d 1 .9 cel l d iv i s ions ( 2 9 . 5 h ) cel ls w e r e r e m o v e d a n d l y s e d in h o t s a r k o s y l - p r o n a s e . A f t e r r e m o v a l o f t h e k i n e t o p l a s t D N A n e t w o r k s b y c e n t r i f u g a - t i o n , t h e n u c l e a r D N A w a s r e c o v e r e d f r o m t h e l y s a t e b y a l c o h o l p r e c i p i t a t i o n a n d p u r i f i e d as d e s c r i b e d p r e v i o u s l y [ 2 ] . C l o s e d m o n o m e r i c min i c i r c l e s w e r e i s o l a t e d f r o m s o n i c a t e d n e t w o r k s b y a lka l ine suc ro se v e l o c i t y g r a d i e n t s as d e s c r i b e d p r e v i o u s l y [ 7 ] . C e n t r i f u g a t i o n c o n d i t i o n s : n D 25° = 1 . 4 0 1 0 , 7 0 h a t 3 9 0 0 0 r e v . / m i n , 2 0 ° C , No . 5 0 r o t o r . T h e t u b e w a s f r a c t i o n a t e d b y d r i p p i n g 5 d r o p f r a c t i o n s o n t o W h a t m a n N o . 3MM f i l t e r d iscs w h i c h were t r i c h l o r o a c e t i c a c i d - p r o c e s s e d a n d c o u n t e d . @~ p a r e n t a l 3 H - l a b e l e d D N A ; o , n e w 3 2 P - l a b e l e d D N A . (a) C l o s e d m o n o m e r i c min i c i r c l e s , 1 .0 cel l d iv is ions . (b) C l o s e d m o n o m e r i c min i - c i rc les , 1 .9 cel l d iv is ions . (c) N u c l e a r D N A , 1 . 0 cel l d iv is ions , (d) N u c l e a r D N A , 1 .9 cell d iv is ions .

171

internal network minicircles, as shown by the identical appearance of the nu- clear DNA patterns in gradients (c) and (d). The relative amounts of the HL and HH peaks do not accurately reflect the expected ratios calculated from the number of cell divisions, but this is probably due to the abnormally long period required for the cell number to double (19.7 h as compared to the normal log phase doubling time of 9--12 h). The fact that the LL--LH--HH transitions seen in the case of the minicircles were identical to those seen in the case of the nuclear DNA suggests strongly that all of the minicircles in dividing cells re- plicated and that they replicated in an apparently normal semi-conservative fashion. This conclusion is obviously dependent on a random recovery of minicircles from all parts of the network structure. That this is likely is indi- cated by the recovery of approx. 23% of the total network DNA in the form of closed monomeric minicircles by the technique employed [7].

Discussion

We have described several aspects of the replication of the kinetoplast DNA networks of two related species of hemoflagellates, L. tarentolae and C. fasciculata. Several salient points have emerged. Networks containing repli- cating minicircles and pulse-labeled minicircles isolated both from networks and from the "free" class of molecules exhibit an equilibrium density in ethidium bromide--CsC1 which is less than that of non-replicating covalently closed networks. These results could be explained either by the presence of nicked minicircles replicating by the rolling circle model [14] or by the pres- ence of closed minicircles replicating by the model of Sebring et al. [15]. Replicating closed circular duplexes have been reported for SV40 DNA [16,17] and also polyoma DNA replication [18]. Apparently similar repli- cative forms have also been reported to occur at high frequency in the kine- toplast DNA of Trypanosoma cruzi cells that were grown in the presence of the drug, berenil [19]. In the cases of L. tarentolae and C. fasciculata, the available evidence is not sufficient to distinguish between these modes of replication.

The topographical limitation of DNA replication in the kinetoplast DNA network to the periphery of the structure is a striking but unexplained phe- nomenon. We speculate that this is an expression of the localization of mem- brane binding sites. Whatever the mechanism, this phenomenon, together with the density transfer results, suggest that there is lateral mobility of molecules within the network structure which allows interior minicircles to pass through the peripheral locus of replication. The redistribution of pulse-labeled DNA throughout the network as observed in the autoradiography chase experiments is another indication of lateral mobility. One possible mechanism could be the existence of a large amount of recombination of minicircles that leads to linear molecules composed of broken and rejoined minicircles. These interdigitated linear molecules would then allow the necessary lateral mobility, and even- tually would undergo another round of breakage and rejoining to reform covalently closed minicircles. Steinert has independently proposed a similar mechanism to explain the appearance of linear molecules during an electron microscopic pulsechase experiment with Crithidia luciliae (personal communi- cation}. Evidence for extensive recombination between kinetoplast DNA mini-

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circles of C. acanthocephali has recently been obtained by Manning and Wol- stenholme [ 20] .

The kinetoplast DNA of L. tarentolae and C. fasciculata have some basic differences in molecular structure and organization, but show many similarities in the mode of replication. An investigation of the precise differences and similarities between these species and others in the order, Kinetoplastida, may lead to a bet ter understanding of the evolution of the mitochondrial DNA in this group of protozoa and perhaps even shed some light on the selective forces involved in such an unusual gene amplification.

Acknowledgements

The research was supported in part by Research Grant AI90102 to L.S. from the National Institutes of Health, by University of California Research Grant 2456 to L.S., and by a Biomedical Sciences Support Grant to the Uni- versity of California. R.D.W. was supported by Training Grants GM-1521 and AI-00070 from the National Institutes of Health.

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