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The kinetoplast DNA of Trypanosoma gambiense: Comparison with the kDNA of Trypanosoma equiperdum
BIOCH1MIE, 1981, 63, 755-765.
The kinetoplast DNA of Trypanosoma gambiense : Comparison with the kDNA of Trypanosoma equiperdum. Guy RIOU <> and Michel BARROIS. (Re~u le 12-6-1981, acceptd le 30-7-1981).
Unitg de Pharmacologie MoHculaire, lnstitut Gustave Roussy, 94800 Ville]uif, France.
R~sum~.
Summary.
Les composants mol~culaires du D N A kin~toplastique ( k D N A ) du Trypanosoma gambiense ont ~tO analys~s et comparLv i~ ceux du Trypanosoma equiperdum, espkce voisine. Le k D N A de T. gambiense comporte environ 80 maxicercles de 20 000 paires de base et 4 000 minicercles de 1 000 paires de base. La carte physique des sites de clivage de 7 endonucl~ases de restriction a ~t~ ~tablie sur le maxicercle de T. gambiense. La plupart de ces sites ont Ot£ observes sur le maxicercle de T. equiperdum, cependant leur position relative est dif[6rente. Des maxicercles pr#sentant des [ourches de r6plication ont ~t~ observes au microscope (lectronique. Des expdriences, d' une part de cin~tique de renaturation et d'autre part d'hybridation aprbs trans[ert du D N A sur [iltre, montrent que les maxicercles de T. gambiense et de T. equiperdum ont une partie de leur s~quence en bases commune. Les minicercles de T. gambiense ont leur s6quence en bases h~t~rogkne contrairement ~ ceIle des minicercles de T. equiperdum. Les minicercles de ces deux espkces ont @alement une partie de leur s6quence commune.
The molecular components of the kinetop!ast D N A ( k D N A ) network of Trypanosoma gambiense have been studied and compared with those o[ the very closely related species T. equiperdum, previously studied in detail. The k D N A o[ T. gambiense contains about 80 maxicircles of 20 kilobase pail's and 4000 minicircles o[ l kilobase pairs. The restriction cleavage sites o[ 7 restriction endonucleases have been mapped on the T. gambiense maxicircle. The majority o[ these sites were also [ound in T. equiperdum maxicircles ; however their relative positions which are di[[erent do not allow us to conclude to relatedness o[ maps. Maxicircles o[ the Cairn 07" of the rolling circle type have been observed and thought to be replicative intermediates. Experiments on renaturation kinetics and hybridization alter blotting trans!er, show that T. gambiense and T. equiperdum maxicircles' have base sequences in common. The T. gambiense minicircles are heterogeneous in base sequence, in contrast to the T. equiperdum minicircles which are homogeneous. The minicircles o[ the two species have also common base sequences.
Mots-clds : DNA mitochondrial / enzymes de restriction / hybridation mol~culaire.
Key-words: mitochondrial DNA / restriction enzymes /molecular hybridization.
Introduction.
and they are morphologically undistinguishable. Like all members of Kinetoplastida, they possess an unique mitochondrion with a specialized portion called kinetoplast, containing DNA (kDNA), which can be isolated as a complex network of high molecular weight. The general properties of the kDNA have recently been reviewed by Borst and Hoeijmakers [1] and by Engtund [2]. In all the species of trypanosomes so far studied, the kDNA is composed of two distinct molecular components, the minicircles and the maxicircles, found in variable proportions among different species. Their biological role remains unknown, although
Trypanosoma gambiense is the agent of sleeping sickness, which constitutes a serious risk to the health and life of million people in Africa. This
flagellate, transmitted by the tse-tse fly, undergoes a complicated cycle of development. T. equiperdum has a quite different infective cell cycie: il is venereally transmitted in Equines. Both trypanosomes belong to the same subgenus, Trypanozoon, © To whom all correspondence should be addressed.
756
G. R i o u and M. Barrois.
recent reports have indicated that, in some species, these molecules are partially transcribed [3, 4]. We had previously reported that T. e q u i p e r d u m a n d T. gambiense possess k D N A networks in nearly the same proportions (6 to 8 per cent of total D N A ) , that the k D N A has the same b u o y a n t density a n d that the minicircles have the same contour length [5, 6]. Analysis by restriction endonucleases a n d molecular h y b r i d i z a t i o n can successfully be applied to characterize the m o l e c u l a r c o m p o n e n t s of k D N A s a n d to establish phylogenetic relationships between two closely related t r y p a n o s o m e species. The k D N A of T. e q u i p e r d u m has previously b e e n studied in detail [6]. I n this work, the molecular c o m p o n e n t s of the T. gambiense k D N A are separated, characterized, a n d c o m p a r e d with the T. e q u i p e r d u m k D N A components.
Materials and Methods.
Strains and isolation of trypanosomes. The original trypanosome strains were obtained from the Institut Pasteur (Paris). T. equiperdum was isolated by Laveran about 60 to 70 years ago and T. gambiense strain Eliane by Vaucel [7] in 1951. These strains were maintained by laboratory mammals transfers. About 106 trypanosomes of each strain were separately injected in rats in order to obtain maximal infection in 3 to 4 days. The trypanosomes were isolated from blood, as described by Lanham and Godfrey [8]. The trypanosomes were washed 3 times with 0.15 M NaC1 0.015 M sodium citrate (SSC). Preparation and purification o] kDNA. The cells (about 200 mg wet weight) were lysed with 5 ml of sarkosyl (1 per cent in SSC), for 15 min at 37°C. Pronase (1 mg/ rnl), preincubated for 1 h at 37°C, was added to the lysate and the mixture was incubated for 2 h at 37°C. The lysate (2 vol) was layered on 20 per cent saacrose in SSC (1 vol) and centrifuged at 22 000 Irom for 1 h at 20°C in a Beckman SW 27 rotor. The collected pellet was then centrifuged in a CsC1-Ethidium bromide (EthBr) gradient (300 ~g/ml EthBr, 0.87 g/ml CsC1 to obtain n D 20-1.3900, 40 000 rpm, 24 h at 20°C in a Beckman 50 Ti rotor). The kDNA was recovered from the lower band of the gradient and the EthBr removed with isopropanol. Analytical ultracentrifugation. A Spinco model E analytical ultracentrifuge was used. Equilibrium density centrifugation in CsC1 was performed during 24 h, at 44 000 rpm and 25°C, using Micrococcus lysodeikticus DNA as density marker. Scanning was done at 265 nm using a Beckman photoelectric system. The sedimentation coefficients were measured by band sedimentation analysis BIOCHIMIE, 1981, 63, n o 10.
in 3 M CsCI, 1 mM Na a EDTA (pH 8.0) at 20°C with an ANJ rotor at 6,000 rpm as described by Brnard et al. [9]. Cleavage of kDNA by restriction endonucleases and gel electrophoresis. The restriction endonucleases were obtained from New England Biolabs (Beverly, Mass.) and used under the digestion conditions recommended for each enzyme [6]. The DNA fragments were separated by electrophoresis in vertical slab gels. Agarose gels (2 per cen0 were used to measure the molecular weight of the fragments up to 1.6 kb, while 0.5 per cent agarose-l.8 per cent polyacrylamide gels were used for larger fragments. The sizes of the fragments were estimated by comparing their migration with that of )~ DNA Hind HI fragments or Hae III T. cruzi k D N A fragments (calibrated by Hae III fragments of • X 174 DNA) [10]. In vitro synthesis o] 8sP-labeled kDNA. Form I minicircles were separated from the linearized maxicircles, after oleavage with Pst I restriction endonuclease, using a CsC1-EthBr gradient [6]. The mini and maxicircles were purified in a CsC1-Hoechst 33258 dye gradient [11]. The kDNA was nick-translated, according to the method of Mackey et al. [12] in the presence of [32p] dCTP and [~P] dATP (Amersham, specific activity 2 000 to 3 000 Ci/mmole). The size of the nick translated DNA fragments was estimated by comparison with sheared, 3H labeled Trypanosoma cruzi nuclear DNA of known size (S°20.w = 5.9). DNA polymerase I was purchased from Boehringer-Mannheim, and DNAse I (electrophoresis grade) from Worthington. The labeled kDNA was treated as described by Pellicer et al. [13] to remove fast reannealing material (15 to 20 per cent of the probe DNA).
Reassociation kinetics. 3zP-labeled DNA was heated for 20 min at 100°C in 44 mM Na phosphate pH 6.8 and reassociation was carried out at 68°C in 0.86 M NaCl, 86 mM Na phosphate pH 6.8 and 0.8 per cent SDS. 100 !~1 fractions were collected at various times and diluted with 0.9 ml of 0.14 M Na phosphate pH 6.8, 0.4 per cent SDS. The fraction of single and double-stranded DNA was determined by hydroxylapatite chromatography, as described by Sharp et al [14]. The labeled mini and maxicircles DNA gave 90 per cent and 85 per cent reassociation at saturation, respectively.
Hybridization experiments by the DNA transfer technique. Mini and maxicireles were cleaved by appropriate restriction enzymes and DNA fragments separated by electrophoresis in agarose gels. They were denatured in situ and blotted onto a nitroceUulose filter and then hybridized by the method of Southern [15]. Hybridization was performed with about 2 × 106 cpm of the a2p labeled DNA probes obtained as described above.
Electron microscopy o] DNA. kDNA networks were spread using the micro-diffusion technique previously described [16].
k D N A of T. gambiense and T. e q u i p e r d u m .
757
Results.
B
1.6906 1.7074 2 1.6909
c
1
rn /\
1.68441.6932 D
!L
rn
j'~
1.6931 FIG. 1. - -
Equilibrium density centri/ugation
o f T. g a m -
biense DNA in CsCl gradient. (A) total DNA; (B) intact kDNA form 1 ; ((2) kDNA form I after cleavage by endonuclease Pst I ; (D) mini¢ircle core-kDNA form I obtained after Pst I cleavage and CsC1-EthBr centrifugation. Micrococcus lysodeikticus DNA (~ = 1.731 g/cm 3) was used as density marker (m).
Fractionation and characterization of the molecular components of k D N A from T. gambiense. W h e n the total D N A extracted from T. gambiense was analyzed by e q u i l i b r i u m density centrifugation in a CsCI gradient at least two c o m p o n e n t s are o b s e r v e d : k D N A with a density 9 = 1.6907 + 0.001 g / c m 3 and a m a i n b a n d of D N A of density 9 = 1.7069 -+ 0.0001 g / c m "~ with a shoulder corresponding to D N A of lower density (figure 1A). T h e k D N A , which accounts for about 8 per cent of the total D N A , was purified by e q u i l i b r i u m density centrifugation in a CsCI gradient containing EthBr. T h e lower b a n d c o n t a i n e d k D N A form I which, after spreading for electron microscopy, presented the typical structure previously described for T. equiperdum [6, 17] (figure 2). Two distinct c o m p o n e n t s could be seen in the n e t w o r k : n u m e r o u s interlocked minicircles of 0.31 lxm long (1 kb) and D N A loops p r o t r u d i n g at the periphery of the n e t w o r k and identified as maxicircles (figure 2) (see below). A linear molecule is connected with some of these maxicircles (figure 2, a to e). After e x a m i n ; n g more than 200 networks of the k D N A preparation, we observed
TABLE 1. Molecular weights, in kb, o[ the fragments of maxicircle k D N A generated by restriction endonucleases from T. gambiense. Restriction endonuclease
Fragment
Bgl II
Bam HI
Pst [
Hpa II
Hae llI
Hind II1
1 2 3 4
20.0
20.0
20.0
10,3 9.2 09
14.0 3.5 1.0
7.4 6.5 6.2
1.0
20.4 Hind 1II Hind III Hind I11 Eco RI Barn HI Hpa II Eco RI Pst I 1
2 3 4 5 6 7
Eco RI
9.3 6 0 4 2
Barn HI Barn HI P~t I Bgl II 14,3 5 7
17 0 3 2
Pst I Bgl 11
Hind III Pst I
11.6 8,7
7,5 6.3 5.3
0.8
1.4
20.4
20 1
20.3
20 0
Eco RI Barn HI
Eco RI Hpa II
Eco RI Hae ill
Hae lIl Pst I
20 2
20.3
Hae III Hpa II Barn HI Barn HI
20 5 Hpa II Pst I
7.5 6.5 4.2 2.0
7.5 6.3 3,4 2.5 0.9
6,3 3.3 3.0 3.0 3,0 1.2 0.8
9.3 4.6 4.2 1.6 0.8
9.3 6.0 3.2 1.0 0.8
6.5 6.2 3.6 2.8 0,8 0.8
9.3 3.4 3.1 2.8 0.8 0.8
13.8 3.5 1.2 1.0 1.0
14.8 3.5 1.0 1.0
10 0 9.3 09
10.3 4.6 4.6 0.9
20.2
20.6
20.6
20.5
20.3
20.7
20.2
20.5
20.3
20.2
20 4
BIOCHIM1E, 1981, 63, n ° 10.
G. R i o u and M. Barrois.
758
FIG. 2. - -
Electron micrograph o] an intact kDNA network )'rom T. gambiense. Magnification of several regions showing three branched maxicircles are s h o w n in inserts a to e.
BIOCHIMIE, 1981, 63, n ° 10.
kDNA o[ T. gambiense and T. equiperdum. 12 networks with at least one three-branched maxicircle. The buoyant density of the purified kDNA network in CsC1 was equal to 1.6907 g/ml (average of 3 determinations) (figure 1B). The networks had a sedimentation coefficient of S~0,w = 2010, as measured by band sedimentation in 3 M CsC1 (average of 3 determinations). When the intact kDNA network was incubated with the restriction endonuclease Pst I two molecular components were obtained and identified by electron microscopy : linear molecules of about 20 kb (table I) identified as linearized maxicircles and a kDNA network containing only interlocked minicircles covalently closed, which we will refer to as minicircle core-kDNA. The linearized maxicircles have a buoyant density of 1.684 g/cm 3, as determined by equilibrium centrifugation in a CsC1 gradient (average of 4 determinations), which corresponds to an AT content of 76 per cent. According to the area under the peak of the scanner tracing (figure 1C), they account for 27 per cent of the intact networks (average of 4 determinations). The core-kDNA minicircles form I which had been recovered from the lower band of a CsCt-EthBr gradient (figure 3) had a buoyant density of 1.6933 g/cm "~ (figure 1 D) (average of 3 determinations).
759
weight were generated (figure 4). The molecular weights of these fragments are presented in table I. The results indicate that the DNA loops are extruding parts of maxicircles, whose uniform length is about 20 kb. Short fragments of about 1 kb corresponding to linearized minicircles were also 1
2
3
4
5
6
7
w
26.2 22.1~
N
8.8 6.1 4.0
2.1~ 1.8_
N
1.45 1.09 0.74
i
0.51 0.36
FIG. 3. - - Photograph oJ CsCI-EthBr gradient centHfugation oJ k D N A # o m T. gambiense. (A) intact k D N A network f o r m I ; (B) after cteavage by Pst I : the upper band is c o m p o s e d o f linearized maxicircles, while the lower band contains c o r e - k D N A minicircle f o r m I.
Cleavage map of maxicircle kDNA [rom T. gambiense. When the intact kDNA networks were digested with the restriction endonucleases Barn HI, Pst I, Bgl II, Hae III, Hpa II, Hind HI, Eco RI and analyzed by gel electrophoresis, one or more fragments of relatively high molecular BIOCHIM1E, 1981, 63, n ° 10.
W
FIG. 4. - - Electrophoresis in 0.5 per cent agarose - 1.8 per cent polyacrylamide gel oJ the [ragments generated by cleavage o] the k D N A network o] T. gambiense by restriction endonucleases. (t) T. cruzi k D N A minicircle fragments obtained by Hae III cleavage ; (2)), D N A fragments obtained by Hind III cleavage and used as size standards (the molecular weights are given in kb) ; (3) B g I I I ; (4) B g l l I + Barn H1 ; (5) Bgl II + Pst I ; (6) Pst I ; (7) Bam H I .
generated in small amounts by the endonucleases (less than 7 per cent of the minicircles). Molecules of the DNA band migrating in the 1.45 kb zone (figure 4) are composed of free minicircles form II as shown by electron microscopy after elution of the DNA band. These minicircles have been released from the kDNA network.
760
G. Riou and M. Barrois.
Since each restriction endonuclease has a limited number of cleavage sites per maxicircle, a physical map of the DNA molecule can be drawn. The unique cleavage site of Pst I is taken as origin. The data of table I obtained from experiments with single or double enzyme digests, enable location of the restriction enzyme cleavage sites on the maxicircles, as drawn in figure 5. However linearized minicircles and free minicircles produce electrophoresis bands which could comigrate with maxicircle DNA fragments. Some restriction sites T. equiperdum
Hae III
Cleavage of the minicircle core-kDNA from T. gambiense by restriction enzymes. When the minicircle core-kDNA was incubated with the restriction endonucleases used above, the minicircles
1
2
3
4
5
6
7
T. g a m b i e n s e
Pstl
_
Pstl
_
EcoRI
_
Eco
1.45 - - H p a II _
B g l II
H i n d Ill _ _
Hpall EcoRI
RI
1.09 *
A
0.74
Hpall_ _
H i n d III
0.36
Bgl II
Bgl I I _
_ H i n d III
_
__ E c o RI _
H i n d III
_
Haelll * BamHt H a e III *
__
H p a II
Hpall Haelll _ E c o RI BamHI--
"~" E c o R I
_ Hae III _ H p a II
_
Eco RI
_
Pst I
1
B Hindlll_
_ Haetll _Pst
Hindlll_
F[G. 6. - - Electrophoresis in 2 per cent agarose gel of the fragments generated by cleavage of minicircle corek D N A ]orm I from T. gambiense by restriction endonucleases. (1) minicir6le k D N A fragments from T. cruzi obtained by Hae III ~leavage ; (2) Xba I ; (3) Taq I + X b a I ; (4) Hinf I + X b a I ; (5) Hin[ I ; (6) Taq I + Hinf I ; (7) Taq I. The molecular weights are given in kb.
I
FIG. 5. - - Restriction maps o] mazicircle k D N A ]rom T. g a m b i e n s e Eliane strain and T. e q u i p e r d u m Institut
Pasteur strain. A and B are the regions of sequence homology, (*) probable restriction sites.
mapped on T. gambiense maxicircle are only probable sites compatible with the length of the maxicircle (figure 5 ). BIOCH1M1E, 1981, 63, n ° 10.
were not significantly cleaved. In contrast, the endonucleases Taq I, Hin[ I and Xba I cleaved all the minicircles in several fragments. Gel electrophoresis of single and double digests with the latter enzymes is presented in figure 6. The results show that the sum of the lengths of the DNA fragments generated is several times higher than the length of each minicircle. This observation indicates that T. gambiense minicircles are heterogeneous in base sequence. Do maxi or minicircle kDNA from T. gambiense have sequences in common with maxi or minicircle kDNA from T. equiperdum ? Maxicircles from T. gambiense were labeled in vitro with [a2p] dCTP and [3._,p] dATP and the reasso-
kDNA of T. gambiense and T. equiperdum. ciation kinetics of the labeled fragments were studied. The reannealing of labeled maxicircles k D N A follows simple second order kinetics, as previously described for T. equiperdum k D N A [6,
761
ments of t 1/2, about 8 per cent of the T. equiperdum maxicircles (23 kb) can hybridize with T. gambiense maxicircle (20 kb) (figure 8). The a'-'P labeled maxicircle k D N A fragments from T. equiperdum were also annealed in the presence of an 80-fold molar excess of unlabeled minicircle k D N A f r o m T. gambiense. The results, presented
A/ 2.0 03 03
2.0
-L r-
1.5
03 03
<.
,/f
1.5
I
10
t
I
t
20
30
40
t
50
time (hours) FIG. 7. Reassociation kinetics o[ s~p labeled kDNA maxicircle from T. gambiense. The linear form of kDNA maxicircles obtained by cleavage with Pst I, was isolated -
-
by equilibrium density centrifugation in CsC1-EthBr gradient and purified in a CsCI Hoechst 33258 gradient. The DNA labeled with 32p had a specific activity of 2.7 X 107 cpmLp,g and a size of about 300 to 400 nucleotides, as measured by alkaline sucrose gradient centrifugation. The reaction mixture contained 70 ng of labeled DNA per ml. Labeled DNA alone (--O--), or with 700 ng of unlabeled kDNA maxicircle from T. equiperdum (--A--). The reciprocal of the fraction of the single-strand DNA (1/f~0 is plotted as a function of time,
18]. The time required for half renaturation of maxicircle k D N A (t 1 / 2 ) was 72 h (figure 7). When reassociation was carried out in the presence of a 9-fold molar excess of unlabeled maxicircle k D N A from T. equiperdum, t 1 / 2 was of 40 h. This result reveals the presence of base sequences c o m m o n to both maxicircle populations (figure 7). We can estimate, from measurements of slopes, at about 10 per cent the base sequences c o m m o n to T. gambiense (20 kb) and T. equiperdum rnaxicircles (23 kb). Other reassociation experiments were done using a2p labeled maxicircle k D N A fragments from T. equiperdum alone, or in the presence of an 11-fold molar excess of unlabeled maxicircle k D N A f r o m T. gambiense. F r o m the measure-
BIOCHIMIE,
1981, 68, n ° 10.
10
20
30
40
time (,hours) FIG. 8. - - Reassociation kbzetics o/ ~P labeled kDNA maxicircle fragments Jrom T. equiperdum. The DNA had a specific activity of 4.0 × 107 cpm/Ixg. The reaction mixture contained 24 ng/ml of ,labeled DNA : alone ( - - 0 - - ) ; or in the presence of 48 ng/ml of unlabeled kDNA of T. equiperdum (--A--) ; or in the presence of 240 ng/ml of unlabeled kDNA maxicircle fragments from T. gambiense (--A--) ; or with 1 t~g/ml of unlabeled kDNA minicircle fragments from T. gambiense (--o--).
in figure 9, show that the rate of the reaction was not significantly modified, indicating that T. equiperdum maxicircles have no base sequence in comm o n with T. gambiense minicircles. These results were confirmed by hybridization after blotting transfer experiments onto nitrocellulose filters. The k D N A maxicircles from T. gambiense were cleaved by Hind III, Hae I I I and Eco R 1 + Pst I. T h e k D N A fragments were separated by electrophoresis in agarose gel, transferred onto nitrocellulose filter and then hybridized with zzp labeled D N A maxicircle from T. equiperdum. The results presented in figure 9 show that 32p D N A from T. equiperdum maxicircles hybridize with T. gambiense maxicircle and
G. Riou and M. Barrois.
762
that the h o m o l o g o u s sequences are d i s t r i b u t e d in two regions along the T. gambiense maxicircle. T h e i r length is a b o u t 8 kb (regions A and B of figure 5).
alone. W e can estimate, f r o m m e a s u r e m e n t s of t 1 / 2 , at a b o u t 10 per cent the base sequence c o m m o n to T. garnbiense and T. equiperdum minicircles ( d a t a not shown).
Eco R I Hind a
b
III c d
H a e III a
+ Pstl
b
a .
.
.
b
.
~i!:ri: ~.. : • ."
i
T h e minicircles f r o m the c o r e - k D N A of T. gambiense were linearized b y cleavage by Xba I. T h e 1 k b fragments g e n e r a t e d were t r a n s f e r r e d onto cellulose filter to be h y b r i d i z e d with ~2p labeled D N A from T. equiperdurn minicircles. T h e resuits p r e s e n t e d in figure 10, show that T. gambiense minicircles h y b r i d i z e with T. equiperdum minicircles, suggesting that only a p a r t of these two minicircles have a c o m m o n base sequence. W h e n r e a s s o c i a t i o n kinetics of ~2p l a b e l e d minicircle t~rom T. equiperdum was c a r r i e d out in the presence of u n l a b e l e d minicircles from T. gambiense, the rate of the reactions was significantly different from that c a r r i e d out with l a b e l e d D N A
BIOCH1MIE, 1981, 63, n ° 10.
FIG. 9. - - Hybridization o] ~P labeled kDNA rnaxicircles ]rom T. equiperdum to restriction enzyme fragments o] T. gambiense kDNA maxicircle : T. gambiense maxicircle was cleaved independently with endonucleases Hind IH, Hae HI and Eco R1 + Pstl. Electrophoresis in 0.5 per cent agarose gel was carried out as previously described [6], after which the DNA bands were denatured and transferred to a nitrocellulose filter and hybridized with 2 × 106 cpm of 32p labeled maxicircle from T. equiperdum according to Southern [15]. Autoradiographs were established. In lanes a UV photographs after EthBr staining, in lanes b corresponding autoradiographs. In lanes c and d, T. equiperdum controls : lane c, UV photograph after EthBr staining of Hind III cleavage fragments from T. equiperdum kDNA maxicircle ; lane d, corresponding autoradiograph after hybridization with T. equiperdum maxicircle probe. In this experiment a ,partial digestion product of the T. gambiense kDNA by Hind III can be noted.
Discussion. T h e c o m p o n e n t s of the k D N A n e t w o r k of T. gambiense, strain E l i a n e , have been a n a l y z e d a n d c o m p a r e d to those of the k D N A f r o m T. equiperdum, strain I.P., a very closely related species p r e v i o u s l y studied in detail [6]. T h e total D N A of this strain of T. gambiense, like the D N A of strain L a r i v i e r e p r e v i o u s l y studied [5], contains two distinct c o m p o n e n t s (figure 1) distinguishable by analytical ultracentrifugation analysis. A third c o m p o n e n t of p = 1.719 g / c m :~, d e s c r i b e d by N e w t o n and Burnett
k D N A o[ T. gambiense and T. equiperdum.
[19] in another strain, was not found. These authors had suggested that the presence of this component characterized T. gambiense and diffe-
763
of the total DNA, has a buoyant density of 1.6907 g / c m 3, a density very similar to that of T. e q u i p e r d u m k D N A (Tables I1 and liD. TABLE II.
Xbal a
b
Hinfl c
d
General properties of k D N A ]rom T. gambiense Eliane strain and T. equiperdum lnstitut Pasteur strain.
kDNA ? (gcm ~) S"20, w per cent from total DNA
FIG. 10. - - Hybridization o] J~P labeled kDNA minicircles Jrom T. equiperdum to Xba 1 ]ragment o] T. gambiense core-kDNA minicircles. T. gambiense core-kDNA minicircle was cleaved in I kb fragment by Xba I and
electrophoresis carried out in 2 per cent agarose gel. The DNA fragments were transferred, hybridized with about 2 × 106 cpm of 32p labeled minicircles from T. equiperdum and then autoradiographed. In lane a, photograph after EthBr staining ; in lane b, corresponding autoradiograph. In lanes c and d, T. equiperdum controls : lane c UV photograph after EthBr staining of Hin] I cleavage fragment from T. equiperdum core-kDNA minicircles ; lane d, corresponding autoradiogr~ph after DNA transfer and hybridization with T. equiperdum minicircle probe.
renciated this species from the other species of the subgenus Trypanozoon. The k D N A network of T. gambiense, which amounts to about 8 per cent BIOCHIMIE, 1981, 63, n o 10.
7". gambiense
T. equiperdum
Eliane Strain
I.P. Strain
1.691 2 000 8
1.691 1 500 6
The k D N A contains two molecular components topologically linked and differing by their base composition, buoyant density, molecular weight and susceptibility to restriction endonucleases. The maxicircles are seen in the electron microscope as long loops at the periphery of the network. In one experiment, 6 per cent of the networks, showed linear D N A molecules connected to maxicircles. We also observed 3 double branched molecules like that shown in figure 2 C. These molecules seem to be replicative intermediates of the Cairns' type with one segment broken during the preparation for electron microscopy or of the rolling circle type. We did not observe, on these molecules, the D-loops and expanded D-loops described in the mitochondrial D N A of other eukaryotic cells [21, 22]. Replicating minicirc!es had previously been seen and isolated [23, 24, 25], but this was the first time that replicating maxicircles were observed, probably due to the fact that these molecules are almost entirely hidden in the network. After incubation with various endonucleases (9 out of 12 tested) the maxicircles were liberated from the n~twork and selectively c!eaved into fragments representing a total homogeneous size of about 20 kb (table I), whereas the m"nicirctes of 1 kb size remained as a core-kDNA form I network, which could be recovered in the lower band of a CsC1-EthBr gradient (figure 3). The k D N A maxicircles have a buoyant density of 1.684 g/ cm a, a density 9 m g / c m :~ lower than that of the minicircles (figure 1 and tables II and III). It is possible to estimate the relative amount of mini and maxicircles in the network by analytical ultracentrifugation in CsCt gradient after selective cleavage by Pst I. The measurement of the area
G. Riou and M. Barrois.
764 under the scanned peak indicates maxicircles account for about 27 intact kDNA network (figure 1). DNA content of T. gambiense had
that the kDNA per cent of the Since the total previously been
species. Moreover experiments of hybridization after DNA transfer by the Southern blot technique show that there are at least two homologous regions. However the absence of restriction sites in
TABLE III.
Molecular components of the kDNA from T. gambiense Etiane strain and T. equiperdum Institut Pasteur strain. Minicircles
Maxicircles
T. gambiense T. equiperdum T. gambiense T. equiperdum Per cent of total kDNA Size (in kb) ? (g/cmu) AT per cent Number per network
73 1 1. 6933 66 (a) 4000
70 1. 012 (b) 1.6915 72.8 (b) 3000
27 20 1. 684 76 (a) 80
30 23 1.6835 76 (a) 50
(a) Values obtained from data of Schildkraut et al. [201. (b) Values obtained after complete nucleotide sequence [32].
found equal to 7.7 × 10 -14 g/cell [26], it can be estimated that one kDNA network contains about 80 maxicircles of 20 kb and 4000 minicircles of 1 kb. The values found for the kDNA network of T. equiperdum are lower (table III). The restriction cleavage sites of 7 restriction endonucleases on the maxicircles of T. gambiense have been mapped and compared with those of the T. equiperdum maxicircles which are about 3 kb longer. The relative positions of the restriction sites are different in these two closely related species. Such a mapping of maxicircles could be a useful method to characterize the trypanosome species. Indeed, the restriction cleavage maps of maxicircles have been determined in several other species of trypanosomes and some phylogenetic relationships have been established [2]. The maxicircle cleavage maps of several strains of T. brucei [27, 28, 29] have certain analogy with that of T. gambiense. Experiments of renaturation kinetics with 3~p labeled DNA have shown that T. gambiense and T. equiperdum maxicircles have a sequence homology of about 2 kb (figures 7 and 8). These results are in good agreement with recent work showing that in vivo maxicircle transcripts (9 S and 12 S RNAs corresponding to about 1500 nucleotides) have been found in several species of trypanosomes and that these transcripts cross-hybridize with maxicircle DNAs of other species [2, 3, 29]. The homologous D N A maxicircle segments in T. gambiense and T. equiperdum could correspond to the DNA regions transcribed in others
BIOCHIM1E, 1981, 60, n ° 10.
a large part of these two regions do not allow to more accurately evaluate the homologous sequences of about 8 kb, as drawn in figure 5. The analysis of T. gambiense minicircles by restriction endonucleases show that these molecules are significantly cleaved by only 3 out of 12 tested restriction endonucleases (figure 6). Furthermore the numerous fragments obtained after cleavage of the minicircles by Hinf I and Taq I + Xba I reveal a certain degree of sequence heterogeneity. Base sequence heterogeneity, initially found in T. cruzi minicircles [30] seems to be a general property of kDNA minicircles [2]. One exception is, however, known so f a r : according to renaturation kinetics and restriction endonucleases cleavage, T. equiperdum minicircles appear homogeneous [6, 31]. Furthermore, our hybridization experiments after DNA transfer on blotting filters (figure 10) show that T. gambiense and T. equiperdum minicircles have an homologous region to about 10 per cent by renaturation kinetics (data not shown). These results are in agreement with those of Barrois et al, [32] who have shown a sequence homology of about 130 nucleotides between T. equiperdum and T. brucei minicircles [33]. The presence of homologous region accounting for about 10 per cent of the minicircles in T. equiperdum, T. brucei and T, gambiense, three closely related species, suggests that this region is sufficiently important to be preserved. It could contain for example the region of initiation of replication.
kDNA o[ T. gambiense and T. equiperdum. From these observations it can be concluded that minicircle sequences change very rapidly from species to species as a consequence of a rapid evolution. This problem has been discussed in detail by Challberg and Englund [34]. Acknowledgements. The authors are undebted to Dr. R. Pautrizel ]or providing T. gambiense, to Dr. A. Kayser 1or ]ruitful discussion and to M. Gabillot [or skill]ul technical assistance. We thank also Dr. E. Delain ]or his help in electron microscopy. REFERENCES. l. Borst, P. & Hoeijmakers, J. H. J. (1979) Plasmid, 2, 20-40. 2. Englund, P. T. (1981) in <~Biochemistry and Physiology o / P r o t o z o a >> (Levandowsky, M. and Hutner, S. H. eds) Second Edition, Academic Press N. Y. Vol. 4, 333-383. 3. Simpson, L. & Simpson, A. M. (1978) Cell, 14, 169178. 4. Fouts, D. L. 8` Wolstenholme, D. R. (1979) Nucleic Acids Res., 6, 3785-3804. 5. Riou, G. & Pautrizel, R. (1967) C. R. Acad. Sei.. Ser. D, 265, 61-63. 6. Riou, G. & Saucier, J. M. (1979) J. Cell Biol., 82, 248-263. 7. Fromentin, H. (1955) Bull. Soc. Path. E~:ot., 48, 651-655. 8. Lanham, S. M. & Godfrey, D. G. (1970) E~ptl. Parasitol., 28, 521-534. 9. B6aard, J., Riou, G. & Saucier, J. M. (1979) N,cleic Acids Res., 6, 1941-1952. 10. Riou, G. $, Yot, P. (1977) Biochemistry, 16, 23902396. 11. Riou, G., Baltz, Th., Gabillot, M. s, Pautrizel, R. (1980) Mol. Biochem. Parasitol., 1, 97-105. 12. Mackey, J. K., Brackmann, K. H., Green, M. R. ,~ Green, M. (1977) Biochemistry, 16, 4478-4483. 13. Pellicer, A., Wigler, M., Axel, R. 8, Silverstein, S. (1978) Cell, 14, 133-141.
BIOCHIM1E, 1981, 63, n ° 10.
765
14. Sharp, F. A., Pettersson, U. & Sambrook, J. (1974) J. Mol. Biol., 86, 709-726. 15. Southern, E. M. (1975) J. Mol. Biol., 98, 503-517. 16. Delain, E., Brack, Ch., Lac6me, A. 8` Riou, G. (1972) in <> (H. Van den Bossche, Ed.) Academic Press Inc. N.Y. 167-184. 17. Riou, G. & Pautrizel, R. (1977) Biochem. Biophys. Res. Commun., 79, 1084-1091. 18. Riou, G., Belnat, P. s: B6nard, J. (1980) 1. Biol. Chem., 255, 5141-5144. 19. Newton, B. A. & Burnett, J. K. (1972) in ¢ Comparatire Biochemistry of Parasites >> (H. Van den Bossche, ed.), Academic Press Inc. N. Y., 127-138. 20. Schildkraut, C. L., Marmur, J. ~ Doty, P. (1962) J. Mol. Biol., 4, 430-443. 21. Kasamatsu, H., Robberson, D. L. & Vinograd, J. (1971) Proc. Natl. Acad. Sci. USA, 68, 2252-2257. 22. Paoletti, C. 8` Riou, G. (1973) in <
The kinetoplast DNA of Trypanosoma gambiense : Comparison with the kDNA of Trypanosoma equiperdum. Guy RIOU <> and Michel BARROIS. (Re~u le 12-6-1981, acceptd le 30-7-1981).
Unitg de Pharmacologie MoHculaire, lnstitut Gustave Roussy, 94800 Ville]uif, France.
R~sum~.
Summary.
Les composants mol~culaires du D N A kin~toplastique ( k D N A ) du Trypanosoma gambiense ont ~tO analys~s et comparLv i~ ceux du Trypanosoma equiperdum, espkce voisine. Le k D N A de T. gambiense comporte environ 80 maxicercles de 20 000 paires de base et 4 000 minicercles de 1 000 paires de base. La carte physique des sites de clivage de 7 endonucl~ases de restriction a ~t~ ~tablie sur le maxicercle de T. gambiense. La plupart de ces sites ont Ot£ observes sur le maxicercle de T. equiperdum, cependant leur position relative est dif[6rente. Des maxicercles pr#sentant des [ourches de r6plication ont ~t~ observes au microscope (lectronique. Des expdriences, d' une part de cin~tique de renaturation et d'autre part d'hybridation aprbs trans[ert du D N A sur [iltre, montrent que les maxicercles de T. gambiense et de T. equiperdum ont une partie de leur s~quence en bases commune. Les minicercles de T. gambiense ont leur s6quence en bases h~t~rogkne contrairement ~ ceIle des minicercles de T. equiperdum. Les minicercles de ces deux espkces ont @alement une partie de leur s6quence commune.
The molecular components of the kinetop!ast D N A ( k D N A ) network of Trypanosoma gambiense have been studied and compared with those o[ the very closely related species T. equiperdum, previously studied in detail. The k D N A o[ T. gambiense contains about 80 maxicircles of 20 kilobase pail's and 4000 minicircles o[ l kilobase pairs. The restriction cleavage sites o[ 7 restriction endonucleases have been mapped on the T. gambiense maxicircle. The majority o[ these sites were also [ound in T. equiperdum maxicircles ; however their relative positions which are di[[erent do not allow us to conclude to relatedness o[ maps. Maxicircles o[ the Cairn 07" of the rolling circle type have been observed and thought to be replicative intermediates. Experiments on renaturation kinetics and hybridization alter blotting trans!er, show that T. gambiense and T. equiperdum maxicircles' have base sequences in common. The T. gambiense minicircles are heterogeneous in base sequence, in contrast to the T. equiperdum minicircles which are homogeneous. The minicircles o[ the two species have also common base sequences.
Mots-clds : DNA mitochondrial / enzymes de restriction / hybridation mol~culaire.
Key-words: mitochondrial DNA / restriction enzymes /molecular hybridization.
Introduction.
and they are morphologically undistinguishable. Like all members of Kinetoplastida, they possess an unique mitochondrion with a specialized portion called kinetoplast, containing DNA (kDNA), which can be isolated as a complex network of high molecular weight. The general properties of the kDNA have recently been reviewed by Borst and Hoeijmakers [1] and by Engtund [2]. In all the species of trypanosomes so far studied, the kDNA is composed of two distinct molecular components, the minicircles and the maxicircles, found in variable proportions among different species. Their biological role remains unknown, although
Trypanosoma gambiense is the agent of sleeping sickness, which constitutes a serious risk to the health and life of million people in Africa. This
flagellate, transmitted by the tse-tse fly, undergoes a complicated cycle of development. T. equiperdum has a quite different infective cell cycie: il is venereally transmitted in Equines. Both trypanosomes belong to the same subgenus, Trypanozoon, © To whom all correspondence should be addressed.
756
G. R i o u and M. Barrois.
recent reports have indicated that, in some species, these molecules are partially transcribed [3, 4]. We had previously reported that T. e q u i p e r d u m a n d T. gambiense possess k D N A networks in nearly the same proportions (6 to 8 per cent of total D N A ) , that the k D N A has the same b u o y a n t density a n d that the minicircles have the same contour length [5, 6]. Analysis by restriction endonucleases a n d molecular h y b r i d i z a t i o n can successfully be applied to characterize the m o l e c u l a r c o m p o n e n t s of k D N A s a n d to establish phylogenetic relationships between two closely related t r y p a n o s o m e species. The k D N A of T. e q u i p e r d u m has previously b e e n studied in detail [6]. I n this work, the molecular c o m p o n e n t s of the T. gambiense k D N A are separated, characterized, a n d c o m p a r e d with the T. e q u i p e r d u m k D N A components.
Materials and Methods.
Strains and isolation of trypanosomes. The original trypanosome strains were obtained from the Institut Pasteur (Paris). T. equiperdum was isolated by Laveran about 60 to 70 years ago and T. gambiense strain Eliane by Vaucel [7] in 1951. These strains were maintained by laboratory mammals transfers. About 106 trypanosomes of each strain were separately injected in rats in order to obtain maximal infection in 3 to 4 days. The trypanosomes were isolated from blood, as described by Lanham and Godfrey [8]. The trypanosomes were washed 3 times with 0.15 M NaC1 0.015 M sodium citrate (SSC). Preparation and purification o] kDNA. The cells (about 200 mg wet weight) were lysed with 5 ml of sarkosyl (1 per cent in SSC), for 15 min at 37°C. Pronase (1 mg/ rnl), preincubated for 1 h at 37°C, was added to the lysate and the mixture was incubated for 2 h at 37°C. The lysate (2 vol) was layered on 20 per cent saacrose in SSC (1 vol) and centrifuged at 22 000 Irom for 1 h at 20°C in a Beckman SW 27 rotor. The collected pellet was then centrifuged in a CsC1-Ethidium bromide (EthBr) gradient (300 ~g/ml EthBr, 0.87 g/ml CsC1 to obtain n D 20-1.3900, 40 000 rpm, 24 h at 20°C in a Beckman 50 Ti rotor). The kDNA was recovered from the lower band of the gradient and the EthBr removed with isopropanol. Analytical ultracentrifugation. A Spinco model E analytical ultracentrifuge was used. Equilibrium density centrifugation in CsC1 was performed during 24 h, at 44 000 rpm and 25°C, using Micrococcus lysodeikticus DNA as density marker. Scanning was done at 265 nm using a Beckman photoelectric system. The sedimentation coefficients were measured by band sedimentation analysis BIOCHIMIE, 1981, 63, n o 10.
in 3 M CsCI, 1 mM Na a EDTA (pH 8.0) at 20°C with an ANJ rotor at 6,000 rpm as described by Brnard et al. [9]. Cleavage of kDNA by restriction endonucleases and gel electrophoresis. The restriction endonucleases were obtained from New England Biolabs (Beverly, Mass.) and used under the digestion conditions recommended for each enzyme [6]. The DNA fragments were separated by electrophoresis in vertical slab gels. Agarose gels (2 per cen0 were used to measure the molecular weight of the fragments up to 1.6 kb, while 0.5 per cent agarose-l.8 per cent polyacrylamide gels were used for larger fragments. The sizes of the fragments were estimated by comparing their migration with that of )~ DNA Hind HI fragments or Hae III T. cruzi k D N A fragments (calibrated by Hae III fragments of • X 174 DNA) [10]. In vitro synthesis o] 8sP-labeled kDNA. Form I minicircles were separated from the linearized maxicircles, after oleavage with Pst I restriction endonuclease, using a CsC1-EthBr gradient [6]. The mini and maxicircles were purified in a CsC1-Hoechst 33258 dye gradient [11]. The kDNA was nick-translated, according to the method of Mackey et al. [12] in the presence of [32p] dCTP and [~P] dATP (Amersham, specific activity 2 000 to 3 000 Ci/mmole). The size of the nick translated DNA fragments was estimated by comparison with sheared, 3H labeled Trypanosoma cruzi nuclear DNA of known size (S°20.w = 5.9). DNA polymerase I was purchased from Boehringer-Mannheim, and DNAse I (electrophoresis grade) from Worthington. The labeled kDNA was treated as described by Pellicer et al. [13] to remove fast reannealing material (15 to 20 per cent of the probe DNA).
Reassociation kinetics. 3zP-labeled DNA was heated for 20 min at 100°C in 44 mM Na phosphate pH 6.8 and reassociation was carried out at 68°C in 0.86 M NaCl, 86 mM Na phosphate pH 6.8 and 0.8 per cent SDS. 100 !~1 fractions were collected at various times and diluted with 0.9 ml of 0.14 M Na phosphate pH 6.8, 0.4 per cent SDS. The fraction of single and double-stranded DNA was determined by hydroxylapatite chromatography, as described by Sharp et al [14]. The labeled mini and maxicircles DNA gave 90 per cent and 85 per cent reassociation at saturation, respectively.
Hybridization experiments by the DNA transfer technique. Mini and maxicireles were cleaved by appropriate restriction enzymes and DNA fragments separated by electrophoresis in agarose gels. They were denatured in situ and blotted onto a nitroceUulose filter and then hybridized by the method of Southern [15]. Hybridization was performed with about 2 × 106 cpm of the a2p labeled DNA probes obtained as described above.
Electron microscopy o] DNA. kDNA networks were spread using the micro-diffusion technique previously described [16].
k D N A of T. gambiense and T. e q u i p e r d u m .
757
Results.
B
1.6906 1.7074 2 1.6909
c
1
rn /\
1.68441.6932 D
!L
rn
j'~
1.6931 FIG. 1. - -
Equilibrium density centri/ugation
o f T. g a m -
biense DNA in CsCl gradient. (A) total DNA; (B) intact kDNA form 1 ; ((2) kDNA form I after cleavage by endonuclease Pst I ; (D) mini¢ircle core-kDNA form I obtained after Pst I cleavage and CsC1-EthBr centrifugation. Micrococcus lysodeikticus DNA (~ = 1.731 g/cm 3) was used as density marker (m).
Fractionation and characterization of the molecular components of k D N A from T. gambiense. W h e n the total D N A extracted from T. gambiense was analyzed by e q u i l i b r i u m density centrifugation in a CsCI gradient at least two c o m p o n e n t s are o b s e r v e d : k D N A with a density 9 = 1.6907 + 0.001 g / c m 3 and a m a i n b a n d of D N A of density 9 = 1.7069 -+ 0.0001 g / c m "~ with a shoulder corresponding to D N A of lower density (figure 1A). T h e k D N A , which accounts for about 8 per cent of the total D N A , was purified by e q u i l i b r i u m density centrifugation in a CsCI gradient containing EthBr. T h e lower b a n d c o n t a i n e d k D N A form I which, after spreading for electron microscopy, presented the typical structure previously described for T. equiperdum [6, 17] (figure 2). Two distinct c o m p o n e n t s could be seen in the n e t w o r k : n u m e r o u s interlocked minicircles of 0.31 lxm long (1 kb) and D N A loops p r o t r u d i n g at the periphery of the n e t w o r k and identified as maxicircles (figure 2) (see below). A linear molecule is connected with some of these maxicircles (figure 2, a to e). After e x a m i n ; n g more than 200 networks of the k D N A preparation, we observed
TABLE 1. Molecular weights, in kb, o[ the fragments of maxicircle k D N A generated by restriction endonucleases from T. gambiense. Restriction endonuclease
Fragment
Bgl II
Bam HI
Pst [
Hpa II
Hae llI
Hind II1
1 2 3 4
20.0
20.0
20.0
10,3 9.2 09
14.0 3.5 1.0
7.4 6.5 6.2
1.0
20.4 Hind 1II Hind III Hind I11 Eco RI Barn HI Hpa II Eco RI Pst I 1
2 3 4 5 6 7
Eco RI
9.3 6 0 4 2
Barn HI Barn HI P~t I Bgl II 14,3 5 7
17 0 3 2
Pst I Bgl 11
Hind III Pst I
11.6 8,7
7,5 6.3 5.3
0.8
1.4
20.4
20 1
20.3
20 0
Eco RI Barn HI
Eco RI Hpa II
Eco RI Hae ill
Hae lIl Pst I
20 2
20.3
Hae III Hpa II Barn HI Barn HI
20 5 Hpa II Pst I
7.5 6.5 4.2 2.0
7.5 6.3 3,4 2.5 0.9
6,3 3.3 3.0 3.0 3,0 1.2 0.8
9.3 4.6 4.2 1.6 0.8
9.3 6.0 3.2 1.0 0.8
6.5 6.2 3.6 2.8 0,8 0.8
9.3 3.4 3.1 2.8 0.8 0.8
13.8 3.5 1.2 1.0 1.0
14.8 3.5 1.0 1.0
10 0 9.3 09
10.3 4.6 4.6 0.9
20.2
20.6
20.6
20.5
20.3
20.7
20.2
20.5
20.3
20.2
20 4
BIOCHIM1E, 1981, 63, n ° 10.
G. R i o u and M. Barrois.
758
FIG. 2. - -
Electron micrograph o] an intact kDNA network )'rom T. gambiense. Magnification of several regions showing three branched maxicircles are s h o w n in inserts a to e.
BIOCHIMIE, 1981, 63, n ° 10.
kDNA o[ T. gambiense and T. equiperdum. 12 networks with at least one three-branched maxicircle. The buoyant density of the purified kDNA network in CsC1 was equal to 1.6907 g/ml (average of 3 determinations) (figure 1B). The networks had a sedimentation coefficient of S~0,w = 2010, as measured by band sedimentation in 3 M CsC1 (average of 3 determinations). When the intact kDNA network was incubated with the restriction endonuclease Pst I two molecular components were obtained and identified by electron microscopy : linear molecules of about 20 kb (table I) identified as linearized maxicircles and a kDNA network containing only interlocked minicircles covalently closed, which we will refer to as minicircle core-kDNA. The linearized maxicircles have a buoyant density of 1.684 g/cm 3, as determined by equilibrium centrifugation in a CsC1 gradient (average of 4 determinations), which corresponds to an AT content of 76 per cent. According to the area under the peak of the scanner tracing (figure 1C), they account for 27 per cent of the intact networks (average of 4 determinations). The core-kDNA minicircles form I which had been recovered from the lower band of a CsCt-EthBr gradient (figure 3) had a buoyant density of 1.6933 g/cm "~ (figure 1 D) (average of 3 determinations).
759
weight were generated (figure 4). The molecular weights of these fragments are presented in table I. The results indicate that the DNA loops are extruding parts of maxicircles, whose uniform length is about 20 kb. Short fragments of about 1 kb corresponding to linearized minicircles were also 1
2
3
4
5
6
7
w
26.2 22.1~
N
8.8 6.1 4.0
2.1~ 1.8_
N
1.45 1.09 0.74
i
0.51 0.36
FIG. 3. - - Photograph oJ CsCI-EthBr gradient centHfugation oJ k D N A # o m T. gambiense. (A) intact k D N A network f o r m I ; (B) after cteavage by Pst I : the upper band is c o m p o s e d o f linearized maxicircles, while the lower band contains c o r e - k D N A minicircle f o r m I.
Cleavage map of maxicircle kDNA [rom T. gambiense. When the intact kDNA networks were digested with the restriction endonucleases Barn HI, Pst I, Bgl II, Hae III, Hpa II, Hind HI, Eco RI and analyzed by gel electrophoresis, one or more fragments of relatively high molecular BIOCHIM1E, 1981, 63, n ° 10.
W
FIG. 4. - - Electrophoresis in 0.5 per cent agarose - 1.8 per cent polyacrylamide gel oJ the [ragments generated by cleavage o] the k D N A network o] T. gambiense by restriction endonucleases. (t) T. cruzi k D N A minicircle fragments obtained by Hae III cleavage ; (2)), D N A fragments obtained by Hind III cleavage and used as size standards (the molecular weights are given in kb) ; (3) B g I I I ; (4) B g l l I + Barn H1 ; (5) Bgl II + Pst I ; (6) Pst I ; (7) Bam H I .
generated in small amounts by the endonucleases (less than 7 per cent of the minicircles). Molecules of the DNA band migrating in the 1.45 kb zone (figure 4) are composed of free minicircles form II as shown by electron microscopy after elution of the DNA band. These minicircles have been released from the kDNA network.
760
G. Riou and M. Barrois.
Since each restriction endonuclease has a limited number of cleavage sites per maxicircle, a physical map of the DNA molecule can be drawn. The unique cleavage site of Pst I is taken as origin. The data of table I obtained from experiments with single or double enzyme digests, enable location of the restriction enzyme cleavage sites on the maxicircles, as drawn in figure 5. However linearized minicircles and free minicircles produce electrophoresis bands which could comigrate with maxicircle DNA fragments. Some restriction sites T. equiperdum
Hae III
Cleavage of the minicircle core-kDNA from T. gambiense by restriction enzymes. When the minicircle core-kDNA was incubated with the restriction endonucleases used above, the minicircles
1
2
3
4
5
6
7
T. g a m b i e n s e
Pstl
_
Pstl
_
EcoRI
_
Eco
1.45 - - H p a II _
B g l II
H i n d Ill _ _
Hpall EcoRI
RI
1.09 *
A
0.74
Hpall_ _
H i n d III
0.36
Bgl II
Bgl I I _
_ H i n d III
_
__ E c o RI _
H i n d III
_
Haelll * BamHt H a e III *
__
H p a II
Hpall Haelll _ E c o RI BamHI--
"~" E c o R I
_ Hae III _ H p a II
_
Eco RI
_
Pst I
1
B Hindlll_
_ Haetll _Pst
Hindlll_
F[G. 6. - - Electrophoresis in 2 per cent agarose gel of the fragments generated by cleavage of minicircle corek D N A ]orm I from T. gambiense by restriction endonucleases. (1) minicir6le k D N A fragments from T. cruzi obtained by Hae III ~leavage ; (2) Xba I ; (3) Taq I + X b a I ; (4) Hinf I + X b a I ; (5) Hin[ I ; (6) Taq I + Hinf I ; (7) Taq I. The molecular weights are given in kb.
I
FIG. 5. - - Restriction maps o] mazicircle k D N A ]rom T. g a m b i e n s e Eliane strain and T. e q u i p e r d u m Institut
Pasteur strain. A and B are the regions of sequence homology, (*) probable restriction sites.
mapped on T. gambiense maxicircle are only probable sites compatible with the length of the maxicircle (figure 5 ). BIOCH1M1E, 1981, 63, n ° 10.
were not significantly cleaved. In contrast, the endonucleases Taq I, Hin[ I and Xba I cleaved all the minicircles in several fragments. Gel electrophoresis of single and double digests with the latter enzymes is presented in figure 6. The results show that the sum of the lengths of the DNA fragments generated is several times higher than the length of each minicircle. This observation indicates that T. gambiense minicircles are heterogeneous in base sequence. Do maxi or minicircle kDNA from T. gambiense have sequences in common with maxi or minicircle kDNA from T. equiperdum ? Maxicircles from T. gambiense were labeled in vitro with [a2p] dCTP and [3._,p] dATP and the reasso-
kDNA of T. gambiense and T. equiperdum. ciation kinetics of the labeled fragments were studied. The reannealing of labeled maxicircles k D N A follows simple second order kinetics, as previously described for T. equiperdum k D N A [6,
761
ments of t 1/2, about 8 per cent of the T. equiperdum maxicircles (23 kb) can hybridize with T. gambiense maxicircle (20 kb) (figure 8). The a'-'P labeled maxicircle k D N A fragments from T. equiperdum were also annealed in the presence of an 80-fold molar excess of unlabeled minicircle k D N A f r o m T. gambiense. The results, presented
A/ 2.0 03 03
2.0
-L r-
1.5
03 03
<.
,/f
1.5
I
10
t
I
t
20
30
40
t
50
time (hours) FIG. 7. Reassociation kinetics o[ s~p labeled kDNA maxicircle from T. gambiense. The linear form of kDNA maxicircles obtained by cleavage with Pst I, was isolated -
-
by equilibrium density centrifugation in CsC1-EthBr gradient and purified in a CsCI Hoechst 33258 gradient. The DNA labeled with 32p had a specific activity of 2.7 X 107 cpmLp,g and a size of about 300 to 400 nucleotides, as measured by alkaline sucrose gradient centrifugation. The reaction mixture contained 70 ng of labeled DNA per ml. Labeled DNA alone (--O--), or with 700 ng of unlabeled kDNA maxicircle from T. equiperdum (--A--). The reciprocal of the fraction of the single-strand DNA (1/f~0 is plotted as a function of time,
18]. The time required for half renaturation of maxicircle k D N A (t 1 / 2 ) was 72 h (figure 7). When reassociation was carried out in the presence of a 9-fold molar excess of unlabeled maxicircle k D N A from T. equiperdum, t 1 / 2 was of 40 h. This result reveals the presence of base sequences c o m m o n to both maxicircle populations (figure 7). We can estimate, from measurements of slopes, at about 10 per cent the base sequences c o m m o n to T. gambiense (20 kb) and T. equiperdum rnaxicircles (23 kb). Other reassociation experiments were done using a2p labeled maxicircle k D N A fragments from T. equiperdum alone, or in the presence of an 11-fold molar excess of unlabeled maxicircle k D N A f r o m T. gambiense. F r o m the measure-
BIOCHIMIE,
1981, 68, n ° 10.
10
20
30
40
time (,hours) FIG. 8. - - Reassociation kbzetics o/ ~P labeled kDNA maxicircle fragments Jrom T. equiperdum. The DNA had a specific activity of 4.0 × 107 cpm/Ixg. The reaction mixture contained 24 ng/ml of ,labeled DNA : alone ( - - 0 - - ) ; or in the presence of 48 ng/ml of unlabeled kDNA of T. equiperdum (--A--) ; or in the presence of 240 ng/ml of unlabeled kDNA maxicircle fragments from T. gambiense (--A--) ; or with 1 t~g/ml of unlabeled kDNA minicircle fragments from T. gambiense (--o--).
in figure 9, show that the rate of the reaction was not significantly modified, indicating that T. equiperdum maxicircles have no base sequence in comm o n with T. gambiense minicircles. These results were confirmed by hybridization after blotting transfer experiments onto nitrocellulose filters. The k D N A maxicircles from T. gambiense were cleaved by Hind III, Hae I I I and Eco R 1 + Pst I. T h e k D N A fragments were separated by electrophoresis in agarose gel, transferred onto nitrocellulose filter and then hybridized with zzp labeled D N A maxicircle from T. equiperdum. The results presented in figure 9 show that 32p D N A from T. equiperdum maxicircles hybridize with T. gambiense maxicircle and
G. Riou and M. Barrois.
762
that the h o m o l o g o u s sequences are d i s t r i b u t e d in two regions along the T. gambiense maxicircle. T h e i r length is a b o u t 8 kb (regions A and B of figure 5).
alone. W e can estimate, f r o m m e a s u r e m e n t s of t 1 / 2 , at a b o u t 10 per cent the base sequence c o m m o n to T. garnbiense and T. equiperdum minicircles ( d a t a not shown).
Eco R I Hind a
b
III c d
H a e III a
+ Pstl
b
a .
.
.
b
.
~i!:ri: ~.. : • ."
i
T h e minicircles f r o m the c o r e - k D N A of T. gambiense were linearized b y cleavage by Xba I. T h e 1 k b fragments g e n e r a t e d were t r a n s f e r r e d onto cellulose filter to be h y b r i d i z e d with ~2p labeled D N A from T. equiperdurn minicircles. T h e resuits p r e s e n t e d in figure 10, show that T. gambiense minicircles h y b r i d i z e with T. equiperdum minicircles, suggesting that only a p a r t of these two minicircles have a c o m m o n base sequence. W h e n r e a s s o c i a t i o n kinetics of ~2p l a b e l e d minicircle t~rom T. equiperdum was c a r r i e d out in the presence of u n l a b e l e d minicircles from T. gambiense, the rate of the reactions was significantly different from that c a r r i e d out with l a b e l e d D N A
BIOCH1MIE, 1981, 63, n ° 10.
FIG. 9. - - Hybridization o] ~P labeled kDNA rnaxicircles ]rom T. equiperdum to restriction enzyme fragments o] T. gambiense kDNA maxicircle : T. gambiense maxicircle was cleaved independently with endonucleases Hind IH, Hae HI and Eco R1 + Pstl. Electrophoresis in 0.5 per cent agarose gel was carried out as previously described [6], after which the DNA bands were denatured and transferred to a nitrocellulose filter and hybridized with 2 × 106 cpm of 32p labeled maxicircle from T. equiperdum according to Southern [15]. Autoradiographs were established. In lanes a UV photographs after EthBr staining, in lanes b corresponding autoradiographs. In lanes c and d, T. equiperdum controls : lane c, UV photograph after EthBr staining of Hind III cleavage fragments from T. equiperdum kDNA maxicircle ; lane d, corresponding autoradiograph after hybridization with T. equiperdum maxicircle probe. In this experiment a ,partial digestion product of the T. gambiense kDNA by Hind III can be noted.
Discussion. T h e c o m p o n e n t s of the k D N A n e t w o r k of T. gambiense, strain E l i a n e , have been a n a l y z e d a n d c o m p a r e d to those of the k D N A f r o m T. equiperdum, strain I.P., a very closely related species p r e v i o u s l y studied in detail [6]. T h e total D N A of this strain of T. gambiense, like the D N A of strain L a r i v i e r e p r e v i o u s l y studied [5], contains two distinct c o m p o n e n t s (figure 1) distinguishable by analytical ultracentrifugation analysis. A third c o m p o n e n t of p = 1.719 g / c m :~, d e s c r i b e d by N e w t o n and Burnett
k D N A o[ T. gambiense and T. equiperdum.
[19] in another strain, was not found. These authors had suggested that the presence of this component characterized T. gambiense and diffe-
763
of the total DNA, has a buoyant density of 1.6907 g / c m 3, a density very similar to that of T. e q u i p e r d u m k D N A (Tables I1 and liD. TABLE II.
Xbal a
b
Hinfl c
d
General properties of k D N A ]rom T. gambiense Eliane strain and T. equiperdum lnstitut Pasteur strain.
kDNA ? (gcm ~) S"20, w per cent from total DNA
FIG. 10. - - Hybridization o] J~P labeled kDNA minicircles Jrom T. equiperdum to Xba 1 ]ragment o] T. gambiense core-kDNA minicircles. T. gambiense core-kDNA minicircle was cleaved in I kb fragment by Xba I and
electrophoresis carried out in 2 per cent agarose gel. The DNA fragments were transferred, hybridized with about 2 × 106 cpm of 32p labeled minicircles from T. equiperdum and then autoradiographed. In lane a, photograph after EthBr staining ; in lane b, corresponding autoradiograph. In lanes c and d, T. equiperdum controls : lane c UV photograph after EthBr staining of Hin] I cleavage fragment from T. equiperdum core-kDNA minicircles ; lane d, corresponding autoradiogr~ph after DNA transfer and hybridization with T. equiperdum minicircle probe.
renciated this species from the other species of the subgenus Trypanozoon. The k D N A network of T. gambiense, which amounts to about 8 per cent BIOCHIMIE, 1981, 63, n o 10.
7". gambiense
T. equiperdum
Eliane Strain
I.P. Strain
1.691 2 000 8
1.691 1 500 6
The k D N A contains two molecular components topologically linked and differing by their base composition, buoyant density, molecular weight and susceptibility to restriction endonucleases. The maxicircles are seen in the electron microscope as long loops at the periphery of the network. In one experiment, 6 per cent of the networks, showed linear D N A molecules connected to maxicircles. We also observed 3 double branched molecules like that shown in figure 2 C. These molecules seem to be replicative intermediates of the Cairns' type with one segment broken during the preparation for electron microscopy or of the rolling circle type. We did not observe, on these molecules, the D-loops and expanded D-loops described in the mitochondrial D N A of other eukaryotic cells [21, 22]. Replicating minicirc!es had previously been seen and isolated [23, 24, 25], but this was the first time that replicating maxicircles were observed, probably due to the fact that these molecules are almost entirely hidden in the network. After incubation with various endonucleases (9 out of 12 tested) the maxicircles were liberated from the n~twork and selectively c!eaved into fragments representing a total homogeneous size of about 20 kb (table I), whereas the m"nicirctes of 1 kb size remained as a core-kDNA form I network, which could be recovered in the lower band of a CsC1-EthBr gradient (figure 3). The k D N A maxicircles have a buoyant density of 1.684 g/ cm a, a density 9 m g / c m :~ lower than that of the minicircles (figure 1 and tables II and III). It is possible to estimate the relative amount of mini and maxicircles in the network by analytical ultracentrifugation in CsCt gradient after selective cleavage by Pst I. The measurement of the area
G. Riou and M. Barrois.
764 under the scanned peak indicates maxicircles account for about 27 intact kDNA network (figure 1). DNA content of T. gambiense had
that the kDNA per cent of the Since the total previously been
species. Moreover experiments of hybridization after DNA transfer by the Southern blot technique show that there are at least two homologous regions. However the absence of restriction sites in
TABLE III.
Molecular components of the kDNA from T. gambiense Etiane strain and T. equiperdum Institut Pasteur strain. Minicircles
Maxicircles
T. gambiense T. equiperdum T. gambiense T. equiperdum Per cent of total kDNA Size (in kb) ? (g/cmu) AT per cent Number per network
73 1 1. 6933 66 (a) 4000
70 1. 012 (b) 1.6915 72.8 (b) 3000
27 20 1. 684 76 (a) 80
30 23 1.6835 76 (a) 50
(a) Values obtained from data of Schildkraut et al. [201. (b) Values obtained after complete nucleotide sequence [32].
found equal to 7.7 × 10 -14 g/cell [26], it can be estimated that one kDNA network contains about 80 maxicircles of 20 kb and 4000 minicircles of 1 kb. The values found for the kDNA network of T. equiperdum are lower (table III). The restriction cleavage sites of 7 restriction endonucleases on the maxicircles of T. gambiense have been mapped and compared with those of the T. equiperdum maxicircles which are about 3 kb longer. The relative positions of the restriction sites are different in these two closely related species. Such a mapping of maxicircles could be a useful method to characterize the trypanosome species. Indeed, the restriction cleavage maps of maxicircles have been determined in several other species of trypanosomes and some phylogenetic relationships have been established [2]. The maxicircle cleavage maps of several strains of T. brucei [27, 28, 29] have certain analogy with that of T. gambiense. Experiments of renaturation kinetics with 3~p labeled DNA have shown that T. gambiense and T. equiperdum maxicircles have a sequence homology of about 2 kb (figures 7 and 8). These results are in good agreement with recent work showing that in vivo maxicircle transcripts (9 S and 12 S RNAs corresponding to about 1500 nucleotides) have been found in several species of trypanosomes and that these transcripts cross-hybridize with maxicircle DNAs of other species [2, 3, 29]. The homologous D N A maxicircle segments in T. gambiense and T. equiperdum could correspond to the DNA regions transcribed in others
BIOCHIM1E, 1981, 60, n ° 10.
a large part of these two regions do not allow to more accurately evaluate the homologous sequences of about 8 kb, as drawn in figure 5. The analysis of T. gambiense minicircles by restriction endonucleases show that these molecules are significantly cleaved by only 3 out of 12 tested restriction endonucleases (figure 6). Furthermore the numerous fragments obtained after cleavage of the minicircles by Hinf I and Taq I + Xba I reveal a certain degree of sequence heterogeneity. Base sequence heterogeneity, initially found in T. cruzi minicircles [30] seems to be a general property of kDNA minicircles [2]. One exception is, however, known so f a r : according to renaturation kinetics and restriction endonucleases cleavage, T. equiperdum minicircles appear homogeneous [6, 31]. Furthermore, our hybridization experiments after DNA transfer on blotting filters (figure 10) show that T. gambiense and T. equiperdum minicircles have an homologous region to about 10 per cent by renaturation kinetics (data not shown). These results are in agreement with those of Barrois et al, [32] who have shown a sequence homology of about 130 nucleotides between T. equiperdum and T. brucei minicircles [33]. The presence of homologous region accounting for about 10 per cent of the minicircles in T. equiperdum, T. brucei and T, gambiense, three closely related species, suggests that this region is sufficiently important to be preserved. It could contain for example the region of initiation of replication.
kDNA o[ T. gambiense and T. equiperdum. From these observations it can be concluded that minicircle sequences change very rapidly from species to species as a consequence of a rapid evolution. This problem has been discussed in detail by Challberg and Englund [34]. Acknowledgements. The authors are undebted to Dr. R. Pautrizel ]or providing T. gambiense, to Dr. A. Kayser 1or ]ruitful discussion and to M. Gabillot [or skill]ul technical assistance. We thank also Dr. E. Delain ]or his help in electron microscopy. REFERENCES. l. Borst, P. & Hoeijmakers, J. H. J. (1979) Plasmid, 2, 20-40. 2. Englund, P. T. (1981) in <~Biochemistry and Physiology o / P r o t o z o a >> (Levandowsky, M. and Hutner, S. H. eds) Second Edition, Academic Press N. Y. Vol. 4, 333-383. 3. Simpson, L. & Simpson, A. M. (1978) Cell, 14, 169178. 4. Fouts, D. L. 8` Wolstenholme, D. R. (1979) Nucleic Acids Res., 6, 3785-3804. 5. Riou, G. & Pautrizel, R. (1967) C. R. Acad. Sei.. Ser. D, 265, 61-63. 6. Riou, G. & Saucier, J. M. (1979) J. Cell Biol., 82, 248-263. 7. Fromentin, H. (1955) Bull. Soc. Path. E~:ot., 48, 651-655. 8. Lanham, S. M. & Godfrey, D. G. (1970) E~ptl. Parasitol., 28, 521-534. 9. B6aard, J., Riou, G. & Saucier, J. M. (1979) N,cleic Acids Res., 6, 1941-1952. 10. Riou, G. $, Yot, P. (1977) Biochemistry, 16, 23902396. 11. Riou, G., Baltz, Th., Gabillot, M. s, Pautrizel, R. (1980) Mol. Biochem. Parasitol., 1, 97-105. 12. Mackey, J. K., Brackmann, K. H., Green, M. R. ,~ Green, M. (1977) Biochemistry, 16, 4478-4483. 13. Pellicer, A., Wigler, M., Axel, R. 8, Silverstein, S. (1978) Cell, 14, 133-141.
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14. Sharp, F. A., Pettersson, U. & Sambrook, J. (1974) J. Mol. Biol., 86, 709-726. 15. Southern, E. M. (1975) J. Mol. Biol., 98, 503-517. 16. Delain, E., Brack, Ch., Lac6me, A. 8` Riou, G. (1972) in <